Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 29 May to 30 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD TG No. 414 without any deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other:
Version / remarks:
OPPTS 870.3800 Reproduction and Fertility Effects, August 1998.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: colourless liquid
- Storage condition of test material: Room temperature, under nitrogen

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, CT9 4LT, England
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 210 g to 319 g.
- Fasting period before study: none
- Housing: individually in solid-floor cages with appropriate bedding provided
- Diet: ad libitum (pelleted rodent diet, VRF1 (manufactured by SDS) supplied by Charles River (UK) Limited, Margate, Kent, CT9 4LT, England)
- Water: ad libitum
- Acclimation period: at least 2 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 23°C
- Humidity (%): 40% to 70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2018-05-29 To: 2018-08-30

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% (high viscosity)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A weighed quantity of test item was added to the final preparation container, then 20 % of the final required volume of vehicle was added and stirred vigorously to form an emulsion. After further addition of vehicle and mixing, the resultant suspension was mixed with a laboratory homogeniser and then stirred for a minimum of 20 minutes thereafter.
Formulations were divided into daily aliquots and were stored refrigerated (2 °C to 8 °C) until at least 20 minutes before dosing on the day of use, when they were stirred before the start of dosing until the completion of their use for dosing, to ensure thorough re-suspension and homogeneity.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on previous toxicology studies
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability: Stability of test item formulations prepared at concentrations of 1 to 100 mg/mL, spanning those used in this study (10 to 100 mg/mL), were examined in an earlier formulation validation study. Those formulations were found to be stable for 8 and 14 days when stored at room temperature (15 to 25 °C) and refrigerated (2 to 8 °C), respectively.
- Homogeneity and achieved concentrations: Duplicate samples were taken from the top, middle and bottom of each test item formulation prepared for use on the first day of dosing and on one occasion towards the end of the dosing period. One set of samples was analysed using a validated method (3) to confirm homogeneity and achieved concentrations by analysis by a gas chromatographic assay using flame ionisation detection. On these occasions, duplicate samples were also taken from the vehicle used to dose Controls and were analysed to confirm absence of test item.
Analysis of the samples confirmed homogeneity and achieved concentrations as well as absence of the test item in the vehicle. All remaining samples were retained as a contingency and stored refrigerated (approximately 2 ºC to 8 °C) and discarded once the final formulation analysis results were accepted.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
From Day 6 to Day 19 of gestation inclusive
Frequency of treatment:
Once daily
Duration of test:
From Day 6 to Day 20 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of results from existing toxicity data
- Rationale for animal assignment (if not random): random

Examinations

Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and morbidity and were given a detailed clinical examination daily from the start of treatment

BODY WEIGHT: Yes
- Time schedule for examinations: by the supplier on Day 0 of gestation. At Sequani, body weights were recorded for all females daily from Day 5 to Day 20 of gestation.

FOOD CONSUMPTION : Yes
- The amount of food consumed by each animal was recorded over Days 6 to 9, 9 to 12, 12 to 15, 15 to 18 and 18 to 20 of gestation

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The cranial and thoracic cavities were opened, a full internal examination was performed, and all macroscopic abnormalities were recorded.
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 (right and left horn)
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Data were processed to give group mean values and standard deviations, where appropriate.
Where the data allowed, the following methods were used for statistical analysis, comparing Groups 2, 3 and 4 against Group 1.
Depending on the nature of the data set that was to be analysed, appropriate tests were applied, Where parametric tests were appropriate they were preceded
by a check for homogeneity of variance using the Levene test and, where available, the Shapiro-Wilks test for normality. If either of these two assumptions failed, a log transformation was applied before retesting. If the transformation failed, appropriate non-parametric tests were applied.
Proportions of foetuses affected were treated as continuous non-parametric data, using onesided step-wise Jonckheere Tests.
Probability values of less than 5 % were regarded as providing sufficient evidence to reject the null hypothesis and therefore statistical significance was identified at the p<0.05 level. For illustrative purposes, significance levels of p<0.01 and p<0.001 were also noted.
Indices:
Pre-implantation loss (%) = (no. of corpora lutea – no. of implantation sites) / no. of corpora lutea x 100
Post-implantation loss (%) = (no. of implantation sites – no. of live foetuses) / no. of implantation sites x 100
Historical control data:
Included in the report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs in the surviving animals following administration of the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were 2 deaths during the study, both were unrelated to the test item. Female 55, given 300 mg/kg/day, was euthanised on Day 12 of gestation due to 9 % body weight loss between Days 11 and 12 of gestation and clinical signs of pale extremities, cold body surface, rapid breathing and hunched postured. At necropsy, the lungs were red and the thoracic cavity had clear fluid contents.
Female 74, given 1000 mg/kg/day, was euthanised on Day 14 of gestation due to red discharge from the vulva. At necropsy, there was red contents in the vagina and the thymus was large.
Due to their isolated nature, these deaths were considered not to be related to the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Following the first day of administration, at 1000 mg/kg/day, there was a slight, but statistically significant, mean body weight loss (p≤0.001). In the group given 300 mg/kg/day, mean body weight gain was also slightly lower than Controls (p≤0.05) but from Day 7 of gestation, mean body weight gain in all groups was similar to, or in excess of, Controls such that by Day 20 of gestation, all body weight values were comparable across all groups.
There was no effect on the terminal body weight adjusted for the weight of the gravid uterus.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, group mean food intake was significantly lower than Controls from Day 6 to Day 9 of gestation (p≤0.001). Thereafter food intake was similar to Controls.
There was no effect on food intake following administration at 100 or 300 mg/kg/day
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:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
On Day 20 of gestation, there were 20, 21, 21 or 20 females pregnant with live foetuses in the groups given 0, 100, 300 or 1000 mg/kg/day, respectively. There was no effect of test item
administration on the pregnancy data.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effect observed

