<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>Warfarin
(CAS No: 81-81-2)
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/112, The Hague, March 30, 2004
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<pre>Preferred citation:
Health Council of the Netherlands: Committee on Updating of Occupational
Exposure Limits. Warfarin; Health-based Reassessment of Administrative
Occupational Exposure Limits. The Hague: Health Council of the Netherlands,
2004; 2000/15OSH/112.
all rights reserved
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<pre>1     Introduction
      The present document contains the assessment of the health hazard of warfarin
      by the Commitee on Updating of Occupational Exposure Limits, a committee of
      the Health Council of the Netherlands. The first draft of this document was
      written by AAE Wibowo, Ph.D. and MM Verberk, Ph.D. (Coronel Institute,
      Academic Medical Centre, University of Amsterdam, Amsterdam, the
      Netherlands).
           The evaluation of the toxicity of warfarin has been based on the reviews by
      the American Conference of Governmental Industrial Hygienists (ACGIH)
      (ACG98) and by Hall et al. (Hal80), Holbrook et al. (Hol96), Palareti and
      Legnani (Pal96), and Sutcliffe et al. (Sut87). Where relevant, the original
      publications were reviewed and evaluated as will be indicated in the text. In
      addition, in September 1998, literature was searched in the databases Medline,
      Embase, Chemical Abstracts, starting from 1966, 1988, and 1970, respectively.
      HSELINE, CISDOC, MHIDAS, and NIOSHTIC (from 1985/1987-1998) and
      POLTOX (Toxline, Cambridge Scient Abstracts, FSTA; from 1990-1994),
      databases available on CD-ROM were also consulted. The following key words
      were used: warfarin and 81-81-2. The final literature search was carried out in
      Medline and Toxline in October 2003.
           In October 2003, the President of the Health Council released a draft of the
      document for public review. No comments were received.
2     Identity
      name                 :   warfarin
      synonyms             :   (RS) 4-hydroxy-3-(3-oxo-1-phenylbutyl) coumarine (IUPAC
                               name); 4-hydroxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-
                               one;(1-(4’-hydroxy-3’coumarinyl)-1-phenyl-3 butanone;3-(alpha-
                               acetonylbenzyl)-4-hydroxycoumarin;coumadin; zoocoumarin
      molecular formula    :   C19 H16O4
      structural formula   :
      CAS number           :   81-81-2
      * Chiral centre.
112-3 Warfarin
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<pre>3     Physical and chemical properties
      molecular weight             :     308.3
      melting point                :     159-161oC
      boiling point                :     decomposes
      flash point                  :     not available
      vapour pressure              :     at 21.5oC: 9 Pa
      solubility in water          :     not soluble (at 20oC: 1.7 mg/100 mL)
      log Poctanol/water           :     2.52, 2.60 (experimental); 2.23 (estimated)
      conversion factor            :     not applicable
      Data from ACG98, NLM02, http://esc.syrres.com.
      Warfarin is a colourless, odourless, and tasteless white crystalline powder. It is
      readily soluble in acetone and dioxane and moderately soluble in alcohols. It is
      acidic. The sodium salts are soluble in water. Contact with strong oxidisers may
      cause fire and explosions. Commercial warfarin is a racemic mixture of
      approximately equal amounts of the R and S enantiomers as either a potassium or
      a sodium salt (Hol96).
4     Uses
      Warfarin is used as an anticoagulant drug as well as a rodenticide. As an oral
      anticoagulant drug, warfarin is effective in the prevention and treatment of deep
      vein thrombosis, and also in the prevention of thromboembolic disease. As a
      rodenticide, warfarin is applied to discrete sites in agriculture and urban rodent
      control in the form of baits containing 0.025% active ingredient. The sodium salt
      is available at 0.5% concentrate for use at a final concentration of 0.05% in liquid
      base (ACG98, Hol96, NLM02).
           According to the database of the Dutch Pesticide Authorisation Board
      (CTB)*, warfarine is at present not permitted in the Netherlands for use as an
      active ingredient in pesticides. It is not registered for use as a drug.
*      At: http://www.ctb-wageningen.nl.
112-4 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>5     Biotransformation and kinetics
      Human data
      In vivo studies
      No qualitative or quantitative data were found on the percentage of pulmonary
      absorption of warfarin in humans. Warfarin caused intoxication in a worker
      following skin contact with the 0.5% concentrate. However, no quantitative data
      on the percentage absorption of warfarin was given (Fri65). Warfarin absorption
      following oral intake was studied in 4 patients on long-term treatment with
      warfarin. Subjects were given single doses of 4-[14C]-racemic warfarin of 0.5
      mg/kg bw. Four and 25 days after treatment, 60% and 92% of the radioactivity,
      respectively, were excreted in the urine. In another experiment by the same
      author, 8 human volunteers were given single oral doses of racemic (rac)
      warfarin of 0.5 mg /kg bw. To assess the percentage of the dose absorbed, the
      same volunteers received an intravenous injection of 0.5 mg/kg, 2 weeks later.
      The subjects were given vitamin K1 prior to warfarin administration to avoid
      signs of toxicity. The percentage of the oral dose absorbed varied between 78 and
      105%, and was complete by 120 minutes after administration. The compound
      reached maximal plasma concentrations (range: 4-7 mg/L) by 25 to 60 minutes
      after administration. The mean half-life of elimination from the plasma was
      about 36 hours. The conclusion of these studies was that warfarin was rapidly
      and extensively (>95%) absorbed from the gastrointestinal tract (Bre73a). In an
      earlier human volunteer study (n=15), a much longer time to reach peak
      concentrations (3 to 9 hours) was found following ingestion of single doses of
      1.5 mg rac-warfarin/kg bw. The maximum plasma warfarin concentrations
      ranged from 8.6 to 17.5 mg/L (mean: 12 mg/L) and the plasma half-life ranged
      from 15 to 52 hours (mean: 42 hours) (ORe63). When 2 patients were given a
      single oral dose of 10 mg rac-warfarin (ca. 0.14 mg/kg bw), maximum plasma
      concentrations were reached by 30 to 60 minutes (McA92). Binding to albumin
      and extensive enterohepatic circulation contribute to the long plasma half-life
      (Jah77). The presence of food slowed the rate of warfarin absorption but did not
      affect its bioavailability (Mus76).
          The relationship between dose levels of warfarin and steady-state warfarin
      concentrations in plasma or serum was reported in 2 studies. When 5 human
      volunteers were given therapeutic daily doses of 10 mg rac-warfarin (ca. 0.14
      mg/kg bw/day) for 26 days, a steady-state plasma concentration (mean: 2.6
112-5 Warfarin
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<pre>      mg/L) was reached after 11 days (Jai79). Daily ingestion of non-therapeutic
      doses of 0.2 or 1.0 mg (ca. 0.003 and 0.015 mg/kg bw/day) rac-warfarin by
      human volunteers (n=7/group), for 3 weeks, resulted in steady-state serum
      concentrations of 0.060 or 0.231 mg/L after 2 weeks (Cho88).
