tert-pentyl 2-ethylperoxyhexanoate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tert-pentyl 2-ethylperoxyhexanoate

EC Number:

211-687-3

EC Name:

tert-pentyl 2-ethylperoxyhexanoate

Cas Number:

686-31-7

Molecular formula:

C13H26O3

IUPAC Name:

2-methylbutan-2-yl 2-ethylhexaneperoxoate

Test material form:

other: liquid

Details on test material:

Name of the test item: LUPEROX® 575
Synonyms : LUPEROX 575 PE-KAN 25K;tert-amylperoxy 2-ethylhexanoate
IUPAC name : 2-methylbutan-2-yl 2-ethylhexaneperoxoate
CAS Number : 686-31-7
Batch number : 11615-616E11-7-13
Peroxide content : 97%
Date of expiry : 01 June 2015
Appearance : colourless or slightly yellow liquid
Storage conditions at RTC : +4°C

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 90 Sprague Dawley [Crl:CD(SD)BR] rats (45 males and 45 virgin females), 7 to 8 weeks old and weighing 220 to 235 g for males and 189 to 195 g for females, were received from Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian. An acclimatisation period of 20 days was allowed before the start of treatment, during which time the health status of the animals were assessed by thorough observations.
he animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22 °C+/-2°C
and 55 % +/- 15 % respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day. From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.

During mating animals were housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Techniplast – Gazzada S.a.r.l.). Each cage tray held absorbent material which was inspected and changed daily. After mating the males were re-caged as they were before mating. The females were transferred to individual polysulfone solid bottomed cages measuring 42.5x26.6x18.5 cm (Techniplast Gazzada S.a.r.l.) for the gestation period, birth and lactation. Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week.

Drinking water was supplied ad libitum to each cage via water bottles. A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum throughout the study.

The animals arrived on 08 January 2015 and were allocated to groups on 21 January 2015. Dosing commenced on 28 January 2015 and the last necropsy was performed on 20 MArch 2015.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment, analyses were performed to confirm that the proposed formulation procedure was acceptable. Samples of the formulations prepared during the study were analysed to check the concentration and homogeneity (the first and the last week of treatment). Chemical analyses were carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study number 88800) in the range from 1 to 200 mg/mL in corn oil. In addition, the stability of formulated samples at 1 and 200 mg/mL was verified after 24 hours and 8 days at +4°C in the same study. The software used for this activity was Empower®2 Build No. 2154.

Duration of treatment / exposure:

Males for 2 consecutive weeks prior to pairing and thereafter through the day before necropsy (Days 29 and 30 of study). Females for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 5 post partum.

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 250 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday a similar procedure was followed except that the final check was carried out at approximately mid-day. This allowed post mortem examinations to be carried out during the working period of that day. A complete necropsy was performed as detailed in section [sub:Necropsy] below.

Clinical signs
All clinical signs were recorded for individual animals. Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (15-30 minutes and 1 - 1.5 hours after dosing).

Observations of the cage tray
Observations of the cage tray, during the pre-mating (males and females) and after mating periods (only males), were performed and recorded three times weekly. During mating and gestation periods (only females until Day 11 post coitum), these observations were performed and recorded daily.

Body weight
Males were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter and at termination.
Females were weighed on the day of allocation to treatment groups, on the day of treatment commencement, weekly thereafter until positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 6 post partum.

Food consumption
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period following allocation. Individual food consumption for the females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 6 post partum starting from Day 1 post partum.

Parturition check and duration of gestation
A parturition check was performed from Day 20 to Day 25 post coitum. Female nos. X0080059 (Group 3) and X0080071 (Group 4) which did not give birth after 25 days of post coitum period were sacrificed shortly after (Days 26 and 27 post coitum, respectively).
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning starting from two weeks before pairing throughout the mating period until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)

Sperm parameters (parental animals):

Not done

Litter observations:

Pups identification, weight and observation
As soon as possible, after parturition was considered complete (Days 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 6 post partum. Pups killed or dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.

Postmortem examinations (parental animals):

Parental animal were euthanised with carbon dioxide. The males were killed after the mating of all females after 29 and 30 days of treatment. The females with live pups were killed on Day 6 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed after 25 days of the last day of the mating session (Days 26 and 27 post coitum).

Gross observation
The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding animals sacrificed for humane reasons) and the required tissue samples preserved in fixative and processed for histopathological examination.
All females were examined also for the following:
• external and internal abnormalities;
• number of visible implantation sites (for pregnant animals);
• number of corpora lutea (if detectable).
Uteri of apparently non-pregnant females or uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Organ weights
From all parental animals completing the scheduled test period the organs indicated in Annex 1 of the study ptotocol were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal. U

Tissues fixed and preserved
Samples of all the tissues indicated in annex 1 of the study protoocol were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are listed in Annex 1 of Study Protocol . After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides of all males in the control and high dose groups were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.
The examination was restricted as detailed below:
Tissues specified in Annex 1 of Study Protocol from all animals in the control and high dose group killed at term
Tissues specified in Annex 1 of Study Protocol from the animal killed during the treatment period
All abnormalities in all groups
On the basis of the treatment-related changes detected in the thymus of high dose treated females, the histopathological evaluation of the thymus was extended to the remaining low and mid-dose females.

Postmortem examinations (offspring):

Pups were euthanised by intraperitoneal injection of sodium thiopenthal.
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities. All live pups sacrificed at termination (Day 6 post partum) were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The non-parametric Kruskal- Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. Further tests were used as considered appropriate.

Reproductive indices:

Males
Copulatory Index (%) = no. of animals mated / no. of animals paired * 100
Fertility Index (%) = no. of males which induced pregnancy / no. of animals paired * 100

Females
Copulatory Index (%) = no. of animals mated / no. of animals paired * 100
Fertility Index (%) = no. of pregnant females / no. of females paired * 100
Pre-birth loss was calculated as a percentage from the formula: no. of visible implantations - total litter size at birth / no. of visible implantations * 100
Pre-implantation loss was calculated as a percentage from the formula: no. of corpora lutea - no. of visible implantations / no. of corpora lutea * 100

Males and females
Pre coital Interval = Mean number of days between pairing and mating

Offspring viability indices:

Pup loss at birth was calculated as a percentage from the formula: Total litter size - live litter size / Total litter size * 100
Cumulative pup loss on Day 6 post partum was calculated as a percentage from the formula: Total litter size at birth - live litter size at Day 6 / Total litter size at birth * 100
Sex ratios were calculated at birth and on Day 6 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

Mortality and fate of females
One female with litter, receiving 50 mg/kg/day was sacrificed for humane reasons on Day 1 post partum.
The day before the unscheduled death the following clinical signs were noted:
- hunched posture, emaciated aspect, piloerection and teeth missing. On the day of sacrifice, decreased activity, cold to touch, staining on perigenital region and pale aspect were recorded. The most relevant changes observed at post mortem examination were distention with gas content in stomach, duodenum, ileum and jejunum; small size of thymus, spleen and pancreas. Histopathological evaluation revealed a severe atrophy of thymus, lymphoid depletion of spleen, mucosal ulceration of forestomach (non glandular region), villous atrophy of jejunum and ileum and cortical vacuolation and nephropathy of kidneys. The above mentioned pathological changes were considered as factors contributory to the illness status of the animal.
One female (X0080071) receiving 1000 mg/kg/day was found not pregnant at necropsy. One female (X0080059) receiving 250 mg/kg/day showed unilateral implantation. This female with unilateral implantation in the right horn had also total resorption in that horn and was not pregnant in the left one. The number of females with live pups on Day 6 post partum was: 10 in the control, 9 in the low dose (50 mg/kg/day), 9 in the mid- dose group (250 mg/kg/day) and 4 in the high dose group (1000 mg/kg/day).

Clinical signs
Matted fur and salivation were the principal clinical signs observed in treated males receiving 1000 mg/kg/day. Occasionally salivation was also noted in one male receiving 250 mg/kg/day. One control male (animal no. X0080010) showed scabs on body surface (thoracic region, lumbar region and upper hindlimb starting from Day 10 of the mating phase) and was isolated an individual cage.
Salivation was mainly noted in females receiving 1000 mg/kg/day before pairing, during gestation and post partum period. Hunched posture was occasionally observed in this group. \vphantom{} Observations of the cage tray Soft faeces, slight, were observed throughout the study in males and females receiving 1000 mg/kg/day. Occasionally this sign was recorded in males receiving 250 mg/kg/day.

Observations of the cage tray
Soft faeces, slight, were observed throughout the study in males and females receiving 1000 mg/kg/day. Occasionally this sign was recorded in males receiving 250 mg/kg/day.

