<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>Nicotine
(CAS No: 54-11-54)
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/105, The Hague, March 30, 2004
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<pre>Preferred citation:
Health Council of the Netherlands: Committee on Updating of Occupational
Exposure Limits. Nicotine; Health-based Reassessment of Administrative
Occupational Exposure Limits. The Hague: Health Council of the Netherlands,
2004; 2000/15OSH/105.
all rights reserved
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<pre>1     Introduction
      The present document contains the assessment of the health hazard of nicotine 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 MA Maclaine Pont, M.Sc. (Wageningen University and Research
      Centre, Wageningen, the Netherlands).
           The evaluation of the toxicity of nicotine has been based on reviews
      published in the monograph ‘Patty's industrial hygiene and toxicology’ (Tro94)
      and by the American Conference of Industrial Hygienists (ACGIH) (ACG99).
      Where relevant, the original publications were reviewed and evaluated as will be
      indicated in the text. In addition, in September 2000, literature was searched in
      the databases Toxline, Medline, and Chemical Abstracts, starting from 1985,
      1966, and 1937, respectively, and using the following key words: nicotine, green
      tobacco disease, green tobacco sickness, 3-(1-methyl-2-pyrrolidinyl)-(S)-
      pyridine, and 54-11-5. Care has been taken to select only those studies dealing
      with nicotine as the only substance. For example, publications on nicotine in
      tobacco smoke have not been considered. The final literature search was carried
      out in Toxline and Medline in March 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                     :   nicotine
      synonyms                 :   (S)-3-(1-methylpyrrilidin-2-yl)pyridine, (S)-3-(1-methyl-2-
                                   yrrolidinyl)pyridine,
                                   1-methyl-2-(3-pyridyl)pyrrolidine, β-pyridyl-α-N-
                                   methylpyrrolidine
      molecular formula        :   C10H14N2
      structural formula       :
      CAS number               :   54-11-5
105-3 Nicotine
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<pre>3     Physical and chemical properties
      molecular weight          :   162.23
      boiling point             :   247oC
      melting point             :   -79oC
      flash point               :   95oC (closed cup)
      vapour pressure           :   at 20oC: 6 Pa
      solubility in water       :   miscible (below 60°C)
      log Poctanol/water        :   1.17 (experimental); 1.00 (estimated)
      conversion factors        :   at 20oC, 101.3 kPa: 1 mg/m3 = 0.15 ppm
                                                          1 ppm = 6.7 mg/m3
      Data from: ACG99, NLM03, Tro94, http://esc.syrres.com.
      Pure nicotine is a colourless oily viscous liquid that darkens readily and develops
      pyridine-like odour on exposure to air. The compound decomposes upon heating
      and combustion, forming corrosive vapours (nitrogen oxides). It reacts
      vigorously with oxidants (ACG99, Tom94).
           Nicotine is a naturally occurring alkaloid found primarily in members of the
      solanaceous plant family, but widely distributed in the plant kingdom through 12
      families and 24 genera (Dav91, Doo95). It is mainly isolated from the leaves of
      Nicotiana tabacum and Nicotiana rustica where it occurs at concentrations up to
      8% (ACG99, Tro94).
4      Uses
      The most widespread use of nicotine is encountered in tobacco (Tro94). Nicotine
      and its salts are used in medicine for the therapy of ulcerative colitis,
      Alzheimer’s disease, Parkinson’s disease, Tourette’s syndrome, sleep apnoea,
      and attention deficit orders (Ben96). Nicotine has therapeutic utility to aid
      smoking cessation.
           Nicotine is also used as a non-systemic insecticide, mostly as a 40% solution
      of the sulphate. This use has declined considerably over the past 3 decades,
      primarily because of its high toxicity (Tro94). According to the database of the
      Dutch Pesticide Authorisation Board (CTB)*, nicotine is at present not permitted
      for its use as an active ingredient in insecticides in the Netherlands.
*     At: http://www.ctb-wageningen.nl.
105-4 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>           The general population might be exposed to nicotine present in foods and
      beverages (Dav91, Sie99). The mean estimated daily dietary intake in the USA is
      approximately 1.4 µg (Sie99).
5     Biotransformation and kinetics
      Human data
      In a human volunteer study, non-smoking women (n=17) were exposed to
      nicotine concentrations in air ranging from 40 to 200 µg/m3 for 1 hour. Nicotine
      concentrations were generated by burning 5 cigarettes every 10 minutes (30
      cigarettes/hour). The respiratory absorption of nicotine was 60-80% (mean
      71.3±10.2%) and was independent of the nicotine air concentration (Iwa91).
          In another human volunteer study, 12 males with a smoking history of at
      least 1 pack of cigarettes per day were treated after 24 hours of smoking
      abstinence with Nicoderm, a commercial nicotine transdermal system. With 24-
      hour time intervals, one transdermal system containing 78 mg nicotine was
      applied to 3 different skin sites (the chest, the arm, or the back), and remained on
      place for 24 hours. Nicotine plasma concentrations increased rapidly after
      application, reaching peak levels after 3.1 to 6.4 hours, and then declined slowly
      until removal of the transdermal system after 24 hours. After removal, the mean
      half-life of nicotine elimination from the plasma ranged between 3.1 and 3.5
      hours. The mean plasma concentrations of the metabolite cotinine increased
      steadily during the 24-hour treatment period and then declined also. There were
      no significant differences in any of the kinetic parameters after application to the
      arm, back, or chest. The average dermal nicotine absorption from Nicoderm was
      14% (Gor92).
          Male human volunteers (n=10), who smoked 15 to 50 cigarettes/day, were
      given a capsule containing 3 mg (7 subjects), 4 mg (2 subjects), or 6 mg
      (1 subject) nicotine base as the bitartrate salt, after overnight abstinence from
      tobacco. Nicotine was absorbed quickly, with a peak level in the plasma
      occurring at about 90 minutes. The oral bioavailability averaged 44% (range: 24-
      59%) (Ben91a).
          Benowitz and Jacob also conducted a kinetic study with deuterium-labelled
      nicotine, which was administered by intravenous injection to 11 male smokers on
      2 occasions at doses of 0.015 and 0.060 mg/kg bw or to 11 non-smokers on 1
      occasion at a dose of 0.015 mg/kg bw. The half-lives of elimination from the
      plasma were 153, 157, or 122 minutes for nicotine and 29.5, 23.9, or 21.0 hours
105-5 Nicotine
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<pre>      for cotinine in the low-dose smokers, high-dose smokers, and non-smokers,
      respectively (Ben93).
          The metabolism of nicotine was studied in 12 male individuals following
      inhalation or dermal exposure. Inhalation exposure took place by smoking of
      cigarettes for 2 days, and dermal exposure by treatment with a nicotine
      transdermal system for 5 days, without smoking. Most of the absorbed doses of
      nicotine (98% and 88%, in case of inhalation or dermal treatment, respectively)
      was excreted as unchanged nicotine and 8 of its metabolites in 24-hour urine,
      collected during day 2 of inhalation exposure, or day 5 of dermal application.
      Following inhalation or dermal exposure, unchanged nicotine accounted for
      10.4% and 11.1% of total urinary excretion products, respectively. The major
      metabolites were trans-3’-hydroxycotinine (39.1% and 37.0% of total urinary
      excretion products, respectively), followed by cotinine glucuronide (15.8% and
      15.4%, respectively), and unconjugated cotinine (13.3 and 14.9%, respectively).
