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

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

603-923-2

Cas Number:

135590-91-9

Molecular formula:

C16H18Cl2N2O4

IUPAC Name:

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 ppm (nominal)

Doses / concentrations 3

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).

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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

2

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

3

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

1

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

2

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

3

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

Results: F2 generation

Effect levels (F2)

1

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

2

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

3

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

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Any other information on results incl. tables

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.