Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02 August 2018 to 19 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study Study performed according to OECD test guideline No. 414 and in compliance with GLP.

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:
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:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
(Inspected between 2017-11-28 and 2017-12-06 / Signed on 2018-01-22)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
- Substance type: Colourless liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light container flushed with nitrogen

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 10-14 weeks old
- Weight at study initiation: 185 and 262 g
- Fasting period before study: no
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm). The cages contained appropriate bedding (Lignocel S8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles.
- Diet (e.g. ad libitum): Prepared powder diets were provided ad libitum throughout the study, except during designated procedures. During the acclimatization period, pre-treatment (Days 0 to 6 postcoitum), animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
The standard powder rodent diet (without the test item) was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.
- Acclimation period: at least 5 days before the commencement of administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 49 to 79%
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2018-08-19 To: 2018-09-06

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently step by step mixed with the remaining required amount of the diet.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the study period. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
The diet was provided in stainless steel containers, covered by a stainless steel grid to prevent spillage.
The same diets remained in the food hopper for a maximum of 5 days. On the day of weighing, the remaining food in the food hopper was replaced with new diet retrieved from the freezer (≤-15°C) and acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag

DIET PREPARATION
Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was used. Diets were prepared freshly for use at room temperature for a maximum of 5 days. Diets were kept in the freezer (≤ -15°C) until use, if not used on the day of preparation for a maximum of three weeks (stability was confirmed under Test Facility Study No. 20153658 (analytical Method Development and Validation Study)). Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 5 days for supplementing food during the respective food consumption measurement interval.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet preparation samples were collected for analysis during Week 1.
- Concentration analysis (all groups)
Duplicate sets of samples (approximately 5 g) were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.
- Homogeneity analysis (groups 2 and 4)
Duplicate sets of samples (approximately 5 g) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was < 10%. After acceptance of the analytical results, backup samples were discarded.
- Stability analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20153658) demonstrated that the test item is stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20153658
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant (arrived on Day 0 or Day 1 post-coitum)
Duration of treatment / exposure:
from Day 6 to Day 21 post-coitum, inclusive
Frequency of treatment:
Ad libitum
Duration of test:
Up to Dat 21 post-coitum, inclusive
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:
22
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
MORTALITY / MORIBUNDITY
- Time schedule: twice daily, in the morning and at the end of the working day

CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy.
Cage debris was examined to detect premature birth.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles/containers.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 (except one female with early delivery)
- Organs examined: external, thoracic and abdominal examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS: Yes
The liver was weighed at necropsy for all scheduled euthanasia animals, with the exception of female no. 48 that delivered early. Organ to body weight ratio (using the body weight on Day 21 post-coitum) were calculated.
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: weight of the (gravid) uterus (not for female no. 48 that delivered early); sex of each fetus based on the ano-genital distance.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [half per litter]
- Visceral examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible.
Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
6.1. Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
6.2. Non-Parametric
Datasets with at least 3 groups were compared using a Steel-test (many-to-one rank test).
Mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and post-implantation loss, and sex distribution were compared using the Mann Whitney test.
Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal
malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences.
6.3. Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level.
The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.
Indices:
Pre-implantation loss (%): (number of corpora lutea - number of implantation sites) / number of corpora lutea x 100.
Post-implantation loss (%): (number of implantation sites - number of live fetuses) / number of implantation sites x 100.
Viable fetuses affected/litter (%): number of viable fetuses affected/litter / number of viable fetuses/litter x 100.
Historical control data:
Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality)
Gestation Day 21
Study Date Range: 2014 - 2017 (37 studies)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Piloerection was noted in 10/22 females at 11000 ppm, starting after four days of treatment (from post-coitum Day 9 onwards) and generally lasting 3-11 days. No other clinical signs were noted in treated females that were considered to be related to treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
On Day 9 post-coitum, a mean body weight loss of 1% was noted in females at 11000 ppm.
One female presented with moderate body weight loss (i.e. 17%), nine other females had a body weight loss of 1-4%. Remaining females gained no weight or had a slight body weight gain. A statistically significantly reduced body weight gain was noted on Days 12 and 15 post-coitum in females at 11000 ppm. At 5000 ppm, a slightly lower body weight gain was noted on Day 9 post-coitum. The resulting differences in mean body weight at 5000 and 11000 ppm compared with concurrent control did not exceed 5%. On Day 21 post-coitum, body weight and body weight gain of animals at 5000 and 11000 ppm had recovered to normal values.
No treatment-related changes were noted in the body weight (gain) of females at 500 ppm and body weight gain corrected for gravid uterus up to 11000 ppm. The statistical significantly increased body weight gain and body weight gain corrected for gravid uterus noted for females at 500 ppm were considered unrelated to treatment in the absence of a dose related response.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 5000 and 11000 ppm, mean food consumption before or after allowance for body weight was statistically significantly reduced with 15 and 42%, respectively, over Days 6-9 postcoitum when compared with control mean. This was followed by recovery in subsequent intervals. At 500 ppm, food consumption before or after correction for body weight remained in the same range as controls over the study period.
Mean test item intake over the study period was as follows:
- Nominal 55 ppm => 52 mg/kg bw/day [42-57]
- Nominal 5000 ppm => 485 mg/kg bw/day [401-552]
- Nominal 11000 ppm => 1012 mg/kg bw/day [767-1144]
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
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):
A treatment-related increase in absolute and relative liver weight was noted in females at 5000 and 11000 ppm. Mean relative liver weight was increased by 5 and 7% when compared with concurrent control for females at 5000 and 11000 ppm, respectively (statistically significant at 11000 ppm only).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of corpora lutea and implantation sites, and pre-implantation loss in the control and test groups were similar and in the range of normal biological variation.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
A higher incidence of early resorptions was noted in females at 11000 ppm when compared with concurrent control (8.0% vs 5.9%). This was mainly caused by two females which had 7/13 and 5/14 early resorptions/implantation sites. The incidence of early resorptions at 11000 ppm remained within the historical control data range1, was observed in the absence of a clear dose-related response and was not statistically significantly different from control.
The higher incidence was therefore considered to represent normal biological variation and unrelated to treatment. Note: as result of the higher incidence of early resorptions, the incidence of post-implantation loss was similarly increased in the 11000 ppm group. The incidence remained within the historical control data range.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
effects observed, non-treatment-related
Description (incidence and severity):
There was one female at 5000 ppm (A048) that delivered offspring on the day of scheduled necropsy (11 viable fetuses). In the absence of a dose-related response, this was considered to be unrelated to treatment.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females were pregnant and had litters with viable fetuses.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 11 000 ppm
Based on:
test mat.
Remarks:
corresponding to an overall mean test item intake of 1012 mg/kg bw/day
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
Description (incidence and severity):
Fetal body weights (both sexes) were considered unaffected by treatment up to 11000 ppm.
Mean combined (male and female) fetal body weights were 5.4, 5.4, 5.2 and 5.2 gram for the control, 500, 5000 and 11000 ppm groups, respectively.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment up to 11000 ppm.
Mean sex ratios (males:females) were 49:51, 46:54, 56:44 and 53:47 for the control, 500, 5000 and 11000 ppm groups, respectively.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on litter size of any group.
Mean litter sizes were 10.4, 9.7, 10.1 and 10.8 fetuses/litter for the control, 500, 5000 and 11000 ppm groups, respectively.
Fetal body weights (both sexes) were considered unaffected by treatment up to 11000 ppm.
Mean combined (male and female) fetal body weights were 5.4, 5.4, 5.2 and 5.2 gram for the control, 500, 5000 and 11000 ppm groups, respectively.
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on external morphology following treatment up to 11000 ppm.
Two externally malformed fetuses were observed in this study. Fetus A028-03 (500 ppm) had a cleft palate and lip and fetus A053-08 (5000 ppm) had short hind limb(s) with brachydactyly, bent tail and no anogenital tubercle. Skeletal examination substantiated the cleft palate (fetus A028-03) and brachydactyly (A053-08) findings, but there was no apparent skeletal origin for the hind limb(s) and tail findings in the latter fetus. Due to the single
occurrence and occurrence in low and mid dose Groups, these malformations were considered to be of spontaneous origin.
External variations were not observed in any of the treatment groups
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related effects on skeletal morphology following treatment up to 11000 ppm.
Aside from the underlying skeletal malformations in fetus A053-08 at 5000 ppm and fetus A028-03 at 500 ppm, the latter fetus also had a costal cartilage anomaly. In addition, two littermates (A037-01 and -05) at 500 ppm were observed with bent limb bones. The low incidence and group distribution of these malformations do not suggest a treatment relationship. Furthermore, as they have been noted previously in historical controls, they were considered to be spontaneous in origin.
Skeletal variations occurred at an incidence of 72.5%, 68.7%, 67.0% and 66.4% per litter in Groups 1, 2, 3 and 4, respectively. All observed skeletal variations occurred in the absence of a dose-related trend, infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data. Therefore, they were not considered treatment related.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no treatment related effects on visceral morphology following treatment up to 11000 ppm.
Visceral malformations were not observed and the visceral variations that were noted occurred in the absence of a dose-related trend, occurred infrequently, and/or at frequencies that were within the range of available historical control data.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 11 000 ppm
Based on:
test mat.
Remarks:
corresponding to a maternal overall mean test item intake of 1012 mg/kg bw/day
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

Any other information on results incl. tables

Dose formulation analysis:

Accuracy

The concentrations analyzed in the diets of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%).

In the Group 1 diets, no test item was detected.

Homogeneity

The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Applicant's summary and conclusion

Conclusions:
Based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) was established as being at least11000 ppm (corresponding to an overall mean test item intake of 1012 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 dietary administration at dose levels of 0, 500, 5000 or 11000 ppm (corresponding to 0, 52, 485 or 1012 mg/kg bw/day) from days 6 through 21 of gestation. The dose levels in this study were based on the results of an OECD 407 and an OECD 421 studies in CD rats.

Chemical analyses of dietary preparations were conducted once during the study to assess accuracy and homogeneity.

The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, liver weights, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations. Dietary analyses confirmed that formulations of test item in diets were prepared accurately and homogenously.

 

No adverse changes were noted in the maternal parameters (i.e. clinical appearance, body weight, food consumption, macroscopic examination, liver weights, number of corpora lutea and implantation sites, (gravid) uterine weight and uterine contents) or in the developmental parameters (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations) examined in this study. There were a few non-adverse findings, which are described below.

 

Ten out of 22 females at 11000 ppm showed piloerection. As this clinical sign was transient in most females and no other signs of toxicity were observed, it was regarded as non-adverse.

Food consumption (absolute and relative) of females at 5000 and 11000 ppm was reduced during the first days of exposure, when compared with control mean. A concurrent body weight loss was noted on Day 9 post-coitum in females at 11000 ppm and a decrease in body weight gain was noted in these females up to Day 15 post-coitum. For females at 5000 ppm, a slightly reduced body weight gain was noted on post-coitum Day 9 only. The resulting differences in mean body weight at 5000 and 11000 ppm compared with concurrent control did not exceed 5%. Based on complete recovery at the end of treatment, the observed differences in food consumption and body weight (gain) were considered non-adverse. Liver weights (absolute and relative) were increased in females at 5000 and 11000 ppm. Mean relative liver weight was increased with 5 and 7% when compared with concurrent control for females at 5000 and 11000 ppm, respectively (statistically significant at 11000 ppm only). As the observed increase remained minimal (<10%), it was considered non-adverse.

 

No developmental toxicity was observed in the 500, 5000 and 11000 ppm groups.

In conclusion, based on the results in this prenatal developmental toxicity study the maternal and developmental No Observed Adverse Effect Level (NOAEL) was established as being at least 11000 ppm (corresponding to an overall mean test item intake of 1012 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.