(2RS,3RS)-3-(2-chlorophenyl)-2-(4-fluorophenyl)-[(1H-1,2,4-triazol-1-yl)methyl]oxirane

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In the two-generation reproduction study in rats, the dietary administration of epoxiconazole to rats in doses of 250 ppm (approximately 23 mg/kg bw/d) caused clear signs of systemic toxicity in the F0 females, the F1 parental animals and their respective progeny. At this dose level, precoital intervals were increased for some mating pairs. However, all parental animals proved to be fertile. Although cyclicity of females has not been examined in the two-generation study, data from a supplementary studies indicate that epoxiconazole prolonged or abolished oestrus cycles in normally cycling rats at dose levels of 1500 ppm and above by decreasing the levels of relevant steroid hormones (for an overview of MoA studies related to reproductive toxicity and developmental effests, please refer to the report attached to IUCLID chapter 13.2). Therefore, this effect observed in the two-generation study at 250 ppm is assumed to indicate interference with hormonal processes.

Doses of 10 and 25 ppm (approximately 0.9 and 2.3 mg/kg/d, respectively) were tolerated by the parental animals of both generations and by all litters without any adverse effect.

In the pre-postnatal toxicity study in guinea pigs, administration of epoxiconazole at the daily dose of 90 mg/kg bw caused maternal toxicity as evidenced by reduced body weight gain and food consumption. Serum levels of progesterone and 21-hydroxyprogesterone were increased at 50 and 90 mg/kg bw/d. Adrenal gland weights were increased, and vacuolization of zona fasciclulata of adrenal glands were seen at 50 and 90 mg/kg bw/d. Liver weights were increased at 90 mg/kg bw/d. Epoxiconazole did not adversely affect gestation length, parturition or postnatal growth and development. Importantly, administration of epoxiconazole did not alter circulating estradiol levels. Histopathological examination of the placentas did not reveal compound-related effects.

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 1990 - Jan 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 87/302/EEC of November 18, 1987 adapting to technical progress for ninth time Council Directive 67/548/EEC. pp. 47 — 50 (1988)

Version / remarks:

1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA/FIFRA Pesticide Assessment Guidelines, Subdivision F, NTIS, § 83—4, pp. 130 - 137, Nov. 1984.

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines for Toxicology Studies" pp. 45 - 48 (Japan/MAFF, 1985).

Version / remarks:

1985

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This strain was selected since extensive experience is available on Wistar rats and the rat is the preferred animal species for reproduction studies according to the different test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl Thomae, Biberach an der Riss, FRG
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 24 days (+/-) 1, 34 days at beginning of treatment
- Weight at study initiation: (P) Males: 129-155 g; Females: 110-133 g
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12 (light from 6 am to 6 pm)
IN-LIFE DATES: From: Feb, 12 1990 To: F0 Generation: Sep 25, Sep 26, and Sep 28 1990. Oct 16 and Nov 6, 1990 for animals that had to be reevaluated for fertility. F1 generation: Jan 3. Jan 4, Jan 8, Jan 9, 1991. Jan 11, Jan 14, Jan 21, Jan 22 and Jan 30, 1991 for animals that had to be reevaluated for fertility

Route of administration:

oral: feed

Vehicle:

other: diet

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): At intervals of not more than 32 days
- Mixing appropriate amounts with (Type of food): Kliba maintenance diet
- Storage temperature of food: not specified

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 3 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After unsuccessful pairing replacement of first male by another fertile male or female animal of the control group each.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each one of the doses were sent for analysis to the analytical laboratory at the beginning of the study and then at approximately three-monthly intervals. The content in the test substance/food mixes was determined by HPLC-method.

Duration of treatment / exposure:

48 weeks

Frequency of treatment:

continuous

Details on study schedule:

- F1 parental animals not mated until 98 days after selected from the F1 litters.
- Selection of parents from F1 generation: after weaning
- Age at mating of the mated animals in the study: 13 weeks

Dose / conc.:

10 ppm (nominal)

Remarks:

average of 0.9 mg/kg bw/day

Dose / conc.:

25 ppm (nominal)

Remarks:

average of 2.3 mg/kg bw/day

Dose / conc.:

250 ppm (nominal)

Remarks:

average of 23 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The doses were chosen on the basis of a previous reproduction toxicity study, which had to be discontinued before schedule for reasons which are explained below. In this study the test substance administered to groups of 25 male and 25 female Wistar rats as a
constant homogeneous addition to the food in doses of 0, 30, 300 and 1,500 ppm. At least 70 days after the beginning of treatment, the (F0) animals were mated to produce a litter (F1a). Groups of 25 males and females selected from the F1a pups of the 0, 30 and 300 ppm groups (no pups survived at 1,500 ppm) were raised for approximately 3 weeks; thereafter, due to the massive signs of systemic toxicity and adverse effects on the
reproductive function especially at the high dose level, the study was discontinued.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: no

Positive control:

not included

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Parameters checked: Signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Parameters checked: nesting. littering. and lactation behavior

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and additionally during gestation and lactation on days 0, 7, 14, and 20, on day of parturition and on days 4, 7, 14, and 21 after birth

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: weekly and additionally during gestation and lactation

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: F0 males and females: Sep 18, 1990 F1 males: Jan 3, 1991 females: Jan 4, 1991
- Animals fasted: No
- Anesthesia: no
- How many animals: 12 (F0 and F1) males and females each
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4/sex/litter as nearly as possible

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF STANDARDIZED PUPS, STILLBORN PUPS, AND DEAD PUPS:
yes, for external and internal abnormalities

DEVELOPMENTAL STAGES: Yes
- Parameters checked: pinna unfolding (PND 4), opening of the auditory canal (PND 13), opening of the eyes (PND 15)

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: yes; Gripping reflex, hearing test, pupillary reflex

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: 200
- Maternal animals: 200

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations
- Organ weights: Liver and adrenal glands

HISTOPATHOLOGY / ORGAN WEIGHTS
- Adrenal glands, coagulating glands, epididymides, liver, ovaries, pituitary gland, prostate gland. seminal vesicles, testes, uterus (including cervix), vagina, and all organs or tissues with macroscopic abnormalities.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at day 4 (culled pups) and day 21 (surplus pups)
- These animals were subjected to postmortem examinations (macroscopic)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined

Statistics:

Dunnett's test, Fisher's Exact test, ANOVA

Reproductive indices:

Male and female mating index, male and female fertility index, gestation index, live birth index

Offspring viability indices:

viability index, lactation index, sex ratio

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 250 ppm: 6 dams with vaginal hemorrhages during gestation for F1a litters: 2 of these dams were unable to deliver and died shortly after the expected delivery date; another dam died during gestation (F1b)

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

- 250 ppm: Three dams died during gestation. Two dams died without delivering their litters on days 23 and 25 p.c. The third dam died on day 7 p.c. of the gestation period for the F1b litter

All intercurrent deaths have to be related to the test substance administration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

All differences are assessed to be within the expected range of biological variation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 250ppm: Reduced food consumption of females during lactation period of F1a litters on days 4-7 and 7-14 post partum

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No effects observed in the nesting, littering, and lactation behavior of dams.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- 250 ppm: prolonged cohabitation time (concerning F1a and F1b) and prolonged duration of the gestation period (concerning F1a only). Lower male and female fertility indices (concerning F1b): however, finally fertility for all F0 sires and dams could be proven. Lower gestation index (73%) due to a high number of dams with only stillborn pups (concerning F1a litter). Clearly lower number of delivered pups/dam (concerning F1a litters)

Especially during the lactation period for the F1a litters, a remarkable number of dams of the 250 ppm group with only stillborn pups appeared, which is assessed as a substance—related effect. For three of these dams, vaginal hemorrhages were noted during the F1a gestation period. Four of the above mentioned dams with stillborn pups had a significantly prolonged F1a gestation period (24 or 25 gestation days).
Moreover, after parturition, umbilical cords were not cut in some F1a and F1b pups by dams of different groups without any relation to the dose.

The male fertility index varied between 96% and 88% (concerning F1a) and between 100% and 78% (concerning F1b) and was lowest for the high dose group males. Even if all males proved their fertiliftu finally, the increased number of 250 ppm males for which fertility could not be proven within the mating period for F1b litters, might be substance-related. The fertility index calculated for this group (78%) is just outside the historical control range (80 - 100%).

The female fertility index varied between 96% and 88% (concerning F1a) and between 100% and 78% (concerning F1b) and was lowest for the high dose group animals. Even if for all dams fertility was confirmed at least in one of the scheduled matings or in the reevaluation of fertility, the increased number of 250 ppm dams which did not become pregnant after mating for F1b litters took place, might be related to the test substance administration. The fertility index calculated for this group (78%) is outside the historical control range.
The mean duration of gestation for the F1a litter was statistically significantly prolonged in the high dose dams (22.8 days) which is mainly caused by dams Nos. 185 and 188 (died delivering on days 23/25 p.c.). This has to be attributed to the test substance administration.

As it was demonstrated that the test substance causes hormonal imbalance at high dose levels, it is reasonable to infer that some of the adverse effect observed in the adults (prolonged cohabitation time and duration of gestation) and possibly in their progency may have been caused by altered hormone concentrations.

Dose descriptor:

NOAEL

Effect level:

2.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

reproductive performance

Critical effects observed:

yes

Lowest effective dose / conc.:

23 mg/kg bw/day (actual dose received)

System:

female reproductive system

Organ:

uterus

Treatment related:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

23 mg/kg bw/day (actual dose received)

System:

endocrine system

Treatment related:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- 250 ppm: only a spontaneous skin lesion at the neck was observed in one male. One female showed vaginal hemorrhage on day 25 p.c. It was not able to deliver the pups, which were palpable around this gestation day in the abdomen. In addition, 3 dams with positive sperm detection did not deliver F2 pups
- 10 ppm: 3 dams with positive sperm detection did not deliver F2 pups

After parturition a few dams of all groups did not cut the umbilical cords; this is a spontaneous finding and not related to the test substance administration.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 250 ppm: clearly lower body weights in comparison to the controls and slightly impaired weight gains in the males (during the whole study period). In total, the weight gain of the 250 ppm males was significantly lower (11%) than that of the male control group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

-250 ppm: reduced food consumption of the males especially at the beginning of the premating period; the dams showed slightly to markedly diminished food intake during gestation and lactation periods of F2 litter

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 250 ppm: increased absolute and relative liver weights in the females without any histopathological correlate

The absolute and relative organ weights of the adrenal glands were decreased in males of groups 10, 25, and 250 ppm (p<.05, p<.05, p<.01, respectively). The absolute liver weight was decreased in males of
group 250 ppm (p<.01). The histologic examination did not reveal any morphologic correlate of these organ weight changes. The significance of the recorded organ weight changes is therefore considered to be of little if any toxicological relevance.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 250 ppm: decreased fatty change in the liver of the males

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

-250 ppm: prolonged cohabitation time and slightly prolonged duration of the gestation period; slightly reduced gestation index. One dam with vaginal hemorrhage during the gestation period; it did not deliver pups after prolonged gestation

For all F1 males (except No. 299 — 250 ppm) which were placed with females to generate F2 pups mating was confirmed, so that the male mating index was 100% in all groups except for test group 13 were it reached only 96%.
Three low dose males and 4 high dose males were not able to prove their fertility during the scheduled matings for F2 litters; however, for all these males fertility was confirmed during the reevaluation of fertility. Therefore, the observable differences concerning the male fertility indices between test groups 0, 10, 25 and 250 ppm are finally assessed as being of spontaneous nature and not related to the test substance administration.

The female mating index reached 100% for test groups 0, 10 and 25 ppm, in test group 250 ppm it was only 96%. The mean duration until sperm could be detected (day 0 p.c.) varied between 2.6 and 3.6 days;it was prolonged for the high dose females.
The female fertility indices varied between 100% and 88%. Because these values are inside the historical range and all females proved their fertility later on, the differences concerning the female fertility index are finally assessed as being of spontaneous nature. The mean duration of gestation was marginally longer in the 250 ppm group, i.e. 22.3 days in comparison to the actual control value (22.0), but was still within the historical control range. A clearer substance-related effect on the duration of gestation was recorded for the first parental generation (F0 females (F1a litter)).

