<b>Bijsluiter</b>. De hyperlink naar het originele document werkt niet meer. Daarom laat Woogle de tekst zien die in dat document stond. Deze tekst kan vreemde foutieve woorden of zinnen bevatten en de opmaak kan verdwenen of veranderd zijn. Dit komt door het zwartlakken van vertrouwelijke informatie of doordat de tekst niet digitaal beschikbaar was en dus ingescand en vervolgens via OCR weer ingelezen is. Voor het originele document, neem contact op met de Woo-contactpersoon van het bestuursorgaan.<br><br>====================================================================== Pagina 1 ======================================================================

<pre>Thiram
(CAS No: 137-26-8)
Health-based Reassessment of Administrative Occupational Exposure Limits
Committee on Updating of Occupational Exposure Limits,
a committee of the Health Council of the Netherlands
No. 2000/15OSH/090, The Hague, 22 October 2003
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<pre>Preferred citation:
Health Council of the Netherlands: Committee on Updating of Occupational
Exposure Limits. Thiram; Health-based Reassessment of Administrative
Occupational Exposure Limits. The Hague: Health Council of the Netherlands,
2003; 2000/15OSH/090.
all rights reserved
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<pre>1     Introduction
      The present document contains the assessment of the health hazard of thiram by
      the Committee on Updating of Occupational Exposure Limits, a committee of
      the Health Council of the Netherlands. The first draft of this document was
      prepared by I Gubbels-van Hal, M.Sc. (NOTOX BV, ‘s-Hertogenbosch, the
      Netherlands).
           The evaluation of the toxicology of thiram has been based on the reviews by
      the American Conference of Industrial Hygienists (ACG99) and Edwards et al.
      in the ‘Handbook of Pesticide Toxicology’ (Edw91). Where relevant, the original
      publications were reviewed and evaluated as will be indicated in the text. In
      addition, in September 1999, literature was searched in the on-line databases
      Medline, Toxline, and Chemical Abstracts, starting from 1965/66, and using the
      following key word: 137-26-8. Data of unpublished studies were generally not
      taken into account. Exceptions were made for studies that were summarised and
      evaluated by the FAO/WHO Joint Meeting of Pesticide Residues (FAO93). The
      final search was carried out in Toxline and Medline in December 2002.
           In April 2003, the President of the Health Council released a draft of the
      document for public review. Comments were received by the following
      individuals and organisations: A Aalto (Ministry of Social Affairs and Health,
      Tampere, Finland) and M Laget (Taminco, Ghent, Belgium). These comments
      were taken into account in deciding on the final version of the document.
2     Identity
      name                  :   thiram
      synonyms              :   tetramethylthiuram disulphide; tetramethylthioperoxydicarbonic diamide;
                                bis(dimethylthiocarbamoyl) disulfide; TMT, TMTD, TMTDS.
      molecular formula     :   C6H12N2S4
      structural formula    :
      CAS number            :   137-26-8
090-3 Thiram
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<pre>3     Physical and chemical properties
      molecular weight                            : 240.4
      melting point                               : 155-156oC
      boiling point                               : decomposes
      flash point                                 : 89oC (closed cup)
      vapour pressure                             : at 25oC: 2 x 10-3 Pa
      solubility in water                         : at 25oC: 3 mg/100 mL
      log P                                       : 1.73
            octanol/water
      conversion factors                          : not applicable
      Data from ACG99, NLM02.
      Thiram is a white to light yellow crystalline powder. It is reported to be stable
      under normal storage conditions, but may loose activity after prolonged exposure
      to air, heat, and moisture (ACG99, Dal88, NLM02).
4     Uses
      Thiram is a protective fungicide applied in agricultural to foliage to control
      Botrytis species on grapes, lettuces, ornamentals, soft fruit, and vegetables. It
      also controls rust on ornamentals, scab, and storage diseases on apple and pear.
      In addition, it is used in seed treatments and as a repellent for rodents and certain
      large animals that cause damage to field crops. In agriculture, thiram is used as a
      wettable powder with up to 800 gram of active ingredient per kg (ACG99,
      Edw91, Lie94, NLM02). Thiram is also an ingredient of medicated soaps,
      suntan, and antiseptic sprays (Dal88). A major use of thiram is as a vulcanising
      accelerator in rubber processing (IARC91, NLM02). It is also used as a
      lubricating oil additive (ACG99).
           According to the database of the Dutch Pesticide Authorisation Board
      (CTB)*, thiram is at present registered in the Netherlands for use as an active
      ingredient in fungicides and paints for specified applications.
*     at: http://www.ctb-wageningen.nl/geel.html.
090-4 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>5     Biotransformation and kinetics
      Human data
      Following oral administration of thiram to human volunteers, CS2 was detected
      in expired air (Lie94).
      Animal data
      Charles River rats (n=5/sex/dose level) were given 14C-thiram (radiochemical
      purity: 100%) as single oral (gavage) doses of 1.9 or 125 mg/kg bw. At both
      doses, 25 and 3% of the administered radioactivity were excreted in the urine and
      faeces, respectively, within 7 days after dosing. At day 7, residues in tissues
      amounted to 3% of the administered radioactivity, most of it in blood, bone, and
      liver. Although excretion of radioactivity in expired breath was not determined,
      the author assumed that 70% of the administered thiram was expired as CO2 or
      other volatile metabolites (Gay87). In another study, male Charles River rats
      (n=5) were given an oral (feed) dose of 14C-thiram (purity: 92.4%) equivalent to
      1.5 mg/kg bw. Within 72 hours after administration, 41% of the administered
      radioactivity was eliminated in expired air (CS2 and CO2), 38% in the urine, and
      20% in the faeces and the gastrointestinal tract. At 72 hours after dosing, 6% of
      the administered radioactivity was retained in the carcass. The total recovery of
      radioactivity averaged 105%. From these data, it was concluded that 85% of the
      dose was absorbed into the systemic circulation of the rat, assuming that no
      absorbed radioactivity was eliminated via the bile into the faeces (Hil89).
          Sprague-Dawley rats (n=5/sex) received a single oral dose of 14C-thiram
      (radiochemical purity: >98%), following 14-day pre-treatment with non-
      radiolabelled thiram at a level of 2 mg/kg bw. Within 96 hours, 41-48, 35-40, and
      2-5% of the dose were excreted in expired air, urine, and faeces, respectively.
      Most of the radioactivity in urine and expired air (amounts not specified) was
      excreted within the first 12 hours after dosing. In males and females, 83.7% and
      89.6% of the administered dose was eliminated from the body, respectively,
      within 4 days. At 96 hours after dosing, traces of radioactivity were found in
      tissues, the highest levels in liver, kidneys, and blood cells, and the lowest in
      brain, plasma, and skeletal muscle. Total recovery was 85 and 93% for males and
      females, respectively (Nom90). The metabolism of thiram was studied in Charles
      River rats (n=3), following a single oral dose of 2.1 to 2.5 mg 14C-thiram/kg bw.
      Within 3 days after administration, 25-43% of radioactivity was excreted in the
090-5 Thiram
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<pre>      urine and an average of 61% in exhaled breath. The total radioactivity in urine
      and expired breath averaged 94% of the dose. Results indicated that metabolites
      in exhaled breath were mainly CO2, CS2, and COS. However, no characterisation
      or quantification of these metabolites could be made. Identification of
      metabolites in urine was not reported (Nor89). In another metabolism study,
      Charles River rats (n=2/sex) were given thiram via the diet at a dose equivalent
      to approximately 2.5 mg/kg bw/day for 9 weeks, followed by a single oral dose
      of 14C-thiram. Within 24 hours after administration, approximately 60% of the
      radioactivity was eliminated in exhaled breath as CS2 and 30% in urine. In the
      urine, 5 polar metabolites were identified: thiosulphenic acid (10% of the dose),
      an alanine conjugate of dimethyldithiocarbamate (9% of the dose), a glucuronide
      conjugate of dimethyldithiocarbamate (7% of the dose), an alanine derivate of
      CS2 (3% of the dose), and the methyl ester of dimethyldithiocarbamate (2% of
      the dose). No unchanged thiram was detected in the urine. Identification of these
      metabolites demonstrated that thiram is biotransformed through reduction of the
      disulphide bond, leading to the formation of dimethyldithiocarbamate, and
      subsequent reactions of the thiol moiety to form oxidative and conjugative polar
      products (McM91). The metabolic pathway of thiram is shown in Figure 1 (see
      Annex I).
           When male Sprague-Dawley rats were given intraperitoneal injections of 15,
      30, or 60 mg thiram/kg bw, CS2 was excreted in exhaled breath in amounts of
      2.6, 26, or 120 nmol within 5 hours after administration (Dal86). In an older
      study, guinea pigs treated with single intraperitoneal doses of 10, 25, or 80
      mg/kg bw excreted CS2 in exhaled breath in amounts of 0.6 to 5.2% of the dose
      within 3 hours after treatment (Mer65).
6     Effects and mechanism of action
      Human data
      Excessive exposure to thiram may produce skin irritation with erythema and
      urticaria, conjunctivitis, mucous membrane irritation, and upper respiratory tract
      irritation (Dal88, Due87, NLM02).
