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Toxicological information

Toxicity to reproduction

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Administrative data

screening for reproductive / developmental toxicity
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2010, April 26 to 2010, July 2
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP with certificate.The study is clear and complete in any parts. The substance is well identified, the method is suitable for the substance.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:

Test material

Details on test material:
Test Item Name: Biodiesel
Alternative Test Item Name: Fatty Acid Methyl Ester
Batch/Lot No: 0912L540
CAS Number: 67762-26-9
Description: Clear, yellow Liquid
Expiry / Retest: A sample of the test item was sent to ASG Analytik Service for
ester content analysis (stability testing) at the end of the
dosing period. The certificate of analysis provided at the end of
the study confirmed that the test item was stable for the duration
of the study.
Purity: 97.3%
Storage Conditions: 2-8°C protected from light

Test animals

Details on test animals and environmental conditions:
One batch of 42 male and 42 female Sprague-Dawley rats (Crl: CD®(SD) strain) were received on 20 April 2010 from Charles River UK Limited, Margate, Kent, UK. The animals were ca 6 weeks of age and weighed 201-239 g for males and 150-179 g for females, on despatch.

Each animal received a subcutaneously implanted electronic chip, which identified it individually within the study, and which corresponded to that animal’s number. Each animal was given a cage card which was colour coded for treatment group, and was marked with the study number, cage and animal numbers, sex and relevant treatment group. In addition, a second cage card was given to each animal identifying that animal by study
number, neurotox identification, animal number and sex. This cage card was not colour coded.

The animals were acclimatised in the Charles River animal room for 12 days prior to commencement of treatment. All the animals were examined on arrival for signs of abnormality or disease. No such signs were found and the animals were accepted for use on the study.

There was automatic control of light cycle, temperature and humidity in the animal room. Light hours were 0700-1900 h. The target ranges for temperature and humidity were 21°C ± 2°C and 55% ± 15% respectively, with a minimum of 15 air changes per hour.
Daily monitoring indicated that temperature remained within the target range of 21°C ± 2°C (actual range 20°C-23°C) and humidity was slightly above the target range on 6 occasions (actual range 35-68%). This deviation from target range was considered to be minor and had no significant impact on the outcome and integrity of the study.

Each day, floors were swept and then mopped with a 0.5% solution of Tego 2000 (Th.Goldschmidt Limited, Ruislip, Middlesex, UK), an amphoteric biocide/cleanser. The room was washed with this solution at approximately weekly intervals.

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 of 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.

Cages, absorbent papers and water bottles were changed at regular intervals, as appropriate. White tissue paper nesting material was changed when it was unacceptably soiled. Clean wood shavings were provided at each change of solid-bottomed cage.

To provide environmental enrichment, wooden chewsticks were made available to the animals as appropriate.

Rat and Mouse Breeder Diet No. 3 (Expanded) SQC supplied by Special Diets Services Limited, Stepfield, Witham, Essex, UK was available to the animals ad libitum. The diet was supplied with a batch analysis for nutritive constituents and a range of significant contaminants. The analytical certificate for a batch of diet used in this study is retained in the study archive.
Due to technical error, Animal 74 (Group 4 female) was fed expired diet from 02-07 June 2010 prior to terminal kill. The diet given expired on the 01 June 2010; therefore this deviation was considered to be minor given that the food had expired on the previous day to use and the short length of time the animal had consumed the expired diet. This protocol deviation had no impact on the outcome or integrity of the study.
The food was not considered to contain any additional substances in sufficient concentration to influence the outcome of the study.
The animals had access to domestic, mains quality water ad libitum. The supply is analysed regularly for dissolved and suspended materials, including a range of significant contaminants. The analytical certificate for a typical recent analysis is retained in the study archive.

Cages were allocated to treatment group by the use of randomly sequenced numbers, in such a way that each complete rack contained representatives from all treatment groups.

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on exposure:
Dose levels were agreed upon with the Sponsor after evaluation of existing relevant toxicological data, including Charles River Study 495299; a one week dose range finding study in rats. Results from this study indicated no adverse effect of treatment at 2000 mg/kg/day. However, for the purposes of this study a high dose level of 1000 mg/kg/day was considered appropriate.

The animals were dosed once daily by oral gavage at a dose volume of 5 mL per kg body weight, using a plastic gavage. The volume to be administered to each animal was determined on each day by the weight of the animal as measured at the time of administration, except during late gestation; from Day 16 of gestation until parturition was complete, the dose volume of the females was determined by the weight of the animal on Day 16 of gestation.
The males were dosed once daily for 4 weeks overall, commencing 2 weeks prior to mating. The females were dosed once daily from 2 weeks prior to mating then continued until at least Day 4 of lactation. Dosing for males and females continued until the day prior to termination.
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:
Duration of treatment / exposure:
4 weeks for males,
2 weeks before pairing, during pairing, during gestation and until 4 days post partum for female
Frequency of treatment:
Once daily
Details on study schedule:
- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks
Doses / concentrationsopen allclose all
Doses / Concentrations:
0 mg/kg/day
actual ingested
Doses / Concentrations:
100 mg/kg/day
actual ingested
Doses / Concentrations:
300 mg/kg/day
actual ingested
Doses / Concentrations:
1000 mg/kg/day
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle

Results and discussion

Results: F1 generation

Effect levels (F1)

Dose descriptor:
1000 mg/kg/day
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

The tested substance revealed no effect in Screening for reproduction for a dose of until 1000 mg/kg/bw
Executive summary:

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