2-(4-tert-butylphenyl)-6-cyano-5-[bis(ethoxycarbonylmethyl)carbamoyloxy]-1H-pyrrolo[1,2-b][1,2,4] triazole-7-carboxylic acid 2,6-di-tert-butyl-4-methylcyclohexylester

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 September 2013 - 02 November 2013

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, done to a valid guideline and the study was conducted under GLP conditions.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

448-050-6

EC Name:

-

Cas Number:

444065-11-6

Molecular formula:

C41 H57 N5 O8

IUPAC Name:

2,6-di-tert-butyl-4-methylcyclohexyl 5-{[bis(2-ethoxy-2-oxoethyl)carbamoyl]oxy}-2-(4-tert-butylphenyl)-6-cyano-1H-pyrrolo[1,2-b][1,2,4]triazole-7-carboxylate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Substance type: Off white, slightly yellow; fine, fluffy powder
- Storage conditions of test material: Room temperature (15 - 30 °C)

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl: WI (SPF)
- Age at study initiation: (P) Young adult rats, approximately 10 weeks old at start and 12 weeks at mating. The age range within the study was kept to the minimum practicable.
- Weight at study initiation: (P) Males: 334 - 397 g; Females: 195 - 231 g; body weights did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
- Fasting period before study: No
- Housing: Rodents were group-housed as practical, up to 4 animals of the same group per cage (during the mating and gestation/delivery period, they were paired or individually housed, respectively). Cages were type II and III polypropylene/polycarbonate.
- Diet: Animals received complete diet for rats and mice, ad libitum.
- Water: Tap water from the municipal supply in 500 mL bottles, ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 – 24.7 °C
- Humidity (%): 33 – 59 % (relative)
- Air changes (per hr): 15 - 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours of light daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

(PEG 400)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Formulation: The test material was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared at up to 4-day intervals, based on the findings of a stability test performed at the analytical laboratory of the testing facility.
- Dosing volume: 3 mL/kg bw; the actual volume administered was calculated and adjusted based on each animal’s most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING
- Concentrations: All three dose formulations (62.5, 250 and 1000 mg/kg bw/day) and the control were sampled for analysis on two occasions during the study.
- Sampling method: Six samples were taken from the dosing formulations at each analytical occasion: two samples from each of the top, middle and bottom of the formulations. Two samples were taken from the control formulations at each analytical occasion.
- Sample preparation: The formulation samples were dissolved and diluted with ACN:water = 9:1 (solution for dilution) prior to HPLC analysis.

IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE
- Method of determination: HPLC-UV
- Conditions:
> Column: Zorbax Eclipse XDB-C8 (150 × 4.6 mm, 5 μm); No.: USRK 054825
> Column temperature: 25 °C
> Mobile phase: ACN:water= 9:1 +0.1 % TFA (isocratic elution). 1 mL of TFA was added into 1000 mL of ACN:water =9:1 solution
> Flow: 1.0 mL/min
> Injection volume: 5 µL
- Detection method: UV (wavelength = 275 nm)
- Retention time: Elution of the test material was observed at retention time of ~7.4 min.

Results of the method validation:
- Selectivity: No interfering component was observed.
- Linearity range: 0.5 – 100 mg/L
- Limit of Quantification: 0.5 mg/L
- Reinjection repeatability (7 injections): Coefficient of Variation ≤ 0.9 %
- Stability of the test material solutions in the autosampler: Stable for at least 20 hours.
- Stock solution stability: Stable for at least 6 days at 5 ± 3 °C.
- Recovery (from formulation): 10 mg/mL = 102 - 106 %; 350 mg/mL = 98 - 106 %
- Stability of the formulations (10 - 350 mg/mL): Stable at room temperature for 24 hours; stable in a refrigerator for 4 days.
- Homogeneity of the formulations: Homogeneous, RSD % ≤5 %
- Calibration: Analytical occasion 1, Correlation coefficient 0.9989; Analytical occasion 2, Correlation coefficient 0.9972. The calibration graph was calculated applying weighted linear regression with 1/concentration as the weighting factor.

