Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991-01-31 to 1991-09-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
other: Toxicological principles for the Safety Assessment of Direct Food Additives and Color Additives used in Food - US FDA -Washington DC
Version / remarks:
1982
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: The rules governing medicinal products in the European Community. Volume III: Guidelines on the Quality, Safety and Efficacy of medicinal products for human use.
Version / remarks:
Revised and completed edition, January 1989
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: Pesticide assessment guidelines - Subdivision F - Hazard evaluation - Human and domestic animals
Version / remarks:
Revised edition. November 1984
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: Guidelines for toxicity of drugs manual - Pharmaceutical Affairs Bureau, Ministry of Health and Welfare - Japan
Version / remarks:
1990
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole
EC Number:
252-615-0
EC Name:
1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole
Cas Number:
35554-44-0
Molecular formula:
C14H14Cl2N2O
IUPAC Name:
(±)-1-(β-allyloxy-2,4-dichloro-phenylethyl) imidazole / (±)-allyl 1-(2,4-dichlorophenyl)-2-imidazol-1-ylethyl ether
Test material form:
solid: crystalline
Details on test material:
- Physical state: crystalline solid
- Appearance: Yellow to brown crystalline solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch No ZE023979G3A 231
- Expiration date of the lot/batch: No data
- Purity test date: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature in closed containers as delivered under the responsibility of a board certified pharmacist
- Solubility and stability of the test substance in the solvent/vehicle: The concentration and stability of the test article in the food were controlled after preparation. The concentration of the test article in the preparation was guaranteed for the period during which the preparation was used. Therefore, it is concluded that the concentration and stability of the test article in the food mixtures complied with the concept of the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: see section diet preparation

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar rat was selected as the rodent species following review of available data on toxicity and pharmacology and also because of the possibility to compare the hereby recorded data with the historical control data of other similar studies conducted with the same test system in the same testing facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: test facility's permanent non-inbred laboratory colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males: 60 days; Females: 2-3 months; (F1;F2) from birth
- Weight at study initiation: (P) Males: 290.8-290.9 g; Females: 182.2-188.3 g; (F1) Males/Females: 6.6 to 6.9 g; (F2) Males/Females: 6.6 to 6.7 g.
- Fasting period before study: no data
- Housing: air conditioned rooms and placed in wire-mesh cages. Cohabitation ws allowed for maximum 3 weeks in wire-mesh cages between one male and one female, equivalently dosed in the first generation, whereas in the 2nd generation, cohabitation was done between 1 male and 3 females. As soon as sperm was observed, the female was housed individually until the end of the pregnancy
- Diet (e.g. ad libitum): ad libitum ; fresh tap-water in drinking bottles
- Water (e.g. ad libitum): ad libitum, Huybrechts rodent food
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): 18 / day
- Photoperiod (hrs dark / hrs light): no data
IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): prepared freshly
- Mixing appropriate amounts with (Type of food): powder rodent food and vitamin premix
- Storage temperature of food: room temperature

