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Toxicological information

Developmental toxicity / teratogenicity

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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
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
405-370-0
EC Name:
-
Cas Number:
6334-25-4
Molecular formula:
C14H28N2O6
IUPAC Name:
N,N,N',N'-tetrakis(2-hydroxyethyl)hexanediamide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch no.: 2010000407
Expiry date: January 2022
Appearance : White granules with powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Healthy young adult female rats (Rattus norvegicus) of the Wistar strain (RccHan: WIST)
The photoperiod in the animal room was 12 hours of lighting and 12 hours of darkness each day with the light hours approximately 06.00 - 18.00 hours.
Temperature: 19-25 °C
Relative humidity: 30-70 %
Feed and water at libitum.
The body weight variation among the female rats was within ±20% of the mean body weight at the beginning of the acclimatisation.
Throughout the experimental period, rats were housed individually except during the mating period. During the mating period, rats were housed in a group of two rats/cage (one male plus one female). Mated female rats were housed individually. Enrichment material (wooden chew block) was provided to all rats. During the study, rats were housed in solid floor polypropylene rat cages .
Each cage was fitted with a stainless-steel top grille having provision for keeping rat pellet food and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5 tier racks. The bottom of the cages was layered with clean sterilised rice husk as the bedding. Cages with bedding material and water bottles were changed twice a week.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Reverse Osmosis (RO) water was selected as the vehicle based on the solubility check performed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC analysis, Column Waters X-bridgeTM C18, 150 x 4.6 mm, 3.5 µm
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: two rats/cage (one male plus one female).
- Length of cohabitation: until the requisite numbers of mated females were obtained.
- Proof of pregnancy: evidence of a copulatory plug in the vagina or by vaginal lavage for sperm
Duration of treatment / exposure:
PROSID was administered orally from gestation day (GD) 5 to 19 to 25 pregnant female rats per group.
Duration of test:
From July 23 to December 26, 2020.
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels have been selected based on the results of the Prenatal Developmental Oral Toxicity Study of PROSID in Wistar Rats (dose range-finding) where no maternal and developmental toxicity were observed up to the dose level of the 1000 mg/kg b. wt./day.
Accordingly, the following dose levels were selected for the present study: Low dose: 100 mg/kg b. wt. /day; Mid dose: 300 mg/kg b. wt./day and High dose: 1000 mg/kg b. wt./day.

Examinations

Maternal examinations:
Rats were observed twice daily for mortality and clinical signs. Maternal body weights and food consumption were recorded throughout the gestation period. All rats were sacrificed on GD 20 and assessed for gross pathological changes.
Ovaries and uterine content:
The uteri were excised, weighed, and examined for the numbers of implantation sites, early and late resorptions, and numbers of live and dead foetuses. The ovaries were excised and the number of corpora lutea counted.
Blood sampling:
Serum thyroid hormones T3 (liothyronine), T4 (levothyroxine), and TSH (thyroid stimulating hormone) were analysed from all female rats during the terminal sacrifice. At the time of terminal sacrifice, the weight of the thyroid gland was recorded from all female rats and preserved for histopathology
Fetal examinations:
The foetuses were identified for sex, weighed, and examined for external findings. The anogenital distance was measured for all foetuses.
Following appropriated fixation, fetuses were examined for visceral abnormalities including razor sectioning of the head, and for skeletal abnormalities.
Statistics:
All raw data were processed to determine group means and standard deviations with statistical significance between the control and treatment groups using validated statistical software.
The parametric data (body weight, body weight change, food consumption, hormones (T3, T4, and TSH) results, organ weight, organ weight ratio, male ratio, percent pre-implantation loss, percent post-implantation loss, percent live foetus, and percent resorption) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. When the data do not meet the homogeneity of variance, statistical analysis was extended following a decision tree (Gad, S.C., 2007).
The non-parametric data (pregnancy rate, foetal observations etc.) were analysed using the Chi-square test.
Count data (viz., foetal count, number of corpora lutea, number of implants, number of live foetuses, number of resorptions (early, late, and total), number of pre-implantation loss, and number of percent post-implantation loss ) were subjected to non-parametric Kruskal-Wallis test.
AGD was normalised (the ratio of AGD to the cube root of body weight) and then subjected for statistical analysis.
Non-pregnant rats were not subjected to statistical analysis.
Flags for significant difference between control and treated groups (single arrow for p≤0.05 and double arrows for p≤0.01) were given in the table along with the footnote.
Historical control data:
The albino rat was selected as a test system because it has been historically shown to be a suitable model for prenatal developmental toxicity studies.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight and corrected body weight of female rats was comparable with that of the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of female rats was comparable with that of the control group.
An incidental decrease in mean food consumption (GD 17-20) was observed at 300 mg/kg b. wt./day dose level.
Food efficiency:
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Endocrine findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
no effects observed
Description (incidence and severity):
Serum T3 and T4 levels of GD 20 female rats, belonging to the 300 and 1000 mg/kg b. wt./day, were statistically significantly increased when compared with that of the control group. However, serum TSH level and other thyroid related parameters such as thyroid weight and thyroid histopathology were comparable with that of the control group. Therefore, this effect is not related to the test item treatment.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Test item treatment did not lead to any alteration in absolute and relative thyroid gland weights and terminal body weight.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal (gross) examination of terminally sacrificed female rats did not reveal any lesion of pathological significance.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histological examination of thyroid gland did not reveal any lesion in rats of the control group as well as the high dose group.
Histopathological findings: neoplastic:
not specified

