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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 October 2020 to 17 February 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
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)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-(cyclohexylimino)bisethanol
EC Number:
224-809-5
EC Name:
2,2'-(cyclohexylimino)bisethanol
Cas Number:
4500-29-2
Molecular formula:
C10H21NO2
IUPAC Name:
2,2'-(cyclohexylimino)diethanol
Specific details on test material used for the study:
Manufactured By
Clariant Iberica Produccion, S.A.
Autovia Tarragona-Salou
3110 La Canonja, Spain

Test animals

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-Q water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulation samples were analyzed for active ingredient concentrations. The results indicated that the analyzed concentrations were within ± 10.0 % of the nominal concentration, and the relative standard deviation (% RSD) was less than 10.0 %.
Dose Level (mg/kg/day) Nominal Con.(mg/mL) Average Calculated Con.(mg/mL) Average %Recoverya, b % Relative Standard Deviationa
0 0.0 0.0 NA NA
45 4.5 4.62 - 4.50 102.7 - 100.0 0.606 - 0.958
90 9 9.24 - 8.9 102.6 - 98.87 0.595 - 0.398
180 18 18.1 - 18.0 100.5 - 99.81 0.416 - 0.287
a Results are the range of values determined from two occasions.
b Average % recovery was calculated from the nominal concentration.
NA – Not Applicable
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.:
45 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
90 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
180 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A preliminary dose range finding study (DRF) in pregnant rats was carried out using 7 rats per group with Genamin CH 020 dosed at 45, 67.5, 100 and
150 mg/kg/day along with the concurrent vehicle control group (Study number N4888). The rats were treated with the dose formulations by oral gavage at a dose volume of 10 mL/kg body weight from GD 5 to 19 and observed for clinical signs and mortality.

In the DRF study, dose levels up to 150 mg/kg/day were tolerated. No test item related clinical signs were noted at any dose. The body weight gains, and food consumption were unaffected up to the highest dose of 150 mg/kg/day. There was no fetal developmental toxicity up to the highest tested dose of 150 mg/kg/day.

Based on the results of the dose range finding study and considering results from additional repeated dose toxicity studies, the following dose levels have been selected for this definitive study in consultation with the Sponsor:

G1 - Vehicle control - 0 mg/kg/day
G2 - Low dose - 45 mg/kg/day
G3 - Mid dose - 90 mg/kg/day
G4 - High dose - 180 mg/kg/day

- Rationale for animal assignment (if not random):

During the mating period, 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. If sperm was detected in a vaginal smear and or if a vaginal plug was observed in the morning, the female was considered to be mated. This day was considered as Day 0 of gestation.

The females were cohabited in batches of required numbers. This procedure was continued until there were sufficient numbers of Day 0 pregnant rats for the study.

Day 0 pregnant rats obtained on each day were randomly distributed to different groups by body weight stratification method using ProvantisTM software.

After grouping and ascertaining the group mean body weight, the rats were given accession number as applicable to the group on each day of assignment.

Note: After confirming the day 0 of gestation, females were housed individually, and the unselected females and males were disposed after mating procedure.

The selected female rats were assigned to vehicle control, and three test item treatment groups.

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
Blood sampling:
At caesarean section, from each rat, blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture for the determination of total Triiodothyronine (T3), Thyroxine (T4) and Thyroid Stimulating Hormone (TSH) hormones.
Blood samples (about 1 mL from each rat) were collected in plain labelled tubes and kept on bench top for approximately 90 minutes before centrifugation. Serum was separated by centrifuging the whole blood samples at 5000 rpm for 5 minutes at 4°C. The serum samples were placed in labeled plastic tubes and stored at approximately -70 °C until they were analysed.
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:
Data captured using the ProvantisTM laboratory information management system (LIMS), parameters such as maternal body weight, body weight change, food consumption, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, Pre/post implantation loss , no. of implantations, sex ratio, Number of corpora lutea, early and late resorptions, hormone analyses (T4, T3, TSH) and the weight of thyroid gland data were evaluated using the Levene test for homogeneity of variances and the Shapiro-Wilks test for normality of distributions. Data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA). Data found to be nonhomogeneous or of nonnormal data was subjected for transformation and ANOVA was done on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.

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.

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 skeletal observations were tested using Cochran Armitage trend test and pair wise comparison were tested by Fisher’s exact test for group association.

