Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 19/06/2018 Experimental Completion Date: 22/10/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2-dimethylpropane-1,3-diyl cyclohex-4-ene-1,2-dicarboxylate
EC Number:
255-180-5
EC Name:
2,2-dimethylpropane-1,3-diyl cyclohex-4-ene-1,2-dicarboxylate
Cas Number:
41026-17-9
Molecular formula:
C21H28O8
IUPAC Name:
6-[(3-{[(6-carboxycyclohex-3-en-1-yl)carbonyl]oxy}-2,2-dimethylpropoxy)carbonyl]cyclohex-3-ene-1-carboxylate
Test material form:
other: Solid
Details on test material:
-Identification: NPG-THPA hemiester (TK 12094).
-Molecular formula: C21H28O8.
-CAS Number: 41026-17-9.
-Description: White to light yellow solid.
-Batch: Colkb03.
-Purity/Composition: >85%.
-Test substance storage: At room temperature protected from light.
-Stability under storage conditions: Stable.
-Expiry date: 28 February 2021.
-Hygroscopic: Yes, store in well-sealed container.
-Volatile: No.
-Reactivity: Use amber-coloured glassware or wrap container in aluminium-foil.
Specific details on test material used for the study:
Identification : 2,2-dimethylpropane-1,3-diyl cyclohex-4-ene-1,2-dicarboxylate
Physical State/Appearance : White solid
CAS Number : 36621-20-2
Alternative Identifier : Aradur HY 225
Chemical Formula : C21H28O8
Purity : >85%
Batch Number : Colkb03
Date Received : 11 December 2017
Storage Conditions : Ambient temperature over silica gel in darkness; used/formulated at ambient humidity in light
Expiry Date : 01 February 2021

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Animal Information
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On the first day of treatment, the females weighed 224 to 323g.

Animal Care and Husbandry
The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, 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 computerized system, and print-outs of hourly mean temperatures and humidity are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20% respectively. There were no deviations from the target range for temperature and transient deviations from the target range for relative humidity were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of the study, the test item was prepared at the appropriate concentrations in Propylene glycol. The stability and homogeneity of the test item formulations were determined. Bulk formulations were therefore prepared one occasion, divided into daily aliquots and stored at approximately 4 °C in the dark until the day of use.

Samples were taken of each test item formulation and were analyzed for concentration of 2,2-dimethylpropane-1,3-diyl cyclohex-4-ene-1,2-dicarboxylate at Envigo Analytical Laboratory, Shardlow. The results indicate that the prepared formulations were within 100 to 103% of the nominal concentration and were acceptable for use on the study.

VEHICLE
- Concentration in vehicle: 0, 20, 60, 150 mg/mL
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were taken of each test item formulation and were analyzed for concentration of 2,2-dimethylpropane-1,3-diyl cyclohex-4-ene-1,2-dicarboxylate. The results indicate that the prepared formulations were within 100 to 103% of the nominal concentration and were acceptable for use on the study.
Details on mating procedure:
Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On the first day of treatment, the females weighed 224 to 323g.
Duration of treatment / exposure:
The test item was administered daily, from Day 3 to Day 19 of gestation
Frequency of treatment:
Daily
Duration of test:
Day 0-6: Mating
Day 6-19: Treatment
Day 20: Necropsy
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected, in collaboration with the Sponsor, based on available toxicity data, including data from a Preliminary Oral (Gavage) Pre-Natal Development Toxicity Study in the Rat (Envigo study TX66FH) and a Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (Envigo Study QK84HX). A dosage of 1000 mg/kg bw/day in both studies was associated with adverse effects on body weight performance and food consumption that were considered to preclude this dosage from further investigation of toxicity. A dosage of 600 mg/kg bw/day in the Preliminary Pre-Natal Development Toxicity Study was associated with equivocal initial effects on body weight and food consumption. In the Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat (QK84HX), a dosage of 750 mg/kg bw/day was well tolerated, with no obvious effect of treatment being apparent. It is considered that the selection of a high dosage of 600 mg/kg bw/day for this Pre-Natal Toxicity Study could have led to insufficient evidence of maternal toxicity being evident, therefore, although 750 mg/kg bw/day was untested in the pregnant female, this dosage was selected as the high dosage for this pre-natal study. Low and intermediate dosages of 100 and 300 mg/kg bw/day respectively, were also selected for use on the study.

- Other: The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Examinations

Maternal examinations:
Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing.
All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 3, 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.


Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The kidneys were weighed and then preserved in buffered 10% formalin. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
Statistics:
The following parameters were analyzed statistically, where appropriate, using the R Environment for Statistical Computing and the test methods outlined below:
Body weight and body weight change, food consumption, gravid uterus weight, caesarean necropsy parameters and fetal external, visceral and skeletal parameters
Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pair-wise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non-parametric analysis of the data was performed incorporating the Kruskal-Wallis test which if significant was followed by the Mann-Whitney "U" test. Dose response relationships were also investigated by linear regression. Where the data were unsuitable for these analyses then pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Due to their distribution, caesarean necropsy parameters and fetal parameters were generally analysed using non-parametric analysis.
Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Pre and Post Implantation Loss

Percentage pre-implantation loss was calculated as:

((Number of corpora lutea - number of implantations) / number of corpora lutea) x 100

Percentage post-implantation loss was calculated as:

((Number of implantations - number of live fetuses) / number of implantations) x 100

Sex Ratio
Sex ratio was calculated as:

% male fetuses (sex ratio) = (Number of males fetuses/ total number of fetuses) x 100

Historical control data:
PLEASE REFER TO THE ATTACHED TABLE "HISTORICAL CONTROL DATA"

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs observed during the study did not indicate any systemic effect of treatment at 100, 300 or 750 mg/kg bw/day.

Increased post-dosing salivation was apparent for three animals at 300 mg/kg bw/day and five animals at 750 mg/kg bw/day. This clinical sign was only present on a single occasion for each animal, with the exception of one animal at 750 mg/kg bw/day, where it was observed on two occasions. Additionally noisy respiration was observed for three animals on one or two occasions during the study at 750 mg/kg bw/day. These clinical signs were considered likely to have resulted from difficulties in dosing isolated animals on occasions during the study and, at the incidence observed, were considered to be of no toxicological significance.

The remaining clinical signs observed during the study were considered to be incidental and unrelated to treatment.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, mean body weight gain was statistically significantly lower than control on the first day of treatment (Day 3 of gestation). However, subsequent mean body weight gain at this dosage was similar or slightly superior to control and no effect of treatment was apparent for mean overall body weight gain at termination (Day 20 of gestation), including when adjusted for the contribution of the gravid uterus.

There was no effect of treatment on body weight gain during gestation, including when values were adjusted for the contribution of the gravid uterus, at 100 or 300 mg/kg bw/day. Observed differences in body weight gain from control during the study were considered to reflect normal biological variation and were unrelated to treatment.

PLEASE REFER TO THE ATTACHED TABLES - "GROUP MEAN BODYWEIGHT VALUES" AND "GROUP MEAN BODYWEIGHT CHANGE VALUES"
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg bw/day, mean food consumption was statistically significantly lower than control from the start of treatment (Day 3 of gestation) until Day 8 of gestation. Thereafter, food intake during gestation was considered to be similar to control until termination on Day 20 of gestation.

There was no effect of treatment on food consumption during gestation at 100 or 300 mg/kg bw/day.
PLEASE REFER TO THE ATTACHED TABLE "GROUP MEAN FOOD CONSUMPTION VALUES"
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
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:
effects observed, treatment-related
Description (incidence and severity):
At 300 or 750 mg/kg bw/day, absolute and body weight relative kidneys weights were statistically significantly higher than control. Kidneys weights were also higher for females receiving 100 mg/kg bw/day but only body weight relative values were statistically significant.
PLEASE REFER TO THE ATTACHED TABLE "GROUP MEAN KIDNEY WEIGHTS"
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic necropsy findings observed for adult animals did not indicate any effect of treatment at 100, 300 or 750 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on litter data, as assessed by pre-implantation loss, number of implantations, subsequent embryofetal survival, litter size or sex ratio on Day 20 of gestation at 100, 300 or 750 mg/kg bw/day.
PLEASE REFER TO THE ATTACHED TABLE "GROUP MEAN LITTER DATA VALUES"
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on litter data, as assessed by pre-implantation loss, number of implantations, subsequent embryofetal survival, litter size or sex ratio on Day 20 of gestation at 100, 300 or 750 mg/kg bw/day.
PLEASE REFER TO THE ATTACHED TABLE "GROUP MEAN LITTER DATA VALUES"
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on litter data, as assessed by pre-implantation loss, number of implantations, subsequent embryofetal survival, litter size or sex ratio on Day 20 of gestation at 100, 300 or 750 mg/kg bw/day.
PLEASE REFER TO THE ATTACHED TABLE "GROUP MEAN LITTER DATA VALUES"
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food efficiency
gross pathology
mortality

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
PLEASE REFER TO THE ATTACHED TABLE "GROUP MEAN GRAVID UTERUS WEIGHT AND ADJUSTED BODYWEIGHTS"
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The type, incidence and distribution of external findings at 100, 300 and 750 mg/kg bw/day did not indicate any effect on fetal development.
One fetus at 300 mg/kg bw/day showed omphalocoele (portion of protruding intestine) and one fetus at 750 mg/kg bw/day showed spina bifida (lower spine exposed). Whilst these findings reflect a notable disturbance of fetal development for the individual fetus, the isolated nature of these findings suggests that their appearance on the study was incidental and unrelated to maternal treatment.
PLEASE REFER TO THE ATTACHED TABLE "SUMMARY INCIDENCE OF SKELETAL EXTERNAL FINDINGS"
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The type, incidence and distribution of skeletal findings at 100, 300 and 750 mg/kg bw/day did not indicate any adverse effect on fetal development.
At 750 mg/kg bw/day, the incidences of fetuses showing incomplete ossification of a number of skull bones (nasal, frontal, parietal, interparietal, squamosal, jugal, zygomatic process of maxilla, zygomatic process of maxilla and hyoid) were statistically significantly lower than control. Additionally statistically significant lower incidences of incomplete ossification of the sacral (neural) arch, pubis and metacarpals were also apparent and there was a statistically significant higher incidence of fetuses with one or more ossified forepaw phalanges at this dosage. Collectively these findings indicated greater developmental maturity of offspring at this dosage compared with control, with the lower incidence of fetuses with incomplete ossification representing a greater proportion of fetus closer to the biological normal for these parameters, compared to control. As these improvements in ossification were not associated with any additional structural variants, these findings are considered to be of no biological significance and unlikely to represent an effect of treatment on embryofetal development.
At 300 mg/kg bw/day, statistically significant differences from control in the incidence of skeletal finding were restricted to lower incidences of fetuses showing incomplete ossification of the parietal, jugal, zygomatic process of squamosal, sacral (neural) arch and pubis. At 100 mg/kg bw/day, the incidences of fetuses showing incomplete ossification of the hyoid and pubis were statistically significantly lower than control. These differences were most probably fortuitous and, as indicated at 750 mg/kg bw/day, do not indicate an effect of treatment on embryofetal development.

PLEASE REFER TO THE ATTACHED TABLE "SUMMARY INCIDENCE OF FETAL SKELETAL FINDINGS"
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The type, incidence and distribution of visceral findings at 100, 300 and 750 mg/kg bw/day did not indicate any obvious effect on fetal development.
For the fetuses selected for visceral examination, one fetus at 300 mg/kg bw/day showed omphalocoele (portion of protruding intestine) and one fetus at 750 mg/kg bw/day showed spina bifida (lower spine exposed). As previously discussed for these fetuses in the assessment of external observations, whilst these findings reflect a notable disturbance of development for the individual fetus, the isolated nature of these findings suggests that their appearance on the study was incidental and unrelated to maternal treatment.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: trunk
external: large intestine
skeletal: skull
Description (incidence and severity):
At 750 mg/kg bw/day, there was no effect of maternal treatment on the survival and growth of the developing conceptus as assessed by number of implantations, implantations loss and fetal, litter and placental weights. External and visceral examination of fetuses revealed one fetus at 300 mg/kg bw/day with omphalocoele (portion of protruding intestine) and one fetus at 750 mg/kg bw/day with spina bifida (lower spine exposed). Whilst these findings reflect a notable disturbance of development for the individual fetus, there was no indication of any similar, but less severe, supporting developmental effects in other fetuses, and the isolated nature of these findings, therefore, suggests that their appearance on the study was incidental and unrelated to maternal treatment.
Skeletal assessments of fetuses at 750 mg/kg bw/day, revealed lower incidences of fetuses with incomplete ossification of a number of skeletal parameters, particularly for bones of the skull. Collectively, the skeletal findings observed indicated greater developmental maturity of offspring at 750 mg/kg bw/day compared with control, with this dosage having a greater proportion of fetuses closer to the biological normal for these parameters, than the control. The improvement in ossification for these treated fetuses were not associated with any additional structural variants, therefore, there was considered to be no indication of any excessive or precocious ossification occurring at this dosage.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on these findings of this pre-natal study, a dosage of 750 mg/kg bw/day was regarded as a No Observed Adverse Effect Level (NOAEL) for the pregnant female.
The findings observed for fetuses at 750 mg/kg bw/day were considered to be unrelated to treatment and this dosage of 750 mg/kg bw/day was considered to represent at least a NOAEL, and most probably a No Observed Effect Level (NOEL), for the survival, growth and development of the conceptus.
Executive summary:

Introduction

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated daily administration by gavage to the pregnant female during Days 3 to 19 of gestation, including the period of organogenesis.

The study was designed to comply with the following guidelines:

·        US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

·        Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 Nousan No 8147, (24 November 2000)

·        OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

·        Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (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 twenty-four time mated Sprague-Dawley Crl:CD®(SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels of 100, 300 and 750 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Propylene Glycol) over the same treatment period to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study. 

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results…….

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

Clinical signs did not indicate any systemic effect of treatment at 100, 300 or 750 mg/kg bw/day.

Body Weight

At 750 mg/kg bw/day, mean body weight gain was statistically significantly lower than control on the first day of treatment, thereafter body weight gain was unaffected by treatment and mean overall body weight gain at termination, including when adjusted for the contribution of the gravid uterus, was similar to control.

There was no effect of treatment on body weight gain during gestation, including when values were adjusted for the contribution of the gravid uterus, at 100 or 300 mg/kg bw/day. 

Food Consumption

At 750 mg/kg bw/day, mean food consumption was statistically significantly lower than control from the start of treatment to Day 8 of gestation. 

There was no effect of treatment on food consumption during gestation at 100 or 300 mg/kg bw/day. 

Water Consumption

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies

Macroscopic necropsy findings observed for adult animals did not indicate any effect of treatment at 100, 300 or 750 mg/kg bw/day.

Litter Data and Litter Placental and Fetal Weights

There was no effect of maternal treatment on pre-implantation loss, number of implantations, subsequent embryofoetal survival, litter size or sex ratio on Day 20 of gestation at 100, 300 or 750 mg/kg bw/day.

Fetal Examination

External Examination

The type, incidence and distribution of external findings at 100, 300 and 750 mg/kg bw/day did not indicate any effect on fetal development, although it was noted that one fetus at 300 mg/kg bw/day showed omphalocoele (portion of protruding intestine) and one fetus at 750 mg/kg bw/day showed spina bifida (lower spine exposed).

Visceral Examination

The type, incidence and distribution of visceral findings at 100, 300 and 750 mg/kg bw/day did not indicate any obvious effect on fetal development. The previous external findings of one fetus at 300 mg/kg bw/day showing omphalocoele (portion of protruding intestine) and one fetus at 750 mg/kg bw/day showing spina bifida (lower spine exposed) were confirmed. 

Skeletal Examination

The type, incidence and distribution of skeletal findings at 100, 300 and 750 mg/kg bw/day did not indicate any adverse effect on fetal development.

At 750 mg/kg bw/day, the incidences of fetuses showing incomplete ossification of a number of skull bones (nasal, frontal, parietal, interparietal, squamosal, jugal, zygomatic process of maxilla, zygomatic process of maxilla and hyoid) were statistically significantly lower than control. Additionally statistically significant lower incidences of incomplete ossification of the sacral (neural) arch, pubis and metacarpels were also apparent and there was a statistically significant higher incidence of foetuses with one or more ossified forepaw phalanges at this dosage. 

At 300 mg/kg bw/day, statistically significant differences from control in the incidence of skeletal findings were restricted to lower incidences of fetuses showing incomplete ossification of the parietal, jugal, zygomatic process of maxilla, sacral (neural) arch and pubis. 

At 100 mg/kg bw/day, the incidences of fetuses showing incomplete ossification of the hyoid and pubis were statistically significantly lower than control.

 

Conclusion

Based on these findings of this pre-natal study, a dosage of 750 mg/kg bw/day was regarded as a No Observed Adverse Effect Level (NOAEL) for the pregnant female.

The findings observed for fetuses at 750 mg/kg bw/day were considered to be unrelated to treatment and this dosage of 750 mg/kg bw/day was considered to represent at least a NOAEL, and most probably a No Observed Effect Level (NOEL), for the survival, growth and development of the conceptus.