Details on mating procedure:
- M/F ratio per cage: 1M/1F for generation 1; 1M/3F for generations 2 and 3
- Length of cohabitation: max 3 weeks
- Proof of pregnancy: The occurence of copulation was established by daily vaginal inspection for sperm. Sperm in the vaginal smear in the morning, arbitrarily dated insemination back to midnight. Thus the day sperm was found was considered to be day 1 of gestation
- Unsuccessful pairing replacement of first male by another male with proven fertility: no data
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): individually until the end of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration and stability of the test article in the food were controlled after preparation. The concentration of the test article in the preparation was guaranteed for the period during which the preparation was used. Therefore, it was concluded that the concentration and tability of the test article in the food mixtures complied with the concept of this study.
Duration of treatment / exposure:
Parents:
- males: 60 days prior to and during mating period
- females: 60 days prior to and during mating and during pregnancy and the weaning period
First and second generation: during growth into adulthood, mating, pregnancy until weaning of the second generation
Frequency of treatment:
daily and continuously until weaning of the second generation
Details on study schedule:
- F1 parental animals not mated until 3 months after selected from the F1 litters.
- Age of the pups when selection of parents from F1 generation: no data
- Age at mating of the mated animals in the study: 3 months + 3 weeks max (mating period)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Actual test article intake during the pre-cohabitation period: 4.2 and 5.0 mg/kg bw/day for males and females
Actual test article intake during pregnancy period (first generation): 4.0 mg/kg bw/day
Actual test article intake during pregnancy period (second generation): 4.3 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Actual test article intake during the pre-cohabitation period: 17.6 and 21.5 mg/kg bw/day for males and females
Actual test article intake during pregnancy period (first generation): 16.3 mg/kg bw/day
Actual test article intake during pregnancy period (second generation): 18.6 mg/kg bw/day
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Actual test article intake during the pre-cohabitation period: 70.5 and 104.3 mg/kg bw/day for males and females (In females, the higher actual dosage rate in the 80 mg/kg bw/day group does not really reflect the real test article intake, since increased waste of food was evidenced in this group.)
Actual test article intake during pregnancy period (first generation): 86.8 mg/kg bw/day (the exact test article intake is considered to be lower due to waste of food)
Actual test article intake during pregnancy period (second generation): 87.8 mg/kg bw/day (waste of food resulted in an apparently higher actual test article intake).
No. of animals per sex per dose:
24 males and 24 females per group, 4 groups
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based upon previously conducted toxicity studies
• Exp 342: rats were dosed during 3 months at 5, 20 and 80 mg/kg bw/day. No adverse effects were noted at 5 mg/kg bw/day whereas dosing at 20 mg/kg bw resulted in altered blood, serum and urinary parameters. At 80 mg/kg bw/day also histological liver changes were found.
• Exp 480: rats were dosed at the same dose level as above. Dosing at 5 mg/kg bw/day did not result in adverse effects whereas dosing at 20 and 80 mg/kg bw/day dosed during 12 and 24 months were considered as slightly toxic dose levels. Altered blood parameters and histological liver changes were present at 20 mg/kg bw/day, whereas at 80 mg/kg bw/day also altered serum and organ weight variables were noted.
• Exp 581: rats were dosed at 2.5, 10 and 40 mg/kg bw/day during either 6, 18 or 30 months. No adverse effects were found at 2.5 mg/kg bw and 10 mg/kg bw/day. At 40 mg/kg bw/day, adverse effects on body weight, organ weight and histological changes were observed.
• Exp 2003: rats were dosed from day 6 until day 16 of pregnancy at doses of 40, 80 and 120 mg/kg bw/day. At all dose levels decreased food consumption and body weight were noted. At 80 and 120 mg/kg bw also the weight of the pups decreased. An increased resorption rate was noted at 120 mg/kg bw/day only.
• In a peri-and postnatal study (Exp 597), no adverse effects were noted at 5 and 20 mg/kg bw/day. At 80 mg/kg bw/day, food consumption and survival rate were decreased while an increase in dead pups was noted.
• In a previously conducted 3 generation study in rats (Exp 736) doses were 5, 20 and 80 mg/kg bw/day during 3 generations. No adverse effects were noted at all dose levels. This study was judged to be repeated mainly because histopathology was not performed and also because the dosing schedule in parent animals was too short.
In the present study, the dose of 80 mg/kg bw/day was determined as the high doe whereas 5 and 20 mg/kg bw/day were chosen as low and medium dose levels. Animals were dosed at least 60 days prior to mating and histopathology was done.
- Rationale for animal assignment (if not random): no data
- Fasting period before blood sampling for clinical biochemistry: no blood samples taken
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (e.g. general condition, chromodacryorrhea, circling animal, piloerection, red stained on external nares, haircoat, skin irritation, subcutaneous mass, food waste)
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Determined for males and females during the 60 days of the pre-cohabitation period and for all females on day 1 and 22 of the presumed pregnancy (days after copulation) and during a 3-week lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, determined for males and females during the 60 days of the pre-cohabitation period and for all females on day 1 and 22 of the presumed pregnancy (days after copulation) and during a 3-week lactation period
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Oestrous cyclicity (parental animals):
not investigated
Sperm parameters (parental animals):
Parameters examined in P/F1 male parental generations:
testes, epididymides and accessory male genital sex glands (control and 80 mg/kg bw/day dosed animals).
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no data
- The selection procedure was designed so that each female which delivered a litter was represented by at least one male and one female pup. The parentage of each litter was recorded so that brother-sister mating in the 2nd generation was avoided.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number of live/dead pups / female, mean litter size, number of implantations, bw of pups at birth and day 4, body weigth of males/females day 14, bw of males/females day 21, survival rate after 4, 14 and 21 days.

