Poly(oxy-1,2-ethanediyl), α,α'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar 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:

Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized

EC Number:

615-231-8

Cas Number:

70983-58-3

Molecular formula:

UVCB

IUPAC Name:

Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized

Test material form:

liquid: viscous

Details on test material:

Name: Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
Product Description: C10-16-alkyletherpropylamine, ethoxylated, DES Quat
CAS No.: 70983-58-3
Physical state: yellowish to amber viscous liquid at 20 °C
Batch No.: PFS-755-173
Re-certification date of batch: 19 April 2018
Purity: 100 % (UVCB)
Color, Gardner 8.8
pH, 5% in water 5.76
Acid Value , mg KOH/g 20.1
Moisture, % 0.127
Total Amine, mg/g 11.18
Viscosity,cps, #4@60,25C 3280
Appearance @25C pass
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Specific details on test material used for the study:

Name: Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-(iminodi-2,1-ethanediyl)bis[.omega.-hydroxy-, N-[3-(C10-16-alkyloxy)propyl] derivs., di-Et sulfate-quaternized
Product Description: C10-16-alkyletherpropylamine, ethoxylated, DES Quat
CAS No.: 70983-58-3
Physical state: yellowish to amber viscous liquid at 20 °C
Batch No.: PFS-755-173
Re-certification date of batch: 19 April 2018
Purity: 100 % (UVCB)
Color, Gardner 8.8
pH, 5% in water 5.76
Acid Value , mg KOH/g 20.1
Moisture, % 0.127
Total Amine, mg/g 11.18
Viscosity,cps, #4@60,25C 3280
Appearance @25C pass
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537) mutations.

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 98: his D 3052; rfa-; uvrB-; R-factor; TA 100: his G 46; rfa-; uvrB-; R-factor; TA 1535: his G 46; rfa-; uvrB-; TA 1537: his C 3076; rfa-; uvrB-; TA 102: his G 428 (pAQ1); rfa-; R-factor;

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test). Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control. The test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 nL/plate

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 316 nL/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:

Experiment I: 0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 nL/plate
(all tester strains with and without metabolic activation)

Experiment II: 0.0200, 0.0632, 0.200, 0.632, 2.00, 6.32, 20.0 and 63.2 nL/plate
(TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation, TA 102 without metabolic activation)

Experiment II: 0.0632, 0.200, 0.632, 2.00, 6.32, 20.0, 63.2 and 200 nL/plate
(TA 102 with metabolic activation)

As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment I.

Vehicle / solvent:

The test item was dissolved in A. dest., and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD; 4-nitro-o-phenylene-diamine (without metabolic activation - TA 98, TA 1537); 2-AA; 2-aminoanthracene (with metabolic activation - TA 98, TA 100, TA 1535, TA 1537 and TA 102)

Evaluation criteria:

Evaluation of Mutagenicity

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).

A test item is considered as mutagenic if:

- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs

in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:

- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher

than the reversion rate of the solvent control.

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

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

Remarks:

A. dest.

Untreated negative controls validity:

valid

Remarks:

A. dest.

Positive controls validity:

valid

Remarks:

4-NOPD (-S9); 2-AA (+S9);

Test results 2

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

Remarks:

A. dest.

Untreated negative controls validity:

valid

Remarks:

A. dest.

Positive controls validity:

valid

Remarks:

NaN3 (-S9); 2-AA (+S9);

Test results 3

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

Remarks:

A. dest.

Untreated negative controls validity:

valid

Remarks:

A. dest.

Positive controls validity:

valid

Remarks:

NaN3 (-S9); 2-AA (+S9);

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

A. dest.

Untreated negative controls validity:

valid

Remarks:

A. dest.

Positive controls validity:

valid

Remarks:

4-NOPD (-S9); 2-AA (+S9);

Test results 5

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

Remarks:

A. dest.

Untreated negative controls validity:

valid

Remarks:

A. dest.

Positive controls validity:

valid

Remarks:

MMS (-S9); 2-AA (+S9);

Remarks on result:

other: Plate-incorporation test (Experiment 1 & 2)

Any other information on results incl. tables

Results

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I and II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:

0.100, 0.316, 1.00, 3.16, 10.0, 31.6, 100 and 316 nL/plate

(all tester strains with and without metabolic activation)

Experiment II:

0.0200, 0.0632, 0.200, 0.632, 2.00, 6.32, 20.0 and 63.2 nL/plate

(TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation, TA 102 without metabolic activation)

0.0632, 0.200, 0.632, 2.00, 6.32, 20.0, 63.2 and 200 nL/plate

(TA 102 with metabolic activation)

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II. In experiment I toxic effects of the test item were observed in tester strains TA 98, TA 100 and TA 1537 at concentrations of 10.0 nL/plate and higher (without metabolic activation) and at concentrations of 100 nL/plate and higher (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were noted at concentrations of 10.0 nL/plate and higher (without metabolic activation) and at concentrations of 31.6 nL/plate (with metabolic activation). In tester strain TA 102 toxic effects of the test item were observed at concentrations of 31.6 nL/plate and higher (without metabolic activation) and at concentrations of 316 nL/plate and higher (with metabolic activation). In experiment II toxic effects of the test item were noted in tester strains TA 98, TA 100 and TA 1537 at concentrations of 6.32 nL/plate and higher (without metabolic activation) and at a concentration of 63.2 nL/plate (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were noted at concentrations of 6.32 nL/plate and higher (with and without metabolic activation). In tester strain TA 102 toxic effects of the test item were observed at a concentration of 63.2 nL/plate (without metabolic activation) and at a concentration of 200 nL/plate (with metabolic activation). No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I and II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 in an OECD 471 Klimisch 1 GLP study. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met. The test item is considered to be non-mutagenic in this bacterial reverse mutation assay.