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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:
7 Sep 1987 - 26 Jul 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
other: Supplementary report
Title:
Unnamed
Year:
1990
Report date:
1990
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted 2001
Deviations:
yes
Remarks:
Mainly missing examinations, please refer to "Principles of methods if other then guideline"
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
adopted 1983
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 83-4 (Reproduction and Fertility Effects)
Deviations:
no
Principles of method if other than guideline:
Sperm parameters (except testes and epididymis weight) were not measured, age of vaginal opening and preputial separation was not determined for F1 pups, anogenital distance was not measured, histopathology was only performed on liver and kidney. Exact days on which animals not chosen for further breeding were killed was not reported.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-dibromo-4-hydroxybenzonitrile
EC Number:
216-882-7
EC Name:
3,5-dibromo-4-hydroxybenzonitrile
Cas Number:
1689-84-5
Molecular formula:
C7H3Br2NO
IUPAC Name:
3,5-dibromo-4-hydroxybenzonitrile

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, England
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 6 wks (mating at ca 20 wks of age); (F1) 3 wks (mating at ca. 17 wks of age)
- Weight at study initiation: (P) Males: 150 - 230 g; Females: 120 - 173 g
- Housing: RC1, RB3 and RB3 modified cages (RC1 and RB3 modified cages had stainless steel grid floors, RB3 cages had solid polypropylene floors and were provided with autoclaved wood shavings for bedding during the littering phase. Pre-mating animals were housed in groups of 5 and separated by sex in RC1 cages. During mating, they were kept 1/1 (m/f) in modified RB3 cages, after that males and females were returned to groups of 5 and divided by sex into RC1 cages (males until termination, females until Day 16-18 post coitum). For littering (from Day 16-18 post coitum to Day 14-18 post partum), females were placed individually into RB3 cages. For lactation, dams and their litter were placed in RC1 cages (Day 14-18 post partum until weaning). Post weaning, animals were housed in groups of 5 and separated by sex in RC1 cages.
- Diet: Labsure Laboratory Animal Diet No. 2 ground, ad libitum (Labsure, Manea, Cambridgeshire, England)
- Water: tap water, ad libitum
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly, divided into 3 and 4 day amounts and sealed in polyethylene bags
- Mixing appropriate amounts with (Type of food): The mixes were prepared by means of a Gardner mixer. The test material was incorporated into the ground diet at the required concentration by initial preparation of a pre-mix, including milling followed by dilution with further quantities of diet and mixing. Diets unused at the end of each week were incinerated.
- Storage temperature of food: room temperature when in use (4 day amounts), otherwise deep frozen (-20 °C, 3 day amounts, these amounts were thawn on fridays for use over the weekends)
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until mating or for 21 days maximum
- Proof of pregnancy: sperm in vaginal smear and ejected copulation plugs, referred to as Day 0 of pregnancy
- After 21 days of unsuccessful pairing replacement of first male by another male with proven fertility: No
- After successful mating each pregnant female was caged: females were returned to groups of 5 and divided by sex into RC1 cages (until Day 16-18 post coitum). For littering (from Day 16-18 post coitum to Day 14-18 post partum), females were placed individually into RB3 cages. For lactation, dams and their litter were placed in RC1 cages (Day 14-18 post partum until weaning).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity of the test substance in the diet was checked twice before commencement of treatment by high-performance liquid chromatography. Six samples taken at regularly spaced positions from the trial mixes of the highest and lowest concentration were analyzed. After the initial assay of individual samples to determine homogeneity further assays were performed on blends of each homogeneity set after one, two, three and four weeks of storage at 21 °C. A further stability test was conducted on mixes stored at -20°C followed by a period at 21°C. The dietary concentrations were determined for the mixes prepared at Weeks 1, 12, 13, 14, and 15, at four weekly intervals over the remaining treatment period, and at commencement of mating and lactation.
Acceptable homogeneity of the test substance in the diet was demonstrated for both trial mixings. Stability tests demonstrated that the test substance was not stable in the diet at room temperature over prolonged time periods, so that the storage regime was changed and the diet was stored deep frozen at -20 °C for four days when they were thawed and transferred to storage at 21°C. This test demonstrated that test substance concentrations remained above 90% of the initial concentration after 4 days at -20 °C and 6 days at 21 °C. Treatment concentrations of analyzed dietary mixes averaged 101 ± 11%, 100 ± 7.2% and 98 ± 6.2% of the intended concentration for the lowest, intermediate and highest dosage levels respectively.
Frequency of treatment:
continously via the diet
Doses / concentrationsopen allclose all
Dose / conc.:
10 ppm (nominal)
Remarks:
corresponding to approximately
F0: 0.69 (males) and 0.81 mg/kg bw/day (females) before pairing
F1: 0.77 (males) and 0.9 mg/kg bw/day (females) before pairing
Dose / conc.:
50 ppm (nominal)
Remarks:
corresponding to approximately
F0: 3.39 (males) and 4.11 mg/kg bw/day (females) before pairing
F1: 3.98 (males) and 4.54 mg/kg bw/day (females) before pairing
Dose / conc.:
250 ppm (nominal)
Remarks:
corresponding to approximately
F0: 17.75 (males) and 21.5 mg/kg bw/day (females) before pairing,
F1: 21.3 (males) and 25.1 mg/kg bw/day (females) before pairing
No. of animals per sex per dose:
(P0) 24
(P1) 24
Control animals:
yes, plain diet
Details on study design:
- Other: F0 parental animals were mated twice (for F1A and F1B litters). Following weaning, 24 male and 24 female offspring from the F1A litters in each group were selected to form the F1 parental generation. They were mated once to produce F2 litters.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Observations: mortality and clinical signs of toxicity
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Not reported