All hypothesis testing was carried out at the 5% (2-sided) significance level. Significant differences are designated throughout the report as below:

*: Statistically significant difference from the control group at p < 0.05
Historical control data:
Refer Annexure 8

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
There was no morbidity or mortality and the treated rats were normal throughout the experimental period at all the doses tested.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The maternal absolute mean body weights, body weight gains and adjusted/corrected body weight gain were unaffected by treatment up to the highest dose of 180 mg/kg/day. The adjusted body weight gain was significantly lower (- 12%) at 90 mg/kg/day which is considered incidental as a similar trend was not observed at the highest dose of 180 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The maternal mean food consumption was unaffected by treatment up to the highest dose of 180 mg/kg/day. At 90mg/kg/day, the food consumption during GD 17-20 was significantly lower (- 9%) which is considered incidental as a similar trend was not observed at the highest dose of 180 mg/kg/day.
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in total Triiodothyronine (T3), Thyroxine (T4) or Thyroid Stimulating Hormone (TSH) hormones at any dose level.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid gland weight was unaffected by treatment. No treatment-related histopathological effects were observed in the thyroid gland.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item related findings in any of the organs on gross examination.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item related changes in organ weights and histopathology of thyroid glands from all treated groups.
The incidences of ectopic thymus and ultimobranchial cysts were randomly distributed across the groups and hence considered as incidental background findings and not related to test item administration.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone levels (T3, T4 and TSH), thyroid weights, and thyroid histology were unaffected by treatment.

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
Description (incidence and severity):
The post implantation loss rate at 45 mg/kg/day was significantly lower which could account for the observed higher total number of fetuses in this dose group. Gross evaluation of placenta revealed no remarkable findings.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Basis for effect level:
other: maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 180 mg/kg/day.

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
Description (incidence and severity):
The litter parameters (mean fetal weight and number of live fetuses) were comparable between the vehicle control group and rats treated up to the highest dose of 180 mg/kg/day, except at 45 mg/kg/day, the total number of fetuses and thus the mean litter size was significantly higher. This was considered incidental and toxicological not relevant as a similar trend was not observed at the higher doses.
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 in male and female fetuses were comparable to the concurrent vehicle control, except at 90 mg/kg/day the mean anogenital distance of male fetuses was significantly higher and this finding was considered incidental as a similar trend was not observed at the highest dose of 180 mg/kg/day.
External malformations:
no effects observed
Description (incidence and severity):
No test item-related changes were observed during external observations of fetuses of rats treated up to 180 mg/kg/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no skeletal malformations observed in any litter at any of the tested dose leve.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test item-related changes were observed during fresh visceral examination of fetuses of rats treated up to 180 mg/kg/day.

Fetal heads in all dose groups were normal when subjected to Wilsons Razor blade sectioning.
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Sex:
not specified
Basis for effect level:
other: fetal resorptions or post implantation loss were comparable to the control, 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 180 mg/kg/d

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no

Any other information on results incl. tables

see attached background materials in "Overall remarks, attachements".

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 180 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 180 mg/kg/day.

• Fetal developmental toxicity and Teratogenicity is 180 mg/kg/day as fetal resorptions or post implantation loss were comparable to the control, 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 180 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the developmental toxicity of Genamin CH 020 in pregnant female Wistar rats and developing embryos/fetuses consequent to treatment of Genamin CH 020 in pregnant rats by oral gavage from gestation day (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 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



45



10



4.5



24



G3



Mid dose



90



10



9



24



G4



High dose



180



10



18



24



*Milli-Q®Water


The following parameters and endpoints 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 the fetuses from each litter were examined for visceral malformations and variations and the remaining half were evaluated for skeletal malformations and variations. In addition, from each dam, the thyroid was weighed and subjected to microscopic evaluation. Thyroid hormones levels were determined from the blood samples collected at terminal sacrifie (on GD 20).


 


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.


Grossly, at necropsy no abnormalities were detected.


·   Maternal Parameters: No treatment-related effects on maternal body weights and food consumption up to the highest tested dose of 180 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 180 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: External, visceral and skeletal examinations revealed no teratogenic effects attributed to the test item.


·  Thyroid hormone levels (T3, T4 and TSH), thyroid weights, and thyroid histology were unaffected by treatment.


 


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


 


• Maternal toxicity is 180 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 180 mg/kg/day.


 


• Fetal developmental toxicity and Teratogenicity is 180 mg/kg/day as fetal resorptions or post implantation loss were comparable to the control, 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 180 mg/kg/day.