      Reports on allergic contact dermatitis have been published in workers in the
      rubber industry using thiram as a catalytic accelerator for the vulcanisation
      process of rubber (Gau57, Sch33), in subjects using medicated soap containing
      thiram (Bae54, Bla56), in subjects following contact with plants (Sau01) or a
      golf green (She64) sprayed with thiram-containing fungicides, and in workers
090-6 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>      who had worn rubber gloves containing thiram (Ket84, Lis87). Contact
      dermatitis has also been reported in haemodialysed patients, probably by contact
      with rubber in haemodialysis equipment (Kru87, Pen76). More recently, a 41-
      year old woman developed acute exudative dermatitis after application of
      eardrops containing thiram to her dogs (Dwy97). In a retrospective study, the
      relative frequency of sensitisation to thiram was investigated in 2933 patients.
      Subjects were patch tested with thiram mix, comprising 0.25% tetraethylthiuram
      disulphide, 0.25% dipentamethylene disulphide, 0.25% tetramethylthiuram
      monosulphide, and 0.25% tetramethylthiuram disulphide, each in petrolatum.
      The observed frequency of thiram sensitisation was 2.8% (Knu96).
          A case report of Henoch-Schönlein purpura was presented of a 23-year-old
      Mexican tree planter, handling seedlings treated with a solution of 42% thiram.
      Initial symptoms included abdominal pain, generalised arthralgias, and
      progressive purpuric rash. Later, he developed fever, malaise, severe nausea and
      vomiting, haematemesis, and melena. After hospitalisation, symptoms consisting
      of hypertension, palpable purpura, tenderness of the joints (thickened),
      abdominal pain, and black stool were noted. Haematological investigations
      showed an increased haemoglobin level and an increased erythrocyte
      sedimentation rate. A high serum IgA level, but a low serum albumin level was
      found. Leukocytoclastic vasculitis and granular deposits outlining or within the
      blood vessel walls were found at skin biopsy. Gastroduodenoscopy revealed a
      small Mallory Weiss tear and diffuse vasculitic erosions in the duodenum. The
      man recovered on corticosteroid therapy, but remained to have asymptomatic
      orthostatic hypotension (Due87).
          In a group of 223 workers (42 men and 181 women), mostly aged between 20
      and 50 years, engaged in the manufacture of thiram for more than 3 years, an
      excess of ocular irritation, coughing, thoracic pain, tachycardia, epistaxis, dermal
      lesions, myocardiodystrophia, liver dysfunction, and asthenia were reported
      compared to a unexposed control group of 193 persons. Thyroid gland disorders
      appeared 7-fold more frequently than in the control group. One case of a
      malignant lesion of the thyroid was reported in a subgroup of 105 thiram workers
      (IARC91).
      In pregnant female operators who had been allegedly exposed to thiram during
      30-90% of their working time, an increased incidence of late gestosis, a
      decreased urinary oestriol level (week 33-37), and an increased number of
      abortions and premature births were reported. Incidence and severity of these
      effects were correlated with duration of employment. Thiram exposure levels
      were not reported (And93).
090-7 Thiram
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<pre>      Intolerance to alcohol has been observed in workers exposed to thiram,
      manifested by flushing of face, palpitation, rapid pulse, dizziness, and
      hypotension. These effects are thought to be due to increased blood acetaldehyde
      levels after alcohol consumption, caused by thiram-induced blocking of the
      metabolism of acetaldehyde (Edw91, Rei66).
      Animal data
      Irritation and sensitisation
      Thiram is irritating to the skin and slightly irritating to the eyes of rabbits. Skin
      sensitisation in rabbits, and sensitisation by the subcutaneous and tracheal routes
      have been reported (ACG99, Edw91). No further details were given.
      Acute toxicity
      Results of acute lethal toxicity tests with thiram are summarised in Table 1.
      Table 1 Summary of acute toxicity studies with thiram in experimental animals.
      exposure route     species (strain; sex)               LC50/LD50 (duration)    reference
                                                                            3
      inhalation          rat                                300-1000 mg/m (4 hours) NLM02
                         rat                                 >100 mg/m3 a            Deb85
      dermal             rat (Sherman; male, female)         >2000 mg/kg bw          Gai69
      oral                rat                                865 mg/kg bw            Leh51
                         rat (Sherman; male)                 640 mg/kg bw            Gai69
                         rat (Sherman; female)               620 mg/kg bw            Gai69
                         rat (Charles River ; male)          4000 mg/kg bw           Lee78
                         rat (Charles River ; female)        1900 mg/kg bw           Lee78
                         mouse (Charles River; male)         4000 mg/kg bw           Lee78
                         mouse (Charles River; female)       3800 mg/kg bw           Lee78
                         mouse (NMRI; female)                2300 mg/kg bw           Mat73
                         rabbit                              210 mg/kg bw            Wor87
      a
           Exposure duration not given.
      The oral LD50 values found for thiram show a broad range of values between and
      within species. A reason for these variations might be the use of different
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<pre>      formulations in the different experiments; however, no details on the
      composition of tested materials were provided.
           Clinical signs of intoxication develop slowly and are characterised by
      rumpled fur and rapid breathing, and, then, ataxia, tremor, and dyspnoea. Death
      is preceded by convulsions (ACG99). Pathological examination after oral
      application revealed hyperaemia, focal ulceration of the gastrointestinal tract,
      focal necrosis of the liver and renal tubules, and patchy demyelination in the
      cerebellum and medulla (Dal88).
      Short-term toxicity
      Groups of 20 male CD rats received thiram via the diet at doses equivalent to 0,
      30, 58, or 132 mg/kg bw for 13 weeks. Mortality occurred in 6/20 and 1/20
      animals of the high- and mid-dose groups, respectively. Body weight gain and
      food intake were reduced dose dependently. No haematological abnormalities
      were reported, but clinical chemistry showed slight elevations in liver function
      tests (alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT))
      at the high dose. In addition, microscopic examination also revealed moderate
      tubular degeneration of the testes with atypical spermatids in the epididymis in
      animals of the high-dose group. No statistical evaluation of the data was
      performed. The LOAEL was 30 mg/kg bw/day, based on reduced food intake
      and body weight gain (Lee78).
           In another study, groups of 6 male Wistar rats were given thiram via the diet
      at doses equivalent to approximately 0, 11, 15, 22.5, 30, 45, or 60 mg/kg bw/day
      for 29 days. Pair-fed control groups (6 rats/group) were assigned to each of the 6
      groups receiving thiram. For each parameter studied and each thiram dose,
      values for treated rats were compared to those for controls. Mean food intake and
      body weight were significantly reduced in a dose-dependent manner at 15 mg/kg
      bw/day and above. Absolute renal and testicular weights were significantly
      decreased at 45 mg/kg bw/day and above, and absolute seminal vesicle weights
      at 22.5 mg/kg bw/day and above. No haematological, clinical chemical, or
      histological data were shown. The NOAEL was 11 mg/kg bw/day, based on
      reduced food intake and body weight (Low80).
           In an unpublished 13-week study, CD rats (n=10/sex/group) received thiram
      via the diet at doses equivalent to 0, 2.5, 25, or 50 mg/kg bw/day. At 50 and 25
      mg/kg bw/day, body weights, body weight gains, and food consumption were
      significantly reduced in both sexes. In these dose groups, female animals had
      reduced haemoglobin, haematocrit, and red blood cell counts. White blood cell
      count was increased in both sexes, and clinical chemical tests showed decreased
090-9 Thiram
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<pre>       total protein and albumin plasma levels and increased urea levels. A reduction in
       absolute liver weight and an increase in relative liver weight were also noted in
       these dose groups, compared to the control group. Macroscopic examination
       showed areas of erosion of the non-glandular stomach, and the mesenteric lymph
       nodes were diffusely red. Microscopically, the mucosa of the stomach had focal
       areas of ulceration and/or mucosal hyperplasia, accompanied by some
       submucosal inflammation and oedema. The NOAEL was 2.5 mg/kg bw/day
       (Keh88).
           In an unpublished 28-day study, Crl:CD mice (n=10/sex/group) were given
       thiram via the diet at doses equivalent to 0, 54, 108, or 201 mg/kg bw/day for
       males and 0, 62, 118, or 241 mg/kg bw/day for females. Food consumption and
       body weights were reduced at all dose levels in a dose-related fashion. Males
       showed a dose-related decrease in haemoglobin, haematocrit, or red blood cell
       count at all dose levels. Females had increased platelet counts at the high- and
       mid-dose level. Relative brain, kidney, and liver weights were increased in a
       dose-related manner at all dose levels. The LOAEL was 54 mg/kg bw/day, the
       lowest dose tested (Keh89a).
           In an unpublished 13-week study, beagle dogs (n=4/sex/group) were fed
       thiram at doses equivalent to 0, 2.2, 6.9, or 12 mg/kg bw/day for males and 0,
       2.3, 7.3, or 13 mg/kg bw/day for females. At the high dose, food consumption
       and body weights were significantly reduced in both sexes. At all dose levels, red
       blood cell count and plasma total protein and albumin levels were reduced in
       both males and females. Haemoglobin and haematocrit levels were decreased in
       females at the high dose only. There were no treatment-related macroscopic or
       microscopic abnormalities. In this study, 2.2 mg/kg bw/day, the lowest dose
       tested , was a LOAEL (Keh89b).
           In another unpublished study, beagle dogs (n=6/sex/group) were given thiram
       via the diet at dose levels equivalent to 0, 0.84, 2.6, or 7.4 mg/kg bw/day for
       males and 0, 0.9, 2.5, or 7.2 mg/kg bw/day for females, for 52 weeks. No signs of
       intoxication were noted. Ophthalmic examination at the end of treatment did not
       show abnormalities. At the high dose, red blood cell count was reduced in males
       and plasma total protein and albumin in both sexes. At 2.6 mg/kg bw/day, plasma
       total protein was lower in males only. Absolute and relative liver weights in
       males were significantly increased at 2.6 mg/kg bw/day and above. In females,
       relative liver weights were increased at the high dose only. No treatment-related
       macroscopic or microscopic changes were found. The NOAELs were 0.84 and
       2.5 mg/kg bw/day for male and female dogs, respectively (Keh91a).
           Beagle dogs (n=4/sex/group) received daily oral (capsule) doses of thiram of
       0, 0.4, 4, or 40 mg/kg bw for 104 weeks. The dogs in the high-dose group had
090-10 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>       severe signs of intoxication, including nausea, vomiting, excessive salivation,
       anorexia, occasional clonic convulsions, and decreased body weights, and all
       were subjected to unscheduled necropsy between weeks 21 and 29. The dogs had
       also ophthalmological changes such as fundal haemorrhage, miosis, and
       desquamation of the retina. Haematological and clinical chemical tests at week
       13 of treatment showed a reduced haemoglobin, haematocrit, and red blood cell
       counts and increased in plasma alkaline phophatase, ALAT, and ASAT levels. At
       necropsy, 2 males and 3 females showed fatty degeneration, atrophy, and focal
       necrosis of hepatocytes at centrilobular and midlobular regions of the liver.
       Kupfer cell pigmentation (haemosiderin) was observed in 1 male and 2 females.
       Other effects observed included swelling and vacuolisation of the proximal
       tubuli of the kidney (2 females), follicular cell and C-cell hyperplasia of the
       thyroid (1 male and 1 female), and retina lesions (2 males and 3 females). At 4
       mg/kg bw, no mortality occurred, but nausea, vomiting, and salivation were
       noted in both sexes, and 1 female showed convulsions from week 37 onwards. At
       the end of treatment, females had reduced haemoglobin, haematocrit, and red
       blood cell values compared to control animals. Plasma alkaline phosphatase and
       cholesterol were increased in both sexes and ALAT in females only. No changes
       in ASAT levels were noted. Absolute and relative liver weights were increased in
       males, but the weights of other organs in the treated groups of both sexes were
       comparable to those in the controls. Microscopic examination revealed
       hepatocellular degeneration in 2 males and 1 female. Swelling and vacuolisation
       of the proximal tubules of the kidney was seen in 2 females. Histological lesions
       in the central or peripheral nervous system were not found. No neoplastic lesions
       were observed in any of the animals at any dose level. The NOAEL was 0.4
       mg/kg bw (Mai91).
       The short-term toxicity studies are summarised in Table 2.
       Long-term toxicity and carcinogenicity
       Groups of rats (n=12/sex/group) were given dietary doses of thiram equivalent to
       4.9, 15, 49, or 125 mg/kg bw/day for 65 weeks. The high dose was fatal to all rats
       within 17 weeks. No treatment-related mortality was observed in any of the other
       groups, but at 49 and 15 mg/kg bw, weakness, ataxia, and various degrees of
       paralysis of the hind legs were observed. Microscopic examination revealed
       calcification of the brain stem and cerebellum and dystrophic changes in the leg
       muscles. The NOAEL was 4.9 mg/kg bw/day (Leh52).
090-11 Thiram
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<pre>Table 2 Summary of short-term oral toxicity studies with thiram in experimental animals.
species                   dose                        exposure     critical effect               NOAEL          reference
(strain; sex)             (mg/kg bw/day)              duration                                   (mg/kg bw/day)
rat                       0, 11, 15, 22.5,            29 d         reduced body weight           11             Low80
(Wistar; male)            30, 45, 60
rat                       0, 30, 58, 132              13 wks       reduced body weight gain      LOAEL: 30      Lee78
(Charles River; male)
rat                       0, 2.5, 25, 50              90 d         reduced body weight;          2.5            Keh88
(Charles River; male,                                              anaemia (female); increased
female)                                                            WBC; decreased plasma
                                                                   albumin and total protein;
                                                                   gastric mucosal irritation
mouse                     males: 0, 54, 108, 201      28 d         reduced body weight;          LOAEL: 54      Keh89a
(Charles River; male,     females: 0, 62, 118, 241                 anaemia (male); increased
female)                                                            relative liver, kidney, brain
                                                                   weights
dog                       males: 0, 2.2, 6.9, 12      90 d         reduced body weight;          LOAEL: 2.2     Keh89b
(beagle; male, female)    females: 0, 2.3, 7.3, 13                 reduced RBC count;
                                                                   decreased plasma albumin
                                                                   and total protein
dog                       males: 0.84, 2.6, 7.4;      52 wks       decreased plasma albumin      males: 0.84    Keh91a
(beagle; male, female)    females: 0.90, 2.5, 7.2                  and total protein; increased  females: 2.5
                                                                   absolute, relative
                                                                   liver weights
dog                       0, 0.4, 4, 40               104 wks      anaemia (females);            0.4            Mai91
(beagle; male, female)                                             renal injury (females);
                                                                   liver injury
              Groups of CD rats (n=24/sex/group; controls: n=48/sex) were given dietary
              doses of technical thiram (purity: not given) equivalent to 5, 20, or 52 mg/kg
              bw/day for males and 0, 6, 26, or 67 mg/kg bw for females for 80 weeks. No
              significant differences in mortality were observed among treated and control
              groups. Body weights were significantly reduced in all male and female dose
              groups. Clinical signs of intoxication were lower body ataxia in 1 high-dose
              female and atrophy of both hind legs in another high-dose female between weeks
              20 and 69. Six more females developed a similar ataxic syndrome, i.e., unusual
              gait with dragging of the hind feet and tail, between weeks 39 and 80. Eventually,
              paralysis posterior to the lumbar region and atrophy of the hind legs developed.
              In the high-dose and some mid-dose rats, patches or wide areas of alopecia
              occurred. No changes were observed in haematological or clinical chemical
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<pre>       parameters. At the end of treatment, relative thyroid and testes weights were
       increased in high-dose males and relative liver, spleen, kidney, thyroid, ovary,
       and brain weights in high-dose females. Microscopic examination revealed an
       increased incidence of squamous metaplasia of the thyroid in both sexes at the
       high dose and a dose-related increased incidence of fatty infiltration of the
       pancreas in females in all treated groups. Examination of the central and
       peripheral nervous systems did not reveal any specific lesions. The incidence and
       severity of nephritis were decreased in treated male or female rats when
       compared to controls. The incidences of tumours were not statistically
       significantly different between the treated and control groups. The LOAEL was 5
       mg/kg bw/day, based on reduced body weight and mild fatty infiltration of the
       pancreas in females (Lee78).
           Male and female Wistar rats (n=32/sex/group) received dietary doses of
       thiram (purity: 99.4%) equivalent to 0, 0.05, 0.5, 5, or 50 mg/kg bw/day for 2
       years. No significant differences were noted in mortality between treated and
       control animals. In the male and female animals of the high-dose group, body
       weights were significantly decreased. Females showed a dose-related decrease in
       haemoglobin level and red blood cell count that was only statistically significant
       for haemoglobin at the high dose. A not statistically significant, dose-related
       decrease in haemoglobin levels was seen in males. No changes were found in
       white blood cell count of animals in treated groups when compared to the control
       group. Clinical chemistry tests showed a significant increase in plasma urea
       levels at the high dose in both sexes, while cholesterol was significantly reduced
       in males only. No changes were noted in plasma total protein, albumin, alkaline
       phosphatase, or ASAT levels. The activity of the enzyme carboxylesterase in
       plasma was significantly increased in females treated at 5 or 50 mg/kg bw/day.
       Although Knapek et al. did not further specify this effect, the committee
       considers it to be adverse. In male and female high-dose animals, relative heart,
       liver, and spleen weights were increased (significantly, except for male liver
       weight). Absolute kidney weight was significantly decreased in high-dose males
       and absolute brain weight in high-dose females, while absolute liver weights
       were increased in high-dose males and females (not statistically significant) and
       absolute heart and spleen weights in females (statistically significant). The
       incidence of macroscopic lesions was not statistically significantly different
       among the treated and control groups. Microscopic examination showed
       treatment-related lesions of the stomach mucosa in all groups. No relationship
       between thiram treatment and incidence of any tumour was observed. The
       NOAEL was set at 0.5 mg/kg bw, based on changes in carboxylesterase activity
       at higher doses (Kna89). According to the committee, the final evaluations were
090-13 Thiram
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<pre>       done with only 8 animals/sex, which was considered to be too low to reach
       statistical power. It is not clear from the report whether the effects reported were
       found in animals surviving until study termination or in interim killed animals.
