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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

Constituent 1
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: 71 to 78 days (at Day 0 of gestation)
- Weight at study initiation: 235-279 g (at Day 0 of gestation)
- Fasting period before study: no
- Housing: 1 animal per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days before commencement of pairing

DETAILS OF FOOD AND WATER QUALITY: SDS VRF1 Certified pelleted diet; potable water from public supply

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed and uniformly mixed with corn oil.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the substance is not soluble in water
- Concentration in vehicle: 0, 4, 6.88, 13.75 mg/ml
- Amount of vehicle (if gavage): 4 ml / kg bw, the low dose group was dosed at 3.4 ml / kg bw
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of dose formulations were analysed by HPLC with UV-detector. Mean concentrations were within an acceptable range of the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1 : 1
- Length of cohabitation: until evidence of mating
- Verification of same strain and source of both sexes: yes, a colony of stud males was maintained
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 6 till day 19 of gestation
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
13.5 mg/kg bw/day (actual dose received)
Dose / conc.:
27.5 mg/kg bw/day (actual dose received)
Dose / conc.:
55 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels selected for investigation in this study (0, 13.5, 27.5, and 55 mg/kg/day) were based on the results of a sub-acute repeated dose study with the reproduction / developmental toxicity screening. In this reproduction screening study there were deaths at 60 mg/kg body weight/day. Therefore dosing was below this dose level. Tolerance of 55 mg/kg/d was confirmed in a preliminary study.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations : visual inspection for ill-health or reaction to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 0, 5, 12, 18, and 20

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 3, and daily from 6-20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: full macroscopic examination of tissues
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
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of fetuses (live and dead)
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations external: Yes: all per litter
Statistics:
The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. For pretreatment 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 comparisons t/The F1 approximate test was applied. This test is designed to detect significant departure from monotonicity of means when the main test for the comparison of the means is a parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The test statistic compares the mean square, NMS, for the deviations of the observed means from the maximum likelihood means, calculated under a constraint of monotonicity with the usual error mean square, EMS. The null hypothesis is that the true means are monotonically ordered. The test statistic is F1 = NMS/EMS which can be compared with standard tables of the F-distribution with 1 and error degrees of freedom. 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.
Indices:
Prenatal losses were separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea – Number of implantations) x 100 / Number of corpora lutea

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations – Number of live fetuses) x 100 / Number of implantations

All group values and SD (as appropriate) were calculated from the individual litter values
Historical control data:
Historical control data from 7 studies performed in the last 10 months preceding the actual study with the same rat strain are provided in the report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Some animals in all dose groups had salivation for a short time after dosing during the second week of treatment that showed dose relationship.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see table of body weight development attached as background material.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Description (incidence and severity):
no differences in body weight gain and food consumption between dosed animals and control animals.
Water consumption and compound intake (if drinking water study):
not specified
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:
no effects observed
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:
effects observed, non-treatment-related
Description (incidence and severity):
The litter from one control animal showed early resorption; therefore the assessment of litter data is based on 19 animals in the control group. In all dose groups the litters of each 20 animals were assessed (see table on litter data attached as background material).
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
The litter from one control animal showed early resorption; therefore the assessment of litter data is based on 19 animals in the control group (see table on litter data attached as background material).
Early or late resorptions:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Dead fetuses:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 55 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: NOAEL = highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
see table on litter and fetal weights attached as background material.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
see table on litter and fetal weights attached as background material.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the high dose group there was an increased incidence of delayed/increased ossification/unossified cranial centers compared to the concurrent control group, but it was within historical control data (see table on skeletal abnormality findings attached as background material).
Visceral malformations:
no effects observed
Description (incidence and severity):
see table on visceral abnormality findings attached as background material.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 55 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
dams were dosed
Sex:
male/female
Remarks on result:
other: NOAEL = highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
no effects of developmental toxicity or teratogenicity observed up to and including the highest dose level of 55 mg/kg body weight/day.