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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Sep 2011 - 25 Apr 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1995
Deviations:
yes
Remarks:
The deviations were considered to have not affected the integrity of the study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 2-methylpropanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2-{[(2-methylpropanoyl)oxy]methyl}-2-{[(3,5,5-trimethylhexanoyl)oxy]methyl}propyl 3,5,5-trimethylhexanoate; 3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate
EC Number:
813-120-0
Cas Number:
1262967-45-2
Molecular formula:
C21H36O8 C26H46O8 C31H56O8 C36H66O8 C41H76O8
IUPAC Name:
3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 2-methylpropanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(2-methylpropanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate; 3-[(2-methylpropanoyl)oxy]-2-{[(2-methylpropanoyl)oxy]methyl}-2-{[(3,5,5-trimethylhexanoyl)oxy]methyl}propyl 3,5,5-trimethylhexanoate; 3-[(3,5,5-trimethylhexanoyl)oxy]-2,2-bis({[(3,5,5-trimethylhexanoyl)oxy]methyl})propyl 3,5,5-trimethylhexanoate
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: JA01YX10
- Expiration date of the lot/batch: Jan 2013
- Purity test date: 95.8 %



RADIOLABELLING INFORMATION (if applicable): N/A




STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL

- Storage condition of test material:bRoom temperature (ca 20 °C), in the dark under Nitrogen
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: stable in the liquid matrix at concentrations of 2 and 200 mg/mL for 24 hours at ambient temperature and for up to 15 days when refrigerated (nominally 2 to 8 ºC). 

- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A



TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: The test material was used as supplied. The test item was prepared for administration as a series of graded concentrations in the vehicle - corn oil.
- Preliminary purification step (if any): Dose range finder was conducted prior to the main experiment ignorer to determine suitable dose levels.

- Final dilution of a dissolved solid, stock liquid or gel: 5 mL/kg
- Final preparation of a solid: N/A



FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

Test animals

Species:
rat
Strain:
CD-1
Remarks:
Crl:CD(SD) rats
Details on species / strain selection:
Crl:CD(SD) rats from Charles River (UK) Ltd.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes

- Age at study initiation: 70 days

- Weight at study initiation: Weight range of 332 to 382 g for males and 238 to 270 g for females.
- Fasting period before study: Not Stated

- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.



DETAILS OF FOOD AND WATER QUALITY:

- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet.
- Water (e.g. ad libitum): Polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 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.

IN-LIFE DATES: From: To: 28 Sep 2011 - 11 Nov 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
 


DIET PREPARATION

- Rate of preparation of diet (frequency): N/A

- Mixing appropriate amounts with (Type of food): N/A

- Storage temperature of food: N/A
 


VEHICLE

- Justification for use and choice of vehicle (if other than water): The test item was insoluble in water hence corn oil was used

- Concentration in vehicle: 2 mg/mL and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bodyweight,
- Lot/batch no. (if required): Not stated

- Purity: Not stated
Details on mating procedure:
Following a minimum of two weeks of treatment males and females were paired on a one-to- one basis from within the same treatment group for a period of up to two weeks.

Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa and the stage of the oestrous cycle. The day on which evidence of mating was found was designated Day 0 of gestation. Once mating occurred, the males and females were separated and smearing was discontinued.

The pre-coital interval was calculated for each female as the time elapsing between initial pairing and detection of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Detailed records of compound usage were maintained. The amount of test material necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Homogeneity and stability of the test material in the vehicle was performed as part of the 13 week study (Huntingdon Life Sciences Study Number: OWH0019). The homogeneity and stability were confirmed following storage at ambient temperature (nominally 21 °C) for 0, 2 and 4 hours, and refrigerated (nominally 2-8 °C) for 1 day, 8 days and 15 days.

Samples of each formulation prepared for administration in first and last formulation occasions were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL) from all groups. Two samples from each group were analysed and the remainder were retained as contingency.
Duration of treatment / exposure:
15 days before pairing until Day 6 after the birth of the F1 generation.
Frequency of treatment:
Daily
Details on study schedule:
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed.

F0 females were not dosed if parturition was in progress at the scheduled time of administration.

Animals received the test material or vehicle control formulations orally at a volume-dosage of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.

All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day, seven days per week. The volume administered to each animal was calculated from the most recently recorded scheduled bodyweight.

A daily record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 Control (Corn oil)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2 (test item)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3 (test item)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 (test item)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Dose selection rationale: Dose selection was based on the result obtained from a 14 day dose range finder study.

- Rationale for animal assignment (if not random): Animal were assigned randomly

- Fasting period before blood sampling for clinical biochemistry: Not stated

- Rationale for selecting satellite groups: Not included

- Post-exposure recovery period in satellite groups: N/A

- Section schedule rationale (if not random): N/A

- Other: Not stated
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

- Cage side observations checked

DETAILED CLINICAL OBSERVATIONS: Yes

- Time schedule: Twice daily

BODY WEIGHT: Yes 

- Time schedule for examinations: The weight of each adult was recorded during acclimatisation, on the day that treatment commenced (Week 0), weekly thereafter and before necropsy. The weight of each F0 female was also recorded on Days 0, 3, 7, 10, 14, 17 and 20 after mating and on Days 1, 4 and 7 of lactation.


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: N/A
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes; The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis from the start of treatment for the F0 adults until the animals were paired for mating. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage. For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled was also recorded for the periods Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.


FOOD EFFICIENCY:

- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes



WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OTHER: Not Specified

Oestrous cyclicity (parental animals):
After pairing until mating, smearing was performed using a pipette lavage.
Sperm parameters (parental animals):
Not specified
Litter observations:
All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. The records maintained were as follows:
Clinical signs: Daily records were maintained for evidence of ill-health or reaction to 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 1-7 of age.
Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and 7 of age.
Bodyweight: Individual offspring bodyweights were recorded on Days 1, 4 and 7 of age.
Postmortem examinations (parental animals):
SACRIFICE: Yes

F0 males were killed after Day 7 of lactation of the females.
F0 females surviving until the end of the scheduled study period were killed on Day 7 of lactation. Group 2 female number 58 failed to produce a viable litter so was killed on Day 25 after mating. Two females and their litters (Group 3 number 67 and Group 4 number 71) were killed on Day 21 and 23 after mating respectively as the dam had poor survival prognosis. A further female and litter (Group 3 number 66) was killed on Day 2 of lactation.

GROSS NECROPSY: Yes

All tissues preserved for examination (as specified above) were examined for all animals of Groups 1 (Control) and 4 (1000 mg/kg/day) sacrificed on completion of the scheduled treatment period and was extended to cover the ovaries of females in Groups 2 and 3 (100 and 300 mg/kg/day).

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes

Epididymides, ovaries and testes were subject to histological processing. Those tissues subject to histological processing included the following regions:
-Epididymides (caput, corpus and cauda)
-Ovaries (qualitative evaluation of one section from each ovary)
-Uterus (uterine body with cervix section and oviducts)
Postmortem examinations (offspring):
SACRIFICE:Yes

Offspring were killed on Day 7 of age.

GROSS NECROPSY: Yes
For offspring surviving to scheduled termination, a careful external examination was performed for gross abnormalities and externally normal offspring were discarded without internal examination. Externally abnormal offspring were internally examined and any abnormal tissues were retained in an appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
Statistics:
The following sequence of statistical tests was used for bodyweight, food consumption, organ weights and litter data: Bartlett's test, Dunnett's test, Shirley's test and Williams’ test

For litter data, if 75 % of the data (across all groups) were the same value, for example c, Fisher’s Exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate (Angervall and Carlstrom, 1963). The treatment comparisons were made on adjusted group means in order to allow for differences in bodyweight which might influence the organ weights.

Sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz et al 1991). Each treated group was compared to control using a Wald chi-square test. The numerator was Number of males, the denominator was Number of live fetuses.

For gestation length, an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step-down’ process was repeated until the test was no longer statistically significant (p≥0.05). If the exact version of the Linear-by-linear test could not be calculated (due to the size of the table containing the data), then the asymptotic version was used instead.
Reproductive indices:
Percentage mating = Number animals mating x 100 Animals paired
Conception rate (%) = Number animals achieving pregnancy x 100 Animals mated
Fertility index (%) = Number animals achieving pregnancy x 100 Animals pairing
Offspring viability indices:
The following were calculated for each litter:
Post-implantation survival index (%) = Total number of offspring born x100 Total number of uterine implantation sites
Post-implantation survival index was expressed as 100 % where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering Total number of offspring born
Viability index (%) = Number of live offspring on Day 7 Number live offspring on Day 1 after littering
Group mean values were calculated from individual litter values.
Percentage males = Number of males in litter x 100 Total number of offspring in litter