       According to the committee this report was insufficiently documented.
           In another 2-year rat study, Wistar rats (n=64/sex/group) received dietary
       doses of thiram (purity: 98.7%) equivalent to 0, 0.1, 1.2, or 11.6 mg/kg bw for
       males and 0, 0.1, 1.4, or 13.8 mg/kg bw for females. The mortality of females
       was dose-dependently increased in the 13.8- and 1.4-mg/kg bw groups during the
       last 8 weeks of the treatment period. Body weight gain and food consumption
       were decreased in both males and females of the high-dose group. During the last
       part of the test period, food consumption was also decreased in males receiving
       1.2 mg thiram/kg bw. In females of the high-dose group, haemoglobin and
       haematocrit levels and red blood cell count were significantly decreased at week
       26, but no differences were found at termination (week 104). At week 104,
       slightly but significantly increased plasma ALAT levels were found in all treated
       groups (both sexes) and a slightly, significantly increased plasma ASAT level in
       high-dose males and in mid-dose females. No changes were observed in plasma
       alkaline phoshatase and urea levels. The committee considered the liver enzyme
       changes to be incidental to treatment. At termination, absolute kidney weight,
       and absolute and relative calf muscle (M. triceps surae) weights were decreased
       in high-dose males. Females showed significantly increased absolute thyroid
       weights in all treated groups, but significantly decreased absolute liver, kidney,
       and calf muscle weights at the high dose only. Microscopic examination revealed
       a significant increase in atrophy and degeneration of this muscle in females of
       the high-dose group. This effect was considered to be secondary to the atrophy
       and degeneration of the sciatic nerve that was also observed in high-dosed
       females (n=15). Pituitary adenomas were observed in 4/5 mid-dose and 7/8
       high-dose females, which were found dead during the last 8 weeks of the
       treatment. Since the incidence of these tumours in these 2 female groups at
       terminal kill was less than in the low-dose or control groups, overall incidences
       of the tumour were comparable in all groups. In addition, this type of tumour is
       often seen in ageing rats, and, therefore, the committee did not consider these
       tumours to be related to thiram treatment. The incidence of fibroadenomas of the
       mammary gland in females was significantly decreased in a dose-related fashion
       in the high-dose group. The incidences of other tumours were comparable to
       those of controls in both sexes. No evidence for carcinogenic effects of thiram
       was found. The NOAEL for long-term toxicity was 1.2 or 1.4 mg/kg bw/day for
       males or females, respectively (Mai91).
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<pre>           In an unpublished study, groups of rats (SD; n=60/sex/group) were given
       dietary doses of thiram (purity: 97.5%) equivalent to 0, 1.5, 7.3, and 15 mg/kg
       bw for males and 0, 1.8, 8.9 and 19 mg/kg bw for females for 104 weeks.
       Compound-related clinical signs included swollen nose, soft faeces, and opaque
       eyes in the high- and mid-dose animals. At these doses, body weight and body
       weight gain were significantly decreased in both sexes. Food consumption was
       significantly lower in all treated groups compared to controls. At the high and
       mid dose, red blood cell count and haemoglobin and haematocrit levels were
       decreased in female rats, compared to controls. In high- and mid-dose male rats,
       extramedullary haematopoiesis of the liver and steatosis and multifocal acinar
       atrophy of the pancreas were observed. In high-dose females, extramedullary
       haematopoiesis and bile duct hyperplasia were reported. Steatosis of the pancreas
       was noted in females receiving 19.5 and 8.9 mg/kg bw/day. Microscopic
       examination did not reveal treatment-related neurological lesions. There was no
       statistically significant increase in the incidence of any tumour in any of the
       treated groups, compared to the control group. Kehoe concluded that thiram was
       not carcinogenic up to doses of 15 and 19 mg/kg bw in males and females,
       respectively. The NOAEL was 1.5 or 1.8 mg/kg bw for males or females,
       respectively (Keh91b).
           In a carcinogenicity study, groups of F344 rats (n=50/sex/group) were fed
       thiram (purity: not given) at doses equivalent to 0,18, or 39 mg/kg bw for males
       and 0, 20, or 42 mg/kg bw for females for 104 weeks. All surviving rats were
       sacrificed at week 112. No differences in survival between the treated and control
       groups became apparent in males, but the high-dose females had shorter survival.
       Body weight gain and food intake were significantly decreased at the high dose,
       especially in females. Red blood cell count was significantly reduced in the high-
       dose males, and white blood cell count was decreased in females at both dose
       levels. Hasegawa et al. considered these changes not to be treatment related.
       Clinical chemistry liver function tests showed slight liver damage in males (not
       further specified). Macroscopic and microscopic examination did not show an
       increased incidence of neoplastic or non-neoplastic lesions in any tissue at any of
       the dose levels. The incidence of leukaemia in both sexes was statistically
       significantly reduced in a dose-related fashion in both treated groups, compared
       to the control group. The incidence of pituitary chromophobe adenomas in
       females was significantly lower in both treated groups and that of C-cell
       adenomas of the thyroid was significantly lower in high-dose females. It was
       concluded that thiram had no carcinogenic effect at doses up to 39 and 42 mg/kg
       bw/day in males and females, respectively. The NOAEL was 18 mg/kg bw/day
       based on reduced body weight gain (Has88).
090-15 Thiram
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<pre>           In a carcinogenicity study, 2 hybrid strains of mice (the offspring of
       C57BL/6 females and C3H/Anf or AKR males) (n=18/strain/sex) were fed
       thiram (purity: not given), beginning at 7 days of age, for 18 months. Until
       weaning, the compound was given at doses of 10 mg/kg bw/day by stomach
       tube, thereafter via the food at doses equivalent to 4.2 mg/kg bw/day. No
       increased incidence of tumours was found in any sex-strain subgroup or in the
       combined sexes of either strain compared to untreated control mice (Inn69).
           In an unpublished study, CD mice (n=50/sex/group were given dietary doses
       of thiram (purity, 97.5%) of 0, 3, 24, or 50 mg/kg bw/day for males and 0, 3, 57,
       or 112 mg/kg bw for females for 97 weeks. No treatment-related increase in
       mortality was observed. The only observed clinical signs of toxicity were sores
       and redness of the skin (mainly of the ears), consistent with bacterial dermatitis,
       in the high- and mid-dose male mice and in the high-dose female mice. A dose-
       related significant decrease in body weight, body weight gain, and food
       consumption was observed in the mid- and high-dose male and female mice.
       Significant decreases in red blood cell count, haemoglobin, and haematocrit
       values were found in the high-dose females. Macroscopic and microscopic
       examination did not show an increased incidence in neoplastic lesions in any of
       the treated groups compared with the control group. Non-neoplastic lesions in
       the mid- and high-dose male and female mice included retinal atrophy,
       intracytoplasmic protein-like droplets in the epithelium of the urinary bladder,
       and necrosis and inflammation of the skin. Increased pigment in the spleen and
       decreased pigment in the inner adrenal cortex was seen in mid- and high-dose
       female mice and hyperkeratosis of the non-glandular stomach in mid- and high-
       dose females and in high-dose males. It was concluded that thiram is not
       carcinogenic up to 50 mg/kg bw/day for male mice and up to 112 mg/kg bw/day
       for female mice. The NOAEL was 3 mg/kg bw (Tru92).
       The tumour-initiating and tumour-promoting potency of thiram were tested in
       Swiss albino mice by the 2-stage skin carcinogenic model. Six groups of male
       mice (n=15/group) were topically treated in the following way. In group 1,
       animals received a single dose of 1 mg thiram in 0.2 mL DMSO. Two weeks
       thereafter, croton oil (a promotor) was applied for 6 weeks, twice weekly. In
       group 2, animals received a single dose of 10 mg thiram in 0.2 mL DMSO.
       Croton oil was applied as in group 1. In group 3, animals received a single dose
       of 7,12-Dimethylbenz[a]anthracene (DMBA), a known initiator of skin
       carcinogenesis, in 0.2 mL DMSO without further treatment. In group 4, animals
       were given a single dose of DMBA in 0.2 mL DMSO. Croton oil was applied as
       in groups 1 and 2. Group 5 received a single dose of DMBA in 0.2 mL DMSO.
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<pre>               2 weeks thereafter, thiram (1 mg/0.2 mL DMSO) was applied for 6 weeks, twice
               weekly. Group 6 received croton oil for 6 weeks, twice weekly. The positive
               control DMBA induced skin tumours in 13 of 15 mice, when DMBA application
               was followed by treatment with croton oil (group 4). No skin tumours developed
               when DMBA application was followed by treatment with thiram (group 5) or
               when DMBA was applied without further treatment (group 3). One mouse
               bearing a skin tumour was found after treatment with thiram at 10 mg, followed
               by croton oil treatment (group 2). No skin tumours were found in animals treated
               with thiram at 1 mg, followed by croton oil treatment (group1), or when croton
               oil was applied without further treatment (group 6). Thus, there was no
               significant increase in skin tumours in the thiram-treated groups, either as
               initiator or promotor (Geo95).
