diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two generation reproduction toxicity (OECD 416), rat: NOAEL reproduction = 396 mg/kg bw/day (males) and 506 mg/kg bw/d (females), NOAEL teratogenicity = 396 mg/kg bw/day (males) and 506 mg/kg bw/d (females), NOAEL systemic = 75 mg/kg bw/day (males) and 99 mg/kg bw/d (females)

Reproduction/developmental toxicity screening (OECD 421), rat: NOAEL reproduction = 392.2 mg/kg bw/day (males) and 494.6 mg/kg bw/d (females), NOAEL systemic = 77.8 mg/kg bw/day (males) and 131.2 mg/kg bw/d (females)

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Dosing of females until weaning (PND 21) and pups for a further 7 days on the respective test diet

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd, Wölferstrasse 4 CH 4414 Füllinsdorf/Switzerland
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) Males: 164-199 g; Females: 133-165 g;
- Fasting period before study: no
- Housing: individually in Makrolon type-3 cages with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, CH 4132 Muttenz/Switzerland).
- Diet (e.g. ad libitum): pelleted standard Kliba 343, (Batches 84/91, 86/91, 87/91) rat/mouse maintenance diet (Klingentalmuehle AG, CH 4303 Kaiseraugst/Switzerland) ad libitum.
- Water (e.g. ad libitum): tap water in bottles ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

other: pelleted food

Details on exposure:

DIET PREPARATION
Dissolved in acetone and mixed to granulated food in a Buehler Mixer, pelleted in a Buehler pelleting machine. Water was added to each feed preparation at an approximate 1:10 volume/weight ratio to ensure pelleting, after which the pellets were dried with warm air for approximately 48 hours
before storage.
- Rate of preparation of diet (frequency): first batch was prepared on April 17, 1991 and the second on May 13, 1991. Thereafter, the diet was prepared at least every 2 weeks.
- Mixing appropriate amounts with (Type of food): granulated feed
- Storage temperature of food: room temperature


VEHICLE
- Justification for use and choice of vehicle (if other than water): admixed to the diet, which is recognized as an efficacious method of absorption.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 8 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- 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:

In this study, analyses of the diet (homogeneity and content) were performed at the start of the prepairing period, at the end of gestation/start of lactation periods and for the last batch prepared. The analyses were performed in the Analytical Laboratories of RCC, Umweltchemie
AG according to an analytical method supplied by the Sponsor.

Duration of treatment / exposure:

P: 50 days (3 weeks prior to pairing, during pairing and until sacrifice), F1 for additional 7 days after weaning

Frequency of treatment:

continuously

Details on study schedule:

- F1 parental animals not mated

Dose / conc.:

1 000 ppm (nominal)

Remarks:

equivalent to 77.8 mg/kg bw/day (males) and 131.2 mg/kg bw/day (females(females during gestation and lactation)

Dose / conc.:

2 000 ppm (nominal)

Remarks:

equivalent to 165.8 mg/kg bw/day (males) and 247.8 mg/kg bw/day (females during gestation and lactation)

Dose / conc.:

4 000 ppm (nominal)

Remarks:

equivalent to 329.2 mg/kg bw/day (males) and 494.6 mg/kg bw/day (females(females during gestation and lactation)

Dose / conc.:

8 000 ppm (nominal)

Remarks:

equivalent to 665.0 mg/kg bw/day (males) and1019.5 mg/kg bw/day (females(females during gestation and lactation)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose levels were based upon a 13-week oral toxicity (feeding) study in rats (RCC Project 285028)

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included: mortality, clinical signs


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: weekly, with the exception of the pairing period. After mating, females were weighed on days 0, 7, 14 and 21 post
coitum. Dams which littered were weighed on days 1, 4, 7, 14 and 21 post partum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly, together with the recording of the body weights, with the exception of the pairing period. During the lactation period, food consumption was recorded only up to day 14 post partum. The relative food consumption ratios and intake of the test article expressed per mg/kg/day were calculated.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

OTHER:
Mating: A record of mating of the females was made by daily vaginal smears for spermatozoa and/or appearance of a vaginal plug throughout the pairing period. The mating data were used to: detect whether or not pregnancy was interrupted after mating, detect marked anomalies of the estrus cycle, determine the mean pre-coital time for the group.

Gestation/parturition:
Towards the end of the gestation period, females were examined twice daily for signs of parturition. Duration of the gestation was calculated. Dams which lost their litter were killed and necropsied together with the dams after weaning of the pups. The uteri of all dams were placed in an aqueous solution of ammonium sulfide to accentuate implantation sites in the dams. The duration of gestation was taken as the time between the day of successful mating and parturition.

Pregnancy rate:
Pregnancy rate was determined as the percentage of surviving paired gravid females.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, on day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield (as near as possible) 4 males and 4 females per litter. The surplus pups were sacrificed and examined macroscopically.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The day 0 of lactation was the day on which the females had delivered all pups. As soon as possible after parturition, the litters were examined for litter size, live birth, stillbirth and any gross anomalies. The sex ratio of pups was recorded on day 0 and day 21 of lactation. Litters were caged together with the dam until weaning on day 21 of lactation. Pups were weighed individually on days 0 (if possible) and/or 1, 4, 7, 14 and 21 of lactation. The dams and pups were observed daily for survival, behavioral abnormalities in nesting and nursing.

Following weaning on day 21 and the removal of the dams, pups which were not selected for organ weight determinations were reared for a further seven days and weighed on day 28 post partum. Food consumption was recorded from day 21 till day 28 post partum.


GROSS EXAMINATION OF DEAD PUPS:
Dead pups were preserved in fixative for possible further examination.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]
All animals were sacrificed and subjected to macroscopic examination. From the dams, implantation sites were counted. Each uterus was placed into a solution of ammonium sulfide to visualize possible hemorrhagic alterations of implantation sites.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations, not further specified.


HISTOPATHOLOGY / ORGAN WEIGHTS
From all parents the following organs were weighed (on day 21 post partum or shortly thereafter) and discarded: prostate, epididymides, pituitary glands, heart, seminal vesicle, spleen, uterus. The body weights were recorded for the calculation of the organ/body weight ratios.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed by CO2 asphyxiation at 28 days of age (7 days after weaning).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: external and internal for abnormalities.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations, not further specified.


HISTOPATHOLOGY / ORGAN WEIGTHS
From each litter two male and two female pups, if possible, (in the average weight of that litter on day 14 post partum) the following organs were weighed (exact on day 21 post partum) and discarded: brain (incl. entire brainstem), prostate, epididymides, pituitary glands heart, seminal vesicle, kidneys, spleen, liver, testes, ovaries, uterus. The body weights were recorded for the calculation of the organ/body weight ratios.

Statistics:

The following statistical methods were used to analyze body weights, food consumption, organ weights, reproduction data and breeding data:
If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the spontaneous mortality of pups data, Fisher's Exact test for 2x2 tables was applied.
Individual values, means, standard deviations and t-statistics were rounded off before printing.

