Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The in-life phase of the study was conducted between 14 February 2012 (first day of treatment) and 06 April 2012 (final necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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 relevant results.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Sponsor's identification : S196439
Description : Black powder
Chemical name : 1,3,5-Naphthalenetrisulfonic acid, 7[[2-[[5-cyano-4- methyl-2,6-bis[(4-sulfophenyl)amino]azo]-4-(2- naphthalenyl)-5-thiazolyl]azo]-, lithium sodium salt
Purity : 93.7%
Batch number : 373
Label : S196439/38 freeze dried sample Date 1/7/11
Date received : 01 September 2011
Storage conditions : Room temperature in the dark
Expiry date : 31 December 2013

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for six days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 294 to 342g, the females weighed 197 to 232g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. 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, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. The diet and drinking water were 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 the Harlan Laboratories Ltd., Shardlow, UK, 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 temperatures and relative humidity controls were set to achieve Study Plan target values of 22 ± 3°C and 50 ± 20% respectively. There were no deviations from these target ranges.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
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 dried Arachis oil BP.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at regular intervals.
Details on mating procedure:
On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
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 dried Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services as part of this study. Results showed the formulations to be stable for at least sixteen days. Formulations were therefore prepared twice monthly and stored at approximately +4ºC in the dark.

Samples of each test item formulation were taken and analysed for concentration of S196439 at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.

Analytical method used:
The concentration of S196439 in the test item formulations was determined spectrophotometrically. The test item formulations were extracted with methanol to give a final, theoretical test item concentration of approximately 0.01 mg/ml. Standard solutions of test item were prepared in methanol at a nominal concentration of 0.01 mg/ml.

The standard and sample solutions were analysed spectrophotometrically using the following conditions:
Spectrophotometer : Camspec M550
Wavelength : max at 611 nm
Cell path length : 1 cm
Reference medium : Methanol

The test item formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for sixteen days.

Acheived concentrations of formulations used during the first week of the study were higher than anticipated (113 to 121% of nominal concentration) but were considered acceptable for use on the study. Acheived concentration of formulations used for dosing during the remainder of the study were considered suitably close to nominal concentration.

Conclusion:
The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.
Duration of treatment / exposure:
Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 15, 100 and 400 mg/kg
Basis:
other: as active ingredient
No. of animals per sex per dose:
10 males and 10 females for each dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (SafePharm Laboratories Limited Project Number 2125-0021). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item.

- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomisation procedure based on
stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was measured daily throughout the study (with the exception of the pairing
phase).

Estrous cyclicity (parental animals):
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964).
Litter observations:
All animals were examined for overt signs of toxicity, ill-health and behavioural change.
Postmortem examinations (parental animals):
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 post partum.

The following was examined:
Organ weights: The epididymides and testes were removed from terminal kill adult males dissected free from fat and weighed before fixation.

Histopathology:
Samples of the following tissues were preserved from all animals from each dose group,
in buffered 10% formalin, except where stated:
Coagulating gland, Prostate, Epididymides, Seminal vesicles, Gross Lesions, Testes, Ovaries Uterus/Cervix, Mammary gland (females only), Vagina and Pituitary
Postmortem examinations (offspring):
Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was considered to be achieved at a level of p<0.05. Statistical analysis was performed as follows:

For data collected on the in-life data capture system, data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).

The following parameters were analysed: Adult Male Body Weight Change, Absolute Organ Weights and Body Weight Relative Organ Weights.
Reproductive indices:
Reproductive Indices:
The following parameters were calculated from the individual data during the mating period of the parental generation:
Pre-coital Interval:
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating. For the female whose scheduled male partner had died, the initial pairing date was regarded as when it was paired with a male rather than the scheduled pairing date of the study.

ii) Fertility Indices
For each group the following were calculated:
Mating Index (%) = Number of animals paired / Number of animals mated x 100

Pregnancy Index (%) = Number of animals mated / Number of pregnant females x 100
Offspring viability indices:
i) Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = Number of offspring born alive on Day 1 / Number of offspring born x 100
Viability Index (%) = Number of offspring alive on Day 4/ Number of offspring alive on Day 1 x 100

