1706 N Ketakonazole

8/12/2019 1706 N Ketakonazole

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Commission of the CLASSIFICATION AND LABELLING OF DANGEROUS SUBSTANCES.European Communities Recommended form to be used for the proposed classification and

labellingDG XI 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

CAS No.65277-42-1

ID No.

1.1 EINECS Name

If not in EINECS IUPAC Name

Ketoconazole

1.2 Synonyms (state ISOname if available)

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

1.3 Molecular formula C26-H28-Cl2-N4-O2

1.4 Structural formula

1.5 Purity (wt/wt) No data.

1.6 Significant impuritiesor 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 inshampoo formulations; particularly in products claiming an antidandruff efficacy.

1


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Used in drugs by oral and topical routes as broad specrum antifugal agent, highlyeffective 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; R48/22Repr. Cat 2; R60

S-phrase(s):

Existing classification -

Existing label

Symbol(s)

-

2


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2. PHYSICO-CHEMICAL CHARACTERISTICS

2.1 Physical formWhite powder or off-white powder

2.2 Molecular weight 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 Not applicable.

2.6 Vapour pressure (Pa(oC)) Not applicable.

2.7 Relative density (D420)Not applicable

2.8 Vapour density (air = 1) Not applicable.

2.9 Conversion factor (11011 hPa at25oC)

2.10 Solubility conc. at sat. (g/l)

Practicallyinsoluble.One part is soluble54 parts.

One part is solublein 2 parts.

One part is solublein 9 parts.

Very slightlysoluble.

solvent

Water

Ethanol

Chloroform

Methanol

Ether

Temperature (oC)

2.11 Partition coefficient (log Pow) Experimental No data

Calculated No data.

2.12 Flammability No data.

Flash point (oC) open cup: closed cup:

Explosivity limits (% v/v) lower limit: upper limit:

Auto-flammability temp. (oC)

2.13 Explosivity No data.

Danger of explosion at result of: shock: friction: ignition:

Explosive properties at hightemperature

2.14 Oxidizing properties No data.

2.15 Other physico-chemical properties No data.

.

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3. OBSERVATIONS OF HUMANS

Where available, human data are considered to be of more relevance in determining the potentialeffects of chemical substances on the human population. (Annex V, Directive 67/548/EEC).

Ref.

3.1 Occupational exposure

3.2 General population

In an epidemiological study the effect of oral ketoconazole treatment on foetal development wasstudied. The use of ketoconazole in the second and third months of gestation was compared betweencases with congenital abnormalities and their matched controls in the large population based data setof the Hungarian Case-Control Surveillance of Congenital Abnormalities, 1980-1996. Birth weightsand gestational age were evaluated in control newborn infants born to mothers with or withoutketoconazole treatment. The case-group comprised 22 843 cases with congenital abnormalities, whilethe control group contained 38 151 newborn infants without any defects. Six infants (0.03%) and 12controls (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 hadmothers with a higher incidence of use of the drug. The mean gestation age was somewhat longerwhile birth weight was somewhat larger in controls with ketoconazole treated mothers. This studytherefore failed to demonstrate a higher rate of congenital abnormalities in infants with mothers whohad received oral ketoconazole treatment during pregnancy.

Ref.

Kazyet al.,2005

4. TOXICOLOGICAL DATA

4.1 ACUTE TOXICITY

4.1.1 Oral

Species LD50(mg/kg)

Observations and Remarks Ref.

Mice 30male30female

M: 786

F: 618

Ketoconazole was delivered at doses of 320, 640 and 1280 mg/kg bw. Theanimals 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.

Ref: 2/1

Rats40males40females

M: 287

F: 166

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.


Ref: 2/1

GuineaPigs40males40females

M: 178F: 226

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.


Ref: 2/1

Dogs15males12females

M: 937

F: 640

Ketoconazole was delivered at doses of 80, 160, 320, 640, and 1280mg/kg bw. The animals were observed during 7 consecutive days afteradministration.

Males: LD50 after 7 days was 937 mg/kg bw.


Ref: 2/1

4.1.2 InhalationNo data available.


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4.1.3 Dermal

Species LD50 (mg/kg) Observations and Remarks Ref.

Cunistarwhite rabbits(20)

Higher than2000 mg/kg bw

In this study 2000 mg/kg bw of ketoconazole was applied underocclusive patch during 24 hours (group 1, 5 males and 5 females).

Group 2, 5 males and 5 females was the control group and receivedthe vehicle. Observations were made daily over a period of 14 days.All animals survived and no general clinical observations were

reported.

Ref: 3/2

Conclusion:According to the results the LD50s 7 days were higher for mice and dogs than for rats and guinea-pigs. Theywere also higher for males than for females in rats and mice. Based on the LD50 values following oraladministration in male rats from 166-309 mg/kg bw, ketoconazole should be classified as acute toxic with the riskphrase 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 iswarranted.