Reproductive indices:

Mating performance, fertility, duration of gestation, implantation rate, birth index, post-implantation loss

Offspring viability indices:

Litter size at first litter check, postnatal loss, viability, weaning indices

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no deaths during the course of this study. No clinical signs of reaction to treatment were noted.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
In all dose groups (1000, 2000, 4000 and 8000 ppm), no test article related effects on the food consumption of the males were noted in both the pre-pairing and after-pairing periods.
Test article related retardation of the body weight gain was noted in the males at 2000 and 4000 ppm during the pre-pairing period and at 8000 ppm during the pre-pairing and after-pairing periods.
No effects of treatment with the test article on the body weight development of the males were evident in the 1000 ppm group.

Females:
At 4000 and 8000 ppm, test article related reduced food consumption was noted between days 7 and 14 of the lactation period. The differences in food consumption between the females of the control group and the 1000 and 2000 ppm groups were not considered to be related to treatment with the test article.
Slight but dose related retardation of the body weight gain was evident in the females at 2000, 4000 and 8000 ppm during the pre-pairing period. Thereafter during the gestation and lactation periods the differences in body weight gain from the control dams were not considered to be related to treatment with the test article.
No effects on the body weight development of the females were noted in the 1000 ppm group.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The mean values for test article intake were based on food consumption and body weight determinations but were not adjusted for the concentrations of HOE 107892 SUBSTANCE TECHNICAL determined upon analysis of the feed.
The mean values for test article intake over the period were based on the mean test article intake or the respective measurement intervals within the defined period.
Mean test article intake values for the males during the pre-pairing and after-pairing periods were:
77.8 mg/kg body weight/day at 1000 ppm;
165.8 mg/kg body weight/day at 2000 ppm;
329.2 mg/kg body weight/day at 4000 ppm;
665.0 mg/kg body weight/day at 8000 ppm.

Mean test article intake for the females during the prepairing period were:
99.9 mg/kg body weight/day at 1000 ppm;
199.1 mg/kg body weight/day at 2000 ppm;
388.0 mg/kg body weight/day at 4000 ppm;
790.1 mg/kg body weight/day at 8000 ppm.

Mean test article intake values during the gestation were:
92.4 mg/kg body weight/day at 1000 ppm;
175.9 mg/kg body weight/day at 2000 ppm;
350.6 mg/kg body weight/day at 4000 ppm;
723.4 mg/kg body weight/day at 8000 ppm.

Mean test article intake values during lactation were:
170.0 mg/kg body weight/day at 1000 ppm;
319.6 mg/kg body weight/day at 2000 ppm;
638.6 mg/kg body weight/day at 4000 ppm;
1315.6 mg/kg body weight/day at 8000 ppm.

The mean test article intake was also calculated for the females during the gestation and lactation periods. These values were:
131.2 mg/kg body weight/day at 1000 ppm;
247.8 mg/kg body weight/day at 2000 ppm;
494.6 mg/kg body weight/day at 4000 ppm;
1019.5 mg/kg body weight/day at 8000 ppm.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Slightly reduced mean number of implantation sites per dam with consequently reduced litter size was noted in the 8000 ppm group (10.4 vs. 11.5 control). This finding was considered to be a slight effect of treatment with the test article. In the 1000, 2000 and 4000 ppm dose groups, there were no adverse effects on the parental reproduction parameters as assessed by the fertility index, conception rate, gestation index, mean precoital time, mean duration of gestation, mean number of implantation sites per dam, postimplantation loss, mean number of living/dead pups per dam and the postnatal loss.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The statistically significant differences were considered to be incidental or to correlate with the reduced mean body weight. The only consistent findings were the significantly increased values for spleen weight at 4000 and 8000 ppm females. This finding was considered to be treatment-related although the respective values in the males at 4000 and 8000 ppm, respectively, did not confirm these findings. This difference between the male and female animals was considered to be caused by the higher test article intake of the females during the lactation period. No statistically significant differences from control values were observed in the 1000 ppm group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Up to and including the highest dose level of 8000 ppm, no abnormal findings were noted at terminal necropsy of the parent animals.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

2 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

4 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

LOAEL

Remarks:

reproduction

Effect level:

8 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: number of implantation sites; litter size

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
In all groups, the postnatal loss before culling (days 0-4 post partum) as well as after culling (days 5-21 post partum) was low and negligible. Health conditions, viability and weaning indexes were similar in all groups. No test article related effects were evident.

CLINICAL SIGNS (OFFSPRING)
In all groups, no treatment related abnormal findings were noted at external examination of the pups. In the 2000 ppm group, irregular development of the coat was noted in all pups of one dam from day 12 post partum up to necropsy day 21 or 28 post partum. This finding was considered to be incidental.

BODY WEIGHT (OFFSPRING)
In the 8000 ppm group, retardation of the body weight gain during the lactation and rearing periods and markedly reduced food consumption during the rearing period (days 21-28 post partum) were noted in the F1 pups. These findings were considered to be test article related. At 4000 ppm, slight retardation of the body weight gain was evident from day 14 post partum through to day 28 post partum. This finding was considered to be a marginal effect of treatment with the test article. No effects on the body weight development of the pups were noted in the 1000 or 2000 ppm groups.

In the 1000, 2000 and 4000 ppm dose groups, food consumption of the F1 pups during the rearing period (days 21-28 post partum) was not affected by treatment with the test article.

SEXUAL MATURATION (OFFSPRING)
Maturation not examined, sex ratios of the F1 pups were not affected by treatment with the test article up to and including the highest dose level of 8000 ppm.

ORGAN WEIGHTS (OFFSPRING)
In both the male and female F1 pups, no test article related effects on the organ weights were evident. The statistically significant differences from the control values were considered to be incidental or to correlate with the reduced terminal body weight for the pups of the 8000 ppm group.

GROSS PATHOLOGY (OFFSPRING)
No test article related abnormal findings were noted at terminal necropsy in the pups of any group.

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

4 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Dose descriptor:

LOAEL

Remarks:

systemic

Generation:

F1

Effect level:

8 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F1

Effect level:

>= 8 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other:

Remarks:

highest dose tested

Reproductive effects observed:

not specified

Conclusion:

A dose level of 1000 ppm was considered to be a NOAEL for systemic toxicity in the parental generation. A dose level of 4000 ppm was considered to be a NOAEL for reproductive toxicity in the parental generation.

In the F1 generation a dose level of 4000 ppm was considered to be a NOAEL for systemic toxicity. No evidence of a teratogenic effect of HOE 107892 substance technical was noted in the F1 generation at any dose level tested (1000, 2000, 4000 or 8000 ppm).