          The kinetics of the enantiomers R- and S-warfarin were also investigated in
      several human studies. When 10 human volunteers received single oral doses of
      rac-warfarin, R-warfarin, and S-warfarin of 1.5 mg/kg bw in separate
      experiments, rac-warfarin and its 2 enantiomers were absorbed from the
      gastrointestinal tract to the same extent. The mean half-lives of elimination from
      the plasma for R-warfarin, S-warfarin, and rac-warfarin were 58, 33, and 42
      hours, respectively (ORe74). In another study, mean plasma half-lives of 45, 26,
      and 42 hours were found in 8 human volunteers receiving single oral doses of
      0.75 mg/kg bw each of R-warfarin, S-warfarin, and rac-warfarin, respectively
      (Lew74). The plasma half-lives of the warfarin enantiomers were compared
      when single oral doses of 0.5 mg/kg bw of each enantiomer were given to 9
      subjects or when daily oral doses of 5-17 mg (ca. 0.07-0.25 mg/kg bw/day) of
      each enantiomer were given to 8 subjects for 17 days. The mean plasma half-life
      of R-warfarin after a single dose was 35 hours and was significantly longer than
      that of S-warfarin (24 hours). After repeated doses to steady-state plasma
      concentrations, the mean plasma half-life of R-warfarin (54 hours) was also
      significantly longer than that of the S-isomer (32 hours). The plasma half-life of
      R- but not of S-warfarin was significantly longer after repeated than after single
      doses. Subjects (n=4) receiving daily doses of 5 mg/day of each enantiomer for
      17 days had mean steady-state plasma concentrations of 1.1 and 0.97 mg/L for
      the R- and S-isomer, respectively (Bre73b). When 5 human volunteers took 1 mg
      of each enantiomer for 2 weeks, steady-state serum concentrations of 0.248 and
      0.175 mg/L were measured for the R- and S-isomer, respectively (Cho86).
          When absorbed, warfarin is bound to plasma proteins, mainly albumin (97.4
      to 99.9%). The anticoagulant activity of warfarin is a function of the
      concentration of the unbound drug in plasma. Albumin-bound warfarin is
      pharmacologically inactive and is not biotransformed and excreted. The small
      apparent volume of distribution of warfarin reflects the high protein binding
      (Hol96). The binding of the S-enantiomer to albumin is greater than for the
      R-enantiomer (Par88). The unbound fraction is distributed mainly to the liver,
      where it binds strongly in a saturable way to the target enzyme, vitamin K-2,3-
      epoxide reductase, in liver microsomes (Pal96).
          Warfarin is metabolised in the smooth endoplasmatic reticulum of the liver,
      involving stereospecific pathways catalysed by a variety of cytochrome P450
      isoenzymes (Hol96, Pal96). The metabolism of rac-warfarin has been studied in
112-6 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>      healthy human volunteers, who were given single oral doses of 1.5 mg/kg bw of
      sodium warfarin. Major metabolites excreted within 72 hours after
      administration were the regioisomers 6- and 7-hydroxywarfarin, formed by
      hydroxylation reactions, and 2 diastereoisomeric warfarin alcohols, formed by
      acetonyl (keto) reduction. No warfarin (<2% of the dose) was excreted in the
      urine. No quantitative data of the amount of excreted products were given
      (Lew70). In a subsequent study, single oral doses of 0.75 mg/kg bw of rac-
      warfarin were given to 3 volunteers, and as a single oral dose of 1.5 mg/kg bw to
      1 subject. The percentage of the dose, excreted in urine collected for 7-10 days
      after administration, was in the range of 33-55%. The main metabolites were 7-
      and 6-hydroxywarfarin (47% and 32% of total urinary metabolites, respectively),
      while RS- and SS-warfarin alcohol were excreted in amounts of 11% and 7.5% of
      total urinary metabolites. Unchanged warfarin represented about 3.5% of total
      urinary metabolites (Lew74). It is likely that some warfarin is eliminated in the
      bile and undergoes enterohepatic circulation (Hol96). No warfarin was detected
      in the faeces of human volunteers given single oral doses of 1.5 mg/kg bw of
      sodium warfarin (ORe63).
      The metabolism of rac-warfarin in humans is presented in Figure 1 (see Annex
      I).
      An examination of the metabolic fate of the R and S-enantiomers of warfarin
      revealed that the 2 enantiomers were metabolised by different routes. In a human
      study, single oral doses of each enantiomer were given to 5 human volunteers in
      separate experiments, in the amount of 0.75 mg/kg bw. Following administration
      of R-warfarin and S-warfarin, 20-31% and 48-71% of the dose were excreted in
      the urine, respectively, within 7 to 10 days. Major metabolites of R-warfarin
      were 6-hydroxywarfarin and (R,S)-warfarin alcohol (50% and 30% of total
      urinary metabolites, respectively). The metabolite 7-hydroxywarfarin was
      formed to a smaller extent (13% of total urinary metabolites). In contrast, the
      main metabolite of S-warfarin was identified as 7-hydroxywarfarin (66% of total
      urinary metabolites). Other metabolites were 6-hydroxywarfarin and S,S-
      warfarin alcohol (Lew74).
      Animal data
      The committee did not find quantitative data on the percentage of pulmonary or
      skin absorption of warfarin in experimental animals. Indirectly, skin absorption
      has been demonstrated in rats and guinea pigs, by the measurement of the effects
112-7 Warfarin
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<pre>      of warfarin on blood coagulation (Fri65, Sag75) (see Section 6). In order to
      assess the amount of absorption via the oral route in rats (Sprague-Dawley),
      urinary excretion following either single oral doses or intravenous injections of
      4-[14C]-warfarin of 1 mg/kg bw was compared. At 100 hours after treatment,
      43% or 41% of the dose were excreted in the urine after oral or intravenous
      administration, respectively. In faeces, 9.3% of the radioactivity could be
      isolated. The same experiment was conducted in rats, fitted with biliary fistula to
      prevent enterohepatic circulation. At 60 hours, 39.4 and 18.9% of the oral dose
      were found in the bile and the urine, respectively, while similar figures were
      obtained (47.5% in bile, and 21.3% in urine) after intravenous administration.
      Thus, elevated levels of radioactivity excreted in urine in animals without biliary
      fistula indicate the existence of an enterohepatic circulation for warfarin and
      metabolites in the rat (Los72).
          The kinetics of 4-14C-labelled S- and R-warfarin have been reported in rats,
      following a single intraperitoneal injection of 0.4 mg/kg bw of each enantiomer.
      At 150 hours after administration, ca. 65% of the R- and 50% of the S-warfarin
      dose were excreted in the urine, and 9% of the dose of either enantiomer in the
      faeces. No radioactivity was detectable in expired air (God69).
          When absorbed, the liver showed the greatest affinity for warfarin in the rat.
      However, distribution and tissue uptake has also been demonstrated in
      extrahepatic tissues, e.g., heart and skeletal muscle, but probably reflected blood
      distribution rather than actual uptake of warfarin (And67). Binding of warfarin to
      plasma albumin is high in the rat, followed by sheep, dog, and horse (Sel77). A
      6-fold greater binding to human, compared to canine plasma albumin has been
      demonstrated (ORe70). However, human and rat are more sensitive to the
      anticoagulant effects of warfarin than the other species investigated, suggesting
      that plasma albumin binding does not account for variations in the
      pharmacological response between species (Sut87). Following single
      intravenous administration of doses ranging from 1 to 12 mg/kg bw, the half-life
      of elimination of warfarin from plasma in rat, monkey, dog, and man was on
      average 9.9, 11.1, 22.5, and 36 hours, respectively (Nag69). The disappearance
      rate of unbound warfarin from plasma, compared to total warfarin, was faster in
      fasted rats (Lal77).
          The metabolism of warfarin has been studied in the rabbit, the rat, and the
      guinea pig. When rabbits were given a single oral dose of racemic 4-[14C]-
      warfarin (1 mg/kg bw), 82% or 90% of the radioactivity were excreted in the
      urine within 48 or 96 hours after administration, respectively. The major urinary
      metabolites were the warfarin alcohols (31% of the dose), 4’-hydroxycoumarin
      (11% of the dose), 6-and 7-hydroxycoumarin (7 and 3% of the dose,
112-8 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>      respectively), and their glucuronides (7.5% of the dose). Unchanged warfarin
      (12% of the dose) was also excreted in the urine. Following an intravenous
      injection of 1 mg/kg bw, the bile was a minor excretory route for warfarin (6.2%
      of the dose after 6 hours), and most metabolites were glucuronide conjugates of
      warfarin and its hydroxylated metabolites (Won80).