ii) Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:
Number of male offspring / Total number of offspring x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical signs in surviving animals were mainly confined to increased post dose salivation and fur staining (by the test item formulation) at 400 mg/kg bw/day AI, and, to a lesser extent, 100 mg/kg bw/day AI).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg, body weight gain was lower than control, as was the food consumption, by termination body weight gain was less than 60% of the control. Body weight gain and food consumption was unaffected by treatment for both the 15 + 100 mg/kg groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg, body weight gain was lower than control, as was the food consumption, by termination body weight gain was less than 60% of the control. Body weight gain and food consumption was unaffected by treatment for both the 15 + 100 mg/kg groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were detected for testes and epididymis weights for treated males when compared to controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No toxicologically significant macroscopic abnormalities were detected.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examinations of control and high dose animals did not reveal any treatment-related effects on the tissues examined.
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
No treatment-related effects were detected.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
No treatment-related effects on fertility were detected.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): At 400 mg/kg bw/day AI, one male (No.: 69) showed a notable body weight loss (32g) during the first week of the study. This animal showed decreased respiration throughout Day 9 although this sign was no longer present the following day. However, decreased respiration rate and dehydration were observed at the early morning check (prior to dosing) on Day 14, and due to continuing body weight loss (33g from Day 8) and the clinical condition of the animal, it was killed. Necropsy also revealed discoloration of most of the gastro-intestinal tract, the mesenteric lymph nodes and kidneys that was considered to represent staining of these tissues by the coloured test item. There were no other unscheduled deaths among adult animals on the study.

For animals surviving to scheduled termination, individual clinical signs were mainly confined to increased post dose salivation and fur staining (by the test item formulation) at 400 mg/kg bw/day AI, and, to a lesser extent, 100 mg/kg bw/day AI. No clinical observations were apparent for either sex at 15 mg/kg bw/day AI.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
For males, initial mean body weight gain at 400 mg/kg bw/day AI was adversely influenced by the male mortality (No.: 69) which showed an atypical body weight loss during the first week of treatment. However, even allowing for this animal, treatment at 400 mg/kg bw/day AI was associated with a noticeably lower body weight gain, compared with control, during the first two weeks of treatment. Thereafter body weight gain tended to be lower than control, although not generally to the same extent, and overall body weight gain by termination was less than 60% of the control. There was no obvious adverse effect of treatment on body weight gain of males at 15 or 100 mg/kg bw/day AI. At 400 mg/kg bw/day AI, food consumption was noticeably lower than control for males during the first two weeks and for females during the first week of treatment. Thereafter, there was no clear adverse effect on food intake for either sex, including for females during the gestation and lactation phases of the study, to termination. There was no obvious adverse effect of treatment on food consumption for either sex at 15 or 100 mg/kg bw/day AI.

Females
At 100 and 400 mg/kg bw/day AI, body weight gain was lower than control throughout the two week pre-pairing period, although statistical significance was only attained during the second week. Pre-pairing body weight gain was unaffected by treatment at 15 mg/kg bw/day AI. No obvious adverse effects on body weight gain were detected during gestation or
lactation at 15, 100 and 400 mg/kg bw/day AI.

REPRODUCTIVE PERFORMANCE:
mating: No treatment-related effects were detected in mating performance with the majority of animals showing a positive indication of mating within the first four days of pairing (i.e. mating at the first oestrus opportunity).

At 400 mg/kg bw/day AI, initial offspring survival following birth was lower than control resulting in a lower mean birth index, although no further offspring mortality occurred after Day 2 of age. This inferior initial survival rate indicates the post partum litter losses observed at this dosage were probably associated with maternal treatment. There was no obvious effect on sex ratio at this dosage indicating that there was no selective effect on survival for either sex. It should be noted that the lower survival was restricted to just a few litters and there was no overall effect on mean litter size on Day 1 or on Day 4 at 400 mg/kg bw/day AI.

There was no obvious adverse effect on offspring survival, mean litter size or sex ratio on Day 1 or Day 4 of age at 15 and 100 mg/kg bw/day AI.

ORGAN WEIGHTS (PARENTAL ANIMALS): There was no obvious adverse effect of treatment on testis and epididymis absolute and
body weight relative organ weights at 15, 100 and 400 mg/kg bw/day AI.

GROSS PATHOLOGY (PARENTAL ANIMALS): Neither the type, incidence or distribution of macroscopic necropsy findings indicated any adverse effect of treatment at 15, 100 and 400 mg/kg bw/day AI. At 400 mg/kg bw/day AI, discolouration of several tissues associated with the gastrointestinal tract and lymphatic system, and in females also the kidneys, was observed and was considered to be attributable to the coloured test item. Similar discoloration of the contents in parts of the gastro-intestinal tract were apparent for one female at 100 mg/kg bw/day AI and another at 15 mg/kg bw/day AI. In the absence of any adverse histopathological change, this discoloration was considered to be of no toxicological significance.