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wölferstrasse 4, CH 4414 Füllinsdorf / Switzerland
- Age at study initiation: (P) 4 wks (at delivery); (F1) x wks
- Weight at study initiation: (P) Males: 134-167 g; Females: 97-135 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study:
- Housing: individually in Makrolon type-3 cages with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/Switzerland). Cages of males were interspersed among those holding females to promote the development of regular estrus cycles.
- Diet (e.g. ad libitum): pelleted standard Kliba 343, rat/mouse maintenance diet (Klingentalmuehle AG, CH 4303 Kaiseraugst/Switzerland) ad libitum (Batch nos. 73/91, 75/91, 76/91, 78/91, 82/92, 84/92, 88/92)
- Water (e.g. ad libitum): Tap water in bottles, ad libitum
- Acclimation period: 10 days under test conditions, after veterinary examination.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

DIET PREPARATION
Dissolved in acetone and mixed to granulated food in a Buehler Mixer, pelleted in a Buehler pelleting machine. Water was added to each feed preparation at an approximate 1:10 volume/weight ratio to ensure pelleting, after which the pellets were dried with warm air for approximately 48 hours
before storage.
- Rate of preparation of diet (frequency): at least every 2 weeks
- Mixing appropriate amounts with (Type of food): granulated food
- Storage temperature of food: room temperature


VEHICLE
- Justification for use and choice of vehicle (if other than water): admixed to the diet, which is recognized as an efficacious method of absorption.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 8 days
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- 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:

The content and homogeneity of the test article in the food pellets was determined at the start of the pre-pairing periods and at the end of the gestation/start of lactation periods. The analyses were performed in the Analytical Laboratories of RCC, Umweltchemie AG according to an analytical method supplied by the Sponsor.

Duration of treatment / exposure:

P: 70-day prepairing period and also during the pairing-, gestation- and lactation periods for breeding the F1 litters (approx. 120 days).
F1: maintainance on their respective diets from the day of weaning (day 21 post partum), during their growth into adulthood (126-days prepairing period) and also during the pairing-, gestation- and lactation periods for breeding the F2 litters (approx. 190 days).

Frequency of treatment:

continuously

Details on study schedule:

- F1 parental animals not mated until weeks after selected from the F1 litters.
- Selection of parents from pups of F1 generation soon after 21 days of age.
- Age at mating of the mated animals in the study: approx. 21 weeks

Dose / conc.:

200 ppm (nominal)

Dose / conc.:

1 000 ppm (nominal)

Dose / conc.:

5 000 ppm (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels were based upon the results of the preliminary study to the two-generation reproduction study in the rat (RCC Project 293422).

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: clinical signs, mortality


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A record of mating of the females was made by examination of the daily vaginal smears for spermatozoa and/or appearance of a vaginal plug throughout the pairing periods. Towards the end of the gestation period, females were examined twice daily for signs of parturition. Duration of the gestation was calculated. As soon as possible after parturition, the litters were examined for litter size, live birth, stillbirth and gross anomalies. The sex ratio of pups was recorded on day 0, 4 and day 21 of lactation. Pups were weighed individually on days 0 (if possible) and/or 1, 4, 7, 14 and 21 of the lactation period. The dams and pups were observed daily for survival and behavioral abnormalities in nesting and nursing.


BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of males and females were recorded at weekly intervals (with exception of the pairing periods). After mating, females were weighed on days 0, 7, 14 and 21 post coitum. Dams which littered were weighed on days 1, 4, 7, 14 and 21 post partum.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The food consumption was recorded weekly, together with the recording of the body weights with exception of the pairing periods. During the lactation periods, food consumption was recorded only till day 14 post partum. The relative food consumption ratios and intake of the test article expressed per mg/kg/day were calculated.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed, examined for possible effects and preserved in neutral phosphate buffered 4 % formaldehyde solution.


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


GROSS EXAMINATION OF DEAD PUPS:
yes, autopsied and/or preserved in fixative for possible further examination.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals when they were no longer necessary for assessment of reproductive effects.
- Maternal animals: All surviving animals when they were no longer necessary for assessment of reproductive effects.


GROSS NECROPSY
From all P and F1 animals selected for pairing, samples of the following tissues and organs were collected at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution:
gross lesions, brain (incl. entire brainstem), heart, lungs, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, testes with epididymides, uterus and cervix (each uterus was first stained according to Salewski and then fixed in neutral phosphate buffered 4 % formaldehyde solution), vagina

Target organs: liver, kidneys, spleen, bone marrow (femur, sternum).

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights:
The following organ weights were recorded from all P animals on day 21 post partum or shortly thereafter:
brain (incl. entire brainstem), epididymides, heart, kidneys, liver, ovaries, prostate, pituitary, seminal vesicles, spleen, testes, uterus.
Organ/body weight ratios were determined.

Histopathology:
Performed from all high dose and control P and F1 animals selected for pairing, and from animals killed in extremis or which died during the study for the following organs: gross lesions, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, testes with epididymides, uterus and cervix (each uterus was first stained according to Salewski and then fixed in neutral phosphate buffered 4 % formaldehyde solution), vagina.
Additionally, from the P and F1 animals of the 200 and 1000 ppm group the spleen was examined histologically.
The reproductive organs of infertile males and females were examined histologically from the control and the high dose groups (P and F1 animals).
Organs with macroscopic abnormalities were examined histologically from the control and the high dose groups (P and F1 animals) and from the low- and mid-dose groups (P animals).

Postmortem examinations (offspring):

SACRIFICE
- Excess F1 and F2 pups after standardization of litter sizes, were sacrificed on day 4 post partum, examined for possible defects and preserved in neutral phosphate buffered 4 % formaldehyde solution. The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning.
- One male and one female each of these animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:


GROSS NECROPSY
From all F1 animals selected for pairing and from one male and one female pup (selected for organ weight recording) of each F1 and F2 litter, samples of the following tissues and organs were collected at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution:
gross lesions, brain (incl. entire brainstem), heart, lungs, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, testes with epididymides, uterus and cervix (each uterus was first stained according to Salewski and then fixed in neutral phosphate buffered 4 % formaldehyde solution), vagina

Target organs: liver, kidneys, spleen, bone marrow (femur, sternum).


HISTOPATHOLOGY / ORGAN WEIGTHS
Organ weights:
The following organ weights were recorded from all F1 parent animals and from one male and one female pup of each F1 and F2 litter (in the average weight of that litter on day 14 post partum), pups exact on day 21 post partum, parent animals on day 21 post partum or shortly thereafter: brain (incl. entire brainstem), epididymides, heart, kidneys, liver, ovaries, prostate, pituitary, seminal vesicles, spleen, testes, uterus.
Organ/body weight ratios were determined.

Histopathology was performed from all high dose and control F1 animals selected for pairing, from animals killed in extremis or which died during the study, and from one male and one female pup (selected for organ weight recording) of high dose and control F1 and F2 litters for the following organs: gross lesions, ovaries, pituitary gland, prostate, seminal vesicles with coagulating gland, testes with epididymides, uterus and cervix (each uterus was first stained according to Salewski and then fixed in neutral phosphate buffered 4 % formaldehyde solution), vagina.
Additionally, from the F1 animals of the 200 and 1000 ppm group the spleen was examined histologically.
The reproductive organs of infertile males and females were examined histologically from the control and the high dose groups (F1 animals).
Organs with macroscopic abnormalities were examined histologically from the control and the high dose groups (F1 animals and F2 pups).