          When male Holtzman rats (n=13) were given a single intraperitoneal
      injection of an aqueous solution containing 1 mg of 4-[14C]-warfarin, 67% of the
      dose was excreted in the urine and 33% in the faeces within 7 days after
      administration. The metabolites excreted in the urine were 7-hydroxywarfarin
      (23% of the dose), 4’-hydroxywarfarin (14% of the dose), 6-hydroxywarfarin
      (10% of the dose), 8-hydroxywarfarin (6% of the dose), a glucuronide of
      7-hydroxywarfarin (2.6% of the dose), and an intramolecular condensation
      product: 2,3-dihydro-2-methyl-4-phenyl-5-oxo-γ-pyrano(3,2-c)(1)benzopyran
      (4.4% of the dose). No warfarin alcohols were detected. Unchanged warfarin
      represented about 4.4% of the dose. Qualitatively, the same metabolites were
      identified in the faeces, but no quantification of metabolites was conducted.
      According to the authors, the similarities in urine and faeces metabolites suggest
      that enterohepatic circulation of metabolites did occur (Bar70).
          Male guinea pigs received a single 1 to 2 mg/kg bw of 4-[14C]-warfarin by
      intraperitoneal injection. About 86% of the administered radioactivity was
      excreted in the urine within 7 days after injection, most of it (50% of the dose)
      during the first 12 hours. Another 9% of the dose was excreted in the faeces
      within 7 days. The major metabolite excreted in the urine during 7 days after
      injection was 4’-hydroxywarfarin (24% of the dose). Other metabolites were
      6-hydroxywarfarin (4% of the dose), salicylic acid (4% of the dose), 7 and 8-
      hydroxywarfarin (2% of the dose each), and 2,3-dihydro-2-methyl-4-phenyl-5-
      oxo-γ-pyrano(3,2-c)(1)benzopyran (4% of the dose). Unchanged warfarin was
      excreted in about 11% of the administered dose. These metabolites were
      detected qualitatively in the faeces, but were not quantified (Dec73). In another
      study, male guinea pigs were given a single intraperitoneal injection of [14C]-
      warfarin (1 mg/kg bw). The maximum blood level was reached within 1 hour,
      and the half-life of elimination from the blood was 9.6 hours. Radioactivity in
      tissues at 6 hours after administration was highest in the kidney, followed by
      bile, and liver. About 70% of the dose was excreted in the urine and 6% in the
      faeces at 48 hours after administration. However, 28% or 42% of the dose were
      excreted in the urine or the bile during the first 12 hours after administration. The
      major metabolite in urine and bile was 4’-hydroxywarfarin, followed by 7- and
      8-hydroxywarfarin. A minor metabolite was 2,3-dihydro-2-methyl-4-phenyl-5-
      oxo-γ-pyrano(3,2-c)(1)benzopyran. Wong et al. concluded that a majority of
112-9 Warfarin
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<pre>       warfarin metabolites, excreted in the bile, underwent enterohepatic circulation
       and was disposed of in the kidney in this species (Won78).
       In vitro studies
       The cytochrome P450 isoenzymes involved in the metabolism of warfarin have
       been studied in in vitro experiments, using either human cDNA-expressed
       cytochrome P450 or isoenzymes isolated from human liver microsomes.
       Cytochrome P450 2C9 has been claimed to be responsible for the metabolism of
       the S-isomer, whereas degradation of the R-isomer was determined mainly by
       cytochrome P450 1A2. Cytochrome P450 3A4 is involved in the metabolism of
       both isomers (Ret92, Wan83).
6      Effects and mechanism of action
       Warfarin inhibits the synthesis of vitamin K-dependent clotting factors II
       (prothrombin), VII, IX, and X. The mechanism of this anticoagulant effect is
       based on interruption of the vitamin K recycling process in the liver, by
       inhibiting the activity of the enzymes vitamin K epoxide reductase and vitamin K
       quinone reductase. Vitamin K epoxide catalyses the conversion of vitamin K 2,3-
       epoxide to vitamin K quinone while vitamine K quinone reductase catalyses the
       subsequent conversion of the quinone to the active form of vitamin K, the
       hydroquinone. The vitamin K quinone can also be reduced to the hydroquinone
       form by a second NADPH-dependent enzyme, which is not inhibited by
       coumarin derivatives. Vitamin K hydroquinone is a substrate in the reaction that
       leads to the formation of γ-carboxyglutamyl residues in precursor proteins, to
       form active vitamin K-dependent clotting factors II, VII, IX, and X. This
       γ-carboxylation of glutamic acid residues at the N-terminal regions of precursor
       proteins, named PIVKAs (proteins induced by vitamin K antagonists), is
       catalysed by a vitamin K-dependent carboxylase enzyme, and requires molecular
       oxygen and carbon dioxide. Concomitantly to this process occuring in the liver
       only, vitamin K hydroquinone is epoxidised to vitamin K 2,3-epoxide. By the
       action of warfarin on the enzymes of the vitamin K cycle, vitamin K epoxide
       accumulates, leading to a reduced supply of vitamin K hydroquinone, and
       subsequently to inhibition of γ-carboxylation of PIVKAs, thereby interrupting
       the supply of functional vitamin K-dependent clotting factors. Depression of the
       plasma concentration of vitamin K-dependent clotting factors will produce a
       prolongation of the prothrombin time. Daily dosing with 2.5 to 15 mg warfarin,
       e.g., patients on long-term anticoagulant therapy, will result in low levels (10 to
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<pre>       30% of normal activity) of vitamin K-dependent clotting factors, with a danger to
       bleeding (Hir92, Hol96, Pal96, Sut87). In the occupational setting, activities
       below 70-80% of normal plasma activity are considered as adverse (Sit94).
       The vitamin K cycle in the liver and its inhibition by coumarins (e.g., warfarin) is
       shown in Figure 2 (see Annex II).
       In extrahepatic tissues (lung, kidney, pancreas, spleen, bone, placenta), vitamin
       K-dependent proteins containing glutamic acid residues, other than clotting
       factors, have been found. Consequently, warfarin may have an effect on these
       proteins by antagonising the action of vitamin K in these tissues (Sut87).
       Human data
       Irritation and sensitisation
       The most important non-haemorrhagic side effect of warfarin is skin necrosis.
       Warfarin-induced skin necrosis occurs predominantly in females (85% of the
       patients), usually in areas rich in subcutaneous fat tissues (breasts, thighs, and
       buttocks) with a sudden onset of painful or cold sensation in the affected areas,
       and the appearance of well-demarcated erythematous lesions that progressed to
       purpuric and haemorrhagic areas. In males with skin necrosis, the distribution of
       lesions was similar to that in females, except that the breasts were usually spared,
       while involvement of the penis was common (Sal97). The first symptoms of skin
       injury usually appear within 10 days of therapy with a peak incidence being
       between days 3 and 6. Cases of rare late-onset warfarin-induced skin necrosis
       have been reported after 15, 16, and 17 days, 3 months, 17 months, and 3 years
       of warfarin therapy (Ess98). Some cases of warfarin-induced skin necrosis are
       reported below. A 64-year-old female patient developed ecchymoses on the
       forearms, hands, left breast, and right thigh and hip, after a total dose of 35 mg
       warfarin during 3 days. The lesions progressed through a blistering stage to a dry
       gangrene with eschar formation. Microscopic examination of the skin showed
       areas of vascular congestion and multiple platelet thrombi in the small arterioles
       and veins (Lac75). Another case dealt with a 53-year-old man who experienced
       2 episodes of skin necrosis on his left flank and buttock, 5 days after initiation of
       warfarin therapy for thrombophlebitis. The lesions formed multiple
       haemorrhagic bullae that ruptured and an eschar that did not heal and eventually
       required skin grafting (Hor81). The same author reported on a 79-year-old
       woman, who developed an area of erythema surrounded by a halo on her left
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<pre>       thigh, 7 days after initiation of warfarin therapy. The erythematic area turned into
       a blue-black colour and rapidly formed an eschar deep into the subcutaneous
       tissue. A 37-year-old woman was admitted with an erythematous area on her
       right thigh that turned black and subsequently formed an eschar. Her
       prothrombin time was 2-fold the control level (Hor81). Late-onset warfarin-
       induced skin necrosis, 16 days after the initiation of warfarin therapy, was
       reported in a 34-year-old female patient. She was treated with 10 mg warfarin/
       day for 4 days, which was decreased to 5 mg/day before discharge from the
       hospital. Petechiae developed in thighs, legs, and left under arm. Lesions
       progressed to blue-black ecchymotic areas with several bullae. Microscopic
       examination revealed subepidermal haemorrhage with adjacent epidermal
       necrosis. Unlike other cases of warfarin-induced skin necrosis, the skin lesion
       was not associated with either a deficiency of protein C, or resistance to activated
       protein C (Ess98).
           Cases of dermatitis have been reported in the older literature. A 50-year-old
       man developed transient urticaria, 40 minutes after the oral administration of 50
       mg of warfarin sodium. The rash completely subsided in 2 days, following
       treatment with diphenhydramine (She59). In another case, a 63-year-old male
       patient developed pruritic, maculopapular, erythematous eruption on the face,
       neck, hands, and forearms after treatment with an initial dose of 75 mg sodium
       warfarin, followed by a daily maintenance dose of 7.5 mg for 27 days. Lesions
       disappeared with steroid therapy, but recurred upon further treatment with
       warfarin (Cra60).
       Short-term toxicity
       There is very little information on health effects of workers engaged in the
       manufacture or use of warfarin. One case has been reported, in which a 23-year-
       old farmer developed signs of poisoning following regular skin contact with a
       0.5% warfarin solution, used for the preparation of baits, during a 4-week period.
       Two days after the last skin contact with warfarin, gross haematuria appeared.
       The next day haematoma were noticed on arms and legs. There was a dull pain in
       both groins. Haematuria subsided after 3 days of rest, but recurred along with
       epistaxis, haemorrhages from the mucosa of palate and mouth, and bleeding
       from the lower lip, 1 day after he returned to work. When admitted to hospital,
       prothrombin time was increased, and haemoglobin and red blood cell count
       decreased. Following treatment with vitamin K1 (phytonadione), the subject
       responded promptly. Two days after treatment, haematology and urine
       parameters did not show abnormalities (Fri65). Cases of a warfarin-induced
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<pre>       haemorrhagic syndrome were reported in 741 Vietnamese infants. The cause was
       identified as dermal exposure to talcum powder, contaminated with 1.7% to
       6.5% warfarin. 177 out of the 741 children died (Mar83).
           Several reports have been published on poisoning cases, when warfarin bait,
       used as a rodenticide, was accidentally or deliberately mixed with food, or taken
       in an attempt to commit suicide. In Korea, a family of 14 persons lived for a
       period of 15 days on a diet consisting almost entirely of corn meal-containing
       warfarin. The first symptoms appeared 7-10 days after the eating of warfarin-
       contaminated food began. A 19-year-old man and a 3-year-old girl died after 15
       days, after having ingested an estimated total warfarin amount of 12.5 and 31
       mg/kg bw, respectively. The other 12 persons, aged between 8 and 70 years,
       survived after having received vitamin K1 therapy. The ingested total amounts of
       warfarin ranged from 4 to 22 mg/kg bw. Symptoms of toxicity were ecchymosis,
       epistaxis, and gum haemorrhages (Lan54). One case dealt with murder on a
       32-year-old man, who was given an estimated daily amount of 60-90 mg
       warfarin in his food for 15 days. On the 4th day after intake started, the victim
       developed severe nosebleeds. Later, he bled from the mouth. Two days before
       death, he complained of pain in his limbs. He died of circulatory failure on day
       15. Macroscopic examination revealed haemorrhages in muscles, intestine,
       lungs, heart, and kidneys. Microscopic examination showed congestion and
       oedema in the lungs and liver injury (Pri66). In another case, a 73-year-old
       woman developed episodes of hypoprothrombinaemia each time she had
       ingested a cough syrup, which had been deliberately contaminated by her
       daughter-in-law with a warfarin-containing rat killer. Symptoms of toxicity were
       backache, haematuria, epistaxis, and bruises on the arms and the legs. The
       estimated ingested amount of warfarin was 20-40 mg/day. Recovery occurred
       after vitamin K treatment (Nil57). In a suicide attempt, a 22-year-old man had
       consumed daily amounts of approximately 20 g of a 0.5% warfarin formulation,
       in the form of a dry powder, over a 6-day period. The total ingested warfarin
       dose was estimated approximately 600 mg. Symptoms of toxicity were back
       pain, abdominal pain, epistaxis, and haematuria. Prothrombin time and Lee-
       White coagulation time were prolonged, but no abnormal haemoglobin level was
       found. Following treatment with vitamin K1, he recovered 14 days after having
       taken the last dose (Hag53, Hol52).
           The above poisoning cases have given some insight in the dose-response
       relationship following ingestion of warfarin. The relationship between warfarin
       dose or plasma warfarin concentration and anticoagulant activity has been
       examined in a series of human volunteer studies and in patients receiving
       therapeutical doses of warfarin. In a human volunteer study, 14 subjects received
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<pre>       a single oral dose of rac-warfarin of 1.5 mg/kg bw (see Section 5). The
       anticoagulant action of warfarin, i.e., inhibition of hepatic synthesis of clotting
       factors II, VII, IX, and X (the prothrombin complex), was determined by the
       measurement of the plasma prothrombin time (PT) by the one-stage prothrombin
       method of Quick. In all subjects, PTs were significantly increased (i.e.,
       prothrombin complex activity significantly depressed) within 24 hours. The
       maximal PT increment was reached between 36 and 72 hours after treatment.
