Mono Dimer Trimer FA TETA PAA

Developmental toxicity / teratogenicity

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

Test material information

Constituent 1

Reference substance name:

TETA – Fatty acids adducts (Mw 600-1000Da)

IUPAC Name:

TETA – Fatty acids adducts (Mw 600-1000Da)

Constituent 2

Reference substance name:

High molecular weight adducts of Fatty acids, C18-unsatd dimers and trimers with amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

High molecular weight adducts of Fatty acids, C18-unsatd dimers and trimers with amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 3

Reference substance name:

lower molecular weight adducts of Fatty acids, C18-unsatd dimers with amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

lower molecular weight adducts of Fatty acids, C18-unsatd dimers with amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 4

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

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): TOFA_DimerFA_TETA_PAA
- Physical state: Yellow liquid with a brown hue.
- Analytical purity: 100%
- Lot/batch No.: BB001030V1
- Expiration date of the lot/batch: 30 May 2013
- Storage condition of test material: When not in use the test article was stored in a sealed container, at room temperature in the dark.

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:

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, 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:

For dose formulation analysis, prepared formulations were sampled at the prior to the initiation (04 Novmber 2019) and at termination (25 November 2019) of treatment period.

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 in the experimental room depending upon the obtained stability results. 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 with back up 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 as the overall mean result (calculated using all the 6 replicate values) of all the layers was 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 was equal to or less than 15.0 %.

The unused back up samples was discarded as the results of first set of analysis were within the acceptable limits

Details on mating procedure:

Cohabitation
During the mating period, females were cohabited randomly with males in a 1:1 ratio.
Proof of pregnancy: Vaginal smear exam / vaginal plug.
Length of cohabitation: 10 Days

Duration of treatment / exposure:

15 days

Frequency of treatment:

GD 5 to GD 19

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

Low Dose

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Mid Dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High Dose

No. of animals per sex per dose:

24

Control animals:

yes

yes, concurrent vehicle

Details on study design:

A preliminary dose range finding study (DRF Study number N4573) in pregnant rats was carried out using 7 rats per group with Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine dosed at 100, 300 and 1000 mg/kg/day along with the concurrent vehicle (corn oil) control group (Study number N4573). The rats were treated with the dose formulations by oral gavage at a dose volume of 4 mL/kg body weight from GD 5 to 19 and observed for clinical signs and mortality.

In this study, dose levels up to 1000 mg/kg/day were evaluated and were tolerated. No test item related clinical signs were noted at any dose. However, significant reduction in maternal body weight and decrease in feed consumption during treatment period was noted at the high dose of
1000 mg/kg/day. There was no fetal developmental toxicity up to the highest tested dose of 1000 mg/kg/day. The high dose of 1000 mg/kg/day was selected based on the decreases in group mean body weight gain and feed consumption. The mid and low-dose levels were selected in order to provide a graded response to the test item.

Based on the results of the dose range finding study and in consultation with the Sponsor, the following dose levels were selected for this definitive study.

Vehicle control (G1) - 0 mg/kg/day
Low dose (G2) - 100 mg/kg/day
Mid dose (G3) - 300 mg/kg/day
High dose (G4) - 1000 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 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/early deaths
• Number of late resorptions/late deaths
• Gross evaluation of placenta

Fetal examinations:

a. Total number of fetuses
b. Number of live fetuses
c. Number of dead fetuses
d. Individual fetal body weight (g)
e. Anogenital distance (mm) from all live fetuses
f. Fetus sex -external determination based on anogenital distance and internal sex based on gonadal examination (internal sex) during visceral 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 fetuses was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female fetuses 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, external, visceral and skeletal observations for variations were analyzed using Kruskal Wallis test for group comparison. Mann-Whitney/ Wilcoxon 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 8 of 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:

effects observed, treatment-related

Description (incidence and severity):

The mean maternal body weights and net body weight gain during the different periods of gestation (GD 0-5; GD 5-20; GD 0-20) were statistically comparable to the vehicle control group at 100 and 300 mg/kg/day.

At 1000 mg/kg/day, there was significant reduction (-23% to -29%) in net body weight gain during GD 5-20 and GD 0-20 as compared to vehicle control group.

There was significant decrease in corrected body weight at 300 mg/kg/day. However, this decrease was not considered adverse as the maternal body weights during different periods of gestation were comparable to vehicle control group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of rats at 100 mg/kg/day were statistically comparable to the vehicle control group.

At 300 mg/kg/day, there was significant decrease (-9% to -13%) in food consumption during GD 11-14, GD 14-17, GD 17-20 and GD 5-20 as compared to vehicle control group.However, this reduction was not considered adverse as there was no concomitant decrease in maternal body weights.

At 1000 mg/kg/day, there was significant reduction (-8% to -15%) in food consumption during GD 5-8, GD 11-14, GD 5-20 and GD 0-20 .This reduction was considered treatment related as this was associated with reduction in maternal body weight gain during different periods of gestation.

