Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Phase: 05 January 2018 to 09 October 2018. Report Issue:
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
There were some minor deviations from the study plan. These deviations were considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Males 70 to 77 days, females 84 to 91 days
- Weight at study initiation: Males 339 to 422g, females 246 to 301g
- Fasting period before study: No
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid were used with the exception of 1) Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering and lactation periods 2) Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum (potable water from the public supply)
- Acclimation period: Males: seven days before commencement of treatment. Females: 21 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis were available for the diet and water. All diet and water used on the study was considered to be of acceptable quality and not to have interfered with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.
- Environmental Enrichment: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary. A plastic shelter was provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cage.

IN-LIFE DATES: From: To: 02 May 2018 (animal arrival) to 12 July 2018 (final necropsy)
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
The test item was administered daily by gavage using a graduated syringe and rubber catheter inserted via the mouth once daily at approximately the same time of day. Females were not dosed if parturition was in progress at the scheduled time of administration. Animals were treated at constant doses in mg/kg/day. Dose volume was 5 mL/kg body weight calculated from the most recently recorded scheduled body weight.
Details on mating procedure:
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 10 and 200 mg/mL were analysed to assess the stability and homogeneity of the test item in the liquid matrix. Formulations were confirmed to be homogeneous and stable for the duration of use on the study.

The mean concentrations of the test substance in test formulations analysed during the study were within ±10% of the nominal concentration, confirming the accuracy of formulation. The difference from the mean value remained within 4%, confirming precise analysis.
Duration of treatment / exposure:
Approximately five weeks for males including two weeks pre-pairing and up to eight weeks for females including a two-week pre-pairing phase, throughout pairing, gestation and until Day 13 of lactation.
Frequency of treatment:
Daily (Animals of the F1 generation were not dosed)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Positive control:
None
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the acclimatisation period, observations of the animals and their cages were recorded at least once per day. During dosing Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED PHYSICAL EXAMINATION AND ARENA OBSERVATIONS
Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, therefore observations were made on these occasions without “blinding”.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 males Weekly during acclimatisation. Before dosing on the day that treatment commenced (Day 1) and weekly thereafter. F0 females Weekly during acclimatisation. Before dosing on the day that treatment commenced (Day 1) and weekly before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7 and 13 of lactation.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption for F0 animals was undertaken weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. For females after mating food consumption was performed to match the body weight recording as follows:
Days 0-6, 7-13 and 14-19 after mating.
Days 1-3, 4-6 and 7-12 of lactation.


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (light general anesthesia induced by isoflurane)
- Collection vessel: collected into tubes containing EDTA as anticoagulant.
- Animals fasted: Not specified
- How many animals: The five lowest numbered surviving males per group. The first five surviving lactating females with a litter per group.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (light general anesthesia induced by isoflurane)
- Collection vessel: collected into tubes containing lithium heparin as anticoagulant.
- Animals fasted: Not specified
- How many animals: The five lowest numbered surviving males per group. The first five surviving lactating females with a litter per group.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation.
- Battery of functions tested: sensory activity and grip strength/ motor activity

IMMUNOLOGY: No


OTHER: THYROID HORMONE ANALYSIS (F0 Adult males nad females)
- Time schedule: At termination all surviving F0 adult males and females (no samples were obtained from animals which failed to litter or with a total litter loss).
Oestrous cyclicity (parental animals):
The incidence and percentage females showing the following classifications of estrous cycles before pairing were assessedas follows:

Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 5 days)
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Extended estrus: At least 4 consecutive days of estrus
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that show estrus during this period and the cycle stage at termination.
Sperm parameters (parental animals):
Sperm parameters were not assessed apart from sperm count estimates from vaginal smears at mating.
Litter observations:
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights: Days 1, 4, 7, 11 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY: Yes

The organs weighed, tissue samples fixed and sections examined microscopically are detailed as follows for F0 animals:

Adrenals
Brain (including cerebrum, cerebellum and pons)
Cecum
Colon
Cowpers glands
Duodenum
Epididymides
Eyes
Glans penis
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys
LABC (levator ani-bulbocavernosus) muscle
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - left axillary
- mesenteric
Ovaries
Peyer’s Patch
Prostate
Sciatic nerve
Seminal vesicles with coagulating glands
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical level)
Spleen
Sternum (with marrow)
Stomach
Testes
Thymus
Thyroid
Trachea
Urinary bladder
Uterus with cervix(weighed with oviducts)
Vagina
Postmortem examinations (offspring):
Offspring Examinations

Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content and particular attention paid to external genitalia was performed. Abnormal tissues were retained.

