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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Remarks:
read-across on structural analogue or surrogate
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD, 2001: The Organisation for Economic Co-operation and Development (OECD) Guidelines for the testing of chemicals, OECD 414, Prenatal Developmental Toxicity Study, adopted by the Council on January 22, 2001
Deviations:
no
Principles of method if other than guideline:
This study was performed to evaluate the prenatal developmental and maternal toxicity potential of Fatty acids, C6-24 and C6-24unsatd., Me esters, distillation Residues when administered through oral gavage to the pregnant Wistar rats in graduated doses from 5th day of gestation to 19th day of gestation. Estimation of the No-Observed-Adverse-Effect Level (NOAEL) and/or No-Observed-Effect-Level (NOEL) was targeted for both developmental and maternal toxicity.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Fatty acids, C6-24 and C6-24-unsatd., Me esters, distn. Residues
IUPAC Name:
Fatty acids, C6-24 and C6-24-unsatd., Me esters, distn. Residues
Test material form:
semi-solid (amorphous): gel
Details on test material:
Fatty acids, C6-24 and C6-24-unsatd., Me esters, distn. residues- Physical state: black , brown semisolid- Analytical purity:100% - Storage condition of test material: room temperature- Solubility: < 10% in water, soluble in acetone, hexane and dichloromethane
Specific details on test material used for the study:
Fatty acids, C6-24 and C6-24-unsatd., Me esters, distn. residues- Physical state: black , brown semisolid- Analytical purity:100% - Storage condition of test material: room temperature- Solubility: < 10% in water, soluble in acetone, hexane and dichloromethane

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
Healthy young adult rats (Rattus norvegicus) of Wistar strain (RccHan:WIST) will be obtained from the Animal Breeding Facility, Jai Research Foundation and used for the experiment. Female rats will be nulliparous and non-pregnant. At the initiation of acclimatization females will be 11-13 weeks old. Mating of siblings will be avoided.
After getting 100 sperm positive females (25 mated females/group), the remaining animals will be returned to the Animal Breeding Facility, JRF or humanly sacrificed without any further examination.
Rats will be maintained in an environment-controlled room at a temperature of 22 ± 3 °C and relative humidity of 30 to 70 percent. The photoperiod will be 12 hours light and 12 hours darkness, light hours being 06.00 - 18.00 hours and air changes will be maintained minimum of 15 per hour.
. Environmental conditions during the study period were as summarized in the table below:
Environmental
Parameters Temperature (°C) Relative
Humidity (%) Air Change (per hour) Mean Light Intensity (LUX)
Minimum Maximum Minimum Maximum
April 2016 22 25 60 68 21 231
May 2016 21 25 66 68 20 173