       The average PT was still abnormal at 144 hours after dosing. There was a
       significant correlation between the plasma warfarin levels at 48, 72, and 96
       hours, and prothrombin complex activity depression. For example, at 48 or 96
       hours after warfarin dosing, average prothrombin complex activities were
       depressed by 70% or 50%, respectively, at an average plasma warfarin
       concentration of 1 mg/L (ORe63). In another study, 5 human volunteers received
       a daily therapeutical oral dose of 10 mg rac-warfarin (ca. 0.14 mg/kg bw) for 26
       days (see Section 5). The mean steady state plasma warfarin concentration (2.6
       mg/L) was associated with a mean PT of 20.9 seconds (normal range: 12-14
       seconds) (Jai79). The effect of low dose administration of rac-warfarin on
       clotting factor activity and vitamin K1 metabolism was studied in groups of 7
       human volunteers, who were given daily oral doses of 0.2 or 1.0 mg (ca. 0.003 or
       0.015 mg/kg bw/day) for 3 weeks (see Section 5). In the 1.0-mg group, at a mean
       steady state plasma warfarin concentration of 0.231 mg/L, there was a
       statistically, but not biologically significant prolongation of the mean PT of 0.9
       seconds. This prolongation was mainly due to one volunteer, who showed a PT
       increment of 2.5 seconds and had a significant decrease in individual clotting
       factor activity. No statistically significant change in mean PT or any clotting
       factor activity was observed in the 0.2-mg group (steady state plasma warfarin
       concentration: 0.06 mg/L). However, when vitamin K1 was given to all 7
       volunteers in both groups, detectable levels of vitamin K 2,3-epoxide were
       observed that peaked approximately 2 hours after treatment. This indicates that
       warfarin inhibits the enzyme vitamin K epoxide reductase, even at 0.003 mg/kg
       bw/day (Cho88). The NOAEL for a biological significant inhibition of clotting
       factor activity was set at 0.015 mg/kg bw/day. In a group of 15 patients receiving
       warfarin at a mean daily dose of 4.6 mg (range: 2 to 7.5 mg) for 3 weeks, a mean
       steady state plasma warfarin concentration of 0.67 mg/L was required to achieve
       a PT ratio of 1.8. The mean free plasma warfarin concentration was 0.014 mg/L
       (Rou79). A clinical study in patients with metastatic breast cancer was conducted
       to investigate which daily warfarin dose was required to maintain a target
       international normalised ratio (INR) of 1.3-1.9. INR is the ratio of the PT in the
       patient to that in a normal person not treated with anticoagulant. This range of
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<pre>       INRs, which is equivalent to only a 1-3 second prolongation of the PT, is
       effective in the prevention of thromboembolic disease and is associated with less
       bleeding. The dose needed to achieve this target INR was 2.6 mg warfarin daily
       for on average 181 days (Lev94). Other therapeutical maintenance doses of
       warfarin as an anticoagulant were reported to be 3-9 mg/day, to keep the INR in
       the range of 2.0-4.5 (Lau92) or 2-10 mg/day (Maj95). It has been reported that
       low-intensity warfarin therapy with an INR of 2.0-3.0, which corresponds with a
       PT ration of 1.35-1.6, is as effective as high-intensity warfarin (INR>3.0) in the
       prevention of thromboembolism and is associated with less bleeding (Hir92).
           The enantiomers of warfarin differ in their anticoagulant potency in man.
       Following single high dose levels (0.75 and 1.5 mg/kg bw), S-warfarin has been
       reported to have 1.6 (Lew74) and 3.4 (ORe74) times the potency of R-warfarin,
       respectively. At steady state therapeutical doses, S-warfarin was 1.6 (Bre73b) or
       2.7 (Win78) times more active as an anticoagulant than the R-enantiomer. Both
       enantiomers produced a significant increase in prothrombin time, when given to
       5 human volunteers at a daily dose of 1 mg each, for 2 weeks. At steady state, the
       increase in prothrombin time with S-warfarin (1.8 seconds) was statistically
       significantly higher than with R-warfarin (1.0 second). The greater anticoagulant
       potency of S-warfarin was reflected by a greater degree of inhibition of the
       enzyme vitamin K epoxide reductase, measured indirectly by increased levels of
       vitamin K 2,3-epoxide following treatment with vitamin K1 (Cho86). The
       LOAEL for a biological significant increase of prothrombin time was set 0.015
       mg/kg bw/day for S-warfarin. For R-warfarin, the NOAEL was 0.015 mg/kg bw/
       day.
           The committee concludes that measurement of enantiomer concentrations
       may better predict anticoagulation than measurement of racemate concentrations.
            A study has been reported on a group of 36 patients receiving long-term rac-
       warfarin therapy at daily doses of 2.5 to 12 mg (mean: 6.1 mg). The PT ratio was
       in the range of 2.0-3.3, at mean steady-state plasma concentrations of 0.48 and
       0.87 mg/L for S- and R-warfarin, respectively. Chan et al. suggested that the
       degree of anticoagulation is best predicted by the concentration of the free
       (unbound) S-isomer of warfarin (Cha94).
           The warfarin metabolites (RS)- and (SS)-warfarin alcohol showed
       anticoagulant activity. However, their potency was much less than for rac-
       warfarin (Lew73).
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<pre>       Reproduction toxicity
       Coumarin derivatives, including warfarin, are teratogenic in man. Warfarin
       therapy specifically between weeks 6 and 9 of gestation may result in ‘warfarin
       embryopathy’, i.e., fetal malformations characterised by nasal hypoplasia and
       stippled epiphyses, while exposure during the second and third trimester is
       associated with disruptional abnormalities of the central nervous system (Hal80,
       Pau93). In a review concerning 418 reported pregnancies with coumarin
       derivative (including warfarin) therapy, one-sixth of these had resulted in
       abnormal liveborn infants, one-sixth in abortion or stillbirth, and two-thirds in
       apparently normal infants. A total of 11 (3%) liveborn infants had primarily
       haemorrhagic manifestations, 16 (4%) had findings consistent with warfarin
       embryopathy (i.e., nasal hypoplasia, with or without stippled epiphyseal
       calcifications that resemble chondrodysplasia punctata as well as defects of the
       bones and deformities of the limbs) whereas 11 (3%) had significant warfarin-
       induced central nervous system abnormalities. Maternal daily doses of warfarin,
       given between 6 and 9 weeks of gestation, ranged between 2.5 and 15 mg. In the
       11 cases with central nervous system abnormalities, not clearly caused by
       intrauterine or perinatal haemorrhage, brain malformation, such as
       hydrocephalus, meningocele, and microcephaly, ophthalmological
       abnormalities, such as microphthalmia and optic atrophy, and other
       abnormalities, such as developmental retardation, and deafness were reported
       (Hal80). In later report, 15 studies published between 1980 and 1989 the effects
       of warfarine on pregnancy outcome were reviewed. Among the total of 635
       pregnancies and 485 liveborns discussed in these studies, 8 (1.3% of the
       pregnancies; 1.6% of the liveborns) certainly had features of the ‘warfarin
       embryopathy’ while broad inclusion criteria resulted in 20 possibly affected
       infants, and 4 (0.6 or 0.8%, respectively) had central nervous system and/or
       ophthalmological abnormalities. From the aforementioned figures of Hall et al.
       (Hal80) and their own figures, Pauli and Haun thought that reasonable ranges of
       risk for ‘warfarin embryopathy’ would be 1.5-5% of exposed infants, only at
       exposure between gestational weeks 6 and 9, and for central nervous system
       effects 0.5-2%, most often resulting from second trimester exposure (Pau93).
       The mechanism giving rise to warfarin embryopathy may be related to the
       reduced γ-carboxyglutamate content of the bone osteocalcin. Inhibition of the
       synthesis of these proteins by coumarin derivatives during a critical
       embryological period of ossification could explain the teratogenic effects seen in
       warfarin embryopathy (Hal80, Hol96, Pau93).
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<pre>            No cases of teratogenicity have been reported following the use of warfarin as a
            rodenticide (WHO95).
            Animal data
            Irritation and sensitisation
            The committee did not find data from experimental animal studies on the skin- or
            eye-irritating or sensitising properties of warfarin.
            Acute toxicity
            The results of acute lethal toxicity tests are summarised in Table 1.