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There was no effect of the test item administration on the overall incidences of minor and variant abnormalities.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were statistically significant increases in a few minor and variant skeletal findings, largely relating to changes in the extent of ossification (p<0.05 and
p<0.01). These abnormalities were largely within the background data range and/or had no dose response. Only 3 isolated skeletal changes were outside the background range and statistically significantly higher than Controls at 300 or 1000 mg/kg/day (incomplete ossification of the cervical vertebral neural arch, interrupted costal cartilage and incomplete ossification of the sternebra). Although statistically significantly higher than Controls at 300 or 1000 mg/kg/day, these findings are isolated and disparate in nature and are considered unrelated to the test item.
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect observed

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Administration of the test item to the pregnant Crl:CD(SD) rat at 100, 300 or 1000 mg/kg bw/day once daily by oral gavage from Days 6 to 19 of gestation inclusive, was generally well tolerated at all dose levels. A mild and transient effect on maternal food consumption and weight gain was observed over the first day of administration on Day 6 of gestation, with no effect on the developing conceptus or foetus. On this basis, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and the No Observed Effect Level (NOEL) for embryo-foetal development were considered to be ≥ 1000 mg/kg bw/day.
Executive summary:

In a developmental toxicity study performed according to OECD TG No. 404 and in accordance with GLP, the test substance was administered to 22 time-mated female CD rats via oral gavage administration at dose levels of 0, 100, 300 or 1000 mg/kg bw/day at a dose volume of 10 mL/kg bodyweight from days 6 through 19 of gestation. The dose levels in this study were based on the results of an OECD 407 and an OECD 415 studies in CD rats.

Body weights, food intake and clinical observations were recorded. The animals were killed on Day 20 of gestation, a necropsy performed, and the internal organs examined for gross abnormalities. The progress and outcome of pregnancy were assessed and maternal dead body weight, gravid uterus and placenta weights were recorded. The foetuses were removed from the uterus, weighed, the sex determined and examined for external, visceral, skeletal and cartilage abnormalities.

There were no test item-related deaths or clinical observations in pregnant female rats that were related to the test item administration at any dose level.

Following the first day of administration on Day 6 of gestation, in the group given 1000 mg/kg bw/day, less food was eaten, accompanied by a slight but statistically significant mean body weight loss. In the group given 300 mg/kg bw/day, mean body weight gain was also slightly lower than Controls after the first day of administration on Day 6 of gestation. From Day 7 of

gestation, however, food intake and mean body weight gain in all groups were similar to, or in excess of Controls, such that by Day 20 of gestation, all body weight values were similar across

the groups, and mean gravid uterus weight when adjusted for body weight was comparable with Controls.

On Day 20 of gestation, there were 20, 21, 21 or 20 females pregnant with live foetuses in the groups given 0, 100, 300 or 1000 mg/kg bw/day, respectively. There was no effect of the test item on any of the pregnancy parameters.

There were no foetal abnormalities that were considered related to the test item administration.

Administration of the test item to the pregnant Crl:CD(SD) rat at 100, 300 or 1000 mg/kg bw/day once daily by oral gavage from Days 6 to 19 of gestation inclusive, was generally well tolerated at all dose levels. A mild and transient effect on maternal food consumption and weight gain was observed over the first day of administration on Day 6 of gestation, with no effect on the developing conceptus or foetus. On this basis, the No-Observed-Adverse-Effect-Level (NOAEL) for maternal toxicity and the No Observed Effect Level (NOEL) for embryo-foetal development were considered to be ≥ 1000 mg/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.