Nearly all pregnant females gave birth to litters with liveborn pups; only one pregnant high dose dam, which had palpable pups in the abdomen, could not deliver. Consequently the gestation index was 100% in all groups. except test group 250 ppm where it was 95%. The number of stillborn pups was markedly increased in the high dose group while the mean number of delivered F2 pups was not influenced by the test substance administration. The percentage of liveborn F2 pups, however, was markedly reduced in test group 250 ppm, whereas there were no substantial differences in this respect between the control and the other substance—treated groups.
Consequently, the live birth index was reduced in the 250 ppm group and the number of stillborn F2 pups was clearly increased.

As it was demonstrated that the test substance causes hormonal imbalance at high dose levels, it is reasonable to infer that some of the adverse effect
observed in the adults (prolonged cohabitation time and duration of gestation) and possibly in their progency may have been caused by altered hormone concentrations.

Dose descriptor:

NOAEL

Effect level:

2.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

reproductive performance

Critical effects observed:

yes

Lowest effective dose / conc.:

23 mg/kg bw/day (actual dose received)

System:

endocrine system

Treatment related:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

23 mg/kg bw/day (actual dose received)

System:

female reproductive system

Organ:

uterus

Treatment related:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

-250 ppm: two F1a litters where pups showed poor general state during the first days after birth, which might be related to the test substance administration

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- 250 ppm: The mean number of delivered F1a pups/dam and the percentage of liveborn F1a and F1b pups were clearly reduced; markedly increased number of F1a and F1b pups which were stillborn; clearly lower percentage of liveborn pups (F1a and F1b). Reduced viability index (concerning F1b litter)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

-250 ppm: slightly reduced pup body weight gains (concerning F1a and F1b litters) during some intervals of the lactation period

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Due to the increased mortality of the high dose pups (F1a and F1b), the numbers of pups with post mortem autolysis (F1a only) and the numbers of partly
cannibalized pups (F1b only) were significantly increased.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- No effects observed regarding sex ratio of live F1a and F1b pups
- statistically significantly higher number of F1a pups of the substance-treated groups with pinna unfolding on time (spontaneous effect).For the F1b pup generation, statistically significantly less pups of the substance—treated groups showed pinna unfolding and/or eye opening on time (spontaneous effect)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

In the highest dose group clear adverse substance induced effects were noted for the progeny of the F0 and F1 parents. The number of liveborn pups was
markedly decreased and the number of stillborn pupsand/or pups which died during the rearing period was significantly increased in all 3 pup generations (F1a,
F1b and F2). Furthermore retarded growth was observed in all pup generations, while slight signs of retarded development were only seen in the F2 pups: however, no signs of substance—induced teratogenic effects were observed for the pups of all pup generations (F1a, F1b, F2), especially no increased incidence of cleft palate was seen.

Dose descriptor:

NOAEL

Generation:

F1a

Effect level:

2.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

gross pathology

Dose descriptor:

NOAEL

Generation:

F1b

Effect level:

2.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

gross pathology

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- 250 ppm: markedly increased number of F2 pups which were stillborn, died or were cannibalized during the rearing period; clearly lower percentage of liveborn pups; consequently reduced viability and lactation indices

The higher number of pups of the 10 ppm and the 25 ppm groups, however, that died during rearing is not assessed as a substance-related but spontaneous finding.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 250 ppm: distinctly lower pup body weight (days 7 - 21 post partum) and impaired pup body weight gains (the mean body weight of the 250 ppm pups was about 12% lower than that of the relevant controls)

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Only very few of the large number of examined pups of all groups showed some spontaneous findings like incisors sloped, wounds by biting, cataract, hernia
diaphragmatica, dilated renal pelvis, hydroureter, anophthalmia and hydrocephaly without a clear relation to dosing: most of these findings can be found at a comparable frequency in the historical control data

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- 250 ppm: higher number of F2 pups with negative test result in one developmental stage monitored (pinna unfolding)
- no effects on sex ratio

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

The statistically significantly higher number of 10 ppm and 25 ppm pups with pinna unfolding on time and the delays in auditory canal opening in all substance treated groups, however, are regarded as spontaneous effects, which are not related to the test substance administration; the relevant values are fully in the range of the control values calculated for the F1a and F1b pups.
In the highest dose group clear adverse substance induced effects were noted for the progeny of the F0 and F1 parents. The number of liveborn pups was
markedly decreased and the number of stillborn pupsand/or pups which died during the rearing period was significantly increased in all 3 pup generations (F1a,
F1b and F2). Furthermore retarded growth was observed in all pup generations, while slight signs of retarded development were only seen in the F2 pups: however, no signs of substance—induced teratogenic effects were observed for the pups of all pup generations (F1a, F1b, F2), especially no increased incidence of cleft palate was seen.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

2.3 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

other: higher number with negative test result in one developmental stage monitored (pinna unfolding)

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

23 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Executive summary:

The test substance was administered to groups of 25 maleand 25 female immature rats (F0 parental generation) as a constant homogeneous addition to the food in different dose levels (0, 10, 25 or 250 ppm). At least70 days after the beginning of treatment, F0 animalswere mated to produce a first litter (F1a) and subsequently remated to produce a second litter (F1b retained only until weaning). Groups of 25 males and 25females selected from F13 pups as F1 parental generation were offered diets containing 0, 10, 25 and250 ppm of the test substance post weaning, and thebreeding program was repeated to produce F2 litter. The study was terminated with the terminal sacrifice of F2 weanlings and F1 adult animals. Test diets containing the test substancewere offered continuously throughoutthe study.

At the 250 ppm level in F0 parental animals, the following findings were observed;reduced food consumption of the females during the

lactation period of F1a litters,6 dams with vaginal hemorrhages during gestation forF1a litters: 2 of these dams were unable to deliverand died shortly after the expected delivery date and another dam died during gestation (F1b), prolonged cohabitation time (concerning F1a and F1b) and prolonged duration of the gestation period (concerning F1a only),lower male and female fertility indices (concerning F1b): however, finally fertility for all F0 sires and dams could be proven,lower gestation index due to a high number of damswith only stillborn pups (concerning F13 litter),clearly lower number of delivered pups/dam (concerning F1a litters). At 250 ppm, F1a and F1b pups showed markedly increased number of F1a and F1b pups which were stillborn with a clearly lower percentage of livebornpups (F13 and F1b) and reduced viability index (concerningF1b litter),slightly impaired pup body weight gains (concerningF1a and F1b litters),two F1a litters where pups showed poor general stateduring the first days after birth. F1 parental animals at 250 ppm showedreduced food consumption of the males especially atthe beginning of the premating period; the damsshowed slightly to markedly diminished food intakeduring gestation and lactation periods of F2 litter,clearly lower body weights in comparison to thecontrols and slightly impaired weight gains in themales (during the whole study period),one dam with vaginal hemorrhage during the gestationperiod; it did not deliver pups after prolongedgestation,prolonged cohabitation time and slightly prolongedduration of the gestation period; slightly reducedgestation index,increased absolute and relative liver weights in the females without any histopathological correlate and decreased fatty change in the liver of the males. At 250 ppm in F2 pups,markedly increased number of F2 pups which werestillborn, died or were cannibalized during therearing period; clearly lower percentage of livebornpups; consequently reduced viability and lactationindices,distinctly lower pup body weight (days 7 – 21 post-partum)and impaired pup body weight gains andhigher number of F2 pups with negative test result inone developmental stage monitored (pinna unfolding).

It can be said in conclusion that the dietary administration of the test substance to rats in doses of 250 ppm(9 approx. 23 mg/kg body weight/day) caused clear signsof systemic toxicity in the F0 females and in the F1 parental animals and their progeny. At this dose level some of the reproductive parameters were impaired (e.g. lower male and female fertility indices in the F0parents (concerning F1b only). prolonged cohabitationtime and/or gestation period), however, fertility ofall parental animals of both generations could befinally confirmed within in the scheduled periods or inthe reevaluation of fertility. There were no indications for any substance—induced teratogenic effect inthe F1a, F1b or F2 pups.

As it was demonstrated that the test substance causeshormonal imbalance at high dose levels, it isreasonable to infer that some of the adverse effectobserved in the adults (prolonged cohabitation time and duration of gestation) and possibly in their progeny

may have been caused by altered hormone concentrations. 10 and 25 ppm (2 approx. 0.9 or 2.3 mg/kg body weight/

day) were tolerated by the parental animals of bothgenerations and by all litters (F1a, F1b, and F2) without any substance—related adverse effects.

Therefore, the NOAEL (no observable adverse effect level) concerning reproductive function and the NOAELconcerning systemic toxicity of the test substance is 25 ppm (approx. 2.3 mg/kg body weight/day) for bothparental generations (F0 and F1 males and females)andfor their progeny (F1a, F1b and F2 pups).

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2011 - Dec 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Jan 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

Aug 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 142

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The potential value of the guinea pig model for assessment of human reproduction toxicity hazards as an alternative to rodent models has long been recognized by the scientific and regulatory community. In the regulatory community this was specifically appreciated for azole compounds, as in 1999, the Scientific Committee on Plants (SCP) concluded in their interpretation of rat and guinea pig 2-generation reproduction toxicity studies conducted with the azole fenarimol that the aromatase inhibition by fenarimol and its specific effects as seen in small rodents is not relevant to the human species and that the guinea pig model seems to be the model of choice for defining the level of risk for the human for the effect of aromatase inhibition. (SCP/FENARI/005 - FINAL; Opinion adopted by the Scientific Committee on Plants on 18 May 1999).
The guinea pig is generally regarded as a most suitable model for elucidating the role of steroid hormones in pregnancy and parturition in humans (Sisk, 1976; cited in Batra et al.,
1980). Also, in more recent publications the applicability of the guinea pig for regulatory reproductive toxicology testing is acknowledged. The authors conclude that the guinea pig can be used successfully for developmental and reproductive toxicology ("DART") studies in cases where traditional animal models are not relevant for human safety assessment (Rocca and Wehner 2009), or preferred the guinea pig as test species because of features "which are more similar to the human than is the case for other rodent species." (Hewitt et al., 2011)
A number of publications from the last 30 years reviewed the characteristics of guinea pig reproductive biology in comparison to a variety of species including humans. There is general consensus that the guinea pig is a promising alternative to murid rodents which displays several key physiological characteristics of gestation that more closely resemble human pregnancy than do currently established standard animal models. Examples of these physiological characteristics comprise hormonal regulation (Batra et al., 1980; Hobkirk et al., 1993), placentation (Carter, 2007) and parturition (Mitchell et al., 2009).

Species:

guinea pig

Strain:

Dunkin-Hartley

Details on species / strain selection:

The Dunkin Hartley (Crl:HA strain) guinea pigs were chosen because of their general acceptance and suitability for toxicity testing and the reliability of the commercial supplier.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: not specified
- Weight at study initiation: (P) Females: 389.6-856.3 g (on GD 0)
- Housing: individually, except Pregnant animals and their litters were housed together until PND 21 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (light: 6 am to 6 pm)
IN-LIFE DATES: From: 08 Feb 2011 To: 18 May , 01 Jun , 17 Jun , 01. Jul , 15 Jul , 05 Aug 2011

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% CMC in highly deionized water

Details on exposure:

- Standard dose volume: 10 ml/kg

PREPARATION OF DOSING SOLUTIONS: aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability.

VEHICLE
- Name: carboxymethyl cellulose
- Concentration in vehicle: 1%

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: several hours or overnight
- Day 0 of pregnancy: The day of observed copulation is referred to as gestation day (GD) 0
- Proof of pregnancy: palpation of uterine contents during weeks 5 - 7 of pregnancy.
- Further matings after two unsuccessful attempts: no. Animals that failed this check were removed from the study (including hitherto recorded data) and were replaced by fresh animals until the number of approved pregnancies reached at least 25 in all groups.
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. Analytical method was HPLC.

Duration of treatment / exposure:

at least 84 days (From GD 6 to end of gestation (about GD 65) and through weaning (post-natal day 21) until one day before necropsy)

Frequency of treatment:

daily

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25-27

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: An independent dose range finding study was performed. Doses of 5, 15, 50, and 180 mg/kg bw/day were used. A dose of 180 mg/kg bw/d killed one third of the female guinea pigs in the respective dose group until mid pregnancy and caused distinct signs of intoxication in the surviving animals of this group. The only remarkable observations were alterations in steroid hormones starting at a dose of 5 mg/kg bw/d. Consequently, the high dose for the definitive study was chosen to be half of the lethal dose. The mid and low dose levels were chosen to describe a dose response of any potential effects.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: fasted

Positive control:

Not included

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, twice daily for moribundity
- The parturition and lactation behavior of the dams was generally evaluated in the mornings

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 6, 14, 21, 28, 35, 42, 49, 56, 63 and 65 as well as on PND 0, 7, 14 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: The food consumption was determined on GD 0 - 6, 6 -14, 14 - 21, 21 - 28, 28 - 35, 35 - 42, 42 - 46, 46 - 49, 49 - 53, 53 - 56, 56 - 60, 60 - 63 and 63 - 65.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day of necropsy
- Animals fasted: Yes
- Anesthesia: isoflurane
- How many animals: All surviving dams
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL)
- Hormones: Estradiol (E2). The following hormones were measured by an in house online SPE-LC-MS/MS (Solid phase extraction-LC-MS/MS) method. Absolute quantification was performed by means of stable isotope-labelled standards: 11-desoxycorticosterone (= 21-Hydroxyprogesterone), 11-desoxycortisol, 18-hydroxycorticosterone, 18-hydroxy-11-deoxcorticosterone, Aldosterone, Androstendione, Corticosterone, Cortisol, Dihydrotestosterone, Progesterone, Testosterone


HEMATOLOGY: Yes
- Time schedule for collection of blood: On day of necropsy
- Animals fasted: Yes
- Anesthesia: isoflurane
- How many animals: All surviving dams
- Parameters checked: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes

Oestrous cyclicity (parental animals):

Twice a day (early morning and late afternoon) the females were inspected for evidence of estrous by checking the vaginal membrane.

Sperm parameters (parental animals):

not applicable

Litter observations:

STANDARDISATION OF LITTERS : No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, weight gain, state of health, viability, sex ratio, stillbirths, live births

GROSS EXAMINATION OF DEAD PUPS:
yes, examined externally, eviscerated and organs were assessed macroscopically.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: No
- Maternal animals: All surviving animals

GROSS NECROPSY: Yes
- Gross necropsy consisted of external and internal examinations
- Organ weights: Adrenal glands, Anesthetized animals, Brain, Kidneys, Liver, Ovaries, Pituitary gland, Spleen, Thyroid glands (with parathyroid glands), Uterus

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
- Light microscopic examination was performed on the following organs:
Adrenal glands, All gross lesions, Cervix uteri, Ovaries, Oviducts, Pituitary gland, Uterus, Vagina

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.

Statistics:

Dunnett's test, Kruskal-Wallis, Wilcoxon-test

Reproductive indices:

Gestation index, live birth index, postimplantation loss,

Offspring viability indices:

Viability index, lactation index

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only relevant clinical observation in the 90 mg/kg bw/day group was a higher number of abortions and abortion-related mortality in comparison to the control (3 cases vs. 1).The abortions may be considered as a consequence of a latent maternal stress at the high dose level which is in line with the mild food consumption/body weight reductions and becomes morphologically evident with the vacuolation of the adrenal cortex. In addition, it was suggested that these three abortions may also possibly be caused by the general stress of daily gavage administration, and not test substance related at all.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

- 90 mg/kg bw/day: One animal died intercurrently (GD 52) after showing piloerection and vaginal discharge and one animal had to be sacrificed in a moribund state (GD 71) after showing hypothermia, piloerection and lateral position.
One control animal, one low-dose female (15 mg/kg bw/d) and one high-dose female were sacrificed prematurely (GD 46, 57, 27, respectively) because of abortion. The slightly higher number of abortion-related decedents in the highdose group may be an indication for treatment-related maternal stress at this dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: Decreased body weights at the end of gestation (approx. 6% below control), Decreased body weight gain during gestation (approx. 15% below control)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: Decreased food consumption during last week of gestation (12 -13% below control)

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In all test groups (groups 1, 2 and 3, 15, 50 and 90 mg/kg bw/d) the mean corpuscular hemoglobin concentration (MCHC) was higher compared to controls. The measured red blood cell parameters (i.e., red blood cell (RBC) counts, hematocrit and hemoglobin values) were not altered. Therefore, MCHC changes were regarded as incidental and not treatmentrelated.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: Increased progesterone and 21-hydroxyprogesterone values
- 50 mg/kg bw/day: Increased 21-hydroxyprogesterone values

The cause for these hormonal changes is not known. Taking into account the changes in the adrenals an effect (direct or indirect) on steroid synthesis is possible. A variable onset of the
sexual cycle after pregnancy and lactation in the females may also be an explanation for this observation on progesterone levels.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: Adrenal cortex: Cytoplasmic change: 14/27 animals (minimal in 3/27, slight in 11/27), Increased vacuolation: 27/27 animals (minimal in 3, moderate in 11, severe in 13)
- 50 mg/kg bw/day: Adrenal cortex: Cytoplasmic change: 10/27 animals (minmal in 8/27, slight in 2/27), Increased vacuolation: 27/27 animals (slight in 2, moderate in 7, severe in 18)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

- The mean duration of gestation was equal in all test groups (69 days).
- All presumed pregnant guinea pigs which were placed into the study delivered pups or had implants in utero.
- All gestational parameters and indices were comparable between control and test substancetreated groups.
- No adverse effects on gestation, parturition or lactation of the parental females up to and including a dose of 90 mg/kg bw/day.

Multifocal areas of cytoplasmic change were detected in the deep zona fasciculata increasing in incidence and severity from test group 2 (50 mg/kg bw/d) to test group 3 (90 mg/kg bw/d).
In these areas the cells of the zona fasciculata lost their large vacuoles and displayed slight basophilic and foamy appearance. Other cells contained small, needle-shape cytoplasmic crystals, resembling cholesterol crystals. In addition, activated sinusoidal cells and minimal mononuclear infiltrating cells were also observed. Compared with the control group 0, the vacuolation of the zona fasciculata in test group 2 and 3 (50 and 90 mg/kg bw/d) was increased. The vacuolation in test group 1 (15 mg/kg bw/d) was comparable to the control group. Altogether, these changes correlated with a significant increase of the relative weight in the adrenal glands in
test groups 2 (50 mg/kg bw/d) and 3 (90 mg/kg bw/d) and might reflect either an alteration in the steroid hormone production of the adrenals or potential consequences of maternal stress at mid- and high-dose level.

Only livers with macroscopic foci (1/25 in the low dose, 6/27 in the mid dose and 5/27 in the high dose) were examined by light microscopy. These findings correlated with acute focal necrosis (coagulation necrosis type accompanied by minimal or no inflammatory response and occasional, minimal dystrophic mineralization). Since no signs of hepatotoxicity were found in the clinical chemistry of these animals, the acute necrosis was attributed to the isoflurane anesthesia performed for the blood sampling before the animals were sacrificed for necropsy.

Ovarian cysts visualized macroscopically in controls animals correlated with cystic rete ovarii. This finding is reported to be commonly observed throughout the normal estrus cycle of the guinea pig. Unlike follicular or luteal cysts, rete ovarii cystic structures lack follicular or luteal cells and display single ciliated cells in their epithelium that ranges from columnar to flattened depending on the dilation grade. They do not secrete hormones but might be hormone-dependent.
One control animal (No. 12), one low-dose female (No. 221, 15 mg/kg bw/d) and one highdose female (No. 243) were sacrificed prematurely because they aborted (GD 46, 57, 27, respectively). One high-dose F0 female (No. 150, 90 mg/kg bw/d) died intercurrently (GD 52), most likely while it aborted as it became obvious by the retention of a dead fetus in its vagina, another high-dose animal (No. 138) was sacrificed in a moribund state (GD 71) because it was unable to deliver. All of these animals showed similar alteration in the uterus, cervix and vagina. Namely, animal No. 150 showed necrosis, hemorrhage and severe inflammatory changes affecting mainly the uterus. Animal No. 138 showed similar but less severe changes predominantly in the cervix. In both animals, large congestive mesometrial vessels invaded by trophoblast were observed in the uterus and in the oviducts. All other animals with abortion (No. 12, 221 and 243) showed similar characteristics, the uterus being more severely affected (hemorrhage, necrosis and presence of trophoblast cells) than the vagina (edema, congestion or infiltrates).

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

System:

endocrine system

Organ:

adrenal glands

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: The number of liveborn pups and subsequent numbers during lactation appeared to be lower. This apparent decrease is attributed to 2 decedents and one abortion in this dose group, which are considered to be a result of maternal toxicity.
- The number of delivered pups and the average litter size was comparable between control, mid and low-dose groups (0, 15 and 50 mg/kg bw/d).
- There were no test substance-related clinical findings in pups of any dose group. None of the following individual observations were considered to be associated to the test compound: an opacity (both eyes), respiratory sounds, unsteady gait, hypothermia, poor general state, gasping and lateral position

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The test substance did not influence pre-weaning pup survival in any of the treated groups

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few F1 pups showed spontaneous findings at gross necropsy, such as partly cannibalized, post mortem autolysis, empty stomach, distended stomach, extended intestine and a blind uterine horn. These findings were not considered to be associated to the test substance.

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

An apparent lower number of liveborn pups is attributed to 2 decedents and one abortion in this dose group, which are considered to be a result of maternal toxicity. This correlation to maternal toxicity is justifiable since neither the average number of implants nor postimplantation loss, gestation and live birth indices were significantly influenced by the high dose of the test material. Thus, this finding is not considered to be associated to the test substance.

All data recorded during gestation and lactation in terms of embryo-/fetal and pup development gave no indications for any developmental toxicity in the offspring up to a dose level of 90 mg/kg bw/day. Up to this dose level, the test substance did not adversely influence pup viability and pup body weights.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Table 1: Summary of weight parameter

	Female parental animals
Test group (mg/kg bw/d)	1 (15)	2 (50)	3 (90)
Adrenal glands	107%	113%**	108%*
Liver	100%	104%	110%**
Uterus	85%	90%	81%**

*: p <= 0.05;**: p <= 0.01

Table 2: Summary of histopathological results

	Female animals
Test group (mg/kg bw/d)	0 (0)	1 (15)	2 (50)	3 (90)
No. of animals examined	27	25	27	27
Cytoplasmic change, (multi)focal	0	0	10	14
·       Grade1			8	3
·       Grade2			2	11
Vacuolation, cytoplasmic, diffuse	27	25	27	27
·       Grade1	1	1	0	3
·       Grade2	6	6	2	0
·       Grade3	16	14	7	11
·       Grade4	4	4	18	13

Effects on developmental toxicity

Description of key information

In the two-generation study in rats, duration of pregnancy was prolonged, probably due to interference with parturition-inducing signals. This resulted in an increased number of pups either being born dead or dying in the early postnatal period. The dose of 250 ppm appears to be a threshold effect dose for teratogenicity as a small number of offspring with generalised oedema was noted in the F2 generation only. Doses of 10 and 25 ppm (approximately 0.9 and 2.3 mg/kg/d, respectively) were tolerated by the parental animals of both generations and by all litters without any adverse effect.

In a developmental toxicity study in rats, dams were treated from day 6 through day 15 post coitum (p.c.) with epoxiconazole doses of 0 (control treated with the vehicle double distilled water with 0.5 % carboxymethyl cellulose), 5; 15 and 45 mg/kg bw/d by gavage. Epoxiconazole caused maternal toxicity at a dose of 45 mg/kg bw/d (reduced food consumption, impaired body weight gain). Placental weights were increased at 15 and 45 mg/kg bw/d, however, this is not considered to be adverse at the 15 mg/kg bw/d dose level since foetal parameters remained in the normal range. Embryo-/foetotoxicity was observed at 45 mg/kg bw/d resulting in a slightly increased number of resorptions, marginally increased postimplantation loss and a markedly increased number of foetuses with skeletal variations (mainly supernumerary ribs).

 

The following NOAELs were achieved:

NOAEL maternal toxicity: 15 mg/kg bw/d

NOAEL embryo-/foetotoxicity: 15 mg/kg bw/d

In a developmental toxicity study in rabbits, epoxiconazole was administered to artificially inseminated Himalayan rabbits from day 7 through 19 post inseminationem (p.i.) in 0.5 % aqueous carboxymethyl cellulose (CMC) preparation. Doses selected were 0 (control), 5; 20 and 80 mg epoxiconazole/kg bw in a constant volume of 10 mL/kg bw.

The test substance caused maternal toxicity at doses of 20 and 80 mg/kg bw in form of reduced food consumption and body weight development. In isolated cases blood in the bedding (1 doe at the high and mid dose level, the latter showing also reduced defecation) and abortion (1 doe at the mid dose level) were noted. Uterus weights at the high dose level were slightly reduced in line with the only effect observed at the high dose level with respect to embryo-/foetotoxicity (markedly increased postimplantation loss/increased resorption rate with 3 does without viable foetuses). No substance-related malformations were observed.

The following NOAELs were derived from this study:

NOAEL maternal toxicity: 5 mg/kg bw

NOAEL embryo-/foetotoxicity: 20 mg/kg bw

Epoxiconazole was administered in a prenatal developmental toxicity study to pregnant guinea pigs at dose levels up to 90 mg/kg bw/d from implantation throughout gestation: GD6 to GD 63. The dose of 90 mg/kg bw/d caused maternal toxicity as shown by signs of anaemia, altered hormone levels and increased weights of the adrenal glands. Some hormone parameters were also altered at 50 and 15 mg/kg/d but were considered adaptive as not adverse effects were observed. No compound-related effects were observed regarding gestation length, numbers of implantations, post-implantation loss, and numbers of live or dead foetuses or sex ratio. Likewise, no external, visceral or skeletal anomalies attributable to the administration of epoxiconazole were observed by foetal pathology examinations.

In a dermal prenatal developmental study in rats, effects noted after administration of 1000 mg/kg/d epoxiconazole included increased placental weights and an increased number of foetuses with skeletal variations (rudimentary cervical and/or accessory 14th ribs). As these findings were also observed in the oral studies they are assessed to be treatment-related. In addition, one foetus with a cleft palate was noted at this dose level.

Thus, the maternal NOAEL is 1000 mg/kg/d and the developmental NOAEL is 400 mg/kg/d.

 

Several prenatal developmental toxicity studies with oral exposure in rats have been performed. Maternal toxicity was observed in the dose range between 20 and 180 mg/kg/d. At 15 mg/kg bw/d, no maternal toxicity was noted. At 20 mg/kg bw/d effects were slight and the signs of maternal toxicity consisted of reduced food consumption and lower corrected body weight gain. Effects became more severe with increasing doses until at 180 mg/kg bw/d maternal toxicity was excessive (see supporting range-finding study).

The dose of 15 mg/kg bw/d is considered a NOAEL for embryofoetal toxicity, equal to that established for maternal effects. At this dose there was no increase in postimplantation loss and the slightly higher number of foetuses with skeletal variants is not assessed as being an adverse effect. Embryofoetal toxicity was observed from a dose of 45 mg/kg bw/d. At this dose level, increased embryo- or foetolethality became apparent and surviving foetuses were found to have a higher rate of skeletal variations (additional cervical and thoraco-lumbar rib elements). At 180 mg/kg bw/d increased postimplantation loss was observed consistently in two studies with different Wistar rat strains. This effect was more pronounced when treatment was extended to cover the foetal as well as the postimplantation embryonic period and thus may have affected the ability to detect malformations. While in the range-finding study most foetuses survived exposure during the embryonal period and were found to have cleft palates at term, this finding was extremely rare in a second high-dose study which compared two different epoxiconazole batches (not reported in IUCLID). In this study, embryo/foetolethality reached about 50% and it must be assumed that conceptuses with oral clefts were preferentially eliminated by death and resorption during the foetal period.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar 31, 1989 - April 27, 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

Nov 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Testing Guidelines for Toxicology Studies, pp. 48-49 (Japan/MAFF)

Version / remarks:

1985

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl Thomae, Biberach an der Riss, FRG
- Age at study initiation: 11-12 weeks
- Weight at study initiation: ca. 242 g
- Fasting period before study: no
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12 (illumination: 6 am to 6 pm)

IN-LIFE DATES: From: March 30/April 5/April 6, 1989 To: April 20/April 26/April 27, 1989

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

doubly distilled water with 0.5% CMC

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Freshly prepared each day before treatment of animals
- Volume of administration: 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in doubly distilled water and its distribution were carried out before the beginning of the study. Furthermore, samples of each preparation of the test substance in . doubly distilled water with 0.5% carboxymethyl were sent to the analytical laboratories twice during the study period for verification of the concentrations.The test substance suspensions were analyzed by HPLC.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1-4
- Length of cohabitation: From about 16.00 hours to 7.30 hours the following morning
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

From Day 6 through Day 15 post coitum

Frequency of treatment:

once daily

Duration of test:

15 days (without acclimatization)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

45 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: An acute oral toxicity study, single intraperitoneal administration, preceding test study in which pregnant rats were dosed, range-finding study
- Rationale for animal assignment (if not random): random
- Rationale for strain selection: This strain was selected since extensive experience is available on Wistar rats and this strain has been proved to be sensitive to substances with a teratogenic potential.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for clinical symptoms, twice daily for mortality

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1. 3. 6. 8. 10, 13. 15, 17 and 20 p.c. Furthermore, the corrected body weight gain was calculated after terminal sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Uterus, ovaries

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead fetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

Dunnett's Test (1955, 1964) was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses.
Fisher's Exact Test (1981) was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings.
Significances resulting from these tests have been indicated in the tables (a for p < 0.05; b for p < 0.01).

Indices:

Conception rate, preimplantation loss, postimplantation loss

Historical control data:

available

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 45 mg/kg bw/day: reduced body weight gains during the first days of the treatment period (days 6 - 8 p.c.)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 45 mg/kg bw/day: reduced food consumption during the treatment period, especially on days 6 — 8 and 13 - 15 p.c.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

15 mg/kg bw and above: slight increase in placental weights; within historical controls; considered non-adverse

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- 5 mg/kg bw/day: one animal, which did not become pregnant, showed hydrometra
Lung edema and/or marginal emphysema (findings. which have to be related to the sacrifice of the animals) were noted for several dams of test groups 0, 5 and 15 mg/kg body weight/day without any dose-response relationship.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- The conception rate varied between 84% (control) and 96% (test group 5 mg/kg body weight)

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

- 45 mg/kg bw/day: marginally increased postimplantation loss (as a consequence of increased resorptions)
- no adverse effects on pre-implantation loss

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

- 45 mg/kg bw/day: slightly increased number of resorptions (especially late ones)

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- number of corpora lutea
- conception rate
- implantation sites

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

early or late resorptions

food consumption and compound intake

pre and post implantation loss

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- mg/kg bw/day: malformations (micrognathis, cleft palate, microtia) observed in just one fetus are assessed as being of spontaneous nature due to a missing dose-response relationship

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

- 45 mg/kg bw/day: Variations: markedly increased number of fetuses with skeletal variations (especially rudimentary cervical and/or accessory 14th rib(s))

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- mg/kg bw/day: Two malformations (dilation of one or both heart ventricles) in two fetuses

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- 45 mg/kg bw/day: statistically significantly increased placental weights
- 15 mg/kg bw/day: statistically significantly increased placental weights (within historical controls; considered non-adverse)

Details on embryotoxic / teratogenic effects:

Taking into account the summarized results of the external, soft tissue and skeletal examinations of the fetuses, the only findings, which are assessed as substance-induced ones, are the increased occurrence of high dose fetuses with rudimentary cervical and/or accessory 14th rib(s).
Due to the high frequency of both findings in the 45 mg/ kg fetuses, the incidence of skeletal and overall variations is statistically significantly increased in comparison to the control group.
A number of publications deals with the finding of supernumerary ribs in fetuses.
Various interpretations and assessments are given in these papers about the significance of increased occurrence of supernumerary ribs in rat fetuses for prenatal toxicity studies.
According to WICKRAMARATNE (1988) an increase in supernumerary ribs "should be considered not as indicative of fetal dysmorphogenesis but as a manifestation of a non—specific stress the dam is placed under in such (i.e. prenatal toxicity) studies.... An increase in frequency of supernumerary ribs could therefore be evidence that a minimal toxic dose had been achieved...."
KIMMEL and WILSON (1973) state that "when an increase in either rudimentary or extra ribs occurs during teratogenicity testing. it may be assumed that the maternal animal is being stressed sufficiently to express the developmental instability inherent in the species. The expectation would be if other teratogenic effects did not occur at that dose level. that at a higher dose, frank embryotoxicity would become evident“.
The latter statement is exactly in—line with the results of the preceding range-finding study in which at the highest dose level (180 mglkg body weight/day) and in the presence of overt maternal toxicity clear signs of embryotoxicity (including teratogenicity) were obvious.
Therefore, the increased number of 45 mglkg fetuses with rudimentary cervical and/or accessory 14th rib(s) in the present study is finally assessed as an embryotoxic effect representing a manifestation of a non-specific stress on the dams: it is, however. not interpreted as being a teratogenic effect of the test substance at this dose level.
All other differences between the actual control group and the substance-treated groups concerning fetal external. soft tissue and/or skeletal observations are without biological relevance and/or appear to about the same extent in the historical control data.

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increase in number of resorptions, postimplantation losses, skeletal variations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

Developmental effects observed:

yes

Lowest effective dose / conc.:

45 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 1989 - 04 July 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Chbb:HM (outbred)
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: 30 and 35 weeks old
- Weight at study initiation: approx. 2553 g (mean)
- Housing: singly in type K 300/8 stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, Germany
- Diet (ad libitum): pelleted Kliba maintenance diet type 23-341-4 for rabbits supplied by KLINGENTALMUEHLE AG, CH-4303 Kaiseraugst, Switzerland
- Water (ad libitum): drinking water of tap water quality
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 -24
- Humidity (%): 30 -70
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

(doubly distilled water with 0.5% CMC)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance suspensions were freshly prepared each day just before treatment of the animals began. For the preparation of the suspensions an appropriate amount of the test substance was weighed and subsequently suspended in doubly distilled water with 0.5% carboxymethyl cellulose using an ultra turrax (supplied by JANKE & KUNKEL KG, Germany). A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.

VEHICLE
- Concentration in vehicle: 0, 50, 200, and 800 mg/100 mL
- Amount of vehicle (if gavage): The volume administered each day was 10 mL/kg body weight. The calculation of the volume administered was based on the individual body weight determined at the beginning of the administration period (day 7 p.i.).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in doubly distilled water and its homogeneous distribution were carried out before the beginning of the study. Furthermore, samples of each preparation of the test substance in doubly distilled water with 0.5% carboxymethyl cellulose were sent to the analytical laboratories twice during the study period for verification of the concentrations. The test substance suspensions were analyzed by HPLC.
The results of the analyses of the suspensions of test substance confirmed the correctness of the prepared concentrations and the homogeneous distribution of the test substance in the carrier.

Details on mating procedure:

- Impregnation procedure: artificial insemination
After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination. This implied that 0.2 mL of a synthetic hormone which releases LH and FSH from the anterior pituitary lobe (Receptalt), trademark of HOECHST AG, Frankfurt) were injected intramuscularly to the female rabbits about 1 hour before insemination. The pooled ejaculate samples used for the artificial insemination were derived from male Himalayan rabbits of the same breed as the females. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.

- The day of insemination was designated as day 0 (beginning of the study) and the following day as day 1 post insemination (p.i.).

Duration of treatment / exposure:

From day 7 to day 19 p.i. (during the period of organogenesis)

Frequency of treatment:

once daily

Duration of test:

From day 7 to day 29 post insemination

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 animals/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a preceding range-finding, pregnant Himalayan rabbits (5/dose) were daily administered the test substance in 0.5% carboxymethyl cellulose suspension by gavage over a period of 13 days (day 7 -19 p.i.) in doses of 0, 50, 150 and 300 mg/kg body weigh/dayt. The following findings were obtained and assessed as substance-related:
150 and 300 mg/kg body weight/day were clearly and dose dependently toxic to dams. During the treatment period (days 7 -19 p.i.) the animals of these two groups consumed clearly less food and lost weight when compared to the control group. At necropsy, 2 dams of the highest dose (300 mg/kg body weight/day) showed cachexia, a degeneration of the liver and ulcerations or erosions of the gastric mucosa. On day 29 p.i., all dams of the 150 and 300 mg/kg groups were shown to be pregnant, but all implantations had already been dead at an early stage of pregnancy, i.e. the test substance administered at doses of 150 and 300 mg/kg body weight/day was 100% embryolethal; thus no live fetuses could be obtained and further examined.
The lowest dose employed, being 50 mg/kg body weight/ day, caused slightly reduced food consumption and body weight gain in the dams during the treatment period (days 7 -19 p.i.). There were no distinct differences from the control concerning the reproduction-specific parameters (number of corpora lutea, of overall implantations, of resorptions and live fetuses, as well as figures of pre-and postimplantation losses) and concerning the placental and fetal weights. External and organ examination of the fetuses revealed no findings that differed from the current or historical control groups in terms of type and number. No examination of the fetal skeletons was carried out in this range-finding study.
Thus, taking into account the results described above, the following doses were fixed for the full-scale prenatal toxicity study in rabbits with the test substance:
- 5 mg/kg body weight: as the no adverse effect level
- 20 mg/kg body weight: as the minimal toxic effect level for does and/or fetuses; however, this dose could also prove to be yet another no effect level
- 80 mg/kg body weight: due to the results of the rangefinding study, this dose was expected to cause signs of maternal toxicity (e.g. an influence on the body weight change); however, a sufficient number of live fetuses should be available with this dose for further examinations.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were examined for clinical symptoms at least once a day. A check on mortality was made twice a day on working days or once a day (Saturday, Sunday or on public holidays).

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 2, 4, 7, 9, 11, 14, 16, 19, 21, 23, 25 and 29 p.i. The body weight change of the animals was calculated from these results. Furthermore, after terminal sacrifice the corrected body weight gain was calculated (body weight on day 29 p.i. minus body weight on day 7 p.i. minus weight of the uterus before it was opened).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice:
On day 29 p.i., the dams were sacrificed by intravenous injection of a pentobarbital and the fetuses were dissected from the uterus.
Dams showing signs of abortion were also sacrificed by intravenous injection of a pentobarbital. These animals as well as the contents of uterus from these animals were investigated, if possible in the same way as at terminal sacrifice (exception: uterus weight).
After the dams had been sacrificed, they were necropsied and assessed by gross pathology.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
- Weight of uterus before it was opened (gravid uterus weight)
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
- live fetuses
- dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Naunyn-Schmiedeberg's Arch. exp. Path. 247, 367, 1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy),
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible),
c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened).

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes:

Details:
At necropsy each fetus was weighed and examined macroscopically for any external findings. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded.

After the fetuses had been sacrificed by CO2, the abdomen and thorax were opened in order to be able to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to assess the internal structure. The sex of the fetuses was determined by internal examination of the gonads. If heads of fetuses revealed severe findings (e.g. anophthalmia, microphthalmia, hydrocephalus, or cleft palate), the heads of these fetuses were severed from the trunk, fixed in BOUIN's solution and later processed and assessed according to WILSON's method (1965). About 10 transverse sections were prepared per head. After the examination the heads treated in this way were discarded.

After the soft tissue examination all fetuses were placed in ethyl alcohol for staining of the skeletons (with the possible exception of the skulls according to a modified method of DAWSON (Stain Technol. 1, 123, 1926). The stained skeletons were placed on an illuminated plate and examined, evaluated and assessed. After the examination the stained skeletons were retained by litter.

Statistics:

Dunnett's Test (1955, 1964) was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses.
Fisher's Exact Test (1981) was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings.
Significances resulting from these tests have been indicated in the tables (a for p < 0.05; b for p < 0.01).

Indices:

- Conception rate (%) = (number of pregnant animals / number of fertilized animals) * 100
- Preimplantation loss (%) = ((number of corpora lutea - number of implantations) / number of corpora lutea) * 100
- Postimplantation loss (%) = ((number of implantations - number of live fetuses) / number of implantations) * 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 80 mg/kg bw/day: One doe was found with blood in bedding on day 16 p.i.
- 20 mg/kg bw/day: One doe was found with blood in bedding (days 20 -21 p.i.) and reduced defecation (days 18 -26 p.i.) were noted.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- 20 mg/kg bw/day: One doe, which aborted on day 18 p.i., was sacrificed on that day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- 80 mg/kg bw/day: Body weight gain of the does was clearly reduced during the treatment period, especially on days 11 -14 p.i.
- 20 mg/kg bw/day: A slight impairment on body weight change of the does was observed during the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- 80 mg/kg bw/day: food consumption of the dams was slightly reduced during the treatment period.
- 20 mg/kg bw/day: food consumption of the does was slightly reduced during the treatment period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 80 mg/kg bw/day: The uterus weight was slightly reduced

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

- 20 mg/kg bw/day: One doe was found with abortion on day 18 p.i.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

- 80 mg/kg bw/day: postimplanation loss was markedly increased due to an increased resorption rate (3 does without any viable fetuses)

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

- 80 mg/kg bw/day: Resorption rate clearly increased, especially due to the fact, that 3 does of this group had no viable fetuses at all but only resorptions (mainly early ones).

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Himalayan rabbits during organogenesis elicited some signs of maternal toxicity (impairment of food consumption and body weight gain. blood in the bedding of one doe and reduced uterus weights) at a dose level of 80 mg/kg body weight/day and to a lesser degree at a dose of 20 mg/kg body weight/day.

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

early or late resorptions

food consumption and compound intake

number of abortions

organ weights and organ / body weight ratios

pre and post implantation loss

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The external examination of fetuses revealed several malformations on 2 fetuses each of test groups 0 and 5 mg/kg bw/day, but none in fetuses of the intermediate and high dose groups.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Only one kind of malformation of the vertebral column (lumbar vertebra absent) was seen in the control and 5 mg/kg bw/day group each. Variations were found in single fetuses of all groups including the controls without any dose-respsonse relationship and without any statistically significant differences between the groups.

Visceral malformations:

no effects observed

Description (incidence and severity):

No malformations in any of the test groups. All soft tissue variations recorded in this study are to be found in the same kind of magnitude in the historical control data.

Other effects:

no effects observed

Description (incidence and severity):

- no effects on weight of placentae

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
In the highest dose group, the administration of the substance caused increased embryolethality, substantiated by an increased resorption rate (mainly early resorptions) and by consequently a markedly increased postimplantation loss. However, no other signs of embryo-/fetotoxicity were noted in the 80 mg/kg group. There were no signs of embryo-/ fetotoxicity in the 5 and 20 mg/kg groups; no indications of any teratogenic effects were present up to and including the highest dose level.

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Food consumption:

Food consumption of the high dose dams (80 mg/kg body weight/day) was slightly reduced during the treatment period (days 7 -19 p.i.); however, statistically significant differences in comparison to the control does appear only on days 8 -9 p.i.. In addition, the food consumption of the does of test groups 1 and 2 (5 and 20 mg/kg body weight/day) during the treatment period (days 7 -19 p.i.) was also slightly impaired in comparison to the controls, but the differences between the groups were not clearly dose-related.

Taking into account the inevitable food spillage of the rabbits and taking into consideration the mean body weight gain of the does of the different groups during the treatment period, the slight reduction of food intake in the 80 and 20 mg/kg groups had to be attributed to the test substance administration, while the reduced food consumption of the low dose group (5 mg/kg body weight/day) was finally assessed as being of spontaneous nature and without any biological significance.

 

Mean maternal food consumption during gestation (grams/animal/day)

		Solvent Control	5 mg/kg bw/d	20 mg/kg bw/d	80 mg/kg bw/d
Days 0 to 7	Mean of means	113.0	112.0	106.1^a	116.0
	SD	4.58	4.69	4.91	4.93
	N	7	7	7	7
Days 7 to 19	Mean of means	90.6	76.4^b	79.0^a	79.1^a
	SD	9.08	13.49	11.58	9.83
	N	12	12	12	12
Days 19 to 29	Mean of means	105.7	101.9	94.6	105.2
	SD	15.82	18.06	17.43	11.23
	N	10	10	10	10
Days 0 to 29	Mean of means	101.2	93.8	90.9	97.0
	SD	14.40	20.41	16.70	18.32
	N	29	29	29	29

a = P < 0.05; b = P < 0.01

 

Body weight/body weight change:

There were no statistically significant differences between the mean body weights of the control and those of the substance-treated groups. The mean body weight change, however, was clearly reduced in the high dose group (80 mg/kg body weight/day) on days 11 -14 p.i. and it was also clearly impaired if the mean body weight gain over the whole treatment period (days 7 -19 p.i.) was calculated. A slight substance-related impairment of the mean body weight change of the intermediate dose animals (20 mg/kg body weight/day) during the treatment period could also be seen, while there were no adverse effects on the weight gains of the 5 mg/kg does.

 

Mean maternal body weight change during gestation (grams)

		Solvent Control	5 mg/kg bw/d	20 mg/kg bw/d	80 mg/kg bw/d
Days 0 to 7	Mean	39.3	36.6	32.1	44.2
	SD	25.91	45.54	39.28	74.80
	N	14	14	15	13
Days 7 to 19	Mean	34.9	35.6	23.1	2.1
	SD	48.09	60.83	54.96	111.22
	N	14	14	14	13
Days 19 to 29	Mean	163.1	185.4	126.8	115.4
	SD	75.39	55.72	86.43	60.07
	N	14	14	14	13
Days 0 to 29	Mean	237.2	257.6	177.9	161.7
	SD	93.17	112.45	122.74	159.24
	N	14	14	14	13

a = P < 0.05; b = P < 0.01

 

 

Clinical symptoms:

No adverse clinical symptoms occurred in any of the does of test groups 0 or 1 (0 and 5 mg/kg body weight/day) during the whole study period (days 0 -29 p.i.). In test group 2 (20 mg/kg body weight/day) one doe (No. 32) aborted on day 18 p.i. and was consequently sacrificed.

Another doe of this test group (No. 44) showed blood in bedding on days 20 -21 p.i. and reduced defecation on days 18 -26 p.i. In test group 4 (80 mg/kg body weight/ day) for one doe (No. 56) blood in bedding was noted on day 16 p.i. These clinical symptoms, recorded only for single does of the intermediate and the high dose groups (20 and 80 mg/kg body weight/day) might be related to the test substance administration, although similar findings occurred sporadically also in control animals.

 

Mortality:

Only one doe (No. 32) of test group 2 (20 mg/kg body weight/day), which aborted on day 18 p.i., was sacrificed on that day.

 

Examinations of the dams at termination:

 

Necropsy findings:

There were no remarkable observations at necropsy in any of the does which could be attributed to the test substance administered. Only some spontaneous findings were recorded for single animals of all dose groups including the controls at necropsy. Most of these findings have to be related to the sacrifice of the animals (lungs with edema and/or marginal emphysema), while agenesia of the gallbladder, which was noted for one control doe, is assessed as being of spontaneous nature.

 

Uterus weight:

There were no substantial differences concerning the uterus weights between the controls and test groups 1 and 2 (5 and 20 mg/kg body weight/day). All these values were within the range of biological variation; however, the mean gravid uterus weight was lowest in the high dose group (80 mg/kg body weight/day), which has to be related to the test substance administration. This is in line with the high number of resorptions and the consequently increased postimplantation loss in this group.

 

Mean gravid uterine weights and net maternal body weight change (grams)

		Solvent Control	5 mg/kg bw/d	20 mg/kg bw/d	80 mg/kg bw/d
Gravid uterus	Mean	309.9	382.1	281.1	242.7
	SD	101.84	69.47	110.27	164.49
	N	14	14	14	13
Carcass	Mean	2512.5	2431.5	2433.5	2486.8
	SD	194.81	154.38	140.75	174.74
	N	14	14	14	13
Net weight change from day 7	Mean	-111.9	-161.1	-131.2	-125.3
	SD	55.26	67.72	91.26	60.45
	N	14	14	14	13

a = P < 0.05; b = P < 0.01

 

 

Reproduction data of dams:

The conception rate varied between 87 and 100%.

Concerning test groups 1 and 2 (5 and 20 mg/kg body weight/day), there were no substance-related and/or statistically significant differences of biological relevance in the conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions or viable fetuses. The differences evident were considered to be incidental and within the normal range of deviations for animals of this strain and age. In test group 3 (80 mg/kg body weight/ day), however, the resorption rate was clearly increased, especially due to the fact, that 3 does of this group (Nos. 56, 57 and 59) had no viable fetuses at all but only resorptions (mainly early ones). As a consequence, the postimplantation loss for the 80 mg/kg group was distinctly increased, which was assessed as a clear substance-induced embryotoxic effect.

 

Summary of reproduction data

		Solvent Control	5 mg/kg bw/d	20 mg/kg bw/d	80 mg/kg bw/d
Females mated	N	15	15	15	15
	N	14	14	15	13
pregnant	%	93	93	100	87
aborted	N	0	0	1	0
Premature births	N	0	0	0	0
Dams with viable fetuses	N	14	14	14	10
Dams with all resorptions	N	0	0	0	3
Female mortality	N	0	0	1	0
	%	0	0	6.7	0
Pregnant at C-section	N	14	14	14	13
	%	93	93	93	87
Corpora lutea	Mean	7.5	8.7^a	7.4	8.4
	SD	1.29	1.20	1.22	1.26
	Total	105	122	103	109
Implantation sites	Mean	6.2	6.8	5.6	7.5
	SD	1.58	1.31	2.06	1.27
	Total	87	95	78	98
Preimplantation loss	Mean %	16.2	21.7	23.9	10.0
	SD	19.09	14.75	25.63	7.70
Postimplantation loss	Mean %	13.6	2.3	10.2	43.0^b
	SD	21.49	6.08	14.76	40.63
Resorptions: total	Mean	0.9	0.1	0.5	3.3^b
	SD	1.61	0.36	0.65	3.25
	Total	12	2	7	43
	Mean %	13.6	2.3	10.2	43.0^b
	SD	21.49	6.08	14.76	40.63
Resorptions: early	Mean	0.6	0.1	0.4	3.2^b
	SD	1.60	0.36	0.63	3.31
	Total	9	2	5	41
	Mean %	10.4	2.3	8.0	40.8^b
	SD	21.48	6.08	14.97	41.41
Resorptions: late	Mean	0.2	0	0.1	0.2
	SD	0.58	0	0.36	0.38
	Total	3	0	2	2
	Mean%	3.2	0	2.2	2.2
	SD	8.54	0	5.64	5.36
Dead fetuses	N	0	0	0	0

a = P < 0.05; b = P < 0.01

 

 

Examination of the fetuses after dissection from the uterus:

 

Sex distribution of fetuses:

The sex distribution in test groups 1 -3 (5, 20 and 80 mg/kg body weight/day) was comparable with the control group. The observable differences were without any biological relevance.

 

Weight of placentae:

The mean placental weights in the test substance-treated groups (5. 20 and 80 mg/kg body weight/day) were not substantially influenced. The differences observed in comparison to the control were without biological relevance and within the range of biological variation.

 

Weight of fetuses:

There were no clearly dose-related differences between the control and test groups 1, 2 and 3 (5, 20 and 80 mg/kg body weight/day) concerning the mean fetal weights. All values were fully within the range of biological variation; therefore, the observable differences between the control and the relevant substance-treated groups were assessed as being of spontaneous nature.

 

External examination of the fetuses:

The external examination of the fetuses revealed several malformations in 2 fetuses each of test groups 0 and 1 (0 and 5 mg/kg body weight/day), but none in the fetuses of the intermediate and high dose groups (20 an 80 mg/kg body weight).

One control fetus showed a cheiloschisis, while for the other one an open eye (unilateral) was noted. The first affected fetus of the low dose (5 mg/kg body weight/day) had micrognathia and macroglossia, while for the second affected fetus of this dose group cleft palate and partial acrania were recorded.

Only one kind of external variations (pseudoankylosis) could be found in one fetus each of the control, the 5 and the 20 mg/kg groups and in 2 fetuses of the high dose group (80 mg/kg body weight/day).

Necrobiotic placentae was the only so-called unclassified observation and occurred in only one high dose fetus (80 mg/kg body weight/day).

 

Soft tissue examination of the fetuses:

The examination of the organs of the fetuses revealed no malformations in anyone of the test groups.

Variations were detected in each group including the control. The very common finding of separated origin of carotids in the rabbit strain used in this study occurred without a clear dose-response relationship. The other variations (hypoplasia of gallbladder, heart with traces of interventricular foramen/septum membranaceum) occurred without any dose-response relationship. All soft tissue variations recorded in this study were to be found in the same kind of magnitude in the historical control data.

Moreover, several fetuses out of test groups 0 and 1 (0 and 5 mg/kg body weight/day) showed focal liver necrosis or blood coagula around the bladder (so-called unclassified observations).

 

Skeletal examination of the fetuses:

Only one kind of malformation of the vertebral column (lumbar vertebra absent) was seen in 1 fetus each of test groups 0 and 1 (0 or 5 mg/kg body weight/day).

The variations elicited were related to the skull (splitting of skull bones, epactal bone between nasal, frontal and parietal bones), the ribs (accessory 13th rib(s)), the clavicula (deformed) and the sternum (sternebra(e) of irregular shape, fused or accessory sternebra) and were found in single fetuses of all groups including the controls without a dose-response relationship and without any statistically significant differences between the groups.

In all groups signs of retardations (incomplete or missing ossification of skull bones, vertebral column and/or sternebra(e)) were found; they occurred without any differences of biological relevance between the groups.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 2010 - Aug 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jan 2001. Since the cited guidelines are designed for rat and rabbit models, the necessary adaptations to guinea pig reproductive biology were made.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

guinea pig

Strain:

Dunkin-Hartley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Females were non-pregnant and nulliparous: no nulliparous females were used for this experiment.
- Age at study initiation: not specified
- Weight at study initiation: 719.0 - 1162.0 g
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least two days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (illumination: 6 am to 6 pm)

IN-LIFE DATES: From: SEp 1/Sep 8/Sep 15/Sep 22/Sep 29, 2010 To: Oct 28/Nov 4/Nov 11/Nov 18/Nov 25, 2010

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% CMC in highly deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The aqueous test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability.
- Applied dose volume: 10 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the beginning and towards the end) for verification of the concentrations. Analytical method was HPLC.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

From GD 6 to GD 63

Frequency of treatment:

once daily

Duration of test:

60 days

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of a preliminary dose range-finding study
- Rationale for animal assignment (if not random): random
- Reason for species selection: The Dunkin Hartley (Crl:HA strain) guinea pigs were chosen because of their general acceptance and suitability for toxicity testing and the reliability of the commercial supplier.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund or dead animals was made twice daily on working days. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: GD 4, 6, 9, 13, 16, 20, 23, 27, 30, 34, 37, 41, 44, 48, 51, 55, 58, 62 and 63. The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 63 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: GD 4 - 6, 6 -13, 13 - 20, 20 - 27, 27 - 34, 34 - 41, 41 - 48, 48 - 55, 55 - 62 and 62 - 63.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

CLINICAL CHEMISTRY/HEMATOLOGY: Yes
- Time schedule for collection of blood: On GD 63
- Animals fasted: No
- Anesthesia: Yes (isoflurane)
- How many animals: all females
- Parameters checked: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, reticulocytes, Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB) , Triglycerides (TRIG), Cholesterol (CHOL), Estradiol. Absolute quantification: 11-desoxycorticosterone, 11-desoxycortisol, 18-hydroxycorticosterone, Androstendione, Corticosterone, Cortisol, Progesterone, Testosterone


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 63
- Organs examined: uterus
- Organ weights: adrenal glands, liver, ovaries
- Histopathology: All placentas and ovaries

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead fetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

Statistics:

Dunnett's test, Fisher's Exact, Wilcoxon test, Kruskal-,Wallis, Mann-Whitney-U-test

Indices:

conception rate, preimplantation loss, postimplantation loss

Historical control data:

not available, lack of historical data for guinea pigs

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: One animals showed reduced defecation. Another showed reduced nutrional state, vaginal hemorrhage, reduced or no
defecation and piloerection.
- 50 mg/kg bw/day: One animal showed reduced defecation.
- 15 mg/kg bw/day: two animals showed reduced or no defecation.
One control animal (No. 11) showed reduced defecation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- 90 mg/kg bw/day: two animals delivered early and were sacrificed thereafter. Two animals were sacrificed moribund
- 50 mg/kg bw/day: Two animals aborted or delivered early and were sacrificed thereafter. Two animals were found dead. Two animals were sacrificed moribund.
- 15 mg/kg bw/day: One animal was sacrificed because an abortion was observed. One animal died during gavaging (no gavage error). One animal was sacrificed after early delivery.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: Decreased red blood cell counts, hemoglobin and hematocrit values

In guinea pigs of all dose groups (test groups 1 – 3, 15 – 90 mg/kg bw/d) mean corpuscular hemoglobin concentrations (MCHC) were higher compared to controls, but they were not dose-dependently increased. Therefore, this alteration was regarded as incidental and not
treatment-related. In guinea pigs of test group 3 (90 mg/kg bw/d) absolute and relative baosphil counts as well as absolute large unstained cell (LUCA) counts were lower compared to controls. Lower absolute LUC counts have no toxicological relevance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: Decreased total bilirubin and cholesterol levels, Increased androstenedione, testosterone, 18-hydroxycorticosterone, corticosterone and 11-desoxycortisol levels
- 50 mg/kg bw/day: Increased androstenedione, testosterone and 11-desoxycortisol levels
- 15 mg/kg bw/day: Increased androstenedione, testosterone and 11-desoxycortisol levels

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: Increase absolute (+ 10%) and relative (+12%) weight of the adrenal glands

In the liver, a statistically significant increase of relative weight was observed in test groups 2 and 3 (50 and 90 mg/kg bw/d). However, the change was only minimal and without a clear dose-dependent relationship (+8% and +6%, at 50 and 90 mg/kg bw/d, respectively).
Therefore the liver weight change was not regarded as treatment-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- 90 mg/kg bw/day: One animal showed a stomach filled with fluid (test substance) and no feces in intestines. Another showed no feces in small intestine, liver pale yellowish and stomach filled with fluid (test substance).
- 50 mg/kg bw/day: One animal showed an empty stomach. Another showed stomach filled with fluid (test substance), multiple erosions in the stomach and no feces in intestines. A third showed no feces in small intestine and rectum and multiple erosions in the stomach.
- 15 mg/kg bw/day: One animal showed a bilobed gallbladder. Another showed stomach filled with gas, no feces in small intestine and rectum.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All findings noted in the ovaries and placentas were either single observations, or were biologically equally distributed between control and treated animals. All of them were considered to be incidental and/or spontaneous in origin.

Details on results:

Regarding pathology, the only noteworthy finding was a statistically significant increase in the absolute and relative weight of the adrenal glands in the high-dose females (90 mg/kg bw/d). No histopathological investigation of this organ was performed, but the weight increase fits well to the increased 18-hydroxycorticosterone and corticosterone levels in these animals and thus a treatment-related effect cannot be ruled out. It is noteworthy, that
other than in rats, histopathology of all placentas revealed no adverse effect caused by the test substance up to and including a dose of 90 mg/kg bw/d.

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Total abortions were recorded for control, 15 mg/kg bw/day, and 50 mg/kg bw/day to be 3, 1, 1, respectively.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The conception rate reached 90% in the control group, 87% in test group 1 (15 mg/kg bw/d), 83% in test group 2 (50 mg/kg bw/d) and 77% in test group 3 (90 mg/kg bw/d). No test substance-related and/or biologically relevant differences with regard to conception
rate, mean number of corpora lutea, implantation sites, pre- and postimplantation loss and
resorptions (total, early and late) were observed.

Details on maternal toxic effects:

Steroid hormone level alterations were observed starting at a dose of 15 mg/kg bw/d. The increases of estradiol precursors such as testosterone and androstenedione may suggest that guinea pigs are sensitive to aromatase inhibition.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: endocrine system (aromatase inhibition)

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Umbilical hernia was the only external malformation which was recorded in fetuses of all dose groups including the controls (0, 15, 50 and 90 mg/kg bw/d).

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations of the fetal skeletons were recorded in fetuses of all dose groups including the controls (0, 15, 50 and 90 mg/kg bw/d). Neither for the individual findings nor the overall incidences a dose-response relationship was present. Thus, these findings were considered to be spontaneous in nature and without a relation to treatment. For all test groups, variations in different skeletal structures were detected with or without effects on the corresponding cartilages. The observed skeletal variations were related to various parts of the fetal skeletons and were statistically significantly increased in the lowand the high-dose groups on a fetus per litter basis (Table 3).
Additionally, isolated cartilage findings without influence on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all groups including the control. The observed unclassified cartilage findings were exclusively related to the sternum and the ribs and do not exhibit a specific pattern or a dose response (Tab. 5). Thus, a toxicological relevance for these findings is not assumed.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No soft tissue malformations were observed in any of the test groups. Malpositioned carotid origin was detected in the low- and mid-dose groups as well as the control group. As there is no dose-response relationship, these findings were considered to be spontaneous in nature and without a relation to treatment. Discolored kidneys were recorded for some fetuses of test group 1 (50 mg/kg bw/d). As there is no dose-response relationship, these findings were considered to be spontaneous in nature and without a relation to treatment.

Details on embryotoxic / teratogenic effects:

One external malformation (umbilical hernia) occurred at litter incidences 1, 1,
2, 3 in all test groups including control (0, 15, 50, 90 mg/kg body weight/day). The presence
of this finding in control animals and the low general incidence indicate that a specific effect
of the test substance on complete closure of abdominal wall at the umbilical ring cannot be
assumed. The lack of historical data for guinea pigs make an assessment, whether the litter
incidence of 3 constitutes a treatment-related increase, difficult. However, this finding
appears to be common in guinea pigs. A recent reference (Rocca et al., 2009) also describes
the presence of omphalocele (which is a slightly more distinct form of umbilical hernia) in 4
different Dunkin Hartley fetuses from 3 separate litters out of 32 control litters. Thus, a
relationship of this finding to treatment seems to be rather unlikely.
No soft tissue malformations were noted in this study and skeletal
malformations were evenly distributed about the test groups and did
not form a specific pattern. Consequently, the incidence of total external, soft tissue and
skeletal malformations showed no dose-response relationship. External variations did not occur in any of the fetuses in this study. Soft
tissue variations, in the form of malpositioned carotid origin occurred in the
low- and mid-dose group and in the control without any relation to dosing. Skeletal
variations consisted primarily of minor delays or disturbances of
ossification. The majority of the skeletal variations are equally distributed among the different
test groups including controls, if normal biological variation is taken into account. However,
two skeletal variations, such as thoracic centrum fused with arch and misshapen sternebra
were noted at significantly higher incidences either in all dose groups (though not doserelated)
or in the high-dose group only. Such slight retardations of the ossification process
occur very frequently in rat and rabbit fetuses and they seem also to be common skeletal
abnormalities in guinea pigs. Rocca et al. (2009) report skeletal abnormalities such as short
rib, discontinuous rib, supernumerary rib, unossified vertebra, bipartite ossification, and extra
sites of ossification to be observable in 35 to 50% of litters (9.6 to 24.7% of fetuses)
examined. An increase of this spontaneously high incidence of skeletal variations is often noted in the
presence of maternal toxicity or maternal stress, as has been substantiated in this study for
the high-dose females displaying microcytic anemia and increased adrenal weights as well
as 18-hydroxycorticosterone and corticosterone serum levels. Taking all this into
consideration, the increased skeletal variations in this study and the subsequently increased
total incidence of variations were regarded to be of no toxicological relevance and are not
classified as adverse events. A spontaneous origin is also assumed for the unclassified cartilage observations which were recorded for several fetuses of all test groups (0, 15, 50, or 90
mg/kg bw/d). Distribution and type of these findings do not suggest any relation to treatment.
Thus, the oral administration of the test substance to pregnant guinea pigs had no
direct and specific effect on morphology of offspring at any dose level tested (15, 50 and
90 mg/kg bw/d).

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Total external malformations - fetuses

		Test group0 0 mg/kgbw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 90 mg/kg bw/d
Litter Fetuses	N N	23 96	21 87	19 79	18 77
Fetal incidence	N	1 (1.0%)	1 (1.1%)	2 (2.5%)	4 (5.2%)
Litter incidence	N	1 (4.3%)	1 (4.8%)	2 (11%)	3 (17%)
Affected fetuses/litter	Mean%	0.9	0.8	2.4	4.3

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Table 2: Total skeletal malformations - fetuses

		Test group0 0mg/kgbw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 90 mg/kg bw/d
Litter Fetuses	N N	23 96	21 87	19 79	18 77
Fetal incidence	N (%)	6 (6.3%)	7 (8.0%)	3 (3.8%)	7 (9.1%)
Litter incidence	N (%)	5 (22%)	6 (29%)	3 (16%)	4 (22%)
Affected fetuses/litter	Mean%	6.2	6.9	4.1	11.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent

Table 3: Total skeletal variations - fetuses

		Test group0 0mg/kgbw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 90 mg/kg bw/d
Litter Fetuses	N N	23 96	21 87	19 79	18 77
Fetal incidence	N (%)	43 (45%)	56 (64%)	54 (68%)	56 (73%)
Litter incidence	N (%)	20 (87%)	20 (95%)	17 (89%)	18 (100%)
Affected fetuses/litter	Mean%	50.4	68.1*	66.1	75.5**

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent

* = p <= 0.05, ** = p<=0.01 (Wilcoxon-Test [one-sided])

Table 4: Statistically significantly increased individual skeletal variations

Finding	0 mg/kg bw/d	15 mg/kg bw/d	50 mg/kg bw/d	90 mg/kg bw/d
Thoracic centrum fused with arch
fetal incidence	7	19	23	25
litter incidence	6	11	12*	13**
mean percentage of affected fetuses/litter	6.6	21.7*	26.3**	38.8**
Misshapen sternebra
fetal incidence	5	11	6	12
litter incidence	4	7	5	9*
mean percentage of affected fetuses/litter	6.6	11.8	7.1	18.8**

* = p<=0.05, ** = p<=0.01 [Wilcoxon-test (one sided)]

Table 5: Total skeletal unclassified cartilage observations

		Test group0 0mg/kgbw/d	Test group 1 15 mg/kg bw/d	Test group 2 50 mg/kg bw/d	Test group 3 90 mg/kg bw/d
Litter Fetuses	N N	23 96	21 87	19 79	18 77
Fetal incidence	N (%)	2 (2.1%)	13 (15%)	6 (7.6%)	8 (10%)
Litter incidence	N (%)	1 (4.3%)	6* (29%)	5 (26%)	5* (28%)
Affected fetuses/litter	Mean%	1.4	12.9*	7.6*	17.0*

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = percent

* = p<=0.05 (Fisher’s exact test[one-sided]; Wilcoxon-Test [one-sided])

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March 1992 - 08 April 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

Nov 1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Testing Guidelines for Toxicology Studies", pp. 48 - 49 (Japan/MAFF)

Version / remarks:

1985

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Chbb:THOM (SPF)
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: 68 -73 days old
- Weight at study initiation: approx. 217.5 g (mean)
- Housing: singly in type DK III stainless steel wire mesh cages supplied by BECKER & CO., Castrop-Rauxel, Germany
- Diet (ad libitum): ground Kliba 343 feed rat/mouse/ hamster supplied by KLINGENTALMUEHLE AG, CH-4303 Kaiseraugst, Switzerland
- Water (ad libitum): drinking water of tap water quality
- Acclimation period: 8 -10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 -24
- Humidity (%): 30 -70
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: March 16/March 18, 1992 To: April 6/April 8, 1992

Route of administration:

dermal

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous CMC solution

Details on exposure:

TEST SITE
- Area of exposure: intact shaven dorsal skin of the animals
- Type of wrap if used: semiocclusive dressing
The patch measured about 4 x 4 cm and consisted of 4 layers. This preparation was fixed to the rat by means of a stretchable bandage (Fixomull@, BEIERSDORF AG, Hamburg, Germany).
- Time intervals for shavings or clipplings: The animals were shaved on the dorsal area of the trunk at least 15 hours before the first treatment (day 6 p.c.) and at additional times during the treatment period as necessary (at least once a week).

REMOVAL OF TEST SUBSTANCE
- Washing: yes
- Time after start of exposure: After removal of the dressing the application site was washed off with luke-warm water and dried.

TEST MATERIAL
Each day the test substance suspensions were freshly prepared shortly before the dermal application of the test substance. For the preparation of the suspensions, an appropriate amount of the test substance was weighed and subsequently suspended in a 0.5% aqueous carboxymethyl cellulose solution using a high speed sonicator (Ultra-Turrax, JANKE & KUNKEL KG, Germany). A magnetic stirrer was used to keep the suspensions homogeneous during treatment of the animals.

VEHICLE
- CMC (Tylose@ CB 30.000; purified carboxymethyl cellulose supplied by Hoechst AG, 6230 Frankfurt/Main, Germany)
- Amount(s) applied (volume or weight with unit): 2 mL/kg bw
- Concentration (if solution): 0, 5, 20, and 50 g/100 mL

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical determinations of the purity and the homogeneity of the test substance itself were carried out before the beginning of the study (method: HPLC).
The stability of the test substance suspensions over a period of up to 24 hours at room temperature could be demonstrated.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: 2 -4 untreated female rats were mated with one untreated fertile male animal of the same breed.
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 6 to day 15 p.c.

Frequency of treatment:

6 hours /day

Duration of test:

day 6 to day 20 p.c.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A range-finding study with rats treated with the test substance orally by gavage and a full-scale prenatal toxicity study in rats was performed.
In a dermal study (1992), the substance was applied daily for 6 hours to the clipped intact dorsal skin of Wistar rats over a period of 3 weeks using a semiocclusive dressing at doses of 1000, 100, and 40 mg/kg bw. A control group was used for comparison. Five male and five female rats were treated at each dose level. Five rats of each sex received 0.5% solution of Tylose@ CB 30.000 (cleaned natrium-carboxymethylcellulose in aqua bidest.) and served as controls. Food consumption and body weight were determined weekly. The animals were carefully inspected twice daily (before and after exposure). Skin findings were recorded daily (about 30 minutes after removal of the dressing). At the end of the study, clinicochemical and hematological examinations were carried out. All animals were assessed by gross pathology, followed by a histopathological examination.
It can be stated that the daily application of the substance to the clipped intact dorsal skin of Wistar rats over a period of 3 weeks caused following findings:
1000 mg/kg body welght:
- slight decrease in red blood cells and hematocrit in the peripheral blood in males
- significantly increased absolute mean liver weights in male and female rats
- slight centrilobular liver cell hypertrophy in male rats
- significantly increased absolute mean kidney weight in female rats, without a morphological equivalent

400 mg/kg body weight:
- significantly increased relative mean liver weight in female rats, without a morphological equivalent

100 mg/kg body weight:
- no substance-induced changes

Based on the results obtained in this dermal study the "no adverse effect level" of the substance is in the range lower than 400 mg/kg body weight and greater than 100 mg/kg body weight.
In a study of the dermal absorption (1991) male rats were exposed to 3.0 and 30.0 mg of the test substance (14C labelled) which was applied to the shaven dorsal skin for 0.5; 1; 2; 4 or 10 hours. Subsequently, each of the animals was killed or observed - only in the case of selected animals with 10-hour exposure - after washing of skin. In each study phase 4 animals were employed.
As the study results reveal, only relatively small amounts of the test substance could be detected in the body of the animals, which were absorbed by the skin. Based on a 10-hour exposure and a 62-hour post-exposure observation period, about 7.81% systemically absorbed substance was found with 30.0 mg/kg and about 3.80% systemically absorbed substance with 3.0 mg/kg.

On the basis of these study results the following doses were established for the study to be carried out on the prenatal toxicity of the substance on rats with dermal application:
- 100 mg/kg body weight/day: with an assumed absorption rate of about 10%, this dose would correspond to about 10 mg/kg body weight after oral intake. Therefore, this dose was expected to be a clear no observable effect level for dams and fetuses.

