O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)

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:

Experimental start date 20 April 2016 Experimental completion date 17 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)

EC Number:

300-947-2

EC Name:

O,O-dibutyl hydrogen thiophosphate, compound with 1-octylamine (1:1)

Cas Number:

93964-99-9

Molecular formula:

C8H19N.C8H19O3PS

IUPAC Name:

octan-1-amine dibutyl sulfanylphosphonate

impurity 1

Reference substance name:

Octylamine

EC Number:

203-916-0

EC Name:

Octylamine

Cas Number:

111-86-4

Molecular formula:

C8H19N

IUPAC Name:

octan-1-amine

Test material form:

liquid

Specific details on test material used for the study:

Identification: X-19575 Phosphorothioic acid, O,O-dibutyl ester, compd. with 1-octanamine, CASRN 93964-99-9
Physical state/Appearance: Amber coloured viscous liquid
Batch: X-019575-00-00
Purity: >95% (treated as 100%)
Expiry Date: 01 June 2017
Storage Conditions: Room temperature in the dark

Method

Target gene:

Salmonella tuphimurium - histidine
Escherichia coli - tryptophan

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 - rat liver homogenate metabolizing system

Test concentrations with justification for top dose:

Experiment 1
Dose selection: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 2 - determined by the results of Exoeriment 1
5, 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix: 2 µg/plate for WP2uvrA 3 µg/plate for TA100 5 µg/plate for TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 Mix: 80 µg/plate for TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9 Mix: 0.2 µg/plate for TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With S9 mix: 1 µg/plate for TA100 2 µg/plate for TA1535 and TA1537 10 µg/plate for WP2uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix: 5 µg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation);

Rationale for test conditions:

The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
Combined historical negative and solvent control ranges for 2014 and 2015 are presented in Appendix 1.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2014 and 2015 are presented in Appendix 1.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

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 will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 1537, TA 98, TA 1535, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.
A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1.
The maximum dose level of the test item in the first experiment was initially selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains at 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level. Consequently the same maximum dose level was used in the second mutation test. The test item induced a slightly stronger toxic response in the second mutation test (pre-incubation method) with weakened bacterial background lawns noted in the absence of S9-mix from 500 µg/plate (TA1535 and TA1537), 1500 µg/plate (TA100 and TA98) and at 5000 µg/plate (WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted from 1500 µg/plate (TA100, TA1535 and TA98) and at 5000 µg/plate (WP2uvrA and TA1537). The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experimental methodology (plate incorporation and pre-incubation). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
There were no 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 (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no 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 (pre incubation method).
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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Any other information on results incl. tables

Table1            Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type							Frameshift type
TA100			TA1535			WP2uvrA			TA98			TA1537
68				31				18				19				14
94		(80)		29		(30)		22		(19)		16		(18)		12		(13)
78				30				17				20				13

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
98		15		28		25		11
92	(94)	18	(19)	25	(25)	8	(16)	10	(9)
93		24		22		16		7

 

  Table2            Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 09 May 2016					To: 12 May 2016
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)	75 67 66	(69) 4.9#	25 25 23	(24) 1.2	18 19 25		(21) 3.8	11 15 20	(15) 4.5	4 11 8	(8) 3.5
	1.5 µg	76 74 75	(75) 1.0	30 20 21	(24) 5.5	28 24 16		(23) 6.1	18 14 20	(17) 3.1	6 5 6	(6) 0.6
	5 µg	77 78 78	(78) 0.6	30 27 20	(26) 5.1	17 19 20		(19) 1.5	11 22 23	(19) 6.7	7 8 7	(7) 0.6
	15 µg	84 76 82	(81) 4.2	25 21 29	(25) 4.0	16 16 13		(15) 1.7	12 16 18	(15) 3.1	7 11 10	(9) 2.1
	50 µg	75 73 65	(71) 5.3	28 25 24	(26) 2.1	19 24 18		(20) 3.2	23 16 21	(20) 3.6	10 12 10	(11) 1.2
	150 µg	67 82 68	(72) 8.4	25 28 25	(26) 1.7	27 19 17		(21) 5.3	21 20 18	(20) 1.5	9 9 10	(9) 0.6
	500 µg	85 76 64	(75) 10.5	28 27 29	(28) 1.0	29 17 15		(20) 7.6	17 16 8	(14) 4.9	5 13 8	(9) 4.0
	1500 µg	71 78 57	(69) 10.7	15 23 25	(21) 5.3	22 16 17		(18) 3.2	15 20 25	(20) 5.0	7 10 7	(8) 1.7
	5000 µg	56 S 47 S 35 S	(46) 10.5	8 S 8 S 11 S	(9) 1.7	16 29 15		(20) 7.8	12 S 2 S 3 S	(6) 5.5	0 S 1 S 2 S	(1) 1.0
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
		481 696 749	(642) 141.9	1180 1161 1058	(1133) 65.6	1179 1198 1127		(1168) 36.8	143 148 151	(147) 4.0	953 1167 818	(979) 176.0

 

ENNG4NQO9AAS#

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

4NQO          4-Nitroquinoline-1-oxide

9AA            9-Aminoacridine

S                Sparse bacterial background lawn

#                Standard deviation

                Table 3         Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 09 May 2016					To: 12 May 2016
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)	67 77 66	(70) 6.1#	29 32 33	(31) 2.1	33 27 30		(30) 3.0	24 18 18	(20) 3.5	12 11 4	(9) 4.4
	1.5 µg	66 63 67	(65) 2.1	24 25 26	(25) 1.0	23 21 24		(23) 1.5	14 15 15	(15) 0.6	6 6 10	(7) 2.3
	5 µg	72 66 65	(68) 3.8	33 25 25	(28) 4.6	18 17 19		(18) 1.0	12 21 19	(17) 4.7	15 10 10	(12) 2.9
	15 µg	61 62 69	(64) 4.4	23 24 34	(27) 6.1	19 23 19		(20) 2.3	22 15 17	(18) 3.6	8 9 9	(9) 0.6
	50 µg	93 76 74	(81) 10.4	32 28 24	(28) 4.0	17 20 20		(19) 1.7	16 14 8	(13) 4.2	12 10 14	(12) 2.0
	150 µg	78 76 70	(75) 4.2	24 31 25	(27) 3.8	17 30 27		(25) 6.8	24 16 25	(22) 4.9	7 17 14	(13) 5.1
	500 µg	70 80 82	(77) 6.4	27 25 25	(26) 1.2	27 14 24		(22) 6.8	24 22 27	(24) 2.5	8 9 6	(8) 1.5
	1500 µg	71 71 80	(74) 5.2	28 25 14	(22) 7.4	23 20 21		(21) 1.5	17 26 19	(21) 4.7	13 12 10	(12) 1.5
	5000 µg	25 S 42 S 41 S	(36) 9.5	9 S 4 S 4 S	(6) 2.9	18 26 23		(22) 4.0	10 S 5 S 14 S	(10) 4.5	0 S 6 S 7 S	(4) 3.8
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
		1273 1226 952	(1150) 173.4	187 195 246	(209) 32.0	285 277 218		(260) 36.6	131 125 161	(139) 19.3	239 289 292	(273) 29.8

  Table4            Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From: 13 May 2016					To: 16 May 2016
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)	102 91 92	(95) 6.1#	18 29 21	(23) 5.7	23 31 27		(27) 4.0	21 17 16	(18) 2.6	23 26 15	(21) 5.7
	5 µg	93 116 99	(103) 11.9	19 21 20	(20) 1.0	21 31 14		(22) 8.5	15 15 15	(15) 0.0	16 26 21	(21) 5.0
	15 µg	88 81 90	(86) 4.7	16 19 23	(19) 3.5	26 28 22		(25) 3.1	15 23 27	(22) 6.1	13 18 20	(17) 3.6
	50 µg	91 85 92	(89) 3.8	18 16 22	(19) 3.1	37 40 25		(34) 7.9	11 18 20	(16) 4.7	17 24 21	(21) 3.5
	150 µg	95 89 92	(92) 3.0	18 18 21	(19) 1.7	23 22 14		(20) 4.9	18 15 10	(14) 4.0	14 17 14	(15) 1.7
	500 µg	69 76 79	(75) 5.1	17 S 13 S 12 S	(14) 2.6	22 28 16		(22) 6.0	12 18 15	(15) 3.0	4 S 12 S 9 S	(8) 4.0
	1500 µg	63 S 51 S 57 S	(57) 6.0	8 S 13 S 13 S	(11) 2.9	24 12 9		(15) 7.9	16 S 9 S 4 S	(10) 6.0	6 S 3 S 6 S	(5) 1.7
	5000 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	18 S 16 S 10 S		(15) 4.2	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	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		508 518 504	(510) 7.2	1178 1227 1218	(1208) 26.1	724 569 594		(629) 83.2	200 152 212	(188) 31.7	157 216 181	(185) 29.7

