2-Naphthalenol, heptyl-1-[2-[3-methyl-4-[2-(3-methylphenyl)diazenyl]phenyl]diazenyl]-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Cas Number:

1619917-05-3

Molecular formula:

Not applicable (UVCB Substance)

IUPAC Name:

Reaction products of 3-Methylaniline with heptyl naphthalen-2-ol

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 78-231-15
- Expiration date of the batch: October 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

Method

Target gene:

histidine

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:

rat liver S9

Test concentrations with justification for top dose:

3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene; Congo Red

Details on test system and experimental conditions:

METHOD OF APPLICATION
The test item was suspended in acetone and diluted prior to treatment. For the plate incorporation
method in experiment I a stock solution of 50 mg/mL was used for preparation of the dilution series.
For the pre-incubation method in experiment II a stock solution of 100 mg/mL was used. So a lower
amount could be applied by performing the pre-incubation and so the solvent was compatible with
the survival of the bacteria and the S9 activity.

Preparation of Bacteria
Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late
exponential or early stationary phase of growth (approx. 109 cells/mL). The nutrient medium consists
per litre:
8 g Nutrient Broth
5 g NaCl
A solution of 125 μL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the
phenotypic characteristics of the strain.

Pre-Experiment for Toxicity
The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a preexperiment.
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 μg/plate

Exposure Concentrations
The test item concentrations to be applied in the main experiments were chosen according to the
results of the pre-experiment (see chapter 12.1.1 Pre-Experiment). 5000 μg/plate was selected as
the maximum concentration. The concentration range covered two logarithmic decades.
Two independent experiments were performed with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
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.

Experimental Performance
For the plate incorporation method with rat liver S9 the following materials were mixed in a test tube
and poured over the surface of a minimal agar plate:
100 μL Test solution at each dose level, solvent control, negative control or reference
mutagen solution (positive control),
500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for
testing without metabolic activation),
100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 μL Overlay agar.
For the pre-incubation method with hamster liver S9 the following materials were mixed in a test tube
and incubated for 30 min. at 30 °C :
50 μL Test extract at each dose level, extract medium control, or
100 μL Negative control or reference mutagen solution (positive control),
500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for
testing without metabolic activation),
100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
After the incubation period (30 min., 30 °C), the overlay agar (2000 μL) was added and poured onto
the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates (in a few cases only two plates
were evaluated, see Table 2: Results Experiment I) were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Data Recording
The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If
precipitation of the test item precluded automatic counting the revertant colonies were counted by
hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and
TA 1537 were counted manually.

Evaluation of Cytotoxicity
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as
"N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a
mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Evaluation criteria:

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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Remarks:

in Exp I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 μg/plate and higer

Test results 2

Species / strain:

S. typhimurium TA 100

Remarks:

in Exp I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 316 μg/plate and higher (without metabolic activation) and at a concentration of 5000 μg/plate (with metabolic activation).

Test results 3

Species / strain:

S. typhimurium TA 1537

Remarks:

in Exp I

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 2500 μg/plate and higher

Test results 4

Species / strain:

S. typhimurium TA 98

Remarks:

in Exp II

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 2500 μg/plate and higher

Test results 5

Species / strain:

other: TA 100, TA1535 and TA1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations of 316 μg/plate and higher

Any other information on results incl. tables

Precipitation of the test item was observed in all tester strains used in experiment I at concentrations of 2500 μg/plate and higher (with metabolic activation).

Toxic effects of the test item were noted in several tester strains evaluated in experiment I and II.

In experiment I toxic effects of the test item were observed in tester strain TA 98 at concentrations of

1000 μg/plate and higher (without metabolic activation). In tester strain TA 100 toxic effects of the

test item were noted at concentrations of 316 μg/plate and higher (without metabolic activation) and

at a concentration of 5000 μg/plate (with metabolic activation). In tester strain TA 1537 toxic effects

of the test item were observed at concentrations of 2500 μg/plate and higher (without metabolic

activation).

In experiment II toxic effects of the test item were noted in tester strain TA 98 at concentrations of

2500 μg/plate and higher (without metabolic activation). In tester strains TA 100, TA 1535 and

TA 1537 toxic effects of the test item were noted at concentrations of 316 μg/plate and higher

(without metabolic activation).

The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment I in

tester strain TA 1535 at a concentration of 3.16 μg/plate (with metabolic activation), in experiment II

in tester strain TA 100 at a concentration of 316 μg/plate (with metabolic activation) and in tester

strain TA 1537 at a concentration of 2500 μg/plate (with metabolic activation) was regarded as not

biologically relevant due to lack of a dose-response relationship. The reduction in the number of

revertants down to a mutation factor of ≤ 0.5 found in experiment I in the negative control of tester

strain TA 1537 (with metabolic activation) was regarded as not biologically relevant due to the fact

that all three numbers of spontaneous revertants were within the historical control data and lack of

concomitant clearing of the background lawn.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were

observed following treatment with RED 2596 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:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, RED 2596 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, RED 2596 is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item RED 2596 was investigated for its potential to induce gene mutations according to the

plate incorporation test with rat liver S9 (experiment I) and the pre-incubation test with hamster liver

S9 (experiment 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:

3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Precipitation of the test item was observed in all tester strains used in experiment I at concentrations

of 2500 μg/plate and higher (with metabolic activation).

Toxic effects of the test item were noted in several tester strains evaluated in experiment I and II.

In experiment I toxic effects of the test item were observed in tester strain TA 98 at concentrations of

1000 μg/plate and higher (without metabolic activation). In tester strain TA 100 toxic effects of the

test item were noted at concentrations of 316 μg/plate and higher (without metabolic activation) and

at a concentration of 5000 μg/plate (with metabolic activation). In tester strain TA 1537 toxic effects

of the test item were observed at concentrations of 2500 μg/plate and higher (without metabolic

activation).

In experiment II toxic effects of the test item were noted in tester strain TA 98 at concentrations of

2500 μg/plate and higher (without metabolic activation). In tester strains TA 100, TA 1535 and

TA 1537 toxic effects of the test item were noted at concentrations of 316 μg/plate and higher

(without metabolic activation).

The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment I in

tester strain TA 1535 at a concentration of 3.16 μg/plate (with metabolic activation), in experiment II

in tester strain TA 100 at a concentration of 316 μg/plate (with metabolic activation) and in tester

strain TA 1537 at a concentration of 2500 μg/plate (with metabolic activation) was regarded as not

biologically relevant due to lack of a dose-response relationship. The reduction in the number of

revertants down to a mutation factor of ≤ 0.5 found in experiment I in the negative control of tester

strain TA 1537 (with metabolic activation) was regarded as not biologically relevant due to the fact

that all three numbers of spontaneous revertants were within the historical control data and lack of

concomitant clearing of the background lawn.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were

observed following treatment with RED 2596 at any concentration level, neither in the presence nor

absence of metabolic activation in experiment I and II.

In conclusion, it can be stated that during the described mutagenicity test and under the

experimental conditions reported, RED 2596 did not cause gene mutations by base pair changes or

frameshifts in the genome of the tester strains used.

Therefore, RED 2596 is considered to be non-mutagenic in this bacterial reverse mutation assay.