A mixture of: esters of C14-C15 branched alcohols with 3,5-di-t-butyl-4-hydroxyphenyl propionic acid; C15 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate; C13 branched and linear alkyl 3,5-bis(1,1-dimethylethyl)-4-hydroxybenzenepropanoate

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:

24 April 2014 to 15 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test". (adopted July 21, 1997).

Deviations:

yes

Remarks:

See "Any other information" for details

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Specific details on test material used for the study:

No further details specified in the study report.

Method

Target gene:

histidine and tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Test system Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale Recommended test system in international guidelines (e.g. OECD, EC and MITI).
Source Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames) (TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006) and (Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK) (WP2uvrA, 2008)

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation system
Rat liver microsomal enzymes (S9 homogenate) was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that had been injected intraperitoneal with Aroclor 1254 (500 mg/kg).

Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per 10 ml: 30 mg NADP (Randox) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Test concentrations with justification for top dose:

Range finding study: 5.4, 17, 52, 164, 512, 1600 & 5000 Source of S9 µg/plate
Main study: 52, 164, 512, 1600 & 5000 µg/plate
No justification detailed in the study report.

Vehicle / solvent:

Solvent used: Ethanol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 6-Chloro-9-(3-N-(2-Chloroethyl-amino)propylamino-2-methoxyacridine dichloride [ICR-191]; 2-aminoanthracene [2-AA]

Details on test system and experimental conditions:

First experiment
Seven concentrations of the test substance, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Second experiment
Based on the results of the first mutation assay, five doses (increasing with approximately half-log steps) of the test substance were selected and tested in triplicate in each strain in the second experiment. The highest concentration of ANOX® 1315 used in the second mutation assay was 5 mg/plate.

Experimental procedure
The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.
The vehicle control and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol, and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.

Colony counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test article precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

Acceptability of the assay
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 historical range for each tester strain.
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 three 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.

Data evaluation and statistical procedures
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
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) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a positive response will be repeated, 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.

Statistics:

No formal hypothesis testing was done.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

First mutation experiment
ANOX® 1315 was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA with concentrations of 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix.

Precipitate
Precipitation of ANOX® 1315 on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 and 5000 μg/plate in all tester strains, except in tester strain TA1535 where the test substance already precipitated on the plates at 512 μg/plate in the absence of S9-mix.

Toxicity
To determine the toxicity of ANOX® 1315, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity
No biologically increase in the number of revertants was observed upon treatment with ANOX® 1315 under all conditions tested.
In strain TA1535, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the absence and presence of S9-mix at several dose levels. However, since the increases were not three-fold (a maximum of 1.4-fold was reached), these increases were not considered to be relevant.
In strain TA100, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the presence of S9-mix at several dose levels. However, since the increases were not two-fold (a maximum of 1.1-fold was reached), these increases were not considered to be relevant.

Second mutation assay
To obtain more information about the possible mutagenicity of ANOX® 1315, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation experiment, ANOX® 1315 was tested up to concentrations of 5000 μg/plate.

Precipitate
Precipitation of ANOX® 1315 on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate and at 1600 and 5000 μg/plate at the end of the incubation period.

Toxicity
The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Mutagenicity
No biologically relevant increase in the number of revertants was observed upon treatment with ANOX® 1315 under all conditions tested.
In strain TA1535, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the presence of S9-mix at several dose levels. However, since no increase above the concurrent vehicle control was seen, these increases were not considered to be relevant.

Any other information on results incl. tables

TABLE OF RESULTS

Range Finding Study

Name of test substance: ANOX® 1315

Date of experiment: from 24 April 2014 to 28 April 2014
S9-mix (-)	Dose level per plate (µg/plate)	Number of Revertants, (Mean), +/- SD
		TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
	Solvent control (ethanol)	29       (30) 30       0.6 30	4         (6) 4         3.5 10	20       (18) 19       3.2 14	124     (130) 121     12.5 144	38       (28) 15       11.8 31
	5.4	34       (31) 38       9.5 20	12       (6) 3         5.2 3	20       (18) 16       2.1 19	136     (131) 141     12.7 117	23       (30) 23       12.1 44
	17	46       (32) 31       13.5 19	8         (9) 14       5.0 4	27       (23) 26       6.1 16	131     (124) 122     6.7 118	31       (34) 30       6.1 41
	52	23       (24) 26       1.7 23	4         (6) 5         3.2 10	19       (19) 16       3.0 22	131     (125) 131     10.4 113	22       (25) 19       8.5 35
	164	30 NP (35) 39 NP 4.5 35 NP	10       (7) 3         3.6 8	14       (19) 24       5.0 19	148     (139) 147     14.7 122	33       (26) 18       7.5 27
	512	34 SP (34) 25 SP 1.2 33 SP	11 NP (8) 7 NP   3.1 5 NP	20 NP (16) 12 NP 4.0 16 NP	150 NP (143) 131 NP 10.4 148 NP	26 NP (36) 48 NP 11.2 33 NP
	1600	23 SP (31) 37 SP 7.2 33 SP	7 SP  (4) 1 SP   3.1 3 SP	26 SP (23) 19 SP 3.6 24 SP	148 SP (134) 137 SP 15.7 117 SP	24 SP (35) 44 SP 10.1 37 SP
	5000	31 n SP (35) 41 n SP 5.1 34 n SP	10 n SP (10) 8 n SP 1.5 11 n SP	33 n SP (26) 19 n SP 7.0 26 n SP	150 n SP (137) 140 n SP 14.7 121 n SP	37 n SP (36) 31 n SP 4.2 39 n SP
S9 mix (-)	Name Dose level No. of revertants	SA	ICR-191	NF	MMS	4-NQO
		5	2.5	10	650	10
		628     (633) 641     7.0 630	324     (306) 313     21.8 282	905     (883) 887     23.7 858	1008   (999) 986     11.5 1003	1355   (1294) 1329   84.7 1197

MMS              methylmethanesulphonate

SA                  sodium azide

4-NQO            4-nitroquinoline N-oxide

NF                  2-nitrofluorene

ICR-191            6-Chloro-9-(3-N-(2-Chloroethyl-amino)propylamino-2-methoxyacridine dihydrochloride

NP                  No precipitate

SP                  Slight precipitate

n                    Normal bacterial background lawn

 

Date of experiment: from 24 April 2014 to 28 April 2014
S9-mix (+)	Dose level per plate (µg/plate)	Number of Revertants, (Mean), +/- SD
		TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
	Solvent control (ethanol)	31       (33) 35       2.1 34	7         (9) 7         2.9 12	24       (31) 31       7.0 38	140     (138) 117     20.6 158	46       (38) 30       8.0 38
	5.4	26       (29) 30       3.1 32	5         (9) 10       3.2 11	22       (25) 27       2.6 26	125     (125) 124     0.6 125	30       (30) 38       8.0 22
	17	35       (36) 37       1.2 35	4         (8) 12       4.0 7	24       (28) 33       4.6 27	146     (141) 139     4.7 137	30       (30) 34       3.5 27
	52	31       (37) 35       6.7 44	11       (10) 8         1.7 11	34       (35) 41       6.0 29	137     (130) 125     6.2 128	38       (33) 26       6.1 34
	164	42       (41) 33       8.0 49	7         (8) 4         4.0 12	33       (28) 16       10.4 35	140     (133) 121     10.2 137	39       (35) 35       4.0 31
	512	29 NP (32) 33 NP 2.3 33 NP	7 NP   (11) 12 NP 3.6 14 NP	27 NP (26) 19 NP 6.1 31 NP	137 NP (133) 146 NP 26.4 151 NP	37 NP (37) 39 NP 2.0 35 NP
	1600	35 SP (43) 48 SP 6.7 45 SP	7 SP  (4) 1 SP   3.1 3 SP	27 SP (26) 19 SP 6.1 31 SP	137 SP (150) 161 SP 12.1 152 SP	31 SP (39) 50 SP 10.0 35 SP
	5000	39 n SP (47) 50 n SP 7.0 52 n SP	19 n SP (22) 18 n SP 5.5 28 n SP	30 n SP (39) 49 n SP 9.5 38 n SP	133 n SP (141) 144 n SP 7.0 146 n SP	38 n SP (43) 46 n SP 4.2 44 n SP
S9 mix (+)	Name Dose level No. of revertants	2AA	2AA	2AA	2AA	2AA
		2.5	2.5	1	1	15
		256     (256) 265     8.5 248	381     (389) 414     22.5 371	705     (667) 624     40.7 672	1189   (1203) 1262   53.0 1159	180     (176) 163     11.9 186

2AA                 2-aminoanthracene

NP                  No precipitate

SP                  Slight precipitate

n                    Normal bacterial background lawn

 

