Anthranilamide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2016 to March 2018 (report issue)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Deviations were considered not to have affected the purpose or validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Anthranilamide

EC Number:

201-851-2

EC Name:

Anthranilamide

Cas Number:

88-68-6

Molecular formula:

C7H8N2O

IUPAC Name:

2-aminobenzamide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Han™:RccHan™:WIST strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for nineteen days during which time their health status was assessed. Following the day of arrival, vaginal smears were performed for all females throughout the acclimatisition period and females considered not showing appropriate estrous cycling activity were excluded from treatment groups at least five days before the start of treatment. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 333 to 396 g and were approximately eleven weeks old. The females weighed 196 to 234 g and were approximately fourteen weeks old.

Animal Care and Husbandry

Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during late gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within a Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Deviations from these targets were considered not to have affected the purpose or integrity of the study.

The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Polyethylene glycol 400. The stability and homogeneity of the test item formulations were determined by analytical investigation. Results showed the formulations to be stable for at least twenty-one days. Formulations were therefore prepared weekly (during the first week as 21 day stability was yet to be confirmed) followed by the remaining formulations being made fortnightly and stored at approximately 4 ºC in the dark.

Samples of test item formulations were taken and analysed for concentration of the test material. The results indicate that the prepared formulations were within ± 8% of the nominal concentration and thus the required content limit of ± 10% with reference to nominal concentrations was met.

Duration of treatment / exposure:

Approximately six weeks for males and up to eight weeks for females including a two week pre-pairing phase, pairing, gestation and early lactation for females.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low Dose

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Intermediate Dose

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

High Dose

No. of animals per sex per dose:

12 male and 12 female per dose

Control animals:

yes, concurrent no treatment

Positive control:

None

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes

Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1, 4 and 7 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1, 4, 7 and 14 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.


WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily throughout the two week pre-pairing treatment period and in the morning of the day of necropsy (deviation from study plan occurred - smear from one female not conducted on day of necropsy). The stage of the estrous cycle was recorded for each day.

Sperm parameters (parental animals):

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination 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. Any cell-or stage-specificity of testicular findings was noted.

Litter observations:

On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1, 4, 7 and 13 post partum
iii. Sex of offspring on Days 1, 4, 7 and 13 post partum
iv. Clinical condition of offspring from birth to Day 13 post partum
v. Individual offspring weights on Days 1, 4, 7 and 13 post partum (litter weights were calculated retrospectively from this data)

Postmortem examinations (parental animals):

GROSS PATHOLOGY
For the repeated dose toxicity assessment part of the study all adult animals including those dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. All offspring were subjected to a full external examination. Surviving adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 44 or 45. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 14 post-partum.


HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in an appropriate preservative.

Adrenals
Aorta (thoracic)
Muscle (skeletal)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland
Colon Salivary glands (submaxillary)
Cowpers glands
Duodenum
Epididymides
Esophagus
Eyes
Glans penis
Gross lesions
Heart
Ileum (including peyer’s patches)
Jejunum
Kidneys
LABC (levator ani-bulbocavernous) muscle
Liver
Lungs (with bronchi)
Lymph nodes (mandibular and mesenteric)
Mammary gland
Ovaries
Pancreas
Prostate
Pituitary
Rectum
Seminal vesicles
Sciatic nerve
Skin
Spleen
Spinal cord (cervical, mid thoracic and lumbar)
Stomach
Testes
Thyroid/Parathyroid
Trachea
Thymus
Urinary bladder
Uterus & Cervix
Vagina

Postmortem examinations (offspring):

Any offspring showing any abnormalities were examined internally.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05.

Data were analysed using the decision tree from the ProvantisTM data capture system tables and statistics module or assessed separately using the R Environment for Statistical Computing.

Offspring viability indices:

The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 13 of age).

i) Implantation Losses (%)

Group mean percentile post-implantation loss was calculated for each female/litter as follows:

Post–implantation loss (%) = [((Number of implantation sites) - (Total number of offspring born)) / Number of implantation sites] x 100


ii) Live Birth and Viability Indices

The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 1 /Number of offspring born) x 100


Viability Index 1 (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100

Viability Index 2 (%) = (Number of offspring alive on Day 13 / Number of offspring alive on Day 4) x100


Viability index 2 takes into consideration the offspring used for blood sampling on Day 4 post partum.


iii) Sex Ratio (% males)

Sex ratio was calculated for each litter value on Days 1, 4, 7 and 13 post partum, using the following formula:

(Number of male offspring / Total number of offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs observed were not considered toxicologically significant and were either incidental in nature or effects often associated with administration of test compounds to rats.

In general, animals of either sex treated with 500 mg/kg bw/day showed incidences of increased salivation from Day 13 until the end of the treatment period (males) or Day 36 (females). Increased salivation was also noted in animals of either sex treated with 150 mg/kg bw/day on Day 19. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and are generally considered to be of no toxicological importance.

Noisy respiration was noted in three, two and one animal from groups of male animals treated with 500, 150 or 30 mg/kg bw/day respectively on one occasion per animal only. However,these clinical signs were considered to reflect occasional difficulties with dosing these animals rather than any underlying toxicological effect of the test item.