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed at commencement of treatment and weekly until termination. Females were weighed at commencement of treatment and weekly until mating was detected, on Days 0, 6, 13 and 20 post coitum and on Days 1, 4, 7, 14, 21 and 25 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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: Yes

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Oestrous cycle was characterized for all females by examining daily vaginal smears over a ten day period prior to mating.
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
- maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, pup weight 24 hours after birth (Day 1), and on Day 4 (before culling), 7, 14, 21 and 25 post partum, postnatal mortality, sexed on Days 1, 4 (before culling), 14 and 25 post partum, physical or behavioral abnormalities,

PHYSICAL DEVELOPMENT
assessed on a litter basis by recording the days on which onset and completion of the following occurred
- Pinna unfolding - detachment of the edge of the pinna
- Hair growth - macroscopic observation of generalized growth of body hair
- Tooth eruption - eruption of upper incisors through the gums
- Eye opening - separation of the upper and lower eyelids.

AUDITORY AND VISUAL FUNCTION
- Time schedule: Day 25 post partum
Parameters checked: Auditory function was assessed using the startle response to a sudden sharp noise, visual function was assessed by examination of the pupil closure response to a bright point source of light and assessment of the visual placing response.

GROSS EXAMINATION OF DEAD PUPS: not reported
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals when the majority of litters (F1B or F2) were weaned were sacrificed by carbon dioxide inhalation.
- Maternal animals: All surviving animals were sacrificed by carbon dioxide inhalation approximately 10 days after their respective litters (F1B or F2) were weaned.
- Females that failed to mate, mated but did not give birth, or whose litters died before weaning were killed with the majority of females by carbon dioxide inhalation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including detailed examination of the cranial, thoracic, abdominal and pelvic cavities and their viscera. The external and cut surfaces of the organs and tissues were examined either before or after weighing, as appropriate. The numbers of uterine implantation sites were recorded in all females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: Epididymides, kidneys, liver, ovaries, prostate, seminal vesicles, testes, and uterus (for details, please refer to Attachment 1)
Paired organs were weighed separately before weights were combined for analysis. Tissues were preserved in buffered 4% formal saline.

The following tissue samples were taken from animals of the control and high dose group and abnormalities from all groups were dehydrated and embedded in paraffin wax, sectioned at 5 µm thickness and stained with haematoxylin and eosin:
Epididymides, mammary glands, ovaries, pituitary, prostate, seminal vesicles, testes, uterus and vagina (for details, please refer to Attachment 1)
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at Day 4 post partum.
- These animals were subjected to postmortem externally and internally examinations for macroscopic abnormalities. Specimens of abnormal tissues were retained.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including detailed examination of the cranial, thoracic, abdominal and pelvic cavities and their viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
No organs and tissues from the weanlings were weighed.
Only organs and tissues with abnormalities were histopathologically examined. Organs and tissues from the adult F1 generation were weighed prepared for microscopic examination (for details, please refer to Attachment 1). Paired organs were weighed separately before weights were combined for analysis. Tissues were preserved in buffered 4% formal saline.
Statistics:
Data were expressed as group mean values and standard deviations. Appropriate tests were used to analyze various parameters, they are indicated in the result tables.
Differences with an associated probability of P < 0.05 were considered to be statistically significant.
Reproductive indices:
Post coital interval: The time elapsing between initial pairing and detection of mating.