Statistics:

The following statistical methods were used to analyze body weights, food consumption, organ weights, reproduction data and breeding data:
If the variables could be assumed to follow a normal distribution, the Dunnett many-one t-test, based on a pooled variance estimate, was used for intergroup comparisons (i.e. single treatment groups against the control group).
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the spontaneous mortality of pups data, Fisher's Exact test for 2x2 tables was applied.
Individual values, means, standard deviations and t-statistics were rounded off before printing.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment-related deaths and clinical signs were observed.
Incidental findings observed in single animals of various dose groups were slight ruffled fur, irregular respiration, rales, slight hunched posture, sedation, body weight loss, frightness, blood in the nose region, ataxia and hairless area on the flanks and/or back.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
In both the P and F1 generations, there were no effects on food consumption of the males in all dose groups (200, 1000 and 5000 ppm). The marginal reduction in food consumption noted in the 5000 ppm males of both generations was related to the reduced mean body weights in these males. This finding was confirmed by the relative food consumption values which were similar or even higher in the 5000 ppm males in comparison with the control males.
At 5000 ppm, test article-related retardation of the body weight gain was evident in the males of both parental generations (P and F1) during the first phase of the pre-pairing period. Thereafter the body weight gain of the 5000 ppm group animals was comparable to that of the respective control animals. In both generations, no test article related effects on the body weight development of the male animals were noted in the 200 or 1000 ppm groups.

Females:
In the P generation females, test article related reduction in food consumption was noted in the females at 5000 ppm during the gestation and lactation periods for breeding F1 pups. No effects on food consumption were noted in the P females at 200 ppm or 1000 ppm or in the females of the F1 generation in all dose groups.
At 5000 ppm, test article related retardation of the body weight gain was evident in the females of the P generation during the first 36 days of the pre-pairing period. Thereafter the body weight gain of the 5000 ppm group animals was comparable to that of the respective control animals. No test article related effects on the body weight development of the female animals were noted in the 200 or 1000 ppm groups of the P generation or in the females of the F1 generation in all dose groups.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Males:
Mean values for test article intake, based on food consumption and body weight determinations but not adjusted for the concentrations of HOE 107892 SUBSTANCE TECHNICAL determined upon analysis of the feed, varied within the following ranges:
P generation:
22.7 - 11.4 (15.0)* mg/kg body weight/day at 200 ppm
115.1 - 57.7 (76.0)* mg/kg body weight/day at 1000 ppm
590.7 - 304.8 (393.8)* mg/kg body weight/day at 5000 ppm

F1 generation:
26.3 - 11.0 (14.8)* mg/kg body weight/day at 200 ppm
130.2 - 55.4 (74.4)* mg/kg body weight/day at 1000 ppm
671.2 - 300.2 (396.7)* mg/kg body weight/day at 5000 ppm

Means of P and F1 generation during the entire study:
15* mg/kg body weight/day at 200 ppm
75* mg/kg body weight/day at 1000 ppm
396* mg/kg body weight/day at 5000 ppm
* = Mean of Means

Females:
Mean values for test article intake, based on food consumption and body weight determinations but not adjusted for the concentrations of HOE 107892 SUBSTANCE TECHNICAL determined upon analysis of the feed, varied within the following ranges:
P generation:
Prepairing and gestation periods
23.8 - 14.3 (18.4)* mg/kg body weight/day at 200 ppm
124.0 - 70.1 (92.2)* mg/kg body weight/day at 1000 ppm
637.2 - 366.8 (466.3)* mg/kg body weight/day at 5000 ppm

Lactation period
38.7 - 24.7 (32.1)* mg/kg body weight/day at 200 ppm
193.4 - 127.0 (161.2)* mg/kg body weight/day at 1000 ppm
944.0 - 621.1 (779.6)* mg/kg body weight/day at 5000 ppm

F1 generation:
Prepairing and gestation periods
27.1 - 14.0 (17.6)* mg/kg body weight/day at 200 ppm
133.0 - 71.1 (87.0)* mg/kg body weight/day at 1000 ppm
670.7 - 364.2 (453.5)* mg/kg body weight/day at 5000 ppm

Lactation period
38.6 - 22.3 (31.0)* mg/kg body weight/day at 200 ppm
180.4 - 110.4 (147.3)* mg/kg body weight/day at 1000 ppm
955.9 - 579.7 (768.6)* mg/kg body weight/day at 5000 ppm

Means of P and F1 generation during the entire study:
20* mg/kg body weight/day at 200 ppm
99* mg/kg body weight/day at 1000 ppm
506* mg/kg body weight/day at 5000 ppm
* = Mean of Means

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In all dose groups (200, 1000 and 5000 ppm), the reproduction data of the P and F1 generation animals - as assessed by: mating performance and fertility, mean precoital time, duration of gestation, mean number of implantation sites per dam, post-implantation loss, mean number of live or dead pups per dam at parturition and the postnatal loss - were not considered to be affected by treatment with the test article.
Post-implantation loss in the P generation was 5.3, 10.3, 12.5 and 11.0 % in the control, 200 ppm, 1000 ppm and 5000 ppm groups, respectively, and was therefore statistically significantly increased in all dose groups in comparison with the control group. As a consequence of this increased post-implantation loss, the birth index (number of pups born alive in percent of number of implantations) was statistically significantly reduced in all dose groups in comparison with the control group. These findings were not considered to be test article related because no dose-relationship was evident and all values were in the normal range of the historical control data.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The only consistent finding which was considered to be test article related was the increase in spleen weight in the 5000 ppm group parent animals of both the P and F1 generations. In both generations, the females had statistically significantly increased absolute and relative spleen-to-body weight ratio whereas in the males only the relative spleen weight was statistically significantly increased from the respective control value. This finding was particularly evident in the female animals probably due to the higher test article intake during the lactation period. All other statistically significant differences from the control values were considered to be incidental or for the 5000 ppm group animals to be correlated with the reduced mean body weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test article related abnormal findings were noted at macroscopic examination in both generations (P and F1) of the parent animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In both parental generations (P and F1), a slight increase in splenic haematopoiesis in the females at 5000 ppm with an equivocal increase in the males of the same dose group was noted at microscopic examination. This finding correlated with the values for spleen weight and was considered to be related to treatment with the test article.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: corresponding to approx. 75 mg/kg bw/day for males and 99 mg/kg bw/day for females

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: corresponding to approx. 396 mg/kg bw/day for males and 506 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

>= 5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: corresponding to approx. 396 mg/kg bw/day for males and 506 mg/kg bw/day for females

Remarks:

highest dose tested

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, treatment-related

VIABILITY (OFFSPRING)
No effects of test article were evident at first external examination of the F1 or F2 pups or during the lactation periods.

CLINICAL SIGNS (OFFSPRING)
No effects of test article were evident at first external examination of the F1 or F2 pups or during the lactation periods.

BODY WEIGHT (OFFSPRING)
In the F1 as well as in the F2 pups of the 5000 ppm group, retardation of the body weight gain was noted during the lactation period. This finding was
considered to be test article related. No effects of treatment with the test article on the body weight development of the F1 or F2 pups were evident in the 200 or 1000 ppm.

SEXUAL MATURATION (OFFSPRING)
No effects of test article were evident at first external examination of the F1 or F2 pups or during the lactation periods. The sex ratios of the pups were not affected by treatment with the test article.

ORGAN WEIGHTS (OFFSPRING)
In both the F1 and F2 pups, the differences in organ weights between the control group and the dose groups were considered to be incidental or for the 5000 ppm group pups to be correlated with the reduced mean body weight. No test article related effects were evident.

GROSS PATHOLOGY (OFFSPRING)
Up to and including the highest dose level of 5000 ppm, there were no test article related findings at macroscopic examination in the F1 or F2 pups.

HISTOPATHOLOGY (OFFSPRING)
Microscopic examination in the F2 pups gave no indications of test article related effects. Test article related effects observed for the F1 generation were a slight increase in splenic haematopoiesis in the parental females at 5000 ppm with an equivocal increase in the males of the same dose group.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F1

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: corresponding to approx. 75 mg/kg bw/day for males and 99 mg/kg bw/day for females

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Generation:

F1

Effect level:

>= 5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: corresponding to approx. 396 mg/kg bw/day for males and 506 mg/kg bw/day for females

Remarks:

highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F1

Effect level:

>= 5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other: corresponding to approx. 396 mg/kg bw/day for males and 506 mg/kg bw/day for females

Remarks:

highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Generation:

F2

Effect level:

1 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: corresponding to approx. 75 mg/kg bw/day for males and 99 mg/kg bw/day for females

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Generation:

F2

Effect level:

5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: corresponding to approx. 396 mg/kg bw/day for males and 506 mg/kg bw/day for females

Remarks:

highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Generation:

F2

Effect level:

>= 5 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed

Remarks on result:

other: highest dose tested

Key result

Reproductive effects observed:

not specified

Conclusion:

In this two-generation reproduction study in rats with HOE 107892 SUBSTANCE TECHNICAL, dietary concentrations of 0 (control), 200, 1000 and 5000 ppm were used.

At 5000 ppm, reduction in food consumption was evident in the females of the P generation during the gestation and lactation periods. Reduction of the body weight gain was noted in the males of both parental generations and in the females of the P generation during the first part of the prepairing period. Retardation of the body weight development was also evident in the Fl and F2 pups during the respective lactation period. At microscopic examination, the parent animals of both generations had an increase in splenic haematopoiesis which correlated with the increase of the spleen weight. These findings were more evident in the females than in the males probably caused by the higher test article intake during the lactation period.

In both generations, the parental reproduction parameters - as assessed by the fertility index, conception rate, gestation index, mean precoital time, duration of gestation, mean number of implantation sites per dam, post-implantation loss, number of living or dead pups at first litter check and postnatal loss - were not affected by treatment with the test article up to and including the highest dietary concentration of 5000 ppm.

Up to and including 1000 ppm, no relevant changes or differences were noted neither in either parent generations or in the respective progeny.

Based on these results, the no-observable adverse effect level (NOAEL) for systemic toxicity in the parent animals and for the progeny was considered to be 1000 ppm corresponding to approximately 75 mg/kg body weight/day* for the males and 99 mg/kg body weight/day* for the females. For the parental reproduction parameters, the no-observable adverse effect level (NOAEL) was considered to be 5000 ppm of the test article in the diet corresponding to approximately 396 mg/kg body weight/day for the males and 506 mg/kg body weight/day* for the females of either generation.

Up to and including the highest dose level of 5000 ppm, no teratogenic effect was noted at external examination of the pups.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

396 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate and reliable studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.7, of Regulation (EC) No. 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A two-generation reproduction toxicity study was performed in rats according to OECD guideline 416 and compliant with GLP. Test item doses of 200, 1000 and 5000 ppm were administered to the P- and the F1-generation in the feed during the prepairing, pairing, gestation and lactation periods, in F1-animals beginning from the day of weaning and during their growth into adulthood. Effects were assessed separately for the parental animals of each generation and those animals of each generation not used for breeding. Based on the systemic effects observed for P- and F1-animals (body weight, food consumption, organ weights, histopathology) and for the F2-animals (body weight) the NOAEL for systemic toxicity was determined to be 1000 ppm (75 mg/kg bw/d males, 99 mg/kg bw/d females). The reproductive parameters of the P- and the F1-generation were not considered to be affected by treatment with the test article, any parameters showing statistically significant differences from the control group were within the normal range of the historical control data and therefore not considered to be test substance-related, the NOAEL for reproductive toxicity was therefore determined to be 5000 ppm (396 mg/kg bw/d males, 506 mg/kg bw/d females). No teratogenic effects were observed in the pups of the F1- and the F2 -generation in this study up to the highest dose level, therefore, the NOAEL for teratogenicity was determined to be 5000 ppm, as well.

In addition, a reproduction/developmental toxicity screening was performed in rats according to OECD guideline 421 and compliant with GLP. In the screening test, NOAELs for systemic toxicity of 1000 (77.8 mg/kg bw/day males, 131.2 mg/kg bw/day females) and 4000 ppm (329.2 mg/kg bw/day males, 494.6 mg/kg bw/day females) were determined for P- and F1-animals, respectively, based on retardations of body weight gain. The NOAEL for reproductive toxicity of the P-animals was determined as 4000 ppm, based on reduced number of implantation sites and reduced litter size at a dose level of 8000 ppm. In this study no teratogenic effects were observed up to and including the highest dose level.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity (OECD 414), rat (GD 7-16): NOAEL maternal = 200 mg/kg bw/day; NOAEL developmental = 200 mg/kg bw/day

Prenatal developmental toxicity (OECD 414), rat (GD 7-16): NOAEL maternal ≥ 1000 mg/kg bw/day; NOAEL developmental ≥ 1000 mg/kg bw/day; NOAEL teratogenicity ≥ 1000 mg/kg bw/day

Prenatal developmental toxicity (OECD 414), rabbit (GD 6-18): NOAEL maternal = 100 mg/kg bw/day; NOAEL embryotoxicity = 100 mg/kg bw/day; NOAEL teratogenicity ≥ 250 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst breeding colony
- Age at study initiation: about 65 - 70 days
- Weight at study initiation: mean 190 ± 12 g
- Fasting period before study: no
- Housing: individual plastic cages on wood-shavings.
- Diet (e.g. ad libitum): Altromin 1310 pellets (Altromin GmbH in D-4937 Lage/Lippe), ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 48 - 55
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: starch mucilage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed freshly every day in starch mucilage.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Easier handling for gavage
- Concentration in vehicle: 200 g/L
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the mixtures were guaranteed for a period of 4 hours (statement from the Analytical Laboratory, dated 06 February 1991).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 16 hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

GD7-16

Frequency of treatment:

daily

Duration of test:

until GD21

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 - 21

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: limit test

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: behaviour, general health conditions


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: continuously, body weight gain once weekly and one day after final application


FOOD CONSUMPTION: Yes, continuously
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Yes


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


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: After caesarean section the dams were dissected and their organs examined macroscopically. Heart, liver, kidneys and spleen were weighed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead foetuses, number of placentae, placenta weights, diameter of resorption sites, foetal weights, crown-rump lengths

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:

Statistical evaluation of the experimental data was carried out by comparing the animals in the dose group with the included control group and also by comparing both groups with the control values from previous studies (normal ranges). For this purpose, body and organ weights, body weight gains, daily food consumption per 100 g body weight, live foetuses and intrauterine deaths, foetal weights, crown-rump lengths and placental weights were taken together as parameter groups. The numbers of implantates and the numbers of corpora lutea were also evaluated.