- 400 mg/kg body weight/day: assuming an absorption rate of 10%, this dose would correspond to about 40 mg/kg body weight/day after oral intake. As in the oral studies on the prenatal toxicity the rats exhibited still clear signs of maternal toxicity at 60 and 45 mg/kg body weight/day and as embryo/fetotoxic, but no teratogenic effects, occurred, this dose may have been the toxic effect level for dams and/or fetuses. In the case of a lower dermal absorption than assumed, this dose could have also been another no observable effect level.

- 1000 mg/kg body weight/day: This dose resulted in non-pregnant female animals after dermal application for 21 times only to marginal increases of the liver and kidney weights without a morphological equivalent. Since it was not to be estimated how much test substance would be absorbed by the skin at this dose, 1000 mg/kg body weight is intended for relatively nontoxic test substances, according to the test guidelines. Therefore, a study with even higher doses was not necessary.

- Rationale for strain selection: This strain was selected since extensive experience is available on Wistar rats and this strain has been proved to be sensitive to substances with a teratogenic potential.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were examined für clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited (days 0 -20 p.c.). A check for mortality was made twice a day on working days or once a day (Saturday, Sunday or on public holidays) (days 0 20 p.c.).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The site of application was examined twice daily (once prior to and once upon termination of each six hour exposure period) (days 6 -15 p.c.). In case of any observed skin reactions, these would have been scored according to the standards published in the Federal Register.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c. The body weight change of the animals was calculated from these results.

FOOD CONSUMPTION: Yes
With the exception of day 0, the consumption of food was determined on the same days as was body weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.
On day 20 p.c., the surviving dams were sacrificed in randomized order by cervical dislocation and the fetuses dissected from the uterus.
After the dams had been sacrificed, they were necropsied and assessed by gross pathology.
- Organs examined: Liver, kidneys, uterus and the ovaries were removed and assessed by gross pathology. Liver and kidney weight were determined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weight of uterus before it was opened (Gravid uterus weight)
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
- live fetuses
- dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (1964) from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single-horn pregnancy)
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No


Details:
At necropsy each fetus was weighed, sexed and examined macroscopically for any external findings. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all fetuses fixed in BOUIN'S solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the appearance of its gonads.
Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded.
After these examinations, approximately one half of the fetuses per dam was placed in ethyl alcohol and the other half was placed in BOUIN's solution for fixation and further evaluation.

Soft tissue examination of the fetuses:
After fixation in BOUIN's solution, approximately one half of the fetuses of the dams of all groups was examined for any findings in the organs according to the method of BARROW and TAYLOR (J. Morph. 127, 291 -306, 1969). After these examinations the relevant fetuses were discarded.

Skeletal examination of the fetuses:
After fixation in ethyl alcohol, the skeletons of approximately one half of the fetuses were stained according to a modified method of DAWSON (Stain Technol. 1, 123, 1926). Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After these examinations the relevant fetuses were retained by litter.

Statistics:

Dunnett's Test (1955, 1964) was used for statistical evaluation of food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), liver weight, kidney weight, weight of the uterus before it was opened, weight of fetuses, weight of placentae, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses.
Fisher's Exact Test (1981) was used for statistical evaluation of conception rate, mortality (of the dams) and all fetal findings.
Significances resulting from these tests have been indicated in the tables (a for p < 0.05, b for p < 0.01).

Indices:

- Conception rate (%) = (number of pregnant animals / number of fertilized animals) * 100
- Preimplantation loss (%) = ((number of corpora lutea - number of implantations) / number of corpora lutea) * 100
- Postimplantation loss (%) = ((number of implantations - number of live fetuses) / number of implantations) * 100

Historical control data:

available

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no substance-related effects on absolute or relative liver or kidney weights. The uterus weights of the animals were not influenced
by the dermal application of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- 100 mg/kg bw/day: Only one spontaneous finding was recorded for one animal, which showed a hydrometra and did not become pregnant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: statistically significantly increased placental weights

- The conception rate varied between 92% (control group) and 100% (1,000 mg/kg group) revealing no treatment-related adverse effect
- number of corpora lutea: no adverse effects observed
- number of implantation sites: no adverse effects observed
- 400 mg/kg bw/day: marginally decreased mean percentage of live female fetuses and increased mean percentage of live male fetuses, however, was assessed as spontaneous in nature

Details on maternal toxic effects:

The dermai appiication of 1,000 mg/kg body weight/day of the test substance to pregnant femaie Wistar rats during organogenesis caused some effects, which were already found in preceding prenatal toxicity studies with oral administration (by gavage), i.e. increased placental weights (an effect concerning dams and fetuses).

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg: One fetus with cleft palate (a malformation being typical for this class of compounds)

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: Statistically significantly increased number of fetuses with skeletal variations (rudimentary cervical and/or accessory 14th rib(s))
- 400 mg/kg bw/day: The sligthiy increased number of fetuses with rudimentary cervica and/or accessory 14th rib(s), however, is assessed as being of spontaneous nature, because it is fully in the range of biological variation

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: statistically significantly increased placental weights

Details on embryotoxic / teratogenic effects:

Thus, under the conditions of this study it could be proven, that the dermai application of 1,000 mg/kg body weight/day of the test substance to pregnant female Wistar rats during organogenesis caused some effects, which were already found in preceding prenatal toxicity studies with oral administration (by gavage), i.e. increased placental weights (an effect concerning dams and fetuses), a very marginal, if any indication for teratogenicity in the form of one cleft palate and an increased occurrence of skeletal variations in the fetuses.

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

other: increased placental weights

Abnormalities:

effects observed, treatment-related

Localisation:

external: face

skeletal: rib

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Food consumption:

The food consumption of all substance-treated groups (100, 400 or 1000 mg/kg body weight/day) did not show any differences of biological relevance if compared to the controls. All values were within the range of biological variation; this includes the marginal, fortuitously reduced food consumption of the 400 and 1000 mg/kg dams during post treatment days 15 -17 p.c. During this interval the dams in question gained more body weight than the concurrent control dams.

 

Body weight data:

There were no adverse effects on body weights or body weight gains which could be attributed to the dermal test substance application. All values are within the range of biological variation.

 

Corrected body weight gain (net maternal body weight change):

The results of the corrected body weight gain (terminal body weight on day 20 p.c. minus weight of the uterus before it was opened minus body weight on day 6 p.c.) of all substance-treated groups (100, 400 or 1000 mg/kg body weight/day) did not show any differences of biological relevance if compared to the controls.

 

Clinical symptoms:

There were no abnormal clinical findings in any dam of anyone group; especially the skin was free from any notable findings during days 6 -15 p.c. before and after the daily treatment.

 

Mortality:

There were no mortalities in any of the groups.

 

Examinations of the dams at termination:

 

Organ weights and necropsy findings:

There were no substance-related effects on absolute or relative liver or kidney weights.

At necropsy only one spontaneous finding was recorded for one animal (No. 44) of test group 1 (100 mg/kg body weight/day), which showed a hydrometra and did not become pregnant.

 

Uterus weight:

The uterus weights of the animals were not influenced by the dermal application of the test substance. The differences between the groups are without biological relevance.

 

Reproduction data of dams:

The conception rate varied between 92% (control group) and 100% (1000 mg/kg group). There were no substance-related and/or statistically significant differences of biological relevance between the groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age; this includes the marginally decreased mean percentage of live female fetuses and the increased mean percentage of live male fetuses in the 400 mg/kg group.

 

 

Examination of the fetuses after dissection from the uterus:

 

Sex distribution of fetuses:

The sex distribution of the fetuses in test groups 1-3 (100, 400 and 1000 mg/kg body weight/day) was comparable with the control fetuses. The differences observed in comparison to the control are without any biological relevance.

 

Weight of placentae:

The mean placental weights in test group 3 (1000 mg/kg body weight/day) were statistically significantly higher in comparison to the controls, whereas the mean placental weights in test groups 1 and 2 (100 or 400 mg/kg body weight/day) were comparable to the actual control values (for details see Table: Mean placental and fetal body weights).

Increased placental weights occurred also in two preceding prenatal toxicity studies in rats with oral administration (by gavage) of the test substance (BASF AG, 34R0035/87005, 1989; BASF AG, 30R0959/88073, 1990). Therefore the increased placental weights in the high dose group (1000 mg/kg body weight/day) are finally assessed as a substance-related effect.

 

Weight of fetuses:

The mean fetal weights were not influenced by the test substance administration. All values are within the range of biological variation.

 

External examination of the fetuses:

Two external malformations (cleft palate and shortened tail) were recorded for two high dose fetuses. Both malformations or very similar findings (acaudia) are also present in the historical control data at low frequency.

Due to the fact that a high incidence of cleft palates was found in a preceding prenatal range-finding study with the test substance with oral administration (by gavage) (BASF AG, 34R0035/87005, 1989), it might be possible that the cleft palate seen in only one high dose fetus was a substance-induced effect. On the other hand cleft palates do also appear in the historical control data at a rather low incidence.

The one high dose fetus with shortened tail (a finding which did not occur in the preceding prenatal toxicity studies in rats with oral administration (by gavage) was finally assessed as being of spontaneous origin.

The external examination of the fetuses revealed no variations in any group.

One so-called unclassified observation (placentae fused) was recorded for one fetus of test group 3 (1000 mg/kg body weight/day).

(For details see Tables: Fetal external malformations; Fetal external varaitions)

 

Soft tissue examination of the fetuses:

The examination of the organs of the fetuses revealed no malformations in any group. Variations (dilated renal pelvis and/or hydroureter) were detected in all groups without any statistically significant differences between the groups. All values are fully in the range of biological variation.

No so-called unclassified observations (like blood coagulum around the bladder) were recorded.

(For details see Table: Fetal soft tissue variations)

 

Skeletal examination of the fetuses:

Various malformations of the sternum (sternebra(e) bipartite, ossification centers dislocated) and/or the vertebral column (thoracic vertebral body/bodies dumbbell-shaped (asym.) or bipartite (asym.), lumbar vertebrae fused and/or of irregular shape) were seen in 8 out of 162 fetuses (in 6 out of 23 litters) of the control, in 11 out of 167 fetuses (in 9 out of 24 litters) of the 100 mg/kg group, in 1 out of 161 fetuses (in 1 out of 24 litters) of the 400 mg/kg group and in 9 out of 178 fetuses (in 6 out of 25 litters) of the 1000 mg/kg group.

These differences did not show any dose-response relationship; the statistically significantly lower number of fetal skeletal malformations in test group 2 (400 mg/kg body weight/day) is without any biological relevance and of spontaneous nature.

 

The varlations elicited were related to the ribs (shortened 13th, accessory 14th ribs or rudimentary cervical ribs), the sternum (sternebra(e) of irregular shape, bipartite, or accessory sternebra) and the clavicula (deformed). Most of the skeletal variations recorded appeared without a clear dose-response relationship (e.g. sternebra(e) of irregular shape, 13th rib(s) shortened), can be found in a similar frequency in the historical control data and/or the differences between the groups are without biological relevance; the statistically significantly increased number of 1000 mg/kg fetuses, however, which showed rudimentary cervical and/or accessory 14th rib(s) has to be related to the dermal application of the test substance. The slightly increased number of intermediate dose (400 mg/kg body weight/day) fetuses with rudimentary cervical and/or accessory 14th rib(s), however, is assessed as being of spontaneous nature, because it is fully in the range of biological variation.

In all groups signs of retardations (incomplete or missing ossification of vertebral bodies/arches, sternebra(e) and/or the hyoid bone) were found without any differences of biological relevance between the groups.

This includes:

- the statistically significantly lower number of 400 mg/kg fetuses/litters with dumbbell-shaped (symmetr.) thoracic vertebral bodies

- the statistically significantly lower number of 100 mg/kg litters with incompletely ossified thoracic vertebral body/bodies

- the statistically significantly higher number of 1000 mg/kg fetuses with incompletely ossified or smaller sternebra(e)

- the statistically significantly lower number of 1000 mg/kg fetuses with sternebra(e) with only one ossification center and the statistically significantly lower number of 400 mg/kg fetuses with overall retardations.

(For details see Tables: Fetal skeletal malformations; Fetal skeletal variations; Fetal skeletal retardations)

Justification for classification or non-classification

Epoxiconazole is classified for reproductive toxicity (cat 1B; H360 Df) according to Regulation (EC) 1272/2008 Annex VI.