GROSS EXAMINATION OF DEAD PUPS:
no, possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not assessed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not assessed
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, at the end of the mating period.
- Maternal animals: All surviving animals, at the end of lactation.

HISTOPATHOLOGY
The testes, epididymides and accessory male genital sex glands, the kidney and liver of P1 and F1 males and the ovaries, uterus, vagina, the kidney and liver of the P1 and F1 females were trimmed, embedded, sectioned, stained and mounted by a procedure as much standardized as possible. The hematoxylin-eosin staining method was used. The female genital tract organs and the liver and lidneys of all dosage groups were examined, while the male genital tract organs were examined of control and 80 mg/kg bw/day dosed animals.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed (on information on age when sacrified).
- These animals were subjected to a complete gross necropsy.

GROSS NECROPSY
- Gross necropsy was conducted on the following organs: vagina, uterus, ovaries, tested, epididymides, seminal sevicles, prostates, kidneys, liver.

HISTOPATHOLOGY / ORGAN WEIGHTS - no data
Statistics:
The Chi-Square test for pairwise copmarison with control according to Siegel (two-tailed, Yates' correction for continuity, was used as the statistical method for clinical observations, mortality, copulation, fertility and gestation rate, survival rate.
The Mann-Whitney U test for paiwise comparison with control accroding to Siegel (two-tailed, correction for ties) was used as the statistical method for body weight and body weight gain, food consumption, cohabitation-mating interval, duration of gestation, live, dead and resorbed fetuses - mean litter size implantations, anomalies, histopathology.
Reproductive indices:
no data
Offspring viability indices:
no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During the pre-cohabitation period, the observations were comparable between groups for males. No adverse effects at 5 and 20 mg/kg bw/day.
Increased presence of wate of food during the precohabitation period, pregnancy and lactation period fo the first and second generation at 80 mg/kg bw/day. Increased incidence of dystocia in the first and second generation. In addition, an increased incidence of animals showing pilo-erection (indicative for a decreased physical condition) was present in animals of the first generation.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortalities were noted in whatever group in males prior to (60 days) and/or during the mating period (max 3 weeks)
Dosing at 80 mg/kg bw resulted in the following sacrifices:
- female 92 was sacrificed on day 14 of the lactation period. Clinical symptoms were: bad condition, piloerection, hypernea and red vaginal discharge. Autopsy revealed hydrothorax and hydroperitoneum, congestion of the liver and fibroendocartis.
- female 113 was sacrificed on day 1 after parturition with clinical symptomsof bad condition, respiratory dificulties, red stained external nares, chromodacryorrhea and red vaginal discharge. Autopsy revealed catarrhal pneumonia of all lobes and a pale liver.
No test article or dose related mortalities noted.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pre-cohabitation period:
- No adverse effects at 5 and 20 mg/kg bw/day
- Males: significant decrease weight change was observed at 80 mg/kg bw/day
- Females: similar but less pronounced decrease at 80 mg/kg bw/day

During pregnancy (day 1 to 22):
- No adverse effects at 5 and 20 mg/kg bw/day
- statistically significant decrease in body weight change noted at 80 mg/kg bw.

During the lactation period (3 weeks):
- No adverse effects at 5 and 20 mg/kg bw/day
- significantly decreased weight change noted at 80 mg/kg bw/day throughout the entire lactation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption:
-during pre-cohabitation period: Comparable in controls and doses animals
-during pregnancy and lactation: comparable between groups in both the first and second generation
Test article intake: the actual intake corresponded well with the proposed dose levels
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No test article related histological changes were noted in the 5 and 20 mg/kg bw/day dosage groups.

Accessory male genital sex glands: comparable findings in the control and the 80 mg/kg bw/day dosed group.
Epididymes: within normal limits
Testes: comparable findings between controls and 80 mg/kg bw/day dosed animals.
Liver: Comparable findings between controls and dosed animals expect for an increased incidence of large vacuoles in the hepatocytes of the 80 mg/kg bw/day in dosed P1 males. This target organ has been observed at the same dose level in previously conducted toxicity studies in rats.