Table 1 Summary of acute toxicity studies for warfarin in experimental animals
exposure route       vehicle         species (sex)             strain          LC50/LD50        reference
sodium warfarin
inhalation                           rat                                       320 mg/m3 a      ACG98
dermal                               rat                                       1400 mg/kg bw    ACG98
oral                 water           rat (male)                Sprague-Dawley  323 mg/kg bw     Hag53
                     water           rat (female)              Sprague-Dawley  58 mg/kg bw      Hag53
                     CMC             rat (male)                Sprague-Dawley  100 mg/kg bw     Bac78
                     CMC             rat (female)              Sprague-Dawley  8.7 mg/kg bw     Bac78
                     peanut oil      rat (male)                Sherman         3.0 mg/kg bw     Gai60
                                     rat                       Sherman         1.6 mg/kg bw     Hay67
                                     rat                       AW 49           3.4 mg/kg bw     Nie73
                     water           mouse (male, female)                      374 mg/kg bw     Hag53
                                     mouse                     NMRI            640 mg/kg bw     Nie73
                     water           guinea pig (male, female)                 182 mg/kg bw     Hag53
                     water           rabbit (male, female)                     800 mg/kg bw     Hag53
                     water           dog (male, female)                        200-300 mg/kg bw Hag53
intravenous          water           rat (male, female)        Sprague-Dawley  186 mg/kg bw     Hag53
                     water           rabbit (male, female)                     100-200 mg/kg bw Hag53
                     water           dog (male, female)                        200-300 mg/kg bw Hag53
acid warfarin
oral                 CMC             rat (male)                Sprague-Dawley  112 mg/kg bw     Bac78
                     CMC             rat (female)              Sprague-Dawley  10 mg/kg bw      Bac78
a
     Exposure time not given.
     Abbreviations: CMC= carboxymethylcellulose.
            There are large differences in lethality of rac-warfarin between species, sexes,
            and strains. In summary, rats were the most sensitive and mice and rabbits the
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<pre>       least sensitive species. In rats, oral LD50s varied from 1.6 to 323 mg/kg bw,
       dependent on sex, strain, and mode of application. Female rats were 5 to 10 times
       more susceptible than males and Sprague-Dawley rats were less susceptible than
       other strains tested. The acute oral toxicity of rac-warfarin in Sprague-Dawley
       rats was higher when the compound was administered in carboxymethylcellulose
       than in water. Sodium warfarin and acid (enol) warfarin exhibited the same range
       of toxicity. When rac-warfarin was intravenously administered to rats or dogs,
       the acute toxicities were in the same range of toxicity.
       When rats were treated with enzyme inducers phenobarbital, chlordane, or DDT
       for 4 days prior to administration of warfarin, a 10-fold increase in the LD50 of
       warfarin was observed (Ike68).
           Signs of intoxication were convulsions in animals that succumbed within
       several hours after dosing. Haemorrhages were not observed in this group.
       Conversely, in animals dying delayedly, convulsions were not observed and
       haemorrhages were found in several tissues. In addition, the animals’ appearance
       was marked by ruffled coat, pallor, and extreme lassitude (Hag53). Autopsy
       revealed haemorrhages in the intestine, thoracic cavity, peritoneal cavity, and
       urinary bladder (Bac78).
           The committee concluded that the differences in acute toxicity of rac-
       warfarin between species, sexes, and strains, as well as differences in acute
       toxicity between enantiomers might be explained by differences in kinetics and
       metabolism on the one hand and by a difference in the inhibition of the synthesis
       of vitamin K-dependent clotting factors on the other hand.
       Several studies have been reported on the anticoagulant effects of sodium
       warfarin following single application via different routes.
           Rabbits or guinea pigs, treated dermally with aqueous sodium warfarin at
       single doses of 0.25 or 1.7 mg a.i./kg bw, respectively, showed a maximum
       inhibition of 32% or 42% of the activity of the prothrombin complex,
       respectively, at day 2 after administration. In guinea pigs, the same anticoagulant
       effect was observed following a single oral dose of 2 mg/kg bw (Fri65). In
       another study, Sprague-Dawley rats (n=4-5/group) were given sodium warfarin
       via the food at doses of 0, 1.3, 4.8, or 32 mg/kg bw for 1 day. On average, mean
       PTs were increased 1.7, 3.8, or 4.9-fold, respectively, compared to the control
       value. No mortality was observed at these doses (Hag53). The anticoagulant
       effect of warfarin was studied in AW49 rats and NMRI mice. A 3-fold increase
       in PT, compared to control animals, was found at a single oral dose of
       1 mg/kg bw for rats and at 10 mg/kg for mice (Nie73).
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<pre>            The effects of organic solvents on the anticoagulant response to warfarin
       treatment were investigated in male Sprague-Dawley rats. A 2.8 to 3.6-fold
       increase in PT compared to control rats was found, 24 hours after a single
       subcutaneous injection of 1 mg/kg bw sodium warfarin in corn oil. Significant
       increments in PT were seen when warfarin (1 mg/kg bw) was administered
       simultaneously with hepatotoxic doses of styrene (4.4- to 6.9-fold) or
       trichloroethylene (4.8- to 7.4-fold). However, no statistically significant changes
       in PT were observed following the simultaneous injection with hepatotoxic doses
       of carbon tetrachloride. Pre-treatment of rats with either styrene,
       trichloroethylene, or carbon tetrachloride, at 24 hours prior to subcutaneous
       injection with warfarin (1 mg/kg bw), also caused an increment of PT compared
       to treatment with warfarin alone. Solvents alone had no effect on PT. The author
       concludes that acute exposure to organic solvents may lead to enhanced
       anticoagulant response to warfarin (Cha86).
            Studies on species differences in anticoagulant response to warfarin showed
       that the rat, mouse, and human are most sensitive, guinea pigs, cats, and dogs
       intermediate, and the rabbit and cow least affected. It was suggested that the
       difference in affinity at the site of action between species and not plasma protein
       binding accounts for differences in pharmacological response (Sut87).
            Differences in the anticoagulant potency of S-warfarin and R-warfarin were
       demonstrated in the rat prothrombin-time assay. A single oral dose of S-warfarin
       was 5.5 times as active as a single oral dose of R-warfarin, as measured at 24
       hours after dosing. Single oral doses of 0.375 mg/kg bw of S-warfarin and 1.9
       mg/kg bw of R-warfarin showed parallel time–response curves between 0 to 48
       hours after treatment. Maximum prothrombin times were increased
       approximately 3.5-fold, compared to control values at 36 hours after dosing
       (Ebl66). In another study, it was demonstrated that following a single
       intravenous administration, S-warfarin was 2 times more potent than R-warfarin
       in its ability to inhibit clotting factor synthesis (Bre72). In rabbits, following a
       single intravenous administration, the minimum plasma concentrations to
       achieve complete inhibition of clotting factor synthesis were 103, 99, and 25 mg/
       L for rac-warfarin, R-warfarin, and S-warfarin, respectively, indicating that the
       S-enantiomer is the most potent anticoagulant (Bre85).
       Short-term toxicity
       When a single dose of 0.4 mg/kg bw of an aqueous solution of sodium warfarin
       was applied to the skin of rabbits for 2 consecutive days, the activity of the
       prothrombin complex was decreased by 62% at post-treatment day 2. At day 5,
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<pre>       the activity was returned to normal (Fri65). In another dermal study, an aqueous
       solution of sodium warfarin was administered to the skin of female Wistar rats
       (n=7/group) at doses of 0,10, 50, or 100 mg a.i./kg bw/day, for 3 consecutive
       days. On the basis of statistically significant increased clotting times at the 2
       higher dose levels, compared to control animals, it was concluded that skin
       absorption did occur. The anticoagulant effect of 3 topical applications at 50
       mg/kg bw/day was about the same as that of 3 oral doses of 0.6 mg/kg bw/day
       (Sag75).