HISTOPATHOLOGY (PARENTAL ANIMALS): Microscopic examinations of control and high dose animals did not reveal any treatmentrelated
effects on the tissues examined.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinically observable signs of toxicity were detected in offspring from treated groups.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
initial offspring survival following birth was inferior to control resulting in a lower mean birth index and two females showed total litter losses, which were considered to treatment related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 400 mg/kg bw/day AI, mean offspring body weight was lower than control on Days 1 and 4 of age resulting in a corresponding reduced mean litter weight.
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were detected for testes and epididymis weights for treated males when compared to controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy. No toxicologically significant macroscopic abnormalities were detected.
Histopathological findings:
no effects observed
Description (incidence and severity):
Microscopic examinations of control and high dose animals did not reveal any treatment-related effects on the tissues examined

Details on results (F1)

Offspring Observations. No clinically observable signs of toxicity were detected in offspring from treated groups.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: This is for reproduction and offspring growth and development.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

DISCUSSION:

The oral administration of the test material to rat for a period of up to eight weeks (including two weeks pre-pairing, gestation and early lactation for females) at dosages of 15, 100 and 400 mg/kg bw/day (incorporating a correction factor for 93.7% purity) resulted in toxicologically significant effects at 400 mg/kg bw/day AI.

One male receiving 400 mg/kg bw/day AI was terminated after showing dehydration, decreased respiration and notable body weight loss on Day 14. For surviving males at this dosage, noticeably lower body weight gain, food consumption and inferior food conversion efficiency was apparent for the first two weeks of treatment, compared to control. Weight gains for males generally continued to be lower, but to a lesser extent, throughout the remainder of the study. For females at this dosage, body weight gain was lower than control during the initial two week pre-paring phase but no subsequent effect on body weight performance was apparent during gestation or lactation. Lower food consumption was apparent for females during the first week of treatment and food conversion efficiency was slightly lower than control during the two week pre-pairing phase.

Both sexes at 400 mg/kg bw/day AI showed an increase in water consumption compared to control, differences being particular marked for males. Consistent with this increase in water intake was a higher than expected incidence of damp/wet bedding (probably due to increased urination) in their cages. Examination of the cage bedding also revealed discolouration of the faeces (probably due to the coloured test item formulations) at this dosage. Clinical signs for at this dosage also included similar fur staining by the test item and post-dosing salivation.

There was no obvious adverse effect of treatment on mating performance, fertility, gestation length, mean number of corpora lutea and implantations, pre- and postimplantation loss or number of offspring born at 400 mg/kg bw/day AI. However, initial offspring survival following birth was lower than control resulting in a lower mean birth index and two females showed total litter post partum. The lower survival was restricted to just a few litters and there was no overall effect on mean litter size on Day 1 or on Day 4. Mean offspring body weights were noticeably lower than control on Day 1 and Day 4 resulting in a corresponding lower mean litter weights. Those litters with the lowest offspring body weights on Day 1 showed the highest incidence of offspring losses compared with other litters, and no offspring mortality occurred after Day 2 of age. It is likely that the lower initial survival rate observed for some litters at this dosage was associated with a treatment related effect on embryofoetal growth rather than any underlying effect on embryofoetal development. Sex ratio was unaffected indicating that there was no selective effect on survival for either sex and offspring clinical signs and necropsy findings were typical for the age observed and did not indicate any specific underlying effect of treatment.

Necropsy examination of adult animals at 400 mg/kg bw/day AI revealed discolouration of tissues associated with the gastro-intestinal tract and lymphatic system, and additionally, for females, the kidneys, but in the absence of any adverse histopathological change, this discoloration was considered to be of no toxicological significance. There were no adverse effects of treatment on testicular or epididymal organ weights and microscopic examination of the reproductive organs and those tissues showing test item staining did not reveal any adverse effect of treatment.

At 100 mg/kg bw/day AI, clinical signs of increased post dose salivation and test item fur staining were observed, but to a lesser extent than 400 mg/kg bw/day AI. Body weight gain and food conversion efficiency for females were slightly lower than control for the two week pre-pairing period, but subsequent body weight performance during gestation or lactation was unaffected, and there were no accompanying effects on food consumption or water consumption.

There was no adverse effect of treatment on mating performance, fertility, gestation length, litter data, offspring body weights or offspring survival at 15 and 100 mg/kg bw/day AI. One pregnant female at 15 mg/kg bw/day AI and another at 100 mg/kg bw/day AI failed to litter but were found to have a single dead foetus in the right horn at necropsy. In the absence of any adverse effect on post-implantation loss for the other females at these dosages, or for females at 400 mg/kg bw/day AI, these isolated occurrences were considered incidental and unrelated to treatment.

