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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Study period:

2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read across substance, well documented, according to GLP and OECD guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

1309959-24-7

Cas Number:

1309959-24-7

IUPAC Name:

1309959-24-7

Test material form:

liquid: viscous

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Charles River Wiga GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 35.5g
- Housing: singly
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

30% DMSO / 70% PEG 400
The vehicle was chosen due to its relative nontoxicity for the animals.

Details on exposure:

Heating (max. 90 °C) will be performed during formulation, for administration the formulation will be kept at body temperature.

Duration of treatment / exposure:

24h and 48h

Frequency of treatment:

single

Post exposure period:

24h treatment:
negative control: 1-5h
low dose: 6-12h
medium dose: 13-19h
high dose: 20-26h
positive control: 27-31h

48h treatment:
negative control: 32-36h
positive control: 37-43h

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500, 1000, and 2000 mg/kg bw
Basis:
actual ingested
24h preparation interval

Doses / concentrations 2

Remarks:

Doses / Concentrations:
200 mg/kg bw
Basis:
actual ingested
48h preparation interval

No. of animals per sex per dose:

7 / test group, 5 for vehicle and positive control group,

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: Cyclophosphamide (CPA)
Supplier: Fisher Scientific GmbH
61130 Nidderau, Germany
Dissolved in: sterile water
Dosing: 40 mg/kg b.w.
Route and frequency
of administration: orally, once
Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:

bone marrow derived erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

DETAILS OF SLIDE PREPARATION:
The animals will be sacrificed using CO2 followed by bleeding. The femora are removed, the epiphyses are cut off and the marrow is flushed out with foetal calf serum, using a syringe. The cell suspension is centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant is discarded. A small drop of the resuspended cell pellet is spread on a slide. The smear is air-dried and then stained with May-Grünwald/Giemsa. Cover slips are mounted with EUKITT. At least one slide is made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides is performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) are analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes is determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis is performed with coded slides.
All surviving animals per test group will be evaluated as described.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (8), p < 0.5) will be used as an aid in evaluating the results, if necessary. The vehicle control group of the respective time point will be used as reference. However, the primary point of consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test (8), p < 0.5) will be used as an aid in evaluating the results, if necessary.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: up to 200 mg/kg bw, 24h and 48h, 2animals /sex/dose
- Clinical signs of toxicity in test animals: no
- Rationale for exposure: limit dose

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): negative
- Ratio of PCE/NCE (for Micronucleus assay): uneffected
- Statistical evaluation: non-parametric Mann-Whitney test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative