Fatty acids, C16, C18 and C18-unsaturated, C12-15 alcohol (linear and branched), esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

11 Mar - 11 May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-octyldodecyl isooctadecanoate

EC Number:

298-361-4

EC Name:

2-octyldodecyl isooctadecanoate

Cas Number:

93803-87-3

Molecular formula:

C38H76O2

IUPAC Name:

2-octyldodecyl isooctadecanoate

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

10, 33, 100, 333 and 1000 µg/plate, with and without metabolic activation

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 9-aminoacridine, daunomycine, methylmethanesulfonate, 4-nitroquinoline-N-oxide, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduction of bacterial background lawn, increase in size of the microcolonies, reduction of the revertant colonies)

OTHER:
the results of the dose range-finding test were included as part of experiment 1

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following
criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain (see Table 1)
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants compared to the solvent control value in any of the tester strains, either with or without metabolic activation. However, any plate with a mean plate count of less than 20 colonies is considered to be not significant and the result will be disregarded.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed in all strains, with and without metabolic activation, from 1000 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
A dose range-finding test with strain TA100 and the WP2uvrA strain (both with and without S9- mix) was performed to select suitable doses for the main experiments. Eight concentrations (3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate) were tested in triplicate. The results were reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the main experiments was the level at which the test substance exhibited limited solubility. Precipitation was observed on the plates from 1000 µg/plate and above in both strains.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the spontaneous mutation rate of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1 under 'Any other information on materials and methods incl. tables'), with the exception of TA 100 without metabolic activation. The value was slightly outside the limit of the range, therefore the validity of the test was considered not to be affected.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed at any concentration level, with or without metabolic activation (see Table 2 and 3).

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 2: Results of experiment 1

 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
-S9	ethanol	55 ± 3	12 ± 3	14 ± 6	16 ± 2	6 ± 1
-S9	3	55 ± 6	-	11 ± 2	-	-
-S9	10	63 ± 7	10 ± 4	10 ± 3	15 ± 6	7 ± 2
-S9	33	49 ± 7	8 ± 3	11 ± 1	16 ± 1	7 ± 4
-S9	100	59 ± 2	8 ± 1	12 ± 2	15 ± 3	5 ± 3
-S9	333	57 ± 7	7 ± 2	11 ± 6	17 ± 4	5 ± 3
-S9	1000 SP	70 ± 6	10 ± 3	10 ± 2	16 ± 1	4 ± 1
-S9	3300 SP	77 ± 9	-	12 ± 2	-	-
-S9	5000 SP	59 ± 13	-	10 ± 4	-	-
Positive controls, –S9	Name	MMS	SA	4-NQO	DM	9-AC
	Concentrations (μg/plate)	650	1	10	4	60
		529 ± 23	121 ± 18	291 ± 26	375 ± 36	185 ± 66
+S9	ethanol	71 ± 3	8 ± 2	10 ± 1	26 ± 2	8 ± 1
+S9	3	66 ± 10		11 ± 4
+S9	10	62 ± 3	9 ± 2	9 ± 4	25 ± 4	8 ± 3
+S9	33	64 ± 3	9 ± 3	14 ± 3	22 ± 2	6 ± 2
+S9	100	50 ± 8	9 ± 3	15 ± 3	24 ± 5	6 ± 2
+S9	333	45 ± 5	13 ± 4	10 ± 3	18 ± 4	6 ± 4
+S9	1000 SP	54 ± 8	12 ± 4	12 ± 6	23 ± 5	8 ± 1
+S9	3330 SP	54 ± 9	-	12 ± 1	-	-
+S9	5000 SP	51 ± 9	-	12 ± 2	-	-
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	1	2.5	5	2.5	2.5
		1182 ± 90	240 ± 8	88 ± 10	1047 ± 57	713 ± 149

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Table 3: Results of experiment 2

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type		Frameshift type
		TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
-S9	ethanol	88 ± 10	10 ± 2	10 ± 6	22 ± 7	5 ± 2
-S9	10	94 ± 13	9 ± 3	9 ± 2	17 ± 2	7 ± 3
-S9	33	93 ± 3	8 ± 3	12 ± 2	17 ± 3	5 ± 1
-S9	100	90 ± 7	10 ± 3	9 ± 2	14 ± 5	5 ± 3
-S9	333	90 ± 9	6 ± 3	11 ± 3	15 ± 6	5 ± 1
-S9	1000 SP	77 ± 5	6 ± 3	9 ± 4	14 ± 4	7 ± 4
Positive controls, –S9	Name	MMS	SA	4-NQO	DM	9-AC
	Concentrations (μg/plate)	650	1	10	4	60
		714 ± 49	152 ± 10	936 ± 118	598 ± 16	251 ± 100
+S9	ethanol	99 ± 8	8 ± 4	11 ± 7	15 ± 1	7 ± 3
+S9	10	98 ± 10	8 ± 3	7 ± 1	17 ± 3	4 ± 2
+S9	33	103 ± 21	6 ± 3	8 ± 2	27 ± 6	7 ± 1
+S9	100	98 ± 9	8 ± 3	13 ± 7	21 ± 7	6 ± 2
+S9	333	93 ± 6	7 ± 5	8 ± 3	20 ± 5	8 ± 2
+S9	1000 SP	112 ± 2	9 ± 3	8 ± 1	23 ± 1	4 ± 1
Positive controls, +S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	1	2.5	5	2.5	2.5
		2235 ± 60	322 ± 15	406 ± 20	1732 ± 115	703 ± 41

 

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Applicant's summary and conclusion

Conclusions:

CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.