Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 21, 2017 to October 05, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Light beige paste
Details on test material:
Identification: Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt
Appearance: Light beige paste
Batch: CH 186527-01-53
Purity/Composition: UVCB
Test substance storage: At room temperature
Stable under storage conditions until: 20 February 2018 (expiry date)

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
- Weight at study initiation: 175 and 266 g
- Fasting period before study:
- Housing: On arrival and following randomization females were housed individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The room in which the animals were kept was documented in the study records. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group and animal number.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 2 d prior to experimentation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (actual mean 21°C)
- Humidity (%): 40 to 70% (actual mean relative humidity 54 to 77%)
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark cycle was maintained.

IN-LIFE DATES: From: To:17 Aug 2017 (Last date of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Trial preparations were performed at the test facility to select the suitable vehicle and to establish a suitable formulation procedure. Trial preparation formulations were not used for dosing and were discarded after the assessment was complete. These trial preparations had a non-GLP status and were carried out in the quality assured environment of the test facility. Test substance dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 5 h after adding the vehicle to the test substance dosing formulations were kept at room temperature until dosing. If practically
possible, the dosing formulations and vehicle were continuously stirred until and during dosing. No adjustment was made for specific gravity of the vehicle and test substance. A factor of 1.88 was used to correct for the purity/composition of the test substance.
Any residual volumes were discarded.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Water
- Source: Elix, Millipore S.A.S., Molsheim, France
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical method: The determination of test susbtnace in formulations Using LC-MS/MS and multisite study for the long term stability. The lower limit of quantitation was 1.00 mg/g of the validated method and the calibration curve ranged from 1.00 to 25.0 mg/L.

Sample injections: The analytical run for the determination of the accuracy of preparation and homogeneity of the test substance formulations was acquired in the following order:
- Analytical blank sample
- Calibration curve
- Analytical blank sample
- Analytical blank sample
- Procedural recovery sample high and low (replicate 1)
- Analytical samples
- Procedural recovery samples high and low (replicate 2)
Calibration solutions were injected in duplicate. Test samples and procedural recovery samples were
analyzed by single injection.

Analytical verification of doses: No test substance was detected in the Group 1 (control) formulation. The concentrations analyzed in the formulations of Groups 2 (100 mg/kg bw), 3 (300 mg/kg bw) and 4 (1000 mg/kg bw) were in agreement with the
target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 prepared were homogeneous (i.e. coefficient of variation ≤ 10%). The reinjection stability was proven for at least 4 d.
Details on mating procedure:
- Impregnation procedure: On 25 Jul 2017 and 27 Jul 2017, time-mated female Wistar Han Rats were received from Charles River Laboratories Deutschland, Sulzfeld, Germany.
- The females arrived on Day 0 or Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating). They were 10-14 weeks old and weighed between 175 and 266 g at the initiation of dosing. A health inspection was performed upon receipt of the animals.
Duration of treatment / exposure:
14 d
Frequency of treatment:
once daily oral gavage 7 days a week
Duration of test:
Day 6 to 20 post-coitum.
Study plan on 16 Jun 2017.
Study was initiated on 30 Jul 2017.
The in-life phase of the main study was completed on 17 Aug 2017.
The experimental start date was 21 Jun 2017.
the experimental completion date was 05 Oct 2017.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
22 (females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range-finding study
- Rationale for animal assignment (if not random): One the day of after receipt, animals were assigned to groups by a computer-generated random algorithm according to body weights

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, in the morning and at the end of the working day.
- Cage side observations checked in table [No.?] were included: yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed at least once daily, beginning on Day 2 post-coitum and lasting up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on 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
- Food consumption was quantitatively measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day # Day 21 post-coitum.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes / No / No data
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
Fetal examinations:
- External examinations: Yes
- Visceral Examinations: Yes
- Skeletal Examinations: Yes
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% and 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.

Comparision of groups:
Group 2 (100 mg/kg bw) vs. Group 1 (Control)
Group 3 (300 mg/kg bw) vs. Group 1 (Control)
Group 4 (1000 mg/kg bw) vs. Group 1 (Control)

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).
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.
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:
No indices were calculated as percentages were used.
Historical control data:
Historical control data attached under attched background material.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
no effects observed
Description (incidence and severity):
Food consumption similar to control group.
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:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
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
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
Mortality: No mortality occurred during the study period.

Clinical Observations: No treatment-related clinical signs were noted during the observation period. Incidental findings noted across the groups during the treatment period included alopecia, scabs and chromodacryorrhoea, which occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered to be unrelated to treatment.

Body Weights and Body Weight Gains: Mean body weights, body weight gain and weight gain corrected for gravid uterus of treated animals remained in the same range as controls over the study period.

Food Consumption: Food consumption before and after correction for body weight of treatment animals was similar to the control level over the study period.

Gross Pathology: Macroscopic observations at necropsy revealed no treatment-related findings.

Maternal Pregnancy Data: Except for two females at 100 mg/kg bw, all females were pregnant and had litters with viable fetuses. One control female and one female at 100 mg/kg delivered their offspring (viable fetuses only) on the day of scheduled necropsy (Day 21 post-coitum). At the incidence observed and in absence of a dose-response, these findings were considered not to be treatment-related. The number of pregnant females, corpora lutea and implantation sites, and pre- and postimplantation loss were unaffected by treatment up to 1000 mg/kg bw.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects up to the highest tested dose level.

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
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
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
effects observed, non-treatment-related
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed up to the highest tested dose level

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions, NOAEL for the prenatal developmental toxicity for the test substance for maternal toxicity and developmental toxicity was determined to be >1000 mg/kg bw.
Executive summary:

A study was conducted to determine the prenatal developmental toxicity of the test substance, according to OECD Guideline 414, in compliance with GLP. Twenty two time-mated female Wistar Han rats per group were treated with test substance, from Day 6 to 20 post-coitum, by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received the vehicle, water, alone. The dose levels in this study were selected to be 0, 100, 300, 1000 mg/kg/day, based on the results of the dose range finder study. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated. Maternal toxicity (F0 -generation), mortality/morbidity, clinical signs, body weights, food consumption, gross necropsy findings, and number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the Fetal (F1 -generation) the number of fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, externa, visceral and skeletal malformations and developmental variations. No mortalit and no treatment-related changes were noted in any of the maternal parameters investigated in this study (i.e. clinical appearance, body weight, food consumption, macroscopic examination) at any dose level. No treatment-related changes were noted in any of the developmental parameters investigated in the study (i.e. the number of fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, external, visceral and skeletal malformations and developmental variations) at any dose level. Under the study conditions, the NOAEL for developmental toxicity was determined to be >1000 mg/kg/day (Van de Ven, 2018).