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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

No adverse effects on mating performance, fertility, fecundity, gestation, partition, or lactation were noted; as such, the NOAEL for reproductive toxicity is 100 mg/kg/day.

Based on the lower F0 female pup body weight gain at 50 and 100 mg/kg/day, and marginally higher number of pup mortality in F0 generation litters following maternal exposure of 100 mg/kg/day, and increased incidence of offspring mortality for the F1 generation following maternal exposure of 50 or 100 mg/kg/day. As such, the NOEL for offspring growth and development is established as 20 mg/kg/day.

No test article immunotoxicity effects were evident in F1 animals; as such, the NOAEL is 100 mg/kg/day for developmental immunotoxicity.

No neurotoxic effects were noted in F1 animals; as such, the NOEL is 100 mg/kg/day for developmental neurotoxicity.

Link to relevant study records

Referenceopen allclose all

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.
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
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.
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)”.
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
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
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: 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.
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.
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.
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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.
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.
Reproductive effects observed:
not specified

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

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.

Endpoint:
extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 October 2017 to 17 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
OECD GLP study requested under decision number CCH-D-2114350480-58-01/F. The full report is appended below.
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
OECD Test Guideline 443 Extended One-Generation Reproductive Toxicity Study (adopted 28 July 2011).
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes
Limit test:
no
Justification for study design:
Study Design
High Dose
A high-dose level of 100 mg/kg/day was anticipated to induce minimal parental toxicity without causing death or affecting parturition, pup growth, or mating of the first generation.

Intermediate Dose
An intermediate-dose level of 50 mg/kg/day was anticipated to be the lowest observed adverse effect level (LOAEL) for parental toxicity.

Low Dose
A low-dose level of 20 mg/kg/day was anticipated to be the no observed effect level (NOEL) for parental and offspring toxicity.

Background Information
A previous OECD 422 study was conducted in the Han Wistar rat at dose levels of 0, 20, 60, and 200 mg/kg/day. Males were dosed up to 31 days. Females were administered the test article formulations for 2 weeks prior to pairing, during pairing, gestation, and up to Lactation Day (LD) 4. Parental toxicity was observed following administration of 60 and 200 mg/kg/day, consisting of changes in body weights; food and water consumption; FOB results; clinical pathology; macroscopic findings (reduced spleen size and enlarged stomachs); and microscopic findings in the thymus, liver, spleen, testes, and epididymides. Females administered 60 mg/kg/day had a trend towards increased water consumption and males at this dose level had toxicologically relevant liver findings following microscopic examination.
Reproductive changes included microscopic changes in the epididymides and testes, characterized by oligospermia, seminiferous cell debris, and degeneration/depletion of spermatocytes along with lower fertility and conception indices following administration of 200 mg/kg/day. Developmental toxicity was observed following administration of 200 mg/kg/day, consisting of postnatal pup mortality and lower pup body weights.
Based on these findings, a NOAEL for parental animals was established as 20 mg/kg/day, and a NOAEL for reproductive and developmental toxicity was established as 60 mg/kg/day.
A prenatal development range-finding study in the female Han Wistar rat has recently been performed (Covance Study 8365910; Covance Laboratories Ltd., 2018) at the same dose levels previously administered in the OECD 422 study. Reduced mean body weights were noted for dams administered 200 mg/kg/day, together with one total in utero litter loss and lower mean fetal weights, compared with controls. These findings supported the findings observed in the previous OECD 422 study.
Due to these equivocal findings, the same dose levels were further explored in a pivotal OECD 414 prenatal development toxicity study (Covance Study 8365912; Covance Laboratories Ltd., 2018). Similar effects were evident following administration of 200 mg/kg/day, together with skeletal variations noted in the fetuses, which were most probably attributable to the lower body weight gains noted for the dams following administration of 200 mg/kg/day.
The oral route of administration was chosen as it is a possible route of human exposure.
Specific details on test material used for the study:
No further details specified in the study report.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animal Specifications and Acclimation
One hundred two male and 102 female Crl:WI(Han) strain rats were obtained from Charles River Laboratories, Margate, United Kingdom, in order to provide sufficient animals for F0 generation study selection.
Upon arrival, animals were approximately 4 weeks old. Males weighed between 125.8 and 234.7 g, females weighed between 116.8 and 182.2 g, and were approximately 6 weeks old at the start of dosing.
Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for 13 days, and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure their suitability for the study.

Environmental Conditions, Diet, and Water
Housing
Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes (Home Office, 2014).
F0 animals were housed in groups of up to four by sex and dose group (pre-pairing and post-pairing phases). During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation, and with their litter during the lactation phase.
F1 animals were housed in groups of up to four by sex, dose group, and cohort.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
Bedding was provided at least weekly to each cage by use of clean Aspen wood chips or European Softwood bedding during gestation and lactation phases (Datesand Ltd; Manchester, United Kingdom).
Each batch of bedding was analyzed for specific constituents and contaminants. No contaminants were present in the bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Water
Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
No contaminants were present in the water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Diet
Animals had ad libitum access to SDS Rat and Mouse Breeder Diet VRF1 (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants.
No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Environment
Animals were housed in exclusive rooms. Rooms were air-conditioned to provide 15 to 20 air changes/hour. The temperature and relative humidity ranges were maintained in the specified ranges of 19 to 25°C and 30 to 70%, respectively.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark.

Environmental Enrichment
Animals were provided with wooden Aspen chew blocks, rodent retreats, and paper wool nesting materials (gestation and lactation phases only) as forms of environmental enrichment.

Animal Identification and Assignment to the Study
Upon arrival, F0 animals were assigned to dose groups based on a total randomization procedure. For F1 animals see Weaning. Animals were individually identified by electronic implant, applicable animals were tail marked when unable to be chipped.
Pups were uniquely identified from Postnatal Day (PND) 1 by back mark with an indelible pen.
For F0 animals, prior to the start of treatment, group mean body weights and standard deviations were calculated and inspected to ensure differences did not exceed 20% of the mean weight of each sex between groups.
During FOB assessments, applicable animals were identified by tail marks.
Cages were placed in dose group order across the batteries, except during FOB assessments.
Relevant data recorded throughout the study for the spare (un-dosed) F0 generation animals are maintained and archived within the raw data, and not reported.
Cages were appropriately identified with study information, including study number and animal number (s).
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
Control Article (Vehicle)
The control article (vehicle) was Propylene Glycol, supplied by Merck.

Test Article Formulation
Formulations were prepared weekly.
The test article was formulated in Propylene Glycol following Dispensary SOPs and the formulation method (Method 8365913_O_01D), as maintained in the study data.
Formulations were stored at room temperature (15 to 25°C) in a sealed container.
Details on mating procedure:
During the pairing phase, one male was housed for up to 14 days with one female of the same dose group.
Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon the confirmation of mating, vaginal washing was discontinued, and the male was removed from pairing/the study/the cage. The day on which mating was confirmed was designated as GD 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and Homogeneity
Formulations of 1 and 100 mg/mL were confirmed to be homogenous and stability for 8 days at 15-25°C (Covance Study 8365911; Covance Laboratories Ltd., 2017).

Achieved Concentration
Formulations prepared for the first and last days of dosing for the F0 and F1 generations (four occasions in total) were analyzed. Triplicate samples were removed from the middle of the test article formulations and were analyzed. A single sample was taken from the middle of control formulations and was analyzed. Samples were analyzed using UPLC.
Duration of treatment / exposure:
F0 Generation Procedures: F0 males were dosed for up to 127 consecutive days (10 weeks prior to pairing, during pairing, and 42/43 days post-pairing); females were dosed for up to 122 days (10 weeks prior to pairing, during pairing, throughout gestation, and up to LD 21).
All F1 offspring were dosed from PND 14 to 21.
Dosing was omitted for any female observed to be in or near parturition at the time of daily dosing.
Formulations were administered at a constant dose volume of 5 mL/kg. Dose volumes will be based on the most recently recorded body weight for each animal.
Frequency of treatment:
F0 Generation Procedures: The test article was administered once daily by oral gavage.
Following selection into appropriate cohorts, the selected F1 animals were administered once daily.
Details on study schedule:
F1 Procedures - Post-Weaning
Test Article Administration
The test article was administered once daily by oral gavage.
Following selection into appropriate cohorts, the selected F1 generation animals were administered once daily as follows:
• Cohort 1A: For up to 73 days
• Cohort 1B: For up to 75 day prior to pairing, throughout pairing, and until LD 4 for females; males were dosed up to 96 days in total.
• Cohort 2A: For up to 56 days
• Cohort 2B: For 1 day
• Cohort 3: For up to 62 days
Dosing was omitted for any Cohort 1B female observed to be in or near parturition at the time of daily dosing.
Formulations were administered at a constant dose volume of 5 mL/kg. Dose volumes will be based on the most recently recorded body weight for each animal.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
Low Dose: A low-dose level of 20 mg/kg/day was anticipated to be the no observed effect level (NOEL) for parental and offspring toxicity.
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Intermediate Dose: An intermediate-dose level of 50 mg/kg/day was anticipated to be the lowest observed adverse effect level (LOAEL) for parental toxicity.
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
High Dose: A high-dose level of 100 mg/kg/day was anticipated to induce minimal parental toxicity without causing death or affecting parturition, pup growth, or mating of the first generation.
No. of animals per sex per dose:
Four groups of 25 male and 25 female Crl:WI(Han) rats were allocated to the study as the parental (F0) generation.
F1 Generation
Cohort 1A: 40 animals per dose group (20 male/20 female)
Cohort 1B: 40 animals per dose group (20 male/20 female)
Cohort 2A: 20 animals per dose group (10 male/10 female)
Cohort 2B: 20 animals per dose group (10 male/10 female)
Cohort 3: 20 animals per dose group (10 male/10 female)
Control animals:
yes, concurrent vehicle
Details on study design:
A previous OECD 422 study was conducted in the Han Wistar rat at dose levels of 0, 20, 60, and 200 mg/kg/day. Males were dosed up to 31 days. Females were administered the test article formulations for 2 weeks prior to pairing, during pairing, gestation, and up to Lactation Day (LD) 4. Parental toxicity was observed following administration of 60 and 200 mg/kg/day, consisting of changes in body weights; food and water consumption; FOB results; clinical pathology; macroscopic findings (reduced spleen size and enlarged stomachs); and microscopic findings in the thymus, liver, spleen, testes, and epididymides. Females administered 60 mg/kg/day had a trend towards increased water consumption and males at this dose level had toxicologically relevant liver findings following microscopic examination.
Reproductive changes included microscopic changes in the epididymides and testes, characterized by oligospermia, seminiferous cell debris, and degeneration/depletion of spermatocytes along with lower fertility and conception indices following administration of 200 mg/kg/day. Developmental toxicity was observed following administration of 200 mg/kg/day, consisting of postnatal pup mortality and lower pup body weights.
Based on these findings, a NOAEL for parental animals was established as 20 mg/kg/day, and a NOAEL for reproductive and developmental toxicity was established as 60 mg/kg/day.
A prenatal development range-finding study in the female Han Wistar rat has recently been performed (Covance Study 8365910; Covance Laboratories Ltd., 2018) at the same dose levels previously administered in the OECD 422 study. Reduced mean body weights were noted for dams administered 200 mg/kg/day, together with one total in utero litter loss and lower mean fetal weights, compared with controls. These findings supported the findings observed in the previous OECD 422 study.
Due to these equivocal findings, the same dose levels were further explored in a pivotal OECD 414 prenatal development toxicity study (Covance Study 8365912; Covance Laboratories Ltd., 2018). Similar effects were evident following administration of 200 mg/kg/day, together with skeletal variations noted in the fetuses, which were most probably attributable to the lower body weight gains noted for the dams following administration of 200 mg/kg/day.
Positive control:
Not required.
Parental animals: Observations and examinations:
Health Monitoring
All animals were observed at the beginning and end (nominal) of each working day for signs of ill health or overt toxicity.

Clinical Examinations
Each animal was given a detailed physical examination once daily from the start of dosing. An individual record was maintained of the clinical condition of each animal.

Postdose Observations
Each animal was observed at approximately 0.5 hours postdose during the first three weeks of dosing (Pre-Pairing Days 1 to 7 and 9 to 15 inclusive). In the absence of any toxicologically significant postdose observations, animals were not observed thereafter.

Weekly Detailed Clinical Observations
All F0 animals were assessed by detailed clinical observations for approximately 30 seconds in an open field arena. Males were assessed once weekly from the start of dosing. Females were assessed once weekly during the pre-pairing and pairing phases; on Gestation Days (GD) 0, 6, 14, and 20; and on LD 1, 7, 14 and 21. Abnormal observations were recorded and reported by exception.
Each animal was observed and evaluated for the following:
Activity; Pain response; Aggression to cage mate; Palpebral reflex; Alertness; Pelage; Approach response; Pinna response; Acoustic startle response; Posture; Bar test; Proprioception (right hind leg); Behavior - other; Pupil status; Behavior - stereotype; Pupillary response; Body tone; Reactivity to handling; Discharge; Respiration; Excretion; Restlessness; Extensor thrust; Righting reflex; Eye closure; Salivation; Eyes; Skin color; Gait; Tail; Grasping loss; Visual response; Grip strength; Vocalization; Involuntary movement; Waxy rigidity; Lacrimation

Body Weights
Body weights were recorded once during acclimation for all animals.
Male body weights were recorded weekly from the 1st day of dosing (Pre-Pairing Day 1).
Female body weights were recorded weekly from the 1st day of dosing (Pre-Pairing Day 1); on GD 0, 7, 14, and 20; and on LD 1, 4, 7, 14, and 21.

Food Consumption
Male food consumption was recorded twice weekly during pre-pairing and post-pairing.
Female food consumption was recorded twice weekly during pre-pairing, and daily from GD 0 to 20 and from LD 1 to 14.
Food consumptions were not recorded during pairing, between GD 21 and LD 0, due to parturition, and from LD 14, due to the start of weaning of pups.
Consumption was calculated as g/animal/day.

Parturition
Animals were observed three times/day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered, or until a potential GD 25, whichever was soonest.
Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, where possible, and marked the end of gestation; when not observed, the end of gestation was the day when the completion of parturition was recorded or on the day prior to when LD 1 observations were made.
Abnormal observations of nesting, parturition, or nursing were recorded. It was important that the dam was disturbed as little as possible on LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or stillborn (by looking for lung inflation).

Clinical Pathology
Sample Collection and Handling
Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.5 mL [trisodium citrate], nominal), and clinical chemistry (1 x 0.6 mL [lithium heparin], nominal) were withdrawn from the abdominal aorta at necropsy on Post-Pairing Day 46/47 (Week 18-19 of dosing) for 10 randomly selected males, and at necropsy on LD 22 for 10 randomly selected females. Samples were collected after animals were fasted overnight.
Blood samples for thyroid hormone (1 x 1.6 mL [serum separator tubes], nominal) were withdrawn from the jugular vein in-life from 10 randomly selected males per dose group on Post-Pairing Day 46/47 (Week 18) or from the abdominal aorta at necropsy for 10 randomly selected females per dose group on LD 22. Samples were collected after animals were fasted overnight. One sample from each animal was analyzed in the first instance, when required the spare sample was analyzed, spare samples not required for additional analysis are retained in storage until report finalization, after which, they will be discarded.
Blood samples for thyroid hormone analysis from unselected F1 generation pups sacrificed on PND 22 (2 x 0.6 mL [serum separator tubes], nominal) were withdrawn by cardiac puncture from two male pups/litter and two female pups/litter. Samples from one male and one female sample were analyzed in the first instance; when required, the spare sample was analyzed. Spare samples not required for additional analysis are retained in storage until report finalization, after which, they will be discarded.
Thyroid hormone sampling was performed at a similar time on each occasion (between 09:00 and 13:00); samples were protected from light until placed in frozen storage.
Each blood sample for thyroid hormone analysis was gently inverted 10 times and was stored at room temperature (15 to 25°C) and centrifuged at 2300g for 10 minutes at approximately 4°C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at <-20°C. Samples were centrifuged and aliquoted within 2 hours of collection, and were analyzed at Covance.
Urine samples from 10 randomly selected males and females in each group were collected overnight the day prior to necropsy. Food was removed during collection.

Hematology Tests
hemoglobin; red blood cell count; packed cell volume; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; reticulocyte count; red cell distribution width; hemoglobin distribution width; total and differential white cell count; platelet count; platelet crit; mean platelet volume; platelet distribution width; Coagulation Tests; prothrombin time; activated partial thromboplastin time; fibrinogen

Clinical Chemistry Tests
aspartate aminotransferase; alanine aminotransferase; alkaline phosphatase; total cholesterol; total bilirubin; total protein; albumin; globulin; albumin:globulin ratio; sodium; potassium; chloride; calcium; inorganic phosphate; creatinine; urea; glucose

Urinalysis Tests
volume; color; turbidity; specific gravity; pH; protein; glucose; ketones; urobilinogen; bilirubin; blood; microscopy of sediment

Thyroid Hormone Tests
thyroxine (Total T4); thyroid stimulating hormone (TSH)
Oestrous cyclicity (parental animals):
Daily vaginal washings were conducted on females for 14 days prior to the start of dosing, and until the confirmation of mating, and on the day of necropsy.
Sperm parameters (parental animals):
For each scheduled male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from the left epididymis (cauda epididymis and vas deferens). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from the first 10 surviving males for each group were read for morphological changes. The left epididymis was discarded following seminology investigations.
Litter observations:
Litter Size and Sex Determination
Litter size and pup sex were recorded on PND 0, 1, 4, 7, 14, and 21.
Daily records of mortality and changes in litter sizes were maintained. Where possible, pups found dead or in a moribund condition were given a macroscopic necropsy for cause of death and possible birth defects.

Clinical Observations
A detailed clinical examination was recorded for each pup on PND 1, 4, 7, 14, and 21.

Body Weights
Offspring body weights were recorded on PND 1, 4, 7, 14, and 21.

Anogenital Distance
The anogenital distance of all pups was measured on PND 4.

Nipple/Areolae Count
The number of nipples/areolae for all male offspring was counted on PND 13.

Weaning
Pups were weaned on PND 21, and 280 pups/sex were randomly selected from the available litters (with the exception that obvious runts [animals with a body weight more than two standard deviations below the mean pup weight of the respective litter] were not included). Animals of Cohorts 1A and 1B were allocated based on a random selection of 20 animals/sex/group from the available litters (1 male and 1 female from each litter). Animals of Cohorts 2A, 2B, and 3 were allocated based on a random selection of 10 animals/sex/group from the available litters (1 male and 1 female from each litter).
Pups not selected had a blood sample for thyroid hormone analysis obtained on PND 22 and were then sacrificed and necropsied.

Clinical Observations
Health Monitoring
All animals were observed at the beginning and end (nominal) of each working day for signs of ill health or overt toxicity.

Clinical Examinations
Each animal was given a detailed physical examination once daily from PND 22, until necropsy. An individual record was maintained of the clinical condition of each animal.

Weekly Detailed Clinical Observations
All adult animals were assessed by detailed clinical observations for approximately 30 seconds in an open field arena. Animals were assessed once weekly from PND 22.
Each animal was observed and evaluated for the following:
Activity; Pain response; Aggression to cage mate; Palpebral reflex; Alertness; Pelage; Approach response; Pinna response; Acoustic startle response; Posture; Bar test; Proprioception (right hind leg); Behavior - other; Pupil status; Behavior - stereotype; Pupillary response; Body tone; Reactivity to handling; Discharge; Respiration; Excretion; Restlessness; Extensor thrust; Righting reflex; Eye closure; Salivation; Eyes; Skin color; Gait; Tail; Grasping loss; Visual response; Grip strength; Vocalization; Involuntary movement; Waxy rigidity; Lacrimation

Body Weights
Body weights for Cohorts 1A, 1B, 2A, and 3 were recorded on PND 22, 24, 26, 28, 30, 32, 34, and 36 and then weekly thereafter.
Body weights for Cohort 2B were recorded on PND 21 and 22 (terminal body weight).
In addition, a body weight was recorded on the day of the occurrence of balano-preputial separation (males) or vaginal opening (females).

Food Consumption
Food consumption for Cohorts 1A, 1B, 2A, and 3 were recorded daily from PND 22.
Consumption was calculated as g/animal/day.

Sexual Maturation
Animals were observed for vaginal opening (females) from PND 30 and cleavage of the balano-preputial gland (males) from PND 40; observations lasted until these milestones were attained.

Estrous Cycle Determination
Daily vaginal lavage (washings) were undertaken for Cohort 1A and 1B females after vaginal opening until the first cornified smear was observed, then from PND 75 to 89, inclusive, and on the day of necropsy.

Acoustic Startle
Acoustic startle was assessed for Cohort 2A animals once on PND 24 (±1day).
Each animal was evaluated for startle habituation using a Kinder Auditory Startle Chamber. Prior to habituation testing, each animal was acclimated to the chamber for 4 minutes under white noise conditions. Each animal underwent one habituation session, consisting of 50 standard trials of 115 dB bursts of noise, with mixed inter-trial intervals.
On a separate day, prior to pre-pulse inhibition (PPI) testing, each animal was acclimated to the chamber for 4 minutes under white noise conditions. Animals underwent testing, which consisted of 41 randomized trials (the first trial was not reported). Thirty were pre-pulse trials (10 trials each of 74, 78, or 86 dB pre-pulse, followed by 115 dB startle) and 10 were standard trials (115 dB startle, with no pre-pulse), with mixed inter-trial intervals and decibel levels. Data are reported as percent pre-pulse inhibition for the PPI session.
Data collected for habituation included the mean maximum amplitude (five blocks of 10 trials for each group). Pre-pulse inhibition (PPI) was calculated as 100 - ([maximum startle amplitude on pre-pulse trials / maximum startle amplitude on 115 dB standard trials] x 100) for each animal.

Functional Observational Battery (FOB)
All Cohort 2A animals were assessed by detailed clinical observations, quantitative assessments, and elicited responses on one occasion between PND 66 and 73 for males and between PND 63 and 73 for females. Animals were observed within the home cage, in the hand, and in the arena for approximately 2 minutes.
Functional observational battery (FOB) assessments were undertaken in a manner so the observer did not know the dose group of animals during testing. Observations were performed at the same time on each occasion, where possible.

Detailed Clinical Observations
Each animal was observed and evaluated weekly as indicated:
Subjective observations:
Activity; Pain response; Aggression to cage mate; Palpebral reflex; Alertness; Pelage; Approach response; Pinna response; Acoustic startle response; Posture; Bar test; Proprioception (right hind leg); Behavior - other; Pupil status; Behavior - stereotype; Pupillary response; Body tone; Reactivity to handling; Discharge; Respiration; Excretion; Restlessness; Extensor thrust; Righting reflex; Eye closure; Salivation; Eyes; Skin color; Gait; Tail; Grasping loss; Visual response; Grip strength; Vocalization; Involuntary movement; Waxy rigidity; Lacrimation
Numerical observations:
Fecal boli; Rears; Latency; Urine pools

Quantitative Assessments
Quantitative assessment parameters are as follows.
Hind limb foot splay; Fore and/or hind limb grip strength

Motor Activity
All Cohort 2A animals were assessed for motor activity in an automated photocell activity recorder for 30 minutes. Assessments were performed between PND 63 and 73.
Activity counts were recorded at 5-minute intervals. The following parameters were determined:
Total activity counts; Total mobile counts; Total rearing

F1 Clinical Laboratory Procedures
Clinical Pathology
Cohort 1A Sample Collection and Handling
Blood samples for hematology (1 x 0.5 mL [EDTA], nominal), coagulation (1 x 0.5 mL [trisodium citrate], nominal), and clinical chemistry (1 x 0.6 mL [lithium heparin], nominal) were withdrawn from the abdominal aorta at necropsy (terminal samples were taken between PND 84 and 97 for males and between PND 89 and 98 for females). Samples were collected after animals were fasted overnight.
Blood samples for thyroid hormone (1 x 1.6 mL [serum separator tubes] nominal) were withdrawn from the jugular vein in-life (10 randomly selected males) or from the abdominal aorta at necropsy (10 randomly selected females); these samples were taken between PND 84 and 97 for males and between PND 89 and 98 for females. Samples were collected after animals were fasted overnight. One sample from each animal was analyzed in the first instance, when required the spare sample was analyzed, spare samples not required for additional analysis are retained in storage until report finalization, after which, they will be discarded.
Blood samples for thyroid hormone analyses were withdrawn from the animal at a similar time on each occasion (between 0900 and 1300 hours); samples were protected from light until frozen storage.
Each blood sample for thyroid hormone analysis was gently inverted 10 times and was stored at room temperature (15 to 25°C) and centrifuged at 2300g for 10 minutes at approximately 4°C. The resultant serum was separated, transferred to uniquely labeled amber polypropylene tubes, and frozen at <-20°C. Samples were centrifuged and aliquoted within 2 hours of collection, and were analyzed at Covance.
Urine samples from 10 randomly selected males and females in each group were collected overnight the day prior to necropsy. Food was removed during collection.

Hematology Tests
hemoglobin; red blood cell count; packed cell volume; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; reticulocyte count; red cell distribution width; hemoglobin distribution width; total and differential white cell count; platelet count; platelet crit; mean platelet volume; platelet distribution width

Coagulation Tests
prothrombin time; activated partial thromboplastin time; fibrinogen

Clinical Chemistry Tests
aspartate aminotransferase; alanine aminotransferase; alkaline phosphatase; total cholesterol; total bilirubin; total protein; albumin; globulin; sodium; potassium; chloride; calcium; inorganic phosphate; creatinine; urea; glucose; albumin:globulin ratio

Urinalysis Tests
volume; color; turbidity; specific gravity; pH; protein; glucose; ketones; urobilinogen; bilirubin; blood; microscopy of sediment

Thyroid Hormone Tests
thyroxine (Total T4); thyroid stimulating hormone (TSH)

Cohort 3 Sample Collection and Handling - Immunotoxicology

Detailed protocol on the TDAR assay using KLH (ELISA):

The immunosuppression of a therapeutic agent can be demonstrated through the analysis of T dependent antibody responses (TDAR). This involves many components of the immune system, that is, antigen presentation, cytokine secretion, T-lymphocyte help and B-cell proliferation. One such method for the investigation of TDAR is the use of the Keyhole Limpet Hemocyanin (KLH) model. This model is a way of testing subjects humoral (antibody) response to immunisation with an antigen, which, in this case is protein (hemocyanin) extracted from a shellfish (keyhole limpet). The resulting response in the subject is to produce immunoglobulin antibodies to KLH (anti-KLH IgG and anti KLH IgM). The anti-KLH IgG and IgM in a sample will be determined using an antibody cut point titration method. An antibody titre is a method of determining how much antibody is present in a sample. A blood serum/plasma sample containing the antibody is diluted serially (for example 1/100, 1/500, 1/2500, 1/12500, 1/62500 etc.). Using an appropriate detection method (for example an ELISA based method), each dilution is analysed for the presence of detectable levels of the antibody in question (e.g. Anti-KLH IgG or IgM). In its most basic form, the assigned titer value can be expressed as the last dilution in which the antibody was detected. Anti-KLH analysis, however, lends itself to a cut-point titration method in that there will always be a background level of non-specific IgG and IgM. This background can easily be defined and used in the assay. Due to the specific response to KLH, normal serum from subjects that have not been inoculated (naïve samples) will not have anti-KLH antibodies. By establishing a background level presented by a pool of naïve matrix a cut point can be defined, whereby sample above the cut-point are positive for anti KLH and those below are considered negative for anti-KLH. Another advantage of using a naïve matrix is that when applied to each plate it can be used to monitor plate coating efficiencies, used to define plate acceptance criteria and offer a degree of continuity between batches. Samples are diluted until the response values are less than the established cut point which is usually defined by running multiple lots of naïve matrix and assigning +3SDs to the mean. The cut-point titer value of the sample will be calculated as the point at which the reading (Y) crosses the dilution axis (X) using the slope of the curve between the points above and below the cut-point.

Attached below is the Analytical Procedures document (AP 11-004), that outlines the validated KLH procedure.

Justification for the selected KLH concentration and concentration of the KLH used per animal per exposure :

The concentration of KLH used for this study run at Harrogate, Covance has been established as the optimal KLH-TDAR response in Cynomolgus monkey, which elicits an immune response (measureable increases in IgG and IgM titers), whilst not effecting standard toxicity endpoints. The dose used are in line with the ranges of KLH used in published literature (Piccotti et al, 2005).

Information on the (concurrent) positive control substance used and its results:

The positive control used in the assay was serum matrix taken from an animal that was exposed to KLH (Positive matrix). The anti-IgG and anti-IgM titers produced in a positive matrix are well above the cut point of the assay, which is calculated using the naïve matrix (derived from an animal sample that is not exposed to KLH).

References:

https://www.ncbi.nlm.nih.gov/pubmed/18958673

Anti KLH
Keyhole Limpet Hemocyanin (KLH) was administered (2 mL/kg intravenously (bolus), via manual injection, at a rate of 4 mL/min) to all F1 cohort 3 animals on PND 56 ± 3 and PND 70 ± 3. Blood samples (0.5 mL - to provide at least 0.2 mL of serum into serum separator tubes) were withdrawn from the jugular vein or abdominal aorta from all F1 cohort 3 animals on PND 61 ± 3, PND 70 ± 3, PND 77 ± 3 and PND 84 ± 3. Samples were allowed to stand for at least 30 minutes at room temperature prior to centrifugation at 2300 g for 10 minutes at 4°C, split into 2 aliquots of 75μL (set 1 and set 2) and the remaining sample was aliquoted as set 3. Analysis for anti-KLH antibodies (IgM and IgG), in the resultant serum, was done using an enzyme-linked immunosorbent assay (ELISA) method.

Immunophenotyping
Immunophenotyping was performed on spleens taken from 10 males and 10 females per dose group from F1 cohort 1A animals. The excised sample was weighed and the weight recorded. The tissue was placed in a Miltenyi Biotec C tube with sufficient volume of appropriate media. A single cell suspension was prepared as soon as possible by means of mechanical disruption of the spleen section using the GentleMACS tissue dissociator. The resultant cell suspension was then transferred to labeled falcon tubes. Assessment of lymphocyte subsets was performed by flow cytometry using the following markers:
CD3+ (total T lymphocytes); CD161a+/CD3- (NK cells); CD4+ (T-helper cells); CD45RA+/CD3- (B lymphocytes); CD8+ (Cytotoxic T lymphocytes)
Postmortem examinations (parental animals):
With the exception of fasting, these procedures were also followed for unscheduled sacrifices and deaths.

Adults Necropsy
Males were sacrificed by isoflurane anesthesia on Post-Pairing Day 46/47 (Week 18) and an overnight period without food. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
Females were sacrificed by isoflurane anesthesia on LD 22 (those that littered) or 26 days post-coitum (those that did not litter), after an overnight period without food. Females with the same day of mating/littering were sacrificed in a controlled randomization order, when possible. Females that did not litter were sacrificed in animal identification order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
The number of implantations sites for all paired females were recorded. The uterus of any apparently non-pregnant female was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.
Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.

Organ Weights
Organ weights were recorded at each scheduled sacrifice, excluding decedents. Organs were dissected free from fat and other contiguous tissue and were weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral-buffered formalin, unless otherwise indicated.

Tissue List for Males that Sired and Pregnant/Littered Females
adrenal; nerve, optic; animal identification; nerve, sciatic; bone marrow smear (femur); ovary; brain (including olfactory bulb); oviduct; cecum; pituitary; colon; prostate; duodenum; rectum; epididymis; seminal vesicle with coagulating glands and fluid; eye; spinal cord, cervical; femur with bone marrow and femorotibial joint; spinal cord, lumbar; gross lesions; spinal cord, thoracic; head; spleen; heart; stomach; ileum; testis; jejunum; thymus; kidney; thyroid with parathyroid; liver; trachea; lungs with main stem bronchi and bronchioles; urinary bladder; lymph node, mandibular; uterus with cervix; lymph node, mesenteric; vagina; mammary gland; Vas deferens (right); muscle, biceps femoris

Tissue List for Males that did not Sire and Non-Pregnant/Non-Mated Females
animal identification; pituitary; epididymis; prostate; gross lesions; seminal vesicle with coagulating glands and fluid; mammary gland; testis; ovary; uterus with cervix; oviduct; vagina

Histology and Microscopic Examinations
All tissues from Groups 1 and 4 and all decedents were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 μm, and stained with hematoxylin and eosin.
All tissues from males that did not sire or had affected sperm assessments or females that were not mated and/or not pregnant were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 μm, and stained with hematoxylin and eosin.
Additional sections of testes and epididymides were also stained with periodic acid-Schiff (PAS) for spermatogenic staging for all males of Groups 1 and 4 and all decedents.
All processed tissues were examined microscopically by the Study Pathologist. Qualitative assessments of stages of spermatogenesis and interstitial testicular cell structure were also performed for males of Groups 1 and 4.
Postmortem examinations (offspring):
Unselected pups on PND 22 and moribund pups were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Sacrifices were carried out in group order. Once a suitable deep plane of anesthesia was established, major blood vessels were severed to exsanguinate the animal.
Full macroscopic examinations with particular attention to the external reproductive genitals were conducted for all decedents and pups sacrificed on PND 22.
The tissues listed were obtained from the unselected PND 22 pups and were retained in 10% neutral-buffered formalin.
animal identification; mammary tissue; brain; spleen; gross lesions; thymus

F1 Generation Terminal Procedures
With the exception of fasting, these procedures were also followed for unscheduled sacrifices and deaths.

F1 Generation Cohort 1A
Necropsy
Animals were sacrificed by isoflurane anesthesia between PND 84 and 97 after an overnight period without food. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.

Organ weights
Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral-buffered formalin, unless otherwise indicated.
F1 Generation Cohort 1A Tissue List
adrenal; nerve, optic; animal identification; nerve, sciatic; bone marrow smear (femur); ovary; brain (including olfactory bulb); oviduct; cecum; pituitary; colon; prostate; duodenum; rectum; epididymis; seminal vesicle with coagulating glands and fluid; eye; spinal cord, cervical; femur with bone marrow and femorotibial joint; spinal cord, lumbar; gross lesions; spinal cord, thoracic; head; spleen; heart; stomach; ileum; testis; jejunum; thymus; kidney; thyroid with parathyroid; liver; trachea; lungs with main stem bronchi and bronchioles; urinary bladder; lymph node, mandibular; uterus with cervix; lymph node, mesenteric; vagina; mammary gland; Vas deferens (right); muscle, biceps femoris

Histology and Microscopic Examinations
All tissues from Groups 1 and 4 and all decedents were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 μm, and stained with hematoxylin and eosin.
The adrenals, thymus, and spleen from all animals in Groups 2 and 3 were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 μm, and stained with hematoxylin and eosin.
The mandibular and mesenteric lymph nodes and femur bone marrow from 10 animals/sex/group (those with the lowest identification numbers) were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 μm, and stained with hematoxylin and eosin.
Females of Groups 1 and 4 had additional slides for ovarian follicle counts; a quantitative evaluation of primordial and small follicles was performed. Then, a qualitative assessment of follicular development to regression (from small growing follicles to corpora lutea) was also performed.
Additional sections of testes and epididymides were also stained with periodic acid-Schiff (PAS) for spermatogenic staging for all males of Groups 1 and 4 and all decedents.
All processed tissues were examined microscopically by the Study Pathologist. Qualitative assessments of stages of spermatogenesis and interstitial testicular cell structure were also performed for males of Groups 1 and 4.
Prenatal and postnatal induced toxic effects were evaluated by assessment of the mandibular and mesenteric lymph nodes and splenic lymphocyte subpopulation analysis from at least 10 animals/sex/group. Additionally, a microscopic evaluation of the thymus, spleen, and adrenal glands was performed.

Sperm Assessments
For each scheduled male, sperm number, motility, and velocity were recorded by Computer Assisted Sperm Analysis (CASA) from a sample of sperm from the left epididymis (cauda epididymis and vas deferens). Each sample was prepared for microscopic evaluation of sperm morphology. Slides prepared from the first 10 surviving males for each group were read for morphological changes. The left epididymis was discarded following seminology investigations.

F1 Generation Cohort 1B
Necropsy
Animals were sacrificed by isoflurane anesthesia from PND 100 to 112. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal.
Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.

Organ weights
Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral-buffered formalin, unless otherwise indicated.
Tissue List for F1 Generation Cohort 1B
animal identification; pituitary; epididymis; prostate; gross lesions; seminal vesicle with coagulating glands and fluid; mammary gland; testis; ovary; uterus with cervix; oviduct; vagina

Histology and Microscopic Examinations
All tissues from animals of Groups 1 and 4, all decedents, and all animals that did not produce a live litter after mating were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 μm, and stained with hematoxylin and eosin.
Tissues were examined from males that did not sire and any female that did not produce a pregnancy.

F1 Generation Cohort 2A
Necropsy
Animals were sacrificed by isoflurane anesthesia on PND 76. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Upon sacrifice, macroscopic examinations were conducted (which included all orifices, cranial cavity, external surfaces of the brain and spinal cord, cervical tissues and organs, thoracic, abdominal and pelvic cavities and viscera, external surface of the body and nasal cavity and paranasal sinuses), and all lesions were recorded.

Organ weights
The brain from all animals (excluding decedents) was retained in 10% neutral-buffered formalin, prior to fixed tissue dissection to remove tissues (see Text Table 3.5). Animals were subjected to whole body perfusion fixation with 50% Karnovsky’s fluid.
Tissue List for F1 Generation, Cohort 2A
Olfactory bulb; Forebrain (including hippocampus); Basal ganglia (including caudate nucleus); Hypothalamus/thalamus; Midbrain; Brain stem (including pons and medulla oblongata); Cerebellum; Cerebral cortex; Entire spinal cord (including cervical [to include enlargement at C4 level], thoracic, and lumbar [to include enlargement at L4/5 level] regions); Cervical dorsal root ganglia; Lumbar dorsal root ganglia; Trigeminal ganglia; Eye including retina; Optic nerve; Proximal and distal sciatic nerve; Proximal and distal tibial nerve; Sural nerve; Anterior tibialis muscle (left side); Gastrocnemius muscle (left side); Macroscopic lesions

Histology and Microscopic Examinations
Tissues were trimmed, processed, and embedded in paraffin wax (all animals); however, neuropathological evaluation by hematoxylin and eosin (H&E) was performed on Groups 1 and 4 only. Or tissues were trimmed, post-fixed in osmium tetroxide, processed, and embedded in epoxy resin (all animals); however, neuropathological evaluation by toluidine blue was performed on Groups 1 and 4 only.

F1 Generation Cohort 2B
Necropsy
Animals were sacrificed by isoflurane anesthesia on PND 22. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.

Organ weights
Organs were dissected free from fat and other contiguous tissue and weighed before fixation. Left and right organs were weighed together. Tissues were retained in 10% neutral-buffered formalin, unless otherwise indicated.
Tissue List for F1 Generation Cohort 2B
Olfactory bulb; Forebrain (including hippocampus); Basal ganglia (including caudate nucleus); Hypothalamus/thalamus; Midbrain; Brain stem (including pons and medulla oblongata); Cerebellum; Cerebral cortex; Macroscopic lesions

Histology and Microscopic Examinations
Those tissues indicated above were trimmed, processed, and embedded in paraffin wax (all animals) but neuropathological evaluation by H&E was performed on Groups 1 and 4 only.

F1 Generation Cohort 3
Animals were sacrificed on PND 84 by isoflurane anesthesia. Sacrifices were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Blood sampling information is detailed in the Immunotoxicology Report. Upon sacrifice, macroscopic examinations were conducted, and all lesions were recorded.
The spleen was weighed and preserved in 10% neutral buffered formalin.

