Fatty acids, C16-C18 (even numbered) and C18 (unsaturated) and Fatty acids, C16-C18 (even numbered) and C18 (unsaturated) methyl esters

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD test guideline 422

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

yes (minor recording errors without any impact on study outcome)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

85049-31-6

Cas Number:

85049-31-6

IUPAC Name:

85049-31-6

Details on test material:

Name: Unsaturated Alkyl Carboxylic Acid Methyl Ester (C10-C18 and C12-C22). The test item is also known as FAME
Batch/Lot No: ASG_161418
Storage Conditions: Ambient room temperature, protect from light/Nitrogen headspace (as indicated from test item data sheet provided by supplier)
Purity: 96.7% prior to study and 96.5% at end of in-life

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: about 8 weeks
- Weight at study initiation: 180-200 g for males and 151-170 g for females (age of 6 weeks)
- Housing:
The animals were initially housed 2 per cage, in polycarbonate cages, with solid bottoms and stainless steel mesh tops and measured ca 48 x 37.5 x 25 cm. A stainless steel food hopper and a polycarbonate water bottle were provided for each cage and sterilised wood shavings were provided as bedding. Male and female cages were racked separately. A few days prior to pairing for mating, males were transferred to individual cages with a stainless steel grid insert measuring ca 48 x 37.5 x 25 cm. Excreta were collected on a tray lined with absorbent paper suspended beneath each cage.
The mated females were transferred to individual solid bottomed cages measuring ca 48 x 37.5 x 25 cm. White paper tissue was supplied as nesting material from Day 20 of gestation. Females with litters retained this cage type until termination. After mating the males remained singly housed until termination.
- Diet (e.g. ad libitum):ad libitum.
- Water (e.g. ad libitum):ad libitum.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 2°C
- Humidity (%): 39-64%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle
- Amount of vehicle (if gavage): 5 mL per kg body weight
The formulations were prepared at approximately weekly intervals and were used with the 13 days stability period at ambient/dark. The test item was formulated by adding the appropriate volume of vehicle (corn oil) to required amounts of test item and mixed manually and magnetically stirred until a visibly homogeneous solution was obtained.

Details on mating procedure:

A few days prior to the initiation of mating, the males were separated into individual grid
bottomed cages.
Pairing was on a 1 male to 1 female basis, within the same treatment group. Each female was
transferred to the cage of an appropriate co-group male near the end of the working day, and
remained there until mating was detected or 14 nights had elapsed.
Vaginal lavages were taken daily early each morning from the day of pairing until mating had
occurred and the stage of oestrus observed in each lavage was recorded. The day of detection
of a copulatory plug in situ and/or of sperm in the lavage was designated Day 0 of gestation.
The time taken for each female to show a positive mating sign was evaluated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of dosing formulations was undertaken with regard to concentration and homogenity. Samples (3 x 1 mL for Groups 1 and 2, 3 x 0.35 mL for Group 3 and 3 x 0.25 mL for Group 4) were taken from formulations (including Control) prepared for use on Week 1 and Week 6 of the study.
The analysis was undertaken by Toxicology Support Laboratory at Charles River, Edinburgh, using a validated method (Charles River Study No. 426246, Method 2624).

Duration of treatment / exposure:

males: 4 weeks
females: 2 weeks prior to mating then continued until at least Day 4 of lactation.

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bodyweight
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations:
Prostration
Lethargy
Writhing
Circling
Breathing abnormalities
Gait abnormalities
Tremor
Fasciculation
Convulsions
Biting (of cage components or self mutilating)
Vocalisations
Piloerection

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

Oestrous cyclicity (parental animals):

All the animals mated within one oestrous cycle

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies

GROSS EXAMINATION OF DEAD PUPS:
no dead pups

Postmortem examinations (parental animals):

The males were sacrificed following 4 weeks of treatment. The females were sacrificed with their litters between Days 5 and Day 6 of lactation.
Adult animals were sacrificed by exposure to carbon dioxide followed by exsanguination.

GROSS PATHOLOGY: Yes
All adults were subject to a detailed necropsy, the necropsy consisted of an external and internal examination which included body orifices (ears, nostrils, mouth, anus, vulva) and cranial, thoracic and abdominal organs and tissues. All gross lesions were recorded in terms of location, size, shape, colour, consistency and number.
HISTOPATHOLOGY: Yes
Histological examination was conducted on 5 males and 5 females from Control and High dose groups, the same animals that were used for Laboratory investigations.

Postmortem examinations (offspring):

Offspring killed or found dead were sexed, then checked for the presence of milk in the
stomach and the presence of externally visible abnormalities. Any abnormal pups were
preserved in 10% formalin or methylated ethyl alcohol as appropriate for possible future
examination. Externally normal decedents were discarded.

Reproductive indices:

Fertility Index (male)
Fertility Index (female)
Gestation Index

Offspring viability indices:

Birth Index
Live Birth Index
Viability Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

dermal irritation

body weight and weight gain

food consumption and compound intake

food efficiency

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

dermal irritation

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a GLP-study according to OECD test guideline 422, the NOEL for reproduction/developmental toxicity of the registered substance was determine to be 1000 mg/kg bw.

Executive summary:

In a GLP-study according to OECD test guideline 422, the repeated dose (28 days) toxicity of the registered substance was determined. 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 vehicle was corn oil. 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. The animals were monitored daily for any signs of ill health or reaction to treatment, body weight and food consumption performance was monitored. Pups were examined externally. At dose levels up to 1000 mg/kg/day, there were no clear toxicological effects of treatment on clinical observations, body weight gain or food consumption performance. Mating performance, pregnancy performance and pup development were similar throughout the groups. There were no necropsy or histology findings that were considered to be related to treatment with the registered substance Under the conditions of this study, the NOEL (No Observed Effect Level) for reproductive effects was 1000 mg/kg/day.