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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

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Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April 2013 to 19 June 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Renewable hydrocarbons (diesel type fraction)
EC Number:
Cas Number:
Molecular formula:
Renewable hydrocarbons (diesel type fraction)
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): NEXBTL renewable diesel
- Substance type: UVCB

Certificate of analysis presented in Appendix 5.

Test animals

ICR (CD-1)
Details on test animals or test system and environmental conditions:
- Source: Harlan Laboratories UK Ltd
- Age at study initiation: approximately 6-10 weeks old
- Weight at study initiation: 23-30g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Group housed of up to 7/cage in solid floor polypropylene cages with wood flake bedding
- Diet: Harlan Teklad 2014C Global Certified Rodent Diet ad libitum
- Water: mains drinking water ad libitum

- Acclimatisation period: at least 5 days
- Temperature: 19 to 25C
- Relative humidity: 30-70%
- Air exchanges: approximately 15 changes per hour
- Light/dark cycles: 12 hours light and 12 hours dark

Administration / exposure

Route of administration:
Vehicle used: arachis oil
Details on exposure:
Animals dosed once via intraperitoneal route. One group of mice from each dose level were terminated 24 hours after treatment and a second group dosed with 2000 mg/kg were killed 48 hours after treatment.
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
Post exposure period:
Treatment with 500, 1000 and 2000 mg /kg: 24 hours
Additional group treated with 2000 mg/kg: 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Groups of 7 male mice per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
5 male mice, single oral administration of cyclophosphamide (50 mg/kg)


Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
Both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension.
Smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giesma, allowed to air dry and a cover slip applied for examination microscopic evaluation.
Evaluation criteria:
2000 PCEs per animal were scored for the presence of micronuclei. The proportion of PCEs among 1000 total erythrocytes was determined for each animal and expressed as the PCE/NCE ratio.
A positive mutagenic repsonse would be demonstrated if a statistically significant dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 hour kill times when compared to vehicle control.
Total polychromatic erythrocytes (PCEs), micronucleated polychromatic erythrocytes, normochromatic, erythrocytes (NCEs) were compared to the control using Students t-test (two tailed)

Results and discussion

Test results
Key result
Clincial sign hunched posture observed in both groups dosed with 2000 mg/kg
Vehicle controls validity:
Negative controls validity:
Positive controls validity:

Any other information on results incl. tables

No statistically significant decreases in the PCE/NCE ratio were observed in any test groups and no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals treated with NEXBTL renewable diesel when compared to control.

The vehicle and positive control groups exhibited a response consistent with the laboratory's historical control data. The negative control mean marginally exceeded the current historical vehicle control upper limit value. However, the numbers of micronucleated polychromatic erythrocytes observed in some of the individual animals were not outside of upper values quoted in the literature for vehicle control animals and therefore were considered acceptable for the purpose of this study. The positive control, cyclophosphamide induced a significant increase in the frequency of micronucleated PCEs (p<0.001)

The test item was found not to produce a toxicologically significant increase on the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

Applicant's summary and conclusion

The test item was considered to be non-mutagenic under the conditions of the test.
Executive summary:

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when adminstered to mice following OECD Guideline 474 (1997).

A range finding test was performed to find suitable dose levels of the test item, route of administration and to investigate if there was a marked difference in toxic response between sexes. There was no marked difference in toxicity of the test item between sexes therefore the main test was performed using male mice only. The micronucleus test was conducted using intraperitoneal route in groups of 7 male mice at the maximum recommended dose of 2000 mg/kg with 1000 and 500 as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were score for the presence of micronuclei.

Vehicle control group of mice were given a single oral dose of arachis oil and a positive control group of mice dosed orally with cyclophosphamide and killed after 24 hours.

There were no premature deaths at any dose level. The clincial sign hunched posture was observed in animals dosed with the test item at 2000 mg/kg in both 24 and 48 hour dose groups. No statistically significant decreases in PCE/NCE ratio were observed in any of the test item dose groups.

There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes confirming the sensitivity of the system to known mutagenic activity of cyclophosphamide under the conditions of the test.

In conclusion, the test item was considered to be non-mutagenic under the conditions of the test.