For comparison of body and organ weights (g) in dams and body weights (g) during gestation standard MANOVA with sequentially rejective multiple comparisons (P<0.05) was performed.
For statistical evaluation of daily food consumption (g)/100 g body weight (relative food consumption) during gestation the nonparametric linear model of PURI/SEN with sequentially rejective multiple comparisons (P < 0.05) was used.
The results at caesarian section (number of implantations, number of corpora lutea, live foetuses and intrauterine deaths, foetuses under resorption) were compared by the Mantel-Haenszel test with sequentially rejective multiple comparisons (P < 0.05).
Differences in body weight, crown/rump length and placental weight in live foetuses were analysed by MANOVA with sequentially rejective multiple comparisons (P < 0.05), the analysis had been corrected for random litter effects.
Morphological findings in foetuses and litters were compared by the exact Fisher Test (1-tailed) (P< 0.05).
Animals not surviving to day 21 or having an early PM on day 21 or were not pregnant or having only resorptions were excluded from the means.

Historical control data:

For comparing the data with historical controls, ranges of normal variation for groups were calculated. These were drawn up in such a way as to include with a probability of 95% at least 75% of the values for a group of control animals. The parallelogram method of ABT (1982) was employed for organ weights and body weights, and the parallelepiped method of WALD (1943) for body weight gains and food consumption among the dams and mean foetal weights, crown-rump lengths and placental weights per litter. Comparison of corpora lutea and implantates was performed with univariate normal ranges by the method of WILKS (1942) . For the ratios of live and dead foetuses and of foetuses under resorption, normal ranges were determined by the triangular method of ROSENKRANZ (1988).

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No toxic effects except for a slightly increased spleen weight.

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No evidence could be found for a teratogenic effect of the test substance.

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Developmental effects observed:

not specified

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Up to postnatal day 47 including behavioural and functional tests of offspring

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Up to postnatal day 47 including behavioural and functional tests of offspring

Principles of method if other than guideline:

The purpose of the study was to supplement an embryotoxicity study conducted earlier in compliance with the aforementioned Guidelines including postnatal development.

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst breeding colony
- Age at study initiation: about 65 - 70 days
- Weight at study initiation: mean 190 ± 7.8 g
- Fasting period before study: no
- Housing: individual plastic cages on wood-shavings.
- Diet (e.g. ad libitum): Altromin 1310 pellets (Altromin GmbH in D-4937 Lage/Lippe), ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 52 - 60
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: starch mucilage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed freshly every day in starch mucilage.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Easier handling for gavage
- Concentration in vehicle: 200 g/L
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the substance in suspension and the homogeneity of the suspension were guaranteed for 6 hours (statement from the Analytical Laboratory of HOECHST AG, dated 24 August 1990).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 16 hours
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

GD7-16

Frequency of treatment:

once daily

Duration of test:

dams, pups taking place in function tests: until days 21 - 23 post partum; pups taking place in behaviour tests up to day 47 post partum

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 - 21

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: limit test

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: behaviour, general health conditions


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: continuously, body weight gain once weekly and one day after final application


FOOD CONSUMPTION: Yes, continuously during gravidity, same intervals as body weights
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Yes


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


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on post natal days # 21 - 23 and 44, respectively
- Organs examined: weights of heart, liver, kidneys and spleen. In addition, the implantation sites were counted in the isolated uterus after fixing in Carnoy's fluid.

OTHER:
Mean duration of gravidity.

Ovaries and uterine content:

The uterine content was examined after termination: Yes
Examinations included:
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes: all per litter (Body weight, sex and any external anomalies)

Statistics:

Simultaneous comparisons were drawn between the substance group and the control group, and comparisons were also carried out with previous historical control values. The procedure of Nemenyi/Dunnett was used to show whether the values for food consumption and body and organ weights of the dams showed any tendency to change as compared with the control. Median and MD68 (sextile differences) for the time periods or organs in question were calculated. The body weight values calculated for offspring were mean weight, variation, median and MD68. Comparisons with the control group were made by the Nemenyi/Dunnett test. The values calculated for the organ weights of the offspring were means and variations, and the data were evaluated statistically by the Wilcoxon test. Implants, live and dead pups and supernumerary implantation sites were given both in absolute numbers and as mean values. Implants and survival times were evaluated by a permutation test, and the other parameters by the procedure of Goodman.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed.

Remarks on result:

other: The slight impairment of body weight gain among the pups indicates that the test dose was near the tolerance limit for the progeny.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
A slight delay of body weight gains was observed from birth up to the end of the lactation period. These pups did not differ from controls in physical development or exhibit any functional or behavioural disturbances. Rearing rate was not adversely affected up to the time of weaning. Autopsy yielded no indications of damage to the internal organs

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

Himalayan

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst breeding colony
- Age at study initiation: about 6 - 10 months
- Weight at study initiation: mean body weight of 2639 ± 152 g
- Housing: individual metal-barred cages on wire-mesh floors
- Diet (e.g. ad libitum): ERKA 6000 standard diet in the form of pellets 4 mm in diameter (manufactured by Robert Koch OHG, Hamm/Westphalia), ad libitum; also 40-50 g autoclaved hay daily.
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-23.5
- Humidity (%): 43-62
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 10/14

Route of administration:

oral: gavage

Vehicle:

other: starch mucilage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspended freshly each day in starch mucilage (20 g potato starch per litre distilled water)

VEHICLE
- Justification for use and choice of vehicle (if other than water): handling at dosing
- Concentration in vehicle: 8, 20, 50 g/L
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the suspensions were guaranteed for a period of at least 4 hours (statements from the Analytical Laboratory, dated 24 August 1990 and 06 February 1991).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: during the morning hours, not further specified
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: Those females with sperm in the vaginal smear were mated again after about 6 hours in case the first mating had not been successful.

Duration of treatment / exposure:

GD 6-18

Frequency of treatment:

once daily

Duration of test:

until GD29

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

15 - 16 mated females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Preliminary range finder study with groups of 2-4 mated Himalayan rabbits administered doses of 200, 320 or 500 mg/kg once daily from the 6th to the 18th day after mating.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: behaviour, general health condition


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: prior to first treatment, once weekly (body weight gains) in the first 3 weeks, GD 19 and GD 29


FOOD CONSUMPTION: Yes, same intervals as body weight
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Yes


WATER CONSUMPTION: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: gall-bladder, heart, intestines, kidney, liver, spleen, ovary, uterus; (heart, liver, kidneys and spleen were weighed)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live and dead foetuses, placentae, placental weights, diameter of conceptuses under resorption, crown/rump length, foetal weight, sex, survival rate after 24h incubator

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter (brain, eyes, heart and both kidneys)
- Skeletal examinations: Yes: all per litter
- Head examinations: No

Statistics:

Comparisons with the control group of body weights and organ weights: standard MANOVA with sequentially rejective multiple comparisons, and relative food consumption by means of the nonparametric linear model of PURI & SEN (1985) with sequentially rejective multiple comparisons.
The Mantel-Haenszel test with sequentially rejective multiple comparisons (MANTEL & HAENSZEL, 1959) was used for analysing implants, corpora lutea, survival rates, ratios of live and dead foetuses, and conceptuses under resorption. The foetal weights, crown-rump lengths and placental weights were evaluated by means of a multivariate variance analysis corrected for litter effects. With each of these methods the probability of error for each group of parameters was 5%.
The findings at autopsy and at the examinations of organ cross-sections and skeletons were evaluated for foetuses and litters separately by the exact Fisher test. This tested whether the relative incidences of these findings in the dose groups differed from those in the control. For comparing the data with historical controls, ranges of normal variation for groups were calculated. These were drawn up in such a way as to include with a probability of 95 % at least 75 of the values for a group of control animals. The parallelipiped method of WALD (1943) was employed for body weight gains and food consumption among the dams and for mean foetal weights, crown-rump lengths and placental weights per litter. Comparisons of corpora lutea, implants and survival rates were performed with univariate normal ranges by the method of WILKS (1942). For the ratios of live and dead foetuses and of conceptuses under resorption, normal ranges were determined by the triangular method of ROSENKRANZ (1988).

Historical control data:

A group of animals of a given size is normal with respect to a parameter, if at least 75 % of the values lie within the ranges of the historical control groups.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
One dam in the 250 mg/kg group aborted during the night from days 22 to 23 of gravidity. Four dams had vaginal haemorrhages, which pointed to an abortion - the first on day 16, the second on day 20, the third on day 23, and the fourth during the night from days 16 to 17 of gravidity. One
other dam delivered prematurely on day 27 of gravidity. In addition to these findings, a decrease or absence of excrement was observed on one or several days between days 11 and 27, involving one dam from the 40 mg/kg group, two dams in the 100 mg/kg group and about half of the dams in the 250 mg/kg group. One dam in the 250 mg/kg group had soft excrement on days 19 and 23 of gravidity. The dams in this group which were killed intercurrently due to abortions had shown marked reductions in food consumption during the one or two weeks prior to killing. The considerably lower
food consumption was matched by reduced quantities of excrement. Lower food consumption was accompanied by reduced water consumption. In the 250 mg/kg group almost all the dams showed generally slight, but in several cases also more marked, body weight losses during the 1st week of treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed

Key result

Dose descriptor:

LOAEL

Remarks:

maternal toxicity

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

pre and post implantation loss

water consumption and compound intake

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
One dam in the 250 mg/kg group had only empty implantation sites in the uterus. There were also five dams in the 250 mg/kg group which had aborted with clinical signs. The animal killed on day 16 of gravidity had 3 empty implantation sites in addition to 6 conceptuses under resorption. Two dams had only conceptuses under resorption in the uterus: 7 in the one which was killed on day 17 of gravidity (having aborted in the night from days 16 to 17) , and 8 in the one which was killed on day 23. The animal killed on day 20 of gravidity had 3 conceptuses under resorption and also 5 dead foetuses, 3 of them severely stunted.
There was no evidence for teratogenic effects.

Key result

Dose descriptor:

NOAEL

Remarks:

embryotoxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Key result

Dose descriptor:

LOAEL

Remarks:

embryotoxicity

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

changes in litter size and weights

Key result

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

>= 250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Remarks on result:

other:

Remarks:

highest dose tested

Key result

Developmental effects observed:

not specified

Selected maternal parameters:

	Dose (mg/kg bw)
Parameter	0	40	100	250
Dams with live foetuses	13	14	15	8
Number of live foetuses (total)	86	82	84	46

Conclusion:

Based on the results of this study, the NOAEL for maternal toxicity and embryotoxicity in the rabbit was 100 mg/kg bw/day Hoe 107892. No teratogenic effect was observed at any dose level tested.

5/6 abortions observed in the high dose group occurred in dams which demonstrated the most severe characteristics of reduced food and water consumption, body weight loss, and reduced defecation in this group.

It is widely known that food deprivation of does during the phase of organogenesis has significant effects on the development of their offspring and the dams themselves, manifesting as reduced body weight gain or even loss, reduced water consumption and increased number of abortions. Publications are available demonstrating this phenomenon: Petrere, J.A. et al. (1993). Fundam. Appl. Toxicol. 21: 517-522; Matsuzawa, T. et al. (1981). Toxicology 22: 255-259.

Therefore, abortions and embryotoxicity observed in this study have to be considered as secondary effects due to maternal toxicity, induced by reduced food uptake and body weight loss of the dams during the phase of organogenesis.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstraße 27, 33178 Borchen, GERMANY
- Age at study initiation: approximately 10-12 weeks
- Weight at study initiation: range of group means: 204.9 ± 6.5 to 211.0 ± 11.2 g
- Housing: in fully air-conditioned rooms in makrolon cages (type III) on soft wood granulate
- Diet (e.g. ad libitum): Ssniff R-Z (V1324), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0-22.0
- Humidity (%): 37-84
- Air changes (per hr): 16-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: starch mucilage

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was mixed freshly every day in starch mucilage (20 g/l). Homogeneity and stability of the test compound in the vehicle were assessed prior to the start of the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): handling at dosing
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each concentration, samples were taken towards the start and end of the dosing period (day 7 to 16) and analysed.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

GD7-16

Frequency of treatment:

once daily

Duration of test:

GD21

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

29, 38, 23 and 29, respectivley (0, 40, 200, 1000 mg/kg dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The substance was tested in a limit test for effects on embryonic and foetal development. No clear maternal NOAEL was established in this study due to slightly increased spleen weights. There was no evidence for embryofoetal toxicity. Two rare malformations of the head (acrania, malformation of the facial skull) were considered to be spontaneous events with regard to the historical control data. The aim of the present study is the verification of the findings from the limit test and to estimate a clear NOAEL. The design of the present study and the rat strain used are nearly identical to those of the limit test.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: several times daily (on weekends and public holidays once daily)
- Cage side observations included: behaviour, general health condition


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: days 1, 4, 7, 10, 14, 17, 19 and 21 of pregnancy