      Minor metabolites (less than 10% of total urinary excretion products) were
      trans-3’-hydroxycotinine glucuronide, nicotine glucuronide, cotinine-N-oxide,
      nicotine-1’-N-oxide, and nornicotine (see Annex I). Benowitz and Henningfield
      concluded that the metabolic profile in man is generally similar when nicotine is
      inhaled or absorbed dermally (Ben94).
          In another metabolism study, 6 non-smoking men received a single
      intravenous dose of 0.190 mg of 14C-labelled racemic nicotine. The half-lives of
      elimination from the plasma were 1.4 and 14.3 hours for nicotine and cotinine,
      respectively. Unchanged nicotine (29% of the dose) and 8 of its metabolites were
      detected in urine collected over a 120-hour period after administration.
      Metabolites were nicotine-1’-N-oxide (0.9% of the dose administered),
      nornicotine (2.4%), cotinine (16.3%), 3-hydroxycotinine glucuronide (11.9%),
      3-hydroxycotinine (1.1%), cotinine-N-oxide (4.9%), norcotinine (5.8%), and
      demethylcotinine ∆2’,3’-enamine (8.4%) (Kye90, Kye91).
          A case of a female with deficient oxidation of nicotine into cotinine has been
      reported. This woman converted only 8% of nicotine into cotinine, resulting in
      an unusually long half-life of nicotine (Ben96).
          The maximum nicotine level in urine of female workers in the tobacco
      industry was reached at the end of the shift. The half-life of nicotine elimination
      from the urine was 8.4 hours (Hu94).
      Animal data
      In a dermal absorption study, a single dose of 14C-labelled nicotine in acetone
      was applied on the clipped backs of a group of young and a group of adult female
105-6 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>      Fischer F344 rats (n=3/group) at doses of 0.016, 0.54, or 2.68 µmol/cm2 (2.6, 87,
      or 435 µg/cm2). Three days after application, the mean absorption percentages
      were 49, 84, or 88%, respectively, in young rats and 75, 83, or 86%, respectively,
      in adult rats (Hal88, Sha87).
           Pulmonary nicotine absorption was studied in mongrel dogs (n=8) after
      installation of 0.5 mg of nicotine at 3 levels of the tracheobronchial tree, i.e., the
      trachea, a subsegmental bronchus site, or a distal site. Each of the 3 applications
      was carried out on a different day with a minimum time interval of 48 hours
      between applications. On a fourth day, the dogs were treated with a single
      intravenous dose of 0.5 mg of nicotine. The absorption of nicotine from the
      subsegmetal bronchus and distal sites was rapid, time-to-reach-peak
      concentrations (tmax) being comparable to that from the intravenous dose.
      Absorption from the tracheal region was slower, tmax being significantly longer
      than those from intravenous and subsegmental administration. Compared to
      intravenous injection, peak plasma concentrations of nicotine (Cmax) following
      application at the distal site were comparable while those following application
      at the subsegmental site and the trachea were significantly lower than those from
      intravenous injection. Total amounts of nicotine absorption, as well as the half-
      life of nicotine elimination from the plasma (84-91 minutes) were the same for
      all routes of administration (Her92).
           Female Swiss-Webster mice were given 14C-nicotine in drinking water at an
      average daily dose of 17 mg/kg bw for 10 days. The amount of radioactivity
      retained in tissues was about 10-fold higher in the adrenal gland and the uterus
      than in the brain or the blood. Other tissues (liver, salivary, ovaries, kidney,
      thymus, and lung) contained about 6 times more radioactivity than blood
      (Row83).
           Male Sprague-Dawley rats were given a dose of 1 mg of 3H-nicotine, either
      by a bolus intravenous injection or a 1-hour constant infusion. One hour after the
      last dose, the highest concentration of radioactivity was found in the kidneys
      (about 50% of the dose) via either route of administration, followed by the
      salivary gland (14 and 16%), the spleen (11 and 13%), and the adrenal gland (11
      and 8%). The lowest concentrations of radioactivity were found in the blood
      (1.2%) after bolus injection and in the caecum (4.7%) after constant infusion.
      Comparing the 2 routes of administration, the retention of radioactivity following
      constant infusion was greater in all tissues, except the adrenal gland. The greatest
      difference was found in the blood, where the level of radioactivity after constant
      infusion was 6-fold higher than after bolus injection (Cho93).
           In another study, male Sprague-Dawley rats were given an intravenous dose
      of 5 mg/kg bw 14C-nicotine. Within 5 to 10 minutes after administration, peak
105-7 Nicotine
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<pre>      concentrations of nicotine were found in plasma, liver, kidney, heart, and brain.
      Concentrations were highest in kidneys, followed by liver, blood, heart, and
      brain. The primary metabolite, cotinine, accumulated in plasma, and by about 30
      minutes, the concentrations of nicotine and cotinine were about equal. While the
      plasma cotinine concentration remained constant at 20 to 60 minutes after
      administration, nicotine was eliminated by the kidneys. The half-life of
      disappearance of nicotine from the blood was approximately 10 minutes and less
      than 20 minutes for the other tissues (Ram95).
          The metabolism of nicotine has been studied in male Sprague-Dawley rats,
      following a single intra-arterial injection of 0.1 mg 14C-labelled nicotine/kg bw.
      The half-lives of elimination from the plasma were 1.0 hour and 5.2 hours for
      nicotine and cotinine, respectively. The total amount of radioactivity excreted in
      the urine over 96 hours was 70% of the administered dose. Nicotine was excreted
      unchanged in amounts of 11.3% of the dose. Nicotine metabolites excreted in the
      urine were cotinine (7.2% of the dose), nicotine-1’-N-oxide (11.6 % of the dose),
      nornicotine (8.9% of the dose), and isomethylnicotinium ion (2.7% of the dose).
      Glucuronidation of nicotine was not reported. Most of the cotinine formed from
      nicotine was further metabolised, and urinary metabolites detected were cotinine
      N-oxide (9.3% of the dose), 3-hydroxycotinine (4.5% of the dose), 3-
      hydroxycotinineglucuronide (‘metabolite A’: 3.1% of the dose), norcotinine
      (1.0% of the dose), γ-(3-pyridyl)-γ-methylaminobutyric acid (4.4% of the dose),
      γ-(3-pyridyl)-γ-oxo-N-methylbutyramide (2.0% of the dose), γ-(3-pyridyl)-γ-
      oxobutyric acid (1.8% of the dose), and 3-pyridylacetic acid (2.2% of the dose)
      (see Annex I). Glucuronidation of 3-hydroxycotinine was not reported (Kye91,
      Kye87).
      In an in vitro study in isolated perfused dog lung, cotinine and nicotine-1’-N-
      oxide were detected in the venous blood and the lung, respectively, following
      administration of 14C-labelled nicotine via the pulmonary artery (Tur75).
      Physiologically based pharmacokinetic (PB-PK) models have been described for
      tissue and plasma kinetics of nicotine and cotinine in man (Rob92) or the
      Sprague-Dawley rat (Plo92). The committee concludes that the rat data do not
      accurately predict the kinetics of nicotine or cotinine in humans.
105-8 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>6     Effects and mechanism of action
      Human data
      Mechanism of action
      The S-isomer of nicotine binds stereoselectively to central and peripheral
      nicotinic cholinergic receptors, while the R-isomer is a weak agonist at
      cholinergic receptors. These receptors are found in the brain, autonomic ganglia,
      and the neuromuscular junction. Nicotine acts by direct stimulation of the
      nicotinic cholinergic receptors, which causes a release of neurotransmitters,
      including acetylcholine, noradrenaline, dopamine, and may result in myriad of
      symptoms, i.e., modulation of neurological, neuromuscular, cardiovascular,
      respiratory, glandular, or gastro-intestinal function. The major effects of nicotine
      are an initial stimulatory effect on these organs due to parasympathetic
      ganglionic stimulation at nicotinic receptor sites. At larger doses, the initial
      stimulatory effect is followed by prolonged ganglionic and neuromuscular
      blockade, which may result in depression and paralysis of the central nervous
      system, all peripheral autonomic ganglia, and motor end-plates in skeletal
      muscles. Fatalities are believed to result from respiratory arrest secondary to
      muscle paralysis (Ben96, Mon94, Lav91).