Body weight
Slight decrease in body weight was noted in treated males receiving 1000 mg/kg/day throughout the study. However this change was always below 10%. Body weight of treated females was, in general, comparable to the control group during the study. Body weight gain of males receiving 1000 mg/kg/day was decreased compared to the control group, both on Day 8 of the study (before pairing) and on Day 15 (mating phase). Changes were of -37% and -79%, respectively.

Food consumption
Food consumption was decreased in females receiving 1000 mg/kg/day before pairing (Day 8 of study) and on Day 6 post partum (-20% and -19%, respectively). Food consumption of treated males were comparable to the control group throughout the study, as well as, for females during the gestation period.

Oestrous cycle, reproductive parameters, pairing combination and mating performance
Oestrus cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups.
Implantation, pre-implantation loss data, pre-birth loss data (or post- implantation loss) and gestation length of females
Gestation periods of control and treated groups were similar and dams gave birth on Day 22 post coitum (mean value). Implantation sites, pre-implantation loss data and total litter size at birth were, in general, comparable between groups. Post- implantation loss was slightly increased in females receiving 1000 mg/kg/day.

Litter data at birth, on Day 1 and on Day 6 post partum and sex ratio of pups
Stillbirths (X0080069) or total litter loss (X0080061, X0080063, X0080077, X0080079) were noted the day of parturition or the day after parturition in females receiving 1000 mg/kg/day. In addition, an increased incidence of pup loss at birth and cumulative loss on Day 6 post partum were noted. Live litter size was reduced at birth, on Days 1 and 6 post partum and consequently litter weight was also decreased. Decreases in the number of male pups and consequently in total number of pups were noted on Day 6 post partum in females receiving 1000 mg/kg/day when compared to controls. Sex ratio on Day 6 post partum was also decreased when calculated as the percentage of males.

Terminal body weight and organ weights
Slight decrease in terminal body weight was observed in high dose animals of both sexes (-6% to -8 %) receiving 1000 mg/kg/day. Some differences, sometimes statistically significantly, were noted in the absolute and/or relative organ weight, such as: - Increased absolute kidneys (+18%) and liver (+21%) weights in males receiving 1000 mg/kg/day - Increased absolute adrenals (+16%), liver (+12%) and uterus weights (+107%) in females receiving 1000 mg/kg/day. Absolute thymus (+23%) weight was increased in females receiving 250 mg/kg/day and decreased in females receiving 1000 mg/kg/day (-18%). - Increase in relative adrenals, kidneys, liver and testes weights in males receiving 1000 mg/kg/day (+24%, +27%, +30%, +9%, respectively). Relative kidneys weight was also increased in males receiving 250 mg/kg/day (+13%). - Increase in relative adrenals, kidneys, liver, uterus weights and decrease relative thymus weight in females receiving 1000 mg/kg/day (+28%, +13%, +21%, +126%, -11%, respectively). No concurrent histological findings were noted in the above mentioned organs with the exception of those observed in the thymus of high dose females.

Macroscopic observations
The most remarkable change noted at post mortem examination was an increased incidence of reduced size of the thymus in high dose females, when compared with the control and low dose females. In addition, enlarged adrenals or kidneys were observed in few treated animals.

Microscopic observations
Treatment-related changes were noted in the thymus of female rats treated at 1000 mg/kg/day. The histopathological change detected in the thymus was atrophy, represented by: a minimal to marked reduction in cortical lymphocytes, shrinkage of the thymic lobules, increased prominence of interlobular septae and an inverse cellular density ratio cortex/medulla. The females treated at 250 and 50 mg/kg/day did not show any remarkable changes in the thymus.

Spermatogenic cycle
A detailed qualitative examination of the testes was performed in all control and high dose group males. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS-H stained sections were used to identify the spermatogenic stages.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. The remaining sporadic lesions, reported in control and treated animals, were considered to be an expression of spontaneous and/or incidental pathology, seen in this species and age of untreated animals.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No treatment-related adverse effects noted

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No treatment-related adverse effects noted

3

Key result

Dose descriptor:

NOAEL

Remarks:

toxicity

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Treatment-related changes in the thymus at 1000 mg/kg/day

4

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

other: Post-implantation loss, stillbirths or total litter loss at 1000 mg/kg/d

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

no effects observed

Details on results (F1)

Clinical signs of pups
Marked mortality of pups or missing pups were noted at 1000 mg/kg/day. Cold to touch, apparently no food intake (milk) and small appearance were noted in the remaining pups of the dams treated at 1000 mg/kg/day and reaching Day 6 post coitum. These clinical signs were also observed in control and treated pups receiving dose levels ≥ 50 mg/kg/day. Found dead and missing pups were observed on control, low and mid-dose groups, with similar incidence.