Ketoconazole is proposed classified with T; R25, Toxic if swallowed.

4.1.4 Skin irritation

Species No. ofanimals

Exposuretime (h/day)

Conc. Dressing:occlusive

semi-occlusive

open

Observations and remarks Ref.

NewZealandwhiterabbits

12 (4groups)

24 hours A shampoowith 1%ketoconazoleand a 15%solution ofthe shampoowith 1%

ketoconazoleVehicle;100% and a15%solution.

Occlusive The test was performed accordingto GLP. 0.3 ml was applied.Erythema and eschar formationwas scored after 24 hours(removal of the occlusive system)and after 48 hours. The shampoowith 1% ketoconazole was

considered moderate irritant sincethe 15% solution was classified asmild irritant. The vehicle wasclassified moderate irritant in bothpreparations.

Ref:14/3

Cunistaralbinorabbits

3 4 hours Ketoconazole 1%shampoo

Occlusive tonon abradedskin

The test was performed accordingto OECD Guideline 404 andaccording to GLP. 0.5 ml wasapplied. Observations of the skinarea were made at 1 hour and 1, 2,3, 4, 7 and 14 days afterapplication. Erythema, eschar andoedema formation was scored

according to the Draize scale. Theprimary dermal irritation index was0.58.No dermal irritation occurredwhen a 15% dilution of the sameketoconazole 1% formulation wasapplied under the same conditions.

Ref:15/4 and16/5


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4.1.5 Eye irritation

Species No. ofanimalsanimals


Conc.(wt/wt) Observations and remarks Ref.

NewZealandmalewhiterabbits

12 The eyes wasscored at 1,24, 48 and 72hours aftertreatment an

further 7, 14and 21 daysaftertreatment

A cream with2%Ketoconazolewas used,single dose of

0.1 ml.

The test was an eye Draize test using the1964 Guideline Guide for grading eyeirritation by Hazardous substances. In thisstudy no changes were reported. It wasconcluded that the ketoconazole 2% cream

was not irritating to eye under theexperimental conditions used.

Ref:17/12


6 A shampoowith 2%Ketoconazolewas used.

The test was performed in compliance withGLP and according to the Federal HazardousSubstances Act Ocular Irritation StudyProtocol. According to the reactions observedin the cornea of all animals and in theconjunctiva of five of six animals,ketoconazole tested full strength wasconsidered irritating to the rabbit eye.

Ref:18/13


6 15% solutionof a shampoowith 2%Ketoconazole.

The same experimental conditions were usedas described above (ref. 18/13). In this studyno ocular irritations were reported in the eye ofthe 6 rabbits.

Ref:19/14


24(6/group)

A shampoowith 1%ketoconazoleand a 15%solution of theshampoo with1%ketoconazole.Vehicle 100%

and a 15%solution.

The same experimental conditions were usedas described above (ref. 18/13). Resultsobtained with the shampoo with 1%ketoconazole were positive and identicalbetween the test product and its vehicle. Bothresults were negative with the 15% dilutedpreparations (ketoconazole 1% shampoo andvehicle). No conclusion was given from thisstudy.

Ref:20/15

Rabbits 3/sex Ocularreactionswere scored 1hour and 1, 2,3, 4, 7, 10, 14and 21 daysafterinstillation

A shampoowith 1%Ketoconazolewas used,single dose of0.1 ml

The study was performed in compliance withGLP and OECD Guideline 405. In this studyall animals had a diffuse redness andchemosis of the conjunctiva within the 48hours following instillation of the product; 2 ofthe rabbits had a slight hyperaemic reaction ofthe iris and a slight opacity of the cornea. Allsigns reversed after 48 hours. It wasconcluded from the study that according to theresults ketoconazole 1% shampoo was notconsidered to be an ocular irritant.

Ref:21/16

Rabbits 3/sex Ocularreactionswere scored 1hour and 1, 2,3, 4, 7, 10, 14and 21 daysafterinstillation

A shampoowith 1%Ketoconazolewas diluted15% , singledose of 0.1 ml

The study was performed in compliance withGLP and OECD Guideline 405. Similarreactions were reported as described above(ref. 21/16) however the effects reversed in 2animals the 2ndday and the 7thday for the thirdanimals.

Ref:22/17

4.1.6 Irritation of respiratory tract

No data available.


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Conclusion:

Skin irritationIn 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 exposedgroup, and in the control group exposed only to the shampoo. Therefore, no clear conclusion can be drawn fromthis study. In a more recent GLP study performed in accordance with OECD Guidelines the primary irritation indexwas 0.58 in the group exposed to a shampoo with 1% ketoconazole, whereas no skin irritation was reported whena 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 todistinguish whether the skin irritation observed is due to the shampoo itself or to the 1% ketoconazole inthe shampoo.

