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Commission of the European Communities DG XI CLASSIFICATION AND LABELLING OF DANGEROUS SUBSTANCES. Recommended form to be used for the proposed classification and labelling of a dangerous substance under Directive 67/548/EEC

ECBI/17/06
Date: February 2006 Health classification prepared by: Norway

The information contained in this form is not regarded as confidential

1. Identification of the substance

INDEX No. EC No. 265-667-4 Ketoconazole CAS No. 65277-42-1 ID No.

1.1 EINECS Name If not in EINECS IUPAC Name 1.2 Synonyms (state ISO name if available)

Chemical name: (+)-cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3dioxolan-4-yl]methoxy]phenyl]piperazine; Fungarest; Fungarol; Ketoderm; Ketoisdin; Nizoral; Orifungal M; Panfungol C26-H28-Cl2-N4-O2

1.3 Molecular formula 1.4 Structural formula

1.5 Purity (wt/wt)

No data.

1.6 Significant impurities or additives, their concentrations (wt/wt)

No data.

1.7 Known uses Industrial General public Currently used in cosmetic products at concentrations of up to 2%, typically in shampoo formulations; particularly in products claiming an antidandruff efficacy.

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Used in drugs by oral and topical routes as broad specrum antifugal agent, highly effective in skin infections caused by dermatophytes and yeast, in dermal candidoses and systemic mycoses. 1.9 Proposed label Symbol(s) R-phrase(s): T; R25 Xn; R48/22 Repr. Cat 2; R60 S-phrase(s):

Existing classification

Existing label Symbol(s)

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2. PHYSICO-CHEMICAL CHARACTERISTICS 2.1 Physical form 2.2 Molecular weight White powder or off-white powder 351.4

2.3 Melting point/range (oC)

148 C to 152 C

2.4 Boiling point/range (oC)

Not applicable.

2.5 Decomposition temperature 2.6 Vapour pressure (Pa( oC))

Not applicable. Not applicable.

2.7 Relative density (D420) 2.8 Vapour density (air = 1) 2.9 Conversion factor (11011 hPa at 25oC) 2.10 Solubility

Not applicable Not applicable.

conc. at sat. (g/l) Practically insoluble. One part is soluble 54 parts. One part is soluble in 2 parts. One part is soluble in 9 parts. Very slightly soluble.

solvent Water Ethanol

Temperature (oC)

Chloroform

Methanol

Ether

2.11 Partition coefficient (log Pow)

Experimental No data Calculated No data.

2.12 Flammability Flash point (oC) Explosivity limits (% v/v) Auto-flammability temp. ( C)
o

No data. open cup: lower limit: No data. shock: friction: ignition: closed cup: upper limit:

2.13 Explosivity Danger of explosion at result of: Explosive properties at high temperature 2.14 Oxidizing properties

No data.

2.15 Other physico-chemical properties No data.

3. OBSERVATIONS OF HUMANS Where available, human data are considered to be of more relevance in determining the potential effects of chemical substances on the human population. (Annex V, Directive 67/548/EEC). 3.1 Occupational exposure Ref.

3.2 General population In an epidemiological study the effect of oral ketoconazole treatment on foetal development was studied. The use of ketoconazole in the second and third months of gestation was compared between cases with congenital abnormalities and their matched controls in the large population based data set of the Hungarian Case-Control Surveillance of Congenital Abnormalities, 1980-1996. Birth weights and gestational age were evaluated in control newborn infants born to mothers with or without ketoconazole treatment. The case-group comprised 22 843 cases with congenital abnormalities, while the control group contained 38 151 newborn infants without any defects. Six infants (0.03%) and 12 controls (0.03%) had mothers who had received oral ketoconazole treatment (prevalence odds ratio. With 95% confidence interval: 0.8, 0.3-2.2) No group of infants with congenital malformations had mothers with a higher incidence of use of the drug. The mean gestation age was somewhat longer while birth weight was somewhat larger in controls with ketoconazole treated mothers. This study therefore failed to demonstrate a higher rate of congenital abnormalities in infants with mothers who had received oral ketoconazole treatment during pregnancy.

Ref. Kazy et al., 2005

4. TOXICOLOGICAL DATA 4.1 ACUTE TOXICITY 4.1.1 Oral Species Mice 30 male30 female Rats40 males40 females Guinea Pigs40 males40 females Dogs15 males12 females

LD50(mg/ kg) M: 786 F: 618

Observations and Remarks Ketoconazole was delivered at doses of 320, 640 and 1280 mg/kg bw. The animals were observed during 7 consecutive days after administration. Males: LD50 after 7 days was 786 mg/kg bw. Females: LD50 after 7 days was 618 mg/kg bw. Ketoconazole was delivered at doses of 80, 160, 320 and 640 mg/kg bw. The animals were observed during 7 consecutive days after administration. Males: LD50 after 7 days was 287 mg/kg bw. Females: LD50 after 7 days was 66 mg/kg bw. Ketoconazole was delivered at doses of 80, 160, 320 and 640 mg/kg bw. The animals were observed during 7 consecutive days after administration. Males: LD50 after 7 days was 178 mg/kg bw. Females: LD50 after 7 days was 226 mg/kg bw. Ketoconazole was delivered at doses of 80, 160, 320, 640, and 1280 mg/kg bw. The animals were observed during 7 consecutive days after administration. Males: LD50 after 7 days was 937 mg/kg bw. Females: LD50 after 7 days was 640 mg/kg bw.