Ataxia was apparent for one day only in one male animal treated with 500 mg/kg bw/day. As no such effects were noted in any other treated animal this was considered to be an isolated incident and not specifically related to systemic toxicity of the test item. No such effects were evident in female animals treated with 30 mg/kg bw/day.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male treated with 150 mg/kg bw/day was found dead on Day 43, no clinical signs were noted in this animal prior to being found dead. At necropsy the lungs and liver were found to be dark, histopathological examinations could not account for the reason for the early death of this animal.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were considered to be no significant effects in body weight development for treated animals of either sex.

Male animals treated with 150 or 500 mg/kg bw/day showed slight reductions in body weight gain during the first two weeks of the treatment period, which achieved statistical significance (p<0.01) in the higher dose group during the first week of treatment. Recovery was evident thereafter as body weight gains were generally similar to controls throughout the remainder of the treatment period. Male animals treated with 500 mg/kg bw/day exhibited statistically significant (p<0.01) higher body weight gains than controls during the final week of treatment. An increase in body weight gain is considered not to represent an adverse effect of treatment, therefore, this intergroup difference is considered to be of no toxicological significance. As overall body weight gains for each of these dose groups were similar to or higher than the control group these effects are considered not to be adverse in nature.

No adverse effects were evident in treated females during maturation or gestation. Female animals treated with 500 mg/kg bw/day actually exhibited a statistically significant (p<0.05) increase in body weight gain when compared to control during the second week of maturation and all treated females exhibited higher body weight gains during the first week of gestation, but without achieving statistical significance. An increase in body weight gain is considered not to represent an adverse effect of treatment, therefore, these intergroup differences are considered to be of no toxicological significance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant effects in food consumption for treated animals of either sex considered to result from treatment.

Male animals treated with 500 mg/kg bw/day showed a reduction in food consumption during the first week of the treatment period, recovery was evident thereafter. No such effects were evident in males treated with 30 or 150 mg/kg bw/day.

Female animals treated with 500 mg/kg bw/day showed a reduction in food consumption during the first week of maturation, with recovery noted thereafter. No such effects were noted in any treated group during gestation.

During lactation, females treated with 500 mg/kg bw/day exhibited a reduction in food consumption during the second half of Week 1. All treated female animals exhibited reductions in food consumption during the final week of lactation. This reduction (during lactation) was only slight for animals treated with 30 or 150 mg/kg bw/day but was more pronounced in the females treated with 500 mg/kg bw/day as statistical significance was achieved (p<0.05) which may be a reflection of the lower litter sizes that needed to be maintained by this treatment group.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the heamatological parameters examined.

Males treated with 150 or 500 mg/kg bw/day showed a statistically significant reduction (p<0.05 - 0.01 respectively) in erythrocytes. Males treated with 500 mg/kg bw/day showed statistically significant (p<0.05 - p<0.01) increases in mean corpuscular volume, mean corpuscular heamoglobin and reticulocytes. Females from all treatment groups also exhibited a statistically significant increase (p<0.05 - p<0.01) in reticulocytes which appeared to be dose related. Although the effect on erythrocytes may be associated with the effects detected microscopically in the spleen, these changes were considered to be non-adverse and representative of minor perturbations in haematopoiesis. The majority of the individual values for all effected parameters were within background control ranges and therefore, in the absence of any associated significant/adverse histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.

Males treated with 500 mg/kg bw/day showed statistically significant increases (p<0.05 - p<0.01) in A/G ratio and bilirubin. A statistically significant reduction (p<0.05) in cholesterol was also apparent for these animals. A statistically significant increase (p<0.05 - 0.01) in alanine aminotransferase was noted for male animals treated with 150 or 500 mg/kg bw/day in a dose related manner. Females treated with 150 or 500 mg/kg bw/day also showed statistically significant increases (p<0.05) in bilirubin and females treated with 500 mg/kg bw/day also showed a statistically significant increase (p<0.01) in bile acids. 500 mg/kg bw/day treated females also exhibited a statistically significant reduction (p<0.05) in chloride concentration.

With the exception of three female animals treated with 500 mg/kg bw/day which exhibited chloride concentration values that were lower than the background control data ranges, the majority of individual values were within background control ranges and as such these intergroup differences were considered not to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional Performance Tests
There were considered to be no treatment related changes in functional performance considered to be related to treatment at 30, 150 or 500 mg/kg bw/day. All treated male animals exhibited a statistically significant (p<0.05-0.01) higher hind limb grip strength, in contrast, all treated female animals exhibited a statistically significant (p<0.05) higher fore limb grip strength. However, as these increases were only noted in one out of the three tests completed and there were no clinical signs observed to signify a neurotoxic effect of the test item, these findings were considered to be incidental and of no toxicological relevance.

There were no treatment-related changes in the behavioural parameters at 30, 150 or 500 mg/kg bw/day.

