Reaction mass of 2-amino-2-methyl-1-propanol hydrochlorid (3207-12-3) and 2-amino-2-methyl-1-propanol (124-68-5)

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:

09 April 2018 - 18 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

(2-hydroxy-1,1-dimethylethyl)ammonium chloride

EC Number:

221-713-5

EC Name:

(2-hydroxy-1,1-dimethylethyl)ammonium chloride

Cas Number:

3207-12-3

Molecular formula:

C4H11NO.ClH

IUPAC Name:

1-hydroxy-2-methylpropan-2-aminium chloride

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver S9 mix

Test concentrations with justification for top dose:

0.05, 0.16, 0.5, 1.6 and 5 µL/plate
Justification for top dose: based on the results of initial cytotoxicity test

Vehicle / solvent:

- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: recommended by guideline

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Aminoanthracene: 4 µg/plate, all strains with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 10^8 viable cells per plate

DURATION
- Preincubation period: 30-45 min (pre-incubation method)
- Exposure duration: 48-72 hrs (plate incorporation and pre-incubation method)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: determination of thinning or disappearance of the bacterial background lawn

Rationale for test conditions:

According to guideline and based on preliminary dose-range finding study.

Evaluation criteria:

A substance is considered positive if the following criteria apply:
There should be a dose related increase over the range tested and/or a reproducible increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test item either in the presence or absence of the metabolic activation system.
The test will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in Salmonella typhimurium strains TA98, TA100 and TA102 or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.

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:

cytotoxicity

Remarks:

Cytotoxicity was observed only at the highest tested concentration (5 µL/plate).

Vehicle controls validity:

valid

Untreated 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:

cytotoxicity

Remarks:

Cytotoxicity was observed only at the highest tested concentration (5 µL/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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:

cytotoxicity

Remarks:

Cytotoxicity was observed only at the highest tested concentration (5 µL/plate).

Vehicle controls validity:

valid

Untreated 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:

cytotoxicity

Remarks:

Cytotoxicity was observed only at the highest tested concentration (5 µL/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed only at the highest tested concentration (5 µL/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was found to be miscible in distilled water at a concentration of 50 µL/mL.
- Precipitation: The test item resulted in no precipitation at and up to 5 µL/plate.

RANGE-FINDING/SCREENING STUDIES:
- Concentrations tested in the mutation assay were selected based on a solubility and precipitation tests as well as based on an initial cytotoxicity test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: observation of back background lawn and number of colonies

Any other information on results incl. tables

Table 1: SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-I Plate Incorporation Method

Treatment	Test Item Concentration  (µL//plate)*		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium						Salmonella typhimurium
			TA 98	TA 100	TA 102	TA 1535	TA 1537		TA 98	TA 100	TA 102	TA 1535	TA 1537
Vehicle Control	100 µL of Distilled water	Mean	26.0	107.3	260.0	22.3	10.7		19.7	100.0	257.3	19.0	10.3
		±SD	2.6	5.9	11.5	2.5	1.5		1.5	8.2	5.5	2.0	2.5
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Catalyst CR	0.05	Mean	28.0	97.0	260.0	20.0	7.7		20.3	94.7	257.7	19.0	6.7
		±SD	2.0	4.6	8.0	3.0	2.1		4.0	4.2	3.5	3.0	2.1
	Fold Increase		1.1	0.9	1.0	0.9	0.7		1.0	0.9	1.0	1.0	0.6
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	27.3	97.7	266.0	20.3	9.0		19.0	96.3	257.0	16.7	9.7
		±SD	1.5	4.7	7.9	2.5	3.0		2.6	8.4	6.6	2.1	1.5
	Fold Increase		1.1	0.9	1.0	0.9	0.8		1.0	1.0	1.0	0.9	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	21.0	98.0	261.0	20.3	8.3		22.0	97.7	264.3	20.0	8.7
		±SD	2.0	7.5	10.1	2.1	2.1		2.0	5.5	8.7	3.6	3.1
	Fold Increase		0.8	0.9	1.0	0.9	0.8		1.1	1.0	1.0	1.1	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	1.6	Mean	23.3	96.0	258.3	18.7	9.0		20.0	93.0	266.0	16.0	7.7
		±SD	3.8	5.0	8.4	2.3	2.0		2.0	2.6	12.1	2.0	3.8
	Fold Increase		0.9	0.9	1.0	0.8	0.8		1.0	0.9	1.0	0.8	0.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	5	Mean	17.3	74.7	204.7	14.0	5.0		15.7	66.3	202.0	13.7	5.0
		±SD	2.5	5.9	2.5	1.0	1.0		1.5	4.0	2.0	0.6	2.0
	Fold Increase		0.7	0.7	0.8	0.6	0.5		0.8	0.7	0.8	0.7	0.5
	Lawn Intensity		3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Control	100 µL of respective Positive Control	Mean	376.7	391.3	602.0	143.0	122.7		348.3	383.0	592.7	142.0	121.7
		±SD	11.2	12.7	12.5	6.2	8.1		21.8	8.9	11.5	8.7	6.7
	Fold Increase		14.5	3.6	2.3	6.4	11.5		17.7	3.8	2.3	7.5	11.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Values of Revertants are in Mean±SD

*Corrected Concentrations are given. As the test item contains 35 % of water and the test substance that is to be registered only contains 15 % of water a correction factor of 1.31 has been applied.

Positive controls:

For with S9:

ForSalmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA102:0.5µg/plate of Mitomycin C

For TA1537: 50 µg/plate of 9-Aminoacridine

Table 2:  SUMMARY OF COLONY COUNTS OF REVERTANTS OF TRIAL-II Preincubation Method

Treatment	Test Item Concentration  (µL//plate) *		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			TA 98	TA 100	TA 102	TA 1535	TA 1537		TA 98	TA 100	TA 102	TA 1535	TA 1537
Vehicle Control	100 µL of Distilled water	Mean	29.3	103.0	260.7	21.7	10.0		28.0	99.7	264.3	19.7	9.7
		±SD	2.5	2.6	8.4	3.2	3.0		2.0	3.8	7.1	2.5	1.5
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
Catalyst CR	0.05	Mean	24.3	97.0	264.0	19.3	8.0		21.0	96.0	262.7	17.7	8.3
		±SD	2.1	7.5	7.5	3.1	2.0		2.6	4.6	10.2	1.5	3.2
	Fold Increase		0.8	0.9	1.0	0.9	0.8		0.8	1.0	1.0	0.9	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.16	Mean	29.7	95.7	262.0	21.0	9.0		28.7	93.3	265.7	18.7	7.3
		±SD	2.5	6.4	7.5	2.0	1.7		4.0	1.5	12.1	1.5	2.1
	Fold Increase		1.0	0.9	1.0	1.0	0.9		1.0	0.9	1.0	0.9	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.5	Mean	29.7	99.0	261.3	17.3	8.7		25.7	100.7	262.0	18.0	8.0
		±SD	4.0	6.2	8.1	2.5	2.5		4.5	2.5	8.2	3.6	2.6
	Fold Increase		1.0	1.0	1.0	0.8	0.9		0.9	1.0	1.0	0.9	0.8
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	1.6	Mean	23.7	97.7	267.3	20.7	6.3		24.3	93.0	263.3	17.7	6.0
		±SD	4.0	5.0	6.0	2.5	2.1		2.5	2.6	5.1	2.5	2.0
	Fold Increase		0.8	0.9	1.0	1.0	0.6		0.9	0.9	1.0	0.9	0.6
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	5	Mean	18.3	74.7	208.3	13.7	3.3		17.7	72.0	211.7	13.0	3.0
		±SD	3.2	5.7	7.5	1.5	0.6		2.5	3.0	11.7	1.0	1.0
	Fold Increase		0.6	0.7	0.8	0.6	0.3		0.6	0.7	0.8	0.7	0.3
	Lawn Intensity		3 +	3 +	3 +	3 +	3 +		3 +	3 +	3 +	3 +	3 +
Positive Control	100 µL of respective Positive Control	Mean	377.3	396.7	606.7	138.3	121.3		354.3	383.7	597.7	133.7	114.7
		±SD	12.1	16.0	14.0	12.1	7.1		14.6	12.1	18.9	6.0	6.7
	Fold Increase		12.9	3.9	2.3	6.4	12.1		12.7	3.8	2.3	6.8	11.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Values of Revertants are in Mean±SD

*Corrected Concentrations are given. As the test item contains 35 % of water and the test substance that is to be registered only contains 15 % of water a correction factor of 1.31 has been applied.

Positive controls:

For with S9:                    

For Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 = 4 µg/plate of 2-Aminoanthracene

For without S9:

For TA98: 2 µg/plate of 2-Nitrofluorene

For TA100 and TA1535: 1 µg/plate of Sodium azide.

For TA102:0.5µg/plate of Mitomycin C

For TA1537: 50 µg/plate of 9-Aminoacridine

Applicant's summary and conclusion

Conclusions:

The test item is considered to be non-mutagenic in bacteria with and without metabolic activation.

Executive summary:

The test item was evaluated for mutagenicity in a Bacterial Reverse Mutation Test according to OECD guideline for testing of chemicals No. 471, “Bacterial Reverse Mutation Test”, adopted on 21st July 1997. The test concentrations used in the mutation assay were selected based on the results obtained in the pre-tests. As the test item contains 35% of water and the test substance that is to be registered only contains 15% of water a purity correction factor of 1.31 has been applied. The mutagenic potential of the test item was investigated in two independent experiments, in plate incorporation test (trial 1) and pre incubation test (trial 2). Each assay was conducted with and without metabolic activation (+S9 mix). Both trials were performed at concentrations of 0.05, 0.16, 0.5, 1.6 and 5 µL/plate (corrected concentrations). All concentrations, including the controls, were tested in triplicate. Vehicle control (distilled water) and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, Mitomycin C for trials without metabolic activation and 2-Aminoanthracene for trials with metabolic activation) were tested simultaneously. The tester strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537.

In the performed trials all of the validity criteria regarding the investigated strains, vehicle and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. All the tester strains treated with the test item at the concentration of 5µL/plate showed slightly reduced lawn (grade 3+) and a reduction in mean number of revertant colonies/plate in the presence and absence of metabolic activation when compared to the vehicle control. No inhibitory effects of the test item were observed at the concentration ≤ 1.6 µL/plate. There was no substantial increase in number of revertant colonies at any of the tested concentrations in both trials. The number of revertant colonies in the positive controls resulted in 2.3 to 17.7 fold increase under identical conditions. Based on the results obtained from the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 5 µL/plate under the test conditions chosen.