      Irritation and sensitisation
      Several studies have been reported on local and systemic skin reactions to
      nicotine patches. In one study, 35-54% of users showed a localised
      erythematous, pruritic skin reaction at the patch site, sometimes associated with
      local oedema. According to Fiore et al., a reaction to the adhesive was the most
      likely cause (Fio92). A case of systemic skin reactions has been described
      concerning a 31-year-old female patient, who developed swelling of the feet,
      hands, face, and throat, and a blisterlike rash under the patch, after using of a first
      set of 21-mg nicotine patches (Fra93). Another report describes 11 cases of local
      and 5 cases of systemic skin reactions to 35 to 52.5-mg nicotine patches. Skin
      reactions usually occur after about 15 days’ use (Kla94). In a number of placebo-
      controlled trials, skin reactions in groups of people treated with nicotine or
      placebo patches were compared. In all studies, a statistically significant increase
      in the incidence of local skin reactions was found in the group treated with
      nicotine patches (Dau91, Hur90, Imp93, Rus93, Ton91). Cases of occupational
105-9 Nicotine
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<pre>       dermatitis have been reported in persons processing tobacco or employed in
       nicotine production (Tro94).
            Sensitisation responses to pure nicotine were studied in 10 males and 4
       females, who had previously experienced adverse skin reactions from the use of
       nicotine patches. Tests were conducted with aqueous solutions of 1%, 10%, and
       50% nicotine base and 5% nicotine sulphate, applied under occlusion to the
       backs of the subjects for 2 to 3 days. The incidences of positive allergic patch test
       reactions to nicotine base were 1/14, 4/14, and 5/14 at concentrations of 1%,
       10%, or 50%, respectively, and 1/14 when exposed to nicotine sulphate (5%). At
       the high concentration, irritant reactions due to occlusion were present in the
       remaining 9 subjects (Bir91). The committee concludes that nicotine is a skin
       irritant and a skin sensitiser.
            Respiratory effects of nicotine were investigated in 13 non-smoking subjects,
       after single-breath inhalation by each subject of 0.01 mL nebulised nicotine
       solution (1, 2, 4, 8, 16, 32, or 64 mg/mL; 15-minute time interval between
       inhalations) on 1 day or on 3 separate days. A concentration-dependent cough
       response and airway obstruction was produced, which was reproducible over 3
       different days. According to Hansson et al, these effects were due to stimulation
       of afferent nerve endings in the bronchial mucosa and mediated by
       parasympathetic cholinergic pathways (Han94). In 12 male non-smoking
       volunteers, airway irritation in the substernal region was induced after inhalation
       of a single puff of cigarette smoke of 3 different types of cigarettes with high
       nicotine content during 3 consecutive days. No or very mild irritation was
       recorded after inhaling cigarette smoke with a low nicotine content or gas-phase
       cigarette smoke without nicotine (Lee91).
       Acute toxicity
       In the older literature, many cases of nicotine poisoning have been recorded, the
       majority from ingestion or skin absorption. Most fatalities occurred in the 1920s
       and 1930s, when concentrated nicotine solutions were commonly used as
       insecticides, and reports of fatal poisoning frequently involved the mistaking of
       nicotine solutions for other medications (Had83). There were 288 fatalities
       reported in the United States between 1930 and 1935. Occupational intoxications
       have also been described in persons engaged in nicotine extraction and in
       spraying insecticides, mainly from percutaneous nicotine absorption. Fatal
       occupational poisoning has been relatively uncommon (Mon94, Tro94).
       Symptoms of nicotine toxicity are manifested by effects on the gastrointestinal,
       central nervous, neuromuscular, cardiovascular, respiratory, and glandular
105-10 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>       systems. The common symptoms of moderate intoxication include nausea,
       vomiting, abdominal pain, diarrhoea, headache, sweating, fatigue, and
       palpitations. More severe symptoms include faintness, dizziness, weakness, and
       confusing, progressing to muscular weakness, collapse, and respiratory arrest.
       Following large doses of nicotine, symptoms develop quickly and death results
       from paralysis of respiratory muscles, produced by peripheral neuromusular
       blockade, and cardiovascular collapse (Lav91, Tro94). Dermal exposure to
       nicotine produced the same range of symptoms that were produced by ingestion
       (ACG99, Tro94).
           A more recent case of fatal nicotine ingestion was reported of a 17-year-old
       boy, who deliberately ingested a solution containing an estimated dose of 5000
       mg nicotine alkaloid. Approximately 1 to 2 minutes after ingestion, he vomited
       and developed cardiopulmonary arrest. During the first few hours following
       admission, the patient had multiple grand mal seizures, cerebral oedema, and no
       cortical function. The boy died at 64 hours after the ingestion (Lav91).
           A 45-year-old male developed atrial fibrillation and seizures following
       inhalation of nicotine, used as a substitute of tobacco smoking (Nun01).
           When a male subject was given a capsule containing 6 mg of nicotine (as the
       bitartrate salt), he complained of nausea and abdominal cramping that began
       about 30 minutes after ingestion and lasted for 1 hour. Subjects receiving doses
       of 3 or 4 mg had no signs or symptoms of toxicity (Ben91a).
           In another study, a nicotine patch (Nicoderm), containing 78 mg nicotine,
       was applied with 24-hour time intervals to 3 different skin sites (the chest, the
       arm, or the back) of 12 male smokers, and remained on place for 24 hours (see
       Section 5). At 4 hours after application, mean heart rate and systolic blood
       pressure had significantly increased compared to baseline values (Gor92).
           When 5 non-smoking subjects (3 males, 2 females) took a single breath
       inhalation of 0.01 mL nebulised nicotine solution at times 0 and 10 minutes, no
       cardiovascular effects were found at concentrations up to 64 mg/mL.
       Cadiovascular effects were also evaluated in 8 non-smoking volunteers (4 men, 4
       women) inhaling nicotine solutions of 0, 2, 4, or 8 mg/mL on 4 different days. A
       single breath was taken every 15 seconds up to 5 minutes (total 21 inhalations),
       giving a dose of 0, 0.4, 0.8, or 1.7 mg nicotine/5 minutes. Heart rate and systolic
       blood pressure were significantly increased in a dose-related fashion compared
       with the vehicle controls (0 mg/ml). Maximal responses were seen within 3
       minutes after inhalation, and the responses lasted between 6 and 10 minutes. No
       significant changes were found in diastolic blood pressure. Nicotine caused a
       decrease in skin temperature, with maximal responses at 5 minutes. Seven of the
105-11 Nicotine
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<pre>       subjects complained of headache that had a maximum at 5-6 minutes and lasted
       for 20 minutes. None of the subjects noticed a tremor or nausea (Han94).