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Underactivity, irregular breathing, dark discharge from the vagina, partially closed eyelids and skin pallor observed in three female at 300 and 1000 mg/kg/day
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were three deaths, all in females at the end of gestation/early lactation:
- Two females in the 300 mg/kg/day group (3F 66 &3F 67) with clinical signs of underactivity, irregular breathing, dark discharge from the vagina, partially closed eyelids and skin pallor. Macroscopic examination at necropsy revealed reduced caecum contents, dark fluid in the caecum and jejunum, inactive mammary tissue, a small spleen, firm, dark material in the stomach and dark fluid in the vagina.
- One female in the 1000 mg/kg/day group (4F 71) showing clinical signs of underactivity, fast breathing, and piloerection. Macroscopic examination at necropsy revealed reduced caecum, duodenum, ileum and jejunum contents, pale and inactive mammary tissue, a small spleen, and a stomach distended with food, placental and fetal material. hree male pups and four female pups were found dead before the adult female was killed for welfare reasons, and three live male pups, one live female pup and one dead female pup were present in the cage with this female when she was killed for welfare reasons.
Body weight and weight changes:
not specified
Description (incidence and severity):
There were no clear adverse effects of treatment of upon bodyweights of males or of females before pairing, with treated animals gaining similar amounts of or slightly more weight than controls.
During gestation there appeared to be a slight reduction in weight gain for females at 1000 mg/kg/day, as a consequence of reduced gain in the later period of measurement (Day 17 -20). Bodyweight at Day 1 post partum was slightly low (95 % of control) but gains were similar in all groups to Day 7 post partum when the study ended.
Food consumption and compound intake (if feeding study):
not specified
Description (incidence and severity):
There were no clear effects on food consumption in the weeks before pairing or, for females, during gestation and lactation at dose levels of up to 1000 mg/kg/day.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Description (incidence and severity):
The pre-coital interval was unaffected by treatment, with all animals mating at the earliest opportunity, when the female came into oestrus.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment. One pairing in the 100 mg/kg/day group was infertile, and could probably be attributed to the male which had small reproductive organs. The isolated nature of this finding suggests that it was unrelated to treatment. With regard to the two females that were killed for welfare reasons around the time of parturition, no live pups were seen for Group 3 female number 67 and was considered to have no live litter because the pups had died before or during parturition. Group 4 female number 71 did have a live litter because there were 4 live pups (as well as 8 dead ones).

The pre-coital interval was unaffected by treatment, with all animals mating at the earliest opportunity, when the female came into oestrus.

The gestation length was within the normal range of 22-23 days, and there was no evidence for a treatment-related shift in the distribution of gestation lengths, although the control group contained a slightly higher proportion of females with a 23 day gestation length. The gestation index was unaffected by treatment.

Details on results (P0)

Reproductive performance in terms of mating, fertility, litter size, growth and survival of the offspring was unaffected by parental treatment with the test item. The microscopic examination of testis, epididymides and ovaries from the F0 generation animals revealed the presence of findings only in the female ovaries, and these changes were considered to be of unclear relationship to treatment with the test item.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the effects seen in utero growth of the offspringon birth weight at 300 and 1000 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adversed effects observed

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Offspring clinical signs were limited to a few instances of cold to touch and bruising. Dark abdomens were observed amongst the offspring of one litter in Group 3 (Litter 69) from Days 2 – 7 of age, and amongst the offspring of two ltters in Group 4 (Litters 74 and 78) on Day 4 or Days 4-5 of age, respectively.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean number of implantations, the live litter size on Day 1 and sex ratio showed no adverse effect of parental treatment. The offspring survival up to Day 7 of age was slightly low for some litters in the treated groups but it was considered that there was no dosage related effect.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Offspring bodyweight on Day 1 of age was slightly low (82 % of control) for litters in the highest dose group (1000 mg/kg/day), but by Day 7 of age there was an improvement in relative offspring weight (88/90 % of male/female weight. Birth weight at 300 mg/kg/day was also slightly low (86-88 % of control).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination of offspring which died or were killed for welfare reasons generally confirmed the last in-life finding of no milk present in stomach. No other macroscopic abnormalities were found amongst these animals.
Macroscopic examination of offspring killed at scheduled termination on Day 7 of age revealed no abnormality.
Histopathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of offspring that died prior to scheduled termination and at scheduled termination on Day 7 of age did not reveal any findings that could be attributed to parental treatment.
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
other: Overal NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: birth weight at 300 and 1000 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects noted

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Tables containing raw data are attached in full study report.

Applicant's summary and conclusion

Conclusions:
Reproductive performance in terms of mating, fertility, litter size, growth and survival of the offspring was unaffected by parental treatment with the test item. Three females were killed for welfare reasons. One at 300 and one at 1000 mg/kg/day were killed during parturition and a second female at 300 mg/kg/day was killed on Day 2 of lactation. Necropsy did not reveal test item related effects, it was concluded that the lower offspring bodyweight and lower maternal weight gain during late lactation seen at these doses and an association with treatment cannot be excluded. The birth weights of litters at 100 mg/kg/day were within the normal control range.

Therefore it was considered that treatment at 300 and 1000 mg/kg/day had biologically significant effects upon the in utero growth of the offspring, but there was no evidence of developmental abnormality. There was no evidence of an effect of treatment upon reproductive performance of the parental generation or that the in utero effect would lead to an effect upon reproductive performance of the F1 generation.

It is concluded that that the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day for reproductive performance and 100 mg/kg/day was considered to be the overall NOAEL because of the effects seen on birth weight at 300 and 1000 mg/kg/day.
Executive summary:

OECD 421 (2012) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 421), Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid was administered to 10 male and 10 female Crl:CD(SD) strain rats in corn oil via oral gavage at dose of 100, 300 and 1000 mg/kg/day for up to 5 weeks. In addition, 10 male and 10 female control rats were administered corn oil only.

 

A summary of adult responses to the test item are described below;

Systemic toxicity (F0)

 

Mortality- There were three deaths, all in females at the end of gestation/early lactation. ne at 300 and one at 1000 mg/kg/day were killed during parturition and a second female at 300 mg/kg/day was killed on Day 2 of lactation. Necropsy did not reveal test item related effects, it was concluded that the lower offspring bodyweight and lower maternal weight gain during late lactation seen at these doses and an association with treatment cannot be excluded.  The birth weights of litters at 100 mg/kg/day were within the normal control range.

 

Clinical signs- Underactivity, irregular breathing, dark discharge from the vagina, partially closed eyelids and skin pallor observed in three female at 300 and 1000 mg/kg/day.

Bodyweight- There were no clear adverse effects of treatment of upon bodyweights of males or of females before pairing, with treated animals gaining similar amounts of or slightly more weight than controls. During gestation there appeared to be a slight reduction in weight gain for females at 1000 mg/kg/day, as a consequence of reduced gain in the later period of measurement (Day 17 -20). Bodyweight at Day 1 post partum was slightly low (95% of control) but gains were similar in all groups to Day 7 post partum when the study ended.

  

Food consumption and efficiency-There were no clear effects on food consumption in the weeks before pairing or, for females, during gestation and lactation at doses up to 1000 mg/kg/day.

  

Histopathological changes and Necropsy-No toxicologically significant effects were detected in terminal kill animals of either sex treated with the test item.  However, Changes of an uncertain relationship to treatment were seen in the ovary of females given the test item at 100, 300 and 1000 mg/kg/day. Prominent corpora lutea of pregnancy in the ovaries were more frequently seen in all treated groups than in controls, particularly in females dosed at 1000 mg/kg/day.

  

Organ weights-Organ weights were not affected by treatment. The testes and epididymides of one male (Group 2 M18) were atypically low, but this was considered unlikely to relate to treatment.

 

Reproductive performance (F0)

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

Estrous cycle- There was no effect of treatment on estrous cycle regularity during the course of the 2-week pre-pairing assessment period for the Reproductive phase females.  

 

Fertility- The pre-coital interval was unaffected by treatment, with all animals mating at the earliest opportunity, when the female came into oestrus.

 

Gestation length- There was no effect of treatment on gestation length, with all gestation lengths within the expected range of 22 to 23 days.  There was no evidence for a treatment-related shift in the distribution of gestation lengths, although the control group contained a slightly higher proportion of females with a 23 day gestation length. The gestation index was unaffected by treatment.

        

Litter size, viability- The mean number of implantations, the live litter size on Day 1 and sex ratio showed no adverse effect of parental treatment. The offspring survival up to Day 7 of age was slightly low for some litters in the treated groups but it was considered that there was no dosage related effect.

Offspring bodyweight on Day 1 of age was slightly low (82 % of control) for litters in the highest dose group (1000 mg/kg/day), but by Day 7 of age there was an improvement in relative offspring weight (88/90 % of male/female weight. Birth weight at 300 mg/kg/day was also slightly low (86-88 % of control).

 

Therefore, it was considered that treatment at 300 and 1000 mg/kg/day had biologically significant effects upon the in utero growth of the offspring, but there was no evidence of developmental abnormality. There was no evidence of an effect of treatment upon reproductive performance of the parental generation or that the in utero effect would lead to an effect upon reproductive performance of the F1 generation. 

It is concluded that that the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day for reproductive performance and 100 mg/kg/day was considered to be the overall NOAEL because of the effects seen on birth weight at 300 and 1000 mg/kg/day. 

This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 421 in the rat.