Hexyl benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 February 2014 - 02 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Hexyl benzoate

EC Number:

229-856-5

EC Name:

Hexyl benzoate

Cas Number:

6789-88-4

Molecular formula:

C13H18O2

IUPAC Name:

hexyl benzoate

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium: histidine gene
Escherichia coli: tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Additional mutations: mutation of the uvrB gene to enhance their sensitivity to some mutagenic compounds and rfa wall mutation to increase permeability to certain classes of chemicals. Tester strains TA98 and TA100 also contain pKM101 plasmid.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: Additional mutation of the UvrA gene to enhance the sensitivity to some mutagenic compounds.

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate (S9) from male Sprague-Dawley rats injected with Aroclor 1254 (200 mg/mL in corn oil)

Test concentrations with justification for top dose:

Initial assay: 5.00, 16.0, 50.0, 160, 500, 1600 and 5000 µg/plate (all strains)
Confirmatory assay: 16.0, 50.0, 160, 500, 1600 and 5000 µg/plate (with S9) and 1.60, 5.00, 16.0, 50.0, 160, 500, 1600 and 5000 µg/plate (without S9) (all strains)
Top dose was chosen according to OECD guideline.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: according to OECD 471

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

See section ''any other information on materials and methods incl. tables''

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 52 +/- 4 hours

NUMBER OF REPLICATIONS: three replicates per dose level

METHODS OF SLIDE PREPARATION: 100 µL of tester strain and 100 µL of test or vehicle control article were added to 2.5 mL of diluted molten selective top agar (maintained at 45 +/- 2°C). After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL minimal bottom agar in a 15 x 100 mm petri dish.

Evaluation criteria:

- A test article is considered to have produced a positive response if it induces a dose-dependent increase in revertant frequency that is ≥2.0-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or ≥3.0-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.
- A test article is considered to have produced a negative response if no dose-dependent, ≥2.0-fold or ≥3.0-fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.

ACCEPTABILITY CRITERIA:
The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number f revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the reliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 160 µg/plate; without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 50 µg/plate; without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 50 µg/plate; without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 50 µg/plate; without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed in any of the strains up to and including the concentration of 5000 µg/plate, with and without S9.

RANGE-FINDING/SCREENING STUDIES: no

HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The substance caused a reduction in the growth of the bacterial background lawn to all Salmonella strains when tested without S9
Initial test: TA1535, TA98 and TA100 ≥160 µg/plate; TA1537 ≥50 µg/plate
Confirmatory test: TA98 and TA100 ≥50 µg/plate; TA1535 and TA1537 ≥160 µg/plate
- No toxicity was observed in Salmonella strains when tested with S9.
- No toxicity was observed in the E. coli strain WP2uvrA with and without S9.