TABLE OF RESULTS

Main Study

Name of test substance: ANOX® 1315

Date of experiment: from 01 May 2014 to 05 May 2014
S9-mix (-)	Dose level per plate (µg/plate)	Number of Revertants, (Mean), +/- SD
		TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
	Solvent control (ethanol)	30       (32) 29       4.9 38	12       (10) 11       2.6 7	20       (19) 20       1.2 18	129     (119) 109     10.0 118	27       (32) 24       10.8 44
	52	24       (18) 22       9.3 7	19       (11) 1         9.1 12	30       (24) 23       5.1 20	113     (105) 90       13.0 112	33       (40) 39       7.5 48
	164	16       (22) 23       5.6 27	10       (7) 5         2.9 5	29       (21) 26       11.9 7	106     (105) 94       10.1 114	27       (30) 33       3.1 31
	512	22 NP (23) 20 NP 3.6 27 NP	7 NP   (9) 10 NP 1.7 10 NP	34 NP (25) 22 NP 7.9 19 NP	118 NP (117) 112 NP 4.2 120 NP	29 NP (36) 48 NP 10.4 31 NP
	1600	20 SP (31) 42 SP 11.0 30 SP	14 SP (9) 10 SP 5.6 3 SP	16 SP (18) 27 SP 8.6 10 SP	109 SP           (97) 93 SP 11.0 88 SP	33 SP (38) 48 SP 8.4 34 SP
	5000	31 n SP (30) 33 n SP 3.1 27 n SP	18 n SP (13) 12 n SP 5.0 8 n SP	24 n SP (22) 20 n SP 2.1 23 n SP	116 n SP (115) 127 n SP 13.1 101 n SP	42 n SP (36) 29 n SP 6.7 38 n SP
S9 mix (-)	Name Dose level No. of revertants	SA	ICR-191	NF	MMS	4-NQO
		5	2.5	10	650	10
		804     (812) 793     23.5 838	517     (553) 584     33.8 558	834     (813) 842     43.5 763	926     (940) 932     19.3 962	1534   (1473) 1291   160.4 1594

MMS              methylmethanesulphonate

SA                  sodium azide

4-NQO            4-nitroquinoline N-oxide

NF                  2-nitrofluorene

ICR-191            6-Chloro-9-(3-N-(2-Chloroethyl-amino)propylamino-2-methoxyacridine dihydrochloride

NP                  No precipitate

SP                  Slight precipitate

n                    Normal bacterial background lawn

 

Date of experiment: from 01 May 2014 to 05 May 2014
S9-mix (+)	Dose level per plate (µg/plate)	Number of Revertants, (Mean), +/- SD
		TA 1535	TA 1537	TA 98	TA 100	WP2uvrA
	Solvent control (ethanol)	38       (36) 45       10.7 24	7         (12) 14       4.4 15	23       (28) 33       5.0 27	143     (142) 144     2.6 139	46       (46) 41       4.5 50
	52	30       (22) 18       6.7 19	14       (11) 14       5.2 5	41       (38) 41       4.6 33	103     (107) 103     11.5 116	37       (42) 44       4.0 44
	164	30       (34) 30       6.9 42	3         (6) 10       3.6 5	38       (40) 50       9.6 31	109     (110) 99       11.5 122	34       (38) 34       6.9 46
	512	34 NP (27) 16 NP 9.5 30 NP	10 NP (9) 11 NP 2.1 7 NP	20 NP (30) 26 NP 12.5 44 NP	141 NP           (122) 116 NP           16.8 109 NP	44 NP (43) 39 NP 3.6 46 NP
	1600	24 SP (27) 35 SP 6.7 23 SP	5 SP  (8) 15 SP 5.8 5 SP	38 SP (35) 31 SP 3.5 35 SP	98 SP (101) 113 SP 11.2 91 SP	39 SP (46) 38 SP 12.4 60 SP
	5000	35 n SP (36) 39 n SP 2.3 35 n SP	10 n SP (16) 22 n SP 6.0 15 n SP	44 n SP (32) 22 n SP 11.1 30 n SP	113 n SP (106) 107 n SP 8.1 97 n SP	50 n SP (54) 46 n SP 11.2 67 n SP
S9 mix (+)	Name Dose level No. of revertants	2AA	2AA	2AA	2AA	2AA
		2.5	5	1	2	15
		220     (194) 192     25.1 170	612     (490) 484     118.6 375	718     (693) 679     21.7 682	1726   (1545) 1490   160.3 1420	288     (277) 249     24.4 294

2AA                 2-aminoanthracene

NP                  No precipitate

SP                  Slight precipitate

n                    Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative control values were within the laboratory historical control data ranges, except for TA1535 in the presence of S9-mix (first and second experiment) and TA100 in the presence of S9-mix (first experiment).
The 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.
Based on the results of this study it is concluded that ANOX® 1315 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of ANOX® 1315 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).

 

ANOX® 1315 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

 

The study procedures described in this report were based on the most recent OECD, EC and Japanese guidelines.

 

Batch WBF3F0004 of ANOX® 1315 was a clear yellow viscous liquid with a purity of 93.5% by GC. The test substance was dissolved in ethanol.

 

In the first mutation assay, ANOX® 1315 was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. ANOX® 1315 precipitated on the plates at dose levels of 1600 and 5000 μg/plate in all tester strains, except in tester strain TA1535 where the test substance already precipitated on the plates at 512 μg/plate in the absence of S9-mix. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

In the second mutation assay, ANOX® 1315 was tested up to concentrations of 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix. ANOX® 1315 precipitated on the plates at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

 

ANOX® 1315 did not induce a biologically significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

The negative control values were within the laboratory historical control data ranges, except for TA1535 in the presence of S9-mix (first and second experiment) and TA100 in the presence of S9-mix (first experiment). However, since these values were just outside the limit of the range, the validity of the test was considered to be not affected.

 

The 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.

 

Based on the results of this study it is concluded that ANOX® 1315 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.