           Several reports have been published on the so-called ‘green-tobacco
       sickness’. This sickness is acute nicotine poisoning caused by the dermal
       absorption of nicotine from mature tobacco plants during cultivation and
       harvesting. Green-tobacco sickness is normally a self-limiting condition from
       which workers recover in 2 to 3 days and is characterised by nausea, vomiting,
       weakness, dizziness, headache, and occasional fluctuations in blood pressure or
       heart rate (Arc01a, Bal95, Geh74). In an epidemiological study among Latino
       farm workers in North Carolina, USA, the workers experienced green-tobacco
       sickness 2 days for every 100 days worked. Factors at risk were lack of work
       experience, picking tobacco leaves late in the season, and working in wet
       clothing (Arc01a, Arc01b). Salivary cotinine levels increased across the season,
       independent of smoking status (Qua01). In a case-control study, crude annual
       incidences in the USA of 10 to 14 per 1000 tobacco workers were reported. The
       study demonstrated that ill workers were younger and were more likely to have
       worked in wet conditions, compared with healthy workers (Bal95).
           In a previous study on tobacco workers in India, the frequency of green-
       tobacco-sickness symptoms was 53%. Mean 24-hour urinary nicotine and
       cotinine concentrations in workers handling tobacco were 4.1 and 3.8 mg/L,
       respectively, and below detection limit in control subjects (Gho86). In a study
       among 100 workers in 2 tobacco-processing factories in India, the main route of
       exposure during tobacco processing was inhalation. The average nicotine air
       concentration in the breathing zone of the workers (10-minute samples) was 1.18
       mg/m3. Urinary nicotine and cotinine concentrations were 3.3 and 2.8 mg/L for
       non-smoking females and males, respectively. Corresponding urinary cotinine
       levels were 3.3 and 4.1 mg/L, respectively. No abnormalities were found in
       pulmonary function tests in workers. However, 69 subjects exhibited one or
       more of symptoms, such as vomiting, giddiness, headache, weakness, or loss of
       appetite (Gho85).
           In a 1-day biological monitoring study on 10 female Italian tobacco
       harvesters, the mean blood nicotine level rose to a peak value of 3.45 µg/L at 2
       hours after the end of the working day. The mean urinary nicotine reached a peak
       value of 158 µg/L in samples collected at the end of the working day. No
       significant trend in mean plasma or urinary cotinine levels was observed at
       different time points during the working day. Plasma cotinine levels were
       approximately 15 µg/L, indicating a daily intake of 1.2 mg of nicotine, and
       urinary cotinine levels were approximately 100 µg/L. No symptoms of nicotine
       poisoning were reported among the workers (Ale01).
105-12 Health-based Reassessment of Administrative Occupational Exposure Limits
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<pre>       Long-term toxicity
       In a case-control study among farmers in the USA, the risk for multiple myeloma
       for farmers who personally mixed, handled, or applied nicotine as an insecticide
       for more than 1 year was studied. Included in the analysis were 6 cases and 22
       controls. Of the total farmer population (111 cases and 378 controls), 84% of the
       cases and 78% of the controls received an in-person interview on the use of
       pesticides. No statistically significant association between multiple myeloma and
       the use of nicotine as an insecticide was found. The Odds Ratio (OR), relative to
       non-farmers (62 cases and 272 controls) was 1.4 (95% confidence interval (CI):
       0.5-3.6) (Bro93). In another case-control study, a statistically significantly
       increased risk for leukaemia was reported for farmers engaged in spraying of
       nicotine. Included in the analysis were 30 cases and 47 controls. The OR was 1.6
       (95% CI: 1.0-2.6). For the group handling nicotine at least 20 years ago (28 cases
       and 36 controls), the OR was 2.0 (95% CI: 1.2-3.4). This result must be viewed
       with caution since of the total farmer population (578 cases and 1245 controls),
       only 25% of the cases and 75% of the controls were interviewed. Furthermore,
       accurate recall of pesticide use probably declines with the passage of time
       (Bro90). The committee is of the opinion that the number of nicotine applicators
       (both cases and controls) was too small to draw a reliable conclusion on the
       association between the handling of nicotine and the emergence of leukaemia or
       multiple myeloma.
           The possible association between long-term exposure to pure nicotine and
       cardiovascular disease has been investigated in 2 studies among smokeless
       tobacco users. During smokeless tobacco use, nicotine is absorbed through the
       buccal mucosa, which results in a larger overall exposure to nicotine than by
       cigarette smoking owing to prolonged absorption. In a cross-sectional study,
       middle-aged and older smokeless tobacco users had a significantly higher
       prevalence of hypertension and cardiovascular symptoms than non-users of
       tobacco (Bol92). A cohort study was conducted among 6297 male smokeless
       tobacco users, employed in the Swedish construction industry, who attended a
       health examination between 1971 and 1974 and were followed regarding cause-
       specific mortality during the period 1974 through 1985. The age-adjusted
       relative risk (RR) of dying from cardiovascular disease compared with non-users
       (n=32,546) was 1.4 (95% CI: 1.2-1.6). Among men aged between 35 and 54
       years at the beginning of the follow-up (n=1672), the RR was 2.1 (95% CI: 1.5–
       2.9). The RR for cardiovascular disease for smokeless tobacco users was lower
       than that for current cigarette smokers, but higher than for ex-smokers. Cancer
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<pre>       mortality was not raised in smokeless tobacco users compared with non-users
       (Bol94).
       Addiction
       Addiction can be defined as the compulsive use of a drug that has psychoactivity
       and that may be associated with tolerance and physical dependence, i.e., may be
       associated with withdrawal symptoms after cessation of drug use (DHH88). For
       smokers, addiction is assumed to involve daily smoking of cigarettes, difficulty
       in not smoking every day, and a high likelihood of withdrawal symptoms after
       cessation of smoking. Smokers who regularly smoke 5 or fewer cigarettes per
       day appear not to be addicted (Shi89). In their report, Benowitz and Henningfield
       stated that the consumption of 5 cigarettes per day corresponds with an average
       serum cotinine level of 70 µg/L. Therefore, they consider a serum cotinine level
       of 50 to 70 µg/L as a cut-off point for the addictive threshold. In a separate study,
       Benowitz and Henningfield estimate the daily intake of nicotine in smokers as
       0.08 times the blood cotinine concentration. Thus, a level of 50 to 70 µg cotinine/
       L corresponds to a daily nicotine intake of 4 to 6 mg. The authors, therefore,
       propose that a daily intake of 5 mg of nicotine is a threshold level, below which
       no addiction occurs (Ben94).
           No data of nicotine addiction have been reported in workers who cultivate
       and harvest tobacco or in workers processing tobacco, where the exposures to
       nicotine were high enough to cause green-tobacco sickness (Arc01a, Arc01b,
       Bal95, Geh74, Gho85, Gho86). From urinary cotinine levels, the committee
       concludes that the daily nicotine intake was 1.2 mg for Italian workers, but
       substantially higher than 5 mg for Indian workers.
           Non-smoking patients with ulcerative colitis did not develop symptoms of
       addiction when treated with nicotine patches that released 15 to 25 mg of
       nicotine daily for 6 weeks (n=35) or 15 mg daily for 6 months (n=40). Mean
       steady-state plasma cotinine levels varied from 102 to 150 µg /L in the 6-week
       study and from 62 to 76 µg /L in the 6-month study (Pul94, Tho95). In another
       study, patients with primary sclerosing cholangitis (n=8) were treated with oral
       doses up to 24 mg/day for 1 year. No patient experienced withdrawal symptoms
       when nicotine administration was discontinued. Due to signs of nicotine
       toxicity, 3 out of the 8 patients completed only 1 to 4 months of treatment
       (Ang99).
           From the above studies, the committee concludes that the addictive
       properties of nicotine were exclusively found in cigarette-smoking subjects.
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<pre>       Developmental toxicity
       Studies of the effects of cigarette smoking and nicotine in humans suggest that
       nicotine may contribute to adverse reproductive outcomes. Mechanisms of
       particular concern include reduction of utero-placental blood flow, leading to
       hypoxia-induced brain damage, peri-natal mortality, and sudden infant death.