Ref. Ref: 2/1

M: 287 F: 166

Ref: 2/1

M: 178 F: 226

Ref: 2/1

M: 937 F: 640

Ref: 2/1

4.1.2 Inhalation No data available.

4.1.3 Dermal Species Cunistar white rabbits (20) LD50 (mg/kg) Higher than 2000 mg/kg bw Observations and Remarks In this study 2000 mg/kg bw of ketoconazole was applied under occlusive patch during 24 hours (group 1, 5 males and 5 females). Group 2, 5 males and 5 females was the control group and received the vehicle. Observations were made daily over a period of 14 days. All animals survived and no general clinical observations were reported. Ref. Ref: 3/2

Conclusion: According to the results the LD50s 7 days were higher for mice and dogs than for rats and guinea-pigs. They were also higher for males than for females in rats and mice. Based on the LD50 values following oral administration in male rats from 166-309 mg/kg bw, ketoconazole should be classified as acute toxic with the risk phrase T; R25 Toxic if swallowed. Based on the dermal acute toxicity data with a LD50 higher than 2000 mg/kg bw in rabbits, no classification is warranted. Ketoconazole is proposed classified with T; R25, Toxic if swallowed. 4.1.4 Skin irritation Species No. of Exposure animals time (h/day)

Conc.

New Zealand white rabbits

12 (4 groups)

24 hours

Cunistar albino rabbits

4 hours

A shampoo with 1% ketoconazole and a 15% solution of the shampoo with 1% ketoconazole Vehicle; 100% and a 15% solution. Ketoconazol Occlusive to e 1% non abraded shampoo skin

Dressing: occlusive semiocclusive open Occlusive

Observations and remarks

Ref.

The test was performed according to GLP. 0.3 ml was applied. Erythema and eschar formation was scored after 24 hours (removal of the occlusive system) and after 48 hours. The shampoo with 1% ketoconazole was considered moderate irritant since the 15% solution was classified as mild irritant. The vehicle was classified moderate irritant in both preparations. The test was performed according to OECD Guideline 404 and according to GLP. 0.5 ml was applied. Observations of the skin area were made at 1 hour and 1, 2, 3, 4, 7 and 14 days after application. Erythema, eschar and oedema formation was scored according to the Draize scale. The primary dermal irritation index was 0.58.No dermal irritation occurred when a 15% dilution of the same ketoconazole 1% formulation was applied under the same conditions.

Ref: 14/3

Ref: 15/4 and 16/5

4.1.5 Eye irritation Species No. of animals animals New 12 Zealand male white rabbits

Exposure time (h/day) The eyes was scored at 1, 24, 48 and 72 hours after treatment an further 7, 14 and 21 days after treatment

Conc.(wt/wt)

Observations and remarks

Ref.

A cream with 2% Ketoconazole was used, single dose of 0.1 ml.

The test was an eye Draize test using the 1964 Guideline Guide for grading eye irritation by Hazardous substances. In this study no changes were reported. It was concluded that the ketoconazole 2% cream was not irritating to eye under the experimental conditions used.

Ref: 17/12

New Zealand white rabbits

A shampoo with 2% Ketoconazole was used.

New Zealand white rabbits New Zealand white rabbits

24 (6/group)

Rabbits

3/sex

Ocular reactions were scored 1 hour and 1, 2, 3, 4, 7, 10, 14 and 21 days after instillation

Rabbits

3/sex

Ocular reactions were scored 1 hour and 1, 2, 3, 4, 7, 10, 14 and 21 days after instillation

The test was performed in compliance with GLP and according to the Federal Hazardous Substances Act Ocular Irritation Study Protocol. According to the reactions observed in the cornea of all animals and in the conjunctiva of five of six animals, ketoconazole tested full strength was considered irritating to the rabbit eye. 15% solution The same experimental conditions were used of a shampoo as described above (ref. 18/13). In this study with 2% no ocular irritations were reported in the eye of Ketoconazole. the 6 rabbits. A shampoo The same experimental conditions were used with 1% as described above (ref. 18/13). Results ketoconazole obtained with the shampoo with 1% and a 15% ketoconazole were positive and identical solution of the between the test product and its vehicle. Both shampoo with results were negative with the 15% diluted 1% preparations (ketoconazole 1% shampoo and ketoconazole. vehicle). No conclusion was given from this Vehicle 100% study. and a 15% solution. A shampoo The study was performed in compliance with with 1% GLP and OECD Guideline 405. In this study Ketoconazole all animals had a diffuse redness and was used, chemosis of the conjunctiva within the 48 single dose of hours following instillation of the product; 2 of 0.1 ml the rabbits had a slight hyperaemic reaction of the iris and a slight opacity of the cornea. All signs reversed after 48 hours. It was concluded from the study that according to the results ketoconazole 1% shampoo was not considered to be an ocular irritant. A shampoo The study was performed in compliance with with 1% GLP and OECD Guideline 405. Similar Ketoconazole reactions were reported as described above was diluted (ref. 21/16) however the effects reversed in 2 15% , single animals the 2nd day and the 7th day for the third dose of 0.1 ml animals.

Ref: 18/13

Ref: 19/14

Ref: 20/15

Ref: 21/16

Ref: 22/17

4.1.6 Irritation of respiratory tract No data available.

Conclusion: Skin irritation In the skin irritation studies 100% ketoconazole was not tested. Only shampoos with 1% ketoconazole or a 15% dilution of this shampoo was tested. In the first study moderate skin irritation was observed both in the exposed group, and in the control group exposed only to the shampoo. Therefore, no clear conclusion can be drawn from this study. In a more recent GLP study performed in accordance with OECD Guidelines the primary irritation index was 0.58 in the group exposed to a shampoo with 1% ketoconazole, whereas no skin irritation was reported when a 15% dilution of the same ketoconazole 1% formulation was applied under the same conditions. Based on the available data no classification for skin irritation is proposed since it is difficult to distinguish whether the skin irritation observed is due to the shampoo itself or to the 1% ketoconazole in the shampoo.

