2-Hexyl-2 ‘-hydroxy-5 ‘methyldecananilide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jul - 28 Jul 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

published June 8, 2000

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): AF-654
- Physical state: white powder
- Lot/batch No.: #CE-201
- Expiration date of the lot/batch: 01 Jan 2005
- Storage condition of test material: at room temperature in the dark

Method

Target gene:

his operon (S. typhimurium strains), trp operon (E. coli strains)

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 (experiment I: S9 mix contained 5% (v/v) S9-fraction; experiment II: S9 mix contained 10% (v/v) S9-fraction)

Test concentrations with justification for top dose:

pre-experiment (with and without S9 mix): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate for TA 100 and E. coli WP2 uvr A
experiment I (with and without S9 mix): 3, 10, 33, 100 and 333 µg/plate for TA 98, TA 1535 and TA 1537
experiment II (with and without S9 mix): 3, 10, 33, 100 and 333 µg/plate for all strains

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycin; 2-aminoanthracene

Remarks:

-S9: sodium azide (SA, 5µg/plate, TA1535); 9-aminoacridine (9AC, 60µg/plate, TA1537); daunomycin (DM, 4µg/plate, TA98), methylmethanesulfonate (MMS, 650µg/plate, TA100); 4-nitroquinoline-N-oxide (4-NQO, 10µg/plate, E. coli); +S9: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction of bacterial background lawn, increase in size of microcolonies, reduction of revertant colonies

OTHER:
The dose range finding test was reported as part of the first experiment of the mutation assay.

Evaluation criteria:

Acceptability criteria:
1) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain.
2) 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.
3) The selected dose range should induce 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.

Evaluation criteria:
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 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 a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

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

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 was observed at concentrations of 100 µg/plate and above in the top agar and on the plates at 333 µg/plate at the start and the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
Selection of an adequate range of doses was based on a dose rane finding test with strain TA 100 and E. coli WP2uvrA in the presence and absence of S9 mix using eight concentrations (3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate). The highest concentration chosen for the main assay was the level at which the test substance showed limited solubility (333 µg/plate). This dose range finding test was reported as a part of the first experiment of the mutation assay.

COMPARISON WITH HISTORICAL CONTROL DATA:
All values are within the range of historical control data, except the response for TA98 in the absence of S9 mix (first experiment, negative control). Since this value was just outside the limit of the range the validity of the test was considered to be not affected.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	E. coli WP2	TA98	TA1537
–	DMSO	138 ± 6	8 ± 2	9 ± 2	11 ± 2	4 ± 1
–	3	139 ± 1	7 ± 2	11 ± 2	10 ± 2	6 ± 3
–	10	137 ± 15	8 ± 2	8 ± 1	11 ± 3	4 ± 2
–	33	156 ± 6	7 ± 2	8 ± 2	11 ± 1	5 ± 3
–	100	145 ± 20	9 ± 1	12 ± 1	14 ± 1	3 ± 1
–	333^SP	149 ± 5	10 ± 3	12 ± 3	12 ± 3	3 ± 2
–	1000^MP	129 ± 4	-	9 ± 2	-	-
–	3330^MP	136 ± 21	-	11 ± 3	-	-
–	5000^MP	128 ± 9	-	13 ± 2	-	-
Positive controls, –S9	Name	MMS	SA	4-NQO	DM	9AC
	Concentrations (μg/plate)	650	5	10	4	60
	Mean No. of colonies/plate (average of 3 ± SD)	962 ± 22	244 ± 16	812 ± 73	516 ± 71	280 ± 46
+	DMSO	140 ± 21	10 ± 1	10 ± 1	17 ± 1	4 ± 4
+	3	142 ± 4	6 ± 2	10 ± 2	14 ± 4	6 ± 2
+	10	140 ± 16	7 ± 4	10 ± 2	13 ± 2	6 ± 2
+	33	140 ± 8	6 ± 1	12 ± 2	17 ± 2	4 ± 2
+	100	155 ± 6	7 ± 2	12 ± 2	20 ± 5	4 ± 2
+	333^SP	149 ± 9	6 ± 1	9 ± 2	15 ± 7	5 ± 2
+	1000^MP	134 ± 20	-	8 ± 2	-	-
+	3330^MP	143 ± 11	-	10 ± 3	-	-
+	5000^MP	151 ± 10	-	11 ± 2	-	-
Positive controls, +S9^1	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	1	5	1	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	717 ± 50	100 ± 4	73 ± 11	520 ± 7	75 ± 16

Table 2. Test results of experiment 2 (plate incorporation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	E. coli WP2	TA98	TA1537
–	DMSO	138 ± 13	10 ± 4	13 ± 3	15 ± 6	5 ± 2
–	3	142 ± 9	11 ± 3	17 ± 4	13 ± 1	4 ± 2
–	10	137 ± 6	9 ± 1	12 ± 3	17 ± 4	6 ± 3
–	33	132 ± 12	11 ± 6	14 ± 2	12 ± 1	5 ± 3
–	100	151 ± 9	9 ± 6	16 ± 4	13 ± 3	5 ± 2
–	333^SP	139 ± 15	9 ± 2	13 ± 4	15 ± 3	5 ± 1
Positive controls, –S9	Name	MMS	SA	4-NQO	DM	9AC
	Concentrations (μg/plate)	650	5	5	4	60
	Mean No. of colonies/plate (average of 3 ± SD)	1025 ± 27	351 ± 20	712 ± 6	499 ± 79	220 ± 34
+	DMSO	141 ± 15	10 ± 3	19 ± 4	23 ± 2	6 ± 2
+	3	137 ± 18	6 ± 1	16 ± 2	21 ± 3	7 ± 3
+	10	127 ± 7	9 ± 5	14 ± 2	18 ± 6	6 ± 3
+	33	139 ± 13	6 ± 4	13 ± 2	20 ± 3	5 ± 1
+	100	130 ± 8	8 ± 3	12 ± 1	19 ± 4	4 ± 2
+	333^SP	121 ± 6	8 ± 2	16 ± 4	21 ± 1	6 ± 2
Positive controls, +S9^2	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	1	5	1	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	766 ± 45	128 ± 11	122 ± 4	436 ± 46	201 ± 19

¹: The S9 mix contained 5% (v/v) S9 fraction

²: The S9 mix contained 10% (v/v) S9 fraction

SP: slight precipitate

MP: moderate precipitate

2AA: 2-aminoanthracene

MMS: methylmethanesulfonate

SA: sodium azide

4-NQO: 4-nitroquinoline-N-oxide

DM: daunomycin

9AC: 9-aminoacridine

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative