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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Remarks:
read-across from a supporting substance (analogue or surrogate)
Adequacy of study:
key study
Study period:
July 2018 - December 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 414 GLP
Justification for type of information:
see attached justification

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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 June 25, 2018
Deviations:
no
Principles of method if other than guideline:
This study was performed to determine prenatal developmental and maternal toxicity potential of Fatty acids, C6-24 and C6-24unsatd., Me esters, Distillation residues when administered, daily, through gavage in mated New Zealand White rabbits, from gestation days (GDs) 6 to 29. The method followed was as per guidelines of the OECD 414 (June 2018) and in GLP compliance. 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 (incl. QA statement)
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:
Batch/Lot Number 20180604

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Healthy, adult rabbits (Oryctolagus cuniculus) of New Zealand White strain were obtained from Sainath Agencies, Hyderabad, India. Female rabbits were nulliparous and non-pregnant. At the initiation of the acclimatisation females rabbits were 6 to 6.5 months old.
A larger number of rabbits than necessary for the study were ordered to permit the selection and/or replacement of rabbits before the start of the cohabitation. A total of 40 male and 110 female rabbits were selected for the acclimatisation. Rabbits were received in the experimental room and allowed to acclimatise for a period of 5 days prior to the cohabitation. During the acclimatisation period, rabbits were observed daily, once, for clinical signs. After obtaining 100 mated females (25/group), the remaining male and female rabbits were returned to the Animal Breeding Facility, JRF.
During the study period, rabbits were kept in the double corridor room (DCR) facility in room N° 504, Department of Toxicology, Jai Research Foundation.
The mean body weight (g) and the standard deviation (SD) of the experimental female rabbits prior to acclimatisation are mentioned in below table:
Total N° of Females Received for Acclimatisation Female Body Weight (g) Body Weight Range (g)
Mean SD
110 2513.92 212.55 2051.2-2940.2
The body weight variation among the rabbits was within ±20% of the mean body weight at the beginning of the acclimatisation.

Environmental Conditions:
The photoperiod of 12 h fluorescent light (06.00 to 18.00 hours) and 12 h dark was maintained. Environmental conditions during the study period are summarised in the table below:

Parameters Temperature (°C) RelativeHumidity (%) All Fresh Air Change (per hour) Mean Fluorescent Light Intensity (LUX)
Month and Year Maximum Minimum Maximum Minimum
July 2018 21 19 61 35 20 226.0
August 2018 21 19 65 39 20 196.6
September 2018 21 19 50 42 20 232.0

Housing
Rabbits were housed individually in stainless steel rabbit cages on a 3-tier rack except during mating. Each cage was fitted with a stainless steel food hopper having provision for rabbit pellet food and a polypropylene water bottle with stainless steel drinking nozzle.

Food and Water
The experimental rabbits were fed with standard rabbit pellet diet ad libitum (Teklad Certified Global High Fiber Rabbit Diet, Batch No 2031C-021418MA, procured from the Envigo, USA) with an unlimited supply of drinking water in polypropylene bottles filtered through Reverse Osmosis water filtration system.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
sorbitan derivative
Remarks:
The Tween 80 and 0.5% CMC (in reverse osmosis water) in the ratio of 1:99 was selected as a vehicle as per solubility check performed at the JRF (JRF Study N° 461-1-04-13828).
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:
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.

