1,1-dimethylprop-3-ynylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-03-08 - 2017-03-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Content: w(C5H9N) = 89.2 g/100 g water content: 7.8 g/100 g
- Purity: 99.2 area %
- Batch Nr.: 85603856P0
- Physical state, appearance: liquid, colorless, clear
- Date of production: 2016-10-19
- Storage conditions: room temperature

Target gene:

Salmonella typhimurium: All used strains have a defective excision repair system (uvrB). Target genes were his G46 (TA 1535 and TA 100), his C 3076 (TA1537), his D 3052 (TA 98).

Escherichia coli: tryptophan auxotrophy/tryptophan independence

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:

Liver S9 mix from induced rats

Test concentrations with justification for top dose:

Standard Plate Test (SPT): 0; 33;100; 333; 1000; 2800 and 5600 µg/plate
Preincubation Test (PIT): 0; 33;100; 333; 1000; 2800 and 5600 µg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. In this study, due to the purity of the test substance 5.6 mg/plate was used as top dose in all experiments.

Recommendations of current guidelines: 5 mg/plate or 5 μL/plate (tested even in the case of relatively insoluble test compounds).
Due to the purity of the test substance 5.6 mg/plate was used as top dose in all experiments.

Vehicle / solvent:

- Vehicle used: ultrapure water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Standard Plate Test (SPT) and Preincubation Test (PIT), both with and without metabolic activation (liver s9 mix from induced rats)

STANDARD PLATE TEST
- Incubation conditions: 37°C, dark conditions
- Incubation period: 48-72 hours
- Number of plates: 3 plates per dose or per control

PREINCUBATION TEST
- Preincubation conditions: 37°C
- Preincubation period: 20 minutes
- Incubation conditions: 37°C, dark conditions
- Incubation period: 48-72 hours
- Number of plates: 3 plates per dose or per control
- Reason: No mutagenicity was observed in the standard plate test

CONTROLS
1) Negative controls:
- Sterility control: with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains
- Vehicle control: only contained the vehicle (ultrapure water) used for the test substance at the same volume for all tester strains (with and wiithout S9 mix)

2) Positive controls:
With S9 mix
• 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA

Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Fluka; 97%)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (Sigma-Aldrich; 98%)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC) (Sigma-Aldrich; 98%)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA


The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using Image Analysis System.

Evaluation criteria:

MUTAGENICITY
The mutagenicity of the test substance was detected by individual plate counts and the mean number of revertant colonies per plate at all dose groups as well as for the positive and negative (vehicle) controls in all experiments.
TOXICITY
The toxicity was detected by a decrease in the number of revertants (factor ≤ 0.6) and by clearing or diminution of the background lawn (= reduced his- or trp- background growth). Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain (see Appendix 5).
• The sterility controls revealed no indication of bacterial contamination (see Appendix 3).
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 109 cells per mL were used.

ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

Remarks on result:

other: Standard Plate Test

Table 1: Results from Standard Plate Test (SPT) without metabolic activation.

Strain	Test group	Dose (μg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water Test item	-	10.7	2.1	-	9, 13, 10
		33	11.3	3.8	1.1	13, 14, 7
		100	12.3	2.5	1.2	10, 12, 15
		333	13.0	3.5	1.2	15, 9, 15
		1000	11.7	3.2	1.1	14, 13, 8
		2800	11.3	3.8	1.1	7, 14, 13
		5600	7.7	1.2	0.7	7, 9, 7
	MNNG	5.0	5811.0	752.0	544.8	6560, 5056, 5817
TA 100	Water Test item	-	109.0	16.5	-	128, 101, 98
		33	117.3	5.5	1.1	120, 121, 111
		100	121.3	6.5	1.1	121, 115, 128
		333	123.0	12.8	1.1	126, 134, 109
		1000	110.0	10.0	1.0	120, 110, 100
		2800	130.0	5.2	1.2	127, 127, 136
		5600	120.3	20.5	1.1	120, 100, 141
	MNNG	5.0	3971.7	673.0	36.4	4318, 3196, 4401
TA 1537	Water Test item	-	9.3	2.3	-	12, 8, 8
33	11.0	2.6	1.2	9, 14, 10
100	8.7	6.7	0.9	3, 16, 7
333	6.7	3.2	0.7	8, 9, 3
1000	8.7	5.1	0.9	10, 13, 3
2800	10.0	1.7	1.1	9, 12, 9
5600	9.0	3.6	1.0	13, 8, 6
	AAC	100	1299.3	176.2	139.2	1271, 1139, 1488
TA 98	Water Test item	-	20.7	4.0	-	23, 16, 23
		33	20.0	5.6	1.0	15, 19, 26
		100	22.3	4.0	1.1	26, 18, 23
		333	14.0	5.6	0.7	15, 8, 19
		1000	20.0	1.0	1.0	19, 21, 20
		2800	21.3	9.0	1.0	30, 12, 22
		5600	18.7	4.5	0.9	14, 19, 23
	NOPD	10	555.3	19.3	26.9	540, 549, 577
E. coli	Water Test item	-	22.7	3.1	-	20, 22, 26
		33	27.7	0.6	1.2	28, 28, 27
		100	26.7	8.0	1.2	26, 19, 35
		333	20.3	9.1	0.9	30, 19, 12
		1000	16.0	3.0	0.7	13, 16, 19
		2800	24.3	9.1	1.1	16, 34, 23
		5600	19.0	3.0	0.8	16, 22, 19
	4-NQO	5	1018.7	16.6	44.9	1017, 1003, 1036

Table 2: Results from Standard Plate Test (SPT) with metabolic activation.

Strain	Test group	Dose (μg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water Test item	-	9.0	2.0	-	11, 7, 9
		33	11.3	4.2	1.3	8, 16, 10
		100	13.3	1.5	1.5	15, 12, 13
	333	7.3	4.2	0.8	6, 12, 4
		1000	6.7	4.0	0.7	9, 9, 2
		2800	9.7	3.5	1.1	6, 13, 10
		5600	12.0	3.6	1.3	13, 8, 15
	2-AA	2.5	300.3	49.1	33.4	282, 356, 263
TA 100	Water Test item	-	116.3	13.7	-	104, 114, 131
		33	114.3	11.2	1.0	106, 127, 110
		100	120.0	16.1	1.0	105, 137, 118
		333	123.3	9.7	1.1	121, 115, 134
		1000	139.3	19.9	1.2	161, 135, 122
		2800	127.3	3.8	1.1	123, 130, 129
		5600	118.3	5.7	1.0	112, 120, 123
	2-AA	2.5	2987.3	220.9	25.7	3241, 2837, 2884
TA 1537	Water Test item	-	6.7	3.1	-	6, 4, 10
		33	6.3	3.1	1.0	7, 3, 9
		100	7.7	2.5	1.2	5, 8, 10
		333	8.7	2.3	1.3	6, 10, 10
		1000	5.3	2.1	0.8	3, 7, 6
		2800	8.7	3.2	1.3	11, 10, 5
		5600	6.7	2.9	1.0	5, 5, 10
	2-AA	2.5	270.0	32.4	40.5	234, 279, 297
TA 98	Water Test item	-	26.7	1.5	-	27, 28, 25
		33	24.7	6.1	0.9	30, 18, 26
		100	25.7	3.1	1.0	29, 23, 25
		333	24.7	6.7	0.9	23, 32, 19
		1000	25.3	3.2	1.0	24, 29, 23
		2800	25.3	3.2	1.0	23, 29, 24
		5600	30.0	4.0	1.1	30, 34, 26
	2-AA	2.5	1398.7	236.8	52.5	1672, 1270, 1254
E. coli	Water Test item	-	22.7	9.9	-	18, 34, 16
		33	24.7	4.5	1.1	29, 25, 20
		100	21.0	2.6	0.9	22, 23, 18
		333	21.7	0.6	1.0	21, 22, 22
		1000	22.3	2.5	1.0	20, 25, 22
		2800	21.0	7.8	0.9	16, 30, 17
		5600	18.7	7.6	0.8	10, 24, 22
	2-AA	60	127.7	5.5	5.6	122, 128, 133

 

Table 3: Results of Preincubation Test (PIT) without metabolic activation.