       Direct effects on neurotransmitter receptors in the developing fetal brain may
       lead to cognitive and learning defects in childhood or adolescence (Ben91b,
       Slo98).
       Animal data
       Irritation and sensitisation
       Nicotine is moderately to severely irritating to the rabbit eye (Sug90). The
       committee did not find data on skin irritation or skin sensitisation properties of
       nicotine
       Acute toxicity
       Results of acute lethal toxicity tests with nicotine are summarised in Table 1.
       Table 1 Summary of acute lethal toxicity studies in mammals.
       exposure route species strain (sex)                          LD50 (mg/kg bw) reference
       intratracheal   rat         Long-Evans                       19.3            Kim84
       dermal          rat                                          140             Tro94
                       rabbit                                       50              Tro94
       oral            rat                                          53              ACG99
                       rat                                          50-60           Tro94
                       rat        Sprague-Dawley (male, female) 71                  Yam90
                       rat                                          188             Ray91
                       mouse                                        3.3             ACG99
                       mouse                                        24              Ray91, Tro94
                       mouse                                        50-60           Tro94
       intraperitoneal rat                                          30              Ray91
                       rat                                          14.6            Tro94
                       mouse                                        5.9             Tro94
                       rabbit                                       14              Tro94
       intravenous     rat                                          7               Tro94
                       mouse                                        7.1             Tro94
                       rabbit                                       9.4             Tro94
                       dog                                          5               Tro94
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<pre>       Signs of intoxication in dogs treated with a lethal intravenous dose were
       vomiting, initial stimulation, followed by depression, seizures, and paralysis of
       the central nervous system, peripheral autonomic nervous ganglia, and skeletal
       muscle endings (Ray91).
       To investigate the role of nicotine in the development of pulmonary emphysema,
       groups of Long-Evans rats (n=13-16) received single intratracheally
       administered doses of 3 or 7.5 mg nicotine/kg bw. Four weeks after treatment,
       ventilatory, mechanical, and gas exchange functions were not significantly
       different compared with control rats. It was concluded that intratracheal
       installation of a single, relatively high dose of nicotine, does not induce the
       development of pulmonary emphysema in the rat (Kim84).
       Short-term toxicity
       In order to investigate a possible role of nicotine in cigarette smoke, which,
       through the mediation of catecholamines, was thought to ‘waste’ a portion of the
       oxygen received by the heart, in being one of the potential causes of increased
       morbidity and mortality from ‘coronary heart disease’, a number of physiological
       and biochemical parameters likely to be affected by repeatedly administered
       nicotine were measured in male Sprague-Dawley rats. Groups of 100 animals
       were given nicotine as the alkaloid in their drinking water at doses of 1.14 or
       4.46 mg/kg bw/day for 34 weeks, and mortalities, gross cardiac lesions,
       haematocrits, and the activities of several heart enzymes, selected as potential
       indices of early cellular injury, were examined. After the end of treatment, half of
       each group were exposed to 6% oxygen for 12 hours, after which aforementioned
       parameters were measured in separate sets of animals at several ‘post-hypoxia’
       intervals The only effects reported in nicotine-only-treated animals were a
       statistically significant increase in the activity of myocardial enzymes isocitric
       dehydrogenase and acid phosphatase and a statistically significant decrease in
       the activity of β-glucuronidase at the high dose (Wen70).
           To assess the effects of long-term treatment with nicotine on several
       behavioural measures, including locomotor activity, exploratory efficiency,
       habituation, short-term and long-term memory, groups of 5-month-old (‘young’)
       and 22-month-old (‘old’) female Sprague-Dawley rats (n=15/group) were given
       nicotine (as its acid tartrate) via the drinking water at concentrations of 0, 20, or
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<pre>       50 mg/L* for 131 days. Mean nicotine plasma concentrations in low- and high-
       dose rats (n=12) were 16.6 and 56.2 µg/L, respectively. Mean terminal body
       weights were statistically significantly decreased at both dose levels in ‘young’
       rats and at the high dose in ‘old’ rats. Water intake was reduced during the first
       half hour of the daily 4.5-hour access to drinking water. Locomotor activity was
       increased throughout the experiment, but dropped immediately to control values
       when treatment was discontinued during a 7-day withdrawal period. In ‘young’
       rats, exploratory efficiency was attenuated. Nicotine did not affect habituation
       and memory tasks (Wel88).
           Five-week-old male NMRI mice were given increasing concentrations of
       nicotine in drinking water for 50 days, equivalent to 60-65 mg/kg bw/day from
       the 3rd week up to the end of the dosing. Locomotor activity was significantly
       increased on the 50th day of nicotine administration compared to control
       animals. However, no difference was observed 12-14 hours after cessation of
       exposure. Concentrations of several brain monoamines were elevated on the 50th
       day, but at 23-25 hours after withdrawal, only hypothalamic dopamine
       concentration was still significantly increased (Gad00).
           When nicotine was administered to Charles River LEW rats by
       subcutaneously implanted miniosmotic pumps at the rate of 1 mg/kg bw/day for
       3 weeks, Con A-induced proliferation of peripheral blood cells and of spleen
       cells was significantly inhibited. Effects persisted for at least 2 weeks after
       termination of exposure. It was demonstrated that short-term nicotine treatment
       regulate T cell proliferation via nicotine acetylcholine receptors, but unlike acute
       treatment, the effects were independent of the hypothalamus-pituitary-adrenal
       axis (Sin00).
       Long-term toxicity and carcinogenicity
       Female Sprague-Dawley rats (n=68) were exposed to an average nicotine
       concentration of about 0.5 mg/m3 (range: 0.40-0.65 mg/m3), 20 hours/day, 5
       days/week, for 103 weeks. The purity of nicotine was >99%. Non-exposed rats
       (n=34) served as a control group. Mean nicotine concentrations in plasma,
       measured after 5 days and at the end of the study, were 108 µg/L and 130 µg/L,
       respectively, giving the plasma concentration found in heavy smokers. Interim
       kills (5-10 nicotine-exposed and 5-6 controls) were performed at 6, 12, and 18
*      Taking a mean body weight of 300 and 400 mg for the ‘young’ and ‘old’ rats, respectively (estimated from data
       presented by Welzl et al.) and assuming the dose amounts were as nicotine (and not as nicotine hydrogen tartrate)
       and a mean water consumption of 22.5 mL/day for both groups, these dose levels could be 1.5 and 3.8 mg/kg bw
       and 1.1 and 2.8 mg/kg bw in ‘young’ and ‘old’ rats, respectively.
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<pre>       months after the beginning of exposure. After 24 months, 7 control rats (21%)
       and 22 nicotine-treated rats (32%) remained. The mean body weight of exposed
       rats was lower compared with the controls throughout the study, but no statistical
       data were given. The proportion of animals withdrawn from the study because of
       general misthriving at observation was 16% in nicotine-exposed rats and 22% in
       controls. Macroscopic and microscopic examination revealed not statistically
       significant increases in incidences of fibroadenomas of the mammary gland, of
       adenomas of the pituitary gland, and of adenocarcinomas of the ovary, compared
       with the control group. Neither lung tumours nor any increase in pulmonary
       neuroendocrine cells were detected. The median absolute heart weights of
       exposed and control animals were not statistically significantly different, and no
       increase in atherosclerotic lesions was found. Macroscopic or microscopic
       examination of other tissues (brain, gastrointestinal tract, liver, kidneys) did not
       reveal treatment-related abnormalities (Wal96).
