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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 Jun 2020 to 19 Aug 2021 (Experimental completion date)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium trifluoroacetate
EC Number:
220-879-6
EC Name:
Sodium trifluoroacetate
Cas Number:
2923-18-4
Molecular formula:
C2HF3O2.Na
IUPAC Name:
sodium trifluoroacetate
Test material form:
solid
Details on test material:
- Batch number: 2019052304
- Appearance: White solid
- Storage conditions: At ambient temperature (15 to 25°C)
- Expiry date: 23 May 2021 (two years after date of manufacture)
- Purity: 99.9%
- Impurity: water, 0.0635%

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK)
- Age at study initiation: 18 to 22 weeks old
- Weight at study initiation: 2.47 to 4.52 kg
- Housing: the animals were single housed in stainless steel cages elevated off the floor. From Day 20 after mating, cage paper was placed into each cage to allow expression of nesting behavior.
- Diet: Teklad 2930, pelleted diet, 200 g/animal/day
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation: Five days before commencement of treatment (Days 1 to 5 of gestation).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 15-21ºC.
- Humidity (%): Monitored and maintained within the range of 45-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 14 hours light: 10 hours dark

IN-LIFE DATES: From: 23 June 2020 To: 24 July 2020

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
Formulations (w/w) were prepared at least once weekly. The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer. No correction was made for the purity of the test substance. The formulation solutions were stored at 2-8°C. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
CONCENTRATION VERIFICATION
The formulations for first and last preparation were sampled, 1 x 10 mL (accurately weighed), from the middle of the formulation. Two aliquots from the sample were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M021/20). The remainder of the samples were retained for contingency.

STABILITY AND HOMOGENEITY
The suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix in Covance Study Number LC59WX. The stability at ambient temperature (15 to 25°C) and refrigerated (2 to 8°C) is 24 hours and 15 days, respectively.
Details on mating procedure:
Natural mating with New Zealand White bucks of established fertility at the supplier’s facility. Males and females were not closely related. After mating each female was injected intravenously with 25 i.u. luteinising hormone. The day of mating was considered Gestation Day 0 (GD0). The study animals arrived at the test facility on GD1 and were acclimated until GD5.
Duration of treatment / exposure:
Females were exposed from GD6 to GD28 by oral gavage.
Frequency of treatment:
Once daily.
Duration of test:
23 treatment days.
Doses / concentrationsopen allclose all
Dose / conc.:
180 mg/kg bw/day
Dose / conc.:
375 mg/kg bw/day
Dose / conc.:
750 mg/kg bw/day
No. of animals per sex per dose:
24 mated females/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for this study were selected based on the results of a preliminary embryo-fetal study in the New Zealand White rabbit with Sodium Trifluoroacetate in water at dose levels of 250, 500 or 750 (1000) mg/kg/day (Covance Study Number YQ44HR). In that study an initial dose at 1000 mg/kg/day resulted in some unanticipated adverse signs (unsteady, underactive behaviour). Consequently, the decision was made to reduce this dose level to 750 mg/kg/day for the remainder of the treatment period; at 750 mg/kg/day these signs improved, until no longer present by Day 12. For females treated at 250 or 500 mg/kg/day, isolated incidences of unsteady gait and decreased activity were similarly observed however to a much lesser magnitude and as such were not considered to be adverse. Therefore a high dose level of 750 mg/kg/day was considered to be a suitable high dose for this main embryo-fetal development study. The low and intermediate dose levels of 180 and 375 mg/kg/day (respectively) were selected to allow evaluation of any dose related trends.