                    A summary of the long-term toxicity of thiram is shown in Table 3.
Table 3 Summary of long-term oral toxicity/carcinogenicity studies with thiram in experimental animals.
species                    dose                       exposure    critical effect                       NOAEL   reference
(strain; sex))             (mg/kg bw/day)             duration                                          (mg/kg
                                                                                                        bw/day)
rat                        0, 4.9, 15, 49, 125        65 wks      ataxia; paralysis of legs             4.9     Leh52
(male, female)
rat                        males: 0, 5, 20, 52        80 wks      reduced body weight; pancreas         LOAEL:  Lee78
(CD; male, female)         females: 0, 6, 26, 67                  injury                                5
rat                        0, 0.05, 0.5, 5, 50        2y          reduced haemoglobin (females);        5       Kna89
(Wistar; male, female)                                            changes in absolute and relative
                                                                  organ weights
                                                                  carboxyesterase                       0.5
rat                        males: 0, 0.1, 1.2, 11.6   2y          reduced body weight; increased        1.2     Mai91
(Wistar; male, female)     females: 0, 0.1, 1.4, 13.8             mortality; anaemia (females)
                                                                   nerve degeneration and muscle
                                                                  atrophy (females)
rat                        males: 0, 1.5, 7.3, 15     2y          anaemia (females); reduced body       1.5     Keh91b
(SD; male, female)         females: 0, 1.8, 8.9, 19               weight; pancreas and liver injury
rat                        males: 0, 18, 39           2y          reduced body weight                   18      Has88
(F344; male, female)       females: 0, 20, 42
mouse                      0, 4.2                     78 wks      -                                     4.2     Inn69
(C57BL/6xC3H/Anf;
C57BL/6xAKR; male,
female)
mouse                      males: 0, 3, 24, 50        97 wks      reduced body weight                   3       Tru92
(CD ; male, female)        females : 0, 3, 57, 112
090-17         Thiram
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<pre>       Based on the above data, the committee concludes that thiram unlikely has
       carcinogenic potential.
       Mutagenicity and genotoxicity
       Mutation assays comprised tests for the detection of gene mutations in bacteria,
       in mammalian cells, and in Drosophila (in vitro), clastogenic effects, e.g., sister
       chromatid exchanges (SCE), chromosomal aberrations, and micronuclei in vitro,
       and dominant lethal mutations and micronuclei in vivo, and other genotoxicity
       assays, e.g., tests for DNA damage and repair (in vivo and in vitro).
       •   In vitro tests:
           • Gene mutation assays. Tests for reverse mutations were positive in S.
              typhimurium strains TA100 and TA1535 at concentrations in the range of
              10 to 120 µg/plate or 10 to 1000 µg/plate, without or with metabolic
              activation by a rat liver microsomal S9 fraction, respectively (And80,
              Cre92, Hed79, Mor83, Pot90, Zdz79). No increased mutation rates were
              found in strains TA98, TA102, TA1537, and TA1538 at concentrations up
              to 1000 µg/plate with or without metabolic activation (And80, Cre92,
              Mor83, Pot90). However, one author found positive results in strains
              TA98 and TA1538 at a concentration of 100 µg/plate in the presence of S9
              (Zdz79). When tested in E. coli strain WP2 uvrA, thiram induced reverse
              mutations at concentrations in the range of 15 to 60 µg/plate in the
              absence or presence of S9. However, under these conditions, the test was
              negative in E. coli strain WP2 (Cre92).
              Thiram did not induce gene mutations in the hprt forward mutation assay
              in cultured Chinese hamster V79 lung cells, when tested at concentrations
              up to 1.6 µg/mL in the presence of S9 (Don83). In another study, negative
              results were obtained when tested at concentrations in the range of 1-10
              µg/mL or 10-56 µg/mL without or with metabolic activation, respectively
              (Deb86). It was positive when tested at a cytotoxic concentration of
              10 µg/mL without metabolic activation (Pas85).
              Thiram induced sex-linked recessive lethal mutations in D. melanogaster
              at concentrations in the range of 1-5 mg/mL. The mutagenicity was
              manifested in the spermatid/spermatocyte stage (3 to 6 days after
              treatment), and no increased mutation rate was found in the pre-meiotic
              stages (6 to 9 days after treatment) (Don83).
           • Cytogenicity assays. Thiram induced a dose-relatedly increased frequency
              of SCEs in cultured human lymphocytes, at concentrations in the range of
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<pre>             1.2 to 1200 µg/L in the absence or presence of S9. At the high dose, a
             2-fold increase over the frequency of SCEs in controls was seen (Pie90).
             A significant dose-dependent increase in SCEs was also found when
             cultured human lymphocytes were treated with thiram concentrations
             ranging from 5 to 25 µg/mL with metabolic activation. However, negative
             results were found in the absence of S9, probably due to the high
             cytotoxicity at 15 µg/mL and higher (Per89). In another study, thiram did
             not induce an increased SCE frequency in cultured Chinese hamster ovary
             (CHO) cells at concentrations in the range of 24 to 240 µg/L without S9,
             or 24 to 2400 µg/L with metabolic activation (Don83).
             The frequency of chromosome aberrations in cultured CHO cells
             remained unaffected following treatment with 3-23 µg/L in the absence of
             S9, or with 200-1500 µg/L in the presence of metabolic activation
             (Put87a). However, positive results were obtained in a chromosomal
             aberration assay (excluding gaps) in cultured CHO cells at concentrations
             ranging from 7.5 to 34.8 µg/L in the presence but not in the absence of S9.
             The increase was not dose dependent (Mos94). A dose related increased
             incidence of micronuclei in cultured human lymphocytes was observed in
             the dose range 0.5-24 µg/mL in the absence or presence of S9 (Vil98).
          • Other assays. Thiram induced unscheduled DNA synthesis in cultured
             human lymphocytes when tested at concentrations of 5-50 µg/L and
             higher, in the presence but not in the absence of metabolic activation. The
             increase was not dose dependent, probably due to cytotoxicity at the
             higher dose levels (Per89).
             When primary rat hepatocytes were treated with thiram at concentrations
             ranging from 30 to 10000 µg/L, no effect on DNA repair was
             demonstrated (Wet85).
             There was no evidence of alkylation activity of thiram towards the
             nucleophiles 4-(p-nitrobenzyl)-pyridine or deoxyguanosine (Hem80).
             Thiram caused a statistically significant increase in the frequency of
             single strand DNA breaks and alkali labile sites in cultured human
             testicular cells at a concentration of 24 µg/mL without metabolic
             activation. In cultured rat testicular cells, a statistically significant
             increase of DNA damage was demonstrated at concentrations in the range
             of 7.2-72 µg/mL (Bjø96). DNA damage (single strand DNA breaks) was
             also demonstrated in cultured human lymphocytes at thiram
             concentrations in the range of 0.1-8 µg/mL in the presence or absence of
             S9 (‘Comet’ assay) (Vil98).
090-19 Thiram
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<pre>       •  In vivo tests:
          • cytogenicity assays. In a dominant lethal mutation assay, 20 male mice
             were fed thiram (purity: 75%) doses equivalent to 150 mg/kg bw/day for 8
             weeks (the entire spermatogenesis cycle). Following mating, pregnant
             mice were sacrificed on day 14 of gestation. A significant increase in dead
             implants compared to controls was found, indicating a thiram-induced
             dominant lethal effect (Agr97).
             When thiram (purity: 99.7%) was orally (gavage) administered to male
             B6C3F1 mice (n=3/group) at 0, 100, 300, and 900 mg/kg bw for 4
             consecutive days, or at 300 mg/kg bw for 8 and 12 days, no statistically
             significant increased incidences of micronuclei in isolated splenocytes
             were found at 24 hours after the last treatment (Vil98).
             No increased incidence was found in bone marrow cells of Chinese
             hamsters (n=1-2/sex/group), 30 hours after single intraperitoneal
             injections of 0, 100, 200, or 500 mg thiram/kg bw (Don83).
             When F1 hybrid mice (n=15/sex) were treated with a single
             intraperitoneal dose of thiram (purity: 80%) of 100 mg/kg bw, a
             significant increase in the incidence of micronuclei in bone marrow
             polychromatic erythrocytes was found at 24 hours after treatment (Pas85).
             In another study, B6C3F1 mice (n=5/sex/group) were given single
             intraperitoneal injections of thiram (purity: 99.7%) of 0, 12.5, 25, or 50
             mg/kg bw. In a second experiment, a dose of 37.5 mg/kg bw was given.
             No increased incidence of micronuclei was found in bone marrow cells of
             either sex, collected at 24 hours after treatment. However, a statistically
             significant increase was found in bone marrow cells of male mice at 25
             mg/kg bw and above, at 48 hours after treament (Cre92).