F2 Generation Pups
F2 pups were sacrificed on PND 4 by an intraperitoneal injection of sodium pentobarbitone (overdose), followed by exsanguination via decapitation. Macroscopic examinations were conducted on all pups, and all lesions were recorded, although no tissues were retained.
Statistics:
Only data collected on or after the first day of dosing were analyzed statistically.
The following data were analyzed using Tox Reporting.
-Food consumption (pre-pairing, post-pairing and maturation) - Procedure I (ANOVA) - Tox Reporting
-Continuous behavioral (FOB) data - latency to first step, number of rears, hindlimb foot splay, forelimb grip strength, hindlimb grip strength -Procedure I (ANOVA) - Tox Reporting
-Locomotor Activity - Procedure I (ANOVA) - Tox Reporting
-Clinical Pathology (adult male and female) Procedure I (ANOVA) - Tox Reporting
The following data were analyzed using Pristima.
-Body weights (adult) - Procedure I (ANOVA) - Pristima
-Body weight gains (adult) - Procedure I (ANOVA) - Pristima
-Food consumption (gestation and lactation) - Procedure I (ANOVA) - Pristima
-Absolute organ weights and organ to terminal body weight ratios for adults - Procedure I (ANOVA)
Reproductive indices:
-The mean number of estrous cycles and mean cycle length - Procedure III
-Male and female mating, fecundity, and fertility indices - Procedure IV (one-sided lower tail)
-Number of pups delivered, live born pups, implantation sites, live birth index, PND 4 viability index, weaning index, duration of gestation, and percent post-implantation loss - Procedure III
-Pup weights (male, female, and combined) - Procedure II (litter size as the covariate).
-Live Pups/Litters with Live Pups - Procedure III
-Percent pregnant, percent delivering, and gestation index - Procedure IV (one-sided lower tail)
-Percent of females with stillborn pups - Procedure IV (one-sided upper tail)
Offspring viability indices:
-Anogenital distance (males only) - Procedure II (cube root of mean pup weight as the covariate)
-Clinical pathology (male, female and combined pups) Procedure I (ANOVA) - Tox Reporting
-Acoustic startle data (mean maximum amplitude for each block of 10 trials and pre-pulse inhibition (PPI) data [%PPI]) - Procedure I (ANOVA) - Pristima
-Seminology data - Procedure III (Acclimation not analyzed)
-Absolute organ weights and organ to terminal body weight ratios for pups - Procedure I (ANOVA).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations were confined to instances of salivation for a few animals administered 100 or 50 mg/kg/day and occasionally following administration of 20 mg/kg/day and in control animals, although a clear dose response was not evident.
All remaining clinical observations (generally confined to thin, or stained fur, fur loss or lesions to the skin) were observed throughout the dose groups, including controls and were unrelated to the test article.
Postdose observations were confined to instances of salivation and mouth rubbing up to 0.5 hours postdose for males administered 100 mg/kg/day.
The only notable finding noted during the weekly detailed observations was the presence of salivation for one or two males administered 50 mg/kg/day; this finding was consistent with observations noted during the daily clinical observations.
One control female was recorded with abnormal gait; however, in the absence of test article administration in this dose group, this finding was incidental.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No unscheduled deaths resulted from test article toxicity.
One control male (Animal Number 1) sent to necropsy on Study Day 5. One male administered 20 mg/kg/day (Animal Number 38) was found dead on Study Day 2, and one male administered 100 mg/kg/day (Animal Number 382) was found dead on Study Day 107. Macroscopic examinations for these animals revealed an esophageal rupture, which was consistent with inappropriate dosing technique (dosing error) and was unrelated to test article toxicity.
One female administered 100 mg/kg/day (Animal Number 590) was sent to necropsy on LD 2 due to a total loss of the litter. The female had only two implantation sites and one pup born. No macroscopic or microscopic abnormalities were noted for this animal. This was an isolated finding and is a low incidence finding in studies of this type; as such, this was considered to have arisen incidentally and unrelated to test article toxicity.
Another female administered 100 mg/kg/day (Animal Number 582) was found dead on LD 5. No macroscopic or microscopic abnormalities were noted to determine the cause of demise for this animal. In isolation, this death was considered to have arisen incidentally.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test article-related effects on body weights were noted for either sex prior to pairing or during gestation or lactation at any administered dose.
Mean body weight and body weight change values for test article-treated groups were essentially similar to controls; as such, any transient instances of statistically significant intergroup differences were considered to have arisen incidentally and did not represent an effect of the test article.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related effects on food consumption were observed during the 10 Week pre-pairing phase.
Mean food consumption was slightly higher during the post-pairing phase for males administered 100 mg/kg/day, compared with controls, with values occasionally attaining statistical significance compared with controls; the mean overall increase over the post pairing phase was +8% compared with controls, and as such, this increase was considered not to have represented an adverse effect of the test article.
Slightly increased food consumption was observed during gestation for females administered 100 mg/kg/day, compared with controls, with statistical significance achieved between GD 7 to GD 15 (P<0.05) and an overall mean of +11% compared with controls during this period. Mean values were essentially similar to controls towards the end of gestation, and as such, these intergroup differences were considered not to represent an adverse effect of the test article.
No test article-related effect on food consumption was evident following administration of 50 or 20 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related hematology changes were observed at all dose levels, with effects more prominent for males.
Hemoglobin (HB), red blood cells (RBC), and packed cell volume (PVC) were significantly lower for male from all test article-treated groups, compared with controls, with a clear dose relationship observed for HB and RBC (P < 0.05 to P < 0.001 with ascending dose, with the exception of PCV). Red blood cells were also significantly lower for females administered 100 or 50 mg/kg/day (P < 0.001 or P < 0.05 respectively), compared with controls. Hemoglobin levels were also marginally lower for females administered 100 mg/kg/day, compared with controls, although statistical significance was not achieved.
Mean reticulocytes (absolute counts and % reticulocytes) were also significantly increased for males administered 100 mg/kg/day (P < 0.001) or 50 mg/kg/day (P < 0.01), compared with controls.
Mean cell volume (MCV) was elevated for males administered 100 or 50 mg/kg/day (P < 0.001 or P < 0.05 respectively), compared with controls. Mean cell hemoglobin (MCH) was also significantly elevated for males administered 100 mg/kg/day, compared with control (P < 0.001). Similar increases were also observed on LD 22 in MCV for females administered 100 or 50 mg/kg/day (P < 0.001 or P < 0.05 respectively), compared with controls, and in MCH for females administered 100 mg/kg/day (P < 0.001) or 50 mg/kg/day (P < 0.05), compared with controls. In addition, HDW was elevated for females administered 100 mg/kg/day, compared with controls (P < 0.01).
Males administered 100 mg/kg/day showed elevated PT (P < 0.001), PLT (P < 0.01), and PCT (P < 0.001), compared with controls, with PCT also increased for males administered 50 mg/kg/day (P < 0.01). Similar increases in PLT (P < 0.05) and PCT (P < 0.01) were observed only for females administered 100 mg/kg/day, compared with controls.
No test article-related changes were noted for females administered 20 mg/kg/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test article-related changes in clinical chemistry parameters were observed for males administered 100 or 50 mg/kg/day.
Alanine aminotransferase (ALT) activity was increased for males administered 100 mg/kg/day, compared with controls (P < 0.05).
Total protein was significantly lower for males administered 100 mg/kg/day, compared with controls (P < 0.001), with reduced globulin (P < 0.001) and increased A:G ratio (P < 0.01) observed for males administered 100 mg/kg/day, compared with controls. Reduced globulin and increased A:G ratio were observed for males administered 50 mg/kg/day, compared with controls (P < 0.01 and P < 0.05 respectively).
No test article-related changes were noted for males administered 20 mg/kg/day or for females at any dose level.
Remaining statistically significant changes for males (elevated cholesterol and creatinine levels following 50 mg/kg/day administration) were minimal and did not show a dose relationship; as such, they were considered not test article-related.
Marginally lower glucose levels (P < 0.05) were observed for females administered 100 mg/kg/day, compared with controls. However, in isolation, this was considered to have arisen incidentally and was unrelated to the test article.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test article-related changes were noted in the urine parameters investigated.
The statistically significantly increase in specific gravity noted for females administered 50 mg/kg/day was isolated, and, in the absence of supporting data or a dose response, was considered to have arisen incidentally and was unrelated to the test article.
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):
Upon microscopic examination, test article-related findings were recorded for the liver, spleen, kidney, and prostate of animals administered 100 mg/kg/day, including three males (Animal Numbers 26, 29, and 55) that did not mate and/or sire litters but which were examined at terminal sacrifice.
In the liver, centrilobular hepatocyte hypertrophy/degeneration was recorded for males administered 20 mg/kg/day and both sexes administered 50 or 100 mg/kg/day. Hepatocellular karyomegaly/multinucleation was recorded for females administered 100 mg/kg/day. Centrilobular hepatocellular hypertrophy/degeneration was characterized by increased size and/or cytoplasmic basophilia of hepatocytes primarily adjacent to central veins, with variable associated single cell necrosis, mitotic figures, and small numbers of mixed inflammatory cells. Hepatocellular karyomegaly/multinucleation was characterized by scattered hepatocytes with increased numbers of nuclei and/or enlarged nuclei and increased amounts of cytoplasm.
Increased pigment was recorded for the spleen of animals administered 20, 50, or 100 mg/kg/day, compared with controls. Splenic pigment was characterized by yellow-brown, granular pigment within the splenic red pulp.
In the kidney, a minor decrease in hyaline droplets was recorded for males administered 50 or 100 mg/kg/day, compared with control males, and was characterized by fewer intra-cytoplasmic accumulations of eosinophilic pigment within proximal tubular cells.
Contraction was noted in the prostate of a few males administered 100 mg/kg/day, and was characterized by decreased secretions within the gland and an overall reduction in glandular size, which corresponded with the decreased organ weights and organ weight ratios noted at necropsy.
No other test article-related microscopic findings were recorded as microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age, and at this stage of the reproductive cycle.
In addition, six females (one control [Animal 508], two [Animals 526 and 529], administered 20 mg/kg/day, one [Animal 555] administered 50 mg/kg/day, and two [Animals 576 and 580] administered 100 mg/kg/day) did not mate and/or produce a pregnancy and/or a litter. No findings were recorded for these animals to suggestive an effect of the test article on reproductive function. Findings in these animals were generally similar to those recorded for animals that produced a litter.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sexual Maturation
No overt differences in age or corresponding body weight at the attainment of sexual maturity were observed in any test article-treated group, compared with concurrent controls.
Statistical analysis of the data did not show any statistically significant intergroup differences.

Thyroid hormone data
No test article-related changes in T4 or TSH were noted at any dose level, compared with controls.
Statistical analysis of the data did not reveal any significant intergroup differences.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No test article-related effect was noted on the mean number or duration of the estrous cycles at any dose level, compared with controls.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No test article-related changes were noted in sperm count, sperm motility, velocity or morphology following administered at the test article at any dose level.
Statistical analysis of the data did not reveal any significant intergroup differences.
Reproductive performance:
no effects observed
Description (incidence and severity):
No test article-related effect was noted on the mean number or duration of the estrous cycles at any dose level, compared with controls.
No test article-related effects on mating, fertility, or fecundity were noted.
All animals mated within 7 days of pairing and 24, 23, 24, or 23 females administered 0, 20, 50, or 100 mg/kg/day, respectively, were pregnant.
Following parturition, 24, 23, 24, and 23 females administered 0, 20, 50, or 100 mg/kg/day, respectively, produced a live litter.
Possible test article-related effects were noted in the group administered 100 mg/kg/day. Viability indices on PND 4 and weaning indices were marginally lower for litters of the group administered 100 mg/kg/day (92 and 95%, respectively), compared with controls (99%), and the number of missing pups (presumed cannibalized) was higher than the numbers of animals in litters of the group administered 100 mg/kg/day (n = 7), compared with controls (n = 3).
Mean gestation lengths for test article-treated females were essentially similar to those of controls.
Live birth index was unaffected by test article administration. Mean numbers of implantation sites or post-implantation losses or litter sizes of test article-treated groups were similar to those of controls. Furthermore, sex ratio was unaffected by maternal test article exposure.
No stillborn pups were noted in the groups administered 50 or 100 mg/kg/day; stillborn pups were only observed at a low incidence in the groups administered control article (vehicle) or 20 mg/kg/day, which was unrelated to the test article.
Once daily oral gavage administration of 20, 50, or 100 mg/kg/day 3,5-Dimethylpyrazole to Han Wistar rats for 10 weeks prior to pairing and throughout pairing and during gestation and throughout lactation for females, and then administered to the F1 offspring until adulthood and into a second generation for some animals, resulted in test article-related effects at all dose levels investigated.
Although the physical health of animals was not impaired and no effects on body weight or food consumption were observed, salivation was observed at all dose levels but was considered attributable to test article palatability and was not a toxic effect of the test article.
A microscopic liver change of hepatocellular degeneration was noted for F0 and F1 animals at all dose levels, although at an overall lower level in the F1 generation compared with F0 animals. In the F0 generation, minimal to moderate hepatocellular degeneration (characterized by variably increased cell size, with cytoplasmic basophilia, and associated single cell necrosis, mitoses, and inflammation that occasionally extended between adjacent central veins) was noted. In the F1 generation, the severity of this finding was predominantly minimal. In males, the primary characteristic was minimal inflammation adjacent to central veins, whereas, in females, centrilobular hepatocellular hypertrophy was more pronounced. This finding in both generations was thought to represent a continuum of hepatocellular hypertrophy that progressed to increased cytoplasmic basophilia, with a few inflammatory cells at a minimal level, to hepatocellular degeneration with associated inflammation, single cell necrosis, and/or mitotic figures at a slight/moderate grade. This was attributed to the test article. Based on the hepatocellular degeneration, a no observed effect level (NOEL) was not established. This finding may have been associated with the minor clinical chemistry changes noted in liver parameters in both generations (i.e., increased ALT and aspartate aminotransferase activities, reduced total protein and albumin levels, and increase A:G ratio for F0 males administered 100 or 50 mg/kg/day and elevated cholesterol level for F1 females administered 100 mg/kg/day).
Hyaline droplets in the kidney appear as eosinophilic cytoplasmic inclusions in the proximal tubular epithelial cells and are a common background finding in male rats, and of no relevance to humans (Hard et al., 1993). The decrease in hyaline droplets in F0 and F1 males was considered of no toxicological significance.
Increases in PT, PLT, and PCT were observed for both sexes administered 50 or 100 mg/kg/day, together with a possible anemic response, evidenced by reductions in hemoglobin, erythrocyte counts, and packed cell volume and corresponding increases in reticulocyte counts, MCV, and MCH. A microscopic splenic change of yellow/brown granular pigment within the splenic pulp was noted in all test article-treated groups; this finding was most likely iron deposits and may have been associated with the hematology changes observed in the red cell mass.
Prostatic contraction was noted in F0 males administered 100 mg/kg/day, which corresponded to decreases in prostate weights and weight ratios in all dose groups. However, no effect on reproductive performance was observed. Similarly, seminal vesicle weights (absolute and body weight ratios) were lower in F0 and F1 males and were characterized microscopically by contraction of the organ in F1 males. These changes were thought to correspond to decreases in secretions and may have represented a direct effect of the test article on the glandular epithelium or may have been secondary to effects on the endocrine and/or reproductive systems. However, no organ weight changes or macroscopic or microscopic findings were noted in other endocrine or reproductive tissues in males, and sperm analysis in the parental generation did not show any effect of the test article. Overall, the significance of these findings in these secondary male reproductive organs was minimal as mating, fertility, and fecundity were unaffected in the F0 generation. A marginally lower mating index was observed for F1 males administered 100 mg/kg/day; however, this did not affect fertility, and was, therefore, considered not to have represented an adverse effect.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive Toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic Toxicity
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Clinical signs:
no effects observed
Description (incidence and severity):
No test article-related clinical observations were noted, and no observations were noted after pups were directly dosed from PND 14.

F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
Notable clinical observations were confined to instances of salivation, which was observed in all test article-treated groups, but not in the controls.
All remaining clinical observations (generally confined to thin or stained fur, fur loss or lesions to the skin) were observed throughout the dose groups and were considered to be unrelated to the test article.
Postdose Observations - No observations were noted following dosing at any dose level.
No test article-related observations were noted following the weekly detailed observations.
One female administered 100 mg/kg/day, was recorded with audible respiration. In isolation, this was considered to have arisen incidentally and was not test article related.

F1 Generation Cohort 1B - Reproductive Toxicity
Notable clinical observations were confined to instances of salivation, which was observed for males administered at 20, 50 or 100 mg/kg/day, and females administered 20 or 50 mg/kg/day. This was not observed for controls.
All remaining clinical observations (generally confined to thin or stained fur, fur loss or lesions to the skin) were observed throughout the dose groups and were considered unrelated to the test article.
Postdose Observations - No observations were noted following dosing at any dose level.
No observations were noted following the weekly detailed observations.

F1 Generation Cohort 2A - Neurotoxicity
Notable clinical observations were confined to isolated instances of salivation, which were noted for a few males and females administered 100 or 50 mg/kg/day and a few females administered 20 mg/kg/day. Hunched posture was also noted for one female administered 100 mg/kg/day.
The remaining clinical observations noted (generally confined to thin, or stained fur, fur loss or lesions to the skin) were observed throughout the dose groups and were considered unrelated to the test article.
Postdose Observations - No observations were noted following test article administration at any dose level.
Weekly detailed clinical assessments did not show any clinical observations.

F1 Generation Cohort 2B - Neuropathology
No test article-related clinical observations were noted. Clinical observations were confined to the presence of thin fur for one 50 mg/kg/day female which was unrelated to the test article.

F1 Generation Cohort 3 - Immunotoxicity
Test article-related clinical observations were confined to salivation, which was noted throughout the dose groups. This finding was also noted for one control male from this cohort.
No postdose observations were noted.
No findings were observed during the weekly detailed clinical observations.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
No test article-related deaths were noted.
One female administered 100 mg/kg/day (Animal 830) was sacrificed on PND 82 due to poor physical condition (subdued/sluggish behavior, raised hair, wet fur). The cause of demise was not determined, but no macroscopic or microscopic findings were observed to suggest that the death was test article related.
One control male (Animal 323) was found dead on PND 25. In the absence of test article administered in this group, this death was considered incidental.

F1 Generation Cohort 1B - Reproductive Toxicity
No test article-related deaths occurred.
One control female (Animal 634) was found dead on PND 16 and another control female (Animal 628) was found dead on PND 59. The cause of death was considered due to inappropriate dosing technique and unrelated to the test article.

F1 Generation Cohort 2A - Neurotoxicity
All Cohort 2A animals survived to the terminal sacrifice.

F1 Generation Cohort 2B - Neuropathology
All Cohort 2B animals survived to the terminal sacrifice.

F1 Generation Cohort 3 - Immunotoxicity
All animals from this cohort survived to the terminal sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test article-related changes on offspring body weight were evident following exposure of 50 or 100 mg/kg/day.
Mean body weights for female offspring exposed to 100 mg/kg/day were essentially similar to control on PND 1, however by PND 14, mean body weights were 6% lower than controls, when the pups were exposed to the test article through maternal milk. Mean body weights were also lower for female offspring exposed with 50 mg/kg/day (-8%).
Following the introduction of direct dosing of the pups, statistically significant lower mean body weights were also noted from PND 16 for male pups exposed to 100 mg/kg/day and on PND 18 for male pups exposed to 50 mg/kg/day. By the end of the pre-weaning period, only marginal differences were observed.

F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
No overt differences in mean body weight or body weight change were noted for test article-treated groups, compared with controls.
Mean body weights for test article-treated offspring on PND 22 at the start of the F1 phase was similar to controls (-4% males and -7% females). Statistical analysis did not reveal any statistically significant intergroup differences.
No test article-related body weight changes were evident following maternal exposure of 20 mg/kg/day.

F1 Generation Cohort 1B - Reproductive Toxicity
No test article-related effects on body weight or body weight change were noted.
Statistically significant intergroup differences were noted during late maturation and into gestation for females administered 50 mg/kg/day, with increases noted on LD 1. An increase in body weight gain was also noted on LD 1 for females administered 100 mg/kg/day, although values did not attain statistical significance. In the absence of a convincing dose-related response, these intergroup differences were considered to have arisen incidentally and were unrelated to the test article.

F1 Generation Cohort 2A - Neurotoxicity
No test article-related effects on body weight or body weight change were noted. Any statistically significant intergroup differences were transient and considered to have arisen incidentally, with no relationship to the test article.

F1 Generation Cohort 2B - Neuropathology
Body weights were unaffected by the test article.

F1 Generation Cohort 3 - Immunotoxicity
No test article-related changes in body weight or body weight change were noted. The statistically significant difference in body weight change noted for males administered 50 mg/kg/day between PND 34 and 46 was an isolated event, and as such, was considered to have arisen incidentally and not related to the test article.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
Mean food consumption for test article-treated groups was essentially similar to controls. Statistical analysis did not reveal any statistically significant intergroup differences.

F1 Generation Cohort 1B - Reproductive Toxicity
No test article-related effects on food consumption were noted. Any statistically significant differences noted were considered to have arisen incidentally and unrelated to the test article.

F1 Generation Cohort 2A - Neurotoxicity
No test article-related changes in food consumption were noted. Statistical analysis of the data did not reveal any significant intergroup differences.

F1 Generation Cohort 3 - Immunotoxicity
No test article-related changes in food consumption were noted. Statistical analysis of the data did not reveal any significant intergroup differences.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
Test article-related changes in hematology parameters were observed following test article administration at any dose level.
Red blood cells were reduced and MCV was elevated for males administered 100 mg/kg/day (P < 0.05), compared with controls. Reticulocytes (absolute and %) were also increased for males administered 100 mg/kg/day (P < 0.001) or 50 mg/kg/day (P < 0.05), compared with controls. Furthermore, males administered 100 mg/kg/day also showed an increase in RDW, compared with controls (P < 0.05).
Similar changes in red cell parameters were observed for females. Red blood cells were reduced and MCV were elevated reduced for females administered 100 mg/kg/day (P < 0.001) or 50 mg/kg/day (P < 0.01), compared with controls. Reticulocytes (%) were also elevated for females administered 100 mg/kg/day, with a reduction in HDW (P < 0.01), compared with controls.
Platelet counts (PLT) and PCT were significantly increased for males from all test article-treated groups, compared with controls, and a dose response was evident (P < 0.01 to P < 0.001). Similar increases were observed for females administered 50 or 100 mg/kg/day (PLT: P < 0.05 to P < 0.01, PCT: P < 0.05), compared with controls. In addition, PDW was significantly lower (P < 0.001) and APTS was elevated (P < 0.01) for females administered 100 mg/kg/day compared with controls. For males administered 100 mg/kg/day, PT was elevated, compared with control (P < 0.001).
Females administrated 100 mg/kg/day also showed an increase in LUC, compared with controls (P < 0.01), although other white cell parameters were unaffected. Although the etiology of this finding was unclear, a relationship to the test article could not be discounted.
Remaining statistically significant intergroup differences were isolated, confined to one sex, or showed no dose relationship; as such, they were considered to have arisen incidentally and were unrelated to test article administration.
Hematology parameters were unaffected for females administered 20 mg/kg/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
Males administered 100 mg/kg/day showed an increase in aspartate aminotransferase and ALT activities (P < 0.001 and P < 0.05, respectively), compared with controls, with a reduction in total protein (P < 0.05).
For females administered 100 mg/kg/day, test article-related changes were confined to an increase in cholesterol (P < 0.001), compared with controls.
No test article-related changes were noted following administration of 50 or 20 mg/kg/day.
Remaining statistically significant differences (reduced sodium and calcium levels noted for females administered 20 mg/kg/day) were minimal and did not show a dose response; as such, these were considered to have arisen incidentally.
Urinalysis findings:
no effects observed
Description (incidence and severity):
F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
No test article-related changes were noted in the urine parameters investigated.
Statistical analysis did not reveal any statistically significant intergroup differences.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
A delay in attainment of sexual maturity was observed for males administered 100 mg/kg/day, with a mean completion day on PND 51, compared with PND 48 for controls, with corresponding higher mean body weight at attainment. Mean values however did not attain statistical significance.
No such effect was noted for males administered 50 or 20 mg/kg/day.
Sexual maturity for test article-treated females was essentially similar to controls.

F1 Generation Cohort 1B - Reproductive Toxicity
Males administered 100 mg/kg/day attained sexual maturity on PND 51, compared to controls, which attained maturity on PND 49, with a corresponding increase in mean body weight on the day of attainment. Mean values however did not attain statistical significance. This finding was observed throughout the majority of the F1 generation males and was considered test article-related.
No such effect was noted for males administered 20 or 50 mg/kg/day, and no effect on sexual maturation was noted for all test article-treated female groups, compared with controls.

F1 Generation Cohort 2A - Neurotoxicity
Sexual maturation for males administered 100 mg/kg/day was delayed by 2 days (PND 50), compared with controls (PND 48), which correlated with the higher body weight at attainment. Statistical analysis, however, did not reveal any significance differences. This was not evident for males administered 50 or 20 mg/kg/day.
No test article-related effects on sexual maturity were evident for females.
The statistically significant earlier age of attainment for females administered 20 mg/kg/day (P < 0.05) was isolated and, in the absence of a dose response or similar findings noted in other F1 generation cohorts, was considered to have arisen incidentally.

