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

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

no

Test guideline 2

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

Test guideline 3

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

Test guideline 4

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

406-850-2

EC Name:

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

Cas Number:

133855-98-8

Molecular formula:

C17 H13 Cl F N3 O

IUPAC Name:

1-{[(2R,3R)-3-(2-chlorophenyl)-2-(4-fluorophenyl)oxiran-2-yl]methyl}-1H-1,2,4-triazole

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 ppm (nominal)

Remarks:

average of 0.9 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

25 ppm (nominal)

Remarks:

average of 2.3 mg/kg bw/day

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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)

Details on results (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

1

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

2

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

endocrine system

Treatment related:

yes

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

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

Details on results (P1)

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.

Effect levels (P1)

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

Target system / organ toxicity (P1)

1

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

endocrine system

Treatment related:

yes

2

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

Results: F1 generation

General toxicity (F1)

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)

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

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.

Effect levels (F1)

1

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

2

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Details on results (F2)

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.

Effect levels (F2)

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)

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

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

Applicant's summary and conclusion

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).