Eye irritationIn 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 eyeirritation studies are equivocal. In one study ketaconazole 2% cream was not irritating to eye. In another testperformed in compliance with GLP ketoconazole 2% shampoo was considered irritating to eye, however, whenusing the same experimental conditions with a 15% diluted ketoconazole 2% shampoo, no eye irritation wasreported. 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 werereported in the first study following installation of ketoconazole 1% shampoo, however all signs reversed after 48hours 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 2ndday 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 todistinguish whether the eye irritation observed is due to the shampoo/cream or to the 1or 2%ketoconazole in the shampoo.


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4.1.7 Skin sensitization

Species Type of test No. ofanimals

(c,t)

Incidence of reactions observed Ref.

Guinea pigs ModifiedMaximisation test

20 of bothsexes

A ketoconazole 2% formulation was used. At theinduction phase, the test formulation was applied at0.5 ml per animal to clipped skin 3 times a week for 3weeks and once at the start of the 4 thweek. The

formulation was maintained each time for 48 hoursunder occlusive patch. After a rest period of 12 daysthe challenge (0.5 ml of test substance for 48 hours)was given under occlusion on a region never beentreated. Readings were made 1, 8, 24 and 48 hoursafter removal of the occlusive patch. Immediate slighterythema was reported at the challenge site on 3animals 1 hour and 8 hours after removal. Noreactions were reported later.

Ref:23/7

Guinea pigs OECD guideline406, MagnussonMaximisation test

20 t female20 c female

A ketoconazole 1% scalp solution was used.According to the results it was concluded thatketoconazole 1% scalp solution is not considered tohave a sensitisation potential.

Ref:24/8

Humans KligmanMaximisation Test

25 healthyadultsvolunteers

A ketoconazole 2% cream and vehicle was used. 0.3 gof the test material was applied under an occlusivedressing for five 48 hours periods. After a 10 day restperiod a challenge patch was applied in different areasfor 48 hours under occlusion. Readings were madeimmediately and 24 hours after removal of the patch.No skin sensitisation was reported.

Ref:25/9

Humans KligmanMaximisation Test

25 healthyadultsvolunteers

Ketoconazole 2%, 1% and 0.5% shampoos versusvehicle were used. 0.1 ml of the test material wasapplied 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 afterremoval of the patch. No skin sensitisation wasreported following application of ketoconazole 0.5%1% or 2% shampoos.

Ref:26/10

Humans 194 healthyadultsvolunteers

A 2% ketoconazole antidandruff shampoo was used.An induction phase of nine 24 hours applicationsunder occlusion of a 1% solution of the original testmaterial (corresponding to 0.02% ketoconazole) wasused (3 times a week for 3 weeks). This was followedby a 2-week rest period. Then a challenge with a 1%solution for 24 hours was used. Readings were made24, 48, 72 and 96 hours after removal of the patch.

Re-challenges were performed on subjects whoexhibited sensitisation to the test material at the firstchallenge, consisting of an additional 24-hour patchperiod on the same site. According to the results andin spite of the high reactions related to the surfactantsystem, the tested solution was considered as notproducing instance of contact sensitisation.

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.


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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

body weight,mg/kg diet

Duration of

treatment


Wistar rats 10rats/sex/group

0, 8, 32, 128mg/kg bw/day inthe diet

13 weeks At the highest dose death of 2/10 males wasreported as well as a significantly decreasein terminal body weight and total weight gainin both males and females. Gross pathologywas reported in 32 mg/kg bw/day femalesincluding discoloured liver, pale adrenalsand slightly larger ovaries. At 128 mg/kgbw/day a discoloured liver was reported inboth sexes and pseudopregnancy andfragile bones of the legs in females. In thefemale 32 mg/kg bw/day dose group a

marginal increase in the absolute andrelative liver and ovaries weight wasreported. In the high dose group an increasein the relative liver and adrenals weight wasreported in both sexes, in the absolute andrelative weight of the testis (males) and inthe relative weight of the spleen and kidneys(females). Histological changes werereported mainly in the high dose group, witha tendency to the same changes in themedium dose group. Changes were seen inthe liver, the kidneys, ovary, uterus andvagina, adrenals and bones. Effects on the

endocrine organs (adrenals and ovaries) aswell as changes in the uterus and vaginaand bones were seen as a result ofhormonal imbalance. There was a goodcorrelation between gross pathology andhistopathology. The NOAEL from this studywas considered 8 mg/kg bw/day.