             When thiram (purity: 75%) was administered to male Swiss albino mice
             (n=6/group) as single intraperitoneal doses of 0, 25, 50, or 100 mg/kg bw,
             a statistically significantly increased incidence of micronuclei in
             polychromatic erythrocytes was found, either at 30 or 48 hours after
             treament (Agr97).
             In an unpublished study, the frequency of micronuclei was not
             significantly changed in CD-1 mice, treated with thiram (purity: 99.7%) at
             single intraperitoneal injections of 38 to 377 mg/kg bw (Put87b).
             Male Swiss albino mice (n=3-5/group) were intraperitoneally treated with
             thiram (purity: not given) at doses of 0, 100, 150, or 200 mg/kg bw. The
             treatment was repeated after 24 hours. No increased incidence of
             micronuclei in bone marrow cells were observed 24 hours after the last
             treatment (Geo95). The effect of thiram at the germ cell level was studied
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<pre>               in groups Swiss albino mice, given thiram at doses of 0, 80, 200, or 320
               mg/kg bw for 3 days by gavage. A significant, dose-dependent increase in
               the frequency of chromosomal polyploidy in primary spermatocytes was
               found (Pra87).
            • Other assays. In a ‘Comet’ assay, thiram induced DNA damage, i.e.,
               single strand breaks or alkali labele sites were found in lymphocytes of
               rats 24 hours following oral treatment with 300 mg/kg bw/day for 8 and
               12 days. Negative results were obtained in splenocytes (Vil98).
       In summary, in vitro, thiram induced gene mutations in sensitive strains of
       S. typhimurium and E. coli and in mammalian cells at cytotoxic concentrations.
       DNA damage was demonstrated in mammalian cultures, but equivocal results
       were obtained for cytogenetic effects. In vivo, thiram induced dominant lethal
       mutations and germ cell abnormalities in mice, but no significant increase in the
       frequency of micronuclei in bone marrow cells was observed after repeated high
       oral doses. Data on the induction of micronuclei in bone marrow cells of mice
       treated by intraperitoneal injection at high doses were conflicting. DNA damage
       was demonstrated in lymphocytes of rats at high oral doses.
            Based on these data, the committee concludes that thiram exhibits mutagenic
       and genotoxic potential in vitro. In vivo, clastogenic effects were only observed
       at levels close to the maximum tolerated dose.
       Reproduction toxicity
       Groups of weanling male CD rats (n=20/group) were given technical-grade
       thiram (purity: not given) via the diet at daily doses equivalent to 0, 30, 58, or
       132 mg/kg bw for at least 13 weeks before mating with untreated females. In
       high-dose males, mortality occurred in 70% of the animals. Body weights were
       reduced in a dose-dependent fashion in all treated groups. At the high- and mid-
       dose levels, animals lost hair and had rough coats. At 132 mg/kg bw/day, thiram
       induced infertility as shown by the inability to fertilise females. Microscopic
       examination revealed testicular hypoplasia, tubular degeneration, and atypical
       spermatids in the epididymides. The NOAEL for effects on the male
       reproductive system was 58 mg/kg bw/day (Sho76). In several studies, thiram
       induced morphologically abnormal sperm in mice. Abnormal sperm heads were
       observed in CFWx C57BL mice, 5 weeks after single intraperitoneal injections
       of thiram at 50 or 100 mg/kg bw or after repeated injections at 30 mg/kg bw/day
       for 5 days. No abnormalities were found at 1 week after treatment (Zdz82). A
       dose-dependent increase in sperm head abnormalities was observed in Swiss
090-21 Thiram
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<pre>       albino mice treated with thiram at doses of 80, 200, or 320 mg/kg bw for 3 days
       by gavage (Pra87). In a more recent study, sperm abnormalities were
       investigated in Swiss mice (n=6/group) after treatment with thiram (purity: not
       given) as single intraperitoneal doses of 500 or 1000 mg/kg bw or as daily
       intraperitoneal doses of 250 mg/kg bw for 5 days. One month after the end of
       treatment, a significant dose-related increased incidence of sperm abnormalities
       was observed, consisting of sperm with acrosomes bent upward, acrosomes bent
       downward, or without acrosomes. Head abnormalities, like double heads, banana
       heads, amorphous heads, microcephaly, and macrocephaly were found. Instances
       of double tails and coiled sperm were also recorded. The incidences of sperm
       abnormalities in mice receiving a single dose of 1000 mg/kg bw or 5 repeated
       doses of 250 mg/kg bw /day were comparable (Hem93). B6C3F1 mice were
       treated intraperitoneally with thiram (purity: 99%) at a single dose of 75 mg/kg
       bw (32 animals) or repeated doses of 25 mg/kg bw/day for 5 days (24 animals).
       Mortality was observed within 5 days in 29% and 15 % of the animals,
       respectively. Reproductive indicators were assessed at days 14, 28, 35, and 56
       after the end of treatment. No effects were observed on body weight, testes
       weight, testicular sperm head numbers, or activities of specific testicular enzyme
       levels at any time after administration. The authors conclude that under the
       conditions of the study, thiram did not cause cytotoxicity on differentiating
       spermatogonia or on late spermatocyte stages of mice gonads (Tra94).
           In 2 separate studies, male rats (n=25/group) received thiram (purity: not
       given) at oral (gavage) doses of 5, 10, and 25 mg/ kg bw in peanut oil for 90 days
       or 360 days, respectively. In both studies, a dose-related increased mortality was
       observed. Clinical signs observed in both studies consisted of diarrhoea,
       salivation, nasal bleedings, and ataxia. Body weight gain was reduced in high-
       dose males in the 90-day study and in a dose-dependent way in all treatment
       groups in the 360-day study. Relative testes weight was significantly increased in
       the 90-day study at 25 mg/kg bw and in the 360-day study at 25 and 10 mg/kg
       bw. In the latter study, the relative epididymis weight was significantly increased,
       and the relative weights of seminal vesicles and prostate were significantly
       decreased at 25 and 10 mg/kg bw, compared to controls. Microscopic
       examination revealed inactive sperm cells, necrosis of seminiferous tubules with
       enlarged interstitium, detachment of germinal cells, and accumulation of debris
       in the lumen of the tubules. The activities of testicular enzymes lactate
       dehydrogenase, alkaline phosphatase, and glucose-6-dehydrogenase were
       significantly increased and that of succinate dehydrogenase and acid phosphatase
       significantly decreased at 10 and 25 mg/kg bw in the 90-day and in all treatment
       groups in the 360-day study. Testicular ATPase activity was increased in all dose
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<pre>       groups in the 360-day study and testicular free sialic acid at all treatment groups
       in both studies. Serum cholesterol was increased and testicular protein was
       reduced in all treated groups in the 360-day study (not determined in the 90-day
       study). The NOAEL was 5 mg/kg bw/day for the
       90-day study, based on changes in activity of testicular enzymes. For the 360-day
       study, a LOAEL of 5 mg/kg bw/day was established, based on decreased body
       weight and changes in activity of testicular enzymes (Mis94, Mis98).
            The effect of thiram on female reproduction was examined in female CD rats
       (n=20/group), given daily dietary doses of technical-grade thiram (purity: not
       given) of 0, 30, or 96 mg/kg bw for periods of time ranging from 14 days to 4.5
       weeks, prior to mating with non-treated males. After mating, all females were fed
       the control diet. At the end of the 2-week dosing period, mean body weight and
       food consumption were significantly decreased in the high-dose animals. The
       number of implants and pups per dam were reduced in low-dose dams. No
       further results on reproductive parameters were shown. At the high dose, the
       dioestrous phase of the oestrus cycle was delayed. This effect was reversible
       after 9 days without thiram. The LOAEL was 30 mg/kg bw/day, the lowest dose
       tested (Sho76).
            Another study was conducted to investigate the mechanism of the acute
       effects of thiram on the hormonal control of ovulation in the rat. A key endocrine
       event in ovulation is the pre-ovulatory surge of luteinising hormone (LH), which
       occurs in the rat during a restricted sensitive period of time just prior to the
       appearance of the LH surge, typically between 4 pm and 7 pm. To establish the
       effect of thiram on the LH surge, ovariectomised oestrogen-primed Long-Evans
       rats were treated with thiram at intraperitoneal doses of 0, 6, 12, 25, 50, or 100
       mg/kg bw. At 100 and 50 mg/kg bw, thiram completely blocked the LH surge in
       all rats tested, while doses of 25 and 12 mg/kg bw blocked the surge in 75% and
       40% of the treated animals, respectively. The NOAEL was 6 mg/kg bw. In a
       second experiment, intact rats were intraperioneally injected with 0, 12, 25, and
       50 mg thiram/kg bw at 1 pm on the day of vaginal pro-oestrus. Thiram at 50
       mg/kg bw blocked the LH surge in all rats examined on the afternoon of vaginal
       pro-oestrus, while 25 mg/kg bw blocked the surge in 60% of the females tested.