The reduction in maternal body weight gain and food consumption at
1000 mg/kg/day was considered treatment related effect indicative of maternal toxicity

Clinical biochemistry findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

There were no gross pathological findings at necropsy in the vehicle control rats and rats treated at 100 mg/kg/day. Grossly, single incidence of stomach distended with gas was observed each at 300 (Rs9376) and 1000 (Rs9396) mg/kg/day dose level

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Early or late resorptions:

no effects observed

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:

body weight and weight gain

food consumption and compound intake

Results (fetuses)

Fetal body weight changes:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In this study, prenatal developmental toxicity of Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine was evaluated in Wistar rats following administration once daily by oral gavage at 0, 100, 300 and 1000 mg/kg/day during gestation days 5 to 19.

Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine up to 300 mg/kg/day had no adverse effect on maternal body weights, weight gain and food consumption. At 1000 mg/kg/day, there was significant decrease in body weight gain and food consumption.The maternal and litter parameters were comparable to vehicle control group at all the doses tested.There were no gross pathological changes at any dose level. Gross evaluation of the placenta revealed no findings. External, visceral and skeletal examination of fetuses revealed no signs of teratogenicity.

Based on the above findings, it is concluded that, Observed- Adverse- Effect- Level (NOAEL) for

• Maternal toxicity is 300 mg/kg/day, due to test item related significant reduction in mean maternal body weight and weight gain and reduction in food consumption at 1000 mg/kg/day.

• Fetal developmental toxicity and Teratogencity is 1000 mg/kg/day, as fetal external, visceral and skeletal examinations revealed no signs of developmental toxicity or teratogenicity up to 1000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the prenatal developmental toxicity of 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) 5 to 19 to presumed pregnant Wistar rats. The results of this study helped to establish the No Observed Adverse Effect Level (NOAEL) of the test item for maternal and developmental toxicity.

The dose levels for this study were selected based on Dose Range Finding study (DRF). In DRF study, Study No. N4573, dose levels of 100, 300 and 1000 mg/kg/day were evaluated. The dose levels up to 1000 mg/kg/day were tolerated. No test item related clinical signs were noted at any dose. There was significant reduction in maternal body weight gains along with decreases in food consumption during treatment period at the highest dose of
1000 mg/kg/day. There was no fetal developmental toxicity up to the highest tested dose of 1000 mg/kg/day.The mid and low-dose levels of300 and 100 mg/kg/day were selected in order to provide a graded response to the test item.

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 volume (mL/kg)	Dose (mg/kg/day)	Concen- tration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control	4	0	0	24
G2	Low dose	4	100	25	24
G3	Mid dose	4	300	75	24
G4	High dose	4	1000	250	24

Test item, Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine was administered at 0, 100, 300 and 1000 mg/kg/day. The control group received the vehicle (corn oil) only.A constant dose volume of 4 mL/kg body weight was administered to all groups.

The identity of the test item was provided by the study Sponsor by a Certificate of Analysis (CoA).The stability and homogeneity of the test item in the vehicle was established under Study No. G18455at concentrations of5and500 mg/mL.The test item was found to be stable and also resuspendable in vehicle for 48 hour at 5 and 500 mg/mL concentration when stored at room temperature.

During the conduct of the experiment, homogeneity and active ingredient analysis was carried out from the dose formulation samples collectedprior toinitiationof treatmentand termination of treatment. The results of analysis of formulations were within the acceptable limits.

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, fetus parameters [sex, weight, anogenital distance, and external, visceral and skeletal findings].All fetuses were examined for gross abnormalities. Approximately half the number of the fetuses from each litter were examined for visceral (soft tissue) malformations and the remaining half of fetuses were evaluated for skeletal malformations. In addition, from each dam the thyroids were weighed and subjected to microscopic evaluation, and thyroid hormone levels were estimated from the blood collected at terminal sacrifice (on GD20).

 

The main findings of the study are presented below:

 

·   There were no mortalities and clinical signs at any of the doses tested. There were no gross necropsy findings at 100 mg/kg/day; while single incidence of stomach distended with gas was observed each at 300 and 1000 mg/kg/day dose level.

·   Mean body weights and gains and food consumption were significantly lower at 1000 mg/kg/day.

·   Mean values of maternal parameters comprising uterine weight and number of corpora lutea, implantations, early resorptions, late resorptions, pre-implantation loss and post implantation loss were statistically comparable to the vehicle control group up to the highest dose of 1000 mg/kg/day.

·   The litter parameters comprising total number of fetuses, number of live fetuses, male and female fetal weights and anogenital distance were statistically comparable to vehicle control group up to the highest tested dose of 1000 mg/kg/day.

·   Fetal external, visceral and skeletal examination revealed no signs of teratogenicity up to the highest tested dose of 1000 mg/kg/day.

·   Thyroid hormone levels (T3, T4 and TSH) , thyroid gland weights and histology of thyroid glands and gross finding (stomach distended with gas) were unaffected by treatment with Fatty acids, C18-unsatd, dimers, polymers with tall-oil fatty acids and triethylenetetramine up to the highest dose of 1000 mg/kg/day.

 

Based on the above findings, it is concluded that, Observed- Adverse- Effect- Level (NOAEL) for

 

·   Maternal toxicity is300 mg/kg/day,due to test item related significant reduction in mean maternal body weight and weight gain and reduction in food consumption at 1000 mg/kg/day.

 

·   Fetal developmental toxicity and Teratogencity is1000 mg/kg/day,as fetal external, visceral and skeletal examinations revealed no signs of developmental toxicity or teratogenicity 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.