Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

HMX

IUPAC Name:

HMX

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): HMX
- Substance type: Organic
- Physical state: Solid
- Analytical purity: 99.9%
- Purity test date: no data
- Lot/batch No.: 12184
- Expiration date of the lot/batch: 31 December 2012
- Stability under test conditions: no data
- Storage condition of test material: Room temperature

Specific details on test material used for the study:

HMX (Octogen): purity > 99.9%, CAS 2691-41-0, batch n°A003

The Test Item was supplied in Dimethyl Sulphoxide (DMSO; VWR, lot 18K08402, CAS 67-68-5) at a concentration of 49.6 mg/mL. Therefore 101 µL of the ‘neat’ Test Item formulation was dosed onto Vogel-Bonner agar plates to achieve a final maximum concentration of 50 mg/mL (equating to 5000 µg/plate). Approximate half-log dilutions were prepared from the ‘neat’ formulation in high purity dimethyl sulphoxide with all subsequent dosing performed using 100 µL aliquots. No correction for purity was required. All test item preparation and dosing was performed under yellow safety lighting.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Salmonella typhimurium
Strains
Genotype
Type of mutations indicated
TA1537
his C 3076; rfa-; uvrB-:
frame shift mutations
TA98
his D 3052; rfa-; uvrB-;R-factor
TA1535
his G 46; rfa-; uvrB-:
base-pair substitutions
TA100
his G 46; rfa-; uvrB-;R-factorCELLS USED
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

MEDIA USED
- Top agar was prepared using 0.6% Bacto agar (lot number 9105946 expiry date 01/2024) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 54834 expiry date 07/2020 and 54898 expiry date 07/2020).

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

MEDIA:
Top agar was prepared using 0.6% Bacto agar (lot number 9105946 expiry date 01/2024) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 54834 expiry date 07/2020 and 54898 expiry date 07/2020).

Metabolic activation:

with and without

Metabolic activation system:

The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Test concentrations with justification for top dose:

Experiement 1: The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Experiment 2: As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix). The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the urgency of testing, the lack of cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

The vehicle control used was as follows:
Identity: Dimethyl sulphoxide
Supplier: ThermoFisher Scientific
Batch number, (purity), expiry 196455, (99.9%), September 2024

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 (2AA)

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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s 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. Values that are statistically significant but are within the in-house historical vehicle/untreated control range are not flagged for statistical significance in the data tables.

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:

valid

True 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

valid

True 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), 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 were 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) and viability 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 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
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 (S9-mix), 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.
Experiment 1 (plate incorporation) – Table 2 and Table 3
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
A test item film (white in appearance) was noted at 5000 g/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
No biologically relevant 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 1 (plate incorporation method). One statistically significant value was noted (TA100 at 5 µg/plate in the absence of metabolic activation (S9-mix), however as the maximum fold increase was only 1.2 times the concurrent vehicle controls and the mean colony count was within the in-house historical vehicle/untreated control range for the relevant strains the response was considered of no biological relevance. Therefore, this value has not been highlighted in Table 2 as it did not meet the required criteria for a positive response.
 
Experiment 2 (pre-incubation) – Table 4 and Table 5
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
A test item film (white in appearance) was noted at 5000 g/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
No biologically relevant 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). Three statistically significant values were noted (WP2uvrA at 1.5, 150 and 1500 µg/plate in the absence of metabolic activation (S9-mix), however as the maximum fold increase was only 1.6 times the concurrent vehicle control and the mean colony counts were within the in-house historical vehicle/untreated control range for the relevant strain the responses were considered of no biological relevance. Therefore, these values have not been highlighted in Table 4 as they did not meet the required criteria for a positive response.

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
113		20		12		22		11
148	(125)	20	(18)	23	(19)	15	(20)	14	(14)
113		15		23		23		18
Viability – Bacterial cells 10^9per mL
2.4		1.8		6.5		1.8		2.3

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
125		18		29		14		13
153	(138)	18	(15)	26	(24)	19	(17)	11	(12)
137		10		18		19		11
Viability – Bacterial cells 10^9per mL
3.3		2.3		4.0		2.0		3.4

 

 

Table 2            Test Results: Experiment 1 – Without Metabolic Activation(Plate Incorporation)