FOOD CONSUMPTION: Yes, between days 1-4, 4-7, 7-10, 10-14, 14-17, 17-19 and 19-21 of pregnancy
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes (per group)

WATER CONSUMPTION: No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: organ weights of heart, liver, kidneys and spleen


OTHER:

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
- Other: Foetuses (gross external abnormalities), placentae, sex

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:

Statistical evaluation: based on the assumption of a monotone dose-response relationship. Statistical comparisons of low dose groups with the control group only if significant effects detectable in the high dose group (Hothorn & Lehmacher, 1991). In the univariate analysis, two-sided questions (body weight of dams, relative food consumption, crown-rump length, foetal weight and placental weight) were generally tested by a two-sided comparison with the high dose group followed by a one-sided test for the low-dose group. Caesarean section data of the foetuses (crown-rump length, foetal weight and placental weight): multivariate statistics were calculated and used in selecting relevant dose groups. Individual parameters: sequential comparisons with the high dose group (Hochberg, 1988) and sequential tests at the 5% level (Hothorn & Lehmacher, 1991) for the low dose. The t-tests and the test statistics of Wilks (Hartung & Elpelt, 1984) were based on common variance estimations for all study groups. For the Wilcoxon test (Hollander & Wolfe, 1973) the exact distribution (Streitberg & Rohmel, 1987) of the meaned ranks was calculated. Daily food consumption of the dams: mean consumption per 100 g body weight always calculated between two successive measurement times and evaluated by the rank sum test after Wilcoxon. Body weights of the dams: weight change determined in comparison to the initial weight. The univariate evaluation was carried out using t-tests. Caesarean section data of the foetuses were used to calculate litter mean values. Multivariate evaluation was carried out using the test statistics of Wilks. In the univariate analysis, t-tests were used. Number of corpora lutea, implantation sites, live foetuses, quotas of dead embryonic primordia undergoing resorption: one-sided Wilcoxon tests. Findings at autopsy, body cross-section and skeletal examination of foetuses evaluated separately for foetuses and litters: Fisher's Exact test (significance levels 5 and 1 %).

Historical control data:

Percentages of the control groups of 5 preceding tests using Wistar rats (June 1996 - June 1997) were given as table (early intrauterine deaths, live fetuses per litter).

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
One female of the 1000 mg/kg group exhibited bloody secretion on the vagina on day 15 of pregnancy and showed only one conceptus undergoing resorption at caesarean section. No other clinical signs were observed in the animals treated with test substance. The animals demonstrated decreased body weight (approx. -20 %) and food consumption and increased spleen weights at a dose of 1000 mg/kg bw/day. Food consumption was slightly decreased at the dose of 200 mg/kg body weight per day (approx. -5 %).

Key result

Dose descriptor:

NOEL

Remarks:

maternal toxicity

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed

Key result

Dose descriptor:

LOAEL

Remarks:

maternal toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Retardations (non-ossified metacarpale 5 of the forepaw and non-ossified phalanx III of the toes of the hindpaw) were observed more frequently in the foetuses at the maternally toxic dose level of 1000 mg/kg bw/day. No embryotoxic effects were observed at 200 mg/kg bw/day. All other findings were not considered to be treatment-related.

Key result

Dose descriptor:

NOEL

Remarks:

developmental

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effect observed

Key result

Dose descriptor:

LOAEL

Remarks:

developmental

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Key result

Developmental effects observed:

not specified

Treatment-related statistically significant increases in the incidences of foetal retardations:

	Dose groups (mg/kg bw/d)
Retardation	0	40	200	1000
Non-ossified metacarpale of forepaw	17/132	26/153	13/129	50/123*
Non-ossified phalanx III of hindpaw toes	1/132	6/153	3/129	21/123*

*statistically significant

Conclusion:

Based on the results of this study, the NOEL and NOAEL were considered to be 40 mg/kg bw/day and 200 mg/kg bw/day, respectively, for maternal toxicity and 200 mg/kg bw/day for developmental toxicity. Embryotoxic effects observed in form of retardations occurred at the maternally toxic dose level of 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Species:

rabbit

Quality of whole database:

The available information comprises adequate and reliable (Klimisch score 1) studies performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex IX-X, 8.6, of Regulation (EC) No. 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There is data from four prenatal developmental toxicity studies available, of which three were conducted in rats and one in rabbits. All studies were conducted according to OECD guideline 414 and in compliance with GLP. Three groups of rats received daily doses of 40, 200 or 1000 mg/kg bw/d from gestation day 7 to 16, the NOAEL for maternal toxicity was determined to be 200 mg/kg bw/d based on the findings of decreased body weight and food consumption and increased spleen weight at the highest dose level. The only developmental effects observed were retardation, in detail non-ossified metacarpale 5 of the forepaw and phalanx III of the hindpaw toes, which occurred in the highest dose group, the maternally toxic dose. No embryotoxic effects were observed at 200 mg/kg bw/d, therefore, this dose level was determined to be the NOAEL for developmental toxicity.

 

The two preceding tests were designed as limit tests, both conducted at a dose level of 1000 mg/kg bw/d applied between gestation days 7 and 16. In both studies 1000 mg/kg bw/d was established as NOAEL, the observed effects were only slight and, therefore, not unequivocally considered toxicologically relevant. One of these studies additionally included a postnatal period of up to 47 days in which behavioural and functional parameters were assessed. Again, no impairments were observed.

 

The fourth study was conducted with rabbits as representatives for non-rodents. Doses of 40, 100 and 250 mg/kg bw/d were applied between GD 6 to 18. In the highest dose group maternal toxicity in form of an increased number of abortions and reduced food and water consumption, accompanied by weight loss and reduced defecation, were observed. Embryotoxic effects manifested as reduction in litter size, empty implantation sites, increased number of conceptuses under resorption and less dams with live foetuses.

However, in the high dose group 6/15 dams aborted, and 5/6 of these dams showed a marked loss of body weight during the second and/or third week of gestation, accompanied by reduced food and water consumption and reduced defecation prior to the abortions, and these effects were observed in further dams of this group, as well.

It is widely known that reduced food uptake of does during the phase of organogenesis, e.g. induced by food deprivation or other conditions preventing them to take up sufficient amounts of food, has significant effects on the development of their offspring and the dams themselves, manifesting as reduced body weight gain or even loss, reduced water consumption and increased number of abortions in the dams. Publications are available demonstrating this phenomenon: Petrere, J.A. et al. (1993). Fundam. Appl. Toxicol. 21: 517-522; Matsuzawa, T. et al. (1981). Toxicology 22: 255-259.

Therefore, embryotoxicity observed at 250 mg/kg bw/d in this study has to be considered as secondary effect due to severe maternal toxicity during the phase of organogenesis.

There was no evidence of teratogenicity. Therefore, the NOAEL for maternal and embryotoxicity in rabbits was determined to be 100 mg/kg bw/d, the NOAEL for teratogenicity was 250 mg/kg bw/d under the conditions of this study.

Justification for classification or non-classification

The available data on toxicity to reproduction and developmental toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.