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
sorbitan derivative
Remarks:
The Tween 80 and 0.5% CMC in the ratio of 1:99 was selected as a vehicle as per solubility check performed at the JRF.
Details on exposure:
Tween 80 (polyoxyethylenesorbitan monooleate) is a nonionic surfactant and emulsifier.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the Test Item in the Dose Formulation
The stability of active ingredient (a.i.) in vehicle was determined in JRF Study N° 228-2-13-13827. The stability of the test item was determined up to 4 hours at room temperature by analysing the dose formulation at 0 and 4 hours. 
6.6 Homogeneity and Active Ingredient Concentration of the Test Item in the Dose Formulations
The samples of control and each of the three dose formulations were collected before start and once during the treatment period and stored at -70 ± 10 °C till analysis. After development and validation of analytical method, the active ingredient concentration was determined and compared to the nominal value. The acceptance criteria is ± 15% from nominal value and a %CV <10. The samples were analysed using a validated analytical method (JRF Study N° 228-2-13-13827). Results are reported in APPENDIX 11.
Samples were analyzed using following instrumental parameters:
Instrument : GC-MS equipped with FID (Agilent Technologies, 7890B)
Column : DB-WAX [30 m x 0.25 mm (i.d.) x 0.25 µm film thickness]
Oven Temperature : 60°C (hold time 2.0 minutes) to 200 °C @ 10 °C/minute to 240 °C @ 5 °C/minute (hold time 7.0 minutes)
Injector Temperature : 250 °C
Detector Temperature : 250 °C
Detector : Flame Ionisation Detector (FID)
Split ratio : 10:1
Carrier N2 Flow : 1.0 mL/minute
Hydrogen Flow : 40 mL/minute
Air Flow : 400 mL/minute
Injection volume : 1.0 mL
Details on mating procedure:
Housing
Throughout the experimental period, the female rats were housed individually, except during the mating period. During the mating period, the animals were housed in group of two animals/cage (one male and one female). Male animals were housed in groups of two animals/cage. Mated female animals were housed individually in clean sterilised solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm) on a rack. Each cage was fitted with a stainless steel top grill having provision for keeping rat pellet food and a polypropylene water bottle with stainless steel drinking nozzle. The bottom of the cages was layered with clean sterilised rice husk, as the bedding material. Cages were changed weekly, thrice. Racks were cleaned daily.
The quality of rice husk was routinely monitored at JRF. The values of potential contaminants (chemical as well as microbial) were within the acceptable limit. The results are presented in APPENDICES 12 and 13.
Cohabitation and Allocation to Group
After the acclimatisation period, female rats were cohabitated with untreated male rats (1:1) until the requisite numbers of mated females (25/group) were obtained. Mating was confirmed by the evidence of a copulatory plug in the vagina or by a vaginal lavage for sperm. After confirmation of mating, females were returned to individual cages, assigned to a group and the day was designated as the day 0 of gestation (GD 0).
Mated females were assigned to dose groups by stratified randomization on the basis of GD 0 body weights. Body weights on GD 0 were arranged in descending order, from the heaviest to the lightest on the first GD 0 date, and assigned in the same order on subsequent days. Beginning with the heaviest weight, one animal was randomly assigned to each stratified group. In the event that the total number of animals inseminated on a given day was not an even multiple of the number of treatment groups, the mated females were assigned to complete the last incomplete stratification group and then assigned to the next stratification group until all GD 0 females for that day were assigned.
5.10 Animal Identification
At the time of acclimatisation a temporary animal number was marked with an indelible non-toxic marker pen on the dorsal surface of the tail. A permanent animal number was marked with a tattoo machine on the dorsal surface of tail of E+ female in a sequential order starting from 1-25 for Group-1, 26-50 for Group-2, 51-75 for Group-3, and 76-100 for Group-4 dose groups. The cages were labeled specifically with labels designating each group with study N°, study code, test item code, group N°, sex, dose, and animal N °.
Duration of treatment / exposure:
Dosing
Dose formulations and vehicle were administered to the pregnant female rats daily, once, through gavage, from GD 5 to 19, approximately, at the same time, each day. The dose volume administered to the female rats was 10 mL/kg body weight. The doses were adjusted according to the most recent body weight recorded. The control group received vehicle (Tween 80 and 0.5% CMC in the ratio of 1:99) only.
Gavage was performed using a cannula (size: 16 G x 7.8 cm), manufactured by arrow stainless steel, India, attached to a BD syringe, which was graduated up to 3 mL.
6.8 Summary of the Experimental Design
Group N° Dose
Levels Dose
(mg/kg b. wt./day) Total N° of Animals Animal
N° Minimum N° of Pregnant Females required
G1 Control 0 (Vehicle) 25 1-25 20
G2 Low Dose 125 25 26-50 20
G3 Mid Dose 500 25 51-75 20
G4 High Dose 1000 25 76-100 20
Frequency of treatment:
once daily
Duration of test:
gestation period GD 20
Doses / concentrations
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
respectively rat group G4
No. of animals per sex per dose:
25 (female) per sex per dose
Control animals:
yes
Details on study design:
Summary of the Experimental Design
Group N° Dose
Levels Dose
(mg/kg b. wt./day) Total N° of Animals Animal
N° Minimum N° of Pregnant Females required
G1 Control 0 (Vehicle) 25 1-25 20
G2 Low Dose 125 25 26-50 20
G3 Mid Dose 500 25 51-75 20
G4 High Dose 1000 25 76-100 20

