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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
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

Test material

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

Test animals

Species:
rat
Strain:
Wistar
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.

Administration / exposure

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
Doses / concentrations
Remarks:
Doses / Concentrations:
20, 60 and 200 mg/kg b.w./day
Basis:
actual ingested
No. of animals per sex per dose:
Ten males and ten females per dose.
Control animals:
yes
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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.

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