F1 Generation Cohort 3 - Immunotoxicity
No test article-related effects on sexual maturity were observed.
Females administered 100 mg/kg/day showed a statistically significant delay in sexual maturity (P < 0.01), with PND 36 being the mean day of attainment, compared with PND 32 for controls. Mean body weights at attainment showed corresponding increases, with statistical significantly increases for females administered 100 mg/kg/day, compared with controls (P < 0.05). However, review of the sexual maturation data for the other F1 cohorts in this study did not show any effect on female sexual maturation; as such, the intergroup differences observed in this cohort were considered to have arisen incidentally.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Mean anogenital distance was unaffected for male or female pups following exposure of the test article at any dose level, compared with controls.
Statistical analysis of the data did not reveal any statistically significance intergroup differences.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Test article administration did not result in the presence of nipples/areolae for male offspring exposed at any administered dose level.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
No test article-related organ weight changes were noted.
Organ weight and organ weight ratio changes recorded for these animals were attributed to biological variation.

F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
Test article-related increases in unadjusted liver weights and weight ratios were recorded for females administered 50 or 100 mg/kg/day, and decreased unadjusted seminal vesicle weights and weight ratios were recorded for males administered 100 mg/kg/day.
Unadjusted liver weights, liver:body weight ratios, and liver:brain weight ratios were higher for females administered 50 or 100 mg/kg/day, compared with concurrent controls. Decreased unadjusted seminal vesicle weights, seminal vesicle:body weight ratios, and seminal vesicle:brain weight ratios were recorded for males administered 50 or 100 mg/kg/day, compared with concurrent controls.
All other organ weight and organ weight ratio changes, including those which were statistically significant, were attributed to biological variation and were considered not test article related as they were small in magnitude, not dose dependent, inconsistent between sexes, attributed to normal inter-animal variability, and/or lacked a microscopic correlate.

F1 Generation Cohort 1B - Reproductive Toxicity
Absolute seminal vesicle weights and body weight ratios were lower for males administered 100 mg/kg/day, and absolute ovary weights were higher in females administered 50 or 100 mg/kg/day, compared with concurrent controls, but without a macroscopic correlate.
All other organ weight and organ weight ratio changes, including those which were statistically significant, were attributed to biological variation and were considered not test article related as they were small in magnitude, not dose dependent, inconsistent between sexes, attributed to normal inter-animal variability, and/or lacked a microscopic correlate.

F1 Generation Cohort 2A - Neurotoxicity
No test article-related changes in brain weight changes were recorded.
Brain Histomorphometry - No test article-related changes in brain measurements were recorded.

F1 Generation Cohort 2B - Neuropathology
No test article-related changes in brain weight changes were recorded.
Brain Histomorphometry - No test article-related changes in brain measurements were recorded.

F1 Generation Cohort 3 - Immunotoxicity
No test article-related spleen weight changes were noted. Splenic weight and weight ratio changes were attributed to biological variation and were considered not test article related as they were small in magnitude, not dose dependent, inconsistent between sexes, attributed to normal inter-animal variability, and/or lacked a microscopic correlate.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test article-related macroscopic findings were recorded.
Most tissues in pups that died or were sacrificed between PND 0 and 22 were macroscopically unremarkable and/or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age, and at this stage of reproductive development.

F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
No test article-related macroscopic findings were recorded.
Most tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age, and at this stage of reproductive development.

F1 Generation Cohort 1B - Reproductive Toxicity
No test article-related macroscopic findings were recorded.
Most tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age and at this stage of reproductive development.

F1 Generation Cohort 2A - Neurotoxicity
No test article-related macroscopic findings were recorded. Most tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.

F1 Generation Cohort 2B - Neuropathology
No test article-related macroscopic findings were recorded. Most tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.

F1 Generation Cohort 3 - Immunotoxicity
No test article-related macroscopic findings were recorded. Most tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age and at this stage of reproductive development.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
Centrilobular hepatocyte degeneration was recorded for the liver of animals administered 20, 50, or 100 mg/kg/day. Centrilobular hepatocellular hypertrophy/degeneration was characterized by increased size and/or cytoplasmic basophilia of hepatocytes adjacent to the central veins, with variable single cell necrosis, mitotic figures, and/or associated small numbers of mixed inflammatory cells. This finding may have correlated with the increased organ weights and weight ratios in females.
Increased pigment was recorded for the spleen of animals administered 20, 50, or 100 mg/kg/day, compared with controls. Splenic pigment was characterized by yellow-brown, granular pigment within the splenic red pulp.
In the kidney, a decrease in hyaline droplets was recorded for males administered 20, 50, or 100 mg/kg/day, compared with control males, and was characterized by fewer intra-cytoplasmic accumulations of eosinophilic pigment within proximal tubular cells.
Contraction was noted for the seminal vesicles of a few males administered 50 or 100 mg/kg/day, although not in a strictly dose-related manner. Contraction was characterized by decreased glandular secretions and an overall reduction in glandular size, which corresponded with the decreased organ weights and weight ratios noted at necropsy.
Microscopic findings recorded for animals that were not pregnant/did not mate/did not sire were generally similar to those in animals examined at the terminal sacrifice in the limited tissue list examined.
No other test article-related microscopic findings were recorded as microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain, age, and stage of reproductive development.

F1 Generation Cohort 1B - Reproductive Toxicity
No test article-related microscopic findings were recorded.
In addition, four males (Animal 204 administered 20 mg/kg/day, Animals 274 and 278 administered 50 mg/kg/day, and Animal 350 administered 100 mg/kg/day) did not mate and/or sire litters, and four females (Animal 705 administered 20 mg/kg/day, Animals 772 and 776 administered 50 mg/kg/day, and Animal 840 administered 100 mg/kg/day) did not mate and/or develop a pregnancy and/or litter. Findings in the limited reproductive tissue list examined for animals that did not mate and/or did not sire and/or did not become pregnant and/or did not litter were generally similar to those recorded for animals that survived to terminal sacrifice.

F1 Generation Cohort 2A - Neurotoxicity
No test article-related neuropathological changes were recorded. Microscopic findings were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.

F1 Generation Cohort 2B - Neuropathology
No test article-related neuropathological changes were recorded. Microscopic findings were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Thyroid Hormone Analysis
Test article-related changes in thyroid hormone levels were noted.
A statistically significant increase in T4 levels was observed for F1 males maternally and directly exposed to 100 mg/kg/day, compared with controls (P < 0.01). No such effect on T4 was evident for F1 males from the lower dose groups or for F1 females at any dose level. Thyroid stimulating hormone (TSH) was unaffected at any dose level. In the absence of a corresponding change in TSH or any macroscopic correlates, this intergroup difference was considered to have arisen incidentally and was unrelated to the test article.

F1 Generation Cohort 1A - Systemic and Reproductive Toxicity
Estrous Cycles - No test article-related effect was noted on the mean number or duration of estrous at any dose level, compared with controls.

Thyroid Hormone Data - No test article-related changes were noted in the thyroid hormones assessed. A statistically significant reduction in TSH was noted for males administered 50 mg/kg/day; however, in isolation, with no corresponding change in T4, this intergroup difference was considered to have arisen incidentally and was considered unrelated to the test article.

Seminology Evaluations - No test article-related changes were noted in sperm count, sperm motility, velocity, or morphology at any dose level. Statistical analysis of the data did not reveal any significant intergroup differences.

Ovarian Follicle Counts - No test article-related changes were noted for ovarian follicles from test article-treated F1 females, compared with controls. A statistically significant increase in primordial follicles was noted following administration of 100 mg/kg/day, compared with controls, which resulted in a statistically significant increase in total number of follicles. The increase was minimal (P < 0.05), and was, therefore, of no toxicological relevance; as such, the marginal increase was considered incidental and unrelated to the test article.

F1 Generation Cohort 1B - Reproductive Toxicity
Estrous Cycles - No test article-related effect was note on the mean number or duration of estrous.
Reproductive Indices - Following pairing, 19, 20, 18, or 19 females administered 0, 20, 50, or 100 mg/kg/day, respectively, were pregnant. Males administered 100 mg/kg/day showed a marginally lower mating index, compared with controls. Animals mated within 7 days of pairing. No test article-related effect on fecundity was noted.
Parturition and Litter Data - Following parturition, 18, 19, 18, or 18 females administered 0, 20, 50, or 100 mg/kg/day, respectively, delivered a live litter. Test article-related effects included a marginally lower number of mean implantation site following administration of 100 mg/kg/day, compared with controls, although % post-implantation loses was unaffected. This resulted in a lower litter size in the group administered 100 mg/kg/day, compared with controls. Furthermore, a higher incidence of missing pups was noted in the groups administered 100 mg/kg/day (n = 6) or 50 mg/kg/day (n = 3), with two dead pups also noted in the litters of the group administered 100 mg/kg/day. This was not observed in controls.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
F1 Generation Cohort 2A - Neurotoxicity
Acoustic Startle - Acoustic startle response was unaffected. No test article-related effect was recorded for mean startle amplitude, and animals showed a trend towards habituation . Pre-pulse inhibition was unaffected for both sexes. Statistical analysis of the data did not reveal any significant differences.
Motor Activity - Motor activity, characterized by mobile activity, total activity (including static activity), and the number of rears, was unaffected by administration of the test article for F1 generation animals. Statistical analysis of the data did not reveal any significant differences.
Functional Observational Battery - No test article-related effects were noted following the functional observation battery or grip strength measurements. Statistical analysis of the data did not reveal any significant differences.
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
F1 Generation Cohort 3 - Immunotoxicity
Immunophenotyping - No test article-related differences in lymphocyte populations were evident within the spleen.
Immunophenotyping analysis of splenocytes revealed that absolute cell counts and relative percentages for T lymphocytes (CD3+, CD3+/CD4+ and CD3+/CD8+) and B lymphocytes (CD3-/CD35RA+) were comparable across all groups, including controls. The evaluation of natural killer (NK) cells (CD3-/CD161a+) revealed slightly reduced cell counts and relative percentages following administration of 100 mg/kg/day, compared to controls. The average difference in cell counts between group administered control article (vehicle) and those administered 100 mg/kg/day was -31% for males and -34% for females, with similar reductions observed for relative percentages. The NK results for all other dose groups were comparable with controls.

Anti-Keyhole Limpet Hemocyanin (KLH) Antibodies - Evaluation of the anti-KLH IgM response showed no toxicologically significant effects at any dose level.
On average, all groups were noted with a moderate IgM response at both time points after the initial administration of keyhole limpet hemocyanin (KLH) - (PND 59 to 65 and 68 to 72). In response to the second challenge with KLH (PND 70 [±3 days]), a marked rise in the titer of specific IgM was observed on PND 76 through 78. The increase was in the region of 7 to 10 times the values observed at the previous time points. On PND 84/85/87, group mean anti-KLH IgM serum titers in all groups, including controls, had returned to values similar to those observed on PND 59 to 65 and 68 to 72 (typically 600 to 2000).
Anti-KLH IgG responses in test article-treated males produced slightly greater titers on PND 76 and 78 in animals administered 50 or 100 mg/kg/day, when compared to controls. Although highly variable within each group, the anti-KLH IgG response observed on PND 59 to 65 and 68 to 72 was generally comparable across all groups. After the second administration of KLH (PND 70 [±3 days]), a moderately higher titer was observed in the groups administered 50 or 100 mg/kg/day, when compared to controls, with the average IgG titer slightly more than twice that of controls on PND 76 to 78. Although a high degree of variability was observed within each group, evaluation of individual results showed that only 2 of 10 control animals had a titer >100,000, compared to 6 of 10 animals/group administered 50 or 100 mg/kg/day. At the final time point (PND 84/85/87), a moderate reduction in specific IgG was observed in the groups administered 50 or 100 mg/kg/day, when compared to PND 76 to 78, whereas the average titer remained unchanged in controls and the group administered 20 mg/kg/day. On PND 84/85/87, average titers in the groups administered 50 or 100 mg/kg/day were approximately +50% above those of controls at the same time point.
In females, the primary anti-KLH IgG responses were highly variable within each dose group after the first dose of KLH (PND 59 to 65 and 68 to 72). However, on average, the titers of KLH-specific IgG were comparable across all dose groups. After the second challenge with KLH (PND 70 [±3 days]), moderately higher titers of anti-KLH IgG were observed in the control group on PND 76 to 78, when compared to all test article-treated groups. An average titer of >18,7401 was observed in controls, with values 30 to 50% lower for test article-treated groups. On PND 84/85/87, control group titers increased further to >239,000, whereas values for all test article-treated groups remained relatively unchanged from PND 76 to 78.
Sexual maturity appeared to be delayed by 2 days for F1 males administered 100 mg/kg/day, compared to controls. The data from all cohorts were collated, and the mean day of completion for all F1 cohorts was PND 48.9 (standard deviation [SD] of 3.53) for controls, compared with PND 50.4 (SD of 5.17) for males administered 100 mg/kg/day. Mean values within each cohort never attained statistical significance, and this delay did not impact overall reproductive performance; as such, this finding was considered not to have represented an adverse effect.
Test article-related effects were observed in F1 offspring following maternal exposure of 100 mg/kg/day. Viability and weaning indices were also lower in the group administered 100 mg/kg/day, with a higher incidence of pup deaths and lower mean pup weights, compared with controls.
No test article-related neurological macroscopic or microscopic findings were noted in the F1 generation on PND 22 or in adulthood.
No marked changes to the immune system, as determined by the T-cell dependent antibody response to KLH and immunophenotyping of splenocytes, were noted in the F1 generation. Test article-related changes were limited to slightly enhanced secondary anti-KLH IgG responses in males administered 50 or 100 mg/kg/day and slightly reduced anti-KLH IgG response in females at all dose levels. None of these observations were considered adverse.
Key result
Dose descriptor:
NOEL
Remarks:
Growth & Development
Generation:
F1
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental Immunotoxicity
Generation:
F1 (cohort 3)
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental immunotoxicity
Key result
Dose descriptor:
NOEL
Remarks:
Developmental Neurotoxicity
Generation:
F1 (cohort 2A)
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity
Key result
Dose descriptor:
NOAEL
Remarks:
Systemic Toxicity
Generation:
F1
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related clinical observations were noted.
The clinical observations noted (generally confined to instances of pale/blue colouration, small size, cold to the touch, mortality) were observed throughout the dose groups, with no dose relationship, and were considered unrelated to the test article.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights on PND 1 or body weight change between PND 1 and 4 for test article-treated animals were essentially similar to controls.
Statistical analysis of the data did not reveal any significant intergroup differences.
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 specified
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test article-related effects on anogenital distance were observed. Statistical analysis of the data did not reveal any significant intergroup differences.
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 test article-related macroscopic findings were recorded F2 offspring.
Most tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age, and at this stage of reproductive development.
Histopathological findings:
not examined
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not specified
Effects were observed in the F2 generation, with marginally fewer implantation sites and smaller litter sizes and an increased incidence of pup mortality in the group administered 100 mg/kg/day. Lower mean offspring body weights were observed for males, although body weights were females were similar to controls on PND 1, body weights gain lower than controls during lactation. A higher incidence of pup mortality was also evident in litters of the group administered 50 mg/kg/day.
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)

F1 Generation Offspring Observations

Pup body weight change based on covariate adjusted mean and percentage change from control

 

Group 1 (controls)

Group 2 (mg/kg/day)

Group 3

(50 mg/kg/day)

Group 4

(100 mg/kg/day)

(percentage change from control)

Males

PND 1

PND 14

PND 20

Change (g)

Percentage change from control

(PND 1-20)

7.03

28.99

44.09

37.06

-

6.98

29.29

43.93

36.95

0%

7.06

27.75

42.11

35.05

-8%

6.96 (-1%)

27.13 (-6%)

40.97 (-7%)

34.01

-8%

Females

PND 1

OND 14

PND 20

Change (g)

Percentage change from control

(PND 1-20)

6.75

28.50

43.76

37.01

-

6.73

28.51

43.29

36.56

-1%

6.62

26.78

40.83

34.21

-8%

6.73 (0%)

26.79 (-6%)

40.91 (-7%)

34.18

-8%

 

F0 Generation Adults

Summary of Organ Weight % - Differences Compared with Concurrent Controls – Terminal Sacrifice

Tissue

Level (mg/kg/day)

Males

Females

2M

3M

4M

2F

3F

4F

20

50

100

20

50

100

 

% difference

Prostate

Group mean unadjusted weights

Body weight ratio

-1.874

-4.601

-6.969

-10.454

-19.511

-20.608

-

-

-

-

-

-

Seminal vesicle

Group mean unadjusted weights

Body weight ratio

-5.636

-7.628

-9.950

-14.342

-15.385

-17.252

-

-

-

-

-

-

Uterus

Group mean unadjusted weights

Body weight ratio

-

-

-

-

-

-

2.632

3.489

22.952

21.666

21.776

24.940

M = Male, F = Female

Note: Organ weights were not taken from decedents.

 

F0 Generation

Incidence of Selected Microscopic Findings: Liver – Terminal Sacrifice

Tissue and Finding

Level (mg/kg/day)

Males

Females

1M

2M

3M

4M

1F

2F

3F

4F

0

20

50

100

0

20

50

100

Liver

No. examined:

24

24

25

24

24

23

24

21

Degeneration/regeneration, hepatocryte, centrilobular

Grade –

1

2

3

24

0

0

0

13

11

0

0

1

18

6

0

0

10

11

3

24

0

0

0

20

3

0

0

12

12

0

0

10

11

0

0

Karyocytomegaly/s multinucleated hepatocyte

Grade –

1

24

0

24

0

25

0

24

0

24

1

24

1

23

2

17

6

- = Finding not present; 1 = minimal; 2 = slight; 3 = moderate; F = Female; M = Male

 

F0 Generation

Incidence of Pigment: Spleen – Terminal Sacrifice

Tissue and Finding

Level (mg/kg/day)

Males

Females

1M

2M

3M

4M

1F

2F

3F

4F

0

20

50

100

0

20

50

100

Spleen

No. examined:

24

24

25

24

24

23

24

21

Pigment

Grade –

1

2

3

15

6

3

0

3

11

10

0

1

6

16

2

0

5

14

5

13

9

2

0

0

11

12

0

0

8

15

1

0

1

14

6

- = Finding not present; 1 = minimal; 2 = slight; 3 = moderate; F = Female; M = Male

 

F0 Generation

Incidence of Hyaline Droplets: Kidney – Terminal Sacrifice

Tissue and Finding

Level (mg/kg/day)

Males

Females

1M

2M

3M

4M

1F

2F

3F

4F

0

20

50

100

0

20

50

100

Kidney

No. examined:

24

24

25

24

24

23

24

21

Hyaline droplets

Grade –

1

2

0

18

6

10

14

0

15

10

0

14

10

0

24

0

0

23

0

0

24

0

0

21

0

0

- = Finding not present; 1 = minimal; 2 = slight; F = Female; M = Male

 

F0 Generation

Incidence of Contraction: Prostate – Terminal Sacrifice

Tissue and Finding

Level (mg/kg/day)

Males

Females

1M

2M

3M

4M

1F

2F

3F

4F

0

20

50

100

0

20

50

100

Prostate

No. examined:

24

24

25

24

0

0

0

0

Contraction

Grade –

Present

24

0

24

0

25

0

19

5

-

-

-

-

-

-

-

-

- = Finding not present; F = Female; M = Male

 

F1 Generation Cohort 1A – Systemic and Reproductive Toxicity

Summary of Organ Weight % - Difference Compared with Concurrent Controls – Terminal Sacrifice

Tissue

Level (mg/kg/day)

Males

Females

2M

3M

4M

2M

3M

4M

20

50

100

20

50

100

 

% difference

Liver

Group mean unadjusted weights

Body weight ratio

Brain weight ratio

2

1

2

1

1

1

3

3

3

2

4

2

10**

10***

8*

19***

19***

17***

Seminal vesicle

Group mean unadjusted weights

Body weight ratio

Brain weight ratio

-8

-8

-8

-16

-16

-16

-21**

-21*

-21*

-

-

-

-

-

-

-

-

-

M = Male, F = Female

* = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001

Note: Organ weights are not taken from decedents.

 

F1 Generation Cohort 1A – Systemic and Reproductive Toxicity

Incidence of Centrilobular Hepatocellular Degeneration: Liver – Terminal Sacrifice

Tissue and Finding

Level (mg/kg/day)

Males

Females

1M

2M

3M

4M

1F

2F

3F

4F

0

20

50

100

0

20

50

100

Liver

No. examined:

20

20

20

19

20

20

20

19

Degeneration, hepatocyte, centrilobular

Grade –

1

2

19

1

0

4

16

0

1

16

3

1

15

3

20

0

0

3

17

0

3

17

0

2

17

0

- = Finding not present; 1 = minimal; 2 = slight; F = Female; M = Male

 

F1 Generation Cohort 1A – Systemic and Reproductive Toxicity

Incidence of Pigment: Spleen – Terminal Sacrifice

Tissue and Finding

Level (mg/kg/day)

Males

Females

1M

2M

3M

4M

1F

2F

3F

4F

0

20

50

100

0

20

50

100

Spleen

No. examined:

20

20

20

19

20

20

20

19

Pigment

Grade –

1

2

3

18

2

0

0

12

8

0

0

14

6

0

0

11

7

1

0

9

8

3

0

0

8

12

0

1

6

11

2

0

1

14

4

- = Finding not present; 1 = minimal; 2 = slight; 3 = moderate; F = Female; M = Male

 

F1 Generation Cohort 1A – Systemic and Reproductive Toxicity

Incidence of Hyaline Droplets: Kidney – Terminal Sacrifice

Tissue and Finding

Level (mg/kg/day)

Males

Females

1M

2M

3M

4M

1F

2F

3F

4F

0

20

50

100

0

20

50

100

Kidney

No. examined:

20

20

20

19

20

20

20

19

Hyaline droplets

Grade –

1

2

2

11

7

10

10

0

10

10

0

2

16

1

20

0

0

20

0

0

20

0

0

19

0

0

- = Finding not present; 1 = minimal; 2 = slight; F = Female; M = Male

 

F1 Generation Cohort 1A – Systemic and Reproductive Toxicity

Incidence of Contraction: Seminal Vesicle – Terminal Sacrifice

Tissue and Finding

Level (mg/kg/day)

Males

Females

1M

2M

3M

4M

1F

2F

3F

4F

0

20

50

100

0

20

50

100

Seminal vesicle

No. examined:

20

20

20

19

0

0

0

0

Contraction

Grade –

Present

19

1

18

2

17

3

15

4

-

-

-

-

-

-

-

-

- = Finding not present; F = Female; M = Male

Conclusions:
Once daily oral gavage administration of 20, 50, or 100 mg/kg/day 3,5-Dimethylpyrazole to Han Wistar rats for up to two consecutive generations resulted in test article-related effects at all dose levels investigated.
No adverse effects on mating performance, fertility, fecundity, gestation, partition, or lactation were noted; as such, the NOAEL for reproductive toxicity is 100 mg/kg/day.
Based on the lower F0 female pup body weight gain at 50 and 100 mg/kg/day, and marginally higher number of pup mortality in F0 generation litters following maternal exposure of 100 mg/kg/day, and increased incidence of offspring mortality for the F1 generation following maternal exposure of 50 or 100 mg/kg/day. As such, the NOEL for offspring growth and development is established as 20 mg/kg/day.
No test article immunotoxicity effects were evident in F1 animals; as such, the NOAEL is 100 mg/kg/day for developmental immunotoxicity.
No neurotoxic effects were noted in F1 animals; as such, the NOEL is 100 mg/kg/day for developmental neurotoxicity.
Systemic effects included changes in hematology parameters and microscopic spleen changes in all groups, clinical pathology changes and correlating microscopic liver changes, and changes in the prostate and seminal vesicles. Based on these findings, neither a no observed effect level (NOEL) nor a no observed adverse effect level (NOAEL) was established for systemic toxicity for males. For females test article-related effects were confined to isolated instances salivation and microscopic splenic changes (pigmentation. These findings were considered not to represent an adverse health effect. As such, a NOAEL for systemic toxicity for females is established as 20 mg/kg/day. Reversibility of these findings was not investigated as part of this study.
Executive summary:

Introduction

The objective of the study was to determine the effects of the test article, 3,5-Dimethylpyrazole, on the gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, lactation, weaning and the growth and development of the offspring when administered orally, by gavage, to two successive generations. Developmental neurotoxicity and developmental immunotoxicity was also assessed in the offspring.

 

Methods

Four groups of 25 male and 25 female Crl:WI(Han) rats were allocated to the study as the parental (F0) generation, and were administered 0 (control article [vehicle]), 20, 50, or 100 mg/kg/day 3,5-Dimethylpyrazole by oral gavage for up to 127 consecutive days for males (10 weeks prior to pairing, during pairing, and approximately 43/44 days post-pairing) or up to 135 days for females (10 weeks prior to pairing, during pairing, throughout gestation, and up to Lactation Day [LD] 21). Direct dosing to the F1 offspring was initiated from Postnatal Day (PND) 14. Formulations were administered at a constant dose volume of 5 mL/kg, and the control article (vehicle) was Propylene Glycol.

 

Assessment of toxicity for the F0 generation was based on mortality, clinical observations, body weights, food consumption, estrous cycles, pregnancy indices, parturition, offspring development, and clinical and anatomic pathology evaluations. Litters were observed from birth, and the number of pups, sexes, body weights, and any clinical observations were monitored. Anogenital distance was measured on Postnatal Day (PND) 4, and areolae (nipple) counts were recorded for male offspring on PND 13. F0 males were sacrificed during Week 19 (Study Days 127/128), and females were sacrificed on LD 22, along with pups not selected for the next phase of the study. Blood samples were taken for thyroid hormone assessments; selected organs were weighed, and macroscopic examinations were performed on the unselected offspring.

 

Pups were selected from F1 litters to form five cohorts of the F1 generation, as follows, and were administered the test article until the day prior to necropsy for each cohort.

 

F1 Generation

Cohort 1A – Systemic and Reproductive Toxicity Testing

Group

Dose Level (mg/kg/day)

Animal Numbers

Males

Females

1 (Control)

2 (Low)

3 (Intermediate)

4 (High)

0

20

50

100

20

20

20

20

20

20

20

20

 

Cohort 1B –Reproductive Toxicity Testing

Group

Dose Level (mg/kg/day)

Animal Numbers

Males

Females

1 (Control)

2 (Low)

3 (Intermediate)

4 (High)

0

20

50

100

20

20

20

20

20

20

20

20

 

Cohort 2A – Developmental Neurotoxicity Testing

Group

Dose Level (mg/kg/day)

Animal Numbers

Males

Females

1 (Control)

2 (Low)

3 (Intermediate)

4 (High)

0

20

50

100

10

10

10

10

10

10

10

10

 

Cohort 2B – Developmental Brain Neuropathy Assessments

Group

Dose Level (mg/kg/day)

Animal Numbers

Males

Females

1 (Control)

2 (Low)

3 (Intermediate)

4 (High)

0

20

50

100

10

10

10

10

10

10

10

10

 

Cohort 2A – Developmental Immunotoxicity Testing

Group

Dose Level (mg/kg/day)

Animal Numbers

Males

Females

1 (Control)

2 (Low)

3 (Intermediate)

4 (High)

0

20

50

100

10

10

10

10

10

10

10

10

 

Clinical observations and body weights were recorded for the F1 animals. Food consumption and sexual maturation was assessed for Cohorts 1A, 1B, 2A, and 3. In addition, estrous cycles, ovarian follicle counts, seminology analysis, and clinical and anatomic pathology assessments were performed for Cohort 1A; estrous cycles, pregnancy indices, parturition, offspring development, and anatomic pathology was assessed for Cohort 1B; acoustic startle, motor activity, learning behavior, and brain histomorphometry was assessed for Cohort 2A; brain histomorphometry assessments was performed for Cohort 2B; and food consumption, sexual maturation and immunotoxicology evaluations were undertaken for Cohort 3.

 

Results

Parental (F0) Generation

No test article-related deaths occurred.

Notable clinical, post-dose, and weekly detailed observations were confined to instances of salivation noted for a few animals administered 100 or 50 mg/kg/day and occasionally following administration of 20 mg/kg/day.

 

No test article-related effects on body weights were noted, and no adverse effect on food consumption was observed.

 

Hemoglobin, red blood cells, and packed cell volume were lower for males from all test article-treated groups, compared with controls. Red blood cells were also lower for females administered 100 or 50 mg/kg/day, compared with controls. Hemoglobin levels were also marginally lower for females administered 100 mg/kg/day, compared with controls. Mean reticulocytes were increased for males administered 100 or 50 mg/kg/day, compared with controls.

 

Mean cell volume (MCV) was increased for males administered 100 or 50 mg/kg/day, compared with controls. Mean cell hemoglobin (MCH) was also increased for males administered 100 mg/kg/day, compared with controls. Similar increases in MCV and MCH were also observed on LD 22 for females administered 100 or 50 mg/kg/day, compared with controls. In addition, hemoglobin distribution width (HDW) was elevated for females administered 100 mg/kg/day, compared with controls.

 

Males administered 100 mg/kg/day showed increased prothrombin time (PT), platelet counts (PLT), and platelet crit (PCT), compared with controls, and PCT was also increased for males administered 50 mg/kg/day. Similar increases in PLT and PCT were only observed for females administered 100 mg/kg/day, compared with controls.

 

No test article-related changes were noted for females administered 20 mg/kg/day.

 

Alanine aminotransferase (ALT) activity was increased, total protein and globulin levels were reduced, and albumin:globulin (A:G) ratio was increased for males administered 100 mg/kg/day, compared with controls. Reduced globulin and increased A:G ratio were also observed for males administered 50 mg/kg/day, compared with controls. This was not evident for males administered 20 mg/kg/day or for females at any dose level.

 

No test article-related changes in thyroxine (T4) or thyroid stimulating hormone (TSH) were noted at any dose level, compared with controls.

 

No test article-related changes were noted in the urine parameters investigated.

 

No test article-related changes in sperm count, sperm motility, velocity, or morphology were noted following administration of any dose level.

 

No test article-related macroscopic abnormalities were detected, although decreases in absolute prostate and seminal weights and body weight ratios were recorded for males from all test article-treated groups, compared with controls. Increases in absolute uterus weights and body weight ratios were recorded for females administered 50 or 100 mg/kg/day, compared with controls.

 

Microscopic changes were noted in the liver, spleen, kidneys and prostate.

 

In the liver, centrilobular hepatocyte hypertrophy/degeneration was recorded for males administered 20 mg/kg/day and both sexes administered 50 or 100 mg/kg/day. Hepatocellular karyomegaly/multinucleation was recorded for females administered 100 mg/kg/day.

 

In the spleen, increased pigment was recorded for animals administered 20, 50, or 100 mg/kg/day, compared with controls.

 

In the kidneys, a minor decrease in hyaline droplets was recorded for males administered 50 or 100 mg/kg/day, compared with control males.

 

In the prostate, contraction was noted in a few males administered 100 mg/kg/day.

 

The animals were not sexually mature on arrival; as such, sexual maturation was assessed from the start of dosing and was found not to be affected by test article administration. Furthermore, estrous cycles were unaffected by the test article at any dose level, compared with controls. No effects on mating, fertility, or fecundity were noted.

 

Following parturition, 24, 23, 24, or 23 females administered 0, 20, 50, or 100 mg/kg/day, respectively, produced a live litter. Possible test article-related effects were noted in the group administered 100 mg/kg/day. Viability indices on PND 4 and weaning indices were marginally lower for litters of the group administered 100 mg/kg/day, compared with controls, and the number of missing pups was higher in litters of groups administered 100 mg/kg/day, compared with controls.

 

F1 Generation Offspring (Pre-Weaning)

No test article-related clinical observations were noted, and no observations were noted after pups were directly dosed from PND 14.

 

Lower mean body weights were noted from PND 4 onwards for both sexes exposed to 50 or 100 mg/kg/day. By the end of the pre-weaning period, only marginal differences were observed. Anogenital distance was unaffected for male or female offspring, and no evidence of the presence of nipple/areolae was noted for male offspring exposed at any administered dose level.

 

No test article-related thyroid hormone level changes, macroscopic findings, or organ weight changes were noted for the unselected offspring on PND 22.

 

F1 Generation Cohort 1A - Systemic and Reproductive Toxicity

No test article-related deaths were noted. Notable clinical observations were confined to instances of salivation, which was observed for all test article-treated groups, but not controls.

 

No effects on body weight, body weight change, or food consumption were noted for test article-treated groups, compared with controls.

 

A delay in attainment of sexual maturity was observed for males administered 100 mg/kg/day, with corresponding higher mean body weight at attainment.

 

No test article-related effect was noted on the mean number or duration of estrous cycles at any dose level, compared with controls.

 

Red blood cells were reduced and MCV was elevated for males administered 100 mg/kg/day, compared with controls, with reticulocyte increases for males administered 100 or 50 mg/kg/day, compared with controls. Furthermore, males administered 100 mg/kg/day also showed an increase in red cell distribution width (RDW) compared with controls. Similar changes in red cell parameters were observed for females. Red blood cells and MCV were reduced for females administered 100 or 50 mg/kg/day, compared with controls. Reticulocytes were elevated for females administered 100 mg/kg/day, with a reduction in HDW, compared with controls.

 

Platelets counts and PCT were elevated for males from all test article-treated groups and for females administered 50 or 100 mg/kg/day, compared with controls. In addition, platelet distribution width was lower for females administered 100 mg/kg/day, with elevated activated partial thromboplastin time (APTS), compared with controls. For males administered 100 mg/kg/day, PT was elevated, compared with controls.

 

Females administrated 100 mg/kg/day also showed an increase in large unstained cells (LUC), compared with controls, although other white cell parameters were unaffected.

 

Hematology parameters were unaffected for females administered 20 mg/kg/day.

 

Males administered 100 mg/kg/day were noted with increases in aspartate aminotransferase and ALT activities, compared with controls, with a reduction in total protein.

 

For females administered 100 mg/kg/day, the only test article-related clinical chemistry change was an increase in cholesterol, compared with controls.

 

No test article-related changes were noted in the urine parameters or thyroid hormone levels assessed.

 

No test article-related changes in sperm count, sperm motility, velocity, or morphology were noted at any dose level.

 

No test article-related changes were noted for ovarian follicles from test article-treated F1 females, compared with controls.

 

Test article-related increases in liver weights (absolute and body weight ratios) were recorded for females administered 50 or 100 mg/kg/day, and decreased seminal vesicle weights (absolute and body weight ratios) were recorded for males administered 50 or 100 mg/kg/day. No test article-related macroscopic findings were recorded.

 

Test article-related microscopic changes were noted in the liver, spleen, kidneys and seminal vesicles. In the liver, centrilobular hepatocyte degeneration was recorded for animals administered 20, 50, or 100 mg/kg/day. In the spleen, increased pigment was recorded for animals administered 20, 50, or 100 mg/kg/day, compared with controls. In the kidneys, a decrease in hyaline droplets was recorded for males administered 20, 50, or 100 mg/kg/day, compared with controls. In the seminal vesicles, contraction was noted for a few males administered 50 or 100 mg/kg/day.

 

F1 Generation Cohort 1B -Reproductive Toxicity

No test article-related deaths occurred. Clinical observations were confined to instances of salivation, which was observed for males administered 100 mg/kg/day and females administered 20 or 50 mg/kg/day. This was not observed for controls.

 

No test article-related effects on body weight, body weight change, or food consumption were noted.

 

Males administered 100 mg/kg/day showed a delay in sexual maturity, compared to controls, with a corresponding increase in mean body weight on the day of attainment.

 

Estrous cycle duration or number was unaffected by test article administration at any dose.

 

Following pairing, 19, 20, 18, or 19 females administered 0, 20, 50, or 100 mg/kg/day, respectively, were pregnant. Males administered 100 mg/kg/day showed a marginally lower mating index, compared with controls. Animals mated within 7 days of pairing, and no effect on fecundity was noted.

 

Following parturition, 18, 19, 18, or 18 females administered 0, 20, 50, or 100 mg/kg/day, respectively, delivered a live litter.

 

Test article-related effects included a marginally lower number of mean implantation site following administration of 100 mg/kg/day, compared with controls, which resulted in smaller litter sizes, compared with controls. Furthermore, increased incidences of missing pups were noted in groups administered 100 or 50 mg/kg/day dose groups, with two dead pups also noted in 100 mg/kg/day litters. No test article-related clinical observations were noted for the F2 generation, and no effects on anogenital distance were observed. Furthermore, no test article-related macroscopic findings were recorded.

 

For the F1 adults, seminal vesicle weights (absolute and body weight ratios) were lower for males administered 100 mg/kg/day, and absolute ovary weights were higher for females administered 50 or 100 mg/kg/day, compared with concurrent controls. No test article-related macroscopic or microscopic findings were recorded.

 

F1 Generation Cohort 2A - Neurotoxicity

All animals allocated to this cohort survived to the terminal sacrifice. Clinical observations were confined to isolated instances of salivation, which was noted for a few males and females administered 100 or 50 mg/kg/day and a few females administered 20 mg/kg/day. Hunched posture was also noted for one female administered 100 mg/kg/day.

 

No test article-related effects on body weight, body weight change, or food consumptions were noted.

 

Sexual maturation was delay for males administered 100 mg/kg/day, compared with controls, with a corresponding higher mean body weight at attainment.

 

No effect on acoustic startle response, motor activity, or grip strength was noted, and no overt differences were noted following functional observational batteries (FOBs). No test article-related changes in brain measurements, macroscopic or neuropathological findings were noted.

 

F1 Generation Cohort 2B - Neuropathology

All animals allocated to this cohort survived to the terminal sacrifice.

 

No test article-related clinical observations were noted, and body weights were unaffected by the test article. Furthermore, no test article-related changes in brain measurements were recorded, and no test article-related macroscopic or neuropathological changes findings were recorded.

 

F1 Generation Cohort 3 - Immunotoxicity

All animals allocated to this cohort survived to the terminal sacrifice.

 

The only test article-related clinical observation was salivation, which was noted throughout the dose groups.

 

No test article-related changes in body weight, body weight change, or food consumption were noted, and no test article-related effects on sexual maturity were observed for this cohort.

 

No test article-related differences in lymphocyte populations were evident within the spleen, and evaluation of the anti-keyhole limpet hemocyanin (KLH) Immunoglobulin M (IgM) response showed no toxicologically significant effects at any dose level.

 

No test article-related macroscopic findings were recorded, and spleen weight changes were unaffected by the test article.

 

Conclusion

Once daily oral gavage administration of 20, 50, or 100 mg/kg/day 3,5-Dimethylpyrazole to Han Wistar rats for up to two consecutive generations resulted in test article-related effects at all dose levels investigated.

 

No adverse effects on mating performance, fertility, fecundity, gestation, partition, or lactation were noted; as such, the NOAEL for reproductive toxicity is 100 mg/kg/day.

 

Based on the lower F0 female pup body weight gain at 50 and 100 mg/kg/day, and marginally higher number of pup mortality in F0 generation litters following maternal exposure of 100 mg/kg/day, and increased incidence of offspring mortality for the F1 generation following maternal exposure of 50 or 100 mg/kg/day. As such, the NOEL for offspring growth and development is established as 20 mg/kg/day.

 

No test article immunotoxicity effects were evident in F1 animals; as such, the NOAEL is 100 mg/kg/day for developmental immunotoxicity.

 

No neurotoxic effects were noted in F1 animals; as such, the NOEL is 100 mg/kg/day for developmental neurotoxicity.

 

Systemic effects included changes in hematology parameters and microscopic spleen changes in all groups, clinical pathology changes and correlating microscopic liver changes, and changes in the prostate and seminal vesicles. Based on these findings, neither a no observed effect level (NOEL) nor a no observed adverse effect level (NOAEL) was established for systemic toxicity for males. For females test article-related effects were confined to isolated instances salivation and microscopic splenic changes (pigmentation. These findings were considered not to represent an adverse health effect. As such, a NOAEL for systemic toxicity for females is established as 20 mg/kg/day. Reversibility of these findings was not investigated as part of this study.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is performed in line with standardised guideline and is sufficient to justify a Klimisch score of 1, thus the dataset is considered to be of high quality.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Extended one generation study with cohorts 1A, 1B, 2A, 2B & 3

The objective of the study was to determine the effects of the test article, 3,5-Dimethylpyrazole, on the gonadal function, the estrous cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition, lactation, weaning and the growth and development of the offspring when administered orally, by gavage, to two successive generations. Developmental neurotoxicity and developmental immunotoxicity was also assessed in the offspring.

Four groups of 25 male and 25 female Crl:WI(Han) rats were allocated to the study as the parental (F0) generation, and were administered 0 (control article [vehicle]), 20, 50, or 100 mg/kg/day 3,5-Dimethylpyrazole by oral gavage for up to 127 consecutive days for males (10 weeks prior to pairing, during pairing, and approximately 43/44 days post-pairing) or up to 135 days for females (10 weeks prior to pairing, during pairing, throughout gestation, and up to Lactation Day [LD] 21). Direct dosing to the F1 offspring was initiated from Postnatal Day (PND) 14. Formulations were administered at a constant dose volume of 5 mL/kg, and the control article (vehicle) was Propylene Glycol.

 

Assessment of toxicity for the F0 generation was based on mortality, clinical observations, body weights, food consumption, estrous cycles, pregnancy indices, parturition, offspring development, and clinical and anatomic pathology evaluations. Litters were observed from birth, and the number of pups, sexes, body weights, and any clinical observations were monitored. Anogenital distance was measured on Postnatal Day (PND) 4, and areolae (nipple) counts were recorded for male offspring on PND 13. F0 males were sacrificed during Week 19 (Study Days 127/128), and females were sacrificed on LD 22, along with pups not selected for the next phase of the study. Blood samples were taken for thyroid hormone assessments; selected organs were weighed, and macroscopic examinations were performed on the unselected offspring.

 

Pups were selected from F1 litters to form five cohorts of the F1 generation, and were administered the test article until the day prior to necropsy for each cohort.

 

Clinical observations and body weights were recorded for the F1 animals. Food consumption and sexual maturation was assessed for Cohorts 1A, 1B, 2A, and 3. In addition, estrous cycles, ovarian follicle counts, seminology analysis, and clinical and anatomic pathology assessments were performed for Cohort 1A; estrous cycles, pregnancy indices, parturition, offspring development, and anatomic pathology was assessed for Cohort 1B; acoustic startle, motor activity, learning behavior, and brain histomorphometry was assessed for Cohort 2A; brain histomorphometry assessments was performed for Cohort 2B; and food consumption, sexual maturation and immunotoxicology evaluations were undertaken for Cohort 3.

 

Results

Parental (F0) Generation

No test article-related deaths occurred.

Notable clinical, post-dose, and weekly detailed observations were confined to instances of salivation noted for a few animals administered 100 or 50 mg/kg/day and occasionally following administration of 20 mg/kg/day.

No test article-related effects on body weights were noted, and no adverse effect on food consumption was observed.

