Methyl 12-hydroxyoctadecanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

other: chromosomal aberrations

Test material

Test material information

Constituent 1

Reference substance name:

Methyl laurate

EC Number:

203-911-3

EC Name:

Methyl laurate

Cas Number:

111-82-0

Molecular formula:

C13H26O2

IUPAC Name:

methyl laurate

Test material form:

liquid

Details on test material:

Dodecanoic acid methyl ester [methyl laurate, CAS No. 111-82-0], manufactured by Tokyo Chemical Industry Co., Ltd., Lot. No. GD 01, content 99.2%, molecular weight 214.35, melting point 5 ° C., boiling point 141 ° C., specific gravity 0.8706 (20 ° C.). Colorless and transparent liquid

Specific details on test material used for the study:

Dodecanoic acid methyl ester (abbreviation: MD, CAS No.:111-82-0, lot number: GB01, manufactured by Tokyo Chemical Industry Co., Ltd.) is a colorless transparent liquid and has a melting point of 5 ° C, a boiling point of 141 ° C. (15 mmHg) , Molecular formula C13 H26 O2, molecular weight 214.35, purity 98% (impurity unknown).

Method

Target gene:

chromosomes

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CHL/IU cells derived from Chinese hamster, obtained from Research · Resource Bank (JCRB) (February 1988, at passage: 4th passage, now 12th) were used in the test within 10 years of thawing succession age.

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from livers of rats induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

A preliminary toxicity test was conducted based on cell proliferation rates. The proliferation inhibitory action of the test substance on CHL/IU cells was determined by measuring the proliferation of each group using a monolayer culture cell densitometer (Monocellater ™ , Olympus Optical Co., Ltd.), and the solvent control group as baseline.

Vehicle / solvent:

acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DURATION
-Exposure duration:
a) Continuous treatment: 24 hrs, 48 hrs
b) Short-term treatment: 6hrs; 18 hrs for recovery
-Fixation time (start of exposure up to fixation or harvest of cells):
a) Continuous treatment: 24 hrs, 48 hrs
b) Short-term treatment: 24 hrs
SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa stain
NUMBER OF CELLS EVALUATED:
Stractural aberrations: 200 cells/group
Polyploidy: 800 cells/group
DETERMINATION OF CYTOTOXICITY:
-Method: relative total growth

Rationale for test conditions:

guideline

Evaluation criteria:

Criteria for a positive call: a statistically significant increase in the frequency of cells with chromosomal aberrations in the treated group compared with that of the solvent control group, and a statistically significant difference in the dose trend test. An inconclusive result will be designated when there is a significant increase in the frequency of cells with aberrations without a postive dose response trend test.
The test will be considered negative when there is no significant difference in frequency of cells with chromosomal aberrations.
The test is inconclusive when the number of cells observed has fewer than 100 structural aberrations, and less than 400 polyploidy due to cytotoxicity.

Statistics:

Fisher's direct probability method is used to distinguish between the solvent background chromosomal aberration levels and those of the test substance- treated group, with a significance level of p <0.05. In addition, when significant differences were found by Fisher's exact stochastic method, the Cochran-Armitage's trend test is applied (p <0.05) to test for significance of the dose dependency.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity screening test, concentrations up to 2.0 mg/ml resulted in significant decrease in cell growth in the 24 and 48 hour continuous cultures with and without S9, and in the 6 hour culture with S9. Cells in the 6-hour culture without S9 tolerated the test material with growth rates above 50%. Therefore, the maximum concentration of the test substance used in the continuous exposure assay was 0.06 mg / ml, and was 0.1 mg / ml in the short-term assay in the presence of S9 mix. In the short term assay without S9 mix, 2.1 mg / ml (10 mM) was the maximum concentration tested. Toxicity was observed (> 50 mitotic index) in the main study of continuous exposure at the high dose of 0.06 mg/ml, but not in the other modules of the study.

Any other information on results incl. tables

Table 1: Continuous

Group	Concentration (mg/ml)	Time of Exposure (h)	No of Cells analysed	No. of structural aberrations							Others^3)	No. of cells with aberrations		Polyploid^4)(%)	Trend test^5)
				gap	ctb	cte	csb	cse	mul^2)	total		TAG (%)	TA (%)		SA	NA
Control			200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.13	NT	NT
Solvent^1)	0	24	200	0	0	0	0	0	0	0	0	0 (0.0)	0(0.0)	0.38
MD	0.015	24	200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.13
MD	0.03	24	200	1	0	0	0	0	0	1	0	1 (0.5)	0 (0.0)	0.25
MD	0.06	24	173	0	1	0	1	0	0	2	1	2 (1.2)	2 (1.2)	0.55^6)
MC	0.00005	24	200	11	40	133	2	3	0	189	2	110 (55.0)	105 (52.5)	0.00
Solvent^1)	0	48	200	0	0	0	0	0	0	0	1	0 (0.0)	0 (0.0)	0.00	NT	NT
MD	0.015	48	200	0	0	0	0	0	0	0	1	0 (0.0)	0 (0.0)	0.25
MD	0.03	48	200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.13
MD	0.06	48	200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.63
MC	0.00005	48	200	6	35	137	4	6	20	208	6	94 (47.0)	94 (47.0)	0.25

Table 2: Short-term

Group	Concentration (mg/ml)	S9 mix	Time of Exposure (h)	No of cells analyzed	No. of structural aberrations							Others^3)	No. of cells with aberrations		Polyploid^4)(%)	Trend test^5)
					gap	ctb	cte	csb	cse	mul^2)	total		TAG (%)	TA (%)		SA	NA
Control				200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.63	NT	NT
Solvent^1)	0	-	6-(18)	200	0	0	1	0	1	0	2	0	2 (1.0)	2 (1.0)	0.63
MD	0.53	-	6-(18)	200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.25
MD	1.1	-	6-(18)	200	1	1	0	0	0	0	2	0	2 (1.0)	1 (0.5)	0.25
MD	2.1	-	6-(18)	200	0	0	1	0	0	0	1	0	1 (0.5)	1 (0.5)	0.25
CPA	0.005	-	6-(18)	200	0	1	0	0	0	0	1	0	1 (0.5)	1 (0.5)	0.13
Solvent^1)	0	+	6-(18)	200	1	0	1	0	0	0	2	1	2 (1.0)	1 (0.5)	0.25	NT	NT
MD	0.025	+	6-(18)	200	0	0	0	0	0	0	0	0	0 (0.0)	0 (0.0)	0.5
MD	0.05	+	6-(18)	200	1	0	0	0	0	0	1	0	1 (0.5)	0 (0.0)	0.75
MD	0.1	+	6-(18)	200	0	0	2	0	0	0	2	0	1 (0.5)	1 (0.5)	1.38
CPA	0.005	+	6-(18)	200	7	40	126	1	2	0	176	0	95 (47.5)	93 (46.5)	0.25

Applicant's summary and conclusion

Conclusions:

A guideline OECD 473-compliant chromosomal aberration test was undertaken with the test material in acetone, with and without rat liver S9 fraction, with valid positive controls. Cytotoxicity was seen at the higher doses in a preliminary dose-range finding study. There were no increase in the number of chromosome aberrations (excluding gaps) or polyploidy observed. The test substance does not induce chromosomal aberrations in CHL/IU cells under the conditions of this study.