The active ingredient (a.i.) concentration and homogeneity of test item in samples of the control and dose formulations were analysed once prior to initiation of treatment and once during the treatment period.
Two sets of samples (10 samples per set) were collected by sampling three aliquots (upper, middle and lower layers) from each concentration except control (only one aliquot). One set of samples was used for analyses and the other set of samples was stored at 2 – 8 °C. On each occasion, the mean concentration was determined and compared with the nominal value. The acceptance criteria was ±15% deviation from nominal value and %CV <10. The samples were analysed at JRF using validated analytical method (JRF Study N° 228-2-13-13827). Results were appended in the study report (APPENDIX 15). Stored samples were disposed of during finalisation of report.
Samples was analyzed by following instrumental parameters:
Instrument : GC-FID
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 µL
Details on mating procedure:
After the acclimatisation period, female rabbits were cohabitated with untreated male rabbits (1:1), until the requisite numbers of mated females (25/group) were obtained. Detection of mating was confirmed by identification of the vulva moist with semen (by visual observation). After confirmation of mating, females were returned to individual cages, assigned to a group and the day was designated as day ‘0’ of gestation (GD 0).
Duration of treatment / exposure:
Dose formulations and vehicle were administered to the mated female rabbits daily, through gavage, from 6th to 29th day of gestation, approximately, at the same time on each day. The dose volume for administration was 5 mL/kg body weight. Gavage was performed using Ryle’s tube and BD syringe, which was graduated up to 20 mL. Doses were adjusted according to the most recent body weight recorded. The control group rabbits were treated in same manner with vehicle alone.
Frequency of treatment:
once a day
Duration of test:
gestation period
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group G4
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group G3
Dose / conc.:
90 mg/kg bw/day (nominal)
Remarks:
Group G2
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
only vehicle for Group G1 (Control)
No. of animals per sex per dose:
25 females for each group. Four groups were tested.
Control animals:
yes
Details on study design:
Group N° Dose Levels Dose
(mg/kg b. wt./day) Total N° of Rabbits Rabbit N°
G1 Control 0 (Vehicle) 25 1-25
G2 Low Dose 90 25 26-50
G3 Mid Dose 500 25 51-75
G4 High Dose 1000 25 76-100

Examinations

Maternal examinations:
OBSERVATIONS
Mortality and Morbidity:
Rabbits were observed twice daily for mortality and morbidity. Aborted female rabbits were weighed, sacrificed, and examined, as per the terminally sacrificed rabbits.

Clinical Signs:
During the gestation period, female rabbits were observed daily, twice, for clinical signs of toxicity including changes in skin, fur, eye, mucous membrane, as well as behaviour pattern and abortion.

Body Weights:
Body weight of all mated female rabbits were recorded individually on GDs 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, and 30.

Food Consumption:
A weighed amount of rabbit pellet food was given daily to each mated female and the feed leftover was recorded on next day to calculate the food consumption for the periods of 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-30, and 0-30 days.

Necropsy:
On day 30th of gestation, all survived female rabbits were weighed, euthanised by intravenous (IV) injection of thiopentone sodium, dissected and examined macroscopically. The non-gravid uteri were immersed in 2% sodium hydroxide solution for one hour for the confirmation of pregnancy status. Ovaries and gravid uteri including the cervix were removed and examined immediately for the following parameters:

Maternal Parameters:
1. Number of corpora lutea
2. Gravid uterus including the cervix weight (g)
3. Number of implants
4. Number of live fetuses
5. Number of dead fetuses
6. Number of early resorptions
7. Number of late resorptions
8. Number of male fetuses
9. Number of female fetuses
10. Pre-implantation loss (%)
11. Post-implantation loss (%)
12. Corrected body weight = Body weight on gestation day 30th – gravid uterine weight
Ovaries and uterine content:
The mean absolute and relative uterine weight of pregnant female rabbits were comparable with that of the control group. The mean number of corpora lutea, implants, live fetuses, dead fetuses, and resorptions were comparable with that of the control group. The mean percent pre-implantation loss, post-implantation loss, live fetuses, and dead fetuses were comparable with that of the control group.
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter ]
- Skeletal examinations: Yes: [50% of litter ]
- Head examinations: Yes: [50% of litter ]

Fetal Parameters :
1. Body weight of male fetuses (g)
2. Body weight of female fetuses (g)
3. Placenta weight (g)
4. Crown rump length (mm)
5. Sex ratio
Statistics:
All raw data was recorded in standard formats. Raw data were processed to get group means and standard deviations with significance among the control and treatment groups using validated statistical software. Data were summarised in tabular form. All parameters, characterised by continuous data such as body weight, body weight change, food consumption, prenatal data, and fetal data, were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test. All analyses and comparisons were evaluated at the 5% (p≤0.05) and 1% (p≤0.01) levels. When data did not meet the homogeneity of variance, F-test was performed followed by the t-test to calculate significance. Non-parametric data such as pregnancy rate, mortality rate, and incidence of fetuses and litters with malformation/variation, were analysed using Chi-square test at 95%.
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.
Indices:
F= female and M= male