Eye irritation In the eye irritation studies 100% ketoconazole was not tested. Only shampoos with 1% or 2% ketoconazole, creams with 2% ketoconazole , or 15% dilutions of these shampoos were tested. The results from the eye irritation studies are equivocal. In one study ketaconazole 2% cream was not irritating to eye. In another test performed in compliance with GLP ketoconazole 2% shampoo was considered irritating to eye, however, when using the same experimental conditions with a 15% diluted ketoconazole 2% shampoo, no eye irritation was reported. Using the same protocol eye irritation was reported in animals exposed to a shampoo with 1% ketoconazole and a 15% diluted ketoconazole 1% shampoo as well as a 100% vehicle and a 15% diluted vehicle. Finally, in two study performed in accordance with GLP and OECD Guideline 405 slight ocular lesions were reported in the first study following installation of ketoconazole 1% shampoo, however all signs reversed after 48 hours in the first study. In the second study following installation of ketoconazole 1% shampoo used as a 15% dilution the same slight ocular lesions were reported, however, they reversed the 2 nd day in two rabbits and the 7 th day for the third rabbit. Based on the available data no classification for eye irritation is proposed, since it is difficult to distinguish whether the eye irritation observed is due to the shampoo/cream or to the 1or 2% ketoconazole in the shampoo.

4.1.7 Skin sensitization Species Type of test No. of animals (c,t) 20 of both sexes Incidence of reactions observed Ref.

Guinea pigs Modified Maximisation test

A ketoconazole 2% formulation was used. At the induction phase, the test formulation was applied at 0.5 ml per animal to clipped skin 3 times a week for 3 weeks and once at the start of the 4 th week. The formulation was maintained each time for 48 hours under occlusive patch. After a rest period of 12 days the challenge (0.5 ml of test substance for 48 hours) was given under occlusion on a region never been treated. Readings were made 1, 8, 24 and 48 hours after removal of the occlusive patch. Immediate slight erythema was reported at the challenge site on 3 animals 1 hour and 8 hours after removal. No reactions were reported later. Guinea pigs OECD guideline 20 t female A ketoconazole 1% scalp solution was used. 406, Magnusson 20 c female According to the results it was concluded that Maximisation test ketoconazole 1% scalp solution is not considered to have a sensitisation potential. Humans Kligman 25 healthy A ketoconazole 2% cream and vehicle was used. 0.3 g Maximisation Test adults of the test material was applied under an occlusive volunteers dressing for five 48 hours periods. After a 10 day rest period a challenge patch was applied in different areas for 48 hours under occlusion. Readings were made immediately and 24 hours after removal of the patch. No skin sensitisation was reported. Humans Kligman 25 healthy Maximisation Test adults volunteers Ketoconazole 2%, 1% and 0.5% shampoos versus vehicle were used. 0.1 ml of the test material was applied under occlusive tape for five 48-hours periods. After a 10 days rest period a challenge of 0.1 ml test material was applied for 48 hours under occlusion. Readings were made 1 hour and 24 hours after removal of the patch. No skin sensitisation was reported following application of ketoconazole 0.5% 1% or 2% shampoos. 194 healthy A 2% ketoconazole antidandruff shampoo was used. adults An induction phase of nine 24 hours applications volunteers under occlusion of a 1% solution of the original test material (corresponding to 0.02% ketoconazole) was used (3 times a week for 3 weeks). This was followed by a 2-week rest period. Then a challenge with a 1% solution for 24 hours was used. Readings were made 24, 48, 72 and 96 hours after removal of the patch. Re-challenges were performed on subjects who exhibited sensitisation to the test material at the first challenge, consisting of an additional 24-hour patch period on the same site. According to the results and in spite of the high reactions related to the surfactant system, the tested solution was considered as not producing instance of contact sensitisation.

Ref: 23/7

Ref: 24/8

Ref: 25/9

Ref: 26/10

Humans

Ref: 27/11

c: control group; t: test group Conclusion: In the sensitisation studies 100% ketoconazole was not tested. Only formulations with 0.5, 1 or 2% ketoconazole was tested. In these studies no skin sensitisation was reported. Based on the available animal data and human data no classification for sensitisation is proposed.

4.2 REPEATED OR PROLONGED TOXICITY GROUPED ACCORDING TO SUBACUTE, SUBCHRONIC AND CHRONIC TOXICITY AND CARCINOGENICITY (ANIMAL EXPERIMENTS)

4.2.1 Oral Species Dose mg/kg Duration of body weight, treatment mg/kg diet Wistar rats 10 0, 8, 32, 128 13 weeks rats/sex/group mg/kg bw/day in the diet Observations and Remarks Ref.

Wistar rats 20 rats/sex/group

0, 4, 16, 70 6 months mg/kg bw/day in the diet

Wistar rats 20 rats/sex/group

0, 4, 15, 71 18 months mg/kg bw/day in the diet

At the highest dose death of 2/10 males was Ref/ 5/18 reported as well as a significantly decrease in terminal body weight and total weight gain in both males and females. Gross pathology was reported in 32 mg/kg bw/day females including discoloured liver, pale adrenals and slightly larger ovaries. At 128 mg/kg bw/day a discoloured liver was reported in both sexes and pseudopregnancy and fragile bones of the legs in females. In the female 32 mg/kg bw/day dose group a marginal increase in the absolute and relative liver and ovaries weight was reported. In the high dose group an increase in the relative liver and adrenals weight was reported in both sexes, in the absolute and relative weight of the testis (males) and in the relative weight of the spleen and kidneys (females). Histological changes were reported mainly in the high dose group, with a tendency to the same changes in the medium dose group. Changes were seen in the liver, the kidneys, ovary, uterus and vagina, adrenals and bones. Effects on the endocrine organs (adrenals and ovaries) as well as changes in the uterus and vagina and bones were seen as a result of hormonal imbalance. There was a good correlation between gross pathology and histopathology. The NOAEL from this study was considered 8 mg/kg bw/day. The study was performed in accordance Ref: 9/19 with OECD Guideline 452. One control male and two high dose females died, however, this was not considered treatment related by the authors. Total food consumption and final body weight was significantly decreased in males at 70 mg/kg bw/day and in females in the mid- and high dose group. No dose-related gross pathology was seen except for broken legs in one medium and 6 high dosed females. Organ weights were mainly normal except for an increase in the relative weight of the liver in high dosed males and females and of the kidneys and brain in the high dosed females. Histological changes were reported mainly in ovary and adrenal in the mid- and high-dose females. The NOAEL from this study was considered 4 mg/kg bw/day. The study was performed in compliance with Ref: 10/20 GLP. 26 animals died during the study and 71 animals had to be sacrificed in a morbid state during the 18th month of the study. There was no difference in mortality between male groups, however, a slight