Examinations

Maternal examinations:
VIABILITY:
A viability check was performed near the start and end of each working day. Animals were killed if they exhibited evidence for pregnancy loss.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. 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. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 1, 6, 12, 18, 24 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 1, 3 and daily from Day 6 to 29 after mating.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Time schedule for examinations: The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 2 to Day 28 after mating.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: All surviving study animals were euthanized and subjected to necropsy and caesarean section on GD 29. 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. The liver was weighed for animals killed at scheduled intervals and preserved in 10% Neutral Buffered Formalin.
Animals that experienced pregnancy lost were sacrificed on the day of detection and also subjected to necropsy.
Ovaries and uterine content:
For females surviving to term, the ovaries and uterine content was examined after termination. Examinations included:
• Gravid uterine weight (including cervix and ovaries)
• Number of corpora lutea
• Number of implantation sites
• Number of resorption sites (classified as early or late)
• Number of fetuses (live and dead).
Blood sampling:
Not included.
Fetal examinations:
All live fetuses were individually weighed and examined for external abnormalities. Fetus with external malformation was sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded. Approximately 50% of eviscerated fetuses were decapitated; heads were fixed in Bouin’s fluid and subject to free-hand serial sectioning for subsequent soft tissue abnormalities. Remaining eviscerated fetuses and torsos were fixed in Industrial Methylated Spirit and stained with Alizarin Red for subsequent skeletal examination.
Statistics:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett, 1937) was not significant at the 1% level. For pre-treatment data, analysis of variance was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using t-tests, with the error mean square from the one-way analysis of variance, were made. For all other analyses the F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1% level, Williams' test for a monotonic trend was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test (Dunnett 1955, 1964) was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. For pretreatment data, Kruskal-Wallis’ test (Kruskal and Wallis 1952, 1953) was used to test for any group differences. Where this was significant (p<0.05) inter group comparisons using Wilcoxon rank sum tests (Wilcoxon, 1945) were made. For all other analyses the H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1% level, Shirley's test for a monotonic trend was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test (Steel, 1959) was performed instead.
For liver weight data, analysis of covariance was performed using terminal body weight as covariate (Angervall and Carlstrom, 1963), unless non-parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the liver weight.
Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Indices:
- Pre-implantation loss (%): (No. of corpora lutea - No. of implantations) / No. of corpora lutea ×100%
- Post-implantation loss (%): (No. of implantations - No. of live fetuses) / No. of implantations ×100%
- Sex ratios of the live fetuses were calculated as the percentage of males per litter.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Signs observed in association with dose administration were limited to one female at 375 mg/kg/day that showed underactive behavior and a dilated pupil on Days 27 and 28 and one female at 750 mg/kg/day that showed irregular breathing and an unsteady gait on GD7. Although these incidences were isolated and inconsistent, these observations are similar to those seen on the preliminary study and as such, a relationship to treatment cannot be discounted.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two females died prior to scheduled termination. On GD27, a control female was observed to have a convulsion shortly after dosing and subsequently died. Macroscopic examination revealed a perforated trachea and consequently this death was attributed to dosing trauma. On GD27, a female receivinmg 750 mg/kg/day was killed for reasons of animal welfare following evidence for pregnancy loss. Macroscopic examination of this female revealed an enlarged spleen and uterine examination revealed eight corpora lutea and two resorbing implantation sites. Enlarged spleen was not apparent for any other females on study and as this pregnancy loss was an isolated incidence it was not attributed administration of Sodium Trifluoroacetate.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Group mean body weight gain during the treatment period (GD6-28) was unaffected by treatment at dose levels up to and including 750 mg/kg/day. Adjusted body weights on Day 29 after mating were calculated from the body weight at termination minus the gravid uterine weight. Body weight change values for the period Day 6-29 were also presented, after being adjusted for the contribution of the gravid uterus. On Day 29 of gestation, the gravid uterine weight for females that received 375 or 750 mg/kg/day was low when compared with Controls (p<0.05 and p<0.01, respectively). A decreased maternal adjusted body weight gain of 20, 48 and 56% of control was observed in the 180, 375 and 750 mg/kg bw/day groups, respectively. Detailed results are provided in Table 1 (see attached document).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
