Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

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:

2017-02-14 to 2017-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Deviation of the study plan: three concentrations of the toxicity control were performed, instead of one concentration. This has no influence on the study results

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

IUPAC Name:

Reaction mass of Reaction product of 1-chloro-3-{[1-chloro-3-(dodecyloxy)propan-2-yl]oxy}propan-2-ol with methyl diethanolamine and [3-(dodecyloxy)-2-hydroxypropyl]bis(2-hydroxyethyl)methylammonium chloride

Test material form:

other: solid, very viscous yellow paste

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

other: uvrB

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: uvrB

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1a (plate incorporation method):
up to concentrations of 5000 μg/plate in the absence and presence of S9; strains TA97a, TA98, TA100, TA102 and TA1535

Experiment 1b (plate incorporation method):
- 500 μg/plate + S9 in the bacteria strains TA98 and TA102
- 150 μg/plate + S9 in the bacteria strains TA97a and TA1535, and - S9 in the bacteria strains TA98 and TA1535
- 50 μg/plate + S9 in the bacteria strain TA100, and - S9 in the bacteria strains TA97a, TA100 and TA102

Experiment 2 (pre-incubation method):
- TA97a without metabolic activation: 15 μg/plate
- TA97a with metabolic activation: 50 μg/plate
- TA98 without metabolic activation: 50 μg/plate
- TA98 with metabolic activation: 150 μg/plate
- TA100 without metabolic activation: 15 μg/plate
- TA100 with metabolic activation: 15 μg/plate
- TA102 without metabolic activation: 15 μg/plate
- TA102 with metabolic activation: 150 μg/plate
- TA1535 without metabolic activation: 50 μg/plate
- TA1535 with metabolic activation: 50 μg/plate

Due to the toxicity in experiment 1a, a repetiton (experiment 1b) of this experiment was performed with lower concentrations.

- Justification: these concentrations were chosen according to the OECD guideline 471 and tested, because test items showing any toxicity below 5 mg/plate should be tested up to the lowest toxic concentration.

Vehicle / solvent:

- Vehicle: demineralized water
- Justification: no precipitates on the agar plates at any of the listed concentrations

Controls

Controls 1

Untreated negative controls:

other: demin. water

Controls 2

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene Diamine

Remarks:

Metabolic activation: none

Controls 3

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Metabolic activation: none

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-Amino-Anthracene

Remarks:

Metabolic activation: S9

Controls 5

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Metabolic activation: S9

Controls 6

Negative solvent / vehicle controls:

other: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1a and 1b: plate incorporation method
- Experiment 2: pre-incubation method

DURATION
- Exposure duration: 48h at 37°C

NUMBER OF REPLICATIONS: 3

ACCEPTANCE CRITERIA:
The assay is considered acceptable if the following criteria are met:
- no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate
- the control of the titre was above the demanded value of 109 bacteria/mL
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data of the laboratory
- all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory
and were increased in comparison with the negative controls

Rationale for test conditions:

These are according to the OECD guideline 471

Evaluation criteria:

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed.
A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, negative control, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction
(mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls.
Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The mean revertant values of the three replicates are presented in the following tables:

Mean Revertants Experiment 1a:

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	90	86	9	12	88	104	265	301	10	12
	sd	21	4.6	2.1	3.1	11.2	5.8	4.6	26.6	0.6	2.1
DMSO	Mean	84	88	10	11	87	79	263	296	15	15
	sd	12	10.4	1	3.6	9.2	4	46.6	66.1	2	3.1
Positive Controls*	Mean	376	245	421	59	483	589	1427	1472	173	121
	sd	114.3	31.1	48.1	5.5	122.9	48.9	91	117.8	34	28
5000 μg/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
1500 μg/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
500 μg/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
150 μg/plate	Mean	1	27	2	11	7	30	64	266	1	2
	sd	0.6	17.0	1.2	1.0	5.1	7.0	34.5	22.7	0.0	1.2
50 μg/plate	Mean	2	83	10	13	1	35	60	313	8	15
	sd	1.2	13.0	1.2	3.8	0.6	1.5	25.1	46.2	0.0	1.5

* Different positive controls were used

Mean Revertants Experiment 1b:

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. Water	Mean	76	95	10	13	99	75	297	304.0	12	13.0
	sd	13.2	13.9	1.5	4.2	10.6	15.0	28.4	50.0	0.6	1.0
DMSO	Mean	84	84	10	10	79	73	277	301	11	12
	sd	18.8	18.8	1.7	0.6	14	12.2	36.3	30.3	2.6	2.3
Positive Controls*	Mean	340	436	235	77	439	643	1341	1205	324	101
	sd	52	131.6	33.2	13	87.3	196.3	146.7	344.3	64	12.2
500 μg/plate	Mean	n.d.	n.d.	n.d.	1	n.d.	n.d.	n.d.	0	n.d.	n.d.
	sd	n.d.	n.d.	n.d.	0.6	n.d.	n.d.	n.d.	0.0	n.d.	n.d.
150 μg/plate	Mean	n.d.	5	1	17	n.d.	n.d.	n.d.	331	1	2
	sd	n.d.	3.1	0.0	4.2	n.d.	n.d.	n.d.	20.1	0.0	1.2
50 μg/plate	Mean	1	72	10	15	0	0	0	265	9	12
	sd	0.6	16.8	0.6	6.1	0.0	0.0	0.0	37.2	1.5	3.8
15 μg/plate	Mean	61	72	10	16	96	73	324	365	12	11
	sd	1.2	11.4	0.6	4.7	19.1	18.2	58.9	67.2	2.6	0.6
5 μg/plate	Mean	79	64	8	10	67	77	252	291	9	12
	sd	12.5	6.9	1.5	4.4	11.5	15.3	48.0	44.1	3.2	0.6
1.5 μg/plate	Mean	71	77	11	11	74	86	289	285	11	8
	sd	10.5	14.8	1.2	0	12.5	18.8	28.4	36.3	1	1.2
0.5 μg/plate	Mean	72	72	8	n.d.	75	71	265	n.d.	12	9
	sd	9.8	8	0.6	n.d.	22.2	12.2	12.9	n.d.	3.2	0.6
0.15 μg/plate	Mean	68	n.d.	n.d.	n.d.	75	77	272	n.d.	n.d.	n.d.
	sd	10.7	n.d.	n.d.	n.d.	4.6	6.4	36.7	n.d.	n.d.	n.d.
0.05 μg/plate	Mean	88	n.d.	n.d.	n.d.	71	76	331	n.d.	n.d.	n.d.
	sd	18.1	n.d.	n.d.	n.d.	16.7	15.6	42.8	n.d.	n.d.	n.d.

n.d. = not determined, due to the toxicity effect in experiment 1a

* Different positive controls were used

Mean Revertants Experiment 2:

Strain		TA98		TA102
Induction		-S9	+S9	-S9	+S9
Demin. Water	Mean	16	20	295	331
	sd	4.9	7.8	32.5	79.4
DMSO	Mean	16	10	297	293
	sd	4.9	1.2	38	49.1
Positive Controls*	Mean	393	74	661	703
	sd	70.2	10.5	100.6	54.3
150 μg/plate	Mean	n.d.	11	n.d.	348
	sd	n.d.	1.2	n.d.	18.3
75 μg/plate	Mean	n.d.	14	n.d.	345
	sd	n.d.	9.2	n.d.	37.8
37.5 μg/plate	Mean	n.d.	19	n.d.	284
	sd	n.d.	1.0	n.d.	0.9
18.8 μg/plate	Mean	n.d.	18	n.d.	352
	sd	n.d.	0.9	n.d.	1.1
9.4 μg/plate	Mean	n.d.	17	n.d.	324
	sd	n.d.	4.6	n.d.	38.2
4.7 μg/plate	Mean	n.d.	20	n.d.	255
	sd	n.d.	2.6	n.d.	74.4
2.3 μg/plate	Mean	n.d.	18	n.d.	195
	sd	n.d.	1.5	n.d.	53.3

n.d. = not determined

* Different positive controls were used

Applicant's summary and conclusion

Conclusions:

The test item is not mutagenic.

Executive summary:

The study procedures described in this report were based on the OECD 471 and EU guidelines B13/14. The study has been performed in compliance to GLP. The test item was tested in the Salmonella typhimurium reverse mutation assay with five bacteria strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of metabolic activation. Due to the toxicity in experiment 1a, a repetition experiment (experiment 1b) was performed with lower concentrations. In experiment 1a and 1b the plate incorporation method was used and in experiment 2 the pre- incubation method.

All of the means of all replicates of the spontaneous revertants (in negative (demin. water) and solvent controls (DMSO)) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative and solvent controls, which demonstrated the mutagenic potential of the diagnostic mutagens.

Based on the results of this study it is concluded, that the test item is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 with or without metabolic activation under the experimental conditions in this study.