RESULTS
- Specificity: No peak was detected in the control samples at the retention time of the test material.
- Homogeneity: The highest RSD % of the measured test material concentrations (based on 6 samples/formulation) was 3 %. As this is lower than the limit of acceptance (10 %), the formulations were considered to be homogenous.
- Measured concentration: The measured test material concentrations were within 96 – 100 % of the nominal concentrations.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Start of cohabitation: 2 weeks after initiation of treatment.
- Length of cohabitation: Up to 14 days. Females remained with the same male until copulation occurred. Successful coitus generally occurred within 7 days of pairing.
- Proof of pregnancy: Presence of a vaginal plug or sperm in vaginal smear was considered as evidence of copulation, referred to as day 0 of pregnancy.
On gestation Day (GD) 13 or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
- After successful mating each pregnant female was caged (how): Individually.

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating).
Females were dosed for 14 days pre-mating, for up to 14 days for the mating period, through gestation and 4 days post-partum (up to and including the day before necropsy).

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 62.5, 250 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were set based on available data and information from previous experimental work performed in a separate laboratory, including the results of a 28-day study where the NOAEL was determined to be 1000 mg/kg bw. A Control group was included and dosed with the vehicle only.
- Exposure route selection rationale: The oral route was selected as it is a possible route of exposure to the test material in humans.
- Rationale for animal assignment (if not random): All parental (P) animals were sorted according to body weight by computer and divided to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomisation to ensure that animals of all test groups were as close as practicable to a uniform weight. The grouping was controlled by SPSS/PC+4.0 software according to the actual body weight, thereby verifying the homogeneity/variability between/within the groups and cages. Males and females were randomised separately.

Examinations

Maternal examinations:

MORTALITY AND MORBIDITY
- Time schedule: Twice daily.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once a day, after treatment.
- Observations: Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, were monitored.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first exposure (to allow for within-subject comparisons), and at least once a week thereafter.
- Location: Observations were made outside of the home cage in a standard arena, at similar times
- Observations: Signs evaluated included monitoring for any changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture, and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition, or bizarre behaviour (e.g. self-mutilation, walking backwards). Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Adults were weighed on Day 0 and at least weekly thereafter until termination. Parent females were weighed on gestation Days (GD) 0, 7, 14 and 20 and on post-partal Days (PPD) 0 (within 24 hours after parturition) and 4 (before termination). The females were additionally weighed on GD 4, 10 and 17 in order to give accurate treatment volumes.

FOOD CONSUMPTION: Yes
Food consumption was determined by re-weighing the non-consumed diet on Day 7 and then at least weekly thereafter.

MATERNAL OBSERVATIONS
- Observations and time scale: Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. All observations were recorded and the animals monitored for any evidence of abnormal deliveries.
The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed for nesting behaviour (whether they made a nest from the bedding material, and covered their new-borns, or not). All observations were recorded.

SACRIFICE
Animals were euthanised under pentobarbital anaesthesia, followed by exsanguination.
- Maternal animals: All surviving animals were euthanised on post-partum Day 4 or shortly thereafter.

GROSS NECROPSY
- Gross necropsy was performed on all animals and consisted of an examination of the external appearance, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
- Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
- Dead animals were submitted for necropsy as soon as possible after death.

ORGAN/BODY WEIGHTS
- Bodyweight: At the time of termination the body weight of each animal was determined.
- The weights of the following organs were recorded: uterus (with and without cervix), vagina, brain, ovaries and the pituitary.
Paired organs were weighed individually; absolute organ weights were measured and reported. Relative organ weights (to body and brain weight) were calculated and reported.
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved.

HISTOPATHOLOGY
- Detailed histological examination was performed on selected retained organs in the Control and High dose groups and any macroscopic findings (abnormalities) observed.
- A detailed histological examination of the ovaries was carried out and covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

PARAMETERS EXAMINED
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Live pups were counted, sexed, weighed individually within 24 hours of parturition (on the first day after parturition was complete, i.e. Day 0 or 1 post-partum) and on Day 4 post-partum. The efficiency of suckling was observed by the presence of milk in the stomach.
Observations were reported individually for each offspring. All the litters were checked and recorded daily for the number of viable and dead pups. All observed abnormalities were recorded and reported.

SACRIFICE
- All surviving offspring were euthanised on post-natal Day 4 or shortly thereafter. The pentobarbital-based product used to terminate adult animals was diluted for pups’ euthanasia as required.