F1 offspring on Day 4 of age: Blood sample was taken. Externally normal offspring were discarded without examination. Externally abnormal offspring were examined, and retained for possible future examination.

F1 offspring on Day 13 of age: Blood sample was taken. All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Thyroid glands were preserved from one male and one female in each litter.
Statistics:
Statistical analyses were performed on the majority of data. For some parameters, including estrous cycles before treatment and stage of estrous cycle at termination, the similarity of the data was such that analysis was not considered to be necessary.

All statistical analyses were carried out separately for males and females. Food consumption before pairing was analysed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit.

For litter/foetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Reproductive indices:
Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.

Mating Performance and Fertility
Group values were calculated for males and females separately for the following:
Percentage mating (%)
Conception rate (%)
Fertility index (%)

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. The Gestation index was calculated for each group.
Offspring viability indices:
The following were calculated for each litter:

Post-implantation survival index % (Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded)

Live birth index (%)

Viability index (%)

Lactation index (%)

Group mean values were calculated from individual litter values
Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
On Day 9 of treatment, one female receiving 330 mg/kg/day was killed for welfare reasons.
On Day 23 of gestation, a second female receiving 330 mg/kg/day was killed for welfare reasons.
On Day 8 of lactation, a Group 4 female receiving 750 mg/kg/day was terminated early due to total litter loss.
On Day 9 of lactation, a third female receiving 330 mg/kg/day was terminated early due to total litter loss.
On Day 25 of gestation, three Group 4 females receiving 750 mg/kg/day were killed due to having surpassed the designated gestational time.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall body weight gains for males given 750 mg/kg/day were moderately low and slightly low for males given 100 or 330 mg/kg/day. During the two-week pre-pairing period the females receiving 750 mg/kg/day body weight gain was higher than Controls. During the gestation and lactation periods body weight gain was slightly or moderately low for females receiving 330 or 750 mg/kg/day. For females receiving 100 mg/kg/day, body weight gain was slightly low during the lactation period, only.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food intake for males receiving 750 mg/kg/day was generally slightly lower compared to Control throughout the treatment period. Mean food consumption during gestation was low for females receiving 750 mg/kg/day and following parturition was low for females receiving 330 or 750 mg/kg/day. Mean food consumption for males receiving 100 or 330 mg/kg/day and for females receiving 100 or 330 mg/kg/day was generally similar to Controls throughout the dosing period, with exception of the lactation period for females at 330 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological examinations at scheduled termination revealed, when compared with Controls, a reduction in red cell mass amongst males and females receiving 330 or 750 mg/kg/day. Mean cell haemoglobin and mean cell volume were slightly lower than that of Controls for males receiving 330 or 750 mg/kg/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Blood chemistry investigations after five weeks of treatment revealed statistically significant changes in the liver enzymes for males at all dose levels where alkaline phosphatase activity was higher compared to Controls and alanine aminotransferase activity was lower compared to Controls. A dose dependent response was observed for alkaline phosphatase only. Liver enzymes changes were observed in females with lower alkaline phosphatase and alanine aminotransferase activity at all dose levels compared to Controls. A dose dependent response was observed for alanine aminotransferase levels only. Electrolyte levels were high for males at 330 and 750 mg/kg/day when compared to Controls although there was no dose-response evident. Bilirubin and bile acids concentrations for males receiving 750 mg/kg/day were higher compared to Controls. Mean albumin concentrations were slightly high at all dose levels in males and in females receiving 750 mg/kg/day, a dose relationship was only apparent in males.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological evaluation of retained tissues revealed treatment related changes in the kidneys, lungs, stomach, mammary gland and liver. In the kidneys, vacuolation of the tubular epithelium was present in all males given 750 mg/kg/day and there was single cell necrosis of the tubular epithelium in three females given 330 mg/kg/day and all females given 750 mg/kg/day. Minimal to slight alveolar macrophage aggregates were distributed at the terminal bronchioles and/or perivascular within the subpleural region affecting all males and females given 750 mg/kg/day and in four females and two males receiving 330 mg/kg/day. This was accompanied by acicular clefting and neutrophilic infiltrate and associated type 2 epithelialisation (particularly for females at 750 mg/kg/day). Hyperplasia and/or hyperkeratosis were present in the mucosa of the non-glandular region of the stomach affecting a single male and female given 750 mg/kg/day and this was accompanied by minimal single erosion of the non-glandular mucosa at the limiting ridge in the male given 750 mg/kg/day; this was associated with gross thickening in some animals. The mammary tissue showed decreased secretory activity in four females receiving 750 mg/kg/day and one female given 330 mg/kg/day. Minimal to slight increase of rarefaction was present in the liver of all males given 330 or 750 mg/kg/day and the majority of females given 750 mg/kg/day and two females given 330 mg/kg/day. Centrilobular hypertrophy was observed in one male and in two females dosed with 750 mg/kg/day
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
An increase in irregular estrous cycles and extended pre-coital intervals were observed for females receiving 750 mg/kg/day, there was no effect on these parameters at 100 or 330 mg/kg/day. A slight shift towards longer gestation lengths was apparent for females receiving 330 or 750 mg/kg/day in comparison with the concurrent Control. The gestational length of females receiving 100 mg/kg/day was unaffected. The number of females with live litters born were lower for females receiving either 330 or 750 mg/kg/day. The rate of conception and fertility index for females receiving 750 mg/kg/day was low compared to Controls. The number of copulation plugs observed in the cages of females receiving 100, 330 or 750 mg/kg/day tended to be low when compared with Controls and indicative of a dose dependent effect. There was no effect of treatment on sperm count estimates and at termination all females were in diestrous.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
In females receiving 750 mg/kg/day two females were not pregnant. For females receiving 750 mg/kg/day, the rate of conception and fertility index were 80% as opposed to Controls and those females receiving 100 or 330 mg/kg/day whereby in all instances the rate of conception and fertility index were 100%. There was no effect of treatment at 100 or 330 mg/kg/day on mating performance and fertility index, all females showing positive evidence of mating.