Test Period		From: 19 June 2020					To: 22 June 2020
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)	113 100 107	(107) 6.5#	14 22 22	(19) 4.6	16 19 27		(21) 5.7	22 10 31	(21) 10.5	19 17 14	(17) 2.5
	1.5 µg	103 101 123	(109) 12.2	11 12 20	(14) 4.9	17 17 21		(18) 2.3	21 15 19	(18) 3.1	20 19 7	(15) 7.2
	5 µg	129 121 142	(131) 10.6	11 15 13	(13) 2.0	21 22 17		(20) 2.6	9 24 22	(18) 8.1	12 12 14	(13) 1.2
	15 µg	113 110 116	(113) 3.0	24 20 20	(21) 2.3	19 24 18		(20) 3.2	25 20 17	(21) 4.0	20 17 10	(16) 5.1
	50 µg	109 108 118	(112) 5.5	13 21 27	(20) 7.0	16 23 22		(20) 3.8	20 23 32	(25) 6.2	21 12 14	(16) 4.7
	150 µg	128 111 112	(117) 9.5	23 24 17	(21) 3.8	17 22 24		(21) 3.6	17 19 21	(19) 2.0	15 15 21	(17) 3.5
	500 µg	115 117 111	(114) 3.1	17 34 10	(20) 12.3	20 20 17		(19) 1.7	21 16 20	(19) 2.6	21 19 21	(20) 1.2
	1500 µg	118 132 107	(119) 12.5	10 19 24	(18) 7.1	15 17 17		(16) 1.2	24 17 27	(23) 5.1	18 17 12	(16) 3.2
	5000 µg	106 F 106 F 104 F	(105) 1.2	19 F 22 F 19 F	(20) 1.7	20 F 17 F 15 F		(17) 2.5	25 F 24 F 21 F	(23) 2.1	13 F 11 F 17 F	(14) 3.1
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
		595 635 681	(637) 43.0	543 792 767	(701) 137.1	867 716 822		(802) 77.5	109 106 133	(116) 14.8	406 631 498	(512) 113.1

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

F                        Test Item Film

#         Standard deviation

 

Table3            Test Results: Experiment 1 – With Metabolic Activation(Plate Incorporation)

Test Period		From: 19 June 2020					To: 22 June 2020
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)	108 122 105	(112) 9.1#	8 13 17	(13) 4.5	28 23 18		(23) 5.0	20 33 21	(25) 7.2	19 15 19	(18) 2.3
	1.5 µg	114 128 133	(125) 9.8	5 13 26	(15) 10.6	18 22 19		(20) 2.1	25 24 35	(28) 6.1	17 16 21	(18) 2.6
	5 µg	134 112 131	(126) 11.9	10 7 13	(10) 3.0	17 28 31		(25) 7.4	16 30 24	(23) 7.0	14 16 21	(17) 3.6
	15 µg	122 119 123	(121) 2.1	12 17 14	(14) 2.5	24 20 29		(24) 4.5	33 28 24	(28) 4.5	15 19 18	(17) 2.1
	50 µg	118 133 114	(122) 10.0	19 11 10	(13) 4.9	19 38 25		(27) 9.7	34 14 25	(24) 10.0	15 14 25	(18) 6.1
	150 µg	121 118 118	(119) 1.7	17 11 12	(13) 3.2	17 23 16		(19) 3.8	32 14 40	(29) 13.3	17 13 11	(14) 3.1
	500 µg	124 112 117	(118) 6.0	9 11 21	(14) 6.4	18 27 15		(20) 6.2	18 16 30	(21) 7.6	16 20 17	(18) 2.1
	1500 µg	122 124 127	(124) 2.5	16 16 13	(15) 1.7	23 28 37		(29) 7.1	16 27 26	(23) 6.1	19 11 14	(15) 4.0
	5000 µg	111 F 106 F 97 F	(105) 7.1	5 F 11 F 18 F	(11) 6.5	19 F 21 F 28 F		(23) 4.7	28 F 33 F 30 F	(30) 2.5	16 F 15 F 15 F	(15) 0.6
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
		2023 1964 1868	(1952) 78.2	339 294 276	(303) 32.4	195 256 186		(212) 38.1	161 149 161	(157) 6.9	315 297 287	(300) 14.2

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

F            Test Item Film

#            Standard deviation

 

Table4            Test Results: Experiment 2 – Without Metabolic Activation(Pre-Incubation)

Test Period		From: 26 June 2020					To: 29 June 2020
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)	137 129 141	(136) 6.1#	16 12 30	(19) 9.5	22 14 16		(17) 4.2	16 24 15	(18) 4.9	9 10 15	(11) 3.2
	1.5 µg	138 145 141	(141) 3.5	13 12 18	(14) 3.2	23 23 29		(25) 3.5	17 23 16	(19) 3.8	9 16 22	(16) 6.5
	5 µg	140 139 118	(132) 12.4	16 12 12	(13) 2.3	19 19 20		(19) 0.6	19 19 19	(19) 0.0	10 8 8	(9) 1.2
	15 µg	136 121 155	(137) 17.0	17 14 13	(15) 2.1	22 16 21		(20) 3.2	14 14 22	(17) 4.6	8 9 6	(8) 1.5
	50 µg	123 116 108	(116) 7.5	16 20 13	(16) 3.5	18 24 16		(19) 4.2	17 20 16	(18) 2.1	9 18 9	(12) 5.2
	150 µg	128 139 138	(135) 6.1	20 15 23	(19) 4.0	26 25 30		(27) 2.6	33 20 28	(27) 6.6	10 9 20	(13) 6.1
	500 µg	129 103 128	(120) 14.7	13 17 23	(18) 5.0	25 17 25		(22) 4.6	16 25 18	(20) 4.7	13 14 14	(14) 0.6
	1500 µg	129 144 124	(132) 10.4	18 15 16	(16) 1.5	28 19 30		(26) 5.9	31 22 16	(23) 7.5	17 12 12	(14) 2.9
	5000 µg	139 F 124 F 120 F	(128) 10.0	18 F 25 F 24 F	(22) 3.8	22 F 22 F 20 F		(21) 1.2	18 F 27 F 24 F	(23) 4.6	10 F 23 F 11 F	(15) 7.2
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
		529 578 672	(593) 72.7	294 530 208	(344) 166.7	406 342 343		(364) 36.7	147 178 160	(162) 15.6	82 84 403	(190) 184.8

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9 -Aminoacridine

F                        Test Item Film

#         Standard deviation

 

Table5            Test Results: Experiment 2 – With Metabolic Activation(Pre-Incubation)

Test Period		From: 26 June 2020					To: 29 June 2020
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)	132 139 150	(140) 9.1#	9 12 11	(11) 1.5	34 30 28		(31) 3.1	34 25 18	(26) 8.0	7 13 10	(10) 3.0
	1.5 µg	152 156 125	(144) 16.9	15 8 12	(12) 3.5	34 38 33		(35) 2.6	21 17 24	(21) 3.5	12 11 6	(10) 3.2
	5 µg	119 126 147	(131) 14.6	14 16 10	(13) 3.1	27 40 26		(31) 7.8	34 37 17	(29) 10.8	16 15 19	(17) 2.1
	15 µg	137 145 126	(136) 9.5	8 12 10	(10) 2.0	31 29 40		(33) 5.9	42 25 28	(32) 9.1	11 11 13	(12) 1.2
	50 µg	138 112 121	(124) 13.2	17 13 8	(13) 4.5	28 39 26		(31) 7.0	37 26 30	(31) 5.6	11 10 17	(13) 3.8
	150 µg	127 128 122	(126) 3.2	16 11 10	(12) 3.2	37 33 23		(31) 7.2	37 27 29	(31) 5.3	18 6 11	(12) 6.0
	500 µg	145 151 158	(151) 6.5	15 19 15	(16) 2.3	25 19 23		(22) 3.1	22 25 27	(25) 2.5	12 10 11	(11) 1.0
	1500 µg	135 101 129	(122) 18.1	12 15 23	(17) 5.7	27 32 19		(26) 6.6	35 24 35	(31) 6.4	8 9 14	(10) 3.2
	5000 µg	120 F 160 F 138 F	(139) 20.0	13 F 10 F 16 F	(13) 3.0	28 F 25 F 43 F		(32) 9.6	22 F 30 F 23 F	(25) 4.4	7 F 8 F 14 F	(10) 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
		1464 1360 1857	(1560) 262.1	296 266 257	(273) 20.4	138 146 131		(138) 7.5	134 132 133	(133) 1.0	216 232 220	(223) 8.3

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

F                   Test Item Film

#            Standard deviation

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471), the test item HMX did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test HMX was considered to be non-mutagenic.

Executive summary:

Mutation Test".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 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). Because of the explosivity of the test item as such, it was provided to the laboratory already diluted in dimethyl sulphoxide (50 g/L), which was the vehicle used for the assay. The dose range for the first experiments (plate incorporation method) was 1.5 to 5000μg/plate, as recommended by the guideline. The same range was used for the second experiment (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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Similarly in the two experiments, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). A test item film (white in appearance) was noted at 5000μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies. 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). One statistically significant value was noted (TA100 at 5μg/plate in the absence of metabolic activation (S9-mix), however it did not meet the criteria for a positive response.

No biologically relevant 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). Three statistically significant values were noted (WP2uvrA at 1.5, 150 and 1500 μg/plate in the absence of metabolic activation (S9-mix), however they did not meet the required criteria for a positive response.

In this Reverse Mutation Assay ‘Ames Test’ using strains ofSalmonella typhimuriumandEscherichia coli(OECD TG 471), the test item HMX did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test HMX was considered to be non-mutagenic.