Hemoglobin, red blood cells, and packed cell volume were lower for males from all test article-treated groups, compared with controls. Red blood cells were also lower for females administered 100 or 50 mg/kg/day, compared with controls. Hemoglobin levels were also marginally lower for females administered 100 mg/kg/day, compared with controls. Mean reticulocytes were increased for males administered 100 or 50 mg/kg/day, compared with controls.

Mean cell volume (MCV) was increased for males administered 100 or 50 mg/kg/day, compared with controls. Mean cell hemoglobin (MCH) was also increased for males administered 100 mg/kg/day, compared with controls. Similar increases in MCV and MCH were also observed on LD 22 for females administered 100 or 50 mg/kg/day, compared with controls. In addition, hemoglobin distribution width (HDW) was elevated for females administered 100 mg/kg/day, compared with controls.

Males administered 100 mg/kg/day showed increased prothrombin time (PT), platelet counts (PLT), and platelet crit (PCT), compared with controls, and PCT was also increased for males administered 50 mg/kg/day. Similar increases in PLT and PCT were only observed for females administered 100 mg/kg/day, compared with controls.

No test article-related changes were noted for females administered 20 mg/kg/day.

Alanine aminotransferase (ALT) activity was increased, total protein and globulin levels were reduced, and albumin:globulin (A:G) ratio was increased for males administered 100 mg/kg/day, compared with controls. Reduced globulin and increased A:G ratio were also observed for males administered 50 mg/kg/day, compared with controls. This was not evident for males administered 20 mg/kg/day or for females at any dose level.

No test article-related changes in thyroxine (T4) or thyroid stimulating hormone (TSH) were noted at any dose level, compared with controls.

No test article-related changes were noted in the urine parameters investigated.

No test article-related changes in sperm count, sperm motility, velocity, or morphology were noted following administration of any dose level.

No test article-related macroscopic abnormalities were detected, although decreases in absolute prostate and seminal weights and body weight ratios were recorded for males from all test article-treated groups, compared with controls. Increases in absolute uterus weights and body weight ratios were recorded for females administered 50 or 100 mg/kg/day, compared with controls.

Microscopic changes were noted in the liver, spleen, kidneys and prostate. In the liver, centrilobular hepatocyte hypertrophy/degeneration was recorded for males administered 20 mg/kg/day and both sexes administered 50 or 100 mg/kg/day. Hepatocellular karyomegaly/multinucleation was recorded for females administered 100 mg/kg/day. In the spleen, increased pigment was recorded for animals administered 20, 50, or 100 mg/kg/day, compared with controls. In the kidneys, a minor decrease in hyaline droplets was recorded for males administered 50 or 100 mg/kg/day, compared with control males. In the prostate, contraction was noted in a few males administered 100 mg/kg/day.

The animals were not sexually mature on arrival; as such, sexual maturation was assessed from the start of dosing and was found not to be affected by test article administration. Furthermore, estrous cycles were unaffected by the test article at any dose level, compared with controls. No effects on mating, fertility, or fecundity were noted.

Following parturition, 24, 23, 24, or 23 females administered 0, 20, 50, or 100 mg/kg/day, respectively, produced a live litter. Possible test article-related effects were noted in the group administered 100 mg/kg/day. Viability indices on PND 4 and weaning indices were marginally lower for litters of the group administered 100 mg/kg/day, compared with controls, and the number of missing pups was higher in litters of groups administered 100 mg/kg/day, compared with controls.

 

F1 Generation Offspring (Pre-Weaning)

No test article-related clinical observations were noted, and no observations were noted after pups were directly dosed from PND 14.

Lower mean body weights were noted from PND 4 onwards for both sexes exposed to 50 or 100 mg/kg/day. By the end of the pre-weaning period, only marginal differences were observed. Anogenital distance was unaffected for male or female offspring, and no evidence of the presence of nipple/areolae was noted for male offspring exposed at any administered dose level.

No test article-related thyroid hormone level changes, macroscopic findings, or organ weight changes were noted for the unselected offspring on PND 22.

 

F1 Generation Cohort 1A - Systemic and Reproductive Toxicity

No test article-related deaths were noted. Notable clinical observations were confined to instances of salivation, which was observed for all test article-treated groups, but not controls.

No effects on body weight, body weight change, or food consumption were noted for test article-treated groups, compared with controls.

A delay in attainment of sexual maturity was observed for males administered 100 mg/kg/day, with corresponding higher mean body weight at attainment.

No test article-related effect was noted on the mean number or duration of estrous cycles at any dose level, compared with controls.

Red blood cells were reduced and MCV was elevated for males administered 100 mg/kg/day, compared with controls, with reticulocyte increases for males administered 100 or 50 mg/kg/day, compared with controls. Furthermore, males administered 100 mg/kg/day also showed an increase in red cell distribution width (RDW) compared with controls. Similar changes in red cell parameters were observed for females. Red blood cells and MCV were reduced for females administered 100 or 50 mg/kg/day, compared with controls. Reticulocytes were elevated for females administered 100 mg/kg/day, with a reduction in HDW, compared with controls.

Platelets counts and PCT were elevated for males from all test article-treated groups and for females administered 50 or 100 mg/kg/day, compared with controls. In addition, platelet distribution width was lower for females administered 100 mg/kg/day, with elevated activated partial thromboplastin time (APTS), compared with controls. For males administered 100 mg/kg/day, PT was elevated, compared with controls.

Females administrated 100 mg/kg/day also showed an increase in large unstained cells (LUC), compared with controls, although other white cell parameters were unaffected.

Hematology parameters were unaffected for females administered 20 mg/kg/day.

Males administered 100 mg/kg/day were noted with increases in aspartate aminotransferase and ALT activities, compared with controls, with a reduction in total protein.

For females administered 100 mg/kg/day, the only test article-related clinical chemistry change was an increase in cholesterol, compared with controls.

No test article-related changes were noted in the urine parameters or thyroid hormone levels assessed.

No test article-related changes in sperm count, sperm motility, velocity, or morphology were noted at any dose level.

No test article-related changes were noted for ovarian follicles from test article-treated F1 females, compared with controls.

Test article-related increases in liver weights (absolute and body weight ratios) were recorded for females administered 50 or 100 mg/kg/day, and decreased seminal vesicle weights (absolute and body weight ratios) were recorded for males administered 50 or 100 mg/kg/day. No test article-related macroscopic findings were recorded.

Test article-related microscopic changes were noted in the liver, spleen, kidneys and seminal vesicles. In the liver, centrilobular hepatocyte degeneration was recorded for animals administered 20, 50, or 100 mg/kg/day. In the spleen, increased pigment was recorded for animals administered 20, 50, or 100 mg/kg/day, compared with controls. In the kidneys, a decrease in hyaline droplets was recorded for males administered 20, 50, or 100 mg/kg/day, compared with controls. In the seminal vesicles, contraction was noted for a few males administered 50 or 100 mg/kg/day.

 

F1 Generation Cohort 1B -Reproductive Toxicity

No test article-related deaths occurred. Clinical observations were confined to instances of salivation, which was observed for males administered 100 mg/kg/day and females administered 20 or 50 mg/kg/day. This was not observed for controls.

No test article-related effects on body weight, body weight change, or food consumption were noted.

Males administered 100 mg/kg/day showed a delay in sexual maturity, compared to controls, with a corresponding increase in mean body weight on the day of attainment.

Estrous cycle duration or number was unaffected by test article administration at any dose.

Following pairing, 19, 20, 18, or 19 females administered 0, 20, 50, or 100 mg/kg/day, respectively, were pregnant. Males administered 100 mg/kg/day showed a marginally lower mating index, compared with controls. Animals mated within 7 days of pairing, and no effect on fecundity was noted.

Following parturition, 18, 19, 18, or 18 females administered 0, 20, 50, or 100 mg/kg/day, respectively, delivered a live litter.

Test article-related effects included a marginally lower number of mean implantation site following administration of 100 mg/kg/day, compared with controls, which resulted in smaller litter sizes, compared with controls. Furthermore, increased incidences of missing pups were noted in groups administered 100 or 50 mg/kg/day dose groups, with two dead pups also noted in 100 mg/kg/day litters. No test article-related clinical observations were noted for the F2 generation, and no effects on anogenital distance were observed. Furthermore, no test article-related macroscopic findings were recorded.

For the F1 adults, seminal vesicle weights (absolute and body weight ratios) were lower for males administered 100 mg/kg/day, and absolute ovary weights were higher for females administered 50 or 100 mg/kg/day, compared with concurrent controls. No test article-related macroscopic or microscopic findings were recorded.

 

F1 Generation Cohort 2A - Neurotoxicity

All animals allocated to this cohort survived to the terminal sacrifice. Clinical observations were confined to isolated instances of salivation, which was noted for a few males and females administered 100 or 50 mg/kg/day and a few females administered 20 mg/kg/day. Hunched posture was also noted for one female administered 100 mg/kg/day.

No test article-related effects on body weight, body weight change, or food consumptions were noted.

Sexual maturation was delay for males administered 100 mg/kg/day, compared with controls, with a corresponding higher mean body weight at attainment.

No effect on acoustic startle response, motor activity, or grip strength was noted, and no overt differences were noted following functional observational batteries (FOBs). No test article-related changes in brain measurements, macroscopic or neuropathological findings were noted.

 

F1 Generation Cohort 2B - Neuropathology

All animals allocated to this cohort survived to the terminal sacrifice.

No test article-related clinical observations were noted, and body weights were unaffected by the test article. Furthermore, no test article-related changes in brain measurements were recorded, and no test article-related macroscopic or neuropathological changes findings were recorded.

 

F1 Generation Cohort 3 - Immunotoxicity

All animals allocated to this cohort survived to the terminal sacrifice.

The only test article-related clinical observation was salivation, which was noted throughout the dose groups.

No test article-related changes in body weight, body weight change, or food consumption were noted, and no test article-related effects on sexual maturity were observed for this cohort.

No test article-related differences in lymphocyte populations were evident within the spleen, and evaluation of the anti-keyhole limpet hemocyanin (KLH) Immunoglobulin M (IgM) response showed no toxicologically significant effects at any dose level.

No test article-related macroscopic findings were recorded, and spleen weight changes were unaffected by the test article.

 

Conclusion

Once daily oral gavage administration of 20, 50, or 100 mg/kg/day 3,5-Dimethylpyrazole to Han Wistar rats for up to two consecutive generations resulted in test article-related effects at all dose levels investigated.

No adverse effects on mating performance, fertility, fecundity, gestation, partition, or lactation were noted; as such, the NOAEL for reproductive toxicity is 100 mg/kg/day.

Based on the lower F0 female pup body weight gain at 50 and 100 mg/kg/day, and marginally higher number of pup mortality in F0 generation litters following maternal exposure of 100 mg/kg/day, and increased incidence of offspring mortality for the F1 generation following maternal exposure of 50 or 100 mg/kg/day. As such, the NOEL for offspring growth and development is established as 20 mg/kg/day.

No test article immunotoxicity effects were evident in F1 animals; as such, the NOAEL is 100 mg/kg/day for developmental immunotoxicity.

No neurotoxic effects were noted in F1 animals; as such, the NOEL is 100 mg/kg/day for developmental neurotoxicity.

 

Systemic effects included changes in hematology parameters and microscopic spleen changes in all groups, clinical pathology changes and correlating microscopic liver changes, and changes in the prostate and seminal vesicles. Based on these findings, neither a no observed effect level (NOEL) nor a no observed adverse effect level (NOAEL) was established for systemic toxicity for males. For females test article-related effects were confined to isolated instances salivation and microscopic splenic changes (pigmentation. These findings were considered not to represent an adverse health effect. As such, a NOAEL for systemic toxicity for females is established as 20 mg/kg/day. Reversibility of these findings was not investigated as part of this study.

OECD 422 Study

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.

Short description of key information:

ORAL

Key 28 day study; Zmarowski (2012), performed to GLP, OECD 422 and EPA OPPT 870.3650, Klimisch 1, NOAEL = 60 mg/kg b.w./ day in the rat.

INHALATION

No data available.

DERMAL

No data available.

Justification for selection of Effect on fertility via oral route:

The key study (Zmarowski, 2012) was performed in compliance with GLP and the standardised guidelines OECD 422 and EPA OPPTS 870.3650. The study was performed to a good standard with a sufficient level of detail to assess the quality of the data presented. The study was assigned a reliability score of 1, in accordance with Klimisch (1997) and considered suitable to fulfil the data requirement.

Justification for selection of Effect on fertility via inhalation route:

Exposure via the oral route is considered an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. Further testing via inhalation is considered less appropriate and has been omitted on this basis.

Justification for selection of Effect on fertility via dermal route:

Exposure via the oral route is considered an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. Further testing via the dermal route is considered less appropriate and has been omitted on this basis.

Effects on developmental toxicity

Description of key information

ORAL

Key; Barraclough, (2018), performed to GLP, OECD 443, Klimisch 1, NOAEL = 60 mg/kg b.w./ day in the rat.

INHALATION

No data available.

DERMAL

No data available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
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.
Qualifier:
according to guideline
Guideline:
other: OECD 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:
yes
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 11 weeks. Females were nulliparous and non-pregnant.
- Weight at study initiation: 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.
Route of administration:
oral: gavage
Vehicle:
propylene 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 meaurment.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was chosen based on trial formulations performed by the research laboratory.
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.
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.
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, gestation, 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.
Duration of test:
28 days
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
Details on study design:
- Dose selection rationale: Dose concentrations were selected based on the findings of a 10 Day dose range finding study performed at 300 and 1000 mg/kg b.w.
Maternal 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: 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.

POST-MORTEM EXAMINATIONS: Yes
- 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;
> 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 1 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 2 for details of examinations. The histopathology data was peer reviewed by a second pathologist.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- 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.
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
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
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.
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.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
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.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.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was increased for females at 200 mg/kg, 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.
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
Description (incidence and severity):
At 200 mg/kg 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.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/kg an enlarged spleen and reduced size of the thymus was seen for 6/10 and 5/10 females.
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. 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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related microscopic findings were seen for animals at 60 and 200 mg/kg, and consisted of:
Thymus: Increased severity of lymphoid atrophy in 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 females treated at 200 mg/kg.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Gestation: The gestation index and duration of gestation were unaffected by treatment. The gestation index was 100% for all groups.
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects on the gestation index and duration, parturition, maternal care and some aspects of early postnatal pup development (clinical signs and macroscopy) were observed up to 200 mg/kg.

Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Early postnatal pup development: There was a statistically significant increase in postnatal loss and a corresponding lower viability index at 200 mg/kg compared to controls. There was a trend towards fewer living pups born at this dose level, though the difference from controls was not statistically significant.
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were treatment related effects on pup mortality and body weights 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.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
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 no. 79 who had a total litter loss by Day 3. A relationship to treatment could not be excluded. Missing pups missing were most likely cannibalized.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The number of dead pups at first litter check and sex ratio were unaffected by treatment, and pup clinical signs and external macroscopy did not reveal toxicologically relevant findings.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Skeletal malformations:
not specified
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Other effects:
no effects observed
Description (incidence and severity):
Clinical signs: 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.
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 3. Corpora Luta and Implantation Sites

 

Dose Group (mg/kg/day)

0

20

60

200

Corpora Lutes

Mean (SD)

16.9 (3.2)

15.6 (2.5)

16.0 (2.9)

14.5 (4.3)

 

No.

8

9

9

6

Implantations

Mean (SD)

13.4 (2.8)

13.1 (2.1)

13.9 (2.4)

11.0 (2.3)

 

No.

8

9

9

6

 

Table 4. Development Data

 

Dose Group (mg/kg/day)

0

20

60

200

Total litters

 

8

9

9

6

Length of gestation

Mean (SD)

21.4 (0.5)

21.2 (0.4)

21.3 (0.5)

22.0 (0.0)

No.

8

9

9

6

No. Dead Pups at First Litter Check

Litters Affected*

2

2

0

2

Total

2

2

0

3

Mean (SD)

0.3 (0.5)

0.2 (0.4)

0.0 (0.0)

0.5 (0.8)

No.

8

9

9

6

Living Pups at First Litter Check

% Males/Females*

44/56

45/55

48/52

51/49

Total

93

111

111

59

Mean (SD)

11.6 (1.4)

12.3 (2.3)

12.3 (2.6)

9.8 (3.1)

No.

8

9

9

6

Postnatal Loss

5 of Living Pups

1.1

0.9

0.9

16.9

Litters Affected*

1

1

1

3

Total*

1

1

1

10**

Mean (SD)

0.1 (0.4)

.01 (0.3)

0.1 (0.3)

1.7 (2.3)

No.

8

9

9

6

Viability Index*

98.9

991

99.1

83.1**

* Significant at 5%

** Significant at 1%

 

Table 5. Body Weight of Pups (g)

Day

Sex

 

Dose Group (mg/kg/day)

0

20

60

200

1

M

Mean (SD)

6.4 (0.6)

6.1 (0.5)

6.2 (0.4)

6.0 (0.4)

No.

8

9

9

6

F

Mean (SD)

6.0 (0.5)

5.8 (0.4)

5.8 (0.5)

5.6 (0.4)

No.

8

9

9

6

M+F

Mean (SD)

6.2 (0.6)

6.0 (0.4)

6.0 (0.4)

5.8 (0.4)

No.

8

9

9

6

4

M

Mean (SD)

9.4 (0.9)

8.9 (0.9)

9.1 (0.9)

7.4 (1.2)**

No.

8

9

9

5

F

Mean (SD)

8.9 (0.8)

8.6 (1.1)

8.6 (1.2)

7.1 (1.1)*

No.

8

9

9

5

M+F

Mean (SD)

9.1 (0.8)

8.7 (1.0)

8.8 (1.1)

7.3 (1.1)**

No.

8

9

9

5

Conclusions:
Under the conditions of the test developmental toxicity was observed at 200 mg/kg, based on treatment related effects observed in pup mortality (postnatal loss) and lower pup body weights at 200 mg/kg.

No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study, i.e. gestation index and duration, parturition, maternal care and clinical signs and macroscopy of pup.

Based on the observed results the developmental NOAEL was determined to be 60 mg/kg b.w./day.

Developmental toxicity was not seen in the absence of parental toxicity, and therefore the material is not classifed as such under Regulation (EC) No. 1272/2008 or Directive 67/548/EEC.
Executive summary:

Toxicity to development 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 developmental toxicity was observed at 200 mg/kg, based on treatment related effects observed on pup mortality (postnatal loss) and lower pup body weights at 200 mg/kg. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study, i.e. gestation index and duration, parturition, maternal care and clinical signs and macroscopy of pup. Based on the observed results the developmental NOAEL was determined to be 60 mg/kg b.w./day.

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

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 August 2017 to 29 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
No further details specified in the study report.
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Animal Specifications and Acclimation
Eighty-eight time-mated female Crl:WI(Han) rats were obtained from Charles River Laboratories, Margate, United Kingdom, in order to provide sufficient animals for study selection.
Animals weighed between 180.2 and 267.1 g at the start of dosing. Upon arrival, all animals were given a clinical inspection for ill health. Acclimation was limited by mated status, and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure their suitability for the study.

Environmental Conditions, Diet, and Water
Housing
Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes (Home Office, 2014).
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom).
Each batch of bedding was analyzed for specific constituents and contaminants. No contaminants were present in the bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Water
Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
No contaminants were present in the water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Diet
Animals had ad libitum access to VRF1 diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants.
No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Covance.

Environment
Animals were individually housed in a single, exclusive room. The room was air-conditioned to provide a minimum 15 to 20 air changes/hour. The temperature and relative humidity ranges were maintained in the specified ranges of 22 ± 3°C and 30 to 70%, respectively.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark, with the exception of when experimental procedures dictated

Environmental Enrichment
Animals were provided with wooden Aspen chew blocks and nesting materials as forms of environmental enrichment.

Animal Identification and Assignment to Study
Animals were assigned to dose groups based on GD 0 body weight data obtained from the Supplier (i.e., all animals confirmed as mated on a specific day were randomly allocated to dose groups). Animals were individually identified by electronic implant.
Cages were placed in dose group order across the batteries. Cages were appropriately identified with study information, including study number and animal number.
Route of administration:
oral: gavage
Details on exposure:
The control article (vehicle) was Propylene Glycol, supplied by Merck.
Formulations were prepared weekly.
The test article was formulated as a suspension in Propylene Glycol following dispensary SOPs and the formulation method (Method 8365912_O_01D), as maintained in the study data.
The formulations were stored at room temperature (15 to 25°C) in a sealed container, protected from light.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations prepared for use for the first and last day of dosing and an interval in between were analyzed to determine the achieved concentration. The mean % nominal concentration should be between 90 to 110% with a relative standard deviation (RSD) ≤5.0%. Results were within this range. Test article was not detected in the Group 1 control samples.

Achieved Concentration
Triplicate samples were removed from the middle of the test article formulations and were analyzed. A single sample was taken from the middle of control formulations and was analyzed.

Analytical Procedure
The analytical procedure (Covance 8365911-01F) was used to determine achieved concentration.
Details on mating procedure:
At the time of mating, females were 8 to 10 weeks old and weighed at least 140 g.
Mating (overnight at the supplier’s laboratory) was confirmed by the presence of a vaginal plug in situ or other evidence of mating, if necessary. The day on which mating was confirmed was designated as GD 0. Animals were delivered to Covance by GD 3.
Duration of treatment / exposure:
15 days
Frequency of treatment:
Daily
Duration of test:
16 days
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
The high-dose level of 200 mg/kg/day was expected to elicit maternal toxicity and possible effects to reproductive performance.
Dose / conc.:
60 mg/kg bw/day (nominal)
Remarks:
The intermediate-dose level of 60 mg/kg/day was expected to elicit mild maternal toxicity with no effects to reproductive performance.
Dose / conc.:
20 mg/kg bw/day (nominal)
Remarks:
The low-dose level of 20 mg/kg/day was the expected maternal no observed adverse effect level (NOAEL).
No. of animals per sex per dose:
22 female animals per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Species Selection and Dose Administration Rationale
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and recommended for reproduction studies because of its reproductive characteristics. The strain of rat was selected due to the extent of background data available at the laboratory.