At 330 and 750 mg/kg/day, the mean number of uterine implantation sites were lower than Control and the historical control data range, resulting in lower mean total litter size but within the scope of this study it was not possible to ascertain the aetiology of these findings. In addition, post implantation survival index, live birth index and lactation index was slightly low at 330 mg/kg/day and moderately low at 750 mg/kg/day and as litter size was outside the expected range, these changes were considered treatment related. The reduced reproductive performance of the animals occurring at doses of 330 and 750 mg/kg/day, was not associated with any histopathological findings; however due to the severity of the effects observed these changes were considered adverse. Offspring survival to Day 13 of age appear unaffected by parental treatment at 100 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
330 mg/kg bw/day (nominal)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
330 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The reduction in the number of uterine implantations at 330 and 750 mg/kg/day (and as a consequence litter size) was associated with reduced survival. These differences, however, were statistically significant and all parameters were outside of the Historical Control Data values (representing 11 OECD TG 422 studies). This effect is considered to be related to treatment and it is likely that the male and/or female reproductive systems have been affected, but the mechanism is undetermined and potentially adverse.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
330 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:
yes
Conclusions:
The reduction in the number of uterine implantations at 330 and 750 mg/kg/day (and as a consequence litter size) was associated with reduced survival. These differences, however, were statistically significant and all parameters were outside of the Historical Control Data values (representing 11 OECD TG 422 studies). This effect is considered to be related to treatment and it is likely that the male and/or female reproductive systems have been affected, but the mechanism is undetermined and potentially adverse.

It was therefore concluded that within the context of this study, the NOAEL for reproductive/developmental toxicity was 100 mg/kg/day.
Executive summary:

Introduction

The purpose of this study was to screen the test substance, KOMAD 710, for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, following repeated oral dosing. The assessment of reproductive effects was made along with assessment of systemic toxicity as part of a Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test which also assessed potential adverse effects of the test item on reproduction. The study was designed to be compatible with the requirements of the following guideline:

  • OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

Method

Three groups of ten male and ten female rats received test substance at doses of 100, 330 or 750 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, propylene glycol,throughout the same relative treatment period.