       Mutagenicity and genotoxicity
       •   In vitro tests:
           • Gene mutation assays. Tests for reverse mutations in 5 strains of
              S. typhimurium (TA97, TA98, TA 100, TA 1535, and TA1537) were
              negative at concentrations up to 5000 µg nicotine/plate both with and
              without metabolic activation by a rat liver microsomal S9 preparation
              (Bra87, Doo95, Flo84, McC75, Rie82). Nicotine metabolites, i.e.,
              nicotine-1’-N-oxide, cotinine, cotinine-N-oxide, and trans-3’-
              hydroxycotinine did not induce reverse mutations in these strains up to
              1000 µg/plate, in the absence or presence of S9 (Doo95). Neither nicotine,
              nor its metabolites, showed missense back mutations in S. typhimurium
              strains TA100, TA7004, TA7005, or TA7006, at concentrations up to
              2000 µg/plate, in the presence or absence of rat liver S9 (Yim01).
              When female Sprague-Dawley rats received a single intraperitoneal
              injection of 0.8 mg nicotine/kg bw (the maximum tolerated dose), 24-hour
              urine samples, either neat or extracts, did not induce reverse mutations in
              S. typhimurium TA98, with and without metabolic activation (Doo91).
           • Cytogenicity assays. Neither nicotine, nor its metabolites nicotine-1’-N-
              oxide, cotinine, cotinine-N-oxide, or trans-3’-hydroxycotinine induced
              sister-chromatid exchanges (SCE) in cultured Chinese hamster ovary
              (CHO) cells at concentrations up to 1000 µg/mL, with and without
              metabolic activation (Doo95). The frequency of SCEs in cultured CHO
              cells was increased in a dose-dependent manner at nicotine or nornicotine
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<pre>              concentrations in the range of 1250- 5000 µg/mL, in the absence of S9.
              The increase was statistically significant at the highest dose only. In the
              presence of S9, no increase in the SCE frequency was found at any of the
              concentrations (Rie83). In another study, a dose-related increase in the
              frequency of SCEs in cultured CHO cells was found at nicotine
              concentrations in the range of 150 to 1000 µg/mL in the absence of
              metabolic activation. Dose-related increases in the frequency of
              chromosome aberrations (excluding gaps) were found at concentrations of
              375 µg/mL and above (Tri90, Tri93).
           • Other genotoxicity assays. In a DNA repair assay with E. coli pol A+/pol
              A- , nicotine at an amount of 40 µg induced repairable DNA damage while
              its metabolites nicotine 1’-N-oxide (400 µg) or cotinine (800 µg) were
              negative (Rie82). In the SOS-chromotest with E. coli strain PQ 37,
              nicotine was negative at concentrations up to 1.62 mg/mL, either without
              or with activation by S9 (Bra87). In a bacterial luminescence test with
              Vibrion fischeri strain NRRL-B-11177, nicotine did not induce significant
              light emission at concentrations of 1250 or 2500 µg/mL, in the presence
              or absence of S9. Cotinine, however, was positive at both concentrations
              in the absence of S9, but not in its presence (Yim95).
       •   In vivo tests:
           Groups of 5 young male Swiss albino mice (n=5/dose/sampling time) were
           given single doses of nicotine of 0.77 and 1.10 mg/kg bw by gavage. Mice
           (n=5) treated with isotonic saline served as a control group. The nicotine-
           dosed animals were sacrificed at 6, 12, 18, and 24 hours after treatment and
           the controls only after 24 hours, and bone marrow chromosome preparations
           were made. Statistically significant, dose-dependent increases in the
           frequency of chromosomal aberrations (excluding gaps) were found for both
           doses at all sampling times, compared with the controls (Sen91).
           When male mice were treated with intraperitoneal injections of doses of
           nicotine of 5 mg/kg bw/day on 2 consecutive days, the percentage of non-
           viable implants was statistically significantly increased in females
           inseminated in weeks 1 and 2 after treatment. This indicates that epididymal
           sperm and spermatids were susceptible to the induction of dominant lethal
           mutations by nicotine (Hem78).
       In conclusion, nicotine has shown clear clastogenic activity (increased frequency
       of SCEs and chromosome aberrations in one in vitro test, and an increased
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<pre>       frequency of chromosome aberrations and of dominant lethal mutations in vivo).
       Nicotine did not induce gene mutations in bacterial systems.
       Reproduction toxicity
       Numerous studies have been conducted on reproductive or developmental effects
       of nicotine by multiple routes of exposure and in several species of animals
       (Tro94). However, the committee could not find a standard 1-, or 2-generation
       reproduction study, or a standard developmental toxicity study.
           Below, a number of studies are reported in which reproductive or
       developmental effects were examined following relevant routes of exposure, i.e.,
       dermal or oral. In addition, some studies using less relevant routes of dosing, i.e.,
       intraperitoneally or subcutaneously are reported. The committee did not find any
       study in which animals were exposed by inhalation.
       Effects on fertility
       Groups of male Swiss albino mice (n=12/group) received nicotine via the
       drinking water at doses equivalent to 0 or 2.7 mg/kg bw/day for 7 weeks or to 0
       or 2.3 mg/kg bw/day for 20 weeks. When females were mated with males at 1 to
       4 weeks after the end of the 7-week exposure, the numbers of litters and
       offspring were similar compared with the control group. However, offspring of
       females mated with males at 1 to 6 weeks after the end of the 20-week exposure,
       showed a statistically significant increase in the incidence of abnormalities of the
       limbs in the first and second week after male treatment (Hem81).
       Groups male albino mice (n= 8/group) received daily intraperitoneal injections
       of 0, 2, 4, or 6 mg nicotine/kg bw for 15 days. There were statistically
       significant, dose-related decreases in relative testicular, epididymis, seminal
       vesicle, prostate, or vas deferens weights and in spermatocyte and spermatid
       counts and not statistically significant, dose-related increases in spermatogonia
       counts. According to Reddy et al., these effects were caused by interference of
       nicotine with the release of pituitary gonadotrophins. A NOAEL was not
       established (Red98).
       Effects on development
       Commercial nicotine transdermal patches, which would deliver 1.75 or 3.5 mg of
       nicotine per day, were applied to the backs of groups of pregnant Sprague-
       Dawley rats (n=2-13/group) either during gestational days 2 through 19 or 2
       through 7. Control animals received either no treatment or were handled daily by
       application of a placebo patch. Mean plasma nicotine levels in animals that
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<pre>       received 1.75 or 3.5 mg/day during gestational days 2 through 19 were 70 or 241
       µg/L, respectively. Corresponding plasma cotinine concentrations were 231 or
       302 µg/L, respectively. None of the animals (n=2) exposed to 3.5 mg/day, 6/13
       animals exposed to 1.75 mg/day, and 11/12 control animals exposed to a placebo
       patch over the full duration of gestation remained pregnant. When exposed
       during gestational days 2-7, 4/8 high-dose animals and 3/4 low-dose animals
       remained pregnant. The average litter size of animals that were exposed to
       nicotine and carried their pregnancy successfully to term was not significantly
       different in any of the groups compared with the control group. Neither was any
       significant difference observed in average pup weight per litter between animals
       exposed to nicotine and the corresponding controls. The offspring was not
       examined. Witschi et al. concluded that nicotine has a pre-implantation effect
       and that continuous exposure to nicotine early during pregnancy may adversely
       affect pregnancy outcome in rats (Wit94). The committee concludes that the
       LOAEL for pregnancy failure was 1.75 mg/day.
            Female Sprague-Dawley rats (n=20) received nicotine in drinking water,
       starting 6 weeks before mating and continuing throughout pregnancy. During the
       first 3 weeks of treatment, the nicotine concentration in the drinking water was
       gradually raised until the daily nicotine intake was 6 mg/kg bw/day. Small, but
       statistically significant decreases in the number of male rats born and in male
       birth weight were found in the exposed group compared with the controls. No
       changes were found in female offspring. After birth, the litters were cross-
       fostered to control dams and behavioural tests were conducted at post-natal days
       25, 45, 60, and 85. Statistically significant decreases in locomotor rearing
       activity were found at post-natal days 60 and 85 in male but not in female
       offspring (Pet82).
            In another study, female Sprague-Dawley rats (numbers not given) received
       nicotine via the drinking water at doses equivalent to 0, 2.4, or 4.5 mg/kg bw/day
       for 1 week before mating and continuing throughout pregnancy and lactation.
       After birth, litters from females treated with nicotine were cross-fostered with
       litters from control mothers to compare the effects of pre-natal and post-natal
       exposure to nicotine. Maternal mean plasma nicotine levels in low- and high-
       dose animals at the time of weaning were 1.0 and 18.5 µg/L, respectively.
       Maternal effects were decreases in water consumption and in body weights
       during lactation in high-dose animals. No significant effects were found in
       average birth weight of offspring. However, the litter size was significantly
       reduced at the high-dose. Offspring exposure to the low and the high dose during
       either gestation or lactation caused no significant change in body weight gain in
       both males and females at post-natal day 10, but statistically significant
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<pre>       reductions were found at post-natal days 20, 30, and 40. Post-natal exposure
       appeared to have a greater effect. The LOAEL was 2.4 mg/kg bw/day (Car85).
           When pregnant Sprague-Dawley rats (n=8-9/group) were given nicotine at
       doses of 0 or 3 mg/kg bw/day by gavage from day 1 to 21 of gestation, maternal
       effects were reductions in average daily food intake and in body weight in the
       exposed animals compared with the controls. Body weight gain throughout
       pregnancy was similar in both groups. Mean litter size and fetal body weight at
       birth were lower in the exposed group, compared with the controls, but the
       differences were not statistically significant. No further data were presented
       (Lei91).
           Nicotine was administered to female Swiss-Webster mice (n=19-26/group
       for controls, mid and high dose; low dose: not given) by addition to the drinking
       water at daily doses equivalent to approximately 0, 5.7, 17.2, or 28.6 mg/kg/day
       for at least 2 weeks before breeding and throughout gestation. The fetuses and
       placentas of all animals were examined on the 17th day of gestation. No signs of
       maternal toxicity were reported. There was a dose-related decrease in fetal
       weight, which was statistically significant at the high and the mid doses. No
       significant changes were observed in average number of pups per pregnancy
       between any of the groups. The mean placental weight and the placental
       accumulation of the amino acid α-aminoisobutyric acid were significantly
       decreased in high-dose animals compared with controls. The NOAEL for
       embryotoxic effects was 5.7 mg/kg bw/day (Row82).
           In a developmental toxicity study, pregnant ICR/SIM mice (n=26/group)
       were given nicotine at oral (gavage) doses of 0 or 35 mg/kg bw/day on days 8
       through 12 of gestation. Maternal mortality was observed in 10/26 treated
       animals. Other maternal effects were body weight reduction and overt signs of
       toxicity. The number of live-born litters was not significantly different in the
       exposed animals compared with the controls, and no resorbed litters were found.
       The number of pups per litter, the number of live animals on post-natal day 3, the
       percentage of pup survival on post-natal days 1-3, and the pup weight on post-
       natal days 1 and 3 were higher than in the control group, but the difference was
       not statistically significant. The NOAEL for embryotoxic effects was 35 mg/kg
       bw/day (Sei86).
           In another study, pregnant Swiss-Webster rats (n=10/group) were given daily
       subcutaneous injections with either vehicle (0.9% saline) or 0.5 mg nicotine/kg
       bw on days 10 to 20 of gestation, and offspring was subjected to developmental
       and behavioural test on post-natal days 1 to 22. Statistically significant effects in
       nicotine-exposed offspring compared with controls were reduced body weights,
       delayed eye opening and appearance of body hairs, and decreased sensory motor
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<pre>       reflexes. However, motor activity was significantly stimulated in early adulthood
       of mouse pups (Aja98).
       In a teratogenicity study, pregnant Sprague-Dawley rats (n=10/group) received
       nicotine, administered subcutaneously by a miniosmotic pump, at a daily dose of
       3.6 mg from day 6 through 12 of gestation. Pair-fed and untreated control
       animals were given physiological saline in a similar manner. Evaluation of the
       fetal skeletal system on gestation day 20 revealed no statistically significant
       differences in the number of complete sternal ossification centres or in the
       ossification of the skull and facial bones in nicotine-exposed rats, when
       compared with the untreated controls. However, when compared with the pair-
       fed control group, a statistically significant increased incidence of ossification in
       sternae and skull was observed. A lower incidence of wavy ribs was observed in
       the nicotine-exposed animals, but the difference with the controls was not
       statistically significant (Nas89). In the same laboratory, another developmental
       toxicity study was conducted, in which groups of pregnant Sprague-Dawley rats
       (n=10) received nicotine subcutaneously by a miniosmotic pump at daily doses
       of 1.8 or 3.6 mg from gestational days 6 through 12. Pair-fed control rats (n=10)
       received physiological saline in a similar manner. Fetuses were examined for
       developmental effects on day 12 of gestation. The embryos treated with the
       higher dose of nicotine were significantly different from control values for
       crown-rump and head lenght, and the development of the embryos was
       significantly delayed for 12 out of 17 developmental endpoints, e.g., heart, brain,
       otic and optic systems, hind limb, and somites. However, the maxillary and
       mandibular processes and the olfactory system were observed to have
       accelerated development. No hind limb development was observed. Embryos
       treated with the lower dose of nicotine showed significant differences from
       controls with respect to yolk sac diameter, crown-rump and head length, and
       development of the olfactory system (delayed). However, the optic and otic
       systems showed accelerated development (Dae91).
           In a study with Rhesus monkeys (n= 3/group), pregnant animals were given 0
       or 1 mg/kg bw/day of nicotine from days 26 to 134 of gestation by subcutaneous
       implantation with miniosmotic pumps. Fetal monkeys were obtained by
       Caesarean section. Nicotine treatment did not affect maternal weight gain or food
       intake compared with controls. Nicotine administration reduced fetal body
       weight by 8% compared with controls. Similar reductions were also seen in body
       length, biparietal, and weights of fetal heart, pancreas, adrenals, kidneys, and
       brain. Fetal lung weight and volume were reduced by 13% and 12%, respectively
       (not significant). The lungs of offspring had hypoplasia and a reduced surface
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<pre>       complexity of developing alveoli. The findings demonstrate that nicotine can
       alter fetal monkey lung development by crossing the placenta to interact directly
       with nicotine receptors on non-neuronal cells in the developing lung (Sek99).
       In summary, in the rat, nicotine caused pregnancy failure after dermal
       application of relatively low levels (1.75 mg/kg bw/day) throughout gestation.
       Embryotoxicity, in the form of reduced pup body weight and number of pups
       born, was observed following oral dosing at 3 mg/kg bw/day before mating and
       throughout pregnancy. Developmental effects were found following continuous
       nicotine administration by miniosmotic pump at 1.8 mg/kg bw/day during
       gestation. Teratogenic effects were not demonstrated. A NOAEL for
       reproductive effects could not be established in any of the rat studies.
           In mice, male fertility was affected at oral doses of 2.7 mg/kg bw/day for 20
       weeks, but not when given for 7 weeks. No embryotoxicity was observed when
       mice were orally dosed with 35 mg/kg bw/day during gestation. A NOAEL of 17
       mg/kg bw/day was found when oral dosing started before mating and continued
       throughout pregnancy.