Duration of gestation: The time elapsing between the detection of mating and commencement of parturition.

Parturition: inspection at approximately 09.00, 13.00 and 17.00 hours during the week and 09.00 and 13.00 hours at weekends from Day 20 post coitum until completion

Percentage mating = (animals mating / animals paired) * 100

Conception rate = (animals achieving a pregnancy / animals mating) * 100

Fertility index = (animals achieving a pregnancy / animals paired) * 100

Gestation index = (number of live litters born / number pregnant) * 100

Group mean live litter size = number of live offspring / number of litters on day of examination
Offspring viability indices:
Post-implantation survival index (F1 generation only) = (total number of offspring born / total number uterine implantation sites)*100

Live birth index = (number of live offspring at Day 1 post partum / total number of offspring at Day 1 postpartum) * 100

Viability index = (number of live pups per litter on day 4 (pre-culling) / number of live pups at Day 1 postpartum) * 100

Lactation index = (number of live pups per litter on day of examination / number of live pups per litter on day 4 (post-culling)) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
The general condition of treated P0 males and females was similar to that of the control animals throughout the study.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- 10 ppm: 1 male was killed for humane reasons during Week 28 of treatment following a history of
weight loss, lethargy and pallor, it was diagnosed with hepatic malignant lymphoma, 1 female was killed for humane reasons on Day 9 post partum (F1A) following a history of piloerection, diarrhoea, weight loss, hunched posture and pale eyes, but no abnormalities were detected at necropsy or during the following histopathological examination.
- 50 ppm: No mortalities occurred.
- 250 ppm: One female of the high dose group was found dead on Day 5 of treatment as described above and was replaced, 1 female was found dead on Day 22 post coitum of the second pregnancy (F1B) without history of ill-health but necropsy revealed thickened mammary tissue, red/brown staining around the eyes, yellow/brown staining around the nares, and 16 dead and autolyzing fetuses In utero.

All of these findings were isolated and therefore not considered treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males
- 10 ppm: body weight gain was slightly, but statistically significantly increased compared to controls
- 50 ppm: body weight gain was slightly, but statistically significantly increased compared to controls
- 250 ppm: No differences observed.

These changes are considered potentially treatment related.

Females
- 10 and 50 ppm: No differences regarding body weight occured up to and including 50 ppm.
- 250 ppm: reduced mean body weight gain compared to controls during maturation (-15%) and reduced body weight at commencement of both F1A and F1B gestation and lactation phases. Weight gain was statistically significantly reduced during the first two weeks of both gestation phases (ca -30%), although some recovery was recorded in the last week of both pregnancies.

Mean body weights during lactation showed some inter- and intra-group variations, but the pattern of body weight change was unaffected by treatment.

Summarized results can be found in Attachment 2 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males
- 10 ppm: marginally increased (in line with increased body weight gain) compared to controls
- 50 ppm: marginally increased (in line with increased body weight gain) compared to controls
- 250 ppm: No differences observed regarding food consumption.

Females:
No difference between control and treatment groups were noted with regard to food consumption.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males:
No differences between control and treatment groups were observed.

Females
- 10 and 50 ppm: No differences regarding food efficiency occurred up to and including 50 ppm.
- 250 ppm: Food efficiency was reduced for females during the first two weeks of treatment compared to controls, but thereafter no consistent adverse effects of treatment were recorded.
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment related changes present following microscopic examination of liver and kidneys for Control and high dose group (250 ppm) animals. The changes that were present were those that are commonly seen in these organs in laboratory rats of that age.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No differences were observed between animals of control and treatment groups.
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
not applicable
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No differences were observed between animals of the control and treatment groups regarding mating performance and fertility.

In the group receiving 250 ppm of the test substance, gestation length was marginally decreased for both F1A and F1B litters (for details, please refer to Attachment 8). However, values remained within the range of previous Control findings and the shift was not confirmed in the F1 generation. Therefore, it was not considered treatment-related. Apart from that, isolated abnormalities like slightly altered gestation length were observed for individual females but no dose-dependency could be established and the findings were not reproducible, so that they were considered incidental.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at this dose level.
Remarks on result:
other: corresponding to approximately 3.39 mg/kg bw/day (males) and 4.11 mg/kg bw/day (females)
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to approximately 17.75 mg/kg bw/day (males) and 21.5 mg/kg bw/day (females)
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level (highest dose tested).
Remarks on result:
other: corresponding to approximately 17.75 mg/kg bw/day (males) and 21.5 mg/kg bw/day (females)

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The general condition of treated animals was similar to that of control animals throughout the study.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- control: 1 male was killed for humane reasons during Week 23, following a short history of a swollen right hind-limb and limping; no other abnormalities were observed at necropsy.
- 10 ppm: 1 male was killed for humane reasons during Week 21 of treatment, following the appearance of edema on the anterior ventral surface.
- 50 and 500 ppm: No mortalities occurred.

These findings were isolated and therefore not considered treatment-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males
- 10 and 50 ppm: In the low and mod dose group, animals achieved statistically significantly higher body weight gain compared to controls throughout the generation. This finding can be considered possibly treatment related.
- 250 ppm: body weight of animals selected to form the P1 generation was significantly lower than that of controls (-19%) and mean bodyweight gain was marginally reduced during the first 11 weeks of treatment.

Females
- 10 and 50 ppm: No significant differences regarding body weight occurred up to and including 50 ppm.
- 250 ppm: body weight of animals selected to form the P1 generation was significantly lower than that of controls (-15%), body weight on Day 0 of pregnancy remained significantly lower compared to controls (-10%), body weight gain was slightly reduced during the first two weeks of gestation.

Bodyweight changes during lactation were unaffected by treatment in all dose groups.

Summarized results can be found in Attachment 4 in the attached background material.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences were observed between animals of control and treatment groups.
Food efficiency:
no effects observed
Description (incidence and severity):
No differences were observed between animals of control and treatment groups.
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males
- 10 and 50 ppm: No differences of toxicological importance occurred up to and including 50 ppm. The increased absolute liver (+11.7%) and kidney (+11.3%) weights at 50 ppm were considered incidental since no dose-dependency could be established and the difference was most probably caused by the general higher body weight described earlier.
- 250 ppm: The increase in relative liver (+5.9%) and kidney (+12.9%) weight compared to controls was considered treatment related.

Females
- 10 and 50 ppm: No differences regarding organ weight occurred up to and including 50 ppm.
- 250 ppm: The increase in relative liver (+13.1%) and kidney (+10.3%) weight compared to controls was considered treatment related.

Summarized results can be found in Attachment 5 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences were observed between animals of control and treatment groups.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males
Histopathological examination of selected tissues from the control and high dose group and of gross abnormalities from any of the groups (including intermediate groups) revealed no treatment related effects.

Females
- 10 and 50 ppm: No differences regarding histopathology occurred up to and including 50 ppm.
- 250 ppm: female rats showed a diminution of nephrocalcinosis possibly implying a slight alteration in mineral balance or increased diuresis. This alteration was considered to be possibly attributed to the test substance treatment (Supplementary report, 1990). However, as this finding represented only a slight alteration, it was not considered adverse.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No differences were observed between animals of control and treatment groups.
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
not applicable
Reproductive performance:
no effects observed
Description (incidence and severity):
No differences were observed between animals of control and treatment groups.

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level.
Remarks on result:
other: corresponding to approximately 3.98 mg/kg bw/day (males) and 4.54 mg/kg bw/day (females)
Key result
Dose descriptor:
LOAEL
Remarks:
general toxicity
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to approximately 21.3 mg/kg bw/day (males) and 25.1 mg/kg bw/day (females)
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
>= 250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at this dose level.
Remarks on result:
other: corresponding to approximately 21.3 mg/kg bw/day (males) and 25.1 mg/kg bw/day (females)

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No differences were observed between animals of control and treatment groups.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
In both the F1A and F1B littering phases, there were some slight inter-group differences in litter size and offspring viability, but there was no evidence of any adverse effect of treatment. For details, please refer to Attachment 9.
Mortality / viability:
no mortality observed
Description (incidence and severity):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 10 and 50 ppm: No differences regarding body weight occurred up to and including 50 ppm.
- 250 ppm: reduced mean body weight and body weight gain in both F1A and F1B litters compared to controls.

Summarized results can be found in Attachment 6 in the attached background material.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Sexual maturation:
not examined
Description (incidence and severity):
not applicable
Anogenital distance (AGD):
not examined
Description (incidence and severity):
not applicable
Nipple retention in male pups:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
not applicable
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All pups that died before weaning lacked milk in their stomach (all dose groups). Necropsy of pups that were culled at Day 4 post partum revealed a greater proportion of small pups in F1A litters of the 250 ppm treatment group but not in F1B litters. The finding was therefore considered incidental.
Offspring not selected to form the F1 generation showed no changes at necropsy that were considered to be related to treatment with the test substance.
Histopathological findings:
not examined
Description (incidence and severity):
not applicable
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Onset and completion of eye opening:
- 10 and 50 ppm: No differences regarding onset and completion of eye opening occurred up to and including 50 ppm.
- 250 ppm: marginally but significantly delayed in F1A and F1B litters

Summarized results can be found in Attachment 6 and 10 in the attached background material.