       The NOAEL was 12 mg/kg bw. No influence of thiram on oestradiol levels was
       reported. Examination of the oviducts 48 hours after the pro-oestrous injection of
       50 mg/kg bw at 1 pm revealed that all rats had ovulated, i.e., ovulation was
       delayed for 24 hours. The conclusion was that thiram is able to block the LH
       surge and inhibits subsequent ovulation if administered during a sensitive period
       prior to the initiation of the surge. According to Stoker et al., thiram most
       probably inhibits noradrenaline synthesis by suppressing the activity of
090-23 Thiram
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<pre>       dopamine-β-hydroxylase. Because of a decreased noradrenaline level in the
       hypothalamus, the release of gonadotropin-releasing hormone, and indirectly the
       release of LH, are lowered (Sto93). In a subsequent study, the effect of a thiram-
       induced delay in ovulation on pregnancy outcome in the rat was investigated.
       Groups of female rats were treated with a single intraperitoneal injection of
       thiram (50 mg/kg bw) at 1 pm on the day of vaginal pro-oestrus and paired the
       following evening (approximately 30 hours later) with untreated males (thiram-
       delayed females). A thiram-treated, non-delayed and an untreated group of
       females were mated on the same day, 6 hours after treatment. A significant
       decrease in the proportion of pregnant females and the litter size on gestational
       day 20 and a significant increase in the number of resorptions were seen in the
       thiram-delayed group, but not in the non-delayed group. According to Stoker et
       al., this indicated that thiram-induced delay in ovulation and not the exposure to
       thiram per se was responsible for altered pregnancy outcome. On gestational
       days 7, 11, and 20, the number of implantations in the delayed females was not
       statistically significantly different from control and non-delayed females.
       However, the number of live embryos was reduced on gestational day 11 in the
       delayed females. The mean developmental score, head length, crown-rump
       length, and somite number in the delayed group was also reduced, indicating
       retarded development of live embryos. According to Stoker et al., the results
       demonstrate that thiram-induced delayed ovulation does not alter the number of
       oocytes released or the number of implants. Effects on the concepti from these
       females occur during mid-gestation (Sto96).
       In an unpublished 2-generation reproduction toxicity study, 63-days-old Crl:CD
       rats (n=26/sex/group) were given thiram (purity: 97.6%) via the diet at levels
       equivalent to 0, 1.5, 2.9, or 8.9 mg/kg bw/day for males and 0, 2.3, 4.6, or 14
       mg/kg bw/day for females for 81 days prior to mating. Three litters (F1a, F1b,
       and F1c) were produced, and F1c rats were fed diets containing thiram at levels
       equivalent to 0, 1.8, 3.8, or 11 mg/kg bw /day for males and 0, 2.4, 5.1, or 16
       mg/kg bw/day for females for at least 105 days after weaning, before being bred
       to produce 2 litters (F2a and F2b). High-dose females showed decreased body
       weights during gestation and lactation of F1 and F2 offspring. Mean maternal
       body weight was also decreased at 4.6 mg/kg bw/day during the F1a-gestation
       period. Food consumption was reduced in high- and mid-dose F0 males and
       females. Mean offspring body weights were significantly reduced in all litters
       across both generations at the high dose. No treatment-related clinical signs of
       toxicity were observed, and no treatment-related abnormalities were seen in male
       and female fertility indices, gestation length, litter size, numbers of viable and
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<pre>       stillborn pups, and offspring survival and growth during lactation. The
       reproductive NOAEL was 4.6 and 2.9 mg/kg bw for males and females,
       respectively, based on reduced body weights in offspring. The parental NOAEL
       was 2.3 and 1.5 mg/kg bw/day, based on reduced food intake (Yor91).
       Developmental toxicity studies were conducted in rats, mice, and rabbits.
       Pregnant CD rats (n=7-18/group; controls: n=28) received oral (gavage) doses of
       technical-grade thiram (purity: not given) of 40, 90, 136, or 164 mg/kg bw/day
       on gestational days 6 to15; a dose of 200 mg/kg bw was given on gestational
       days 7 to 12. Animals were sacrificed on gestation day 20. Mortality was 67% in
       the high-dose group. Food consumption and body weight gain during gestation
       were reduced in all treated groups compared to controls. Litter size was
       decreased at doses 136 mg/kg bw. The number of implants per dam was
       significantly decreased at 164 and 200 mg/kg/day. At 136 mg/kg bw and above,
       the number of live fetuses was significantly decreased, and a corresponding
       increase in resorptions was seen, amounting to 100% at 164 and 200 mg/kg bw.
       Fetal body weights were significantly reduced in all treated groups. At 136
       mg/kg bw, anomalies including domed cranium, hydrocephalus, unossified
       sternebrae, and incomplete ossified supraoccipital were observed. The
       committee considered these changes the result of maternal toxicity. The LOAEL
       for maternal as well as for fetotoxicity was 40 mg/kg bw/day. For teratological
       effects, a NOAEL of 90 mg/kg bw was established (Sho76).
            In an unpublished study, groups of 25 pregnant CD rats received oral
       (gavage) doses of thiram (purity: 99.8%) of 0, 7.5, 15, and 30 mg/kg bw/day on
       gestational days 6 to 15. Animals were sacrificed on day 20 of gestation. No
       deaths were observed and the only clinical sign observed was a dose-related
       increase of alopecia on various parts of the body. A transient, dose-related loss of
       maternal body weight was observed at 15 and 30 mg/kg bw/day. Body weight
       gain was significantly reduced during the treatment period in females of the low-
       dose group, but subsequent weight gain was similar to that of the controls. No
       effects on implantation or fetal survival were reported. Fetal weights were
       decreased at 15 and 30 mg/kg bw/day (reaching statistical significance at 30
       mg/kg bw). Placental weights were significantly decreased at all dose levels.
       Fetal immaturity was observed at 30 mg/kg bw/day, and an increased incidence
       of thirteenth ribs of reduced size was observed at 15 and 30 mg/kg bw/day. These
       effects were considered a result of maternal toxicity. The NOAEL for
       developmental effects was 7.5 mg/kg bw. For maternal effects, a LOAEL of
       7.5 mg/kg bw/day was established (Tes88a).
090-25 Thiram
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<pre>           Pregnant NMRI mice (n=19-23/group) received oral (gavage) doses of
       thiram (purity: not given) of 0, 357, and 714 mg/kg bw/day on gestational days 6
       to 17. Treatment-related maternal mortality or signs of toxicity were not
       reported. No differences in the number of implantations per female were
       observed between the treated and the control groups at any dose level. A dose-
       dependent decrease in the frequency of life fetuses at birth and a corresponding
       increase in the frequency of early and late resorptions and of fetal deaths were
       seen. These changes were statistically significant at the high dose only. Fetal
       body weight was significantly decreased in the high-dose group. An increased
       frequency of fetal malformations was seen at both doses compared to controls,
       especially at the high dose (55.2% vs. 0.9%). Dose-dependent fetal
       malformations were characterised by wavy ribs, cleft palate, distorted bones, and
       micrognatia. Retarded fetal development was also observed at the high dose. In a
       parallel experiment, pregnant NMRI mice were treated with thiram on specific
       days of gestation in order to find out the most susceptible phase of embryonic
       development. Pregnant SW mice were used for comparison. NMRI and SW mice
       were given oral doses of 357, 714, and 1071 mg/kg bw, and 250, 500, 1000, and
       1500 mg/kg bw, respectively, on gestational days 10 and 11, or 12 and 13. The
       12th and 13th day of embryonic development, i.e., the end of embryonal and the
       beginning of the fetal development phase, proved to be the most susceptible
       phase. Embryotoxic effects in NMRI and SW strain were comparable and
       increased in a dose-dependent fashion. At doses of 10 mg/kg bw/day and higher,
       NMRI mice were more susceptible to the induction of cleft palates and SW mice
       to the induction of wavy ribs. A NOAEL for developmental effects in SW mice
       was 250 mg/kg bw/day. For NMRI mice, a LOAEL was 357 mg/kg bw/day was
       established (Rol71).
           In another study, pregnant Swiss-Webster mice (n=18-19/group) received
       oral (gavage) doses of technical-grade thiram (purity: not given) of 0, 100, or 300
       mg/kg bw/day on gestational days 6 to 14. Animals were sacrificed on
       gestational day 18. Mortality was 22% in the high-dose group. The treatments
       did not significantly change body weight gain during gestation, litter size,
       number of implants per dam, number of live fetuses, or incidence of resorptions.
       No significant differences in body weights of fetuses from treated or control
       dams were found. At the high dose, anomalies including hydronephrosis,
       collapsed cranium, thick atrium wall, malaligned sternebrae, and the presence of
       fibrous material connecting lens and retina were observed. No statistical
       treatment of these data was shown. Short et al. did not consider the changes a
       specific, thiram-induced teratogenic effect. The NOAELs for maternal and
       developmental toxicity were 100 and 300 mg/kg bw/day, respectively (Sho76).
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<pre>            In an unpublished study, groups of 15 to 20 pregnant New Zealand white
       rabbits received oral (gavage) doses of thiram (purity: 99.5%) of 0, 1.0, 2.5, or
       5.0 mg/kg bw/day on gestational days 6 to 19. The animals were killed on day 29
       of gestation. No compound-related mortality was recorded. Maternal body
       weight gain was decreased in high-dose females. Reproductive parameters, i.e.,
       number of implantation sites, number of resorption sites, number and distribution
       of live and dead fetuses, were unaffected by treatment, as well as growth and
       morphological development. The NOAELs for maternal and developmental
       toxicity were 2.5 and 5 mg/kg bw, respectively (Tes88b).