Any other information on results incl. tables

Table 1 Initial Mutagenicity Assay Results with S9

		Dose	Mean		Ratio
		level	revertants		treated/	Individual revertant
Strain	Compound	( g/plate)	per plate	SD	vehicle	colony counts
TA98	Test substance	5000	22.0	1.0	0.8	22 N, 21 N, 23 N
		1600	20.0	5.3	0.7	16 N, 26 N, 18 N
		500	23.0	6.2	0.9	28 N, 16 N, 25 N
		160	23.7	5.1	0.9	18 N, 28 N, 25 N
		50.0	21.7	3.5	0.8	18 N, 25 N, 22 N
		16.0	23.0	5.2	0.9	26 N, 26 N, 17 N
	Dimethyl	5.00	21.3	3.5	0.8	25 N, 21 N, 18 N
			27.0	4.6		28 N, 31 N, 22 N
	Sulfoxide
TA100	Test substance	5000	101.7	11.4	0.7	105 N, 111 N, 89 N
		1600	125.7	14.3	0.9	129 N, 138 N, 110 N
		500	125.3	2.5	0.9	123 N, 128 N, 125 N
		160	134.7	17.8	1.0	155 N, 127 N, 122 N
		50.0	152.7	13.3	1.1	138 N, 156 N, 164 N
		16.0	130.7	18.6	0.9	110 N, 146 N, 136 N
	Dimethyl	5.00	138.7	15.6	1.0	153 N, 141 N, 122 N
			140.7	15.0		153 N, 145 N, 124 N
	Sulfoxide
TA1535	Test substance	5000	10.3	2.3	1.0	13 N, 9 N, 9 N
		1600	13.3	0.6	1.3	13 N, 14 N, 13 N
		500	14.3	4.6	1.4	17 N, 17 N, 9 N
		160	13.3	4.0	1.3	9 N, 14 N, 17 N
		50.0	12.0	3.5	1.2	10 N, 10 N, 16 N
		16.0	14.0	8.5	1.4	23 M N, 13 N, 6 N
	Dimethyl	5.00	13.3	3.5	1.3	17 N, 10 N, 13 N
			10.3	3.2		14 N, 8 N, 9 N
	Sulfoxide
TA1537	Test substance	5000	12.7	1.2	1.8	12 M N, 12 N, 14 N
		1600	8.0	2.6	1.1	5 N, 9 N, 10 N
		500	7.0	1.7	1.0	8 N, 8 N, 5 N
		160	9.7	2.1	1.4	8 N, 12 N, 9 N
		50.0	9.0	3.6	1.3	13 N, 8 N, 6 N
		16.0	8.3	2.1	1.2	10 N, 9 N, 6 N
	Dimethyl	5.00	7.7	2.3	1.1	5 N, 9 N, 9 M N
			7.0	2.6		10 N, 5 N, 6 N
	Sulfoxide

Table 1 (continued) Initial Mutagenicity Assay Results with S9

		Dose	Mean		Ratio
		level	revertants		treated/	Individual revertant
Strain	Compound	( g/plate)	per plate	SD	vehicle	colony counts
WP2uvrA	Test substance	5000	12.0	2.6	0.9	14 N, 9 N, 13 N
		1600	11.3	3.1	0.9	12 N, 8 N, 14 N
		500	13.3	4.2	1.0	18 N, 12 N, 10 N
		160	15.0	1.7	1.1	14 N, 17 N, 14 N
		50.0	17.7	1.5	1.3	16 M N, 18 N, 19 N
		16.0	14.7	5.1	1.1	16 N, 19 N, 9 N
	Dimethyl	5.00	15.0	1.7	1.1	16 N, 16 N, 13 N
			13.3	3.5		17 N, 10 N, 13 N
	Sulfoxide
TA98	BP	2.5	563.0	33.2	20.9	586 N, 578 N, 525 N
TA100	2AA	2.5	1753.3	97.0	12.5	1849 N, 1756 N, 1655 N
TA1535	2AA	2.5	242.3	23.8	23.5	268 N, 221 N, 238 N
TA1537	2AA	2.5	139.7	6.7	20.0	138 N, 134 N, 147 N
WP2uvrA	2AA	25.0	291.0	58.1	21.8	231 N, 295 N, 347 N

