Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The tested substance revealed no effect in combined Repeated Dose Toxicity Study on rats and in the reproduction/Developmental Toxicity screening Test on Rats and on rabbits for a dose of until 1000 mg/kg/bw

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

From the available data of the Prenatal Developmental Oral Toxicity Study in Wistar Rats and in New Zeland White rabbits; OECD 414 studies, 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.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
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. certificate)
Limit test:
no
Specific details on test material used for the study:
Batch/Lot Number 20180604
Species:
rabbit
Strain:
New Zealand White
Details on test animals 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.

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
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
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
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
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
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
number of abortions
total litter losses by resorption
effects on pregnancy duration
early or late resorptions
dead fetuses
changes in pregnancy duration
changes in number of pregnant
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
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
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
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.

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:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
see attached justification
Qualifier:
according to
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
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
Species:
rat
Strain:
Wistar
Details on test animals 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
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
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
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.
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
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):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:

no adverse effects
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
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
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

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.

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.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Toxicity to reproduction: other studies

Additional information

Further studies on reproduction are not proposed based on the followings:

The substance is a complex residue resulting from the distillation of C6-24 and C6-24 unsatd. fatty acids methyl esters which is derived from natural fats having a carbon range of C6 -24. It consists predominantly of mono, di, triglycedides of satd. and unsatd. fatty acids, free fatty acids and corresponding methyl esters , dimerisation products, and wax esters.

The basis for a weight of evidence approach consists of the following cumulative sources of confirmation:

·               The substance is of low toxicological activity.

·               The metabolism of the substance does not lead to reproductively toxic metabolites.

·               Evidence from chronic toxicity studies.

·               Evidence from reproductive toxicity studies.

The substance is of low toxicological activity;

There is no evidence of toxicity seen in any of the tests available, either short or long term

 The metabolism of the substance does not lead to reproductively toxic metabolites.

Fatty acid methyl esters are metabolised as other dietary fats to fatty acids and methanol, neither of which have been shown to be reproductive toxins. Higher molecular weight aliphatic esters are readily hydrolysed to the corresponding alcohol and acid and then generally oxidised to carbon dioxide and water[1]. In addition there is data from human and animal studies that shows rapid absorption in the liver and breakdown of the substance into methanol and fatty acids; there is absence of the substance itself in the plasma/blood and in the urine. Methanol has been shown to not be a reproductive toxin.

 There is no direct evidence that exposure of people to methanol adversely affects reproduction or development. The NTP[3]states that there is minimal concern for adverse developmental effects when humans are exposed to methanol levels that result in low blood methanol concentrations, i.e., < 10 mg/L blood. Blood methanol levels of 10 mg/L or greater are not expected to result from normal dietary or occupational exposures.

Evidence from reproductive toxicity studies.

For the analogues substance fatty acids methyl esters (component and a metabolic precursor), in a

combined Repeated Dose Toxicity Study with the Reproduction/Development Toxicity Screening Test in Rats, four groups of 10 male and 10 female Sprague-Dawley rats were dosed orally by gavage once daily at levels of 0, 100, 300 and 1000 mg/kg/day. The males were treated for 2 weeks prior to mating, through until necropsy after at least 4 weeks of treatment. Females were treated for 2 weeks prior to mating, then through mating, gestation until at least Day 4 of lactation.[3]

At dose levels up to 1000 mg/kg/day, there was no obvious effects of treatment on clinical observations, necropsy findings, body weight gain or food consumption. There was no obvious indication of an effect of treatment on neurotoxicity observations or haematology, coagulation and clinical chemistry parameters. Mating performance, fertility, duration of gestation, survival, litter size and litter and pup weights did not indicate any obvious effect of treatment any of the dose levels applied.

Under the conditions of the study, the NOEL (No Observed Effect Level) for both parental and reproductive effects was 1000 mg/kg/day.

In the 90 day study with methyl oleate on rats, where also reprodiction has been studies on 20 females, there were also no adverse effects seen at ca. 3500 mg/kg bw/day.[4]

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.

Conclusion

There are no indications of effects related to reproductive toxicity:


[1]         Mattison et al (J. Nutrition 102, 1171 (1972), J. Lipid Res 13, 325 (1972).

[2]         NTP-CERHR Monograph on the Potential Human Reproductive and Developmental Effects of Methanol September 2003 NIH Publication           No. 03-4478 National Toxicology Program (NTP).

[3]         C16-C18 and C18 Unsaturated Alkyl Methyl Ester: Combined Repeated Dose Toxicity Study with the Reproduction/Development Toxicity            Screening Test in Rats. Test Facility Study No. 495325, Report No. 31335

[4]        MurrayT. K.,J. A.,C. Y., Chisholm M. J. (1058). The effect of mono-enoic fatty acid esters on the growth and fecal lipides of rats. Journal            of the American Oil Chemists' Society, 35, 156-158.

Justification for classification or non-classification

No classification for reproductive toxicity is warranted at present under 67/548/EEC or Regulation 1272/2008.