Sex ratio, auditory and visual responses:
Sex ratio, auditory and visual responses were comparable between animals of the control and treatment groups.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined
Description (incidence and severity):
not applicable

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at this dose level.
Remarks on result:
other: corresponding to approximately 3.39 mg/kg bw/day (males) and 4.11 mg/kg bw/day (females)
Key result
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: physical development particularly with respect to eye opening
Remarks on result:
other: corresponding to approximately 17.75 mg/kg bw/day (males) and 21.5 mg/kg bw/day (females)

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 differences were found between animals of the control and treatment groups.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality / viability:
no mortality observed
Description (incidence and severity):
Litter size and offspring viability were unaffected by treatment with the test substance.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 10 and 50 ppm: No differences regarding body weight occurred up to and including 50 ppm.
- 250 ppm: body weight gain from Day 1 until weaning at Day 25 post partum was significantly reduced

Summarized results can be found in Attachment 7 in the attached background material.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
Sexual maturation:
not examined
Description (incidence and severity):
not applicable
Anogenital distance (AGD):
not examined
Description (incidence and severity):
not applicable
Nipple retention in male pups:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
not applicable
Gross pathological findings:
no effects observed
Description (incidence and severity):
All pups that died before weaning lacked milk in their stomach (all dose groups). No differences were observed between animals of the control and treatment groups.
Histopathological findings:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
Onset and completion of eye opening:
- 10 and 50 ppm: No differences regarding onset and completion of eye opening occurred up to and including 50 ppm.
- 250 ppm: marginally but significantly delayed in F2 litters

Summarized results can be found in Attachment 7 and 10 in the attached background material.

Sex ratio, auditory and visual responses:
Sex ratio, auditory and visual responses were comparable between animals of the control and treatment groups.

Offspring development:
- 10 and 50 ppm: No differences regarding offspring development occurred up to and including 50 ppm.
- 250 ppm: Offspring development as assessed by completion of pinna unfolding, hair growth and tooth eruption was marginally delayed in F2 pups at 250 ppm without reaching statistical significance. This was in agreement with the delay in eye opening and the slightly lower rate of body weight gain.
For details, please refer to the Attachment 10.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined
Description (incidence and severity):
not applicable

Effect levels (F2)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
50 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level.
Remarks on result:
other: corresponding to approximately 3.98 mg/kg bw/day (males) and 4.54 mg/kg bw/day (females)
Key result
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Generation:
F2
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: physical development particularly with respect to eye opening
Remarks on result:
other: corresponding to approximately 21.3 mg/kg bw/day (males) and 25.1 mg/kg bw/day (females)

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The study was conducted according to Guideline EPA OPP 83-4, and similar to OECD Guideline 416 (adopted 1983). It was performed under GLP conditions and was considered valid. The quality of the data is sufficient to classify it as a key study.
Based on the findings of this study, it was concluded that continuous dietary administration of the test substance at a level of 250 ppm to male and female rats throughout two successive generations resulted in slight adverse effects on somatic growth, as evidenced by reduced bodyweight gain of P1 males, P0 and P1 females prior to pairing and during the first two-thirds of each gestation period, and F1A, F1B and F2 offspring during the lactation periods. A NOAEL for general toxicity of 50 ppm for P0 and P1 parental animals (corresponding to 3.39 and 4.11 mg/kg bw/day for P0 males and females, and to 3.98 and 4.54 mg/kg bw/day for P1 males and females, respectively) was established based on decreased body weights and increased liver weights. Body weight-relative liver weights were slightly increased in P0 males and P0 and P1 females. In P1 males and females body weight-relative kidney weights were slightly increased as well. Physical development of offspring reflected the slight retardation in bodyweight gain, particularly with respect to eye opening. The NOAEL for effects on neonatal parameters was also 50 ppm (corresponding to 3.39 and 4.11 mg/kg bw/day for F1 males and females, and to 3.98 and 4.54 mg/kg bw/day for F2 males and females, respectively) based on decreased pup body weight and delayed physical development. The NOAEL for reproductive effects was ≥ 250 ppm in both parental generations (corresponding to 17.75 and 21.5 mg/kg bw/day for P0 males and females and 21.3 and 25.1 mg/kg bw/day for P1 males and females, respectively), the highest dose tested as no adverse effects upon reproductive performance and fertility of either the P0 or P1 generation have been observed.