From Day 6 of gestation (Day 1 of treatment) food consumption was low in all treated groups when compared with Controls. Food intake remained low at 180 mg/kg/day until GD14, at 375 mg/kg/day until GD15 and at 750 mg/kg/day until GD16. At the end of the treatment period females receiving TFA showed high food consumption when compared with Controls (GD26-28). Detailed results are provided in Table 2 (see attached document).
Food efficiency:
not examined
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:
effects observed, treatment-related
Description (incidence and severity):
Body weight adjusted liver weights for females at scheduled termination on GD29 were high when compared with Controls (p<0.05 at 180 mg/kg/day; p<0.01 at 375 and 750 mg/kg/day); a dose response was apparent.
Detailed results are provided in Table 3 (see attached document).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination on Day 29 after mating revealed an irregular surface of the liver in one female, that received 375 mg/kg/day; there were no other findings that could be attributed to treatment with Sodium Trifluoroacetate.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the liver, minimal to moderate bile duct hyperplasia/fibrosis and minimal generalized hepatocellular hypertrophy was seen in females of all treated groups and exhibited a dose relationship. Bile duct hyperplasia can be a response to insult to the bile duct epithelium (Sahota et al., 2019), and is indicative of test article excretion in the bile. The bile duct finding appeared to have no adverse effect on maternal clinical condition, but in the absence of blood chemistry it is unknown whether there were any liver changes possibly attributable to the bile duct change. Therefore the hyperplasia/fibrosis, at the moderate severity which was observed at the mid and high dose is considered as adverse. Detailed results are provided in Table 4 (see attached document).
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female, administered 750 mg/kg/day, was killed on Gestation Day 27 due to suspected abortion. At necropsy, an enlarged spleen was noted. At microscopic examination of the liver, slight multifocal bile duct hyperplasia/fibrosis and minimally decreased generalized hepatocyte cytoplasmic rarefaction in the liver were seen. Bile duct hyperplasia/fibrosis was seen as a test article related finding in other females of this dose group. These findings were not considered to have contributed to the death of this animal, with the main contributory factor being due to suspected abortion.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Mean corpora lutea, the subsequent number of implantations and litter size were low for females that received 750 mg/kg/day; however, as treatment commenced at or around the time of implantation, this difference is considered unrelated to treatment. Post-implantation loss (%) was slightly high at 750 mg/kg/day but this difference did not achieve statistical significance. Detailed results are provided in Table 5 (see attached document).
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Mean number of resorptions (early or late) showed no effect of treatment. Detailed results are provided in Table 5 (see attached document).
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male fetal weights were significantly reduced at 375 and 750 mg/kg/day (p<0.01) and female fetal weights were significantly reduced at 750 mg/kg/day (p<0.05); a dose response was apparent.
Detailed results are provided in Table 5 (see attached document).
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Mean live litter size at 750 mg/kg bwt/day was slightly but significantly lower than that of the controls (6.1** cf. 8.3, p<0.01). This was due to a combination of the lower mean number of ova shed, as indicated by the numbers of corpora lutea, which occurred before the start of treatment (8.3** cf. 10.5, p<0.01) and was therefore not relevant for the interpretation of the study, and a slight non-significant increase in the extent of post-implantation loss (14.9% cf. 9.7%). Detailed results are provided in Table 5 (see attached document).
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Detailed results are provided in Table 5 (see attached document).
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Mean live litter size at 750 mg/kg bwt/day was slightly but significantly lower than that of the controls (6.1** cf. 8.3, p<0.01). This was due to a combination of the lower mean number of ova shed, as indicated by the numbers of corpora lutea, which occurred before the start of treatment (8.3** cf. 10.5, p<0.01) and was therefore not relevant for the interpretation of the study, and a slight non-significant increase in the extent of post-implantation loss (14.9% cf. 9.7%). Overall mean fetal weights and consequently mean total litter weights were reduced at 375 and 750 mg/kg/day (p<0.01); a dose response was apparent. Detailed results are provided in Table 5 (see attached document).