Test item discoloration similar to that observed at 400 mg/kg bw/day AI was observed for the content of parts of the gastro-intestinal tract of one female at 100 mg/kg bw/day AI and another female at 15 mg/kg bw/day AI, but as previously described, this discoloration was considered to be of no toxicological significance.

Applicant's summary and conclusion

Conclusions:
Overall, treatment at 400 mg/kg bw/day AI was associated with clear adult toxicity in both sexes and, although reproduction in terms of mating and fertility appeared unaffected, there was a clear decrease in early neonatal survival probably due to an adverse effect on embryofoetal growth. While lower body weight gain for females was observed during the initial two week pre-pairing at 100 mg/kg bw/day AI, effects on body weight performance did not persist and, at the level observed, were not considered to exclude this dosage from classification as a No Observed Adverse Effect Level (NOAEL) for maternal toxicity. Within the confines of this study, this dosage of 100 mg/kg bw/day AI is therefore considered to be the NOAEL for adult toxicity and the No Observed Effect Level (NOEL) for reproduction and offspring growth and development.
Executive summary:

Introduction:

The study was performed to screen for potential adverse effects of the test item on reproduction including embryo/foetal development and provides an initial hazard assessment for effect on reproduction. The study was designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 421 “Reproduction/Developmental Toxicity Screening Test” (adopted 27 July 1995). 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).

Methods: The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 15, 100 and 400 mg/kg bw/day (incorporating a correction factor for 93.7% purity). A control group of ten males and ten females was dosed with vehicle alone (dried Arachis oil BP).

Observations:

Clinical signs, body weight change, dietary intake and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Surviving adult males were terminated on Day 43, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not litter was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Results.

Adult Responses:

Mortality. There was one unscheduled adult death at 400 mg/kg bw/day

Clinical Observations. Clinical signs in surviving animals were mainly confined to increased post dose salivation and fur staining.

Body Weight. At 400 mg/kg bw/day AI, body weight gain of males was generally lower than control, with differences being particularly marked during the first two weeks of treatment, and by termination body weight gain was less than 60% of the control. Body weight gain was unaffected by treatment for both sexes at 15 mg/kg bw/day and males at 100 mg/kg bw/day AI.

Food Consumption. At 400 mg/kg bw/day AI, lower food consumption was apparent for both sexes during the first week of treatment and this continued for males into the second week of treatment. Food consumption was unaffected by treatment at 15 or 100 mg/kg bw/day AI.

Water Consumption. At 400 mg/kg bw/day AI, water consumption was increased for both sexes throughout the study when compared with control. Water intake was unaffected by treatment at 100 or 15 mg/kg bw/day AI.

Reproductive Performance:

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

Fertility. No treatment-related effects on fertility were detected.

Gestation Lengths. No treatment-related effects were detected in the length of gestation between control and treated groups.

Litter Responses::

Offspring Litter Size, Sex Ratio and Viability. At 400 mg/kg bw/day AI, there was no adverse effect of treatment on mean number of corpora lutea and implantations, pre- and post-implantation loss or number of offspring born. However, initial offspring survival following birth was inferior to control resulting in a lower mean birth index and two females showed total litter losses, which were considered to treatment related. Lower offspring survival was restricted to a few litters, no further offspring mortality occurred after Day 2 of age and mean litter size on Day 4 was still similar to control. Sex ratio was unaffected indicating that there was no selective effect on survival for either sex.

There was no adverse effect of treatment on the mean number of corpora lutea and implantations, pre- and post-implantation loss, number of offspring born, offspring survival, litter size or sex ratio on Day 1 or Day 4 of age at 15 and 100 mg/kg bw/day AI.

Offspring Observations. No clinically observable signs of toxicity were detected in offspring from treated groups.

Pathology:

Necropsy: No toxicologically significant macroscopic abnormalities were detected.

Organ Weights: No treatment-related changes were detected for testes and epididymis weights for treated males when compared to controls.

Histopathology: Microscopic examinations of control and high dose animals did not reveal any treatment-related effects on the tissues examined.

Conclusion:

Overall, treatment at 400 mg/kg bw/day AI was associated with clear adult toxicity in both sexes and, although reproduction in terms of mating and fertility appeared unaffected, there was a clear decrease in early neonatal survival probably due to an adverse effect on embryofoetal growth. While lower body weight gain for females was observed during the initial two week pre-pairing at 100 mg/kg bw/day AI, effects on body weight performance did not persist and, at the level observed, were not considered to exclude this dosage from classification as a No Observed Adverse Effect Level (NOAEL) for maternal toxicity. Within the confines of this study, this dosage of 100 mg/kg bw/day AI is therefore considered to be the NOAEL for adult toxicity and the No Observed Effect Level (NOEL) for reproduction and offspring growth and development.