Ref/ 5/18



6 months The study was performed in accordancewith OECD Guideline 452. One control maleand two high dose females died, however,this was not considered treatment related bythe authors. Total food consumption andfinal body weight was significantlydecreased in males at 70 mg/kg bw/day andin females in the mid- and high dose group.No dose-related gross pathology was seenexcept for broken legs in one medium and 6high dosed females. Organ weights weremainly normal except for an increase in therelative weight of the liver in high dosedmales and females and of the kidneys andbrain in the high dosed females. Histologicalchanges were reported mainly in ovary andadrenal in the mid- and high-dose females.The NOAEL from this study was considered4 mg/kg bw/day.

Ref: 9/19



18 months The study was performed in compliance withGLP. 26 animals died during the study and71 animals had to be sacrificed in a morbidstate during the 18 thmonth of the study.There was no difference in mortalitybetween male groups, however, a slight

Ref: 10/20


10/22

increase in mortality rate was reported inhigh dosed females. A significant decreasein body weight was reported in high dosedanimals. Gross pathology was reported inthe salivary glands and abdominal fat insome medium and several high dosedmales and females. Furthermore, in theadrenals in some high dosed males andmedium and high dosed females. Fragile

bones were reported in all dose groups,including controls. An absolute and relativeincrease in adrenal weights was reported inhigh dosed animals. Histopahtologicalchanges were reported in the parenchymaland lymphoid tissues in medium and highdosed animal, and in the adrenals in thehigh dosed group. The NOAEL from thisstudy was considered 4 mg/kg bw/day.

Beagle dogs3/dogs/sex/group

0, 2.5, 10, 40mg/kg bw/dayby gavage

12 month One control animal died during the study. Inthe high dosed group food consumption wasdecreased, and a significantly lower bodyweight gain was reported. Gross pathology

was reported in the high dosed groupincluding a brownish discoloration of severalorgans and tissues. As a result of the lowerbody weight gain in the high dosed group,the absolute weight of several organsdecreased, with an increase in absolute andrelative liver weight. The NOAEL from thisstudy was considered 10 mg/kg bw/day.

Ref: 11/21

Beagle dogs3/dogs/group

20 mg/kgbw/day for 2weeks, 40mg/kg bw/dayfor 2 additional

weeks and then60 mg/kgbw/day for 9weeks. Onemale and onefemale wascontinuouslytreated with 60mg/kg bw/daythe following 11weeks (week14-24)

24 weeks In the high dosed animals the following wasreported: a slight body weight loss related toreduced appetite, small-sized thymus andswollen liver, decrease in absolute andrelative thymus weight and increase in

absolute and relative liver weight. All effectswere reversible in the dogs not treated fromweek 14-24 (2/dogs/sex/group).

Ref: 12/21bis


11/22

4.2.2 Inhalation

No data available.

4.2.3 Dermal

Species Dosemg/kg/day


Duration oftreatment


New

Zealandwhiterabbits4rabbits/sex/group

Cream with

2%Ketoconazole wasused. Thetest articlewas appliedat 0.5 1and 2 g/kgbw. Thevehicle(crem) was2 g/kg bw

Once daily 30 days In this study the skin was clipped weekly,

left intact in 2 males and 2 females of eachgroup, and abraded on the others. In thisstudy no adverse effects were reported,however, no gross examination wasperformed at the end of the study.

Ref: 4/6

New

Zealandwhiterabbit4rabbits/sex/group

0, 2, 20 and

50 mg/kgbw

Ketoconazol

e 2%shampoowas applieddaily toabraded andintact clippedskin for 1hours andthenremoved

28 days The study was performed according to GLP.

No mortality was reported. No skin irritation,no histological changes, and no changes inbody weight, food consumption and organweights were reported. The NOAEL from thestudy was the highest dose tested 50 mg/kgbw/day.

Ref: 6/22

NewZealandwhiterabbit4rabbits/sex/group

0, 1, 10 and25 mg/kgbw

Ketoconazole 1%shampoowas applieddaily to nonabradedclipped skinfor 1 hours ofcontact,openexposure

28 days (7days/week)

The study was performed according to GLP.No mortality was reported. No skin irritation,no histological changes, and no changes inbody weight, food consumption and organweights were reported. The NOAEL from thestudy was the highest dose tested 25 mg/kgbw/day.

Ref: 7/25

NewZealandwhiterabbit

5rabbits/sex/group

0, 10, 20and 40mg/kg bw

Ketoconazole 2%shampoowas applied

daily to nonabradedclipped skinfor 1 hours ofcontact,openexposure.

28 days (5days/week)

The study was performed according to GLP.Erythemal reactions reversible within 24hours were reported in dosed animals aswell as in the vehicle control animals. No

mortality was reported. No skin irritation, nohistological changes, and no changes inbody weight, food consumption and organweights were reported. The NOAEL from thestudy was the highest dose tested 25 mg/kgbw/day.

