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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:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

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
Chemical structure
Reference substance name:
N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(2-methoxyphenyl)azo]-3-oxobutyramide
EC Number:
279-914-9
EC Name:
N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(2-methoxyphenyl)azo]-3-oxobutyramide
Cas Number:
82199-12-0
Molecular formula:
C18H17N5O4
IUPAC Name:
2-[(1E)-2-(2-methoxyphenyl)diazen-1-yl]-3-oxo-N-(2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)butanamide
Test material form:
solid: bulk

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Source: Hylasco Biotechnology (India) Pvt. Ltd.
Plot 4B, MN Park, Shameerpet Mandal,
Turkapally Village,
Medchal District, Telangana -500078

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for analysis of homogeneity and active ingredient (a.i.) were collected from the dose formulations intended for first treatment and last treatment (-2 days). The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was stored at ambient condition. For each set, duplicate sample were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The samples collected were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the homogeneity and concentration of the dose formulation.
Details on mating procedure:
The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug we re examined in the morning hours of the subsequent day to confirm mating
Duration of treatment / exposure:
Gestation day 5 to gestation day 19
Frequency of treatment:
Daily from gestation day 5 to gestation day 19
Duration of test:
Upto gestation day 20
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
111 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
24 day 0 pergnant rats per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Group Nos. Groups Dose
(mg/kg/day) Dosage volume (mL/kg) Concent-ration (mg/mL) No. of Day 0 pregnant rats Rat numbers
From To
G1 Vehicle control 0 10 0 24 Rz121 Rz144
G2 Low dose 111 10 11.1 24 Rz145 Rz168
G3 Mid dose 333 10 33.3 24 Rz169 Rz192
G4 High dose 1000 10 100 24 Rz193 Rz216

Examinations

Maternal examinations:
CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included
Ovaries and uterine content:
The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta
Fetal examinations:
• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)
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 was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were 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 were 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 was 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 observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the
group differences were significant. The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s
exact test for group association. The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.
Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.
Indices:
Refer Table 6 of the final report
Historical control data:
Refer Annexure 8 of the final report

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
Food consumption and compound intake (if feeding study):
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with Novoperm-Gelb F2G (C.I. Pigment Yellow 194) up to the highest dose of 1000 mg/kg/day.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions in the study
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
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): 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.
Basis for effect level:
other: There were no adverse effects at all

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
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.
Sex:
male/female
Basis for effect level:
other: There were no effects at all

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 above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as
- the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.
- fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights.
- the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day
Executive summary:

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item C.I. Pigment Yellow 194 to cause adverse effects on the pregnant rats and development of the embryo and fetus consequent to exposure of pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for  a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.


A total of 96 Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:














































Group Nos.



Groups



Dose


(mg/kg/day)



Dosage volume (mL/kg)



Concentration (mg/mL)



No. of Day 0 pregnant rats



G1



Vehicle control*



0



10



0



24



G2



Low dose



111



10



11.1



24



G3



Mid dose



333



10



33.3



24



G4



High dose



1000



10



100



24



*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q®Water


 


The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half the number of fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroid glands were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifice (GD20).


 


Results of the study are summarized below:


 


·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected yellow coloured faeces was observed in the test item treated groups which was attributed to the physical nature of the test item.


·        Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item. 


 ·       Maternal Parameters: There were no treatment-related effects on maternal body weights and food consumption up to the highest tested dose of 1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no findings.


·        Litter Parameters: There were no treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights and anogenital distance in male and female foetuses.


·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day.


·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with C.I. Pigment Yellow 194 up to the highest dose of
1000 mg/kg/day.


 


Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for  Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as


-            the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.


-            fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights. 


The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day