Female genital tract (ovary, uterus and vagina):
In the 80 mg/kg bw/day dosed P1 female rats a decreased number of animals with corpora lutea of lactation was noted. This is a secondary consequence to the increased incidence of stillborn pups and a decrease in survival rate of pups and therefore decreased lactation stimulus at this dose level. The slight increase of metestrus stage as observed in the ovaries and vagina of the 20 mg/kg bw/day dosed P1 females is considered as a coincidental finding. For the uterus all findings were within normal limits.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Pregnancy: No adverse effects at 5 and 20 mg/kg bw/day. A decreased gestation rate is noted at 80 mg/kg in the first generation.
Copulation rate and fertility rates are comparable between groups while a decreased gestation rate is present at 80 mg/kg bw/day.

Duration of gestation:
No adverse effects at 5 and 20 mg/kg bw/day. The duration of gestation is significantly increased at 80 mg/kg bw/day. As a result of this a higher percentage of dystocia was observed in the 80 mg/kg bw/day dosed group.

Cohabitation-mating interval:
Cohabitation-mating intervals remained comparable between groups in both the first and the second generation.

A higher incidence of stillborn pups and associated herewith a lower incidence of live pups was observed at 80 mg/kg bw/day.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Remarks:
parental toxicity
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
LOAEL
Remarks:
reproductive toxicity
Effect level:
80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
80 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortalities were noted in whatever group in males prior to (60 days) and/or during the mating period (max 3 weeks)
Dosing at 80 mg/kg bw resulted in the following deaths and sacrifices:
- female 605 from the control groups died on day 15 of the lactation period after delivering 10 live pups. Autopsy revealed congestion and emphysema of the lungs.
- female 702 of the 80 mg/kg bw/day group died on day 13 of the lactation period, without previous clinical signs. Autopsy revealed endocarditis.

No test article or dose related mortalities noted.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During pregnancy (day 1 to 22): statistically significant decrease in body weight change noted at 80 mg/kg bw.

During the lactation period (3 weeks): significantly decreased weight change noted at 80 mg/kg bw/day throughout the entire lactation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
comparable in all groups
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Accessory male genital sex glands: comparable findings in the control and the 80 mg/kg bw/day dosed group.
Epididymes: within normal limits
Testes: comparable findings between controls and 80 mg/kg bw/day dosed animals.
Kidney: In the 20 mg/kg bw/day dosed male rats of the F1 generation, a slight decrease in basophilic tubuli was noted. This finding is considered coincidental.
Liver: Comparable findings between controls and dosed animals

Female genital tract (ovary, uterus and vagina):
Differences in the number of animals with corpora lutea of lactation between control and dosed groups of the female F1-generation were not detected. In all dosed groups of the female F1-generation a decrease in number of corpora lutea generations and a decrease in clear aspect of the interstitial tissue was observed in comparison to the controls. this is due to the individual variation in moment of sleughtering with respect to the weaning of pups. Other statistical differences observed in the ovaries (slight increase of proestrus and of estrus stage) and the uterus (high epithelium and healing of tissue at mesanteric site) of the female F1 generation are occasional findings.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Copulation rate, fertility and gestation rates are comparable between groups.
The duration of gestation is significantly increased at 80 mg/kg bw/day. As a result of this a higher percentage of dystocia was observed in the 80 mg/kg bw/day dosed group.
Cohabitation-mating intervals remained comparable between groups.

A higher incidence of stillborn pups and associated herewith a lower incidence of live pups was observed at 80 mg/kg bw/day. A lower number of implantations sites was noted at the same dose level.

