Triglycerides, C16-C18 (even numbered) and C18 (unsaturated), maleated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

According to the guideline and under GLP tested with the source substance CAS 85711-46-2. As the tested substance is a breakdown product of the target substance, this study is considered valid for read-across. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C14-18 and C16-18-unsatd., maleated

EC Number:

288-306-2

EC Name:

Fatty acids, C14-18 and C16-18-unsatd., maleated

Cas Number:

85711-46-2

IUPAC Name:

85711-46-2

Details on test material:

- Name of test material (as cited in the study report): WS400104
- Appearance: Light brown to brown liquid (semi-solid or wax-like at 20°C)
- Analytical purity: not applicable (UVCB substance)
- Lot/batch No.:KL-10-104
- Expiration date of the lot/batch: 21th Dec. 2015
Storage: Room temperature (15 - 25ºC, below 70 RH%)

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Wistar Crl:WI rats
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Age at study initiation: 11 weeks
- Weight at study initiation: males: Males: 350 g – 382 g, Females: 183 g - 218 g
- Fasting period before study: overnight prior to treatment
- Housing: 5 animals of the same sex and group/cage with the exception of the mating and gestation/delivery period, when they
were paired or individually housed, respectively. (cage: Type II and/or III polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20,1-25
- Humidity (%): 36-70
- Air changes (per hr):15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

VEHICLE
- Concentration in vehicle: 20, 60, 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

The test material and the vehicle was warmed up on a water bath to 50 C for approximately 10 minutes separately and mixed up on a hot stirrer plate. Once a suitable formulation was obtained, the container was removed from the plate. Pending administration to the animals, the dose formulations were stirred on a magnetic stirrer at room temperature and were protected from light.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of WS400104 formulations for concentration and homogeneity was performed using validated HPLC method (CiToxLAB study code 11/352-316AN). The concentration analysis was performed on 3 occasions, during the first, fourth and last weeks of the treatment period. Recovery of WS400104 from propylene glycol ranged between 92% and 106%.

Details on mating procedure:

M/F ratio per cage: 1/1
- Length of cohabitation: until copulation occurred, for up to 7 days.
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged individually.
- Mating of siblings was avoided.

Duration of treatment / exposure:

Main males: 35 days (14 days pre-mating, 14 days mating/post-mating period followed by an additional week)
Main females: ca. 47 days (14 days pre-mating, for up to 7 days mating period, through gestation til PPD 4)
Satellite females (nulliparous and nonpregnant): 35 days

Frequency of treatment:

daily, 7 days/week

Duration of test:

Main males: 35 days
Main females: ca. 47 days (necropsy on PPD5)
Offspring: from birth to PND4

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 animals/sex/dose
(Satellite female group: 5 animals/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose finding toxicity study with WS400104 (administered via oral gavage to Wistar rats for 7 consecutive days at dose levels of 100, 300 and 1000 mg/kg bw) showed no overt adverse effects related to the test material (7.5.1 Dose Range Finding Study 7 days_WS400104).
Based on these results, the dose levels selected for the main study were 0, 100, 300 and 1000 mg/kg bw/day.

This study (OECD Guideline 422) was conducted to examine both repeated dose toxicity and reproductive/developmental toxicity as an OECD screening combined study . Therefore, animals initially entering the study were divided into toxicity subgroup animals (Satellite females) and reproductive subgroup animals (Main females and males), whereby 5 of the 12 Main males (used for pairing) per dose group formed the toxicity male Subgroup A.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality.
Delivery process was observed as carefully as possible. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly, observations in a standard arena

BODY WEIGHT: Yes
- Time schedule: Parent females were weighed on gestation Days GD 0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition), and PPD5 (before termination).

SACRIFICE
- Maternal animals: All surviving animals on Day 5 of lactation (PND5).

GROSS NECROPSY
- Gross necropsy consisted of external examinations.
- Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea were recorded in the Main females as applicable.

ORGAN WEIGHTS
Weight of the following organs of all adult animals were determined:
- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain
- With a precision of 0.001 g: ovaries, pituitary

FOOD CONSUMPTION: yes
-Time schedule: at least weekly

HISTOPATHOLOGY :
Detailed histological examinations was performed in all female adults of control and high dose groups.
Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Other examinations: yes, clinical chemistry , haematology and urinalysis in the toxicity groups (satellite females and subgroup A - males), see: Combined repeated dose toxicity_gavage_rat_WS400104

Ovaries and uterine content:

The ovaries and uterine content was examined after termination of the dams on Day 5 post-partum
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
Post implantation survival was determined.

In addition, gestation length (time elapsing between detection of mating and commencement of parturition) was recorded.

Fetal examinations:

- External examinations: from PPD0 to PPD4, all pubs per litter
- Parameters observed: number and sex of pups, stillbirths, live births; postnatal mortality; presence of gross anomalies, weight gain (PPD0-PPD4), physical or behavioural abnormalities

Statistics:

Performance with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test,the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Indices:

Female Mating Index: Number of sperm-positive females : Total Number of females cohabited x 100
Female Fertility Index: Number of pregnant females : Number of sperm-positive females x 100
Gestation Index: Number of females with live born pups : Number of pregnant females x 100

Formulas for Calculation of Pups’ Mortality and Sex Ratio Indices
Survival Index: Number of live pups (at designated time) : Number of pups born x 100
Pre-implantation mortality: (Number of Corpora lutea − Number of Implantations) : Number of Corpora lutea x 100
Intrauterine mortality: (Number of implantations - Number of liveborns) : Number of implantations x 100
Total mortality: (Number of implantations - Number of viable pups (d4)) : Number of implantations x 100
Post-natal mortality: (Number of viable pups (d0) - Number of viable pups (d4)) : Number of viable pups (d0) x 100
Sex ratio: (Number of pups examined − Number of males) : Number of pups examined x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Dose levels of 1000 mg/kg body weight to Wistar rats for 35 consecutive days caused a minimal/mild hyper/paraceratosis of the nonglandular gastric mucosa in stomach in 4 of 5 males and 2 of 5 females. This finding is considered to be indicative of local irritation in a structure of the stomach that does not exist in man. Systemic effects were not observed.
No adverse effect was found on reproduction parameters at this dose level.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
WS400104 administered to parental generation at up to 1000 mg/kg bw/day did not lead to mortality or any adverse effects considered related to treatment or
toxicologically significant in the F1 generation. No abnormal behaviour of the pups was noted. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups.
The number of viable pups on PND4 as well as pups survival indices on PND0 and PND4 were comparable to control values at up to and including 1000 mg/kg bw/day.
The sex ratios were similar in the Control and treated groups.
There was no effect of treatment on the offspring body weight or body weight gain.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Daily oral gavage administration of WS400104 at dose levels of 100, 300 and 1000 mg/kg day was not associated with signs of developmental toxicity and the NOAEL was considered to be 1000 mg/kg /day.