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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
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

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

Test material

Constituent 1
Reference substance name:
dimethoxyphosphinic acid; methoxyphosphonic acid; phosphoric acid
EC Number:
908-996-7
IUPAC Name:
dimethoxyphosphinic acid; methoxyphosphonic acid; phosphoric acid
Test material form:
other: liquid
Details on test material:
Reaction mass of methyl dihydrogen phosphate and orthophosphoric acid and dimethyl hydrogen phosphate
EC No.: 908-996-7

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Test Animals
Source - Vivo Bio Tech Ltd, Sy # 349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana, India

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation analysis was carried out from samples prior to the initiation of treatment and before the termination.

The prepared formulations was sampled in duplicate sets wherein one set was used for analysis and another was kept as back up set which was stored at room temperature in the experimental room. For each set, composite samples were drawn in three triplicates for each dose formulation. In case of control, duplicate composite samples were drawn. Dose formulations were sent for formulation analysis to determine the concentration of the test item using a validated analytical method (Study No.: G16490).
Formulations were considered acceptable if the overall mean result (calculated using all the 3 replicate values) is within ± 15.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 3 replicate values) is equal to or less than 10.0 %.

The back up samples were either used for reanalysis when the results of first set of analysis were outside the acceptable limits or discarded if the results of first set of analysis were within the acceptable limits
Details on mating procedure:
Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 12 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0] of pregnancy
Duration of treatment / exposure:
15 days
Frequency of treatment:
Gestation day 5 to Gestation day 19
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day
Remarks:
Group 2 (Low dose)
Dose / conc.:
120 mg/kg bw/day
Remarks:
Group 3 (Mid dose)
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 3 (High dose)
No. of animals per sex per dose:
Day '0" Pregnant rats : 24
Control animals:
yes, concurrent vehicle
Details on study design:
Dose Selection rationale
Based on the dose range finding study carried out by the sponsor at the doses of 100, 300 and 1000 mg/kg/day, the dose levels of 50 (G2), 120 (G3) and 300 (G4) mg/kg/day have been selected for this study in consultation with the Sponsor.

Vehicle control - 0 mg/kg/day
Low dose - 50 mg/kg/day
Mid dose - 120 mg/kg/day
High dose - 300 mg/kg/day

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day (pre dose and post dose)
- Cage side observations checked in table [No.2] were included
- At necropsy Thyroid gland was collected and weighed and subjected to histopathology
- Thyroid harmone analysis [T3, T4, TSH]
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Gross evaluation of placenta: Yes
Fetal examinations:
External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
- Ano-genital distance: Yes: [all per litter]
- Fetus sex (external determination based on anogenital distance and internal sex based on gonadal examination)
Statistics:
The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland were analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances are heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences are found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female were analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss, external, visceral and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon (Mann-Whitney) pairwise comparison of the treated groups with the control group was performed, when the group differences were significant.

The incidence of dams with and without resorptions was tested using Cochran Armitage trend test followed by Fisher’s exact test for group association.

Statistically significant differences (p<0.05) were designated as * throughout the report
Historical control data:
Refer: Annexure 7 iin the Report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs findings at any of the doses tested.
Mortality:
no mortality observed
Description (incidence):
There were no mortalities at any of the doses tested
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and gains were comparable across the tested doses.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption were comparable across the tested doses.
Clinical biochemistry findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid gland weights were unaffected by treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross necropsy findings at any of the doses tested
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Histopathology of thyroid gland were unaffected by treatment.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone profile (T3, T4 and TSH) were unaffected by treatment.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
Mean values of maternal parameters comprising uterine weight and number of corpora lutea, implantations, early deaths, late deaths,
pre-implantation loss and post implantation loss were statistically comparable to the vehicle control group up to the highest dose of
300 mg/kg/day
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation loss and post implantation loss were statistically comparable to the vehicle control group up to the highest dose of
300 mg/kg/day
Early or late resorptions:
no effects observed
Description (incidence and severity):
Early deaths and late deaths were statistically comparable to the vehicle control group up to the highest dose of
300 mg/kg/day

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
maternal abnormalities
other: Teratogeneicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Male and female fetal weights were statistically comparable to vehicle control group up to the highest tested dose of 300 mg/kg/day
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
Fetal external examination revealed no signs of teratogenicity up to the highest tested dose of 300 mg/kg/day
Skeletal malformations:
no effects observed
Description (incidence and severity):
Fetal skeletal examination revealed no signs of teratogenicity up to the highest tested dose of 300 mg/kg/day
Visceral malformations:
no effects observed
Description (incidence and severity):
Fetall visceral examination revealed no signs of teratogenicity up to the highest tested dose of 300 mg/kg/day

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: ne effects observed up to the 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:
Based on the findings of the OECD 414 study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and fetal developmental toxicity is 300 mg/kg/day (highest dose tested).
Executive summary:

An OECD 414 study was performed with the test substance. Ninety six presumed pregnant Wistar rats were assigned to four groups bybody weight stratification (Group 1 to Group 4) and each group (G1: control, G2: low dose, G3: mid dose and G4: high dose) consisted of 24 presumed pregnant rats (gestation day 0). Day `0' of gestation for each individual female rat in the study was considered as the day on which vaginal smear was found positive for sperm.

The test substance in vehicle (Milli-Q water) was administered at 0, 50, 120 and 300 mg/kg/day. The control group received the vehicle only. A constant dose volume of 10 mL/kg body weight was administered to all groups.

All rats were observed for clinical signs, morbidity and mortality, body weight changes and food consumption. Prior to caesarean section, blood was collected for Thyroid hormone analysis. At caesarean section on GD 20, dams were examined for gross pathological changes and thyroid gland was collected, weighed and subjected to histopathological examination. The uterus was removed by laparotomy, weighed and the contents were examined for number of implantation sites, early and late resorptions and number of fetuses. The number of corpora lutea in ovaries was counted. All the fetuses were sexed, weighed and examined for external malformations. Approximately half the number of fetuses from each dam was examined for visceral malformations and the remaining half was evaluated for skeletal malformations.

The main findings of the study are presented below:

- There were no mortalities, clinical signs or gross necropsy findings at any of the doses tested.

- Mean body weights and gains, food consumption were comparable across the tested doses.

- Mean values of maternal parameters comprising uterine weight and number of corpora lutea, implantations, early deaths, late deaths, pre-implantation loss and post implantation loss were statistically comparable to the vehicle control group up to the highest dose of 300 mg/kg/day.

- The litter parameters comprising total number of fetuses, number of live fetuses, male and female fetal weights, anogenital distance in male and female fetuses were statistically comparable to vehicle control group up to the highest tested dose of 300 mg/kg/day.

- Fetal external, visceral and skeletal examination revealed no signs of teratogenicity up to the highest tested dose of 300 mg/kg/day.

- Thyroid hormone profile (T3, T4 and TSH), thyroid gland weights and histopathology of thyroid gland were unaffected by treatment with the test item up to the highest dose of 300 mg/kg/day.

In conclusion, based on the above findings, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity and fetal developmental toxicity is 300 mg/kg/day.