Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

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:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: approx. 71 days (at Day 0 of gestation)
- Weight at study initiation: 229-287 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:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amounts of test material were weighed and dissolved in water.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0, 10, 30, 100 mg/ml
- Amount of vehicle (if gavage): 10 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.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 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, 100, 300 and 1000 mg/kg/day) were based on the results of a sub-acute repeated dose study with the reproduction / developmental toxicity screening.

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:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female receiving 300 mg/kg/day (No. 50) was killed for welfare reasons on Day 19 of gestation due to sudden and marked decline in general clinical condition. After dose administration on Day 19 of gestation, signs of salivation, piloerection, laboured respiration and dry rales were apparent. Having previously shown normal body weight gain to Day 18 of gestation, between Day 18 to 19 of gestation bodyweight loss of 12g was observed (3% total body weight). Food consumption had been unaffected. Macroscopic examination revealed that the esophagus was distended and contained clear/viscous fluid and food material; the uterus contained 18 implantation sites, all with live young. In the absence of similar events in this or higher dose groups, this isolated premature death was considered not to be associated with THEIC administration.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see figure 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:
no effects observed
Description (incidence and severity):
see table on litter data attached as background material.
Total litter losses by resorption:
no effects observed
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:
>= 1 000 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):
The incidence of major and minor abnormalities and skeletal variants showed no relationship to maternal treatment with THEIC.
At 1000 mg/kg/day there was a slight increase in the fetal and litter incidence of medially thickened/kinked ribs compared to concurrent control and outside of Historical Control Data (HCD) range. Medially thickened/kinked ribs are widely accepted as a skeletal variant which are likely repairable via post-natal skeletal remodeling; in the absence of any other fetal abnormalities this skeletal variant is not adverse in isolation, would be of no consequence to the long term survival of the fetuses and was therefore considered likely to be an incidental finding with no relationship to maternal treatment.

see table on abnormality findings attached as background material.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg/day there was a slight increase in incidence of left umbilical artery compared to concurrent control but within HCD range. During prenatal development a spontaneous closure of either the left or right umbilical artery occurs, and most commonly this occurs in the left umbilical artery. After birth their function of facilitating blood flow from/to the placenta is redundant and the artery then forms the medial umbilical ligament. The retention of the left umbilical artery (rather than the right) is classified as a non-adverse variation, and since the incidence at 1000 mg/kg/day was unaffected the slight increase in incidence of left umbilical artery in this study was considered an incidental finding and of no relationship to maternal treatment.

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:
> 1 000 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 1000 mg/kg body weight/day.