Beagle dogs 3/dogs/sex/grou p

0, 2.5, 10, 40 mg/kg bw/day by gavage

12 month

Beagle dogs 3/dogs/group

20 mg/kg 24 weeks bw/day for 2 weeks, 40 mg/kg bw/day for 2 additional weeks and then 60 mg/kg bw/day for 9 weeks. One male and one female was continuously treated with 60 mg/kg bw/day the following 11 weeks (week 14-24)

increase in mortality rate was reported in high dosed females. A significant decrease in body weight was reported in high dosed animals. Gross pathology was reported in the salivary glands and abdominal fat in some medium and several high dosed males and females. Furthermore, in the adrenals in some high dosed males and medium and high dosed females. Fragile bones were reported in all dose groups, including controls. An absolute and relative increase in adrenal weights was reported in high dosed animals. Histopahtological changes were reported in the parenchymal and lymphoid tissues in medium and high dosed animal, and in the adrenals in the high dosed group. The NOAEL from this study was considered 4 mg/kg bw/day. One control animal died during the study. In Ref: 11/21 the high dosed group food consumption was decreased, and a significantly lower body weight gain was reported. Gross pathology was reported in the high dosed group including a brownish discoloration of several organs and tissues. As a result of the lower body weight gain in the high dosed group, the absolute weight of several organs decreased, with an increase in absolute and relative liver weight. The NOAEL from this study was considered 10 mg/kg bw/day. In the high dosed animals the following was Ref: 12/21 reported: a slight body weight loss related to bis reduced appetite, small-sized thymus and swollen liver, decrease in absolute and relative thymus weight and increase in absolute and relative liver weight. All effects were reversible in the dogs not treated from week 14-24 (2/dogs/sex/group).

4.2.2 Inhalation No data available. 4.2.3 Dermal Species New Zealand white rabbits4 rabbits/se x/group Dose mg/kg/day Cream with 2% Ketoconazo le was used. The test article was applied at 0.5 1 and 2 g/kg bw. The vehicle (crem) was 2 g/kg bw 0, 2, 20 and 50 mg/kg bw Exposure Duration of time (h/day) treatment Once daily 30 days Observations and Remarks In this study the skin was clipped weekly, left intact in 2 males and 2 females of each group, and abraded on the others. In this study no adverse effects were reported, however, no gross examination was performed at the end of the study. Ref. Ref: 4/6

New Zealand white rabbit 4 rabbits /sex/group

Ketoconazol e 2% shampoo was applied daily to abraded and intact clipped skin for 1 hours and then removed New 0, 1, 10 and Ketoconazol Zealand 25 mg/kg e 1% white bw shampoo rabbit was applied 4 daily to non rabbits abraded /sex/group clipped skin for 1 hours of contact, open exposure New 0, 10, 20 Ketoconazol Zealand and 40 e 2% white mg/kg bw shampoo rabbit was applied 5 daily to non rabbits abraded /sex/group clipped skin for 1 hours of contact, open exposure. New 0, 2, 20 and Ketoconazol Zealand 50 mg/kg e 2% white bw shampoo rabbit was applied 4 daily to males/gro abraded and up, 5 intact skin for female/gro 1 hours of up contact, open exposure.

28 days

The study was performed according to GLP. Ref: 6/22 No mortality was reported. No skin irritation, no histological changes, and no changes in body weight, food consumption and organ weights were reported. The NOAEL from the study was the highest dose tested 50 mg/kg bw/day.

28 days (7 days/week)

The study was performed according to GLP. Ref: 7/25 No mortality was reported. No skin irritation, no histological changes, and no changes in body weight, food consumption and organ weights were reported. The NOAEL from the study was the highest dose tested 25 mg/kg bw/day.

28 days (5 days/week)

The study was performed according to GLP. Ref: 8/23 Erythemal reactions reversible within 24 hours were reported in dosed animals as well as in the vehicle control animals. No mortality was reported. No skin irritation, no histological changes, and no changes in body weight, food consumption and organ weights were reported. The NOAEL from the study was the highest dose tested 25 mg/kg bw/day. The study was performed according to GLP. Ref: 13/24 No dose-related mortality was reported in dosed and control rabbits. No histological changes, and no changes in body weight, food consumption and organ weights were reported. The NOAEL from the study was the highest dose tested 50 mg/kg bw/day.

26 weeks

Conclusion: Oral exposure In Wistar rats exposed from 13 weeks to 18 month with doses ranging from 4 to 128 mg/kg bw/day gross pathology were reported in both male and female rats in the liver, adrenals, ovary and bones. A correlation in the effects reported was observed between the studies. Histopathological changes were also reported in both male and female rats in the liver, kidney, ovary, uterus, vagina, adrenals, bones and lymphoid tissue. Organ weigh changes were reported as well. These effects were reported at doses below 50 mg/kg bw/day and higher. More severe effects were seen at higher doses. A good correlation was found between gross pathology and histopathological changes. Effects on the endocrine organs (adrenals, testes and ovaries) as well as changes in the uterus and vagina and bones were seen as a result of hormonal imbalance.

Based on the effects reported in Wistar rats ketoconazole should be considered classified with Xn; R48/22. Dermal exposure Following dermal exposure to New Zealand White rabbits at doses up to 50 mg/kg bw /day no significant changes were reported. Based on the available data no classification for dermal repeated exposure is warranted.

4.3 GENOTOXICITY Test In vitro Bacterial reverse mutation assay (Ames test) Species Conc. (mg/l) Metabolic activ. +/Observations and Remarks Ref.

HGPRT locus test (hypoxanthineguanine phosphoribosyl transferasetest) Transformation test

Salmonella Typhimurium, strain: TA 98, TA 100, TA 1535, TA 1537, TA 1538. V79 cell line of the Chinese Hamster

0, 0.5, 2.5, 10.0 and 50.0 g/plate

No genotoxicity was reported up to 50.0 g/plate.

Ref:39/2 6

0, 20, 40 and 60 g/ml

+/-

Ketoconazole did not induce mutations in V79 cells up to 60 g/ml. Higher concentrations could not be applied due to the low solubility of the compound.

Ref: 40/27

Balb/3T3 mice fibroblasts in vitro

Transformation test. Transformation response to nitrogen stimulation. In vivo In vivo micronucleus test

Lymphocytes from human healthy young adult donors

0, 0.45, 0.91, +/1.82, 3.65, 7.30 g/ml without met.act, 0, 1.05, 2.10, 4.20, 8.40 and 16.80 g/ml with met. act. 0, 1.0, 2.5, 5.0 or 10.0 g/ml

Ketoconazole or its metabolites did not induce transformation in Balb/3T3 mice fibroblasts at the concentrations tested.

Ref: 41/28

Ketoconazole did not cause significant Ref: depression in lymphocyte 42/29 transformation responses in either unstimulated or nitrogen stimulated (PHA or Con A) cell cultures. At the concentrations used ketoconazole was considered non mutagenic. At the concentrations tested, ketoconazole was considered non mutagenic. Male mice were exposed to ketoconazole before mating to undosed female mice. Mutations were evaluated by counting of embryonic deaths and of reduction in number of implants in females necropsied 15 days after mated to the males. No induction of dominant lethals was reported. Female mice were exposed to ketoconazole before mating to undosed male mice. Mutations were evaluated by counting of embryonic deaths and of reduction in number of implants in females necropsied 15 days after mated to the males. No induction of dominant lethals was reported. After 3 days exposure Berlin K males, were mated to 3 sequential groups of virgin Muller 5- females to rise F1 progeny. F1 females were then mated with their brothers and their progeny were examined for recessive lethal mutations. No induction of recessive lethal mutations was reported. Ref:43/3 0

Mice

Dominant lethal Male mice

0, 20, 40 and 80 mg/kg bw, intraperitoneally injection 0, 20, 80 or 320 mg/kg bwsingle oral administration

Ref: 44/31

Dominant lethal Female mice

0, 20, 80 or 320 mg/kg bw single oral administration

Ref: 45/32

Sex linked recessive lethal test

Drosophila 200 and 2000 melanogaster ppm for 3 days (males)

Ref: 46/33

Conclusion:

Based on the negative in vitro data and negative in vivo data ketoconazole is considered not mutagenic, and no classification for mutagenicity is proposed.

4.4 FERTILITY Species Route Dose Exposure Number of time (h/day) generations exposed 8 weeks (30 1 generation male rats) or 13 weeks (39 male) rats. Each group was then divided into subgroups 10/group and mated with untreated females Observations and Remarks Ref.

SpragueDawley rats

Oral

0, 25 and 75mg/kg bw

Female Rats

Oral

0, 10, 30 and 100 mg/kg/day

Gestation day 1-8

The males were paired for a maximum of 3 weeks (treatment continued during pairing) with untreated females (69 females). Caesarean section was performed on day 20. No clinical signs were reported. At 75 mg/kg /day reduced spermatozoa motility (19 to 26 %) was reported which may have been related to reduced plasma testosterone levels (41 to 68 %) or/and to a direct effect of ketoconazole on spermatozoa. Furthermore, an increase in the number of abnormal spermatozoa (74 to 81 %) mainly due to an increased incidence of detached sperm heads (74 to 223 %) was reported as well. After 13 weeks of treatment decreased sperm count was noted at 75 mg/kg /day, which may have been related to changes in male fertility as indicted by lower pregnancy rate (30 to 34 %). Histological changes were noted at both doses in testes and adrenals such as increased incidence of focal tubular atrophy and hyperplasia of Leydig cells, and vacuolization of zona fasciculasta. The severity of these histological changes was directly related to the dose level and treatment duration. No adverse effects were noted in the untreated females mated with treated males. No information regarding effects in the live offspring was included. In this study the effect of Ketoconazole on early pregnancy was studied in rats. In the 100 mg/kg/day dose group a 50 % pre-implantation loss and a 100 % rate of resorptions were reported on gestation day 9. Ovarian weight was double of control (142 vs 76 3 mg). Ovaries from treated animals were white as opposed to pink in control animals. The white colour of the ovaries suggests an accumulation of lipid. Serum progesterone was significantly decreased at 100 mg/kg/day relative to controls (6.2 0.6 vs. 66.0 5.2 ng/ml) while serum LH was significantly higher

Delonge as et al., 1995

Cummin gs and Metcalf, 1996

Adult SpragueDawley rats

Oral by Increasing gavag doses from e 10 to 300 mg/kg bw/day

One single dose. Samples were collected 4 hours after dosing

Male Sprague- Oral by 0, 200 Dawley rats gavag and 400 (18-19/group) e mg/kg

3 days. The results represent three separate experiments. There were no significant differences between the data for the 3 experiments. All data were combined to represent one set of data.

in treated dams (12.5 1.4 vs 3.5 0.4 ng/ml), estradiol remained unchanged. It was concluded by the authors that the failure of pregnancies was due to the drastic decline in progesterone and that the rise in LH is a feed back effect from the CNS in response to the lower progesterone levels. No information regarding maternal toxicity was included. In this study the effect of ketoconazole on testosterone secretion, testicular interstitial fluid (TIF) formation and TIF testosterone levels were studied. A dose dependent decrease in serum testosterone, TIF testosterone and TIF volume was reported. In other studies Ketoconazole have also been shown to inhibit testosterone secretion (Pont et al., 1982; Schurmeyer and Nieschlag, 1984; De Coster et al., 1985 and Oftebro et al., 1994). TIF formation, like testosterone secretion, is a major regulatory aspect of testicular function. TIF testosterone levels reflect testosterone secretion inside the testis, where it exerts paracrine and autocrine effects on steroidogenesis and spermatogenesis (Sharpe, 1988; Sharpe, 1990). The results indicate that Ketoconazole suppress the two major regulatory aspects of testicular function, testosterone secretion and TIF formation, supporting the hypothesis that Ketoconazole can suppress male reproductive function and fertility. Each male was paired with an untreated female immediately after the administration of the third dose. All paired females were euthanized 10 days after pairing. Seven animals in the high dose group and 2 animals in the low dose group died during dosing. The control group and the low dose group appeared normal in behavior and appearance after dosing, whereas the high dose group was lethargic and generally had reduced motor activity when compared to the other groups. 93%, 44% and 0% of the females in the control, 200 and 400 mg/kg dose group, respectively became pregnant. There were no significant differences in implantation sites, normal fetuses and number of corpora lutea between the control and low dose

Adams et al., 1998

Waller et al., 1990

Male mice (15/group)

Oral

0, 400 mg/kg/bw

60 days

Male Beagle dogs (5 treated and 4 control)

Oral

0, and 25 mg/kg/day

4 weeks (In the exposed group, and the control group 2 dogs were kept for a 4week recovery period

group, however, there tended to be reduced in the low dose group. In the males no changes were reported in testicular and epididymal weight and sperm count. However, a dose-related decrease in motility (74.9, 54.4 and 39.2 in the control, 200 and 400 mg/kg dose group, respectively) and forward progression of epididymal spermatozoa (70.3, 37.5 and 21.3 in the control, 200 and 400 mg/kg dose group, respectively) was reported between the three groups. No significant changes in body weight were reported (25 2 vs. 282 in controls). The weight of the testes (1656 vs. 1907 in controls), epididymis (601 vs. 751 in controls) , seminal vesicle (1008 vs. 1457 in controls), and ventral prostate (80.4 vs. 100.5 in controls) were significantly reduced. Furthermore, a significant declined in sperm motility in cuda epididymis (464 vs. 653 in controls) as well as a decrease in sperm density in testis (10.1 vs. 1.20.1 in controls) and cuda epididymis (291 vs. 381 in controls) was also reported. A significant reduction in fertility was reported in ketoconazole treated mice (50% vs 90% in controls). A reduction in food intake resulting in a transient loss of body weight was reported during the first two weeks. No blood chemistry alterations were reported. Exposure was associated with a decrease in the sperm motility beginning during the 1st week of treatment, an increase in the total mean number of abnormal spermatozoa beginning during the 2nd week of treatment, and a slight decrease in total sperm count at the end of the dosing period. These changes were considered to be related to a slight increase in plasma progesterone levels. The plasma levels of testosterone were slightly lower in the treated dogs but remained within the range of control values. No organ weight changes were noted. The histopathological changes reported were marked increase in the incidence of minimal focal tubular degeneration and/or atrophy of the testes when compared to controls. None of these changes were seen after

Joshi et al., 1994

Delonge as et al., 1996

Endocrine effects

Oral

0, 30 and Day 1 to 8 of 100 mg/kg pregnancy bw/day

the recovery period. In a variety of mammalian test Ref: 53/0 systems ketoconazole was shown to completely inhibit the key steroidogenic cytochroms P 450 17-hydroxylase/17, 20-lyase, P 450 11-hydroxylase, and P 450 aromatase in a dose-dependent manner. As a result, adrenal and gonadal androgen secretion was reduced, to a lesser degree the production of oestrogens and corticosteroids. Oral ketoconazole administration was therefore shown to induce endocrine imbalance.

Conclusion: A statistically significant effect on sperm quality as well as histopathological changes in the testis was reported in rats following exposure to 75 mg/kg/day for 8 or 13 weeks. The effect on testis was accompanied with a lower pregnancy rate in untreated females. In mice statistically significant effects were reported on the weight of the testes, epididymis, ventral prostate and seminal vesicle, as well as on sperm density and motility and fertility following exposure to 400 mg/kg/day for 60 days. Effects on sperm quality as well as histopahtological changes in testis were also reported in a study with Beagle dogs following exposure to 25 mg/kg/day for 4 weeks. Ketoconazole was also shown to induce 50 % pre-implantation loss as well as 100 % resorptions in pregnant rats exposed to 100 mg/kg/day the first 8 days of gestation (see the developmental section for further details). In several studies Ketoconazole was shown to inhibit testosterone synthesis, and in one study testicular interstitial fluid (TIF) volume which indicates that Ketoconazole may suppress two major regulatory aspects of testicular function, and thereby may have effect on male reproductive function and fertility. The effects of Ketoconazole on the male reproductive organs have been discussed in the book; Comprehensive toxicology, Volume 10, Reproductive and endocrine toxicology (Kelce, 1997; Cook et al., 1997), and the following is written: Ketoconazole is an example of a chemical that directly alters Leydig cell function without affecting Leydig cell viability. Ketoconazole was fist suspected to inhibit Leydig cell steroidogenesis following the development of gynecomastia in humans (Pont et al., 1982; De Felice et al., 1981). Clinical studies revealed that Ketoconazole administration reduced serum testosterone levels (Pont et al., 1982) in the absence of alterations in serum gonadotropins, suggesting a direct effect on Leydig cells steroidogenesis (Schurmeyer and Nieschlag, 1984). Similar in vivo effects were observed in adult male rats treated with Ketoconazole (Vawda and Davis, 1986). Ketoconazole is one of the best studied and most widely known testosterone biosynthesis inhibitors (Feldman, 1986).

Based on the reported effects on fertility/reproductive organs, Classification with Repr. Cat. 2; R60, May impair fertility is warranted.

4.5 DEVELOPMENTAL TOXICOLOGY Species Route Dose Exposure Exposure period: mg/kg/day time number of geneppm (h/day) rations or- number Conc(mg/l) of days during pregnancy 0, 25 and 75 mg/kg/day 8 weeks (30 male rats) or 13 weeks (39 male) rats. Each group was then divided into subgroups 10/group and mated with untreated females Observations and Remarks Ref.

Sprague -Dawley rats

Oral

Female rats

oral

0, 30 and 100 mg/kg /day

From pregnancy day 1 to 8

No No informati inform on ation

From 12 to 100 mg/kg/day

No information

Rat

Oral in the diet

No 1 information

The treated males were paired for a maximum of 3 weeks (treatment continued during pairing) with untreated females (69 females). Caesarean section was performed on day 20, the uterine content was examined and the fetuses observed for external alterations. At 75 mg/kg /day reduced sperm quality and quantity was reported, which may have been related to changes in male fertility as indicted by a lower pregnancy rate (30 to 34 %). No adverse effects were noted in the untreated females mated with treated males. No information regarding effects in the live offspring was included. In the 100 mg/kg/day dose group a 50 % preimplantation loss and a 100 % rate of resorptions were reported on day 9. No information regarding maternal toxicity was included. For further details see the fertility section. Ketoconazole was studied together with other suspected antiandrogens for a possible effect on sexual differentiation. Anogenital distance, nipple/areola development and reproductive organ weight were measured in male offspring. However, Ketoconazole failed to induce any of these endpoints in the male offspring (only abstract available). Ketoconazole has been shown to be teratogenic [syndactylia (congenital webbing of digits) and oligodactylia (the presence of fewer than five digits on a hand or foot)] in rat when given in the diet at 80 mg/kg/day. However these effects may be related to maternal toxicity, evidence of which has been reported at this and higher dose levels. Ketoconazole was also found to be embrytoxic to the rat when given in the diet at doses higher than 80 mg/kg during the first trimester of gestation. In addition dystocia (difficult labor) was noted in rats administrated oral Ketoconazole during the third trimester of gestation. This occurred when Ketoconazole was administered at doses higher than 10 mg/kg bw.

Delong eas et al., 1995

Cummi ngs and Metcalf, 1996

Gray et al., 1999

Ref: 28/0

Rat

Oral in the diet

No No First trimester of information information gestation

The malformation and the embryotoxicity observed may be due to maternal toxicity, however, no more information from these studies was available in the TOXNET HSDB database.

Ref: 28/0

Conclusion: Ketoconazole induced a 50 % pre-implantation loss as well as 100 % resorption in pregnant rats exposed to 100 mg/kg/day the first 8 days of gestation. Alterations in ovarian weight and hormone levels were also reported in the 100 mg/kg/day dose group. Ketoconazole was also reported to be teratogenic and embryotoxic in rats, however, due to limited information from these studies it is difficult to conclude if the effects reported in the pups were related to maternal toxicity or not. In the study by Gray et al., 1999 only effects on sexual differentiation was studied, related to a possible antiandrogen effect of ketoconazole, however, no changes in sexual differentiation were reported. In an epidemiological study they failed to demonstrate a higher rated of congenital abnormalities in infants with mothers who had received oral ketoconazole treatment during pregnancy (Kazy et al., 2005). Since limited information is available to conclude if the teratogenicity/embryotoxicity reported in rats was related to maternal toxicity, and limited information is available on the magnitude of the maternal toxicity, a final conclusion on classification for developmental toxicity is difficult. Due to the limited information related to maternal toxicity and the magnitude of the teratogenicity/embryotoxicity reported in rats no clear conclusion can be drawn regarding a classification for developmental toxicity following exposure to ketoconazole.

References:
The references given numbers from 2/1 up to 53/0 is from the document; Opinion of the scientific committee on cosmetic products and non-food products intended for consumers concerning ketoconazole , adopted by the SCCNFP during the 8 th plenary meeting of 23 June 1999. This document is attached, to the agenda. Adams ML, Meyer ER and Cicero TJ. 1998. Imidazoles suppress rat testosterone secretion and testicular interstitial fluid formation in vivo. Biology of reproduction, 59; 248-254. Cook JC, Frame SR and Obourn JD. 1997. Leydig cell tumours. In: Comprehensive toxicology. Volume 10. Reproductive and endocrine toxicology. Boekelheide K, Chapin RE, Hoyer PB and Harris C (eds). Pergamon, Elsevier Science Ltd. UK. Cummings Amand Metcalf JL. 1996. Effect of ketoconazole on early pregnancy and the decidual cell response. Biology of Reproduction, 54 (suppl 1): 181. De Coster R, Caers I, Haelterman C and De Broye M. 1985. Effect of a single administration of ketoconazole on total and physiologically free plasma testosterone and 17 -oestradiol levels in healthy male volunteers. Eur. J. Clin. Pharmacol., 29: 489-493. De Felice R, Johnson DG and Galgiani JN. 1981. Gynecomastia with ketaconezole. Antimicrob. Agents Chemother., 19: 1073-1097. Delongeas JL, Rangara R, Leonard JF, Albaladejo V, Peric C and Guittin P. 1995. Effects of repeated administration of ketoconazol on genital organs and reproductive performance in the male rat. Teratology, 51 (3): 183 Delongeas JL, Justice C, Labbe V, Falda-Buscaiot F, Sarsat JP, Leonard JF, Albaladejo V and Guittin P. 1996. Effects of repeated administration of ketoconazol on genital organs and semen in the male Beagle dog. Teratology, 53 (2): 190 Feldman D. 1986. Ketoconazole and other imidazole derivates as inhibitors of steroidogenesis. Endocrinol. Rev., 7: 409-420. Gray LE Jr, Price M, Lambright C, Wolf C, Hotchkiss A, Parks L, Ostby J. 1999. Environmental antiandrogens: the malformation pattern varies with the mechanism of antiandrogenic action. Biol. Reprod., 60 (suppl 1): 201. Joshi SC, Jain GC and Lata M. 1994. Effects of ketoconazole (an imidazole antifugal agent) on the fertility and reproductive function of male mice. Acta Europaea fertilatis, Volume 25 (No. 1); 55-58. Kazy Z, Puh E and Czeizel E. 2005. Population-based-case-control study of oral ketocoanzole treatment for birth outcomes. Congenital Anomalies, 45; 5-8. Kelce WE. 1997. The Leydig cell as a target for toxicants. In: Comprehensive toxicology. Volume 10. Reproductive and endocrine toxicology. Boekelheide K, Chapin RE, Hoyer PB and Harris C (eds). Pergamon, Elsevier Science Ltd. UK. Oftebro H, Jensen J, Mowinckel P and Norli HR. 1994. Establishing a ketoconezole suppression test for verifying testosterone administration in the doping control of athletes. J. Clin. Endocrin. And Metabol., 78: 973-977. Pont A, Williams PL, Azhar RE, Reitz et al. 1982. Ketoconezole blocks testosterone synthesis. Arch.Intern. Med., 142: 2137-2140. Ref: 2/1. Experiment n N13375: Acute intravenous and oral toxicity of R41400 in mice, rats, guinea pigs and dogs. Ref: 3/2: Experiment n 3214: Acute dermal toxicity study in albino rabbits (limit test). Ref: 4/6: Experiment n 1109: Subacute dermal toxicity study in New Zealand white rabbits (dermal administration). Ref: 5/18: Experiment n 711: Oral toxicity in Wistar rat (repeated dosage, 13 weeks). Ref: 6/22: Experiment n 1683: 28-day dermal irritation study in New Zealand white rabbits. Ref: 7/25: Experiment n 1849: 28-day dermal irritation study in New Zealand white rabbits (repeated dosage for