Examinations

Maternal examinations:
No mortality or toxic clinical signs were observed up to the dose level of 1000 mg/kg b. wt./day. No toxicological effects were observed on maternal body weight, body weight change, corrected body weight, and food consumption during gestation period.
Ovaries and uterine content:
The mean numbers of corpora lutea, implantation, resorptions, live fetuses, dead fetuses, the mean percent pre-implantation loss, post-implantation loss were comparable between the control and the treatment groups.
Fetal examinations:
The mean fetal count and mean fetal body weight of male, female, and total fetuses (male + female) were comparable between the control and the treatment groups.
No significant incidence of malformation/variation was observed in external and visceral observation of fetuses of the Fatty acids, C6-24 and C6-24unsatd., Me esters, distillation Residues treated groups.
Statistics:
Data Evaluation and Statistical Analysis
The data on the number of sperm positive animals in each group and number of animals, which were found pregnant at term, were compiled and the pregnancy rate was expressed in terms of percentage. The percentage of dead animals (mortality rate) and percentage of sacrificed animals (survived) at term were evaluated. The maternal food consumption was calculated for the gestational intervals. Similarly, the percent maternal body weight change, during gestational intervals, was also calculated from the gestational body weights.

The gravid uterine weight was recorded and the percent relative uterine weight was calculated along with 20th day corrected maternal body weight. Pre-implantation loss was estimated from the total implants and the number of corpora lutea. Post implantation loss was calculated from the total implantation and resorption sites including dead fetuses.
The mean of the female fetal weights, male fetal weights, and fetal weights composite of both sex, the mean count of male fetus, female fetus, and of both sexes were calculated for each litter. The group means were calculated from litters mean and compared with control. The male to female (male/female) sex ratio was also calculated for all groups
Indices:
The test parameters (numerical results) were analysed using appropriate statistical techniques as follows:
Parameter Statistical Technique
1. Mean body weight
2. Mean body weight change (%)
3. Mean food consumption
4. Mean gravid uterine weight
5. Prenatal data
6. Fetal data Bartlett test, Student’s t-test, ANOVA with Dunnet’s t-test
7. Mortality rate
8. Pregnancy rate
9. Incidence of fetuses with malformation/variation
10. Incidence of litters containing fetuses with malformation/variation Chi-Square test.
Flags for significant difference between control and treated groups (single arrow for p≤0.05 and double arrows for p≤0.01) were given in the table along with the footnote.
Historical control data:
All the study variations are incidental in nature and within range of the historical control data.

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
Description (incidence and severity):
The mean body weight, body weight change, and 20th day corrected body weight were comparable between the control and the treatment groups except statistically significant decrease in mean body weight change during gestation period 11-14 in 500 mg/kg b. wt./day dose groups
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the pregnant female rats was comparable between the control and the treatment groups except statistically significant increase in mean food consumption during gestation period 8-11 in 1000 mg/kg b. wt./day dose groups
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
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
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:

no adverse effects

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: none adverse effect

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:

no adverse effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: none adverse effect

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Maternal Data

8.2.1   Mortality and Clinical Signs(TABLES 2 and 3; APPENDIX 1)

No mortality and clinical signs of toxicity were observed up to the dose level of 1000 mg/kg b. wt./day during experimental period.

8.2.2   Pregnancy Data(TABLE 2)

Pregnancy rate was 100.0%, 88.0%, 88.0%, and 100.0% in the control, 125, 500 and 1000 mg/kg b. wt./day dose groups, respectively.

8.2.3   Body Weight, Body Weight Change and 20thDay Corrected Body Weight(TABLES 4, 5 and 7; APPENDICES 2 and3;FIGURES 1, 2 and 3)

The mean body weight, body weight change, and 20thday corrected body weight were comparable between the control and the treatment groups except statistically significant decrease in mean body weight change during gestation period 11-14 in 500mg/kg b. wt./day dose groups.