 

ENNG4NQO9AASV#

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

4NQO          4-Nitroquinoline-1-oxide

9AA            9-Aminoacridine

S                Sparse bacterial background lawn

V               Very weak bacterial background lawn

#                Standard deviation

Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 13 May 2016					To: 16 May 2016
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)	85 84 86	(85) 1.0#	26 24 15	(22) 5.9	27 25 26		(26) 1.0	19 31 18	(23) 7.2	17 13 14	(15) 2.1
	5 µg	106 75 92	(91) 15.5	18 20 29	(22) 5.9	27 18 23		(23) 4.5	18 20 31	(23) 7.0	24 13 17	(18) 5.6
	15 µg	76 101 81	(86) 13.2	19 17 32	(23) 8.1	31 23 22		(25) 4.9	18 29 21	(23) 5.7	13 5 7	(8) 4.2
	50 µg	83 100 86	(90) 9.1	19 13 22	(18) 4.6	26 26 33		(28) 4.0	14 19 27	(20) 6.6	16 16 19	(17) 1.7
	150 µg	92 89 92	(91) 1.7	21 25 23	(23) 2.0	31 28 22		(27) 4.6	20 22 30	(24) 5.3	12 8 12	(11) 2.3
	500 µg	94 114 97	(102) 10.8	32 19 20	(24) 7.2	22 27 26		(25) 2.6	24 30 24	(26) 3.5	10 12 17	(13) 3.6
	1500 µg	79 S 75 S 82 S	(79) 3.5	11 S 16 S 10 S	(12) 3.2	26 19 22		(22) 3.5	25 S 31 S 24 S	(27) 3.8	11 19 16	(15) 4.0
	5000 µg	45 S 55 S 52 S	(51) 5.1	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V		(0) 0.0	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
		404 498 472	(458) 48.5	174 180 180	(178) 3.5	199 225 207		(210) 13.3	108 92 111	(104) 10.2	170 159 177	(169) 9.1

 

BP2AASV#

BP           Benzo(a)pyrene

2AA        2-Aminoanthracene

S                    Sparse bacterial background lawn

V                   Very weak bacterial background lawn

#            Standard deviation

Applicant's summary and conclusion

Conclusions:

X-19575 Phosphorothioic acid, O,O-dibutyl ester, compd. with 1-octanamine, CASRN 93964-99-9 was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to 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). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The second experiment was performed at a later date (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate. Seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Results

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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was initially selected as the maximum recommended dose level of 5000 µg/plate. In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of all of theSalmonellatester strains at 5000 µg/plate in both the absence and presence of S9-mix. No toxicity was noted toEscherichia colistrain WP2uvrAat any test item dose level. Consequently the same maximum dose level was used in the second mutation test. The test item induced a slightly stronger toxic response in the second mutation test (pre-incubation method) with weakened bacterial background lawns noted in the absence of S9-mix from 500 µg/plate (TA1535 and TA1537), 1500 µg/plate (TA100 and TA98) and at 5000 µg/plate (WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted from 1500 µg/plate (TA100, TA1535 and TA98) and at 5000 µg/plate (WP2uvrAand TA1537). The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experimental methodology (plate incorporation and pre-incubation). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

There were no 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 (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no 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 (pre‑incubation method). 

Conclusion

X-19575 Phosphorothioic acid, O,O-dibutyl ester, compd. with 1-octanamine, CASRN 93964-99-9 was considered to be non-mutagenic under the conditions of this test.