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
80 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No external abnormalities were seen.
A decreased incidence of pups showing a ringtail was present in both the 20 and 80 mg/kg bw/day dosed groups. An increased incidence of pups with bad condition and hypothermia was noted at 80 mg/kg bw/day. This observation is associated with the decreased survival seen in this group.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The survival rate was lower at all doses and at all time intervals.
Based upon the fact that in the present study survival rate in the second generation of this study was comparable between controls and animals dosed at 5 and 20 mg/kg bw/day and that in a previously conducted study this was also the case the decreased survival rate in the first generation of the present study is considered as coincidental.
No adverse effects noted on the survival rate at doses of 5 and 20 mg/kg bw/day in both generations; at 80 mg/kg bw/day survival rate was decreased during lactation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Birth weight, body weight and weight gain comparable between groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see results described in parental generation P1
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Litter data:
- Litter size: decreased number of live pups at 80 mg/kg bw/day in the first generation
- weight at birth and during lactation: comparable between groups
- survival rate of pups during lactation: decreased survival at 80 mg/kg bw/day
- abnormalities: no teratogenic potential up to 80 mg/kg bw/day

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Generation:
F1
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no teratogenic effects reported

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
No external abnormalities were seen.
An increased incidence of bad condition and hypothermia was recorded at 5 mg/kg bw/day but not at 20 and 80 mg/kg bw/day. Therefore, the relevance of these observations is considered coincidental.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Survival rate was, although statistically seen lower, considered to be normal at 5 mg/kg bw/day. This also was the case at 20 mg/kg bw/day. At 80 mg/kg bw:day, survival rate was decreased.
Description (incidence and severity):
Birth weight, body weight and weight gain comparable between groups.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F2)

Litter data:
- litter size: decreased litter size, due to decreased number of implantations, and an increased incidence of stillborn pups at 80 mg/kg bw/day in the second generation
- weight at birth and during laction: comparable between groups
- survival rate of pups during lactation: decreased survival at 80 mg/kg bw/day
- abnormalities: no teratogenic potential up to 80 mg/kg bw/day

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Remarks:
teratogenicity
Generation:
F2
Effect level:
>= 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no teratogenic effects reported

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Dosing at 5 and 20 mg/kg bw/day during pre-cohabitation period of 60 days and during mating period and pregnancy during 2 generations did not result in test article related advserse effects. Dosing at 80 mg/kg bw/day resulted in test article related maternal toxicity. Increased presence of waste of food and a descreased bodyweight gain were noted during the pre-cohabitation period, during pregnancy and lactation of both the first and second generation.
histological examination of kidneys, male and female genital tract organs did not reveal primary test article-related changes. in the liver, an increased presence of large vacuoles in the hepatocytes was noted at 80 mg/kg bw/day in the pretreated males of the first generation. The duration of gestation was increased in both the first and second generation further resulting in an increased incidence of dystocia. related to these maternal effects at 80 mg/kg bw, a decreased number of live pups and an increase in number of stillborn pups were present in both generations. The survival rate of pups during lactation was considered to be decreased i both generatoins. No teratogenic potential was found.

Applicant's summary and conclusion

Conclusions:
In this two-generation study, parental toxicity was seen at the highest dose (reduced body weight and body weight gain, increased incidence of pilo-erection and, in P1 males, vacuolisation of hepatocytes). At the high dose 80 mg/kg bw/day, a reduced gestation rate and increased duration of gestation were also seen in females, the latter considered responsible for the concurrent increased rate of dystocia. At the highest dose, reproductive toxicity was seen as a slightly reduced number of implantations, reduced number of live pups and offspring survival and increased number of stillborn pups. No teratogenic effects were reported.
Executive summary:

From the above data, it can be concluded that dosing at 5 and 20 mg/kg bw/day during pre-cohabitation period of 60 days and during mating period and pregnancy during 2 generations did not result in test article related adverse effects. Dosing at 80 mg/kg bw/day resulted in test article related maternal toxicity. Increased presence of waste of food and decreased body weight gain were noted during the pre-cohabitation period, during pregnancy and lactation of both the first and the second generation. Histological examination of kidneys, male and female genital tract organs did not reveal primary test article-related changes. In the liver, an increased presence of large vacuoles in the hepatocytes was noted at 80 mg/kg nw/day in the pretreated males of the first generation. The duration of gestation was increased in both the first and second generation further resulting in an increased incidence of dystocia. Related to these maternal effects at 80 mg/kg, a decreased number of live pups and an increase in number of stillborn pups were present in both generations. The survival rate of pups during lactation was considered to be decreased in both generations. No teratogenic potential was found.