During the study, clinical condition, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. 

Results (Reproductive Effects)

Mortality

On Day 9 of treatment, one female receiving 330 mg/kg/day was killed for welfare reasons.

On Day 23 of gestation, a second female receiving 330 mg/kg/day was killed for welfare reasons.

On Day 8 of lactation, a Group 4 female receiving 750 mg/kg/day was terminated early due to total litter loss. 

On Day 9 of lactation, a third female receiving 330 mg/kg/day was terminated early due to total litter loss. 

On Day 25 of gestation, three Group 4 females receiving 750 mg/kg/day were killed due to having surpassed the designated gestational time.

Clinical Signs

There were no clinical signs observed among the offspring which were considered to be related to parental treatment with the test substance.

There were no clinical signs observed following dose administration or signs at routine clinical examination that were considered to be associated with treatment.

Body Weight

Overall body weight gains for males given 750 mg/kg/day were moderately low and slightly low for males given 100 or 330 mg/kg/day. During the two-week pre-pairing period the females receiving 750 mg/kg/day body weight gain was higher than Controls.

During the gestation and lactation periods body weight gains was slightly or moderately low for females receiving 330 or 750 mg/kg/day. For females receiving 100 mg/kg/day, body weight gain was slightly low during the lactation period, only.

Food Consumption

Mean food consumption during gestation was low for females receiving 750 mg/kg/day and following parturition was low for females receiving 330 or 750 mg/kg/day. Mean food consumption for males receiving 100 or 330 mg/kg/day and for females receiving 100 or 330 mg/kg/day was generally similar to Controls throughout the dosing period, with exception of the lactation period for females at 330 mg/kg/day.

Estrous Cycle

An increase in irregular estrous cycles and extended pre-coital intervals were observed for females receiving 750 mg/kg/day, there was no effect on these parameters at 100 or 330 mg/kg/day. A slight shift towards longer gestation lengths was apparent for females receiving 330 or 750 mg/kg/day in comparison with the concurrent Control. The gestational length of females receiving 100 mg/kg/day was unaffected. The number of females with live litters born were lower for females receiving either 330 or 750 mg/kg/day. The rate of conception and fertility index for females receiving 750 mg/kg/day was low compared to Controls. The number of copulation plugs observed in the cages of females receiving 100, 330 or 750 mg/kg/day tended to be low when compared with Controls and indicative of a dose dependent effect. There was no effect of treatment on sperm count estimates and at termination all females were in diestrous

F1 litter responses

A reduction in mean number of implantation sites of females receiving 330 or 750 mg/kg/day resulted in lower mean litter size at both dose levels when compared with Control; all values were outside the historical control data. The mean number of implantation sites and litter size were unaffected at 100 mg/kg/day.

The post implantation survival index and group mean live birth indices were also low at 330 and 750 mg/kg/day. There was also a suggestion of slightly low lactation survival indices at 330 or 750 mg/kg/day. Offspring survival to Day 13 of age appear unaffectedby parental treatment at 100 mg/kg/day.

Body weight gain of male and female offspring receiving test substance at a dose level of 750 mg/kg/day were consistently lower than that of Control. Mean offspring body weights on Day 1 of age and subsequent body weight gain up to Day 13 of age was similar to Controls at 100 or 330 mg/kg/day.

The clinical condition, sex ratio and ano-genital distances of the F1 offspring were unaffected by parental treatment and at scheduled termination, there were no findings associated with treatment.

Thyroxine Levels

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in male and female offspring.

Conclusion

The reduction in the number of uterine implantations at 330 and 750 mg/kg/day (and as a consequence litter size) was associated with reduced survival. These differences, however, were statistically significant and all parameters were outside of the Historical Control Data values (representing 11 OECD TG 422 studies). This effect is considered to be related to treatment and it is likely that the male and/or female reproductive systems have been affected, but the mechanism is undetermined and potentially adverse. It was therefore concluded that within the context of this study, the NOAEL for reproductive/developmental toxicity was 100 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Additional information