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two of 173 fetuses in two litters of the mid-dose group had omphalocele as external malformation. Gastroschisis was observed in one mid-dose fetus and one high-dose fetus. These findings were considered incidental due to low incidence (within the range of historical control data) and since there was no correlation to the dose. Detailed results are provided in Table 6 (see attached document).
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Multiple cervical/thoracic/lumbar/caudal vertebral and rib abnormalities; with associated minor abnormalities affecting the vertebrae, ribs, sternum and costal cartilages were increased at 375 and 750 mg/kg/day. However, most of the abnormalities showed no dose-correlation and/or were within the range of historical control data. Therefore, the relation to treatment is uncertain.
There was also an increased incidence of the minor findings 20 thoracolumbar vertebrae, unossified 5th sternebra and epiphyses, incompletely ossified cervical/thoracic vertebrae and metacarpals and forepaw flexure compared to concurrent control. As most of these abnormalities are variants within the population and were mostly within or just outside of the historical control range they are not considered adverse but may still be related to treatment.
Detailed results are provided in Table 6 (see attached document).
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
A number of visceral fetal abnormalities were recorded in all groups. Treatment at 375 or 750 mg/kg/day was associated with clear increase in the numbers of fetuses/litters with major abnormalities compared with Controls:
• An high incidence of the major eye abnormalities, multiple folded retina and absent aqueous/vitreous humour was associated with treatment at 375/750 mg/kg/day and the incidences were well above that in the concurrent controls and the historical control data (HCD) range. There were also single instances of retina ruptured into surrounding tissues, small/misshapen lens and microphthalmia in each of these groups with the ruptured retina not present in the HCD. There were also low incidences of the minor abnormalities small eye and haemorrhage aqueous/vitreous humour at both doses, and folded retinas at 750 mg/kg/day only, above the HCD range. At 180 mg/kg/day, one fetus had microphthalmia, with multiple folded retina and absent aqueous/vitreous humour; although these incidences were within the HCD range, since higher incidences of fetuses had these extremely rare eye abnormalities at higher doses a relationship to treatment could not be ruled out.
• Cardiovascular abnormalities, comprising of transposition of ascending aorta/pulmonary trunk; narrow/dilated ascending aorta and pulmonary trunk; double outlet ventricle(s) and ventricular septal defect were recorded in one or maximum two fetuses across all dose levels. Because these abnormalities occurred at low incidence, in concurrent controls (Transposition of ascending aorta/pulmonary trunk and Muscular ventricular septal defect) and/or were noted previously in historical controls and no clear dose response relationship was noted, the relationship with treatment is uncertain.
• Kidney abnormalities in fetuses of treated dams consisted of fused/displaced kidney (one fetus at 180 and 750 mg/kg/day), absent kidney and absent ureter (one fetus at 375 mg/kg/day). Because these abnormalities occured singly and/or are within the range of historical control data, a relationship with the treatment is uncertain.
• Cleft plate was observed in one high-dose fetus. This finding was considered incidental due to low incidence and with no dose-relationship. This is also covered by historical control data.
Detailed results are provided in Table 6 (see attached document).
Details on embryotoxic / teratogenic effects:
Fetal Eye Histopathology
Fetal eyes from the Bouin’s fixed tissue for fetuses that showed abnormality were subject to histopathological examination. Examination revealed slight to marked retinal folds in the majority (5 of 8) of fetuses whose dams were administered 375 mg/kg/day and in all fetuses (11 of 11) whose dams were administered 750 mg/kg/day and demonstrated a relationship to dose in terms of relative group incidences. Marked retinal folds were seen in the one fetus whose dam was administered 180 mg/kg/day. Lens degeneration was seen in several fetuses (6 of 20), and hemorrhage in the vitreous chamber was seen in a few fetuses (2 of 20), that had retinal folds. The majority of the fetuses that had retinal folds also had absence of aqueous and/or vitreous humor (13 of 20, generally bilateral) reported at macroscopic examination.
At microscopy, structural (anatomical) disorganisation of the eyes was seen in one male fetus and female fetus of each treated group whose dams were administered 375 or 750 mg/kg/day. The retinal rupture through the vitreous chamber reported at macroscopic examination could not always be reliably identified and/or confirmed at microscopy.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
< 180 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
external malformations