            In another unpublished study, thiram (purity: not given) was administered to
       groups of 20 pregnant rabbits at doses of 0, 1.0, 5.0, and 10 mg/kg bw on
       gestational days 7 to 19. The animals were sacrificed on day 29 of gestation. No
       compound-related mortality or clinical signs of toxicity were seen. Treatment did
       not induce maternal effects. No adverse effects on fetal development were
       observed at the teratological examination. The NOAELs for maternal and
       developmental toxicity were 10 mg/kg bw (Yor92).
       In summary, thiram is able to induce delayed ovulation in female rats and sperm
       abnormalities and testicular necrosis in male rats. The lowest NOAEL for
       reproductive toxicity in the rat was 2.9 mg/kg bw/day. Mice were much less
       sensitive to developmental toxicity than rats or rabbits. The lowest NOAEL for
       developmental effects in the latter species was 5.0 mg/kg bw. Effects on
       reproduction and development were noted at dose levels which caused maternal
       toxicity.
7      Existing guidelines
       The current administrative occupational exposure limit (MAC) for thiram in the
       Netherlands is 5 mg/m3, 8-hour TWA.
            Existing occupational exposure limits in some European countries and in the
       USA are summarised in Annex II.
8      Assessment of health hazard
       Workers can be exposed to thiram through inhalation of dust or aerosols or by
       direct skin contact with the compound or a formulation. The committee did not
       find data on the respiratory or dermal absorption of the chemical. The extent of
       absorption following oral administration to rats ranged from 80 to 95%.
       Following absorption, the majority of the dose was eliminated in the urine
090-27 Thiram
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<pre>       (25-43%) and exhaled breath (41-61%), most of it within the first 12 hours after
       dosing. About 2-6% of the dose is retained in the carcass at 72 hours after dosing,
       most of it in the liver, the kidneys, and the blood. There is no evidence of
       cumulation of the compound in any of these tissues. Thiram is biotransformed
       into dimethyldithiocarbamate, which is further metabolised into carbon
       disulphide and a range of oxidative and conjugative polar products. According to
       the committee, the toxicity of thiram can most probably be attributed to the
       parent compound and the metabolites carbon disulphide and
       dimethyldithiocarbamate.
           Case studies in humans demonstrated thiram-induced allergic contact
       dermatitis in workers in the rubber industry, and more recently, in workers who
       had worn rubber gloves containing thiram and in haemodialysed patients. In
       factory workers, skin and eye irritation and thyroid disorders have been reported.
       Reproductive effects have been reported in female operators, allegedly due to
       exposure to thiram. Thiram induced alcohol intolerance, but to a lesser extent
       than disulfiram, the ethyl analogue of thiram.
           In experimental animals, the compound is slightly irritating to the eyes and
       the skin, but not a skin sensitiser. Based on the results of acute lethal toxicity
       studies in test animals, the committee considers the compound as toxic via the
       inhalation route. When applied via the dermal or oral route, the acute toxicity is
       of a low order, although large variations were seen between species, strains, and
       sexes.
           Effects of thiram observed in short-term or long-term oral toxicity studies in
       rats, dogs, and mice included: reduced body weight, anaemia, and degenerative
       changes of the sciatic nerve in a 2-year study in rats (NOAEL: 1.2 mg/kg
       bw/day), reduced body weight in a 97-week study in mice, and anaemia and liver
       and kidney injury in a 104-week study in dogs (NOAEL: 0.4 mg/kg bw/day).
           In vitro mutagenicity/genotoxicity tests showed both positive and negative
       results for gene mutations in bacteria or cultured mammalian cells as well as for
       cytogenetic effects in cultured mammalian cells. DNA damage (single strand
       DNA breaks) was demonstrated in cultured testicular cells and peripheral
       lymphocytes. In vivo, 8-week oral administration of thiram at 150 mg/kg
       bw resulted in dominant lethal mutations in mice, but no increase in the
       frequency of micronuclei in bone marrow cells of mice was observed at the
       maximum tolerated dose of 300 mg/kg bw for 8 to 12 days. The committee
       considers thiram to be mutagenic/genotoxic in vitro. In vivo, clastogenic or DNA
       damaging effects were only observed at levels close to the maximum tolerated
       dose. Carcinogenicity studies in rats and mice did not show treatment-related
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<pre>       increased incidences of neoplastic lesions. The committee concluded that thiram
       was not carcinogenic in these species.
           Effects on the male reproductive system included testicular injury and
       inactive sperm cells in a 1-year oral toxicity study in rats (NOAEL: 5 mg/kg
       bw/day). The LOAEL was 5 mg/kg bw/day, based on reduced body weight and
       changes in activity of testicular enzymes. Thiram induced a delay of the
       dioestrous phase of the oestrus cycle in rats at a dose of 96 mg/kg bw/day for
       14 days. It was demonstrated that an acute dose of thiram is able to block the pre-
       ovulatory surge of luteinising hormone and to inhibit subsequent ovulation
       (NOAEL: 12 mg/kg bw). In a 2-generation reproduction toxicity study in rats,
       the parental and reproductive NOAELs were 1.5 and 2.9 mg/kg bw/day,
       respectively. In developmental toxicity studies in rats and mice, embryotoxicity
       was only demonstrated at dose levels which caused maternal toxicity. A recent
       study in rats showed a NOAEL for developmental toxicity of 7.5 mg/kg bw/day
       and a LOAEL of 7.5 mg/kg bw for maternal toxicity. Teratogenicity was not
       observed. In rabbits, NOAELs of 5.0 and 2.5 mg/kg bw/day were found for
       developmental and maternal toxicity, respectively.
       The committee takes the 2-year oral dog study with a NOAEL of 0.4 mg/kg bw
       as starting point in establishing a health based recommended occupational
       exposure limit (HBROEL). Since workers are exposed for 5 days a week, this
       NOAEL from a continuous study (i.e., 7 days a week) is adjusted by multiplying
       with a factor of 7/5 resulting in a no-adverse-effect level (NAEL) of 0.56 mg/kg
       bw. For the extrapolation to a HBROEL, a factor of 1.4 for allometric scaling
       from dogs to humans, based on caloric demand, and an overall factor of 9,
       covering inter- and intraspecies variation, are applied, resulting in a NAEL for
       humans of 0.04 mg/kg bw/day. Assuming a 70-kg worker inhales 10 m3 of air
       during an 8-hour working day and a retention of 100%, and applying the
       preferred value approach, a HBROEL of 0.2 mg/m3 is recommended for thiram.
       The committee considers a ‘skin notation’ not necessary.
       The committee recommends a health-based occupational exposure limit for
       thiram of 0.2 mg/m3 as inhalable dust, as an 8-hour time-weighted average
       (TWA).
090-29 Thiram
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<pre>Zdz79  Zdzienicka M, Zielenska M, Tudek B, et al. Mutagenic activity of thiram in Ames tester strains of
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090-35 Thiram
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<pre>       Annex I
       Figure 1 Metabolism of thiram in rats (from FAO93).
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<pre>              Annex II
  Occupational exposure limits for thiram in various countries.
country                             occupational             time-weighted            type of              notea         referenceb
- organisation                      exposure limit           average                  exposure limit
                                    ppm       mg/m3
the Netherlands
- Ministry of Social Affairs and    -         5              8h                       administrative                     SZW03
Employment
Germany
- AGS                               -         5c             8h                                                          TRG00
                                    -         20c            15 min
- DFG MAK-Kommission                -         5c             8h                                            sens, e, f    DFG02
                                    -         5c             15 mind
Great Britain
- HSE                               -         5              8h                       OES                                HSE02
                                    -         10             15 min
Sweden                              -         -                                                                          Swe00
Denmark                             -         1              8h                                                          Arb02
USA
- ACGIH                             -         1              8h                       TLV                  A4g           ACG03b
- OSHA                              -         5              8h                       PEL                                ACG03a
- NIOSH                             -         5              8h                       REL                                ACG03a
European Union
- SCOEL                             -         -                                                                          EC03
a
     S = skin notation, which means that skin absorption may contribute considerably to body burden; sens = substance can
     cause sensitisation.
b
     Reference to the most recent official publication of occupational exposure limits.
c
     Measured as inhalable fraction of the aerosol.
d
     Maximum number per shift: 4, with a minimum interval between peaks of 1 hour.
e
     Classified in pregnancy categorie D, i.e., classification in one of the groups A-C is not yet possible because, although the
     data available may indicate a trend, they are not sufficient for a final classification.
f
     Classified in carcinogen category A4, i.e., not classifiable as a human carcinogen: agents which cause concern that they
     could be carcinogenic for humans but which cannot be assessed conclusively because of lack of data. In vitro or animal
     studies do not provide indications of carcinogenicity which are sufficient to classify the agent into one of the other
     categories.
090-37        Thiram
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<pre>090-38 Health-based Reassessment of Administrative Occupational Exposure Limits</pre>

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