Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Toxic effect type:

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
short-term repeated dose toxicity: oral
combined repeated dose and reproduction / developmental screening
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
Reason / purpose for cross-reference:
reference to other study
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Limit test:
Details on test animals or test system and environmental conditions:
One batch of 42 male and 42 female Sprague-Dawley rats (Crl: CD®(SD) strain) were received on 30 March 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.

Route of administration:
oral: gavage
corn oil
Details on oral 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.
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
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
Observations and examinations performed and frequency:
All the animals were checked for viability early in the morning and again as late as possible on each day.

All the animals were examined for reaction to treatment on each day from commencement of dosing. The onset, intensity and duration of any signs were recorded. In addition, once each week (starting during Pretrial), all adult animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.
For both sexes, body weights were recorded one week prior to the start of treatment then daily throughout the dosing period until termination.
For brevity of reporting, only weekly weights for males and for females during the pre-mating period have been presented. Additionally, weights for females on Days 0, 7, 14, 16 and 20 of gestation and Days 1 and 4 of lactation have been presented.

For all animals, the weight of food consumed by each cage was recorded once weekly commencing during the week prior to the start of dosing, until pairing for mating. For mated females, the amount of food consumed was recorded over Days 0-7, 7-14 and 14-20 of gestation, and Days 0-4 of lactation.

Water consumption was monitored by visual inspection of the water bottles on a weekly basis throughout the study. No intergroup differences were noted, therefore measuring water consumption gravimetrically twice weekly was not considered necessary.

The eyes were examined using indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®). The following areas were evaluated: anterior, lenticular and fundic areas. An ophthalmic examination was undertaken from all animal during Pretrial, all males during Week 4 and all females shortly prior to sacrifice.

Once during the pre-treatment week (Week -1) and weekly thereafter, a more detailed examination was made on all animals. These examinations were conducted by a technician not involved in the dosing procedures or in the collection of body weight and food consumption data, and were performed at an approximately standardised time of day. Before the independent technician entered the animal room on each occasion to perform the examinations, the cage card showing treatment group location was removed from each cage, leaving the second pre-prepared card as the neurotox animal identifier. In each cage one or two animals will have their tail marked to allow the independent technician to identify each animal.

Posture/condition on first approach (animal undisturbed), checking for:
Breathing abnormalities
Gait abnormalities
Biting (of cage components or self mutilating)
Ease of removal from cage.
Body temperature:
This was taken and recorded from the implanted electronic identification chip. If the electronic chip was not functioning, a rectal temperature was recorded.
Condition of the eyes, checking for:
Pupillary function (reaction to visual stimulus)
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Test Facility Study No. 495325
Condition of the coat.
Presence of salivation.
Overall ease of handling.
Observations in a standard arena (2 min observation period):
Latency (time to first locomotory movement).
Level of mobility.
Arousal (level of alertness).
Posture, tremor/convulsions, vocalisation, piloerection – recorded as for
cageside observatons.
Palpebral closure.
Gait abnormalities.
Stereotypy (excessive repetition of behaviours) and/or unusual behaviours.

The following additional functional assessments were performed on 5 males and 5 females per group during Week 4 for males (prior to blood sampling) and during lactation for females. For males, the animals selected were the first 5 males in the group. For females, the animals
selected for assessment were the first 5 females to reach at least Day 3 of lactation. Again, these assessments were performed at an approximately standardised time of day. Reaction to sudden sound (click above the head) Reaction to touch on the rump with a blunt probe

This was measured using a method derived from that of Meyer et al (1979). A strain gauge was used, to which was attached a wire pull-bar. Once the animal gripped the bar, the body was pulled until its grasp was broken; the strain gauge recorded the force required. The procedure was repeated 3 times for the forelimbs and 3 times for the hindlimbs, and the mean fore and hind grip strengths calculated.

This was assessed by measurement of the tail flick response, using a technique based on the method devised by D’Amour and Smith (1941). The apparatus used shined a calibrated infrared heat source onto the tail and automatically measured the reaction time of the animals (accurate to 0.1 s). It was ensured that no visible injury was caused to the tail by this test.

Maize oil was applied to the hind paws of each animal. The animal was then held in a horizontal, prone position with the nose ca 30 cm above a bench surface covered with absorbent paper. When the animal was calm, it was dropped. The distance between the prints of the central footpads was measured. The procedure was repeated 3 times. If the rat did not land properly on its feet, it was recorded.

Each animal was placed in an individual monitoring cage, scanned by a motion sensor utilising infra-red pyroelectric detectors. Movement was detected in 3 dimensions anywhere in the cage, and was differentiated into large and small movements. Each animal was monitored for one session for 1 h for males and for females it was 0.5 h. Activity counts were recorded over successive periods of 5 min each.

Any other abnormality not already recorded in the above screening battery.

Sacrifice and pathology:
The males were sacrificed following 4 weeks of treatment. The females were sacrificed with their litters between Days 5 and Day 7 of lactation.
Adult animals were sacrificed by exposure to carbon dioxide followed by exsanguination. The pups were sacrificed by intra-peritoneal or intra-thoracic injection of sodium pentobarbitone.


Samples were obtained during Week 4 for males, and on or after Day 3 of lactation for females.
The animals were not deprived of food overnight prior to sampling.

Blood samples were obtained from 5 males and 5 females from each dose group. For males, the first 5 animals in each group were tested. For females, the first 5 animals to have reared their litter to at least Day 3 of lactation were tested.

Samples for haematology, coagulation and clinical chemistry were collected via the tail vein after careful cleaning of the sampling site to avoid any possible contamination. Sampling was by sterile, disposable plastic syringes and vygon infusion sets. Blood was then transferred into plastic tubes containing anticoagulant as follows:

Ca 0.5 mL was taken into tubes containing EDTA and assayed for:
Red Blood Cell Count
White Blood Cell Count
Mean Cell Volume
Mean Cell Haemoglobin
Mean Cell Haemoglobin Concentration
Red Cell Distribution Width
Differential White Blood Cell Count:
Large Unclassified Cells
A blood film smear was produced from each haematology specimen. Blood smears were labelled, stained for possible examination and stored and archived. No abnormal haematological findings were obtained or treatment related effects observed. Blood cell morphology, therefore, was not performed.

Ca 0.9 mL blood into tubes containing 0.1mL 3.8% (w/v) trisodium citrate. The final sample volume will be as close as possible to 1.0 mL to give a final concentration of 0.38% (blood to citrate ratio of 9:1). The citrated blood samples will be centrifuged and the plasma separated into plain plastic tubes and analysed for:
Prothrombin Time
Activated Partial Thromboplastin Time


Ca 1.0 mL into tubes containing lithium heparin which were then centrifuged and assayed for:
Aspartate Aminotransferase
Alanine Aminotransferase
Alkaline Phosphatase
Gamma Glutamyl Transferase
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Test Facility Study No. 495325
Glutamate Dehydrogenase
Lactate Dehydrogenase
Total Protein
Globulin – derived
AG Ratio – derived
Creatine Kinase
Total Bilirubin

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.

The females were allowed to litter normally. The day of birth of the litter was designated Day 0 of lactation. The duration of gestation in days was calculated and evaluated. The numbers of live and dead pups born in each litter were recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined for the presence of milk in the stomach and for any externally visible abnormalities daily up
to Day 4 of lactation. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation. Pups killed on Day 7 of lactation were also weighed en masse by sex; this additional data has not been reported and will be retained in the raw data. This protocol deviation had no significant impact on the outcome or integrity of the study.
When possible, any pups that were found dead or killed during lactation were sexed and appropriately examined as above. Any externally normal decedent pups were discarded. Any deficiencies in maternal care were recorded. Points looked for were inadequate construction and cleaning of the nest, pups left scattered and cold, physical harm of pups, or apparently inadequate lactation or feeding. Detailed information is retained in the study data.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations in the males or females that were attributable to treatment with Biodiesel
no mortality observed
Description (incidence):
There were no clinical observations in the males or females that were attributable to treatment with Biodiesel
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males treated at 100 mg/kg/day had a statistically significant reduction in mean body weight and food consumption, which was evident from pre-trial and remained throughout the study. As these differences were evident during pre-trial and as there was no s
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Group mean food consumption for females prior to mating and throughout gestation and lactation was similar to Control
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ophthalmic findings in males or females which were considered to be related to treatment with Biodiesel
Haematological findings:
no effects observed
Description (incidence and severity):
In males, slight intergroup differences in the haematology and coagulation parameters were too small to be attributed to treatment
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The type and distribution of the clinical chemistry parameters in males and for females during lactation did not indicate any association with treatment
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no neurotoxicity clinical observations in males or females that were considered to be related to treatment with Biodiesel
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Males treated at 100 mg/kg/day had a statistically significant reduction in mean brain and heart weight, compared with Control. When adjusted for body weight (covariance analysis) these mean organ weights did not achieve statistical significance and was c
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no necropsy or histology findings that could be attributed to treatment with Biodiesel
Dose descriptor:
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
The tested substance revealed no effect in Repeated dose oral toxicity 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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
1 000 mg/kg bw/day
Study duration:

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A sub-chronic toxicity study (90 days) is not proposed by the registrant because the frequency and duration of human exposure indicates that a longer term study is not appropriate; the exposure to the substance is likely in the working scenario, where exposure of worker to biodiesel is pertinent only for accidental dermal route, and RMM are already applied. Inhalation route is not considered due to the physical chemical properties of the substance.

Furthermore the substance don't have dangerous properties that cannot be detected in a short-term toxicity study; accumulation of the substance or its metabolites in certain tissues or organs which would possibly remain undetected in a shortterm

toxicity study don't are liable to result in adverse effects after prolonged exposure. They enter in the endogen metabolism of fatty acids and fatty acid methyl esters that can be accumulated or released by tissues on demand.

A NOAEL can be established because of the absence of adverse effect both in 28 and 90 days. Route to route estrapolation can be done from oral to dermal.

Justification for classification or non-classification

Classification for STOT is relevant where significant toxic effects are seen from single or repeated dose studies. No such significant toxicity is seen in all available studies

No classification for STOT is warrented at present under 67/548/EEC or Regulation 1272/2008.