           In monkeys, reduced fetal body and organ weights and effects on lung
       development were observed following continuous nicotine administration by
       miniosmotic pump at 1.0 mg/kg bw/day during gestation.
       The committee concludes that nicotine may cause reproductive and
       developmental effects in different species when administered via various routes
       (dermal, oral, subcutaneous). Mice seem to be less sensitive for such effects than
       rats or monkeys.
7      Existing guidelines
       The current administrative occupational exposure limit (MAC) for nicotine in the
       Netherlands is 0.5 mg/m3 (0.07 ppm), 8-hour TWA, with a skin notation.
           Existing occupational exposure limits for nicotine in some European
       countries and in the USA are summarised in Annex II.
8      Assessment of health hazard
       Workers can be occupationally exposed to nicotine through inhalation of dust or
       aerosols or by direct skin contact with tobacco leaves or a formulation of the
       compound. Nicotine is absorbed through the lungs, the skin, the gastrointestinal
       tract, and the buccal and nasal mucosa. In humans, the percentage of uptake of
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<pre>       the compound through the lungs is 60 to 80%. The average dermal absorption of
       nicotine, measured in human volunteers who were treated with nicotine patches,
       was 18% over 24 hours. In the rat, the mean percentage of dermal absorption
       varied between 49 and 88%. The bioavailability of nicotine in humans following
       oral intake ranged from 24 to 59%. Following absorption, peak concentrations of
       nicotine in human plasma were found at 3-6 hours after dermal application and at
       90 minutes after oral administration. Nicotine disappears rapidly from the blood,
       with a half-life of 2-3 hours. In the rat, at 1 hour after an intravenous injection,
       the highest concentrations of nicotine residues or metabolites were found in the
       kidneys and the lowest in blood. In humans, the majority of absorbed nicotine
       (about 80%) is biotransformed into cotinine, which has a much longer half-life in
       blood (20-30 hours) than nicotine. Cotinine and the remaining nicotine are
       further biotransformed into a range of products that are mainly excreted in the
       urine. The major metabolite identified in human urine is trans-3’-
       hydroxycotinine. The metabolic profile of nicotine is generally similar following
       inhalation or dermal exposure.
           Case studies in humans show that nicotine induces both local and systemic
       skin reactions and skin sensitisation following the application of nicotine
       patches. Occupational dermatitis has also been reported in workers processing
       tobacco or employed in nicotine production. In the older literature, many cases of
       fatal acute nicotine poisoning have been reported. More recent cases of non-fatal
       acute toxicity have been reported in tobacco workers in the field who had direct
       skin contact with tobacco plants especially under wet circumstances or in
       tobacco-factory workers who inhaled nicotine-containing dust. This so-called
       ‘green-tobacco sickness’ produces mild symptoms of intoxication, such as
       nausea, vomiting, weakness, and dizziness. In case-control studies, there was no
       association between spraying nicotine and the incidence of multiple myeloma in
       farmers, neither was there convincing association between spraying nicotine and
       the incidence of leukaemia. The committee, however, is of the opinion that the
       number of subjects (both cases and controls) using nicotine was too small to
       draw a reliable conclusion. In none of the occupational studies, there were
       reliable exposure data available. Experience on the long-term toxic effects of
       nicotine has also been obtained from users of smokeless tobacco. Among this
       group of tobacco users, there was an increased risk of dying from cardiovascular
       disease, but not from cancer. In cigarette smokers, a daily intake of 5 mg of
       nicotine has been suggested as a threshold level, below which no addiction
       occurs. However, no signs of addiction were reported in patients treated with oral
       doses of up to 25 mg nicotine/day for 6 weeks to 1 year. In humans, nicotine may
105-25 Nicotine
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<pre>       contribute to adverse reproductive outcomes. Mechanisms include reduction of
       uteroplacental blood flow and direct effects on the developing fetal brain.
       Based on the results of acute lethal toxicity studies in experimental animals, the
       committee concludes that the compound is toxic after dermal and oral exposure.
       No data were found of acute inhalation studies.
           In a rat study, it was demonstrated that both acute and short-term nicotine
       exposures had an effect on the immune system, probably via different
       mechanisms. The committee only found one short-term (18-week) oral
       neurobehavioural and one 2-year inhalation toxicity study, both in rats, which
       could be used in deriving a health-based occupational exposure limit. Decreased
       body weights, increased locomotor activity, and attenuated exploratory
       efficiency were observed in female rats (males not tested), being 5-month old at
       the start of the experiment, receiving daily doses of 20 or 50 mg/L in their
       drinking water. In the 2-year inhalation study, in which female rats were exposed
       to a nicotine concentration of 0.5 mg/m3, the only concentration tested, 20 hours/
       day, 5 days/week, for 2 years, there were only decreased body weights but no
       treatment-related increases in mortality and atherosclerosis or in the incidences
       of neoplastic or non-neoplastic lesions.
           Nicotine did not induce gene mutations in in vitro tests, but positive results
       were obtained in in vitro and in vivo cytogenicity tests. According to the
       committee, nicotine has clear clastogenic activity.
           Nicotine has been shown to have adverse effects on fertility, reproduction,
       and fetal development by multiple routes of exposure and in several species of
       animals. In rats, fetotoxicity was mainly characterised by a reduced number of
       pups born, and by reduced fetal weight, but no teratogenic effects were observed.
       Based on the above data, the committee could not establish a critical effect. The
       committee takes the NOAEL of 0.5 mg/m3 from the 2-year inhalation rat study as
       a starting point in deriving a health-based recommended occupational exposure
       limit (HBROEL). The committee notes that the actual no-adverse-effect level
       (NAEL) might be higher since exposure was for 20 hours/day and only one
       concentration was tested. Since workers are supposed to be exposed for
       maximally 8 hours/day, this NOAEL is adjusted, resulting in a NAEL of 1.25
       mg/m3. For the extrapolation to a HBROEL, the committee establishes an overall
       assessment factor of 9. This factor covers the following aspects: intra- and
       interspecies variation. Thus, applying this factor of 9 and the preferred-value
       approach, a health-based occupational exposure limit of 0.1 mg/m3 is
       recommended for nicotine.
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<pre>       The committee recommends a health-based occupational exposure limit for
       nicotine of 0.1 mg/m3, as an 8-hour time-weighted average (TWA).
            Because of the high skin absorption potential of nicotine, the committee
       advices a skin notation.
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<pre>       Annex I
        Figure 1 Proposed metabolism for nicotine (from Kye91)
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<pre>              Annex II
Occupational exposure limits for nicotine 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       0.07     0.5            8h                 administrative      S          SZW03
Employment
Germany
- AGS                                  0.07     0.5                                                   S          TRG00
                                       0.28     2.0
- DFG MAK-Kommission                   -        -c                                                    S          DFG03
Great Britain
- HSE                                  -        0.5            8h                 OES                 S          HSE02
                                                1.5            15 min             STEL
Sweden                                 -        -                                                                Swe00
Denmark                                -        0.5            8h                                     S          Arb02
USA
- ACGIH                                -        0.5            8h                 TLV                 S          ACG03b
- OSHA                                 -        0.5            8h                 PEL                 S          ACG03a
- NIOSH                                -        0.5            10h                REL                 S          ACG03b
European Union
- SCOEL                                -        0.5            8h                 ILVd                           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
     Listed among compounds for which studies of the effects in man or experimental animals have yielded insufficient
     information for the establishment of MAK values.
d
     Listed among compounds for which OELs are already included in Commission Directives.
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