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 30, 150 or 500 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The following treatment-related microscopic abnormalities were detected:

Spleen
There was a minor increase in haematopoiesis in the spleen of males treated with 150 and 500 mg/kg bw/day, 3/5 with mild haematopoiesis compared to 0/5 in controls. Mild haematopoiesis was present in one male treated with 30 mg/kg bw/day. As the finding in the spleen occurred at a low grade without a corresponding organ weight increase and was only noted in male animals, this is considered likely to be a non-adverse change.

Thyroid Glands
Follicular hypertrophy was present in 3/5 males and one female treated with 500 mg/kg bw/day at a minimal severity. It was also present in 1/5 males treated with 30 mg/kg bw/day. These changes are generally considered to be an adaptive response and often occur in conjunction with liver enzyme induction in response to the administration of chemicals (Capen et al, 2002). The occurrence in one male in the low dose group and one female in the high dose group is considered incidental, although there was an increase in thyroid weight noted in the high dose group females.

Kidneys
An increase in hyaline droplets was present in 2/5, 3/5 and all 5/5 males treated with treated with 30, 150 or 500 mg/kg bw/day respectively. This is consistent with the accumulation of α-2u-globulin, a common finding in animals treated with various test items as well as untreated male rats (Khan KNM et al., 2002). Hyaline droplet accumulation is specific to the male rat and in this case as there was no accompanying adverse pathological changes it is considered to be without toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

General (statistically significant) reductions (p<0.05- p<0.001) in the following parameters were observed: litter size at birth/Day 1, offspring viability (Day 4), body weight, body weight gain and litter weights. These offspring also exhibited higher incidences of being small, weak, no milk in stomach and cold when compared to control. Instances of pups missing or found dead were also higher than control.

Mating performance and fertilty were unaffected by treatment.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

reproductive performance

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Statistically significant reductions (p<0.05- p<0.001) in offspring viability on day 4 after birth.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reductions (p<0.05-p<0.001) in the following parameters were observed: body weight, body weight gain and litter weights.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Pups exhibited higher incidences of being small, weak, no milk in stomach and cold when compared to control. Instances of pups missing or found dead were also higher than control.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

other: These offspring also exhibited higher incidences of being small, weak, no milk in stomach and cold when compared to control. Instances of pups missing or found dead were also higher than control.

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

no

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

Due to effects noted on offspring body weight, body weight gains and general development, the ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive/developmental toxicity was considered to be 150 mg/kg bw/day.

Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development), to evaluate some endocrine disruptor relevant endpoints and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Method

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for approximately six weeks (males) and up to eight weeks (females) (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 150 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Polyethylene glycol 400).

Results

Mortality: There were no unscheduled deaths during the study that were considered to be related to treatment with the test item. One male treated with 150 mg/kg bw/day was found dead on Day 43, no clinical signs were noted in this animal prior to being found dead. At necropsy the lungs and liver were found to be dark.

Clinical Observations: Clinical signs observed were not considered toxicologically significant and were either incidental in nature or effects often associated with administration of test compounds to rats.  

Reproductive Performance

Estrus Cycle: There was no effect of treatment with the test item at any dose level on the nature of estrus cycle with most females showing regular cycles over the pre-pairing phase of the study. There were also no intergroup differences in the stage of estrus on the day of necropsy.

Mating: No treatment-related effects were detected in mating performance.

Fertility: No treatment-related effects were detected in fertility.

Gestation Lengths: Gestation lengths were between 22 and 24½ days, the distribution of gestation lengths for treated females was essentially similar to control.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was considered to be no effect of treatment on the mean numbers of implantations present in any treated female.  However, post-implantation losses were higher than control across all treated groups.

Litter size throughout lactation and offspring survival at Day 1 of age at 500 mg/kg bw/day were lower than control. Two females treated with 500 mg/kg bw/day also had a total litter loss by Day 2 post-partum. Sex ratio for the offspring was similar in all groups and did not indicate any selective effect of maternal treatment on survival for either sex.

There was no detrimental effect of treatment with the test item on litter size or offspring survival to Day 13 of age at 30 or 150 mg/kg bw/day.

Offspring Growth and Development

For females treated with 500 mg/kg bw/day, offspring body weight, body weight gain and litter weights were lower than control from Days 1 to 13 post-partum. There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights up to Day 13 of age at 30 or 150 mg/kg bw/day.

There was no detrimental effect of treatment with the test item indicated by offspring anogenital distance on Day 1 post-partum or visible nipple count in male offspring on Day 13 post-partum in any treated group. The clinical signs noted in litters from females treated with 500 mg/kg bw/day showed a higher incidence of small, weak and cold offspring when compared to control. Instances of pups missing or found dead were also higher than control. Necropsy findings also showed a slightly higher instance of small pups when compared to control.

The clinical signs and necropsy findings apparent for offspring from females treated with 30 or 150 mg/kg bw/day on the study were typical for the age observed.

Conclusion

Due to effects noted on offspring body weight, body weight gains and general development, the ‘No Observed Adverse Effect Level’ (NOAEL) for reproductive/developmental toxicity was considered to be 150 mg/kg bw/day.