GROSS NECROPSY
- Gross necropsy consisted of at least an external examination for gross abnormalities.
- The dead pups found were subjected to necropsy with macroscopic examination. Some of the pups that were found dead were cannibalised, thus, they were counted and sex determined (when it was possible), but were not further examined macroscopically.

Statistics:

Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.
The statistical evaluation of appropriate data was performed with the statistical program package SPSS/PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Upon getting a significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. The Chi² test was performed if feasible.

Indices:

Reproductive Indices:
- Male mating index: (Number of males with confirmed mating / Total number of males cohabited) x 100
- Female mating index: (Number of sperm-positive females / Total number of females cohabited) x 100
- Male fertility index: (Number of males impregnating a female / Total number of males cohabited) x 100
- Female fertility index: (Number of pregnant females / Number of sperm-positive females) x 100
- Gestation index: (Number of females with live-born pups / Number of pregnant females) x 100

- Pre-implantation mortality: [(Number of corpora lutea - Number of implantations) / Number of corpora lutea] x 100
- Intrauterine mortality: [(Number of implantations - Number of live-born) / Number of implantations] x 100

Offspring Viability Indices:
- Survival index: (Number of live pups at designated time / Number of pups born) x 100
- Total mortality: [(Number of implantations - Number of viable pups on Day 4) / Number of implantations] x 100
- Sex ratio: [(Number of pups examined - Number of males) / Number of pups examined] x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There was no unscheduled mortality during the study.
No treatment related adverse effects or systemic clinical signs were noted following daily administration of the test material by oral gavage.
Thin fur of both forelimbs was observed in 1 Control and 4 Low dose females. Scar on right forelimb in 1 Control female and on the neck on the dorsal area in 1 Low dose female was also noted during the observation period. These observations were considered to be incidental.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No treatment related effects were noted on the mean body weight and body weight gain values following daily administration of the test material at dose levels up to and including 1000 mg/kg bw/day.
There were no treatment related differences in the mean daily food consumption in any treated group (62.5, 250 or 1000 mg/kg bw/day) when compared to the Control.
Minor differences to Control were noted, or variations within the group, and were generally associated with mating or other scheduled activities, and were unrelated to treatment.

OESTROUS CYCLE, REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no significant differences between the Control and treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation with test doses.
In all groups, the mating indices were 100 %. The fertility indices were 100, 92, 92 and 100 % for the Control, Low, Mid and High dose groups, respectively. The incidence of non-pregnant females, in the Low and Mid dose groups were within the historical control range. Gestation indices were 100 % at the Low and High dose, and 82 % in the Mid dose. See Table 1.
Test material administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within 7 days of pairing (cohabitation).
There were no effects on the duration of gestation, or abnormalities in gestation outcome, that could be ascribed to treatment.
The mean duration of gestation (22 to 23 days) was similar between the Control and treated groups. All parturitions were normal. See Table 2.
The number of corpora lutea and implantation sites in the treated groups were comparable with the values recorded for the Control group.
There were no toxicologically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 1000 mg/kg bw/day. See Table 3.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no toxicologically significant effects on organ weights.
Compared to Controls, slightly higher absolute weight of the brain was recorded for females at 1000 mg/kg bw/day (High dose). The difference was approximately 7 % and attained statistical significance (p<0.01). There were no differences in the relative values. The changes were not associated with any findings in clinical pathology or microscopic changes and were regarded as normal physiological variation.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment related findings were observed at necropsy.
An enlarged spleen was observed in 1 Control female and was regarded as incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment related microscopic findings were observed in the experimental animals.
The follicular, luteal and interstitial compartments of the ovary, as well as the epithelial capsule and stroma, were similar in histological structure between both Control and High dose females. The primordial, secondary and tertiary follicles and corpora lutea were also bilaterally present.
Extramedullary haematopoiesis was detected in the spleen of a Control female. This observation was considered to be incidental or background.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
No mortality or adverse effects considered to be treatment related were observed in the offspring from dose groups up to 1000 mg/kg bw/day.
The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were comparable to control values at up to and including 1000 mg/kg bw/day.