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:
not specified
Changes in number of pregnant:
not specified
Details on maternal toxic effects:
The mean absolute and relative uterine weight of the pregnant female rats were comparable between the control and the test item treated groups.
The mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, the mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions were comparable between the control and test item treated groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
dead fetuses
early or late resorptions
gross pathology
histopathology: non-neoplastic
mortality
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean body weight of male, female, and total foetuses (male + female) was comparable between the control and the test item treated groups.
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
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
The mean anogenital distance of female foetuses and the male sex ratio were comparable between the control and the test item treated groups.
A statistically significantly increase in AGD was observed in males of the 100 mg/kg b. wt./day dose level. This effect did not show any dose dependency; therefore, it was considered a biological variation without any toxicological relevance
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
A total of 273, 244, 293, and 288 foetuses were examined in 0, 10, 300, and 1000 mg/kg b. wt./day dose groups, respectively.
No treatment-related external anomalies were observed in foetuses of the treatment groups up to the dose level of 1000 mg/kg b. wt./day except two runt foetuses from the 100 mg/kg b. wt./day dose group and a runt foetuses each from the 300 and 1000 mg/kg b. wt./day dose groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
A total of 144, 127, 154, and 149 foetuses were observed for the skeletal evaluation in 0, 100, 300, and 1000 mg/kg b. wt./day dose groups, respectively.
No treatment-related skeletal anomalies were observed in foetuses of the treatment groups up to the dose level of 1000 mg/kg b. wt./day.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 129, 117, 139, and 139 foetuses were examined in 0, 100, 300, and 1000 mg/kg b. wt./day dose groups, respectively.
No treatment-related visceral anomalies were observed in foetuses of the treatment groups up to the dose level of 1000 mg/kg b. wt./day. Some spontaneous findings such as situs inversus (one foetus from the 100 mg/kg b. wt./day dose group) and dilated ureter (four foetuses from the control; two foetus from the 100 mg/kg b. wt./day dose group and two foetus from the 1000 mg/kg b. wt./day dose group) were observed in the foetuses.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations
skeletal malformations
visceral malformations

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

The dose formulation analysis results revealed that the mean recovery was within the acceptance level of ±10% of nominal value, and %CV was < 10. The dose formulation analysis demonstrated that the dose formulations were homogeneous.

Applicant's summary and conclusion

Conclusions:
Based on the result of this study, it is concluded that the “No Observed Adverse Effect Level (NOAEL)” of PROSID for the maternal and developmental toxicity is 1000 mg/kg b. wt./day.