Strain	Test group	Dose (μg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water Test item	-	11.0	1.0	-	12, 11, 10
		33	10.3	3.1	0.9	11, 13, 7
		100	10.0	1.0	0.9	10, 11, 9
		333	10.0	1.7	0.9	9, 9, 12
		1000	8.7	4.6	0.8	14, 6, 6
		2800	9.0	2.0	0.8	9, 11, 7
		5600	8.0	2.6	0.7	5, 9, 10
	MNNG	5.0	6627.3	155.7	602.5	6637, 6778, 6467
TA 100	Water Test item	-	140.7	4.7	-	137, 146, 139
		33	130.3	5.0	0.9	135, 131, 125
		100	132.3	5.9	0.9	130, 139, 128
		333	126.3	6.4	0.9	119, 131, 129
		1000	121.7	8.5	0.9	112, 125, 128
		2800	115.3	11.2	0.8	103, 118, 125
		5600	127.0	24.0	0.9	100, 135, 146
	MNNG	5.0	4926.0	242.6	35.0	4646, 5058, 5074
TA 1537	Water Test item	-	12.0	1.7	-	10, 13, 13
		33	9.3	3.1	0.8	10, 12, 6
		100	9.0	2.6	0.8	10, 6, 11
		333	10.0	4.6	0.8	14, 5, 11
		1000	14.0	3.6	1.2	15, 17, 10
		2800	9.7	4.0	0.8	14, 6, 9
		5600	10.7	4.7	0.9	16, 9, 7
	AAC	100	777.7	39.7	64.8	749, 761, 823
TA 98	Water Test item	-	24.7	7.2	-	21, 20, 33
		33	25.0	2.6	1.0	28, 23, 24
		100	31.0	6.1	1.3	35, 34, 24
		333	26.0	2.0	1.1	26, 28, 24
		1000	28.0	2.0	1.1	30, 28, 26
		2800	27.0	4.4	1.1	24, 25, 32
		5600	25.7	6.7	1.0	29, 18, 30
	NOPD	10	790.0	174.5	32.0	702, 677, 991
E. coli	Water Test item	-	26.3	3.1	-	29, 27, 23
		33	23.3	4.9	0.9	20, 29, 21
		100	24.3	5.8	0.9	21, 31, 21
		333	21.0	1.0	0.8	21, 20, 22
		1000	30.0	2.0	1.1	30, 28, 32
		2800	24.0	4.6	0.9	19, 28, 25
		5600	22.0	2.0	0.8	24, 22, 20
	4-NQO	5	757.3	69.5	28.8	688, 757, 827

Table 4: Results from Preincubation Test (PIT) with metabolic activation.

Strain	Test group	Dose (μg/plate)	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Water Test item	-	11.0	1.0	-	10, 11, 12
		33	9.0	1.0	0.8	8, 10, 9
		100	8.7	0.6	0.8	9, 8, 9
		333	9.0	2.6	0.8	11, 6, 10
		1000	12.7	2.5	1.2	10, 13, 15
		2800	10.3	0.6	0.9	10, 11, 10
		5600	11.0	3.6	1.0	7, 12, 14
	2-AA	2.5	275.7	43.8	25.1	285, 314, 228
TA 100	Water Test item	-	150.0	12.3	-	141, 145, 164
		33	128.3	5.1	0.9	127, 134, 124
		100	133.7	18.9	0.9	155, 127, 119
		333	139.3	10.1	0.9	138, 150, 130
		1000	133.0	12.8	0.9	130, 147, 122
		2800	118.0	3.0	0.8	121, 115, 118
		5600	136.0	13.5	0.9	122, 137, 149
	2-AA	2.5	2766.0	290.7	18.4	2755, 3062, 2481
TA 1537	Water Test item	-	9.0	1.0	-	8, 10, 9
		33	8.3	3.2	0.9	7, 6, 12
		100	8.3	2.1	0.9	9, 10, 6
		333	7.3	2.5	0.8	5, 7, 10
		1000	8.0	4.4	0.9	13, 6, 5
		2800	9.7	4.0	1.1	6, 9, 14
		5600	7.7	2.9	0.9	6, 11, 6
	2-AA	2.5	280.3	32.1	31.1	317, 257, 267
TA 98	Water Test item	-	26.0	2.6	-	24, 25, 29
		33	24.7	6.7	0.9	23, 32, 19
		100	23.0	1.7	0.9	21, 24, 24
		333	28.0	4.6	1.1	23, 32, 29
		1000	21.0	2.0	0.8	21, 23, 19
		2800	23.0	1.7	0.9	25, 22, 22
		5600	26.3	2.5	1.0	24, 26, 29
	2-AA	2.5	2258.3	140.0	86.9	2356, 2321, 2098
E. coli	Water Test item	-	24.3	3.2	-	23, 28, 22
		33	26.7	7.6	1.1	32, 18, 30
		100	19.0	2.6	0.8	22, 18, 17
		333	26.3	8.1	1.1	30, 32, 17
		1000	25.3	4.0	1.0	26, 29, 21
		2800	33.7	8.3	1.4	31, 27, 43
		5600	30.7	4.6	1.3	28, 36, 28
	2-AA	60	116.3	14.6	4.8	100, 128, 121

Table 5: Results from sterility control, 1st experiment.

Components	Results
2.0 mL soft agar	No growth
0.5 mL S9 mix	No growth
0.5 mL buffer	No growth
0.5 mL vehicle	No growth
5600μg test substance	No growth

Table 6: Results from sterility control, 2nd experiment.

