Octanoic acid, 2-phenoxyethyl ester

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:

28 Nov - 8 Dec 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-phenoxyethyl octanoate

EC Number:

607-240-0

Cas Number:

23511-73-1

Molecular formula:

C16H24O3

IUPAC Name:

2-phenoxyethyl octanoate

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:

Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and beta-naphtoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate with and without metabolic activation for TA100 and WP2uvrA

Main study (experiment 1 and 2):
10, 33, 100, 333 and 1000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance is soluble and stable in the vehicle throughout the test

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: 2-nitrofluorene (NF; 10 μg/plate in DMSO, -S9, TA98); 2-Amino-anthracene (2-AA; 1 to 10 μg/plate in DMSO, +S9, all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test substance is considered positive if the total number of revertants is greater than 2 (TA 100) or three (TA 98, TA 1535, TA 1537, WP2uvrA) times the concurrent control. Positive responses should be reproducible in at least one additional independent experiment. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.

Statistics:

Mean values and standard deviation were calculated.

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:

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

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 4

Key result

Species / strain:

S. typhimurium 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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 1000 μg/plate and above, the test material precipitated in the medium.

RANGE-FINDING/SCREENING STUDIES: In the range finding test, the test substance was tested up to 5000 µg/plate in the absence and presence of S9 mix in TA100 and WP2uvrA. No reduction of the bacterial lawn and no biologically relevant decrease in the number of revertants were observed. Due to the precipitation of the test substance in medium at a concentration of 1000 µg/plate and above, this concentration was for the main experiment (TA1535, TA1537 and TA98 in experiment 1 and all strains for experiment 2).

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain specific positive control values were within laboratory historical control data ranges (table 3, any other information on results)

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation) and range finding study (TA100 and WP2 uvrA)           

With or without S9-Mix (5%)	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		cross-linking type	Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0	137 ± 1	12 ± 3	25 ± 6	29 ± 3	9 ± 3
–	3	139 ± 14	-	28 ± 6	-	-
–	10	133 ± 16	10 ± 3	24 ± 4	31 ± 2	6 ± 2
–	33	137 ± 9	8 ± 2	23 ± 4	34 ± 5	4 ± 2
–	100	131 ± 9	11 ± 3	24 ± 5	26 ± 8	6 ± 2
–	333	132 ± 7	7 ± 4	26 ± 7	27 ± 10	5 ± 2
–	1000	137 ± 19P	7 ± 1P	21 ± 3P	28 ± 4P	6 ± 3P
–	3330	140 ± 9P	-	17 ± 1P	-	-
–	5000	129 ± 13P	-	23 ± 4P	-	-
Positive controls, -S9	Name	MMS	SA	4NQO	NF	9AA
	Concentrations (μg/plate)	650	5	10	10	60
	Mean No. of colonies/plate (average of 3 ± SD)	1132 ± 23	933 ± 31	1271 ± 22	1122 ± 62	357 ± 38
+	0	140 ± 11	9 ± 2	26 ± 9	35 ± 6	6 ± 3
+	3	127 ± 8	-	24 ± 3	-	-
+	10	125 ± 10	9 ± 3	27 ± 5	37 ± 6	6 ± 2
+	33	130 ± 8	9 ± 2	25 ± 2	38 ± 7	4 ± 1
+	100	136 ± 6	9 ± 4	32 ± 7	34 ± 4	6 ± 1
+	333	117 ± 4	7 ± 2	26 ± 4	34 ± 5	6 ± 2
+	1000	125 ± 10P	5 ± 1P	26 ± 1P	34 ± 11P	6 ± 1P
+	3330	106 ± 7P	-	24 ± 6P	-	-
+	5000	102 ± 9P	-	21 ± 2P	-	-
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	2.5	10	1	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	1442 ± 40	406 ± 27	586 ± 62	1075 ± 21	539 ± 15

 

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

With or without S9-Mix (10%)	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type		cross-linking type	Frameshift type
		TA 100	TA1535	WP2 uvrA	TA98	TA1537
–	0	106 ± 11	12 ± 1	23 ± 5	29 ± 6	6 ± 1
–	10	123 ± 24	9 ± 2	23 ± 2	20 ± 5	6 ± 1
–	33	130 ± 20	8 ± 1	23 ± 2	21 ± 4	5 ± 2
–	100	103 ± 4	9 ± 1	26 ± 4	20 ± 5	3 ± 1
–	333	115 ± 6	10 ± 3	26 ± 4	18 ± 7	7 ± 4
–	1000	112 ± 9P	10 ± 4P	21 ± 5P	21 ± 5P	6 ± 2P
Positive controls, -S9	Name	MMS	SA	4NQO	NF	9AA
	Concentrations (μg/plate)	650	5	10	10	60
	Mean No. of colonies/plate (average of 3 ± SD)	921 ± 74	1022 ± 31	937 ± 20	1094 ± 8	322 ± 28
+	0	107 ± 4	8 ± 3	31 ± 5	36 ± 8	6 ± 2
+	10	94 ± 10	11 ± 1	29 ± 4	33 ± 9	4 ± 1
+	33	100 ± 5	10 ± 2	27 ± 4	27 ± 9	6 ± 1
+	100	105 ± 8	4 ± 1	34 ± 3	28 ± 2	7 ± 1
+	333	116 ± 9	8 ± 4	30 ± 4	23 ± 5	4 ± 1
+	1000	82 ± 5P	9 ± 1P	36 ± 3P	13 ± 6P	5 ± 2P
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	10	1	5
	Mean No. of colonies/plate (average of 3 ± SD)	1261 ± 141	260 ± 43	317 ± 67	541 ± 123	526 ± 12

                                  

MMS = methylmethanesulfonate

4NQO = 4-nitroquinoline-N-oxide

9AA = 9-aminoacridine

2AA = 2-Aminoanthracene

SA = sodium azide

NF = 7-nitrofluorene

P = Precipitate

Table 3. Laboratory historical control data

	TA1535		TA1537		TA98		TA100		WP2uvrA
Negative control	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Min	3	3	3	3	12	12	63	60	4	4
Max	28	29	19	21	45	51	194	195	44	44
Mean	12	11	7	6	21	26	119	99	19	20
3xSD	23	23	92	83	22	19	10	9	14	14
	TA1535		TA1537		TA98		TA100		WP2uvrA
Positive control	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Min	375	58	79	57	286	186	458	228	67	56
Max	1923	538	927	833	1790	1703	1593	2061	1403	1146
Mean	1061	202	352	370	1059	736	1057	1056	849	315
3xSD	596	229	389	383	545	935	530	991	892	377

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative