6-methyl-1,3,5-triazine-2,4-diyldiamine

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:

August - October 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliable without restrictions; Guideline study (OECD 471) according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4-Diamino-6-methyl-1,3,5-triazine

IUPAC Name:

2,4-Diamino-6-methyl-1,3,5-triazine

Constituent 2

Reference substance name:

6-methyl-1,3,5-triazine-2,4-diyldiamine

EC Number:

208-796-3

EC Name:

6-methyl-1,3,5-triazine-2,4-diyldiamine

Cas Number:

542-02-9

Molecular formula:

C4H7N5

IUPAC Name:

6-methyl-1,3,5-triazine-2,4-diamine

Details on test material:

- Name of test material (as cited in study report): Acetoguanamine
- Physical state: white powder
- Lot./batch-no.:10/08/89 AG
- Purity: 99.2 %
- Storage condition of test material: In the dark at room temperature

Method

Target gene:

- genes involved in histidine synthesis

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: histidine requiring

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: histidine requiring

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: histidine requiring

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: histidine requiring

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: histidine requiring

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

Concentrations of Treatment Solution (mg/mL):
Toxicity range-finder experiment: 0.016, 0.080, 0.400, 2.000, 10.00 mg Acetoguanamine /ml.
Mutation experiment 1: 0.016, 0.080, 0.400, 2.000, 10.00 mg Acetoguanamine /ml.
Mutation experiment 2: 2.0, 4.0, 6.0, 8.0, 10.0 mg Acetoguanamine /ml.

Final Concentration (µg/plate):
Toxicity range-finder experiment: 8, 40, 200, 1000, 5000 µg Acetoguanamine /plate.
Mutation experiment 1: 8, 40, 200, 1000, 5000 µg Acetoguanamine /plate.
Mutation experiment 2: 1000, 2000, 3000, 4000, 5000 µg Acetoguanamine /plate.

Vehicle / solvent:

Test chemical solutions:
- prepared by dissolving Acetoguanamine in distilled water.

Stock solution of positive control chemicals:
- 2-nitrofluorene, 9-aminoacridine and 2-aminoanthracene were dissolved in DMSO
- sodium azide was dissolved in distilled water

Controls

Controls 1

Positive controls:

yes

Remarks:

500 µg/ml (stock concentration), 50.0 µg/plate (final concentration); for strains TA98 and TA1538; induced in quintuplicate without S-9 mix

Positive control substance:

2-nitrofluorene

Controls 2

Positive controls:

yes

Remarks:

20 µg/ml (stock concentration), 2.0 µg/plate (final concentration); for strains TA100 and TA1535; induced in quintuplicate without S-9 mix

Positive control substance:

sodium azide

Controls 3

Positive controls:

yes

Remarks:

500 µg/ml (stock concentration), 50.0 µg/plate (final concentration); for strain TA1537; induced in quintuplicate without S-9 mix

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 500µ

Controls 4

Positive controls:

yes

Remarks:

50 µg/ml (stock concentration), 5.0 µg/plate (final concentration); for strains TA98, TA1538, TA100, TA1535, TA1537; induced in quintuplicate with S-9 mix

Positive control substance:

other: 2-aminoanthracene

Controls 5

Untreated negative controls:

yes

Remarks:

sterile distilled water, included in quintuplicate without and with S-9 mix

Evaluation criteria:

EVALUATION CRITERIA:
A test compound was considered to be mutagenic if:
i) the assay was valid (see acceptance criteria)
ii) two or three-fold increase (depent on strain: TA98 and TA100 two-fold increase; TA1535, TA1537 and TA1538 three-fold increase) in revertant numbers, were accompanied by significant F-statistics and dose response correlations
iii) the positive resposes described in (ii) were reproducible

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met:
i) the mean negative control counts fell within the normal range (compared to historical data)
ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation.
iii) no more than 5% of the plates were lost through contamination or some other unforseen event.

Statistics:

no data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- carried out in TA100 only
- using final concentrations of Acetoguanamine at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and positive control
- no toxicity was observed following these treatments
- same dose range was therefore used for treatment of the remaining test strains in experiment 1
- for experiment 2 a concentration of 5000 µg/plate was retained as a maximum test dose, but with a narrowed dose range utilised in order to investigate more closely those concentrations of Acetoguanamine most likely to exhibit a mutagenic effect.

Any other information on results incl. tables

Table 1: Results of the AMES Test with the test substance Acetoguamine.

Sample	Dose µg/plate		TA98		TA100		TA1535		TA1537		TA1538
			- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Mutation Experiment 1
Acetoguanamine	0	mean sd	29 21 23 26 23   24.4 3.1	35 34 31 28 32   32.0 2.7	77 89 86 105 82   87.8 10.6	71 85 70 87 94   81.4 10.5	44 27 36 29 36   34.4 6.7	25 33 13 28 26   25.0 7.4	5 5 9 8 3   6.0 2.4	6 11 9 14 13   10.6 3.2	12 8 12 6 11   9.8 2.7	36 32 21 28 25   28.4 5.9
Acetoguanamine	8	mean sd	18 16 26   20.0 5.3	29 35 31   31.7 3.1	78 72 80   76.7 4.2	99 116 122   112.3 11.9	20 28 31   26.3 5.7	32 29 C   30.5	9 8 3   6.7 3.2	17 10 17   14.7 4.0	9 12 13   11.3 2.1	25 23 29   25.7 3.1
Acetoguanamine	40	mean sd	19 28 24   23.7 4.5	33 24 39   32.0 7.5	69 102 88   86.3 16.6	84 82 104   90.0 12.2	23 27 29   26.3 3.1	27 29 C   28.0	5 2 2   3.0 1.7	17 20 16   17.7 2.1	16 11 9   12.0 3.6	25 41 33   33.0 8.0
Acetoguanamine	200	mean sd	14 19 26   19.7 6.0	47 40 41   42.7 3.8	84 82 95   87.0 7.0	80 108 107   98.3 15.9	43 34 34   37.0 5.2	19 21 28   22.7 4.7	8 11 9   9.3 1.5	20 14 18   17.3 3.1	11 19 11   13.7 4.6	23 28 18   23.0 5.0
Acetoguanamine	1000	mean sd	23 18 20   20.3 2.5	39 34 33   35.3 3.2	80 92 86   86.0 6.0	123 112 104   113.0 9.5	33 32 36   33.7 2.1	23 19 28   23.3 4.5	4 3 3   3.3 0.6	12 23 12   15.7 6.4	12 5 14   10.3 4.7	28 19 28   25.0 5.2
Acetoguanamine	5000	mean sd	20 21 18   23.0 7.0	26 25 26   25.7 0.6	94 87 67   82.7 14.0	84 99 104   95.7 10.4	31 31 C   31.0 C	18 23 31   24.0 6.6	10 2 8   6.7 4.2	11 10 12   11.0 1.0	9 10 17   12.0 4.4	C 20 24   22.0
Positive Controls (Mutation Experiment 1)
2-nitrofluorene	50.0	mean sd	1152 1021 969 1293 1144   1115.8 126.5	-	-	-	-	-	-	-	219 268 246 177 170   216.0 42.6	-
Sodium azide	2.0	mean sd	-	-	453 427 441 439 444   440.8 9.4	-	305 345 332 343 339   332.8 16.3	-	-	-	-	-
9-aminoacridine	50.0	mean sd	-	-	-	-	-	-	1127 1145 887 899 926   996.8 128.0	-	-	-
2-aminoanthracene	5.0	mean sd	-	339 360 365 360 369   358.6 11.6	-	1042 886 781 834 819   872.4 102.0	-	194 157 156 183 154   168.8 18.4	-	65 50 61 59 43   55.6 8.9	-	227 210 202 209 192   208.0 12.8
Mutation Experiment 2
Acetoguanamine	0	mean sd	25 35 32 29 18   27.8 6.6	43 39 48 35 44   41.8 5.0	110 107 122 108 86   106.6 13.0	130 150 138 107 115   128.0 17.3	47 37 44 46 41   43.0 4.1	28 31 33 23 21   27.2 5.1	11 8 11 14 6   10.0 3.1	23 25 29 23 13   22.6 5.9	14 13 12 19 17   15.0 2.9	20 29 32 33 37   30.2 6.4
Acetoguanamine	1000	mean sd	29 29 25 27.7 2.3	44 37 36   39.0 4.4	102 116 115   111.0 7.8	138 127 130   131.7 5.7	28 25 20   24.3 4.0	16 28 31   25.0 7.9	9 5 10   8.0 2.6	9 9 13   10.3 2.3	13 13 25   17.0 6.9	39 36 26   33.7 6.8
Acetoguanamine	2000	mean sd	29 13 10   20.7 9.7	49 36 27   37.3 11.1	92 93 90   91.7 1.5	143 140 117   133.3 14.2	24 28 25   25.7 2.1	28 34 32   31.3 3.1	13 5 6   8.0 4.4	16 16 16   16.0 0.0	12 18 9   13.0 4.6	32 29 33   31.3 2.1
Acetoguanamine	3000	mean sd	27 19 13   19.7 7.0	36 37 37   36.7 0.6	118 89 120   109.0 17.3	128 141 158   142.3 15.0	31 25 24   26.7 3.8	29 48 27   34.7 11.6	13 8 6   9.0 3.6	35 13 11   19.7 13.3	18 9 10   12.3 4.9	26 20 28   24.7 4.2
Acetoguanamine	4000	mean sd	31 25 13   23.0 9.2	36 25 34   31.7 5.9	122 130 102   118.0 14.4	112 142 127   127.0 15.0	31 26 37   31.3 5.5	34 36 24   31.3 6.4	4 12 9   8.3 4.0	23 16 18   19.0 3.6	11 17 21   16.3 5.0	32 35 35   34.0 1.7
Acetoguanamine	5000	mean sd	29 12 28   23.0 9.5	41 29 46   38.7 8.7	103 112 111   108.7 4.9	138 107 161   135.3 27.1	28 35 19   27.3 8.0	32 26 37   31.7 5.5	11 4 13   9.3 4.7	20 19 19   19.3 0.6	14 16 21   17.0 3.6	37 18 31   28.7 9.7
Positive Controls (Mutation Experiment 2)
2-nitrofluorene	50.0	mean sd	1321 1285 1224 1297 1108   1247.0 85.5	-	-	-	-	-	-	-	289 309 268 280 276   284.4 15.7	-
Sodium azide	2.0	mean sd	-	-	926 776 875 907 864   869.6 57.9	-	831 776 833 758 739   787.4 42.8	-	-	-	-	-
9-aminoacridine	50.0	mean sd	-	-	-	-	-	-	1076 1183 1310 1022 1035   1125.2 121.1	-	-	-
2-aminoanthracene	5.0	mean sd	-	301 348 355 316 318   327.6 22.9	-	781 853 715 683 735   753.4 66.1	-	203 158 158 143 125   157.4 28.9	-	65 78 69 84 55   70.2 11.3	-	275 263 224 444 242   289.6 88.5

 S9: liver homogenate from rats treated with Arcolor

mean: average number of revertants per plate

sd: standard deviation

C: Contaminated plate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

From the findings of this study it is concluded that Acetoguanamine was unable to induce mutation in five strains of Salmonella Typhimurium when tested up to 5000 µg/plate in the absence and presence of a rat liver metabolic activation system.

Executive summary:

Acetoguanamine was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA1538) of Samonella typhimurium, both in the absence and presence of metabolic activation by an Arcolor 1254 induced rat liver post-mitochondrial fraction (S-9), in two seperate experiments. The study was carried out according to OECD 471.

An initial toxicity range-finder experiment was carried ot in TA100 only, using final concentrations of Acetoguanamine at 8, 40, 200, 1000 and 5000 µg/plate plus solvent and positive control. No toxicity was observed following these treatments and the same dose range was therefore used for treatment of the remaining test strains in experiment 1 (again no toxicity was observed). For experiment 2, 5000 µg/plate was retained as the maximum test dose, but with a narrowed dose range utilised in order to investigate more closely those concentrations of Acetoguanamine most likely to exhibit a mutagenic effect.

Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

No Acetoguanamine treatment of any of the test strains, either in the absence or presence of S-9, resulted in increases in revertant numbers sufficient to be considered evidence of mutation iduction.

It is concluded that Acetogunamine was unable to induce mutation in five strains of Salmonella typhimurium when tested up to 5000 µg/plate in the absence and presence of a rat liver metabolic activation system.