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
180 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
not specified

Any other information on results incl. tables

Results of Dose Formulations Analysis

The mean achieved concentrations of formulation samples taken from the first and last dose preparations were within 2% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 1%, confirming precise analysis.

Applicant's summary and conclusion

Conclusions:
The No-Observed-Adverse-Effect-Level (NOAEL) of sodium trifluoroacetate for maternal toxicity in rabbits was considered to be 180 mg/kg/day, and the NOAEL of sodium trifluoroacetate for embryo-fetal developmental toxicity in rabbits was considered to be <180 mg/kg/day.
Executive summary:

A GLP compliant prenatal developmental toxicity study with sodium trifluoroacetate was conducted in New Zealand White rabbits according to OECD Guideline 414. Groups of 24 time-mated female New Zealand White rabbits were treated daily with Sodium trifluoroacetate by oral gavage from gestation day (GD) 6 to GD28 at dose levels of 0, 180, 375 and 750 mg/kg bodyweight/day. Clinical condition, food consumption and bodyweights of the dams were recorded during the study and the females were killed on GD 29. At termination, the ovaries were examined for the numbers of corpora lutea, the gravid uteri were weighed and the contents were examined for numbers of implantations, early and late resorptions, and live and dead foetuses. Foetal and placental weights were recorded. At necropsy, all foetuses were examined externally and internally for thoracic and abdominal visceral changes. The heads were removed from one half of the foetuses and fixed to permit examination of internal head structures. The torsos and the remaining intact foetuses were processed, stained with alizarin red S and examined for skeletal changes.

In this study, 19/24, 21/24, 24/24, and 24/24 mated females were pregnant at 0, 180, 375 or 750 mg/kg/day, respectively. There were no treatment-related adverse effects upon group mean bodyweights or body weight gain; food consumption showed a slight dose-related reduction during the first part of the treatment period but was similar or slightly greater than that of the controls during the latter part of treatment. Adjusted body weights on Day 29 after mating were calculated from the body weight at termination minus the gravid uterine weight. On Day 29 of gestation, the gravid uterine weight for females that received 375 or 750 mg/kg/day was low when compared with Controls (p<0.05 and p<0.01, respectively). The maternal adjusted body weight gain was decreased by 20, 48 and 56% compared to control at 180, 375 and 750 mg/kg bw/day, respectively. Body weight adjusted liver weights for females at scheduled termination on GD29 were high when compared with Controls; a dose response was apparent. Histopathological examination of the liver revealed minimal to moderate bile duct hyperplasia/fibrosis and minimal generalized hepatocellular hypertrophy in all treated group; a dose response was apparent.

Litter data as assessed by the number of implantations, resorptions, live young and sex ratio, and the extent of pre-/post-implantation loss did not show any differences that could be attributed to maternal treatment. Mean placental weights were unaffected by maternal treatment. Mean live litter size at 750 mg/kg bwt/day was slightly but significantly lower than that of the controls (6.1** cf. 8.3, p<0.01). This was due to a combination of the lower mean number of ova shed, as indicated by the numbers of corpora lutea, which occurred before the start of treatment (8.3** cf. 10.5, p<0.01) and was therefore not relevant for the interpretation of the study, and a slight non-significant increase in the extent of post-implantation loss (14.9% cf. 9.7%). Overall mean fetal weights and consequently mean total litter weights were reduced at 375 and 750 mg/kg/day (p<0.01); a dose response was apparent.

Detailed fetal examination revealed an increased incidence of major abnormalities at 375 or 750 mg/kg bw/day, predominantly affecting the eyes. The incidences of multiple folded retina and absent aqueous/vitreous humor were above the concurrent and historical control data (HCD) range. At 180 mg/kg/day one fetus had microphthalmia, with multiple folded retina and absent aqueous/vitreous humour; although these incidences were within the HCD range, since higher incidences of fetuses had these extremely rare eye abnormalities at higher doses a relationship to treatment could not be completely ruled out.

Based on the maternal liver pathology that showed moderate bile duct hyperplasia/fibrosis at 375 and 750 mg/kg/day, 180 mg/kg/day was considered to be the maternal no observed adverse effect level (NOAEL). Due to the fetal abnormalities that were observed at all dose levels, a NOAEL for embryo-fetal developmental toxicity could not be established in this study.