Ref: 8/23

NewZealandwhiterabbit4males/gro

up, 5female/group

0, 2, 20 and50 mg/kgbw

Ketoconazole 2%shampoowas applieddaily toabraded and

intact skin for1 hours ofcontact,openexposure.

26 weeks The study was performed according to GLP.No dose-related mortality was reported indosed and control rabbits. No histologicalchanges, and no changes in body weight,food consumption and organ weights werereported. The NOAEL from the study was

the highest dose tested 50 mg/kg bw/day.

Ref: 13/24


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Conclusion:

Oral exposureIn Wistar rats exposed from 13 weeks to 18 month with doses ranging from 4 to 128 mg/kg bw/day grosspathology were reported in both male and female rats in the liver, adrenals, ovary and bones. A correlation in theeffects reported was observed between the studies. Histopathological changes were also reported in both maleand female rats in the liver, kidney, ovary, uterus, vagina, adrenals, bones and lymphoid tissue. Organ weighchanges were reported as well. These effects were reported at doses below 50 mg/kg bw/day and higher. Moresevere 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 inthe 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 exposureFollowing dermal exposure to New Zealand White rabbits at doses up to 50 mg/kg bw /day no significant changeswere reported.

Based on the available data no classification for dermal repeated exposure is warranted.


13/22

4.3 GENOTOXICITY

Test Species Conc. (mg/l) Metabolicactiv.


In vitro

Bacterialreversemutation assay(Ames test)

SalmonellaTyphimurium,strain: TA 98,TA 100, TA

1535, TA1537, TA1538.

0, 0.5, 2.5, 10.0and 50.0

g/plate

+/- No genotoxicity was reported up to

50.0 g/plate.

Ref:39/26

HGPRT locustest(hypoxanthine-guaninephosphoribosyltransferase-test)

V79 cell lineof theChineseHamster

0, 20, 40 and 60

g/ml

+/- Ketoconazole did not induce

mutations in V79 cells up to 60 g/ml.

Higher concentrations could not beapplied due to the low solubility of thecompound.

Ref:40/27

Transformationtest

Balb/3T3micefibroblasts invitro

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.

+/- Ketoconazole or its metabolites didnot induce transformation in Balb/3T3mice fibroblasts at the concentrationstested.

Ref:41/28

Transformationtest.Transformationresponse tonitrogenstimulation.

Lymphocytesfrom humanhealthy youngadult donors

0, 1.0, 2.5, 5.0 or

10.0 g/ml

Ketoconazole did not cause significantdepression in lymphocytetransformation responses in either un-stimulated or nitrogen stimulated(PHA or Con A) cell cultures. At theconcentrations used ketoconazolewas considered non mutagenic.

Ref:42/29

In vivo

In vivomicronucleustest

Mice 0, 20, 40 and 80mg/kg bw,intraperitoneallyinjection

At the concentrations tested,ketoconazole was considered nonmutagenic.

Ref:43/30

Dominant lethal Male mice 0, 20, 80 or 320mg/kg bwsingleoraladministration

Male mice were exposed toketoconazole before mating to un-dosed female mice. Mutations wereevaluated by counting of embryonicdeaths and of reduction in number ofimplants in females necropsied 15days after mated to the males. Noinduction of dominant lethals wasreported.

Ref:44/31

Dominant lethal Female mice 0, 20, 80 or 320mg/kg bw singleoraladministration

Female mice were exposed toketoconazole before mating to un-dosed male mice. Mutations wereevaluated by counting of embryonicdeaths and of reduction in number ofimplants in females necropsied 15days after mated to the males. Noinduction of dominant lethals wasreported.

Ref:45/32

Sex linkedrecessive lethaltest

Drosophilamelanogaster(males)

200 and 2000ppm for 3 days

After 3 days exposure Berlin K males,were mated to 3 sequential groups ofvirgin Muller 5- females to rise F1

progeny. F1 females were then matedwith their brothers and their progenywere examined for recessive lethalmutations. No induction of recessivelethal mutations was reported.

Ref:46/33

Conclusion:


14/22

Based on the negative in vitrodata and negative in vivodata ketoconazole is considered not mutagenic,and no classification for mutagenicity is proposed.

4.4 FERTILITY

Species Route Dose Exposuretime (h/day)

Number ofgenerations

exposed


Sprague-Dawley rats

Oral 0, 25 and75mg/kgbw

8 weeks (30male rats) or13 weeks(39 male)rats. Eachgroup wasthen dividedinto sub-groups10/groupand matedwithuntreatedfemales