Background Information
In an OECD 422 study, 3,5-Dimethylpyrazole was administered by oral gavage to Crl:WI(Han) rats at dose levels of 20, 60, or 200 mg/kg/day. A reduction in parental bodyweight was noted at 60 and 200 mg/kg/day. Effects on pup survival and a reduction in pup body weight were also noted at 200 mg/kg/day.
In the dose-range finding study females administered 200 mg/kg/day were observed to have reduced body weight gain and food consumption, incidences of piloerection were also noted. Effects on fetal body weight were also noted in litters from this dose group. Females administered 60 mg/kg/day showed slight reduction in food consumption between GD 6 to 10; no other associated adverse observations were noted.
The oral gavage route of administration was chosen to ensure the level of dosing, as oral consumption is a possible route of human exposure.
Maternal examinations:
Health Monitoring
All animals were observed at the beginning and the end of the working day for signs of ill health or overt toxicity.

Clinical Examinations
Each animal was given a detailed physical examination daily from the start of dosing until necropsy. An individual record of the clinical condition of each animal was maintained.

Postdose Observations
Animals were observed immediately after dosing upon return to the home cage and approximately 2 hours postdose from GD 6 to 8.

Body Weights
Body weights were recorded on GD 3, 6, 9, 12, 15, 17, 19, and 21.

Food Consumption
The amount of food consumed was determined daily from GD 6. Food consumption was calculated as g/animal/day.

Maternal Necropsy
Females were sacrificed in replicate order. Food was not removed prior to scheduled necropsies.
Females were sacrificed on GD 21 by cervical dislocation following isoflurane anesthesia; death was confirmed by exsanguination, and animals were examined macroscopically. All lesions were recorded and retained in the relevant fixative.
Ovaries and uterine content:
The ovaries and uteri were removed and examined, and the following data were recorded.
-Pregnancy status
-Gravid uterus weight
-Number of corpora lutea
-Terminal body weight (recorded for adjusted gravid uterus weight calculations only and have not been reported)
-Number and intrauterine position of implantations subdivided into:
-Live fetuses
-Early intrauterine deaths
-Late intrauterine deaths
-Dead fetuses

Early intrauterine deaths were classified as those which showed decidual or placental tissue only. Late intrauterine deaths showed embryonic or fetal tissue in addition to placental tissue. Dead fetuses were classified as those which appeared to have died shortly before necropsy.
Implantations were allocated numbers relating to their position in utero.
Fetuses were allocated numbers relating to their position in utero.
The uterus of any apparently non-pregnant female (two out of 88) was immersed in a 10% ammonium sulphide solution to reveal any evidence of implantation.
Fetal examinations:
Live fetuses were sacrificed by a subcutaneous injection of sodium pentobarbitone followed by confirmation of cessation of circulation.
Following the injection of sodium pentobarbitone, individual fetal and placental weights were recorded, and fetuses were examined externally and sexed.
Approximately one-half of the fetuses in each litter, selected by systematic sampling, were dissected, and the visceral abnormalities were examined. They were then eviscerated, and the carcasses were processed for skeletal examination. The skeletons were examined in 50% glycerol and retained in glycerol/propylene glycol.
The remaining fetuses were fixed in Bouin's fluid and examined. The fetuses fixed for visceral examination were retained in the relevant fixative.
Fetal abnormalities were classified as follows: malformations (rare and/or potentially lethal defects) and variations (commonly occurring non-lethal abnormalities).
Statistics:
Data from treated animals were compared with control data. Statistical analyses were performed where appropriate.
Data for each sex were analyzed separately. Only data collected on or after the first day of dosing were analyzed statistically.

Procedure I (Analysis of Variance [ANOVA])
Levene’s test was conducted to test for equality of variances between groups. When Levene’s test was significant (P > 0.05), a statistical comparison across treated and control groups was conducted using a one-way ANOVA. If the group effect of the ANOVA was not significant (P > 0.05), no further analyses were conducted. If the ANOVA was significant (P ≤ 0.05), Dunnett’s test was used for pairwise comparisons between each treated group and the control group. If Levene’s test was significant (P ≤ 0.05), a Kruskal-Wallis nonparametric ANOVA was conducted.

Procedure II (Analysis of Covariance [ANCOVA])
Analysis of covariance (ANCOVA) was performed. When the group effect from the ANCOVA was significant (P ≤ 0.05), Dunnett’s test was used for pairwise comparisons between each treated group and the control group.

Procedure III
A Kruskal-Wallis nonparametric ANOVA was conducted. If the Kruskal-Wallis test was significant (P ≤ 0.05), pairwise comparisons of each treated group with the control group was made using the Wilcoxon Rank Sum Test.

Procedure IV
Pairwise comparisons of each treated group with the control group was made using Fishers exact test.
Indices:
The following data were analyzed:
-Body weights
-Body weight gains
-Food consumption
-Mean fetal weight (male, female, and combined)
-The number of corpora lutea, implantation sites, early and late resorptions, dead fetuses, live fetuses, percent pre-implantation and post-implantation loss, and percent male fetuses
-Gravid uterine weights and corrected body weights (carcass weights)
-The percentage of fetuses affected (mean litter percent)
-Proportion of litters affected
Historical control data:
Findings compared to control and historical control data.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Yellow staining in the uro-genital region was noted from GD 12 to 17 for one female administered 200 mg/kg/day (Animal R0317).
All other clinical observations including; thinning fur, minimal lesions, and fur staining and pale teeth, were considered to be incidental as they presented in controls, showed no dose relationship and/or are common background finding for animals of this age and strain.
No postdose observations were noted.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No unscheduled mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
An initial reduction in body weight gain was noted for all test article treated groups, compared with controls between GD 6 and 9, and a dose-related response was evident, with statistical significance achieved for each dose group (20 mg/kg/day; P<0.05, 60 or 200 mg/kg/day; P<0.001), compared with controls.
Thereafter, lower body weight gains were evident for females administered 200 mg/kg/day, with a statistically significant reduction in overall mean body weight gain (GD 6 - 21) evident, when compared with controls (P<0.001).
Although overall body weights from GD 6 to 21 were lower for females administered 20 or 60 mg/kg/day, a clear dose response was not evident. As such, the slightly lower body weights and body weight gains noted in these dose groups were considered not to represent an adverse effect of treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A test article-related effect on food consumption was observed in dose groups administered 3,5-Dimethylpyrazole, compared with control.
Significantly lower food consumption was evident for females administered 200 mg/kg/day, with statistical significance achieved between GD 6-9, GD 11-16 and GD 20-21 (P<0.005 - P<0.001), compared with controls. Mean food consumption was reduced by approximately 16% lower than control values over the duration of the study.
An initial reduction in food consumption was noted for females administered 60 mg/kg/day during the first three days of dosing, compared with control (P<0.05 -P<0.01). Although mean values were slightly lower than controls for the remainder of the study, mean food consumption over the study duration was only 8% lower than control values, and as such, was considered not to represent an adverse effect of treatment.
Slightly lower food consumption was also evident for females administered 20 mg/kg/day, with minor statistical significance achieved on two occasions (P<0.05), compared with controls. Overall mean food consumption was only 7% lower than controls. And as such, was not considered to represent an adverse effect of treatment.
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
Description (incidence and severity):
Test article-related effects on gravid uterine weight, carcass weight, corrected weight change and total weight change were noted for dams administered 200 mg/kg/day, compared with controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic observations noted were considered due to 3,5-Dimethylpyrazole.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Females administered 200 mg/kg/day showed a persistent reduction in food consumption and body weight/body weight gain for the duration of the study.
Corrected body weight/body weight change were also noted to be lower than controls, confirming body weight reduction was a maternal effect and not an effect of reduction in fetal body weight gains. These observations are considered attributed to test article administration and were adverse.
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No effect on pre-implantation or post-implantation loss was noted.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No effect on resorptions was noted.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No effect on resorptions was noted.
Dead fetuses:
not examined
Description (incidence and severity):
No effect on number or sex ratio of live fetuses was noted.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There were 22, 21, 22 or 21 pregnant females administered 0, 20, 60 or 200 mg/kg/day with live fetuses on GD 21.
One female administered 200 mg/kg/day (Animal R0303) was noted to have no viable fetuses. Although instances of total in utero litter loss are occasionally noted in background findings, an association to test article administration at this dose level cannot be disregarded.
One female administered 20 mg/kg/day (Animal R0115) was supplied non-pregnant from the animal supplier, and is unrelated to test article administration.
Other effects:
not specified
Details on maternal toxic effects:
Females administered 200 mg/kg/day showed a persistent reduction in food consumption and body weight/body weight gain for the duration of the study.
Corrected body weight/body weight change were also noted to be lower than controls, confirming body weight reduction was a maternal effect and not an effect of reduction in fetal body weight gains. These observations are considered attributed to test article administration and were adverse.
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean male and female fetal weights were significantly lower than control fetuses (P<0.001).
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No effect on number or sex ratio of live fetuses was noted.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effect on number or sex ratio of live fetuses was noted.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
Test article-related malformations were noted in litters maternally administered 200 mg/kg/day.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Incidences of bent scapula blade were noted in fetuses from dams administered 20 or 200 mg/kg/day. This observation was noted in one fetus from one dam administered 20 mg/kg/day and six fetuses from three dams administered 200 mg/kg/day. Incidences of bent scapular in fetuses from dams administered 200 mg/kg/day also exceeded background control ranges of 1.5% litters / 0.3% fetuses, compared with 14% litters / 4.81% fetuses in 200 mg/kg/day litters.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Supernumerary digit/polydactyly was noted in three fetuses from two litters maternally administered 200 mg/kg/day. Diaphragmatic cyst, and short humerus were noted in fetuses maternally administered 200 mg/kg/day. Incidences of these observations have not been noted in background data.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Male and female fetuses maternally administered 200 mg/kg/day weighed significantly less than control fetuses. These lower body weights were considered to represent an adverse effect of test article administration. It is also worth noting that one female administered 200 mg/kg/day had a total in utero litter loss (no viable fetuses). Furthermore, the number of fetuses showing malformations was greatly increased in the 200 mg/kg/day litters, compared with controls. The etiology of these findings is unclear, although an effect of test article administration cannot be disregarded.
Polydactyly was noted in three fetuses from two dams administered 200 mg/kg/day.
Fused ribs, diaphragmatic cyst and shortened humerus were also noted in fetuses from this dose group. The occurrence of these findings in the high dose group, and in the absence in the control group, lower dose groups or the absence in the historical background control data, this finding is considered to represent an effect on development of the fetus at this dose.
Higher incidences of bent scapular were noted in females administered 200 mg/kg/day; this was associated with thick humorous and an increased incidence of wavy ribs. Unossified and incomplete ossification of parts of the skeleton and abnormal pelvic girdle alignment were also noted. The increased incidence of these observations was considered secondary to maternal and developmental toxicity.
However, these observations are considered to be transient and, therefore, considered non-adverse (Kimmel et al., 2014).
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
visceral malformations
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
60 mg/kg bw/day (nominal)
Treatment related:
no

Fetal Malformations

Dose Level

Dam Number

Fetus Identity

Malformation

0 (Control)

R0012

R0013

 

R0013

R0016

L5

R5

 

R9

R10

Eye: anophthalmia (right)

Kidney: hydronephrosis (left)

Ureter – hydroureter (left)

Sternebra–partially split–6

Blood vessel: Descending aorta–right-sided

Blood vessel: Aorta/pulmonary trunk–common origin

Blood vessel: Ductus arteriosis – right sided

Blood vessel: Pulmonary trunk-right sided, passing behind ascending aorta

Blood vessel: Subclavian artery-malpositioned-right, arising from descending aorta

Heart: Ventricular septum defect-membranous region

20 mg/kg/day

R0104

 

 

 

 

 

 

 

 

 

 

R0114

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

R0117

L5

 

 

 

 

 

 

 

 

 

 

R10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

L1

Head: Micrognathia-lower jaw

Mouth: Microglossia

Mouth: Microstomia

Mouth: Palate-high arched

Rib: 11, right-entire length branched

Rib: 10/11, left-articulated supernumerary, full

Skull: Mandible fused

Skull: Mandible short-bilateral

Skull: Maxilla misshapen, palatine aspect-bilateral

Skull: Palatine malpositioned-bilateral

Skull: Tympanic annulus supernumerary site

Great blood vessels: transposition

Head: Micrognathia-lower jaw

Mouth: Aglossia

Mouth: Microstomia

Mouth: Palate – high arched

Heart: Ventricular septum defect

Lung: lobe-abnormal lobulation

Skull: Mandible fused

Skull: Mandible short-bilateral

Skull: Palatine misshapen-bilateral

Skull: Maxilla misshapen-bilateral

Skull: Basisphenoid/tympanic annulus misshapen, with supernumerary site-bilateral

Skull: Basisphenoid misshapen and fused to tympanic annulus-bilateral

Skull: Tympanic annulus misshapen

Skull: Tympanic annulus misshapen, supernumerary site-bilateral

Pectoral girdle: Right scapula-blade bent

200 mg/kg/day

R0301

R0302

R0305

R0307

R0307

 

R0308

R0308

 

R0309

R0310

 

 

 

R0310

 

 

R0310

R0311

R0322

 

R0322

 

 

R0322

R0322

L5

R8

L2

R5

R9

 

R9

R12

 

R7

L1

 

 

 

R7

 

 

R8

L5

L1

 

L4

 

 

R10

R11

Pectoral girdle: Right scapula-blade bent

Ribs-fused-left, 7/8, proximal

Left kidney-hydronephrosis

Sternebra: 4/5/6-split

Sternebra: 1-misshapen

Supernumerary sternebra-between 5/6 sternebra

Pectoral girdle: Right scapula-blade bent

Hind limb: Right paw-supernumerary digit(s)-1, one fused

Left ureter-hydroureter

Hind limb: Right paw-supernumerary digit(s)-2, two fused

Paw: hind limb-polydactyly-two additional

Sternebra: 3/4-misaligned

Hind limb: Right paw-supernumerary digit(s)-2, one fused

Paw: hind limb-polydactyly-one additional

Diaphragm-cyst-tendinous region, adhering to left lung

Left ureter-hydroureter

Right humerus-thick

Pectoral girdle: Right scapula-blade bent

Right humerus-short

Right humerus-thick

Pectoral girdle: Right scapula-blade bent

Pectoral girdle: Right scapula-blade bent

Pectoral girdle: Right scapular-blade bent

 

Noteworthy Fetal Variations

Tissue /Organ

Variation

 

Dose Level (mg/kg/day)

Historical Control background data

0

20

60

200

Blood Vessel

Umbilical artery – left sided

%Litter

%Fetal

41

8.54

43

9.41

45

10.74

86**

29.27

59.34

17.09

Skull

Presphenoid-incomplete ossification

%Litter

%Fetal

5

0.79

15

3.00

0

0.00

24

6.63

Not present

Skull

Suture-sutural bone

%Litter

%Fetal

5

0.68

5

1.67

0

0.00

33*

7.30

Not present

Pelvic Girdle

Iliac alignment-abnormal

%Litter

%Fetal

19

3.62

20

3.83

18

5.91

95***

89.59

15

5

Sternebra

Additional ossification site

%Litter

%Fetal

0

0.00

0

0.00

0

0.00

24*

7.30

0.82

0.15

Sternebra

Incomplete ossification

%Litter

%Fetal

0

0.00

10

1.83

5

1.82

38**

18.87

14.50

3.43

Vertebra

Cervical centrum-incomplete ossification

%Litter

%Fetal

43

10.60

55

18.08

36

12.66

81*

30.71

37.50

10.67

Vertebra

Cervical centrum-unossified

%Litter

%Fetal

67

26.29

80

37.17

45

19.70

86

59.16

30

9.59

Vertebra

Thoracic centrum-incomplete ossification

%Litter

%Fetal

19

4.85

5

0.83

14

2.47

48

12.32

27.50

6.11

Hind limb

Metatarsal-incomplete ossification

%Litter

%Fetal

5

0.95

10

3.00

14

2.69

33*

8.15

17.50

4.02

Hind limb

Metatarsal-unossified

%Litter

%Fetal

19

3.89

30

8.33

32

11.82

43

16.43

2.50

0.63

Rib

Nodulated

%Litter

%Fetal

57

18.44

60

17.67

36

16.93

76

37.40

26.19

8.39

Rib

Wavy

%Litter

%Fetal

19

4.54

35

8.00

14

5.00

43

11.08

6.07

1.69

Statistically significant from control group: * P<0.05, ** P<0.01, *** P<0.001

Summary of Reproductive Performance

Treatment Group

Control

20 mg/kg

60 mg/kg

200 mg/kg

Total Females

Pregnant Females

Non-Pregnant Females

Pregnant with Total Litter Loss (NVF)

Females with Live Fetuses

22

22

0

0

22

22

21

1

0

21

22

22

0

0

22

22

22

0

1

21

NVF = No Viable Fetuses

 

Summary of Clinical Observations

Group #

Category, Observations

Day of Phase:

Gestation

6

7

8

9

10

11

12

13

Control

 

 

Skin and Fur, sores/lesions, minimal, neck

Skin and Fur, staining, neck, orange

Skin and Fur, thinning fur, abdomen

Skin and Fur, thinning fur. neck

Skin and Fur, thinning fur, paws fore both

Skin and Fur, thinning fur, shoulder left

Skin and Fur, thinning fur, shoulder right

Skin and Fur, thinning fur, shoulders both

Teeth, appearance, lower, pale

Number Examined:

Number Normal:

22

17

1

1

0

0

0

1

1

1

1

22

16

1

1

0

0

0

1

2

1

1

22

16

1

1

0

0

0

1

2

1

1

22

14

1

1

1

0

1

1

2

1

1

22

14

1

1

1

0

1

1

2

1

1

22

15

1

1

1

0

0

1

2

1

1

22

16

1

1

1

0

0

0

2

1

1

22

16

0

1

1

1

0

0

2

1

1

2

 

 

Skin and Fur, sores/lesions, minimal, neck

Skin and Fur, staining, neck, orange

Skin and Fur, thinning fur, neck

Skin and Fur, thinning fur, shoulder left

Skin and Fur, thinning fur, shoulders both

Number Examined:

Number Normal:

22

18

1

0

0

1

2

22

17

1

0

0

1

3

22

17

1

0

0

1

3

22

18

0

1

0

1

2

22

18

0

1

0

1

2

22

17

0

1

0

2

2

22

17

0

1

0

2

2

22

17

0

1

0

2

2

3

 

 

Skin and Fur, hair loss, paws fore left

Skin and Fur, staining, neck, orange

Skin and Fur staining, neck, pink

Skin and Fur, thinning fur, abdomen

Skin and Fur, thinning fur, legs fore both

Skin and Fur, thinning fur, legs hind both

Skin and Fur, thinning fur, neck

Skin and Fur, thinning fur, shoulder left

Teeth, appearance, lower, pale

Number Examined:

Number Normal:

22

18

0

0

0

0

0

0

2

1

1

22

18

0

0

0

0

0

0

2

1

1

22

18

0

0

0

0

0

0

2

1

1

22

19

0

0

0

0

0

0

1

1

1

22

18

0

1

0

0

0

0

1

1

1

22

18

1

1

0

0

0

0

1

0

1

22

16

1

1

0

0

0

0

3

0

1

22

17

1

1

0

0

0

0

2

0

1

4

 

 

Skin and Fur, sores/lesions, minimal, neck, small

Skin and Fur, staining, uro-genital area, yellow

Skin and Fur, thinning fur, abdomen

Skin and Fur, tinning fur, neck

Skin and Fur, thinning fur, shoulder left

Skin and Fur, thinning fur, shoulders both

Teeth, appearance, lower, pale

Number Examined:

Number Normal:

22

18

1

0

0

1

1

1

1

22

16

2

0

0

2

1

1

1

22

15

1

0

0

2

2

1

1

22

16

0

0

0

2

2

1

1

22

16

0

0

0

3

2

1

1

22

16

0

0

0

3

2

1

1

22

17

0

1

0

3

1

0

1

22

17

0

1

0

3

1

0

1

 

Group #

Category, Observations

Day of Phase:

Gestation

14

15

16

17

18

19

20

21

Control

 

 

Skin and Fur, staining, neck, red

Skin and Fur, staining, neck, orange

Skin and Fur, thinning fur, abdomen

Skin and Fur, thinning fur. neck

Skin and Fur, thinning fur, shoulder left

Skin and Fur, thinning fur, shoulder right

Skin and Fur, thinning fur, shoulders both

Teeth, appearance, lower, pale

Number Examined:

Number Normal:

22

16

0

1

1

0

0

2

1

1

22

16

0

1

1

0

1

2

0

1

22

16

0

1

1

0

1

2

0

1

22

17

0

1

1

0

1

1

0

1

22

18

0

0

1

0

2

1

0

1

22

17

0

0

2

1

1

1

0

1

22

17

0

0

2

1

1

1

0

1

22

18

1

0

1

1

1

1

0

1

2

 

 

Excretion, discharge, minimal, uro-genital area, red

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, thinning fur, neck

Skin and Fur, thinning fur, shoulder left

Skin and Fur, thinning fur, shoulders both

Number Examined:

Number Normal:

22

17

0

0

1

0

2

2

22

17

0

0

1

1

2

2

22

18

0

0

1

1

2

1

22

20

0

0

0

1

1

1

22

20

0

0

0

1

1

1

22

20

0

0

0

1

1

1

22

19

1

0

0

1

1

1

22

19

0

1

0

1

1

1

3

 

 