% F Mortality:No of F died during the study X 100 /No of mated F at the start
Pregnancy Rate:No of F with implantation site at term X 100 /No of mated F sacrificed at term
30th Day Corrected Body Weight: Body weight on gestation day 30 – gravid uterine weight
Body Weight Change (%) at each Interval: (Body weight on a particular interval - Body weight on previous interval)X 100/Body weight on previous interval
Relative Uterine Weight (%):Gravid uterine weight X 100/ 30th day female body weight
Pre-implantation Loss (%): (No of corpora lutea – No of implantation sites)X 100/ No of corpora lutea
Post-implantation Loss (%):(No of implantation sites – No live fetuses)x100/No of implantation sites
Live Fetus (%):No of live fetuses x100/No of implantation sites
Dead Fetus (%):No of dead fetuses/No of implantation sites x100
Sex Ratio : N° of male fetuses/ No of female fetuses

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical sign of toxicity was observed up to the dose level of 1000 mg/kg b wt./day during experimental period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality and morbidity were observed up to the dose level of 1000 mg/kg b. wt./day during experimental period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight, body weight change, and 30th day corrected body weight of the pregnant female rabbits were comparable between the control and the test item treated groups except statistically significant increase in mean body weight change during gestation period 12-15 in 90 mg/kg b. wt./day dose group.
This increase in mean body weight change could be considered as incidental and unrelated to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the pregnant female rabbits was comparable between the control and the test item treated groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean absolute and relative uterine weight of pregnant female rabbits were comparable with that of the control group. The mean number of corpora lutea, implants, live fetuses, dead fetuses, and resorptions were comparable with that of the control group. The mean percent pre-implantation loss, post-implantation loss, live fetuses, and dead fetuses were comparable with that of the control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
External and internal examination of aborted and terminally sacrificed female rabbits did not reveal any abnormality.
Neuropathological findings:
no effects observed
Description (incidence and severity):
External and internal examination of aborted and terminally sacrificed female rabbits did not reveal any abnormality.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
External and internal examination of aborted and terminally sacrificed female rabbits did not reveal any abnormality.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
External and internal examination of aborted and terminally sacrificed female rabbits did not reveal any abnormality.
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
G1: 1/25 (4%)
G2: 2/25 (8%)
G3: 1/25 (4%)
G4: 2/25 (8%)
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
pre-implantation loss mean: G1: 9.56 ; G2: 12.05; G3: 8.73; G4: 16.01

post-implantation loss mean: G1: 2.08 ; G2: 1.41; G3: 0; G4: 2.10

Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
total n. of resorption mean: G1: 0.10 ; G2: 0.11; G3: 0; G4: 0.20
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
early resorption mean: G1: 0 ; G2: 0.11; G3: 0; G4: 0.05

late resorption mean: G1: 0.10 ; G2: 0; G3: 0; G4: 0.15
Dead fetuses:
no effects observed
Description (incidence and severity):
Dead Fetus%: G1: 0 ; G2: 0; G3: 0; G4: 0
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Pregnancy rate was 84.0%, 84.0%, 80.0%, and 88.0% in the control, 90, 500, and 1000 mg/kg b. wt./day dose groups, respectively.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rate was 84.0%, 84.0%, 80.0%, and 88.0% in the control, 90, 500, and 1000 mg/kg b. wt./day dose groups, respectively.
Other effects:
not examined