           Sprague-Dawley rats (n=4-6/group) were given sodium warfarin via the diet
       at doses equivalent to approximately 0.13, 0.35, 0.4, 1.0, 1.5, 3.0, 6.0, or 18
       mg/kg bw/day for various time periods. Doses of 0.4 mg/kg bw/day and above
       caused death of all animals within about 7 to 11 days. One out of 4 rats given
       0.35 mg/kg/day for 6 months died, but all 4 rats given 0.13 mg/kg bw for 8
       months survived. In another experiment by the same authors, rats (n=10/group)
       received daily doses of sodium warfarin via the diet, varying from 1.2, 6.9, or 28
       mg/kg bw for 2 days to 1.1, 4.4, or 17 mg/kg bw for 5 days. Mortality was 50%
       or more in the high-dose group after 2 to 5 days feeding and in the mid-dose
       group after 3 to 5 days feeding. In the low-dose group, 2 or 1 out of 10 animals
       died after 4 or 5 days feeding respectively. In a separate experiment, the authors
       also determined the PTs of animals (n=2-4/group) treated with the same doses
       for 1 to 5 days. After 2 days of feeding, mean PT values were increased 2.8, 4.9,
       or 5.7-fold and after 4 days feeding 7.7, 23, or 33-fold, respectively, compared to
       non-treated animals (Hag53). In another study, 5-day cumulative oral LD50s of
       2.1 and 25 mg/kg/day were found in AW49 rats and NMRI mice, respectively
       (Nie73). When groups of Sprague-Dawley rats (n=110) were given warfarin via
       the diet for 90 days, a cumulative oral LD50 of 0.077 mg/kg/day was found
       (Hay67). For R- and S-warfarin, 10-day cumulative oral (diet) LD50 values of
       17.7 and 2 mg/kg diet (i.e., ca. 0.9 and 0.1 mg/kg bw/day, assuming a 200-g rat
       consumes 10 g diet/day), respectively, were calculated in male Sprague-Dawley
       rats (Elb66).
           Dogs (greyhound; n=3/sex) were given warfarin via the diet at a dose
       equivalent to 10 mg/kg bw/day for 4 or 5 days. Signs of intoxication were apathy
       and reduced food intake by day 4 or 5 and anorexia and vomiting by day 7 or 8.
       Two dogs developed severe respiratory distress and 2 died suddenly from
       intraperitoneal bleeding. Blood urea levels were significantly increased after
       treatment, but no changes were observed in liver function tests. None of the
       animals showed evidence of gastro-intestinal bleeding. At the end of treatment,
       the average PT was increased more than 18-fold compared to pre-exposure
       values. Levels of factors II, IX, and X at the end of treatment were reduced to
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<pre>       7%, 3.5%, and 3% of pre-exposure levels. Platelet count, platelet aggregation,
       and platelet adhesiveness were not changed significantly after treatment.
       Average warfarin concentrations in plasma were 32.6 and 11.9 mg/L at days 4
       and 7, respectively (For73).
       Long-term toxicity and carcinogenicity
       The committee did not find data from studies on the long-term toxicity, including
       carcinogenicity, of warfarin.
       Mutagenicity and genotoxicity
       The committee did not find data from studies on the mutagenicity or genotoxicity
       of warfarin
       Reproduction toxicity
       In a developmental toxicity study, pregnant F1 mice (n=8-14/group) were
       intraperitoneally given doses of sodium warfarin of 0, 1, 2, or 4 mg/kg bw/day on
       days 3 to 11 of gestation. The mice were killed on gestational days 12 through
       17. In the groups treated with 2 and 4 mg/kg bw/day, there was a very high
       incidence of haemorrhaged placentas and fetal deaths. Maternal deaths were also
       increased in these groups compared with the control group. PTs were 3.5- to 5-
       fold the control values when measured 24 hours after the final injection. No
       significant effects were found in mice treated with 1 mg/kg bw/day. No increase
       in the frequency of malformations of offspring was observed in any of the treated
       groups. When pregnant mice were given a single intraperitoneal dose of warfarin
       of 4 mg/kg bw on gestational day 10 or 11 and killed on day 18, the incidence of
       fetal deaths and the frequency of minor fetal malformations was significantly
       increased. Malformations included open eyelid and minor skeleton and
       ossification abnormalities, particularly of the sternum. Administration of vitamin
       K1 to mice treated with warfarin on day 10 reduced the incidence of fetal deaths
       to that of the control mice. It was suggested that the embryotoxicity of warfarin
       is related to its vitamin K antagonism, rather than to its direct toxicity (Kro74).
            In another study, Sprague-Dawley rats (n=5) were given warfarin at daily
       oral doses of 0 or 100 mg/kg bw on gestational days 1 through 12. Concurrently,
       daily intramuscular injections of 10 mg/kg bw of vitamin K1 were given. Other
       groups of rats (n=6/group) received oral doses of warfarin of 0, 1, 3, 6, 12, 25,
       50, or 100 mg/kg bw and concurrent daily intramuscular injections of 10 mg/kg
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<pre>       bw of vitamin K1, on gestational days 9 through 20. The concurrent
       administration of vitamin K1 creates an extrahepatic vitamin K deficiency while
       it allows normal synthesis of vitamin K-dependent clotting factors in the liver.
       Therefore, the experimental design is an animal model of the warfarin
       embryopathy observed in humans. None of these dosing regimens had any
       apparent deleterious effect on the dams. Maternal PTs were the similar among
       exposed and control animals. There were no haemorrhages in any of the dams,
       and all treated animals maintained their pregnancies. No significant
       abnormalities were observed in the fetuses when warfarin and vitamin K1 were
       administered from day 1 to day 12 of pregnancy. However, similar treatment
       from day 9 to day 20 of gestation caused a significant decrease in mean litter size
       and an increase in resorptions at 100 mg/kg bw/day and a high incidence (28-
       37%) of haemorrhages in the fetuses examined on day 21 of gestation at 3 mg/kg
       bw/day and above. There were no haemorrhages in the control fetuses from dams
       receiving vitamin K1 only and at 1 mg/kg bw, the incidence of haemorrhages was
       not significantly different from controls. Haemorrhages affected the fetal brain,
       face, eyes, and ears, and, occasionally, the limbs. Brain haemorrhages were often
       intraventricular and caused various degrees of hydrocephaly. Bony defects were
       not a feature of prenatal exposure to warfarin, probably because the vitamin K-
       dependent components of bone development occur post-natally in the rat. The
       NOAEL was estimated at 1 mg/kg bw/day (How90).
            In a consecutive study, the same authors gave daily subcutaneous doses of
       sodium warfarin (100 mg/kg bw) and vitamin K1 (10 mg/kg bw) for up to 12
       weeks to Sprague-Dawley rats, starting on the day after birth. All rats survived
       without any sign of haemorrhage. However, the warfarin-treated rats showed a
       marked maxillonasal hypoplasia associated with an 11-13% reduction in the
       length of the nasal bones compared with controls, large calcified areas in the
       septal cartilage of the nasal septum (not present in the controls), and abnormal
       calcium bridges in the epiphysial cartilages of the vertebrae and long bones. The
       authors suggested that the septal growth retardation occurs because the warfarin-
       induced extrahepatic vitamin K deficiency prevents the normal formation of the
       vitamin K-dependent matrix carboxyglutamylic acid (GLA) protein in the
       embryo (How92).
            The committee concludes that with the above rat model, most of the features
       of warfarin teratogenicity in the human have been demonstrated.
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<pre>7      Existing guidelines
       The current administrative occupational exposure limit (MAC) for warfarin in
       the Netherlands is 0.1 mg/m3, 8-hour TWA.
           Existing occupational exposure limits for warfarin in some European
       countries and the USA are summarised in Annex III.
