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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:
2020
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:
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
Reference substance name:
High moecular weight adducts of Fatty acids, C16-18 sat. C18 unsat., linear, dimers, and trimers, with Amines, polyethylenepoly-, triethylenetetramine fraction
IUPAC Name:
High moecular weight adducts of Fatty acids, C16-18 sat. C18 unsat., linear, dimers, and trimers, with Amines, polyethylenepoly-, triethylenetetramine fraction
Constituent 2
Reference substance name:
Higher molecular weight adducts of C18 Fatty acids linear (unsat & sat) with amines,polyethylenepoly-, tetraethylenepentamine fraction
IUPAC Name:
Higher molecular weight adducts of C18 Fatty acids linear (unsat & sat) with amines,polyethylenepoly-, tetraethylenepentamine fraction
Constituent 3
Reference substance name:
Amines, polyethylenepoly-, tetraethylenepentamine fraction
EC Number:
292-587-7
EC Name:
Amines, polyethylenepoly-, tetraethylenepentamine fraction
Cas Number:
90640-66-7
IUPAC Name:
Amines, polyethylenepoly-, tetraethylenepentamine fraction
Constituent 4
Reference substance name:
lower molecular weight adducts of C18 Fatty acids linear (unsat & sat) amines,polyethylenepoly-, tetraethylenepentamine fraction
Molecular formula:
for example : C26H53N5O, C26H55N5O...
IUPAC Name:
lower molecular weight adducts of C18 Fatty acids linear (unsat & sat) amines,polyethylenepoly-, tetraethylenepentamine fraction
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report):TOFA DimerFA TEPA PAA
- Physical state:Yellow-red, transparent, viscous liquid
- Analytical purity: 9% free amine
- Storage condition of test material:Room temperature (15°C to 30°C)
-Sponsor batch: BB000649V1
Specific details on test material used for the study:
Source of Test Material
Huntsman Advanced Materials, Ernst-Schering-Straße 14, 59192 Bergkamen, Germany

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Test Animals
Source
In-house, Department of Safety Assessment, Eurofins Advinus Limited, Post box no. 5813, Plot Nos. 21 & 22 Phase-II, Peenya, Industrial Area, Bengaluru 560 058, India

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
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 of treatment.

The prepared formulations were 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. For each set, one replicate sample was drawn from top, middle and bottom layers for each dose formulation. In case of control, one sample from middle layer was drawn and processed similar to dose formulation concentration. The dose formulation samples along with back up samples were sent for formulation analysis to determine the concentration and homogeneity of the test item.

Samples were analyzed for Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine using analytical method validated under Study No. G18455.

Formulations were considered acceptable if the overall mean result (calculated using all the 6 replicate values) of all the layers is within ± 20.0 % of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 6 replicate values) of assay of top, middle and bottom layers is equal to or less than 15.0 %.

The unused back up samples were disposed.
Details on mating procedure:
Cohabitation
During the mating period, females were cohabited randomly with males in a 1:1 ratio.
Proof of pregnancy: Confirmation of mating visually.
Length of cohabitation: 8 Days

Duration of treatment / exposure:
23 days
Frequency of treatment:
GD 6 to GD 28
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
G2 (Low Dose)
Dose / conc.:
35 mg/kg bw/day (nominal)
Remarks:
G3 (Mid Dose)
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
G4 (High Dose)
No. of animals per sex per dose:
Day '0' Pregnant rabbits: 23
Control animals:
yes
yes, concurrent vehicle
Details on study design:
A preliminary dose range finding study (DRF) in pregnant female New Zealand White rabbits was carried out under the study number N4574. Six rabbits per group were treated with Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine. The rabbits were treated with the dose formulations by oral gavage at a dose volume of 2 mL/kg body weight from GD 6 to 28 and observed for clinical signs and mortality. The animals in the vehicle control group were handled in an identical manner as in the treatment groups and treated with vehicle (corn oil).

The initial doses tested in the study (DRF-I) were 100, 300, 600 and 1000 mg/kg/day, along with the vehicle control (G1). Considering treatment related deaths at all the test item treated groups, the surviving rabbits in these treatment groups were euthanized on GD 9 without further evaluation. The vehicle administration for control group was continued and these rabbits were sacrificed on GD 29. Due to treatment related deaths in DRF-I, additional rabbits were treated at 15 and 50 mg/kg/day, (DRF-II). These additional lower doses were well tolerated without any toxicity. Hence an additional higher dose of 75 mg/kg/day was tested (DRF –III) to arrive at the maximum tolerated dose.

Based on the reduction in body weight at 75 mg/kg/day and decrease in food consumption along with increase in post implantation loss at 50 and 75 mg/kg/day, the following doses are selected for the definitive study in consultation with sponsor:

Vehicle control - 0 mg/kg/day
Low dose - 15 mg/kg/day
Mid dose - 35 mg/kg/day
High dose - 75 mg/kg/day

Examinations

Maternal examinations:
CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose)
- Cage side observation checked in table 2 were included

Ovaries and uterine content:
The ovaries and uterine contents were examined after termination GD 29.
• Pregnancy status
• Gravid uterine weight (from all rabbits 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:
Data were captured and compiled using Provantis Integrated Preclinical Software System.

The data on maternal body weight and food consumption, interval body weight changes, gravid uterine weight, body weight change corrected to gravid uterine weight were 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.

Number of corpora lutea, number of implantations, early and late resorptions/deaths, pre-implantation and post-implantation loss, external, soft tissue and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon test pair wise comparisons 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.

The incidence of external, visceral and skeletal findings for fetus and litter was tested using Cochran Armitage trend test and pair wise comparison was tested 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 6 of the final report

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
• There were no mortalities at any of the doses tested.
• There was a total of three abortions observed one each in vehicle control, 15 and 75 mg/kg/day doses on GD 21 (RBa6177), GD 25 (RBa6199) and GD 21 (RBa6256) respectively
Mortality:
no mortality observed
Description (incidence):
There were no mortality at any of the doses tested.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were statistically non-significant reductions in maternal body weight gain at 75 mg/kg/day when compared to the vehicle control data.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was statistically significant reduction reductions in food consumption at 75 mg/kg/day when compared to the vehicle control data.
Clinical biochemistry findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed (including reproductive organs) in all rabbits

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
As rabbits are sensitive animals to handling and environmental stress, the observed number of abortions in the presented study were considered to have occurred incidentally and were within the historical control range.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 75 mg/kg/day, there was reduction in uterine weight (16%), significant increase in percent late resorptions (12%), post implantation loss (18%) and dams with resorptions. Increase in late resorptions/post implantation loss can be correlated to lower food consumption and decreased body weights, indicative of maternal stress and hence considered secondary to maternal toxicity.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
At 75 mg/kg/day, there was reduction in uterine weight (16%), significant increase in percent late resorptions (12%), post implantation loss (18%) and dams with resorptions. Increase in late resorptions/post implantation loss can be correlated to lower food consumption and decreased body weights, indicative of maternal stress and hence considered secondary to maternal toxicity.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
35 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
At 75 mg/kg/day, there was significant reduction in mean fetal weights (12 to 15%) of males, females and combined sex. The reduced fetal weights were associated with decrease in uterine weight which may be due to reduced maternal body weight and food consumption caused by general toxicity rather than a direct developmental effect. The reduction in fetal weights (upto 15%) was within the historical control data range and is considered secondary to maternal toxicity. No test item related changes were observed in other litter parameters of total number of fetuses and mean litter size.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
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 75 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 75 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 75 mg/kg/day

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects in abscence of maternal toxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Test item had no effect on maternal body weight, weight gain, food consumption, uterine and litter parameters at the doses of 15 and 35 mg/kg/day.

At 75 mg/kg/day, there was reduction in maternal body weight gain and food consumption. The decrease in uterine weight, increase in late resorptions and lower fetal weights correlated to lower food consumption and decreased body weights indicative of maternal stress and hence were considered secondary to maternal toxicity. Gross evaluation of the placenta revealed no findings. There were no gross pathological changes at any dose level.

External, visceral and skeletal examination of fetuses revealed no signs of teratogenicity up to the highest dose of 75 mg/kg/day.

In conclution, based on the above findings, under the test conditions and doses employed in this study, it is concluded that No-Observed-Adverse-Effect Level (NOAEL) for:

• Maternal systemic toxicity is 35 mg/kg/day due tendency towards lower body weight gains and significant reduction in food consumption at
75 mg/kg/day.

• Developmental toxicity and Teratogenicity is 75 mg/kg/day as decrease in uterine weight, increase in number of late resorptions and reduction in fetal weights were considered secondary to maternal toxicity and fetal external, visceral and skeletal examinations revealed no signs of teratogenicity up to the high dose of 75 mg/kg/day
Executive summary:

The objective of this study was to evaluate the prenatal developmental toxicity of the test item, Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine when administered daily by oral gavage during gestation days (GD) 6 to 28 to presumed pregnant New Zealand White rabbits. This study evaluated the maternal toxicity and adverse effects on development of the embryo and fetus in pregnant female rabbits. This study provided a rational basis for risk assessment in humans and helped to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rabbits.

 

The dose levels for this study were selected based on a Dose Range Finding study (DRF) (Study Number N4574). The initial doses tested in the study (DRF-I) were 100, 300, 600 and 1000 mg/kg/day, along with the vehicle control (G1). Considering treatment related deaths at all the test item treated groups, the surviving rabbits in these treatment groups were euthanized on GD 9 without further evaluation. The vehicle administration for control group was continued and these rabbits were sacrificed on GD 29. Due to treatment related deaths in DRF-I, additional rabbits were treated at 15 and 50 mg/kg/day, (DRF-II). These additional lower doses were well tolerated without any toxicity. Hence an additional higher dose of 75 mg/kg/day was tested
(DRF –III) to arrive at the maximum tolerated dose. The rabbits were treated with test item formulations by oral gavage from GD 6 to GD 28 and observed for clinical signs and mortality. The animals in the vehicle control group were handled in an identical manner as the animals in the treatment groups and treated with vehicle (corn oil).

 

At 75 mg/kg/day there was reduction in body weight and lower food consumption along with increase in post implantation loss at 50 and
75 mg/kg/day. Based on the results, high dose of 75 mg/kg/day was selected for the definitive study. Mid dose of 35 mg/kg/day and low dose of
15 mg/kg/day were selectedto provide a graded response to the test item.

 

In the definitive study, 92 mated female rabbits were assigned to four groups. Each group consisted of 23 mated rabbits (G1: vehicle control, G2: low dose, G3: mid dose and G4: high dose). Day 0 of gestation for each individual female rabbit in the study was considered as the day on which mating had occurred.

 

The test item, Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetraminewas suspended in corn oil and administered orally from GD6to28at the dose volume of2mL/kg body weight.Rabbits in the control group werewere handled in an identical manner as the animals in the treatment groupsandweretreatedwith vehicle.

Rabbitswere observed daily for clinical signs, morbidity, mortality and body weightchanges andfood consumption.All the surviving rabbits were sacrificed on GD 29 and subjected to gross pathological examination. Gross pathological examinations were also performed for rabbits that aborted during the course of study. The uteri from all the animals sacrificed on GD 29 were removed (by laparo-hysterectomy).The uteri were weighed and examined for the number of implantation sites, early and late resorptions and number of live fetuses. The number of corpora lutea was counted. All the fetuses were individually weighed and observed for external malformations. All the live fetuses were examined forvisceral and skeletal variations and malformations. 

The main findings of the study are presented below:

·           There were no mortalities at any of the doses tested.

·           There was a total of three abortions observed one each in vehicle control, 15 and 75 mg/kg/day doses on GD 21 (RBa6177), GD 25 (RBa6199) and GD 21 (RBa6256) respectively. As rabbits are sensitive animals to handling and environmental stress, the observed number of abortions in the presented study were considered to have occurred incidentally and were within the historical control range.

·           There were statistically non-significant reductions in maternal body weight gain and significant reductions in food consumption at 75 mg/kg/day when compared to the vehicle control data.

·           There were no gross pathological changes observed (including reproductive organs) in all rabbits.

·           At 75 mg/kg/day, there was reduction in uterine weight (16%), significant increase in percent late resorptions (12%), post implantation loss (18%) and dams with resorptions and significant reduction in mean fetal weights (12 to 15%) of males, females and combined sex. These changes were associated with decrease in maternal body weight gain and food consumption indicating general toxicity and hence considered secondary to maternal toxicity.Gross evaluation of placenta revealed no remarkable findings.

·           Fetal external, visceral and skeletal examinations revealed no signs of teratogenicity up to the highest tested dose of 75 mg/kg/day. 

 

 

 

 

 

In conclusion, based on the above findings, under the test conditions and doses employed in this study, it is concluded that No Observed-Adverse-Effect Level (NOAEL) for:

 

·           Maternal systemic toxicity is 35 mg/kg/daydue to tendency towards lower body weight gains and significant reduction in food consumption at 75 mg/kg/day.

 

Developmental toxicity and Teratogenicity is 75 mg/kg/dayas decrease in uterine weight, increase in number of late resorptions and reduction in fetal weights were considered secondary to maternal toxicity and fetal external, visceral and skeletal examinations revealed no signs of teratogenicity up to the high dose of 75 mg/kg/day.