Skin and Fur, hair loss, paws fore left

Skin and Fur, staining, neck, orange

Skin and Fur staining, neck, pink

Skin and Fur, thinning fur, abdomen

Skin and Fur, thinning fur, legs fore both

Skin and Fur, thinning fur, legs hind both

Skin and Fur, thinning fur, neck

Teeth, appearance, lower, pale

Number Examined:

Number Normal:

22

17

1

1

0

0

0

0

2

1

22

17

1

0

1

0

0

0

2

1

22

17

1

0

1

0

0

0

2

1

22

17

1

0

1

1

0

0

1

1

22

16

1

0

1

1

0

1

2

1

22

16

1

0

0

1

1

1

2

1

22

16

1

0

0

1

1

1

2

1

22

16

1

0

0

1

1

1

2

1

4

 

 

Skin and Fur, staining, uro-genital area, yellow

Skin and Fur, thinning fur, abdomen

Skin and Fur, tinning fur, neck

Skin and Fur, thinning fur, shoulder left

Teeth, appearance, lower, pale

Number Examined:

Number Normal:

22

18

1

0

3

1

0

22

17

1

0

2

2

0

22

19

1

0

2

0

0

22

19

14

0

2

0

0

22

20

0

0

2

0

0

22

20

0

1

2

0

0

22

20

0

1

2

0

0

22

21

0

0

1

0

0

 

Summary of Body Weight by Day (g)

Group #

 

Gestation

Day: 3

Session: 1

Day: 6

Session: 1

Day: 9

Session: 1

Day: 12

Session: 1

Day: 15

Session 1

Day: 17

Session: 1

Day: 19

Session: 1

Day: 21

Session: 1

Control

(n)

Means

Sdevs

22

207.5

16.94

22

220.2

19.09

22

230.8

20.66

22

244.0

21.44

22

258.4

22.79

22

276.8

23.77

22

298.5

27.01

22

322.5

29.96

2

(n)

Means

Sdevs

21

211.1

15.29

21

222.9

16.70

21

230.7

16.86

21

243.6

18.05

21

256.0

19.45

21

272.6

24.02

21

291.9

29.16

21

314.2

34.18

3

(n)

Means

Sdevs

22

208.4

16.80

22

218.7

15.65

22

223.9

16.21

22

238.1

17.53

22

253.4

18.93

22

271.2

20.95

22

289.9

22.58

22

314.4

26.96

4

(n)

Means

Sdevs

21

208.2

17.62

21

221.5

19.48

21

222.7

19.79

21

232.9

20.16

21

243.5

21.70

21

261.0

23.83

21

283.5

26.51

21

302.4

29.15

 

Summary of Body Weight Gain per Interval (g)

Group #

 

GD:3-GD:6

GD:6-GD:9

GD:9-GD:12

GD:12-GD:15

GD:15-GD:17

GD:17-GD:19

GD:19-GD:21

GD:6-GD:21

Control

(n)

Means

SD

22

12.7

6.56

22

10.6

4.97

22

13.2

3.88

22

14.4

3.92

22

18.4

3.36

22

21.7

5.90

22

24.0

5.60

22

102.3

16.65

2

(n)

Means

SD

21

11.7

4.39

21

7.8*r

3.38

21

12.9

3.91

21

12.4

4.52

21

16.6

6.27

21

19.3

6.11

21

22.3

8.03

21

91.3

24.34

3

(n)

Means

SD

22

10.3

4.75

22

5.2#r

2.32

22

14.1

4.11

22

15.4

3.77

22

17.8

4.66

22

18.7

4.14

22

24.4

7.23

22

95.6

15.20

4

(n)

Means

SD

21

13.3

7.59

21

1.2#r

5.93

21

10.2

5.08

21

10.6*H

5.58

21

17.5

6.54

21

22.5

5.26

21

18.9

7.91

21

80.9#H

16.48

GD – Gestation

*r = Wilcoxon Rank Sum Test Significant at 0.05 level.               #r = Wilcoxon rank Sum Test Significant at 0.001 level.

*H = Dunnett Exact Homogeneous Test Significant: 0.05 level #H = Dunnett Exact Homogeneous Test Significant: 0.001 level

 

Summary of Food Consumption (g)

Group #

 

GD:6-GD:7

GD:7-GD:8

GD:8-GD:9

GD:9-GD:10

GD:10-GD:11

GD:11-GD:12

GD:12-GD:13

GD:13-GD:14

Control

(n)

Mean

SD

22

18.1

2.58

22

17.8

2.32

21

18.5

3.26

22

19.4

3.74

22

19.4

2.82

22

20.5

4.04

22

21.4

3.70

22

21.4

2.74

2

(n)

Mean

SD

21

16.4

2.40

21

16.6

3.09

21

16.4*H

2.78

21

17.5

2.33

21

18.9

2.70

21

18.7

3.60

21

19.4

2.56

21

19.9

2.76

3

(n)

Means

SD

22

15.8+H

2.18

22

15.5*H

2.18

22

15.7+H

2.18

22

17.4

2.34

22

17.7

3.22

22

18.6

3.05

22

19.4

1.99

22

20.0

2.76

4

(n)

Means

SD

21

14.5#H

2.49

21

14.4#H

2.71

21

15.5+H

2.80

21

17.4

2.82

21

17.3

2.79

21

16.3#H

3.59

21

17.5#H

2.74

21

17.8#H

2.50

GD – Gestation

+H = Dunnett Exact Homogenous Test Significant: 0.01 level   #H = Dunnett Exact Homogenous Test Significant: 0.001 level

*H = Dunnett Exact Homogenous Test Significant: 0.05 level

Group #

 

GD:14-GD:15

GD:15-GD:16

GD:16-GD:17

GD:17-GD:18

GD:18-GD:19

GD:19-GD:20

GD:20-GD:21

Control

(n)

Mean

SD

22

19.3

2.62

21

22.6

4.49

22

24.5

9.15

22

22.3

3.62

22

22.1

3.81

22

20.2

4.44

22

20.7

3.57

2

(n)

Mean

SD

21

18.8

2.81

21

20.0+H

2.46

21

22.5

3.21

21

22.5

7.77

21

20.9

3.79

21

19.1

3.66

21

19.1

3.11

3

(n)

Means

SD

22

19.9

3.26

22

20.1

2.50

22

22.3

2.89

22

21.6

3.93

22

20.6

2.77

22

20.2

3.32

22

19.8

2.64

4

(n)

Means

SD

21

16.9*H

2.81

21

17.7#H

3.96

21

20.1

4.06

21

19.3

4.02

21

20.0

3.88

21

17.7

2.98

21

17.2+H

3.67

GD – Gestation

+H = Dunnett Exact Homogenous Test Significant: 0.01 level   #H = Dunnett Exact Homogenous Test Significant: 0.001 level

*H = Dunnett Exact Homogenous Test Significant: 0.05 level

 

Summary of Cesarean Section Data

 

Group

Control

20 mg/kg

60 mg/kg

200 mg/kg

Summary of Cesarean Section Data – Including Female with No Viable Fetuses

Number of females pregnant at cesarean section

(n)

22

21

22

22

Corpora Lutea

(n)

Mean

SD

22

12

1.8

21

12

1.7

22

12

2.3

22

12

1.7

Implantation Sites

(n)

Mean

SD

22

11

2.9

21

10

3.0

22

10

2.3

22

11

2.2

Pre-implantation Loss

(n)

Mean

SD

22

1

2.0

21

2

2.6

22

1

1.9

21@

1

1.2

Pre-implantation Loss (%)

(n)

Mean

SD

22

11.4

20.15

21

15.5

24.62

22

10.5

14.36

21@

5.7

11.27

Early Resorptions

(n)

Mean

SD

22

0

1.1

21

1

1.3

22

1

1.1

22

1

2.1

Late Resorptions

(n)

Mean

SD

22

0

0.0

21

0

0.0

22

0

0.0

22

0

0.0

Total Resorptions

(n)

Mean

SD

22

0

1.1

21

1

1.3

22

1

1.1

22

1

2.1

Dead Fetuses

(n)

Mean

SD

22

0

0.0

21

0

0.0

22

0

0.0

22

0

0.0

Post-implantation Loss

(n)

Mean

SD

22

0

1.1

21

1

1.3

22

1

1.1

22

1

2.1

Post-implantation Loss (%)

(n)

Mean

SD

22

3.6

8.04

21

8.9

15.32

22

6.0

11.35

22

10.7

20.79

Live Fetuses

(n)

Mean

SD

22

11

2.7

21

9

3.3

22

10

2.7

22

10

3.1

Gravid Uterine Weight (g)

(n)

Mean

SD

22

72.0

17.21

21

63.1

21.31

22

67.3

16.73

21

65.2

12.35

Corrected Body Weight

(Carcass Weight) (g)

(n)

Mean

SD

22

246.7

18.82

21

247.5

17.07

22

243.7

18.54

21

235.9

22.54

Corrected Weight Change (g)

Day 3 to 21

(n)

Mean

SD

22

39.2

10.07

21

36.3

9.75

22

35.3

10.65

21

27.7

12.90

Total Weight Change

(Weight Change) (g)

Day 3 to 21

(n)

Mean

SD

22

111.2

19.16

21

99.4

25.33

22

102.6

15.93

21

92.9

19.06

Number of Females with Live Fetuses

(n)

22

21

22

22

Mean Number of Male Fetuses per Litter

(n)

Mean

SD

22

6

2.1

21

5

2.7

22

5

1.9

22

5

2.5

Mean Number of Female Fetuses per Litter

(n)

Mean

SD

22

5

1.8

21

4

2.1

22

5

1.8

22

5

2.2

% Male Fetuses

(n)

Mean

SD

22

54

16.4

21

50

22.4

22

49

13.2

22

43

19.2

Mean Fetal Weight (g)

(n)

Mean

SD

22

5.17

0.358

21

5.12

0.377

22

5.18

0.390

21

4.62

0.358

Mean Weight

- Male Fetuses (g)

(n)

Mean

Adj Mean

SD

22

5.27

5.30

0.381

20

5.31

5.28

0.356

22

5.35

5.33

0.452

21

4.74

4.75#H

0.374

Mean Weight

- Female Fetuses (g)

(n)

Mean

Adj Mean

SD

21

4.99

5.02

0.213

20

4.89

4.87

0.316

22

5.02

5.00

0.363

21

4.53

4.53#H

0.371

@Number examined reduced due to excluded data

#H = Dunnett Exact Homogeneous Test Significant: 0.001 level

 

Summary of Cesarean Section Data

 

Group

Control

20 mg/kg

60 mg/kg

200 mg/kg

Summary of Cesarean Section Data – Excluding Female with No Viable Fetuses

Number of females pregnant at cesarean section

(n)

22

21

22

21

Corpora Lutea

(n)

Mean

SD

22

12

1.8

21

12

1.7

22

12

2.3

21

12

1.7

Implantation Sites

(n)

Mean

SD

22

11

2.9

21

10

3.0

22

10

2.3

21

11

2.3

Pre-implantation Loss

(n)

Mean

SD

22

1

2.0

21

2

2.6

22

1

1.9

20@

1

1.2

Pre-implantation Loss (%)

(n)

Mean

SD

22

11.4

20.15

21

15.5

24.62

22

10.5

14.36

20@

5.9

11.49

Early Resorptions

(n)

Mean

SD

22

0

1.1

21

1

1.3

22

1

1.1

21

1

0.6

Late Resorptions

(n)

Mean

SD

22

0

0.0

21

0

0.0

22

0

0.0

21

0

0.0

Total Resorptions

(n)

Mean

SD

22

0

1.1

21

1

1.3

22

1

1.1

21

1

0.6

Dead Fetuses

(n)

Mean

SD

22

0

0.0

21

0

0.0

22

0

0.0

21

0

0.0

Post-implantation Loss

(n)

Mean

SD

22

0

1.1

21

1

1.3

22

1

1.1

21

1

0.6

Post-implantation Loss (%)

(n)

Mean

SD

22

3.6

8.04

21

8.9

15.32

22

6.0

11.35

21

6.5

6.04

Live Fetuses

(n)

Mean

SD

22

11

2.7

21

9

3.3

22

10

2.7

21

10

2.2

Gravid Uterine Weight (g)

(n)

Mean

SD

22

72.0

17.21

21

63.1

21.31

22

67.3

16.73

21

65.2

12.35

Corrected Body Weight

(Carcass Weight) (g)

(n)

Mean

SD

22

246.7

18.82

21

247.5

17.07

22

243.7

18.54

21

235.9

22.54

Corrected Weight Change (g)

Day 3 to 21

(n)

Mean

SD

22

39.2

10.07

21

36.3

9.75

22

35.3

10.65

21

27.7

12.90

Total Weight Change

(Weight Change) (g)

Day 3 to 21

(n)

Mean

SD

22

111.2

19.16

21

99.4

25.33

22

102.6

15.93

21

92.9

19.06

Number of Females with Live Fetuses

(n)

22

21

22

21

Mean Number of Male Fetuses per Litter

(n)

Mean

SD

22

6

2.1

21

5

2.7

22

5

1.9

21

5

2.4

Mean Number of Female Fetuses per Litter

(n)

Mean

SD

22

5

1.8

21

4

2.1

22

5

1.8

21

6

1.9

% Male Fetuses

(n)

Mean

SD

22

54

16.4

21

50

22.4

22

49

13.2

21

45

17.1

Mean Fetal Weight (g)

(n)

Mean

SD

22

5.17

0.358

21

5.12

0.377

22

5.18

0.390

21

4.62

0.358

Mean Weight

- Male Fetuses (g)

(n)

Mean

Adj Mean

SD

22

5.27

5.30

0.381

20

5.31

5.28

0.356

22

5.35

5.33

0.452

21

4.74

4.75#H

0.374

Mean Weight

- Female Fetuses (g)

(n)

Mean

Adj Mean

SD

21

4.99

5.02

0.213

20

4.89

4.87

0.316

22

5.02

5.00

0.363

21

4.53

4.53#H

0.371

@Number examined reduced due to excluded data

#H = Dunnett Exact Homogeneous Test Significant: 0.001 level

Conclusions:
Once daily oral (gavage) administration of 0 (control), 20, 60, or 200 mg/kg/day 3,5 Dimethylpyrazole to the pregnant rat from implantation to the day before parturition, resulted in test article-related changes at all dose levels.
Test article-related effects on the maternal animal and subsequent litters following 200 mg/kg/day administration were considered adverse. Maternal effects following 20 or 60 mg/kg/day were considred not to represent an adverse health effect, and no effect on the fetuses was evident. As such, the maternal and fetal No Observed Adverse Effect Level (NOAEL) is considered to be 60 mg/kg/day.
Executive summary:

The objective of the study was to determine the effects of the test article, 3,5-Dimethylpyrazole, on the embryonic and fetal development of the rat.

 

The test article is an industrial chemical.

 

Four groups of 22 time-mated female Crl:WI(Han) rats/group were administered 0 (control article [vehicle]), 20, 60, or 200 mg/kg/day by oral gavage from Gestation Day (GD) 6 to 20 at a volume of 5 mL/kg. The control article (vehicle) was Propylene Glycol.

 

Assessment of toxicity was based on clinical observations, body weights, and food consumption. On GD 21, animals were sacrificed and the progress and outcome of pregnancy was evaluated. Complete necropsies were performed on all animals, and any macroscopic abnormalities were recorded. Fetuses were evaluated for variations and malformations.

 

Test article related effects on mean body weight, body weight change and food consumption values were noted in all test article-treated females up to GD 9. Females administered 200 mg/kg/day continued to show a reduction in food consumption and body weight gain for the duration of the study, with body weight and food consumption effects following 20 or 60 mg/kg/day regressing to normal.

 

A reduction in gravid uterine weight correlated with a statistically significant reduction in the fetal body weight of both sexes (P<0.001) and an overall reduction of mean fetal body weight in litters from dams administered 200 mg/kg/day. The reduction in body weight and markers of delayed development are considered to have a relationship to maternal toxicity; reduced weight gain and reduction in food consumption, and were therefore considered to be associated to the test article.

 

There were 22, 21, 22 or 21 pregnant females administered 0, 20, 60 or 200 mg/kg/day with live fetuses on GD 21. One female administered 200 mg/kg/day was noted to have no viable fetuses.

 

An increased incidence of fetuses with malformations was noted of dams administered 200 mg/kg/day, compared with controls. Test article related malformations including supernumerary digit/polydactyly, fused ribs, diaphragmatic cyst, and short humorous were all noted. Fetal findings often observed in females administered a maternally toxic dose level including; bent scapula, and increased incidence of incomplete ossification and unossified bones were also noted at this dose

level.

 

Clinical observations and post-dose observations were generally unaffected and macroscopic tissue examination revealed no test article effects and there were no effects on fertility; Pre-implantation or post-implantation loss, resorptions, and number or sex ratio of live fetuses were all similar to control values and within expected background ranges.

 

Once daily oral (gavage) administration of 0 (control), 20, 60, or 200 mg/kg/day 3,5-Dimethylpyrazole to the pregnant rat from implantation to the day before parturition, resulted in test article-related changes at all dose levels.

 

Test article-related effects on the maternal animal and subsequent litters following 200 mg/kg/day administration were considered adverse. Maternal effects following 20 or 60 mg/kg/day were considered not to represent an adverse health effect, and no effect on the fetuses was evident. As such, the maternal and fetal No Observed Adverse Effect Level (NOAEL) is considered to be 60 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
60 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is performed in line with standardised guideline and is sufficient to justify a Klimisch score of 1, thus the dataset is considered to be of high quality.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The objective of the study was to determine the effects of the test article, 3,5-Dimethylpyrazole, on the embryonic and fetal development of the rat. 

Four groups of 22 time-mated female Crl:WI(Han) rats/group were administered 0 (control article [vehicle]), 20, 60, or 200 mg/kg/day by oral gavage from Gestation Day (GD) 6 to 20 at a volume of 5 mL/kg. The control article (vehicle) was Propylene Glycol.

Assessment of toxicity was based on clinical observations, body weights, and food consumption. On GD 21, animals were sacrificed and the progress and outcome of pregnancy was evaluated. Complete necropsies were performed on all animals, and any macroscopic abnormalities were recorded. Fetuses were evaluated for variations and malformations.

 

Test article related effects on mean body weight, body weight change and food consumption values were noted in all test article-treated females up to GD 9. Females administered 200 mg/kg/day continued to show a reduction in food consumption and body weight gain for the duration of the study, with body weight and food consumption effects following 20 or 60 mg/kg/day regressing to normal.

A reduction in gravid uterine weight correlated with a statistically significant reduction in the fetal body weight of both sexes (P<0.001) and an overall reduction of mean fetal body weight in litters from dams administered 200 mg/kg/day. The reduction in body weight and markers of delayed development are considered to have a relationship to maternal toxicity; reduced weight gain and reduction in food consumption, and were therefore considered to be associated to the test article.

There were 22, 21, 22 or 21 pregnant females administered 0, 20, 60 or 200 mg/kg/day with live fetuses on GD 21. One female administered 200 mg/kg/day was noted to have no viable fetuses.

An increased incidence of fetuses with malformations was noted of dams administered 200 mg/kg/day, compared with controls. Test article related malformations including supernumerary digit/polydactyly, fused ribs, diaphragmatic cyst, and short humorous were all noted. Fetal findings often observed in females administered a maternally toxic dose level including; bent scapula, and increased incidence of incomplete ossification and unossified bones were also noted at this dose level.

Clinical observations and post-dose observations were generally unaffected and macroscopic tissue examination revealed no test article effects and there were no effects on fertility; Pre-implantation or post-implantation loss, resorptions, and number or sex ratio of live fetuses were all similar to control values and within expected background ranges.

 

Once daily oral (gavage) administration of 0 (control), 20, 60, or 200 mg/kg/day 3,5-Dimethylpyrazole to the pregnant rat from implantation to the day before parturition, resulted in test article-related changes at all dose levels.

Test article-related effects on the maternal animal and subsequent litters following 200 mg/kg/day administration were considered adverse. Maternal effects following 20 or 60 mg/kg/day were considered not to represent an adverse health effect, and no effect on the fetuses was evident. As such, the maternal and fetal No Observed Adverse Effect Level (NOAEL) is considered to be 60 mg/kg/day.

Justification for selection of Effect on developmental toxicity: via oral route:

The key study ( Barraclough, 2018) was performed in compliance with GLP and the standardised guidelines OECD 414. The study was performed to a good standard with a sufficient level of detail to assess the quality of the data presented. The study was assigned a reliability score of 1, in accordance with Klimisch (1997) and considered suitable to fulfil the data requirement.

Justification for selection of Effect on developmental toxicity: via inhalation route:

Exposure via the oral route is considered an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. Further testing via inhalation is considered less appropriate and has been omitted on this basis.

Justification for selection of Effect on developmental toxicity: via dermal route:

Exposure via the oral route is considered an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. Further testing via inhalation is considered less appropriate and has been omitted on this basis..

Toxicity to reproduction: other studies

Additional information

Under the conditions of the test developmental toxicity was observed at 200 mg/kg, based on treatment related effects observed on pup mortality (postnatal loss) and lower pup body weights at 200 mg/kg. No treatment-related changes were noted in any of the remaining developmental parameters investigated in this study, i.e. gestation index and duration, parturition, maternal care and clinical signs and macroscopy of pup. Based on the observed results the developmental NOAEL was determined to be 60 mg/kg b.w./day.

Justification for classification or non-classification

The key study submitted to evaluate reproductive toxicity of the test material via the oral route. This is considered an appropriate route of exposure for the submitted substance, as it is known to be readily absorbed following oral administration. The study indicated that the test material requires classification in accordance with Regulation EC 1272/2008 and the test material meets the criteria for classification as a reproductive toxicant category 2 (H361: Suspected of damaging fertility or the unborn child) .

Additional information