CLINICAL SIGNS (OFFSPRING)
No treatment related adverse effects were observed in any of the offspring from dose groups up to 1000 mg/kg bw/day.
Any pups found cannibalised were counted and the sex determined where possible, but were not examined macroscopically. A few surviving pups were cold, not suckled, grey or haemorrhagic. No external abnormalities ascribable to treatment were detected at the clinical or external macroscopic examinations of the pups. The incidence of any findings was low, and within the physiological range expected in Wistar rats, hence they were considered incidental, and unrelated to treatment. See Table 3.
The sex ratios were similar in the Control and treated groups, with no statistically significant differences observed. See Table 4.

BODY WEIGHT (OFFSPRING)
There were no adverse effects on the weight or weight gain of offspring following administration of the test material at 62.5, 250 or 1000 mg/kg bw/day to the parental generation.
When evaluated on a per-litter basis, the mean litter body weights (PND 0 and 4) and/or weight gains of pups (PND 4) showed no statistically or toxicologically significant differences compared to Controls.
When evaluated on a whole group mean basis, statistically significant increases were noted in the mean body weights for all groups at PND 0 and the Low and High dose groups at PND 4. Mean body weight gain values for all treated groups were also statistically significantly greater than that of the Control group. All values were in the normal range however, and no differences were considered to be related to treatment with the test material. See Table 5.

GROSS PATHOLOGY (OFFSPRING)
No macroscopic changes were seen in the F1 offspring generation examined at scheduled termination. Of the pups found dead, the floating test was positive in 1/4 Low dose, 1/2 Mid dose and 1/3 High Dose pups. There were no treatment related effects.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Summary of Reproductive Performance

	Dose (mg/kg bw/day)
	Control	62.5 (Low)	250 (Mid)	1000 (High)
Paired males - females	12/12	12/12	12/12	12/12
Mated males - females	12/12	12/12	12/12	12/12
Non-pregnant females	0/12	1/12	1/12	0/12
Pregnant females, but not delivered	0/12	0/11	2/11	0/12
Female Mating Index (%)	100	100	100	100
Fertility Index (%)	100	92	92	100
Gestation Index (%)	100	100	82	100

Table 2: Summary of Gestation, Parturition and Post-Partal Period

Females	Dose (mg/kg bw/day)
	Control	62.5 (Low)	250 (Mid)	1000 (High)
Number of delivered dams / dams with implantations	12/12	10/10#	9/9##	12/12
Duration of pregnancy (days, mean)	22.25	22.40	22.44	22.67
Number of corpora lutea / dams (mean)	17.83	18.20	17.56	16.58
Number of implantations / dams (mean)	17.25	16.70	16.33	16.00
Number of dams with live pups day 0 / number of dams delivered	12/12	10/10	9/9	12/12
Number of dams with live pups day 4 / number of dams delivered	12/12	10/10	9/9	12/12
Pre-implantation mortality (%)	3.28	7.96	8.56	3.54
Intrauterine mortality (%)	4.92	7.53	3.11	6.47
Post-natal mortality (%)	3.11	0.53	0.56	0.46
Total mortality (%)	7.87	8.03	3.67	6.91

#: Two dams excluded (No. 2501: outlier - only one live pup delivered; No. 2511: non-pregnant)

##: Three dams excluded (No. 3507: non pregnant; No. 3501 and 3511: Total intrauterine mortality)

Table 3: Summary of Offspring Data

	Dose (mg/kg bw/day)
	Control (12 dams)	62.5# (Low) (10 dams)	250## (Mid) (9 dams)	1000 (High) (12 dams)
Number of pups born (total)	197	159	143	182
Number of pups born (mean)	16.42	15.90	15.89	15.17
Number of dead pups (total) PND0	0	4	1	3
Number of live births (total)	197	155	142	180
Number of live births (mean)	16.42	15.50	15.78	15.00
Number of viable pups (total) PND0	197	155	142	179
Cannibalised pups PND0 - 4	4	1	0	0
Dead pups (found dead, intact) PND0 - 4	3	4	2	3
Number of viable pups (total) PND4	190	154	141	179
Number of viable pups (mean) PND4	15.83	15.40	15.67	14.92
Viability indices (%)	96.4	99.4	99.3	100.0
Not suckled	1	0	0	0
Survival index PND0	100.00	97.36	99.31	98.56
Survival index PND4	96.89	96.86	98.75	98.56

