Acetic acid, C8-10-branched alkyl esters, C9-rich

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected June 2015; signature: September 2016

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acetic acid, C8-10-branched alkyl esters, C9-rich

Cas Number:

108419-33-6

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Acetic acid, C8-10-branched alkyl esters, C9-rich

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: liquid
- Storage condition of test material: Approximately 4 °C in the dark.
- Other: Clear colourless liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Experiment 1 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method):
Details are provided in the following tables given in 'any other information on results incl. tables'.
Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic limit of the test item.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration. Dimethyl sulphoxide was selected as the vehicle. No correction was made for purity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1 and 2. in medium; in agar (plate incorporation)

DURATION
- Exposure duration:
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were performed, predominantly due to interference in the base agar e.g. minor precipitation of salts/dust particles and marks on the base of the plates slightly distorting the counts

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.

Statistics:

Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

and or tested up to guideline limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

cytotoxicity

Remarks:

and or tested up to guideline limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

N/T : Not tested at this dose level

2AA: 2-Aminoanthracene

S: Sparse bacterial background lawn

V: Very weak bacterial background lawn

**: p < or = 0.01

#: Standard deviation

 

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains								Frameshift strains
		TA100		TA1535			WP2uvrA			TA98			TA1537
	Solvent Control (DMSO)	69 75 67	(70) 4.2#	10 14 9		(11) 2.6	19 15 11		(15) 4.0	13 17 13		(14) 2.3	5 5 15		(8) 5.8
	1.5 µg	80 65 70	(72) 7.6	13 9 13		(12) 2.3	12 17 9		(13) 4.0	19 11 17		(16) 4.2	9 13 12		(11) 2.1
	5 µg	66 61 65	(64) 2.6	13 15 16		(15) 1.5	9 16 12		(12) 3.5	21 8 20		(16) 7.2	7 7 12		(9) 2.9
	15 µg	63 69 60	(64) 4.6	9 16 15		(13) 3.8	19 25 15		(20) 5.0	13 20 23		(19) 5.1	8 11 5		(8) 3.0
	50 µg	61 61 60	(61) 0.6	13 9 11		(11) 2.0	20 17 21		(19) 2.1	19 13 13		(15) 3.5	4 4 4		(4) 0.0
	150 µg	64 66 62	(64) 2.0	43 S 67 S 14 S		** (41) 26.5	15 12 15		(14) 1.7	19 16 8		(14) 5.7	3 3 5		(4) 1.2
	500 µg	62 64 62	(63) 1.2	10 S 7 S 5 S		(7) 2.5	20 11 15		(15) 4.5	15 13 5		(11) 5.3	3 S 7 S 3 S		(4) 2.3
	1500 µg	65 S 66 S 61 S	(64) 2.6	0 V 0 V 0 V		(0) 0.0	16 12 15		(14) 2.1	8 S 12 S 5 S		(8) 3.5	1 S 3 S 4 S		(3) 1.5
	5000 µg	43 S 49 S 65 S	(52) 11.4	0 V 0 V 0 V		(0) 0.0	13 S 20 S 20 S		(18) 4.0	12 S 15 S 13 S		(13) 1.5	0 V 0 V 0 V		(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG			ENNG			4NQO			9AA
		3 µg		5 µg			2 µg			0.2 µg			80 µg
		438 314 330	(361) 67.4	519 445 652		(539) 104.9	337 326 310		(324) 13.6	103 86 126		(105) 20.1	231 184 335		(250) 77.3
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
	Solvent Control (DMSO)	80 72 61	(71) 9.5#	14 10 11	(12) 2.1		27 15 27	(23) 6.9		11 10 14	(12) 2.1		8 9 7	(8) 1.0
	1.5 µg	71 74 60	(68) 7.4	11 11 8	(10) 1.7		31 35 27	(31) 4.0		12 13 14	(13) 1.0		8 13 5	(9) 4.0
	5 µg	61 65 65	(64) 2.3	13 12 8	(11) 2.6		24 21 15	(20) 4.6		11 19 21	(17) 5.3		12 4 3	(6) 4.9
	15 µg	71 69 63	(68) 4.2	10 11 9	(10) 1.0		20 28 25	(24) 4.0		20 11 20	(17) 5.2		5 9 3	(6) 3.1
	50 µg	69 69 66	(68) 1.7	12 15 9	(12) 3.0		12 24 29	(22) 8.7		15 13 10	(13) 2.5		5 7 17	(10) 6.4
	150 µg	71 73 61	(68) 6.4	16 10 17	(14) 3.8		12 25 19	(19) 6.5		13 20 12	(15) 4.4		4 7 5	(5) 1.5
	500 µg	65 60 61	(62) 2.6	12 S 11 S 13 S	(12) 1.0		23 23 17	(21) 3.5		25 S 17 S 16 S	* (19) 4.9		7 3 11	(7) 4.0
	1500 µg	60 S 50 S 50 S	(53) 5.8	0 V 0 V 0 V	(0) 0.0		12 S 15 S 23 S	(17) 5.7		0 V 0 V 0 V	(0) 0.0		0 V 0 V 0 V	(0) 0.0
	5000 µg	44 S 50 S 45 S	(46) 3.2	0 V 0 V 0 V	(0) 0.0		15 S 5 S 8 S	(9) 5.1		0 V 0 V 0 V	(0) 0.0		0 V 0 V 0 V	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA			2AA			BP			2AA
		1 µg		2 µg			10 µg			5 µg			2 µg
		235 237 278	(250) 24.3	152 134 214	(167) 42.0		195 265 315	(258) 60.3		98 79 96	(91) 10.4		204 159 168	(177) 23.8

 

Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535			WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	94 76 89	(86) 9.3#	7 12 8	(9) 2.6		27 35 19	(27) 8.0	12 11 12	(12) 0.6	7 6 9	(7) 1.5
	0.15 µg	N/T		11 9 15	(12) 3.1		N/T		N/T		N/T
	0.5 µg	N/T		9 12 7	(9) 2.5		N/T		N/T		9 6 8	(8) 1.5
	1.5 µg	74 76 84	(78) 5.3	7 17 11	(12) 5.0		23 28 20	(24) 4.0	11 16 20	(16) 4.5	9 7 12	(9) 2.5
	5 µg	68 72 72	(71) 2.3	7 7 11	(8) 2.3		16 24 19	(20) 4.0	17 21 9	(16) 6.1	9 8 6	(8) 1.5
	15 µg	59 77 66	(67) 9.1	14 14 16	* (15) 1.2		20 19 19	(19) 0.6	16 14 10	(13) 3.1	3 4 8	(5) 2.6
	50 µg	61 65 67	(64) 3.1	17 S 15 S 12 S	(15) 2.5		21 9 17	(16) 6.1	8 9 9	(9) 0.6	5 3 6	(5) 1.5
	150 µg	48 59 58	(55) 6.1	11 S 12 S 14 S	(12) 1.5		20 13 19	(17) 3.8	9 8 11	(9) 1.5	3 S 2 S 0 S	(2) 1.5
	500 µg	39 48 49	(45) 5.5	11 V 13 V 12 V	(12) 1.0		11 21 13	(15) 5.3	11 S 12 S 8 S	(10) 2.1	2 S 3 S 4 S	(3) 1.0
	1500 µg	33 S 56 S 48 S	(46) 11.7	N/T			11 23 7	(14) 8.3	11 S 9 S 8 S	(9) 1.5	2 S 3 S 5 S	(3) 1.5
	5000 µg	42 S 27 S 40 S	(36) 8.1	N/T			16 S 13 S 20 S	(16) 3.5	6 S 10 S 10 S	(9) 2.3	N/T
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG			ENNG		4NQO		9AA
		3 µg		5 µg			2 µg		0.2 µg		80 µg
		428 470 404	(434) 33.4	600 521 577	(566) 40.6		358 319 382	(353) 31.8	262 204 261	(242) 33.2	299 564 500	(454) 138.3
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
	Solvent Control (DMSO)	90 91 89	(90) 1.0#	9 9 7		(8) 1.2	41 39 48	(43) 4.7	16 19 12	(16) 3.5	11 6 13	(10) 3.6
	0.5 µg	N/T		6 8 10		(8) 2.0	N/T		21 14 16	(17) 3.6	10 12 8	(10) 2.0
	1.5 µg	83 91 96	(90) 6.6	6 6 8		(7) 1.2	20 32 32	(28) 6.9	18 22 24	(21) 3.1	6 10 7	(8) 2.1
	5 µg	87 84 90	(87) 3.0	8 13 8		(10) 2.9	32 36 19	(29) 8.9	14 17 19	(17) 2.5	7 11 8	(9) 2.1
	15 µg	85 67 80	(77) 9.3	13 6 8		(9) 3.6	19 36 27	(27) 8.5	14 18 19	(17) 2.6	9 15 6	(10) 4.6
	50 µg	84 83 69	(79) 8.4	7 14 7		(9) 4.0	35 20 31	(29) 7.8	14 17 12	(14) 2.5	8 6 5	(6) 1.5
	150 µg	84 82 79	(82) 2.5	10 11 6		(9) 2.6	39 20 28	(29) 9.5	13 20 25	(19) 6.0	10 7 6	(8) 2.1
	500 µg	65 S 55 S 63 S	(61) 5.3	15 S 7 S 12 S		(11) 4.0	15 21 13	(16) 4.2	16 S 10 S 13 S	(13) 3.0	3 4 4	(4) 0.6
	1500 µg	35 S 38 S 45 S	(39) 5.1	0 V 0 V 0 V		(0) 0.0	12 S 8 S 5 S	(8) 3.5	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0
	5000 µg	51 S 51 S 51 S	(51) 0.0	N/T			21 S 11 S 12 S	(15) 5.5	N/T		N/T
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA			2AA		BP		2AA
		1 µg		2 µg			10 µg		5 µg		2 µg
		331 382 343	(352) 26.7	156 139 146		(147) 8.5	210 182 214	(202) 17.4	96 104 110	(103) 7.0	174 211 212	(199) 21.7

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test material was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). Since the test item had unknown volatility all testing was performed using the pre-incubation modification. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended slightly, based on these results. Eight test item dose levels were again selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test, the test item induced avisible reduction in the growth of the bacterial background lawns and/or a substantial reduction in revertant colony frequency to all of the tester strains, initially from 150 μg/plate in the absence of S9-mix and 500 μg/plate in the presence of S9-mix. Consequently the same maximum dose level (5000 μg/plate) or the toxic limit was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA1535), 150 μg/plate (TA1537), 500 μg/plate (TA98), 1500 μg/plate (TA100) and at 5000 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns and/or substantial reductions in revertant colony frequency were noted to all of the tester strains from 500 μg/plate. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1. Small, statistically significant increase in TA1535 revertant colony frequency were noted in the absence of S9-mix at 150 μg/plate and to TA98 at 500 μg/plate in the presence of S9-mix. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted at the same concentrations. Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. A small, statistically significant increase in TA1535 revertant colony frequency was also observed in the absence of S9-mix at 15 μg/plate in this test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. the individual revertant colony counts at 15 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control. Under the conditions of this study, the test substance was considered non-mutagenic.