1 generation The males were paired for amaximum of 3 weeks (treatmentcontinued during pairing) withuntreated females (69 females).Caesarean section was performedon day 20. No clinical signs werereported. At 75 mg/kg /dayreduced spermatozoa motility (19to 26 %) was reported which mayhave been related to reducedplasma testosterone levels (41 to68 %) or/and to a direct effect ofketoconazole on spermatozoa.Furthermore, an increase in thenumber of abnormal spermatozoa(74 to 81 %) mainly due to anincreased incidence of detachedsperm heads (74 to 223 %) wasreported as well. After 13 weeksof treatment decreased spermcount was noted at 75 mg/kg /day,which may have been related tochanges in male fertility asindicted by lower pregnancy rate(30 to 34 %). Histological changeswere noted at both doses in testesand adrenals such as increasedincidence of focal tubular atrophyand hyperplasia of Leydig cells,and vacuolization of zonafasciculasta. The severity of thesehistological changes was directlyrelated to the dose level andtreatment duration. No adverseeffects were noted in theuntreated females mated withtreated males. No informationregarding effects in the liveoffspring was included.

Delongeas et al.,1995

Female Rats Oral 0, 10, 30and 100mg/kg/day

Gestationday 1-8

In this study the effect ofKetoconazole on early pregnancywas studied in rats. In the 100mg/kg/day dose group a 50 %pre-implantation loss and a 100 %rate of resorptions were reportedon gestation day 9. Ovarianweight was double of control (142

vs 76 3 mg). Ovaries from

treated animals were white asopposed to pink in control

animals. The white colour of theovaries suggests an accumulationof lipid. Serum progesterone wassignificantly decreased at 100mg/kg/day relative to controls (6.2

0.6 vs. 66.0 5.2 ng/ml) while

serum LH was significantly higher

Cummings andMetcalf,1996


15/22

in treated dams (12.5 1.4 vs 3.5

0.4 ng/ml), estradiol remained

unchanged. It was concluded bythe authors that the failure ofpregnancies was due to thedrastic decline in progesteroneand that the rise in LH is a feedback effect from the CNS inresponse to the lower

progesterone levels. Noinformation regarding maternaltoxicity was included.

AdultSprague-Dawley rats

Oral bygavage

Increasingdoses from10 to 300mg/kgbw/day

One singledose.Sampleswerecollected 4hours afterdosing

In this study the effect ofketoconazole on testosteronesecretion, testicular interstitial fluid(TIF) formation and TIFtestosterone levels were studied.A dose dependent decrease inserum testosterone, TIFtestosterone and TIF volume wasreported. In other studiesKetoconazole have also been

shown to inhibit testosteronesecretion (Pont et al., 1982;Schurmeyer and Nieschlag, 1984;De Coster et al., 1985 and Oftebroet al., 1994). TIF formation, liketestosterone secretion, is a majorregulatory aspect of testicularfunction. TIF testosterone levelsreflect testosterone secretioninside the testis, where it exertsparacrine and autocrine effects onsteroidogenesis andspermatogenesis (Sharpe, 1988;

Sharpe, 1990). The resultsindicate that Ketoconazolesuppress the two major regulatoryaspects of testicular function,testosterone secretion and TIFformation, supporting thehypothesis that Ketoconazole cansuppress male reproductivefunction and fertility.

Adams etal., 1998

Male Sprague-Dawley rats(18-19/group)

Oral bygavage

0, 200and 400mg/kg

3 days.

The resultsrepresent

threeseparateexperiments.There werenosignificantdifferencesbetween thedata for the3experiments.All datawere

combined torepresentone set ofdata.

Each male was paired with anuntreated female immediatelyafter the administration of the thirddose. All paired females wereeuthanized 10 days after pairing.Seven animals in the high dosegroup and 2 animals in the lowdose group died during dosing.The control group and the lowdose group appeared normal inbehavior and appearance afterdosing, whereas the high dosegroup was lethargic and generallyhad reduced motor activity whencompared to the other groups.93%, 44% and 0% of the females

in the control, 200 and 400 mg/kgdose group, respectively becamepregnant. There were nosignificant differences inimplantation sites, normal fetusesand number of corpora luteabetween the control and low dose

Waller etal., 1990


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group, however, there tended tobe reduced in the low dose group.In the males no changes werereported in testicular andepididymal weight and spermcount. However, a dose-relateddecrease in motility (74.9, 54.4and 39.2 in the control, 200 and400 mg/kg dose group,

respectively) and forwardprogression of epididymalspermatozoa (70.3, 37.5 and 21.3in the control, 200 and 400 mg/kgdose group, respectively) wasreported between the threegroups.

Male mice(15/group)

Oral 0, 400mg/kg/bw

60 days No significant changes in bodyweight were reported (252vs.282in controls). The weight ofthe testes (1656vs. 1907 incontrols), epididymis (601vs.751in controls) , seminal vesicle

(1008 vs. 1457 in controls), andventral prostate (80.4vs. 100.5in controls) were significantlyreduced. Furthermore, asignificant declined in spermmotility in cuda epididymis (464vs. 653in controls) as well as adecrease in sperm density intestis (10.1vs. 1.20.1incontrols) and cuda epididymis(291vs. 381in controls) wasalso reported. A significantreduction in fertility was reported

in ketoconazole treated mice(50% vs 90% in controls).

