Isodecyl pivalate

Genetic toxicity: in vitro

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Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Apr - 15 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(adopted July 21, 1997).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Method

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640, supplemented with 20% (v/v) heat-inactivated foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) and 30 U/ml heparin.

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

3 h exposure time: 10, 33 and 100 µg/mL (with and without S9 mix)
24 h exposure time: 66, 150 and 250 µg/mL (without S9 mix)
48 h exposure time: 3, 125 and 150 µg/mL (without S9 mix)
(At a concentration of 100 µg/mL isopropyl laurate precipitated in the culture medium)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide (10 µg/mL, with S9); mitomycin C (0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period without S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3, 24 and 48h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h for 3 h treatment period; 48 h for 48 h treatment period

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µL/mL medium
STAIN (for cytogenetic assays): Giemsa 5% (v/v) in tap water

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test; it induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations; a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Chi-square test, one-sided, p < 0.05

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: cultured human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 150 µg/mL (exposure period 24 h, fixation time 24 h, -S9 mix) and at 125 µg/mL (exposure period 48 h, fixation time 48 h, -S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 100 µg/mL isopropyl laurate precipitated in the culture medium
- Polyploidy did also not occur in a significant way and was also observed in control group.

RANGE-FINDING/SCREENING STUDIES: The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h and 24 h continuous exposure) or on toxicity, inhibition of the mitotic index of about 50% or greater (48 h continuous exposure).

COMPARISON WITH HISTORICAL CONTROL DATA: Negative controls were in range of the historical control data from experiments performed between January 2007 and December 2009.

Any other information on results incl. tables

Table 1: Cytotoxic and Genotoxic observations

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 3h, fixation time 24h, without S9 mix
Ethanol	1.0% (v/v)	100	6	4
MMC	0.5	97	56	56
Test substance	10	97	6	1
	33	92	4	2
	100	81	2	2
Exposure period 3h, fixation time 24h, with S9 mix
Ethanol	1.0% (v/v)	100	7	3
CP	0.5	59	52	50
Test substance	10	101	7	7
	33	96	2	2
	100	91	5	3
Exposure period 24h, fixation time 24h, without S9 mix
Ethanol	1.0% (v/v)	100	3	3
MMC	0.2	86	45	45
Test substance	66	96	1	1
	150	55	4	4
	250	41	2	2
Exposure period 48h, fixation time 48h, without S9 mix
Ethanol	1.0% (v/v)	100	3	3
MMC	0.1	89	56	56
Test substance	3	94	6	6
	125	70	3	3
	150	46	5	5
Exposure period 3h, fixation time 48h, with S9 mix
Ethanol	1.0% (v/v)	100	2	2
CP	10.0	--	42	42
Test substance	10	99	0	0
	33	96	0	0
	100	95	1	1

MMC: Mitomycin          

CP: Cyclophosphamide

Applicant's summary and conclusion

Conclusions:

negative