#: Two dams excluded (No. 2501: outlier - only one live pup delivered; No. 2511: non-pregnant)

##: Three dams excluded (No. 3507: non pregnant; No. 3501 and 3511: Total intrauterine mortality)

Table 4: Offspring Sex Ratio

	Dose (mg/kg bw/day)
	Control (12 dams)	62.5# (Low) (10 dams)	250## (Mid) (9 dams)	1000 (High) (12 dams)
Sex ratio PND0	41.38	42.80	49.36	49.62
Sex ratio PND4	42.24	42.42	48.98	49.62

#: Two dams excluded (No. 2501: outlier - only one live pup delivered; No. 2511: non-pregnant)

##: Three dams excluded (No. 3507: non pregnant; No. 3501 and 3511: Total intrauterine mortality)

Table 5: Summary of Offspring Body Weight

	Dose (mg/kg bw/day)				Remarks
	Control	62.5 (Low)	250 (Mid)	1000 (High)
Litter mean:
Mean body weight (g), PND0	12 litters 6.45	10 litters 6.61	9 litters 6.82	12 litters 6.90	NS
Mean body weight (g), PND4	12 litters 10.41	10 litters 11.30	9 litters 11.25	12 litters 11.68	NS
Body weight gain (g), PND0 - 4	3.96	4.67	4.44	4.77	NS
Mean Litter Weight PND0	104.9	104.2	105.5	104.0	NS
Mean Litter Weight PND4	162.4	172.3	169.4	172.0
Mean for all pups:
Mean body weight (g), PND0	197 pups 6.39	158 pups 6.59**	143 pups 6.64**	182 pups 6.86**	U
Mean body weight (g), PND4	190 pups 10.26	154 pups 11.19**	142 pups 10.74	179 pups 11.53**	U
Body weight gain (g), PND0 - 4	3.86	4.58**	4.10*	4.67**	DN

* = p<0.05, ** = p<0.01

NS = not significant; DN = Duncan's Multiple Range Test; U = Mann-Whitney U-test versus Control

Applicant's summary and conclusion

Conclusions:

Under the conditions of the test there were no adverse effects ascribed to test material administration up to dose levels of 1000 mg/kg bw/day on F1 offspring development or maternal toxicity. Therefore the NOAEL for both developmental toxicity and maternal toxicity was determined to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

The potential reproductive toxicity of the test material was determined using Wistar strain rats in a screening study performed under GLP conditions and in accordance with the standardised guideline OECD 421.

Groups of 12 rats per sex per dose were exposed to the test material at concentrations of 62.5, 250 and 1000 mg/kg bw/day formulated in polyethylene glycol and delivered via oral gavage. Control animals were treated with the vehicle only. Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating). Females were dosed for 14 days pre-mating, for up to 14 days for the mating period, through gestation and up to and including the day before necropsy, 4 days post-partum.

The following parameters were evaluated in adults throughout the dosing period: mortality, clinical signs, body weight and food consumption. Special attention was paid to evaluation of the mating, pregnancy, parturition and post-partum periods, and relevant parameters and/or indices were measured and/or calculated. Following sacrifice, all adults were submitted for the following post-mortem examinations: gross necropsy, organ weights, gross abnormalities and detailed histological examination. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

After delivery, all pups from each litter were counted, sex was established, body weights were recorded on post-natal days (PND) 0 and 4. Offspring were evaluated for presence of stillbirths, live births, runts (pups that are significantly smaller than normal pups) or any gross abnormalities, then examined clinically at least daily. Pups were sacrificed on PND4 or shortly thereafter.

Under the conditions of the study, treatment up to 1000 mg/kg bw/day did not result in test material related mortality, clinical adverse effects, or changes in body weight or food consumption. No treatment related or adverse effects were noted at evaluation of the reproductive parameters, either during the mating, gestation, delivery or post-partum/lactation period up to post-partum Day 4. Furthermore there were no treatment related changes in organ weights, gross findings or histopathology of the adult animals. There were no adverse effects ascribed to test material administration on the F1 offspring viability, clinical signs, development or at observation following euthanasia.

Therefore the NOAEL for both developmental toxicity and maternal toxicity was determined to be 1000 mg/kg bw/day, the highest dose level tested.