Table 2 Initial Mutagenicity Assay Results without S9

		Dose	Mean		Ratio
		level	revertants		treated/	Individual revertant
Strain	Compound	( g/plate)	per plate	SD	vehicle	colony counts
TA98	Test substance	5000	9.0	0.0	0.5	9 R, 9 R, 9 R
		1600	13.3	2.3	0.7	12 R, 12 R, 16 R
		500	11.7	3.2	0.6	8 R, 13 R, 14 R
		160	15.7	5.5	0.8	13 R, 22 R, 12 R
		50.0	16.3	2.5	0.8	19 N, 14 N, 16 N
		16.0	14.0	2.6	0.7	17 N, 13 N, 12 N
	Dimethyl	5.00	14.3	5.7	0.7	19 N, 8 N, 16 N
			19.7	1.2		19 N, 19 N, 21 N
	Sulfoxide
TA100	Test substance	5000	85.3	3.8	0.7	81 R, 88 R, 87 R
		1600	71.7	0.6	0.6	72 R, 72 R, 71 R
		500	90.0	7.5	0.7	89 R, 83 R, 98 R
		160	97.0	10.8	0.8	88 R, 109 R, 94 R
		50.0	108.7	11.5	0.9	102 N, 122 N, 102 N
		16.0	112.7	13.2	0.9	110 N, 127 N, 101 N
	Dimethyl	5.00	117.0	13.0	0.9	109 N, 132 N, 110 N
			127.3	15.0		136 N, 110 N, 136 N
	Sulfoxide
TA1535	Test substance	5000	11.0	6.6	0.7	5 R, 18 R, 10 R
		1600	12.0	3.5	0.7	14 R, 14 R, 8 R
		500	10.0	2.0	0.6	8 R, 12 R, 10 R
		160	14.0	2.0	0.8	16 R, 12 R, 14 R
		50.0	10.3	6.1	0.6	9 N, 17 N, 5 N
		16.0	17.0	1.0	1.0	17 N, 16 N, 18 N
	Dimethyl	5.00	13.7	3.5	0.8	10 N, 17 N, 14 N
			16.7	2.3		14 N, 18 N, 18 N
	Sulfoxide
TA1537	Test substance	5000	2.0	1.0	0.2	1 R, 3 M R, 2 M R
		1600	7.0	2.6	0.8	4 R, 9 R, 8 R
		500	3.0	2.0	0.4	1 R, 3 M R, 5 R
		160	5.7	3.1	0.7	5 R, 9 R, 3 R
		50.0	7.0	2.6	0.8	6 R, 10 R, 5 R
		16.0	8.3	0.6	1.0	8 N, 8 N, 9 M N
	Dimethyl	5.00	6.7	1.2	0.8	6 N, 8 N, 6 N
			8.3	4.0		9 N, 4 N, 12 N
	Sulfoxide

Table 2 (continued) Initial Mutagenicity Assay Results without S9

		Dose	Mean		Ratio
		level	revertants		treated/	Individual revertant
Strain	Compound	( g/plate)	per plate	SD	vehicle	colony counts
WP2uvrA	Test substance	5000	18.7	2.1	1.4	18 M N, 17 N, 21 N
		1600	19.3	1.5	1.5	18 N, 19 N, 21 N
		500	16.7	2.5	1.3	14 N, 19 N, 17 N
		160	14.7	2.3	1.1	16 N, 12 N, 16 N
		50.0	13.7	3.5	1.0	14 N, 17 N, 10 N
		16.0	12.7	3.1	1.0	12 N, 10 N, 16 N
	Dimethyl	5.00	20.3	2.1	1.5	21 N, 22 N, 18 N
			13.3	5.1		12 N, 9 M N, 19 N
	Sulfoxide
TA98	2NF	1.0	480.0	43.0	24.4	524 N, 478 N, 438 N
TA100	SA	2.0	1513.7	32.5	11.9	1547 N, 1512 N, 1482 N
TA1535	SA	2.0	1081.7	65.5	64.9	1146 N, 1084 N, 1015 N
TA1537	ICR	2.0	130.7	14.5	15.7	140 N, 114 N, 138 N
WP2uvrA	4NQO	1.0	114.3	8.4	8.6	109 N, 124 N, 110 N

Table 3 Confirmatory Mutagenicity Assay Results with S9

		Dose	Mean		Ratio
		level	revertants		treated/	Individual revertant
Strain	Compound	( g/plate)	per plate	SD	vehicle	colony counts
TA98	Test substance	5000	19.3	6.1	0.8	18 N, 14 N, 26 N
		1600	20.0	9.6	0.8	31 N, 16 N, 13 N
		500	22.3	0.6	0.9	22 N, 22 N, 23 N
		160	22.0	9.0	0.9	31 M N, 22 N, 13 N
		50.0	19.0	7.0	0.7	27 N, 14 N, 16 N
	Dimethyl	16.0	19.0	2.6	0.7	17 N, 22 N, 18 N
			25.7	3.2		28 N, 27 N, 22 N
	Sulfoxide
TA100	Test substance	5000	109.3	16.5	0.9	114 N, 91 N, 123 N
		1600	116.7	7.1	0.9	118 N, 109 N, 123 N
		500	122.0	11.5	1.0	133 N, 123 N, 110 N
		160	135.7	8.5	1.1	127 N, 144 N, 136 N
		50.0	136.0	3.5	1.1	134 N, 140 N, 134 N
	Dimethyl	16.0	140.0	6.0	1.1	146 N, 134 N, 140 N
			126.7	13.7		111 N, 136 N, 133 N
	Sulfoxide
TA1535	Test substance	5000	13.7	3.5	1.1	14 M N, 10 N, 17 N
		1600	12.7	3.1	1.0	12 N, 16 N, 10 N
		500	18.3	4.0	1.4	22 N, 19 N, 14 N
		160	15.3	1.2	1.2	16 N, 14 N, 16 N
		50.0	13.0	3.6	1.0	12 N, 17 N, 10 N
	Dimethyl	16.0	19.0	0.0	1.5	19 N, 19 N, 19 N
			13.0	3.6		17 N, 10 N, 12 N
	Sulfoxide
TA1537	Test substance	5000	7.0	2.6	0.7	10 N, 6 N, 5 N
		1600	5.3	1.2	0.5	6 N, 6 N, 4 N
		500	8.0	3.5	0.8	10 N, 4 N, 10 N
		160	8.3	4.0	0.8	12 N, 4 N, 9 N
		50.0	7.0	1.7	0.7	8 N, 8 N, 5 N
	Dimethyl	16.0	8.0	2.6	0.8	9 N, 10 N, 5 N
			10.7	4.2		14 N, 6 N, 12 N
	Sulfoxide
WP2uvrA	Test substance	5000	10.3	4.7	0.6	14 N, 5 N, 12 N
		1600	11.7	1.5	0.7	13 N, 10 N, 12 N
		500	16.3	0.6	0.9	16 N, 17 N, 16 N
		160	10.0	2.6	0.6	8 N, 13 N, 9 N
		50.0	15.7	6.5	0.9	9 N, 22 N, 16 N
	Dimethyl	16.0	17.0	3.6	1.0	14 N, 21 N, 16 N
			17.3	1.5		17 N, 19 N, 16 N
	Sulfoxide

Table 3 (continued) Confirmatory Mutagenicity Assay Results with S9

		Dose	Mean		Ratio
		level	revertants		treated/	Individual revertant
Strain	Compound	( g/plate)	per plate	SD	vehicle	colony counts
TA98	BP	2.5	615.7	28.3	24.0	632 N, 632 N, 583 N
TA100	2AA	2.5	2083.3	161.6	16.4	2205 N, 2145 N, 1900 N
TA1535	2AA	2.5	243.0	28.6	18.7	275 N, 220 N, 234 N
TA1537	2AA	2.5	192.7	5.9	18.1	186 N, 195 N, 197 N
WP2uvrA	2AA	25.0	423.0	66.2	24.4	468 N, 347 N, 454 N

Table 4 Confirmatory Mutagenicity Assay Results without S9

		Dose	Mean		Ratio
		level	revertants		treated/	Individual revertant
Strain	Compound	( g/plate)	per plate	SD	vehicle	colony counts
TA98	Test substance	5000	11.7	5.1	0.8	13 R, 16 R, 6 R
		1600	9.0	4.6	0.6	14 R, 5 R, 8 R
		500	10.0	4.4	0.7	13 R, 5 R, 12 R
		160	12.7	3.5	0.8	13 R, 9 R, 16 R
		50.0	14.3	3.8	1.0	16 R, 17 R, 10 R
		16.0	12.7	3.1	0.8	12 N, 10 N, 16 N
		5.00	14.0	6.6	0.9	21 N, 8 N, 13 N
	Dimethyl	1.60	15.3	6.0	1.0	16 N, 21 N, 9 N
			15.0	6.2		13 N, 10 N, 22 N
	Sulfoxide
TA100	Test substance	5000	70.3	8.1	0.6	79 R, 69 R, 63 R
		1600	63.3	7.5	0.6	71 R, 63 R, 56 R
		500	74.3	5.0	0.7	69 R, 75 R, 79 R
		160	77.3	3.2	0.7	81 R, 75 R, 76 R
		50.0	89.0	14.9	0.8	106 R, 78 R, 83 R
		16.0	111.3	15.8	1.0	125 N, 94 N, 115 N
		5.00	120.7	17.5	1.1	106 N, 140 N, 116 N
	Dimethyl	1.60	115.0	6.2	1.0	113 N, 122 N, 110 N
			111.7	7.6		110 N, 120 N, 105 N
	Sulfoxide
TA1535	Test substance	5000	11.7	5.1	0.5	13 R, 16 R, 6 R
		1600	13.3	4.2	0.6	18 R, 10 R, 12 R
		500	16.0	9.5	0.7	25 R, 6 R, 17 R
		160	17.3	1.5	0.8	17 R, 16 R, 19 R
		50.0	17.7	3.5	0.8	21 N, 14 N, 18 N
		16.0	18.0	4.0	0.8	14 N, 18 N, 22 N
		5.00	17.0	2.6	0.8	18 N, 19 N, 14 N
	Dimethyl	1.60	11.0	4.4	0.5	13 N, 14 N, 6 N
			21.7	5.5		28 N, 19 N, 18 N
	Sulfoxide
TA1537	Test substance	5000	4.0	0.0	0.6	4 R, 4 R, 4 R
		1600	5.7	2.9	0.8	4 R, 4 R, 9 R
		500	6.3	1.5	0.9	5 R, 6 R, 8 R
		160	7.0	4.6	1.0	3 R, 6 R, 12 R
		50.0	8.0	5.2	1.1	5 N, 5 N, 14 N
		16.0	8.7	3.1	1.2	12 N, 6 N, 8 N
		5.00	5.0	1.7	0.7	3 N, 6 N, 6 N
	Dimethyl	1.60	9.0	3.0	1.3	12 N, 6 N, 9 M N
			7.0	5.3		13 N, 5 N, 3 N
	Sulfoxide

Table 4 (continued) Confirmatory Mutagenicity Assay Results without S9

		Dose	Mean		Ratio
		level	revertants		treated/	Individual revertant
Strain	Compound	( g/plate)	per plate	SD	vehicle	colony counts
WP2uvrA	Test substance	5000	12.3	2.9	0.8	14 N, 9 N, 14 N
		1600	11.3	6.4	0.8	14 N, 4 N, 16 N
		500	13.0	3.6	0.9	14 N, 16 N, 9 N
		160	14.0	4.0	0.9	10 N, 18 N, 14 N
		50.0	10.3	3.8	0.7	12	N, 6 N, 13 N
		16.0	13.3	3.1	0.9	14	N, 16 N, 10 N
		5.00	13.0	3.0	0.9	10	N, 13 N, 16 N
	Dimethyl	1.60	13.0	3.0	0.9	10	N, 16 N, 13 N
			15.0	5.3		17 N, 9 N, 19 N
	Sulfoxide
TA98	2NF	1.0	307.3	53.4	20.5	291 N, 264 N, 367 N
TA100	SA	2.0	1431.7	107.4	12.8	1477 N, 1509 N, 1309 N
TA1535	SA	2.0	1165.7	55.1	53.8	1183 N, 1210 N, 1104 N
TA1537	ICR	2.0	364.3	23.2	52.0	349 N, 391 N, 353 N
WP2uvrA	4NQO	1.0	300.7	33.5	20.0	299 N, 335 N, 268 N

Key to Positive Controls		Key to Plate Postfix Codes
2NF	2-nitrofluorene	R	Reduced background bacterial lawn
SA	sodium azide	N	Normal background bacterial lawn
ICR	ICR-191	M	Plate counted manually
4NQO	4-nitroquinoline-N-oxide
BP	Benzo{a}pyrene
2AA	2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

The substance is negative in the Bacterial Reverse Mutation Assay, performed according to OECD 471 guideline and GLP principles, when tested up to 5000 µg/plate in the presence and absence of S9.

Executive summary:

A Bacterial Reverse Mutation Assay test was conducted to assess the mutagenic activity of the substance. The study was conducted according to OECD 471 guideline and GLP principles. One initial and one confirmatory test was performed with doses up to and including 5000 µg/plate, in the absence and presence of S9-mix. Adequate solvent and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that the substance is not mutagenic in the Bacterial Reverse Mutation Assay.