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Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2012 to 04 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, performed to valid guidelines and conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Protocol Deviations:

1. The mean measured concentration of Group 2 samples was 111% of the target concentration and was outside the acceptance criteria for solutions (90-110%).
Evaluation: The deviation is only slightly outside the criterion. Since the variation coefficient was small, this was considered acceptable.
2. Temporary deviations from the maximum level of daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
3. On 02 and 21 August the functional observations and motor activity tests were performed before clinical observations and on 9 July and 10 August the clinical observations were performed outside the 1-3 hour window after dosing.
Evaluation: A difference in the observation order on these two days has no impact on the study. Furthermore, clinical signs would likely be observable outside of the 1-3 hour window, and thus there was no impact on the study’s integrity.
5 During the lactation period, no clinical observations were registered in the computer for pup 4 of litter 45 (Group 1) on Day 3 and the coagulating glands from male no. 35 were not available for histopathology.
Evaluation: Sufficient data is available for a thorough evaluation.
6. Animal nos. 63, 78 and 42 are necropsied after dosing on Day 27 (nos. 63 and 78) and Day 26 (no. 42) post-coitum.
Evaluation: These animals did not require fasting beforehand; this does not affect the study’s integrity.
7. Several animals were necropsied later than after a maximum of 20 hours fasting, i.e. with a maximum of approximately 3.25 hours.
Evaluation: The fasting period was only slightly longer and was considered not to have adversely affected any findings. Current animal welfare approved practice allows a maximum fasting of 24 hours, which was not yet effective at the time of issuing the protocol.
8. Functional observational and motor activity data were collected for female no. 60 though they were not needed.
Evaluation: This just adds extra information and has no adverse impact on the study. These data are reported in Appendices 1 and 2.

The study integrity was not adversely affected by the deviations.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,5-dimethylpyrazole
EC Number:
200-657-5
EC Name:
3,5-dimethylpyrazole
Cas Number:
67-51-6
Molecular formula:
C5H8N2
IUPAC Name:
3,5-dimethyl-1H-pyrazole
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) Approximately 11 weeks. Females were nulliparous and non-pregnant.
- Weight at study initiation: (P) Males: 287-326 g; Females: 187-218 g; All parent animals were within ± 20% of the sex mean.
- Housing: Animals were housed in plastic cages, approximately 18 cm high. Cages contained sterilised sawdust as bedding material with paper as cage enrichment and nesting material.
> Pre-mating: Animals were housed by dose group in groups of 5 animals/sex/cage.
> During mating: Animals were housed in mating pairs, 1:1 by dose group.
> Post mating: Males were house in groups of 5/cage, females were housed individually.
- Diet: Pelleted rodent diet, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: At least five days prior to treatment.
- Water, diet, bedding and cage enrichment was evaluated for contaminants and/or nutrients. There were no findings which could affect the results of the study.
- Health: The health of each individual was checked prior to study initiation, to ensure all animals were in good state health.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24°C.
- Humidity: 40 to 70%.
- Air changes: Approximately 15 room air charges per hour.
- Photoperiod: A 12 hour light/12 hour dark cycle was maintained.

IN-LIFE DATES: From: 09 June 2012 To: 03 September 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
- Method: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustments were made for specific gravity of the vehicle (1.036). No corrections were made for the purity of the test material.
- Dose volume: 5 mL/kg b.w. based on the latest body weight measurment.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was chosen based on trial formulations performed by the research laboratory.
Details on mating procedure:
- Impregnation procedure: Rats were mated by cohabitation, after a minimum of 14 days of exposure, sibling mating was avoided. After mating, pairs were separated.
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days.
- After 14 days of unsuccessful pairing the first male was replaced by another male with proven fertility, for a further 3 days.
- Further mating after two unsuccessful attempts: No
- Proof of pregnancy: Pregnancy was confirmed by the presence of a vaginal plug or sperm in vaginal smear, this was referred to as Day 0 post-coitum.
- Parturition: Females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed, i.e. when membranes and placentas were cleaned up, nest build up and/or feeding of pups has started. Females that were littering were left undisturbed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Sampling: Test solutions were sampled and analysed once during the course of the study. All concentrations were sampled to confirm the accuracy of each preparation. Samples of the highest and lowest concentration were analysed for homogeneity and stability in the vehicle. Stability was confirmed at room temperature over a 6 hour period.
- Criteria: The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
- Results:
> Accuracy of preparation: The mean concentration of the 20 mg/kg b.w/day test solution was slightly outside the criterion, i.e. 111%. This was considered acceptable. The concentrations analysed in the 60 and 200 mg/kg b.w./day test solutions were in agreement with target concentrations, i.e. the mean accuracies were between 90% and 110%. No test substance was detected in the 0 mg/kg b.w./day formulation.
> Homogeneity: The 20 and 200 mg/kg b.w./day were homogeneous, i.e. the coefficient of variation was ≤ 10%.
> Stability: Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.
Duration of treatment / exposure:
Total exposure ≥ 28 days.
Males: Exposed for 29-31 days, i.e. 2 weeks prior to mating, during mating and up to the day prior to scheduled necropsy.
Females: Exposed for 45-56 days, i.e. 2 weeks prior to mating, during mating, coitum, and at least 4 days of lactation (up to the day prior to scheduled necropsy). Two of the females were not dosed during littering.
Pups: Were not exposed directly, but were potentially exposed to the test material in utero and through lactational transfer.
Frequency of treatment:
Animals were dosed daily 7 days a week, at approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Details on study schedule:
Males: 2 weeks premating, during mating and up to the day prior to scheduled necropsy, a minimum of 28 days.
Females: 2 weeks premating, during mating, gestation, and at least 4 days of lactation up to the day prior to scheduled necropsy. The appropriate sacrifice time can be seen in the field “Postmortem examinations (Parental animals)”.
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 60 and 200 mg/kg b.w./day
Basis:
actual ingested
No. of animals per sex per dose:
Ten males and ten females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose concentrations were selected based on the findings of a 10 Day dose range finding study.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
MORTALITY/VIABILITY OBSERVATIONS: Yes
- Time schedule: Recorded at least twice daily.

CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations were made daily; detailed clinical observations were made 1-3 hours after dosing, including once prior to study initiation and at weekly intervals during the treatment period. Observations were performed outside of the cage in a standard arena.
- Scoring: The onset, grade and duration of each observation were recorded. Observations were graded 1 to 4 dependant on severity; 1 slight, 2 moderate, 3 severe, 4 very severe. Some clinical signs were scored dependant on their presence 1, or their absence 0.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were measured on the first day of exposure and then at weekly intervals thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and Day 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Measurements were recorded weekly, except during mating and for females without evidence of mating. Consumption for mated females were recorded on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Examinations were recorded daily starting from Day 12, except when animals were housed in pairs during the mating period.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
- Macroscopic examinations were performed on the epididymis, testes, prostate gland of all parental males.
- Organ weights were recorded for the epididymis and testes of all males, whereas the prostate gland was measured in five selected males of each dose group.
- Histopathology was performed on five selected males from each dose group, examining the testes for staging of spermatogenesis. Additional slides from the 0 and 200 mg/kg b.w./day groups and all males suspected to be infertile were examined.
- Histopathology was performed on the epididymis and testes of all selected males in the 20 and 60 mg/kg b.w./day. These examinations were based on treatment related changed observed in the 200 mg/kg b.w./day dose group.
- Histopathology was performed on the reproductive organs of all males which failed to sire.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Number and sex of pups, determined on Days 1 and 4 of lactation.
- Number of dead and live pups, determined first on Day 1 of lactation and then daily thereafter.
- Weights were recorded for live pups on Days 1 and 4 of lactation.
- Detailed clinical observations were performed at least once daily for all animals.
Postmortem examinations (parental animals):
SACRIFICE:
All males and the five selected females/group were subjected to necropsy. Animals were fasted for a maximum of 23.5 hours prior to necropsy then anaesthetised using isoflurane and subsequently exsanguinated.
Schedule;
> Males: Following completion of the mating period, a minimum of 28 days of exposure.
> Females which delivered pups: Lactation Days 5-7.
> Females which failed to deliver pups with evidence of mating: Post-coitum Days 25-27.
> Females which failed to deliver pups without evidence of mating: Approximately 21 days after the last day of the mating period.
> Females with total litter loss: Within 24 hours of litter loss.

GROSS PATHOLOGY: All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissue and organs, with special attention being made to the reproductive organs. Tissues were collected and fixed in 10% buffered formalin, see Table 2 descriptions on the tissues and organs examined.

ORGAN WEIGHTS: The following organs were examined organ weights and terminal body weight were recorded in five selected animals/sex/group; adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus (including cervix), prostate, seminal vesicles (including coagulating glands) and the thyroid (including parathyroid). The epididymides and testes were examined in all remaining males.

HISTOPATHOLOGY: All organ and tissue samples were processed and embedded and cut at a thickness of 2-4 mm and then stained with haematoxylin and eosin, see Table 3 for details of examinations. The histopathology data was peer reviewed by a second pathologist.
Postmortem examinations (offspring):
SACRIFICE
- All F1 pups surviving to termination were sacrificed by decapitation on Days 5 or 7 of lactation.

GROSS NECROPSY
- All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were also evaluated.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
- Mating Index (%)
(Number of females mated/Number of females paired) x 100

- Fertility Index (%)
(Number of pregnant females/Number of females paired) x 100

- Conception Index (%)
(Number of pregnant females/Number of females mated) x 100

- Gestation Index (%)
(Number of females bearing live pups/Number of pregnant females) x 100

- Duration of Gestation
Number of days between confirmation n of mating and the beginning of parturition
Offspring viability indices:
- Percentage of Live Males at First Litter Check
(Number of live male pups at first litter check/Number of live pups at first litter check) x 100

- Percentage of Live Females at First Litter Check
(Number of live female pups at first litter check/Number of live pups at first litter check) x 100

- Percentage of Postnatal Loss Days 0-4 of Lactation
(Number of dead pups on Day 4 of lactation/Number of live pups at first litter check) x 100