8      Assessment of health hazard
       The major use of warfarin is as an anticoagulant drug in the prevention and/or
       treatment of thromboembolic disease. As a rodenticide, it is used in agriculture
       as a tracking dust and in urban rodent control as a bait, containing 0.025-0.05%
       active ingredient. Warfarin consists of a racemic mixture of equal amounts of 2
       distinct enantiomers (S and R). Occupational exposure may occur during
       manufacture, formulation, and bait application. Workers can be exposed to
       warfarin through inhalation of dust or by direct skin contact with the compound.
       The committee did not find qualitative or quantitative data on the uptake of the
       compound through the lungs. In one occupational worker, signs of poisoning and
       prolongation of the prothrombin time indirectly demonstrated skin absorption of
       warfarin, but there were no quantitative data of the percentage of dermal
       absorption. In a human study, an oral dose of 4-[14C]-rac-warfarin of 0.5 mg/kg
       bw was completely absorbed within 120 minutes after administration. Rac-, S-,
       and R-warfarin are absorbed to the same extent. When absorbed, warfarin is
       bound to plasma proteins, mainly albumin. The binding of S-warfarin to albumin
       is greater than that of R-warfarin. The elimination half-life of rac- warfarin from
       human plasma varies greatly among individuals and ranges from 15 to 52 hours
       (mean: 42 hours). The average plasma half-life of the S-enantiomer is shorter (33
       hours) than that of the R-enantiomer (58 hours). Steady-state blood levels are
       reached after 7-10-day daily warfarin administration. The committee concludes
       that warfarin is a cumulative agent due to its long half-life. Binding to albumin
       and extensive enterohepatic circulation contribute to the long half-life. Warfarin
       is extensively, but slowly metabolised in the smooth endoplasmatic reticulum of
       liver cells and slowly excreted in the urine. In man, only 60% of total urinary
       metabolites were excreted within the first 4 days after a single oral
       administration. Major urinary metabolites of rac-warfarin are the regioisomers
       6- and 7-hydroxywarfarin, and 2 diastereoisomeric warfarin alcohols. No
       warfarin was detected in the faeces. Metabolic patterns of the enantiomers differ
       in that the major metabolite of S-warfarin is 7-hydroxywarfarin and major
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<pre>       metabolites of R-warfarin are 6-hydroxywarfarin and (R,S) warfarin alcohol.
       Substantial differences in the metabolic pathways exist in various species,
       including rat, guinea pig, rabbit, and man.
           Cases of warfarin-induced skin effects, including skin necrosis and
       dermatitis, have been reported when the drug was used as an anticoagulant in the
       clinical treatment of patients. Warfarin is highly toxic for mammalian species,
       including humans. The primary mechanism of warfarin toxicity is inhibition of
       the synthesis of vitamin K-dependent blood clotting factors. A cumulative total
       dose of about 1 g of warfarin consumed in 15 days, equivalent to about 60-90
       mg/kg bw/day, has been reported to be fatal to a 32-year-old man. The main
       symptoms of warfarin poisoning in less severe cases are excessive bruising, nose
       and gum bleeding, pallor, haematomas around joints and on the buttocks, and
       blood in the urine and faeces. Bleeding from several organs within the body,
       leading to shock and possibly death, occurs in the more severe cases. The onset
       of the symptoms of warfarin poisoning may not be evident until a few days after
       exposure. A clinical study revealed that the lowest maintenance dose used in the
       prevention of thromboembolic disease, associated with minimal bleeding, was
       2.5 mg warfarin/day (ca. 0.04 mg/kg bw), for on average 181 days. In a 3-week
       toxicity study in human volunteers taking low, non-therapeutical daily doses of
       rac-warfarin of 0.2 and 1 mg (ca. 0.003 and 0.015 mg/kg bw), no biologically
       significant inhibition of the activity of vitamin-K dependent clotting factors, i.e.,
       below 70 to 80% of normal plasma activity, was observed. Numerous cases on
       developmental effects, the so-called ‘warfarin embryopathy’, which is
       characterised by nasal hypoplasia and stippled epiphyses, as well as disruptional
       abnormalities of the central nervous system, have been reported from the use as a
       therapeutical drug at daily doses between 2.5 and 15 mg. From reviews
       comprising 418 and 635 pregnancies, respectively, Pauli and Haun (Pau93)
       estimated that ‘warfarin embryopathy’ could occur in 1.5-5% of infants if only
       exposed during gestational weeks 6 to 9, and central nervous system effects in
       0.5-2% infants if exposed in the second trimester.
           In experimental animals, the compound is not irritating to the skin. The
       committee did not find data on the eye-irritation or sensitisation potential of
       warfarin. Based on the results of acute lethal oral toxicity studies in the rat, the
       committee considers the compound as very toxic. Large variations in the acute
       oral toxicity of warfarin between species were found. No reliable data on the
       acute inhalation or dermal toxicity of warfarin were available. Embryotoxicity
       and developmental effects were found in one study in rats following oral
       exposure to warfarin. The NOAEL was 1 mg/kg bw/day. No information could
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<pre>       be found on standard short- or long-term toxicity, including carcinogenicity, or
       mutagenicity or genotoxicity studies of warfarin.
       The committee considers that the teratogenic effects reported in women treated
       therapeutically with warfarin doses of 2.5 to 15 mg are the critical findings in
       deriving a health-based recommended occupational exposure limit (HBROEL)
       and that 2.5 mg or 0.04 mg/kg bw is the LOAEL to be used as a starting point.
       For extrapolation to a HBROEL, the committee applies an assessment factor of
       30, taking into account the absence of a NOAEL and the type and severity of the
       effects. Assuming a worker inhales 10 m3 of air during an 8-hour working day
       and a retention of 100%, and applying the preferred-value approach, a health-
       based occupational exposure limit of 0.01 mg/m3 is recommended for warfarin.
       The committee recommends a health-based occupational exposure limit for
       warfarin of 0.01 mg/m3, as inhalable dust, as an 8-hour time-weighed average
       (TWA). In view of the potential for dermal penetration, a skin notation is
       recommended.
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<pre>       Annex I
        Figure 1 Metabolic pathways for warfarin in the rat and man (from Sut87).
        Indicated figures are the percentage of dose excreted.
        * = chiral centre
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<pre>       Annex II
        Figure 2 The vitamin K cycle (from Hol96).
        (1) and (2) are dithiol-dependent reductase enzymes that are inhibited by warfarin. In the
        presence of warfarin, vitamin K epoxide accumulates.
        (3) is an NADPH-dependent reductase that is relatively insensitive to the effects of warfarin.
        The carboxylase enzyme (4) is responsible for the γ-carboxylation of glutamine residues to γ-
        carboxyglutamyl residues.
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<pre>              Annex III
Occupational exposure limits for warfarin 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 -                 0.1             8 hour               administrative             SZW03
Employment
Germany
- AGS                              -               0.5c            8 hour                                          TRG00
                                   -               2.0c            15 min
- DFG MAK-Kommission               -               0.5c            8h                                              DFG03
                                   -               1.0c            15 mind
Great Britain
- HSE                              -               0.1             8 hour               OES                        HSE02
                                                   0.3             15 min
Sweden                             -               -                                                               Swe00
Denmark                            -               0.1             8 hour               OEL                        Arb02
USA
- ACGIH                            -               0.1             8 hour               TLV                        ACG03b
- OSHA                             -               0.1             8 hour               PEL                        ACG03a
- NIOSH                            -               0.1             10 hour              REL                        ACG03a
European Union
- SCOEL                            -               -                                                               EC04
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
     As inhalable fraction.
d
     Maximum number per shift: 4, with a minimum interval between peaks of 1 hour.
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