Reaction products of naphthalene, benzyl alcohol, sulphuric acid, formaldehyde and ammonia

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:

October 09, 2017 to November 03, 2017

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Diammonium 1,1'-methylenebis[(phenylmethyl)naphthalene-2-sulphonate]

EC Number:

284-353-8

EC Name:

Diammonium 1,1'-methylenebis[(phenylmethyl)naphthalene-2-sulphonate]

Cas Number:

84852-41-5

Molecular formula:

C35H38N2O6S2.2H3N

IUPAC Name:

Diammonium 1,1'-methylenebis((phenylmethyl)naphthalene-2-sulphonate)

Constituent 2

Reference substance name:

Benzyl methylene-bis-napthalene-2-sulphonic acid ammonium salt

IUPAC Name:

Benzyl methylene-bis-napthalene-2-sulphonic acid ammonium salt

Constituent 3

Reference substance name:

Isomers of Benzyl methylene-bis-naphthalene trisulphonic acid ammonium salt

Molecular formula:

Not applicable

IUPAC Name:

Isomers of Benzyl methylene-bis-naphthalene trisulphonic acid ammonium salt

Constituent 4

Reference substance name:

Ammonium sulphate

EC Number:

231-984-1

EC Name:

Ammonium sulphate

Cas Number:

7783-20-2

Molecular formula:

(NH4)2SO4

IUPAC Name:

diammonium sulfate

Constituent 5

Reference substance name:

Unidentified isomers formed during the reaction

Molecular formula:

Not applicable

IUPAC Name:

Unidentified isomers formed during the reaction

Constituent 6

Reference substance name:

Formaldehyde

EC Number:

200-001-8

EC Name:

Formaldehyde

Cas Number:

50-00-0

Molecular formula:

C H2 O

IUPAC Name:

formaldehyde

Test material form:

solid

Method

Target gene:

Cytotoxicity test (range-finding study): Salmonella typhimurium TA100.
Mutation assay: S. typhimurium TA98, TA100, TA1535, and TA1537; Escherichia coli WP2 uvrA (pKM101).

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA100

Remarks:

Cytotoxicity test

Species / strain 2

Species / strain / cell type:

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

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-naphthoflavone induced rat liver S9 homogenate

Test concentrations with justification for top dose:

Cytotoxicity test (range-finding study) concentrations selected according to the results of a precipitation test: 0.7, 0.8, 0.9, 1, 2, 3, 4, and 5 mg/plate.
Mutation assay: 0.05, 0.16, 0.5, 1.6, and 5 mg/plate (with half-log dose interval).

Vehicle / solvent:

- Vehicle used: Distilled water (Batch number: 7461).
- Justification for choice of vehicle: The test item at the given concentration of 50 mg/mL was soluble in distilled water.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

On the basis of test item solubility and precipitation tests, an initial cytotoxicity experiment was performed at 0.7, 0.8, 0.9, 1, 2, 3, 4, and 5 mg/plate. The cytoxicity experiment was performed with Salmonella typhimurium TA100 both in the presence and absence of metabolic activation system.
The test strains used in the mutation assay were Salmonella typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia Coli WP2 uvrA (pKM101).
For the ultimate genetic mutation assay, concentrations were selected according to the results of solubility, precipitation, and the cytotoxicity test: 0.05, 0.16, 0.5, 1.6, and 5 mg/plate. Two independent trials (trial I and II) were carried out by plate incorporation for a 67-hour and 30-minute incubation duration and pre-incubation for a 69-hour incubation duration in the presence and absence of metabolic activation. A vehicle control (distilled water) and five appropriate positive controls were tested simultaneously.

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:

other: not applicable

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not applicable

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not applicable

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: not applicable

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

No significant increase in the number of revertant colonies was observed in any bacterial strain relative to the vehicle control following exposure to aryl sulphonate condensate, with and without metabolic activation. The registered substance can subsequently be regarded as non-mutagenic under the conditions of the test.

Executive summary:

OECD Guideline 471 (Bacterial Reverse Mutation Assay), in conjunction with Good Laboratory Practise (GLP) standards, were followed in order to determine the potential genetic toxicity of aryl sulphonate condensate. The in vitro gene mutation experiment consisted on an initial cytotoxicity test at 0.7, 0.8, 0.9, 1, 2, 3, 4, and 5 mg/plate with Salmonella typhimurium TA100, with and without metabolic activation, that was based on the results of a previous precipitation (no precipitation ≤5 mg/plate) and solubility test (soluble in distilled water at 50 mg/mL). A concurrent vehicle control consisting of distilled water was prepared. The test item did not induce cytotoxicity up to 5 mg/plate. On the basis of the cytotoxicity test, 5 mg/plate was considered to be the highest concentration suitable for the reverse mutation assay. The following test concentrations were subsequently selected for use in the main test: 0.05, 0.16, 0.5, 1.6, and 5 mg/plate, using bacterial strains S. typhimurium TA98, TA100, TA1535, and TA1537 and Escherichia coli WP2 uvrA, without and without metabolic activation. A vehicle control and five positive controls were used alongside the test. An initial trial was performed using the plate incorporation method and a second trial using the preincubation method.

The vehicle and positive controls were considered valid and the acceptability criteria of the test fulfilled. The results obtained in trial I (plate incorporation) and trial II (preincubation) for all strains and test concentrations closely resembled the vehicle controls when tested in both the presence and absence of metabolic activation, with no significant increase in the mean number of revertant colonies/plate. The treated bacterial background lawns were not observed to be different to the vehicle control. A viable plate count demonstrated that all bacterial strains were within an acceptable range of 1 to 2x10^9 Colony Forming Units (CFU)/mL. It can be concluded that, under the conditions of the experiment, Aryl sulphonate condensate was non-mutagenic up to 5 mg/plate.