Necropsy findings in decedent pups and in pups sacrificed on Day 6 post partum.
Autolysed abdominal/thoracic organs were generally observed in control and treated pups which died during the lactation period. No milk in stomach and dark staining (abnormal colour) on the abdominal region were also noted in pups at 1000 mg/kg/day. No necropsy findings were observed in all pups of control and treated groups, sacrificed on Day 6 post partum.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Pups mortality at 1000 mg/kg/d

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for parental toxicity and fertility could be considered to be 1000 mg/kg/day for males and 250 mg/kg/day for females. The NOAEL for the toxicity on development could be considered to be 250 mg/kg/day.

Executive summary:

The possible effects of CAS# 686 -31 -7 on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus, parturition and early lactation of the offspring (Day 6 post partum) was evaluated in an OECD 421 study (Sisti, 2015). The test item was given to Sprague Dawley rats by oral administration (gavage) before and during mating and throughout the gestation period until Day 6 post partum at dosages of 50, 250 and 1000 mg/kg/day. One female with litter, receiving 50 mg/kg/day was sacrificed for humane reasons on Day 1 post partum. Histopathological evaluation revealed a severe atrophy of thymus, lymphoid depletion of spleen, mucosal ulceration of forestomach (non glandular region), villous atrophy of jejunum and ileum and cortical vacuolation and nephropathy of kidneys. These changes were considered as factors contributory to the illness status of the animal. The major clinical signs noted in treated males receiving 1000 mg/kg/day were matted fur and salivation, while only salivation was recorded in females receiving the same dose level. Soft faeces were observed in the cage tray of animals of both sexes receiving 1000 mg/kg/day and sometimes in males receiving 250 mg/kg/day. Body weight of males receiving 1000 mg/kg/day was slightly lower (<10%) than the control group, as well as, body weight gain on Days 8 (before pairing) and 15 (mating phase) of the study. Food consumption was decreased in females receiving 1000 mg/kg/day before pairing (Day 8 of study) and on Day 6 post partum. Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day) and the copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage). The resulting copulatory index and fertility index did not show intergroup differences. Post-implantation loss was slightly increased in females receiving 1000 mg/kg/day when compared to the control group. Stillbirths or total litter loss were noted in 5 females receiving 1000 mg/kg/day the day of parturition or the day after parturition. Increased incidences of pup loss at birth and cumulative loss on Day 6 post partum were also noted. Decreases in the number of males and consequently in the total number of pups were noted on Day 6 post partum in females receiving 1000 mg/kg/day when compared to controls. Sex ratios on Day 6 post partum was also decreased when calculated as the percentage of males. Marked mortality of pups or missing pups were noted at 1000 mg/kg/day. Cold to touch, apparently no food intake (milk) and small appearance were noted in the surviving pups of dams receiving 1000 mg/kg/day, in control pups and in those receiving the dose levels ≥ 50 mg/kg/day. Necropsy findings observed in decedent control and treated pups, were similar. No necropsy findings were observed in all pup of control and treated groups, sacrificed on Day 6 post partum. Slight decrease in terminal body weight was observed in high dose animals of both sexes receiving 1000 mg/kg/day. Some changes in absolute and relative organ weights (adrenals, liver, kidneys, uterus, testes and thymus) were noted in treated animals mainly in those receiving 1000 mg/kg/day. However, the differences were not accompanied by histological findings, with the exception of those observed in thymus of high dose females. At macroscopic observations the most remarkable change was an increased incidence of reduced size of the thymus in high dose females. At microscopic observations treatment-related atrophic changes were noted in the thymus of female treated at 1000 mg/kg/day. On the basis of the results obtained, the NOAEL (No Observed Adverse Effect Level) for parental toxic ity and fertility could be considered to be 1000 mg/kg/day for males and 250 mg/kg/day for females. The NOAEL for the toxicity on development could be considered to be 250 mg/kg/day.