Joshi etal., 1994

Male Beagledogs (5treated and 4control)

Oral 0, and 25mg/kg/day

4 weeks (Inthe exposedgroup, andthe controlgroup 2dogs werekept for a 4-weekrecoveryperiod

A reduction in food intakeresulting in a transient loss ofbody weight was reported duringthe first two weeks. No bloodchemistry alterations werereported. Exposure wasassociated with a decrease in thesperm motility beginning duringthe 1stweek of treatment, anincrease in the total mean numberof abnormal spermatozoa

beginning during the 2ndweek oftreatment, and a slight decreasein total sperm count at the end ofthe dosing period. These changeswere considered to be related to aslight increase in plasmaprogesterone levels. The plasmalevels of testosterone were slightlylower in the treated dogs butremained within the range ofcontrol values. No organ weightchanges were noted. Thehistopathological changesreported were marked increase inthe incidence of minimal focaltubular degeneration and/oratrophy of the testes whencompared to controls. None ofthese changes were seen after

Delongeas et al.,1996


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the recovery period.

Endocrineeffects

Oral 0, 30 and100 mg/kgbw/day

Day 1 to 8 ofpregnancy

In a variety of mammalian testsystems ketoconazole was shownto completely inhibit the keysteroidogenic cytochroms P 450

17-hydroxylase/17, 20-lyase, P

450 11-hydroxylase, and P 450

aromatase in a dose-dependentmanner. As a result, adrenal and

gonadal androgen secretion wasreduced, to a lesser degree theproduction of oestrogens andcorticosteroids. Oral ketoconazoleadministration was thereforeshown to induce endocrineimbalance.

Ref: 53/0

Conclusion:

A statistically significant effect on sperm quality as well as histopathological changes in the testis was reported inrats following exposure to 75 mg/kg/day for 8 or 13 weeks. The effect on testis was accompanied with a lowerpregnancy rate in untreated females. In mice statistically significant effects were reported on the weight of thetestes, epididymis, ventral prostate and seminal vesicle, as well as on sperm density and motility and fertilityfollowing exposure to 400 mg/kg/day for 60 days. Effects on sperm quality as well as histopahtological changes intestis 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 ratsexposed to 100 mg/kg/day the first 8 days of gestation (see the developmental section for further details). Inseveral studies Ketoconazole was shown to inhibit testosterone synthesis, and in one study testicular interstitialfluid (TIF) volume which indicates that Ketoconazole may suppress two major regulatory aspects of testicularfunction, 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; Comprehensivetoxicology, Volume 10, Reproductive and endocrine toxicology (Kelce, 1997; Cook et al., 1997), and the followingis written:Ketoconazole is an example of a chemical that directly alters Leydig cell function without affecting Leydig cellviability. Ketoconazole was fist suspected to inhibit Leydig cell steroidogenesis following the development ofgynecomastia in humans (Pont et al., 1982; De Felice et al., 1981). Clinical studies revealed that Ketoconazoleadministration reduced serum testosterone levels (Pont et al., 1982) in the absence of alterations in serumgonadotropins, suggesting a direct effect on Leydig cells steroidogenesis (Schurmeyer and Nieschlag, 1984).Similar in vivoeffects 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.


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4.5 DEVELOPMENTAL TOXICOLOGY

Species Route Dosemg/kg/day

ppmConc(mg/l)

Exposuretime

(h/day)

Exposure period: -number of gene-rations or- numberof days duringpregnancy


Sprague-Dawleyrats

Oral 0, 25 and75mg/kg/day

8 weeks(30 malerats) or 13weeks (39male) rats.Eachgroup wasthendividedinto sub-groups10/groupand mated

withuntreatedfemales

The treated males were paired fora maximum of 3 weeks (treatmentcontinued during pairing) withuntreated females (69 females).Caesarean section was performedon day 20, the uterine content wasexamined and the fetusesobserved for external alterations.At 75 mg/kg /day reduced spermquality and quantity was reported,which may have been related tochanges in male fertility as indictedby a lower pregnancy rate (30 to

34 %). No adverse effects werenoted in the untreated femalesmated with treated males. Noinformation regarding effects in thelive offspring was included.

Delongeas etal.,1995

Femalerats

oral 0, 30 and100mg/kg /day

Frompregnancyday 1 to 8

In the 100 mg/kg/day dose group a50 % preimplantation loss and a100 % rate of resorptions werereported on day 9. No informationregarding maternal toxicity wasincluded. For further details seethe fertility section.