8.2.4   Food Consumption(TABLE 6; APPENDIX 4; FIGURES 4 and 5)

The mean food consumption of the pregnant female rats was comparable between the control and the treatment groups except statistically significant increase in mean food consumption during gestation period 8-11 in 1000 mg/kg b. wt./day dose groups


8.2.5   Pathological Findings(APPENDIX 9)

External and visceral examination of the terminally sacrificed female rats did not reveal any lesion of pathological significance.

8.2.6   Prenatal Data(TABLE 7; APPENDIX 5)

The mean absolute and relative uterine weight of the pregnant female rats was comparable between the control and the treatment groups.

The mean number of corpora lutea, implantation, resorptions, live fetuses, dead fetuses, the mean percent pre-implantation loss, post-implantation loss, live fetuses, and dead fetuses were comparable between the control and the treatment groups.

8.3      Fetus Data
8.3.1   Litter Data(TABLE 8, APPENDIX 8)

The mean count of male, female and total fetuses (male + female) was comparable between the control and the treatment groups.

8.3.2   Fetus Body Weight(TABLE 8, APPENDICES 6 and 7)

The mean body weight of male, female and total fetuses (male + female) was comparable between the control and the treatment groups.

8.3.3   External Observations(TABLE 9; APPENDIX 10)

A total of 303, 275, 266, and 302 fetuses were examined in 0, 125, 500, and 1000 mg/kg b. wt./day dose groups, respectively.

No external anomalies were noted in fetuses of the control and the various treatment groups up to the dose level of 1000 mg/kg b. wt./day.

8.3.4   Visceral Observations(TABLE 10; APPENDIX 10)

A total of 145, 131, 127, and 145 fetuses were examined for the fetal visceral observations in 0, 125, 500, and 1000 mg/kg b. wt./day dose groups, respectively.

No gross treatment related visceral anomalies were noted in fetuses of the control and the various treatment groups up to the dose level of 1000 mg/kg b. wt./day except misshaped heart in one fetus belonging to the 1000 mg/kg b. wt./day dose group. This variation was considered as incidental variation.

 

8.3.5   Head Razor Observations(TABLE 11; APPENDIX 10)

A total of 145, 131, 127, and 145 fetuses were examined for the fetal head razor observations in 0, 125, 500, and 1000 mg/kg b. wt./day dose groups, respectively.

Nochangeswere observed in fetal head razor section of the fetus, belonging to either the control group or the treatment groups, up to the dose level of 1000 mg/kg b. wt./day.

8.3.6   Skeletal Observations(TABLE 12; APPENDIX 10)

A total of 158, 144, 139, and 157 fetuses were observed for fetal skeletal evaluation in 0, 125, 500, and 1000 mg/kg b. wt./day dose groups, respectively. The type of skeletal malformations/variations and the incidence of number of fetuses and number of litters affected with these malformations/variations were recorded.

Statistically significant increase innumber of fetuses with ‘Xiphisternum:Unossified’was observed in 125 mg/kg b. wt./day dose group.

Statistically significant increase innumber of fetuses with2ndSternebra:Absentand5th Sternebra:Dumbbell ossificationwas observed in 500 mg/kg b. wt./day dose group.

Statistically significant decrease innumber of fetuses with5thSternebra:Unossifiedwas observed in 500 mg/kg b. wt./day dose group.

Statistically significant increase innumber of litters with5thSternebra:Dumbbell ossification andstatistically significant decrease innumber of litters with5thSternebra:Unossified was observed in 500 and 1000 mg/kg b. wt./day dose group, respectively.

However, these variations are incidental in nature and within range of the historical control data.

Applicant's summary and conclusion

Conclusions:
From the available data of the Prenatal Developmental Oral Toxicity Study in Wistar Rats; OECD 414 study, it is concluded that the expected “No Observed Adverse Effect Level (NOAEL)” of Fatty acids, C6-24 and C6-24unsatd., Me esters, distillation Residues for maternal and fetal toxicity is = 1000 mg/kg b. wt./day.