coconut oil, reaction products with glycerol esters of 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoic acid

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:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD, GLP, QAU)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26, 1983

Deviations:

yes

Remarks:

only 1000 polychromatic erythrocytes/animal were counted, the guideline was updated in 1997, here the analysis of 2000 polychromatic erythrocytes/animal is designated

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-400-6

EC Name:

-

Cas Number:

179986-09-5

Molecular formula:

UVCB substance

IUPAC Name:

methyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate; propane-1,2,3-triol; tetradecanoic acid

Details on test material:

- Physical state: liquid, colourless
- Analytical purity: 95%
- Storage condition of test material: room temperature
No further information.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf
- Age at study initiation: 8-12 weeks
- Weight at study initiation: males/females mean value: 31.9/25.7 g
- Assigned to test groups randomly: yes
- Fasting period before study: approximately 18 hours before treatment the animals received no food but water ad libitum.
- Housing: single in Makrolon type I, with wire mesh top
- Diet (ad libitum): pelleted standard diet (Altromin 1324)
- Water (ad libitum): tap water
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol 400
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: prior to administration

Frequency of treatment:

once

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
200, 670, 2000 mg/kg bw
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

polychromatic erythrocytes

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Animals were treated once orally. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KENDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

The test substance is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
The test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose: 2000 mg/kg bw
- Solubility: the test substance was formulated in PEG 400
- Clinical signs of toxicity in test animals: reduction of spontaneous activity, apathy

Any other information on results incl. tables

Table 1: Micronuclei in polychromatic erythrocytes in male and female mice (5 animals/sex) scoring 24 and 48 hours after treatment

Sex (m/f)	Hours after treatment	Dose (mg/kg bw)	Micronucleated cells per 1000 PCEs per animal
M	24	Vehicle	4
F	24	Vehicle	3
M	24	200	6
F	24	200	4
M	24	670	3
F	24	670	3
M	24	2000	6
F	24	2000	3
M	24	Positive control	97
F	24	Positive control	62
M	48	2000	3
F	48	2000	4