Cummings andMetcalf,1996

Noinformation

Noinformation

From 12 to100mg/kg/day

Noinformation Ketoconazole was studied togetherwith other suspectedantiandrogens for a possible effecton sexual differentiation.Anogenital distance, nipple/areoladevelopment and reproductiveorgan weight were measured inmale offspring. However,Ketoconazole failed to induce anyof these endpoints in the maleoffspring (only abstract available).

Gray etal.,1999

Rat Oral inthe

diet

Noinformation

1 Ketoconazole has been shown tobe teratogenic [syndactylia

(congenital webbing of digits) andoligodactylia (the presence offewer than five digits on a hand orfoot)] in rat when given in the dietat 80 mg/kg/day. However theseeffects may be related to maternaltoxicity, evidence of which hasbeen reported at this and higherdose levels. Ketoconazole wasalso found to be embrytoxic to therat when given in the diet at doseshigher than 80 mg/kg during thefirst trimester of gestation. In

addition dystocia (difficult labor)was noted in rats administratedoral Ketoconazole during the thirdtrimester of gestation. Thisoccurred when Ketoconazole wasadministered at doses higher than10 mg/kg bw.

Ref:28/0


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Rat Oral inthediet

Noinformation

Noinformation

First trimester ofgestation

The malformation and theembryotoxicity observed may bedue to maternal toxicity, however,no more information from thesestudies was available in theTOXNET HSDB database.

Ref:28/0

Conclusion:

Ketoconazole induced a 50 % pre-implantation loss as well as 100 % resorption in pregnant rats exposed to 100mg/kg/day the first 8 days of gestation. Alterations in ovarian weight and hormone levels were also reported in the100 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 wererelated to maternal toxicity or not. In the study by Gray et al., 1999 only effects on sexual differentiation wasstudied, related to a possible antiandrogen effect of ketoconazole, however, no changes in sexual differentiationwere reported. In an epidemiological study they failed to demonstrate a higher rated of congenital abnormalities ininfants 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 tomaternal toxicity, and limited information is available on the magnitude of the maternal toxicity, a final conclusionon classification for developmental toxicity is difficult.

Due to the limited information related to maternal toxicity and the magnitude of theteratogenicity/embryotoxicity reported in rats no clear conclusion can be drawn regarding a classificationfor developmental toxicity following exposure to ketoconazole.


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References:

The references given numbers from 2/1 up to 53/0 is from the document; Opinion of the scientificcommittee on cosmetic products and non-food products intended for consumers concerningketoconazole, adopted by the SCCNFP during the 8 thplenary meeting of 23 June 1999. This document isattached, to the agenda.

Adams ML, Meyer ER and Cicero TJ. 1998. Imidazoles suppress rat testosterone secretion and testicularinterstitial 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. AgentsChemother., 19: 1073-1097.

Delongeas JL, Rangara R, Leonard JF, Albaladejo V, Peric C and Guittin P. 1995. Effects of repeatedadministration 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: themalformation 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 andreproductive 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 birthoutcomes. Congenital Anomalies, 45; 5-8.

Kelce WE. 1997. The Leydig cell as a target for toxicants. In: Comprehensive toxicology. Volume 10. Reproductiveand endocrine toxicology. Boekelheide K, Chapin RE, Hoyer PB and Harris C (eds). Pergamon, Elsevier ScienceLtd. UK.

Oftebro H, Jensen J, Mowinckel P and Norli HR. 1994. Establishing a ketoconezole suppression test for verifyingtestosterone 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 anddogs.

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


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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 60and 80 mg/kg.

Ref: 13/24: Experiment n 1703: Chronic dermal toxicity study in New Zealand white rabbits (Repeated dosage for6 months).

Ref: 14/3: Experiment no reference: A primary dermal irritation study of full strength and 15% MEDIC formula1116-77, 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 albinorabbits.

Ref: 20/15: Experiment no reference: ocular irritation study of full strength and 15% MEDIC, formula 1116-77 andfull 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 formulatedas a 10 mg/g shampoo.

Ref: 22/17: Experiment n 3308: Primary eye irritation study in albino rabbits R041400: Ketoconazole formulatedas 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 Maximisationtest.

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 re-challenge of subjects to 2% ketoconazole anti-dandruff shampoo and its breakout products using the RepeatedInsult 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/MicrosomalActivation Assay.

Ref: 40/27: Experiment n LMP 027: Mutations affecting the hypoxanthine-guanine-phosphoribosyl transferaselocus in V79 cells HGPRT-test.

Ref: 41/28: Experiment n 82400: Transformation/liver-microsome test (in vitro test for transformation usingproperties in mammalian fibroblasts).

Ref: 42/29: Experiment n 667: Ketoconazole and lymphocyte transformation.

Ref: 43/30: Experiment n 910: Micronucleus test in mice.


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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 andtoxicology 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 Wistarrats. 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.

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1706 N Ketakonazole