Components	Results
2.0 mL soft agar	No growth
0.5 mL S9 mix	No growth
0.5 mL buffer	No growth
0.5 mL vehicle	No growth
5600μg test substance	No growth

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1999-11-17

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Not in full compliance with GLP principles, study conducted according to good scientific practice

Qualifier:

no guideline followed

Version / remarks:

No guideline was available until the experiment had been carried out. The test was performed based on the description of Gee, P et al.: Comparison of responses of base-specific Salmonella tester strains with the traditional strains for identifying mutagens: The results of a validation study. Mut. Res., 412, 115 -130 (1998) and on the "Users Manual" prepared by XENOMETRIX, Inc., Colorado, USA (without date).

Principles of method if other than guideline:

PRINCIPLE OF TEST
Mutations were detected by the reversion of mutations present in the amino acid requiring bacterial strains, which Ieads to a restoration of the functional capability of bacteria to synthesize the essential amino acid and thus to the ability to grow in the absence of the amino acid required by the parent strains.

TEST CONDITIONS
Liquid fluctuation test both with and without metabolic activation (Aroclor-induced rat liver S9 -Mix).

ANALYZED PARAMETERS
Mutagenicity detected by reversion of mutations present in amino acid requiring bacterial strains (Salmonella typhimurium TA 98, TA-Mix).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Solubility : no precipitation of the test substance was found
- Date of manufacture: 1999-03-02
- Degree of purity: 91,8% (main component)
- Batch Nr.: Abl.-Nr. 99-0031
- Storage conditions: room temperature (N2 conditions)

Remark: The stability of the test substance throughout the study period has not been verified by reanalysis. The stability of the test substance in the vehicle water has not been determined analytically.

Target gene:

TA 98: his D 3052
TA 7001: hisG 1775
TA 7002: hisC 9138
TA 7003: hisC 9074
TA 7004: hisG 9133
TA 7005: hisG 9130
TA 7006: hisG 9070

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium, other: TA Mix (Mixed strains TA 7001 - TA 7006)

Metabolic activation:

with and without

Metabolic activation system:

Exogenous, Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

0; 4; 20; 100; 500; 2500 and 5000 µg/mI

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

other: 2-aminoaanthraacene (2-AA)

Details on test system and experimental conditions:

TEST DESIGN
- Strains: TA 98, TA Mix (TA 7001-7006)
- Type of test: liquid fluctuation test with and without S-9 mix
- Number of microtiter plates: triplicate plates per dose, control chemical or vehicle
- Doses: 0; 4; 20; 100; 500; 2,500 and 5,000 µg/mL

Evaluation criteria:

The test chemical was considered positive if a dose-related and reproducible increase in the number of positive wells by a factor of about 2 (calculated primarily on the basis of baseline data) in at least one tester strain either without S-9 mix or after adding a metabolizing system were met. A test substance was generally considered non-mutagenic in this test if the number of revertant wells for all tester strains were within the historical negative control range under all experimental conditions.

Toxicity was detected by a
- decrease in the number of positive wells
- clearing or diminution of the background lawn (= reduced his- background growth) leading from turbid to non-turbid purple wells
is recorded for all test groups both with and without S-9 mix and indicated in the tables

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

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA-Mix (TA 7001-7006)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no precipitation of the test substance was found

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, but without confidence interval)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

STERILITY CONTROL
160 µI S-9 mix/ml and 5,000 µg test substance/mI negative (no yellow wells)

TOXICITY
- bacteriotoxic effect (clearing of the background Iawn, decrease in the number of yellow wells): no

Abbreviations: RCSP = Revertant Colony Selection Plate (384 -Well), SD = Standard Seviation, REM = Remarks, MEAN = Mean of Replicates, MEAN^c= Mean Corrected: < 1 = 1, F = Factor, 1F = Based on MEAN^c, 2F = Baseline Data/Based on MEAN^c + SD, 3F = Baseline data/ Based on MEAN + SD of a run, 4 -NQO + 2 -NF = 4 -Nitroquinnoline: 0.0625 pgtml + 2 -nitroflourene 0.25 pgImnl, 2 -AA = 2 -Amninoanthracene: 5.0 pglml.

Strain: TA 98, without S9-Mix.

