2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]benzoic acid

Developmental toxicity / teratogenicity

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

Test material information

Constituent 1

Reference substance name:

2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

EC Number:

235-462-4

EC Name:

2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

Cas Number:

12236-62-3

Molecular formula:

C17H13ClN6O5

IUPAC Name:

2-[(4-chloro-2-nitrophenyl)diazenyl]-3-oxo-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)butanamide

Test material form:

solid: nanoform

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 for the first batch of rats 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 were 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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

111 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

333 mg/kg bw/day (nominal)

Remarks:

Mid dose

Doses / concentrations 4

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 Rz221 Rz244
G2 Low dose 111 10 11.1 24 Rz245 Rz268
G3 Mid dose 333 10 33.3 24 Rz269 Rz292
G4 High dose 1000 10 100 24 Rz293 Rz316

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, orange contents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.

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-Orange HL (C.I. Pigment Orange 36) 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:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

mortality

necropsy findings

pre and post implantation loss

total litter losses by resorption

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:

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

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 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 developmental toxicity and Teratogencity is 1000 mg/kg/day as fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further 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 Orange 36 to cause adverse effects on the pregnant female rats and development of the embryo and fetus consequent to exposure of C.I. Pigment Orange 36 to pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for risk assessment in humans and to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

 

A total of 96Day 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 of the fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroids were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifie (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 light to dark orange coloured faeces were observed in the test item treated groups which can be accounted for the physical nature of the test item.

Grossly, orangecontents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.

·        Maternal Parameters: 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 remarkable findings.

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

·        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 Orange 36 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 is1000 mg/kg/dayas the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

 

Fetal developmental toxicity and Teratogencity is1000 mg/kg/dayas fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

[1]The day of confirmed mating (sperm positive vaginal smear or presence of vaginal plug) was designated as GD 0.