- Viability Index
(Number of live pups on Day 4 post partum/Number of pups born alive) x100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically relevant findings.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significant reduction observed at 200 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A significant reduction observed at 200 mg/kg.
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):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment related effects were observed at 60 and 200 mg/kg.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Treatment related effects observed at 200 mg/kg.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Fertility and conception were reduced at 200 mg/kg.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Mortality: No treatment related mortalities occurred during the course of the study. One female was euthanized in the highest concentration group, 200 mg/kg, this was due to total litter loss.
- Clinical signs: Piloerection was noted for three females from the 200 mg/kg exposure group and one female at 20 mg/kg, however this was only noted for a limited number of days in each animal and the reaction was slight.
Incidental findings include alopecia, salivation (60 mg/kg) and pale appearance (200 mg/kg). These effects occurred in low incidences and thus were not considered to be toxicologically relevant.
No clinical signs were reported for males in either the control group or any of the treatment groups.
Females from the control group displayed alopecia, this was first observed during the preproduction period on Day 1 of Week 3. The reaction lasted until Day 2 of Week 5 and was assigned a grade of 1, slight. The reaction was observed in 5 to 15% of the animals, which rose to between 15% and 25% on the last day (Day 2 of Week 5).
Piloerection was observed for females in the 20 mg/kg exposure group, the reaction was observed in the reproduction period on Day 4 of Week 2. The reaction was recorded for one day and was assigned a grade of 1, occurring in between 5% and 15% of the animals.
Salivation was observed in the females of the 60 mg/kg treatment group during premating on Day 1 of Week 1. Salivation was assigned a grade of 1 and observed in 5 to 15% of the animals.
Females from the 200 mg/kg group were first observed for piloerection on Day 2 of Week 2 lasting until Day 5 of week 4, the median value for the highest individual reaction never exceeded 1, slight. The reaction was observed in between 5 and 15% of the animals with the exception of Day 4 and 7 of Week 2 where the reaction was observed in between 15 and 25% of the animals. A pale appearance was observed in between 5 and 15% of the animals on Day 3 of Week 4.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weights and body weight gains were significantly lower for males at 200 mg/kg than controls on Day 8 of the premating and through the entire mating period. Body weight gain was significantly lower for females on Day 8 of the premating period and on Day 11 of the post coitum period. Body weight gain was also slightly (not significantly) lower on Days 7, 14-17 and 20 of the post coitum period compared to controls.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- At 200 mg/kg, microscopic changes in the epididymides and testes, characterized by oligospermia, seminiferous cell debris and degeneration/depletion of spermatocytes were seen.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- At 200 mg/kg the fertility index of 60% and the conception index of 66.7% were considerably lower than the 80% seen for controls. There were 8, 9, 9 and 6 pregnant females in the control, 20, 60 and 200 mg/kg treatment groups, respectively. The mating index, precoital time and numbers of corpora lutea and implantation sites were unaffected by treatment up to 200 mg/kg.
- No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study, i.e. the mating index, precoital time, and numbers of corpora lutea and implantation sites.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- At 200 mg/kg lower terminal body weights, testes (absolute and relative), seminal vesicles (absolute and relative), prostate (absolute), epididymides (absolute) and increased spleen (absolute and relative), thyroids (relative) and kidney (relative) weights were noted for males while lower thymus (absolute and relative) and higher spleen (absolute and relative) weights were noted for females.
- Absolute and relative spleen weights were significantly increased for animals at 20 and 60 mg/kg as well. As no corresponding adverse effects in the spleen were noted for these groups during the microscopic examination, these increased weights were not considered toxicologically relevant.
- The reduced absolute brain weights seen for females at 200 mg/kg were considered secondary to their slightly lower body weights since their brain to body weight ratios were not significantly different than controls.
- The significant increase in seminal vesicle weight seen for males at 60 mg/kg was not considered toxicologically relevant as it occurred in the absence of a dose response (these weights were reduced for males at 200 mg/kg) and remained within the range considered normal for this age and strain.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- At 200 mg/kg an enlarged spleen and reduced size of the thymus was seen for 6/10 and 5/10 females. Reduced size of the epididymides, testes and seminal vesicles was noted for single males. Despite the limited incidence, microscopic examination confirmed that these were treatment related.
- Incidental findings seen for animals of the control and treated groups included uterus contains fluid, pelvic dilation of the kidney, alopecia on the foreleg, tan focus or discoloration on the left clitoral gland, yellow or soft nodule on the head of the epididymides. These observations were within the background range of findings that are encountered among rats of this age and strain. As they did not show a dose related incidence trend, they were not considered to be toxicologically relevant.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related microscopic findings were seen for animals at 60 and 200 mg/kg, and consisted of:
- Thymus: Increased severity of lymphoid atrophy in males (3/5, minimal) and females (5/5, up to moderate) treated at 200 mg/kg.
- Liver: Hepatocellular basophilia (up to slight) and/or apoptosis/single cell necrosis (up to marked) in the area directly around the central veins in males treated at 60 mg/kg and in both sexes treated at 200 mg/kg. Additionally, males treated at 200 mg/kg showed hepatocellular karyomegaly (5/5, up to slight) in the same area and midzonal hepatocellular vacuolation (3/5, up to slight).
- Spleen: An increase in severity of hematopoietic foci in males (6/6, up to marked) treated at 200 mg/kg.
- Epididymides: Oligospermia (1/6, marked) and an increased incidence and/or severity of seminiferous cell debris (3/6, up to slight) treated at 200 mg/kg.
- Testes: Degeneration/depletion of spermatocytes (6/6, up to massive) and an increase in incidence and/or severity of spermatidic giant cells (5/6, up to moderate) treated at 200 mg/kg.

Treatment-related effects on reproductive performance in the 200 mg/kg treated rats were seen, including degeneration/depletion of spermatocytes in the testes and oliogospermia in the epididymides, which underlie the poor reproductive results. Spermatogenic staging profiles were aberrant due to degeneration/depletion of spermatocytes.

FOOD CONSUMPTION
- Absolute food consumption was lower for males at 200 mg/kg from Days 1-8 of the premating period while relative food consumption was lower during the entire premating period. For females, relative food consumption was slightly lower during Days 1-8 of the premating period, and Days 17-20 of the post coitum period. Absolute and relative food consumption were both significantly lower than controls from lactation Days 1-4.

WATER CONSUMPTION
- At 200 mg/kg water consumption was increased for males during the entire treatment period (with the exception of Days 3-5 of the mating period) while water consumption was increased for females during the entire premating and post coitum periods (not always statistically significant).
Water consumption was also higher for females at 60 mg/kg over several days during the post coitum period, though the difference from controls was never statistically significant, a relationship to treatment could not be excluded.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive toxicity, epididymides and testes.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No toxicologically relevant findings.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Treatment related effects on pup mortality at 200 mg/kg.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment related effects on pup body weight at 200 mg/kg.
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:
no effects observed
Description (incidence and severity):
No toxicologically relevant findings.
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
- There were treatment related effects on pup mortality at 200 mg/kg.
- Three, three, one and thirteen pups of the control, 20, 60 and 200 mg/kg groups were found dead or went missing during the first days of lactation. Seven of the thirteen dead pups at 200 mg/kg were attributable to female who had a total litter loss by Day 3. A relationship to treatment could not be excluded. Missing pups missing were most likely cannibalized.

CLINICAL SIGNS (OFFSPRING)
- Incidental clinical symptoms of pups consisted of lean, pale appearance, a wound or scab on the flank, swelling on the neck or abdomen, blue discoloration, cold, swelling of the abdomen, no milk in the stomach, blue hind legs or lumbar region and blue spot on the neck. These were most commonly noted for pups that were later found dead or went missing. As the nature and incidence of these clinical signs remained within the range considered normal for pups of this age, they were therefore not considered toxicologically relevant.

BODY WEIGHT (OFFSPRING)
- There were treatment related effects on pup body weight at 200 mg/kg.
- At 200 mg/kg pup body weights were significantly lower than controls on Day 4 of lactation. There were no other effects on pup body weights.

GROSS PATHOLOGY (OFFSPRING)
- Small lower jaw, an external malformation, was noted for a single pup at 60 mg/kg (pup 10, litter 61). Due to its single occurrence in the mid dose, it was considered a chance finding and was not attributable to treatment. Incidental macroscopic findings of pups that were found dead included moderate autolysis, absence of milk in the stomach, partial cannibalism (abdominal organs missing) and/or autolysis. The only incidental macroscopic finding noted for surviving pups was scab on the left flank. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered toxicologically relevant.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fertility index, conception indices and histopathology of the epididymides and testes.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 3. Reproductive Data Summary

Parameter

Dose Group (mg/kg/day)

0

20

60

200

Females paired

10

10

10

10

Females mated

10

10

10

9

Females non-mated

0

0

0

1

Pregnant/non pregnant females

8/2

9/2

9/1

6/3

Females with living pups on Day 1

8

9

9

6

Mating Index (%)

100.0

100.0

100.0

90.0

Fertility Index (%)

80.0

90.0

90.0

60.0

Conception index (%)

80.0

90.0

90.0

66.7

Gestation index (%)

100.0

100.0

100.0

100.0

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test parental toxicity was observed at 60 and 200 mg/kg At 200 mg/kg, treatment related effects on body weights, food and water consumption, functional observations, clinical pathology, macroscopic findings and microscopic findings in the thymus, liver spleen, testes and epididymides were seen. Females at 60 mg/kg had a trend towards increased water consumption and males at this dose level had toxicologically relevant liver findings at the microscopic examination. There was no parental mortality in the study.

Toxicity to reproduction was observed at 200 mg/kg, where microscopic changes were recorded in the epididymides and testes, characterized by oligospermia, seminiferous cell debris and degeneration/depletion of spermatocytes. Lower fertility and conception indices were also observed in the 200 mg/kg exposure group.
Executive summary:

Toxicity to reproduction was determined in a 28 day oral repeat dose toxicity study performed in line with GLP and the standardised guidelines OECD 422 and EPA OPPTS 870.3650.

Both male and female wistar rats were exposed to the test material at 0 (control), 20, 60 and 200 mg/kg b.w./day administered via oral gavage for ≥ 28 days. Test solutions were analysed once during the course of the study and the accuracy of the preparations, homogeneity and stability were confirmed.

Under the conditions of the test parental toxicity was observed at 60 and 200 mg/kg. At 200 mg/kg, treatment related effects on body weights, food and water consumption, functional observations, clinical pathology, macroscopic findings and microscopic findings in the thymus, liver spleen, testes and epididymides were seen. Females at 60 mg/kg had a trend towards increased water consumption and males at this dose level had toxicologically relevant liver findings at the microscopic examination. There was no parental mortality in the study. Toxicity to reproduction was observed at 200 mg/kg, where microscopic changes were recorded in the epididymides and testes, characterized by oligospermia, seminiferous cell debris and degeneration/depletion of spermatocytes. Lower fertility and conception indices were also observed in the 200 mg/kg exposure group. Based on these observations the NOAEL for reproduction was determined to be 60 mg/kg b.w./day and the parental NOAEL was determined to be 20 mg/kg b.w./day.

On the basis of the effects seen, the material should be classified as Repr. 2: H361: Suspected of damaging fertility or the unborn child in accordance with Regulation (EC) No. 1272/2008.