28 days). Ref: 8/23: Experiment n 3699: Subchonic dermal toxicity study in albino rabbits. Ref: 9/19: Experiment n 837: Oral toxicity in Wistar rats (Repeated dosage, 6 month). Ref: 10/20: Experiment n 838: Chronic oral toxicity study in Wistar rats (Repeated dosage for 18 months). Ref: 11/21: Experiment n 726: Oral toxicity in Beagle dogs (Repeated dosage for 1 months). Ref: 12/21 bis: Addendum to the experiment n 726: Oral toxicity study in Beagle dogs supplemental doses of 60 and 80 mg/kg. Ref: 13/24: Experiment n 1703: Chronic dermal toxicity study in New Zealand white rabbits (Repeated dosage for 6 months). Ref: 14/3: Experiment no reference: A primary dermal irritation study of full strength and 15% MEDIC formula 1116-77, and full strength and 15% MEDIC placebo, formula 1116-80 in albino rats. Ref: 15/4: Experiment n 3212: Primary dermal irritation study in albino rabbits. Ref: 16/5: Experiment n 3307: Primary dermal irritation study in albino rabbits. Ref: 17/12: Experiment n 1103: Acute eye irritation study in New Zealand white rabbits. Ref: 18/13: Experiment n 825: Ocular irritation study of MEDIC, formula B(G14) in albino rabbits. Ref: 19/14: Experiment n 822: Ocular irritation study of a 15% v/v solution of MEDIC, formula B(G14) in albino rabbits. Ref: 20/15: Experiment no reference: ocular irritation study of full strength and 15% MEDIC, formula 1116-77 and full strength and 15% MEDIC placebo, formula 1118-80 in albino rabbits. Ref: 21/16: Experiment n 3213: Primary eye irritation study in albino rabbits R041400: Ketoconazole formulated as a 10 mg/g shampoo. Ref: 22/17: Experiment n 3308: Primary eye irritation study in albino rabbits R041400: Ketoconazole formulated as a 10 mg/g shampoo used as a 15% dilution. Ref: 23/7: Experiment n 1106: Sensitising potential study in Guinea Pig. Ref: 24/8: Experiment n 2841: Dermal sensitisation study according to the Magnusson Guinea Pig Maximisation test. Ref: 25/9: Experiment no reference: Sensitising potential study in Guinea pig. Ref: 26/10: Experiment no reference: Maximisation test (skin sensitisation potential) of 2% ketoconazole (R41400) shampoo. Ref: 27/11: Experiment n 90-122: Evaluation of the induction of contact dermal sensitisation by challenge and rechallenge of subjects to 2% ketoconazole anti-dandruff shampoo and its breakout products using the Repeated Insult Patch (RIPT). Ref: 28/0: Reproductive effects data: RETCS data base. Ref: 39/26: Experiment n 935: In vitro mutagenicity screening of ketoconazole by Ames Salmonella/Microsomal Activation Assay. Ref: 40/27: Experiment n LMP 027: Mutations affecting the hypoxanthine-guanine-phosphoribosyl transferase locus in V79 cells HGPRT-test. Ref: 41/28: Experiment n 82400: Transformation/liver-microsome test (in vitro test for transformation using properties in mammalian fibroblasts). Ref: 42/29: Experiment n 667: Ketoconazole and lymphocyte transformation. Ref: 43/30: Experiment n 910: Micronucleus test in mice.

Ref: 44/31: Experiment n 787: Dominant lethal test in male mice (single oral dose). Ref: 45/32: Experiment n 788: Dominant lethal test in female mice. Ref: 46/33: Experiment n 1047: Sex-linked recessive lethal test on D. Melanogaster. Ref: 53/0: PDR (1999) Physicans Desk Reference Nizoral (ketoconazole) Medical Economics Company Inc. Schurmayer T and Nieschlag E. 1984. Effect of ketoconezole and other imidazole fungizides on testosterone biosynthesis. Acta Endocrinol. (Copenh.), 105: 275-280. Sharpe RM. 1988. Endocrinology and paracrinology of the testis. In: Lamb JC, Foster PMD (eds), Physiology and toxicology of male reproduction. San Diego: Academic press, Inc.: 71-102. Sharpe RM. 1990. Intratesticular control of steroidogenesis. Clin. Endocrinol., 33: 787-807. Vawda AI and Davis AG. 1986. An investigation into the effects of ketoconazole on testicular function in Wistar rats. Acta Endocrinol. (Copenh.), 111: 246-251. Waller DP, Martin A, Vickery BH and Zaneveld LJD. 1990. The effect of ketoconazole on fertility in male rats. Contraception, 41 (No. 4); 411-417.