Dose [µg/ml]	REM	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^c	SD	1F	2F	3F
Water		7	2	4	4.3	4.3	2.05	1.0	0.7	0.9
4		1	2	1	1.3		0.47	0.3	0.2	0.3
20		2	3	6	3.7		1.70	0.8	0.6	0.8
100		1	2	2	1.7		0.47	0.4	0.3	0.4
500		2	2	3	2.3		0.47	0.5	0.4	0.5
2500		2	4	2	2.7		0.94	0.6	0.4	0.6
5000		4	3	0	2.3		1.70	0.5	0.4	0.5
4-NQO + 2-NF		25	27	30	27.3		2.05	6.3	4.3	6.0

Strain: TA 98, with S9-Mix.

Dose [µg/ml]	REM	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^c	SD	1F	2F	3F
Water		7	4	2	4.3	4.3	2.05	1.0	0.7	0.8
4		4	4	2	3.3		0.94	0.8	0.5	0.6
20		0	3	2	1.7		1.25	0.4	0.3	0.3
100		9	6	3	6.0		2.45	1.4	0.9	1.1
500		2	2	2	2.0		0.00	0.5	0.3	0.4
2500		2	2	2	2.0		0.00	0.5	0.3	0.4
5000		1	1	3	1.7		0.94	0.4	0.3	0.3
2-AA		48	48	48	48.0		0.00	11.1	7.5	9.2

Strain: TA-Mix, without S9-Mix.

Dose [µg/ml]	REM	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^c	SD	1F	2F	3F
Water		0	0	1	0.3	1.0	0.47	1.0	0.7	0.6
4		2	2	1	1.7		0.47	1.7	1.1	1.0
20		2	1	1	1.3		0.47	1.3	0.9	0.8
100		0	1	0	0.3		0.47	0.3	0.2	0.2
500		0	0	0	0.0		0.00	0.0	0.0	0.0
2500		1	0	1	0.7		0.47	0.7	0.5	0.4
5000		0	0	1	0.3		0.47	0.3	0.2	0.2
4-NQO + 2-NF		22	25	22	23.0		1.41	23.0	15.6	14.4

Strain: TA-Mix, with S-9 -Mix

Dose [µg/ml]	REM	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^c	SD	1F	2F	3F
Water		0	1	0	0.3	1.0	0.47	1.0	0.7	0.7
4		0	0	1	0.3		0.47	0.3	0.2	0.2
20		0	0	1	0.3		0.47	0.3	0.2	0.2
100		0	0	1	0.3		0.47	0.3	0.2	0.2
500		0	1	0	0.3		0.47	0.3	0.2	0.2
2500		1	3	1	1.7		0.94	1.7	1.1	1.1
5000		0	1	0	0.3		0.47	0.3	0.2	0.2
4-NQO + 2-NF		45	47	43	45.0		1.63	45.0	30.6	30.2

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic Toxicity In Vitro

Key information

The test substance was assessed for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay in the Standard Plate Test and in the preincubation test with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA (BASF SE, 2017). The study was conducted according to the guidelines OECD TG 471, EU Method B 13/14 and US EPA OPPTS 870.5100. The dose range chosen was 0; 33; 100; 333; 1000; 2800 and 5600 µg/plate (vehicle = ultrapure water), whereby three test plates per dose or per control were used. No test substance precipitation was found with and without S9 mix. In both tests negative controls (sterility control with S9 mix, vehicle control with and without S9 mix) and positive controls (with and without S9 mix) were performed. After incubation at 37°C for 48-72 hours in the dark, the bacterial colonies of his+ (Salmonella typhimurium) respectively trp+ revertants (Escherichia coli) were counted. In the Preincubation Test, 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were pre-incubated beforehand at 37°C for the duration of about 20 minutes using a shaker.

In result, the test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). The results of the negative as well as the positive controls performed in parallel indicated the validity of this study. Under the experimental conditions chosen, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Supporting Information

The substance 1,1-Dimethylpropin-2-ylamin was tested for its mutagenic potential based on the ability to induce point mutations in selected Ioci of several strains of Salmonella typhimurium (TA 98, TA-Mix) in a modified version of the traditional Ames test, i.e. the Ames II Assay (microtiter Version), both with and without the addition of a metabolizing system (S-9 mix) obtained from rat liver (BASF SE, 1999).

In result, an increase in the number of positive wells (his+ revertants) was not observed either without S-9 mix or after the addition of a metabolizing system. Furthermore, no bacteriotoxic effect was observed. Regarding solubility, no precipitation of the test substance was found. All established validity criteria were met. Therefore it can be concluded that the test substance is not mutagenic in the Ames II Assay (Salmonella typhimurium reverse mutation assay) under the experimental conditions chosen. According to the results of the study, the test substance is not mutagenic in the Ames II Assay (Salmonella typhimurium reverse mutation assay) under the experimental conditions chosen.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.