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
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
mortality
number of abortions
organ weights and organ / body weight ratios
total litter losses by resorption
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weight of male, female and total fetuses (male + female) was comparable between the control and the test item treated groups except statistically significant increase in mean female fetus body weight in 90 mg/kg b. wt./day dose groups. This increase in the mean female fetus body weight could be considered as incidental and has no toxicological importance.
The mean placental weight and crown rump length of male, female, and total fetuses (male + female) were comparable between the control and the test item treated groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The mean count of male, female, and total fetuses (male + female) was comparable with that of the control group. The mean percent male ratio was comparable with that of the control group
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
A total of 122, 119, 138, and 129 fetuses were observed for external abnormalities belonging to 0, 90, 500, and 1000 mg/kg b. wt./day dose groups, respectively.
No treatment related external anomalies were observed in fetuses of all treatment groups.
However, a total of 2, 1, 2, and 2 short fetuses were observed in 0, 90, 500, and 1000 mg/kg b. wt./day dose groups, respectively. This finding was comparable with that of the control group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of 122, 119, 138, and 129 fetuses were observed for the skeletal abnormalities belonging to 0, 90, 500, and 1000 mg/kg b. wt./day dose groups, respectively. No treatment related variation/malformation was observed in fetuses belonging to all treatment groups.
However, Statistically significant decrease in number of fetuses with 5th Sternebrae: Absent was observed in 90 mg/kg b. wt./day dose groups. Statistically significant decrease in number of fetuses with 5th Sternebrae: Hypoplastic was observed in 90, 500, and 1000 mg/kg b. wt./day dose groups.
These decrease in number of incidences has no toxicological importance.
Visceral malformations:
no effects observed
Description (incidence and severity):
A total of 122, 119, 138, and 129 fetuses were observed for visceral abnormalities belonging to 0, 90, 500, and 1000 mg/kg b. wt./day dose groups, respectively.
No treatment related variation/malformation was observed in fetuses belonging to all treatment groups. However, left kidney and ureter was absent in one fetus belonging to the control group.
Other effects:
not examined

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
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
external malformations
skeletal malformations
Remarks on result:
not determinable due to absence of adverse toxic effects

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 results of this study, the No-observed-adverse-effect-level (NOAEL) of Fatty acids, C6-24 and C6-24unsatd., Me esters, Distillation residues for maternal and developmental toxicity is 1000 mg/kg b. wt./day. The test item was found to be non-teratogenic up to the dose level 1000 mg/kg b.wt./day.
Executive summary:

This study was performed to determine prenatal developmental and maternal toxicity potential of Fatty acids, C6-24 and C6-24unsatd., Me esters, Distillation residueswhen administered, daily, through gavage in mated New Zealand White rabbits, from gestation days (GDs) 6 to 29. The method followed was as per guidelines of the OECD 414 (June 2018).

Method

Fatty acids, C6-24 and C6-24unsatd., Me esters, Distillation residues was administered dailythroughgavage from GDs 6 to 29, to 25 mated rabbits per group at dose levels 90, 500, and 1000 mg/kg b. wt./day. The control group receivedvehicleonly.Dose formulations were analysed, for homogeneity and active ingredient concentration, prior to initiation of treatment and once during the treatment period.Rabbits were observed daily for mortality and clinical signs. Maternal body weight was recorded and food consumption was calculated from food input and food left over throughout the gestation period.All survived females were sacrificed on GD 30 and assessed for gross pathological changes.The uteri were excised, weighed, and examined for the number of implantation sites, early and late resorptions, and number of live and dead fetuses. Placenta was weighed and crown rump length was measured, individually. Ovaries were removed and the number of corpora lutea counted. Fetuses were determined for sex, weighed, and examined for external, visceral, head razor, and skeletalabnormalities.

Results

The meanrecovery (%)of the test item invehicle prior to initiation of treatment was 112.81, 104.47, and 88.74 at dose levels90, 500, and 1000mg/kg b. wt./day, respectively.The meanrecovery (%)of the test item invehicle during treatment was 108.76, 91.50, and 108.91 at dose levels90, 500, and 1000mg/kg b. wt./day, respectively.At each dose level and at each interval, the active ingredient concentration of the test item invehiclewas within acceptance criteria of±15.0% of nominal concentrationand %CV < 10.