1-Naphthol, reaction products with formaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames, Groot (2018)

Under the conditions of this study the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay or the Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 January 2018 to 07 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Agar plates (ø 9 cm) containing 25 mL glucose agar medium. Glucose agar medium contained per litre: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine.
- Top agar: Milli-Q water containing 0.6 % (w/v) bacteriological agar and 0.5 % (w/v) sodium chloride was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3 °C.
- Properly maintained: Yes. The Salmonella typhimurium strains are checked at least every year to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).
- All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0 °C
(actual range 34.7 - 39.0 °C).

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Agar plates (ø 9 cm) containing 25 mL glucose agar medium. Glucose agar medium contained per litre: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan.
- Top agar: Milli-Q water containing 0.6 % (w/v) bacteriological agar and 0.5 % (w/v) sodium chloride was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3 °C.
- Properly maintained: Yes. The strain is checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year. Stock cultures were stored in liquid nitrogen (-196 °C).
- All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0 °C
(actual range 34.7 - 39.0 °C).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced by Aroclor 1254)

Test concentrations with justification for top dose:

Dose-Range Finding Test: 1.7, 5.4, 17, 52, 164, 512. 1600 and 5000 µg/plate
Experiment 1: 17, 52, 164, 152 and 1600 µg/plate
Experiment 2: 154, 275, 492, 878 and 1568 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test material formed a dark blue formulation in Milli-Q water. Due to the colour of the test material it could not be observed if the test material was dissolved or suspended.
- A correction factor of 5 for the purity/composition of the test item was applied in this study.
- The stock solution was treated with ultrasonic waves. Test material concentrations were used within 2.5 hours after preparation. Any residual volumes were discarded.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DOSE RANGE FINDING TEST/ MUTATION ASSAY
- Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5 % (v/v) S9-mix.
- Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of the test material used in the subsequent mutation assays was the level at which the test material exhibited limited solubility.

MUTATION ASSAY
- At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain. The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test material was tested both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains. Initially the second experiment was rejected due to a technical error. This part of the study was repeated.
- The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
- Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in Milli-Q water and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
- In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
- A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535 & TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING TEST/ FIRST MUTATION EXPERIMENT
Precipitate
Precipitation of the test material on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 μg/plate and upwards. Except for tester strains TA100 and WP2uvrA where precipitation of the test material at the start of the incubation period was already observed at the concentration of 512 μg/plate.

Toxicity
To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in tester strain TA1537, in the presence of S9-mix, at the concentration of 1600 μg/plate.
In tester strains TA1537 (absence and presence of S9-mix) and TA98 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, the amount of revertants was just below the historical control database, these reductions are not considered to be caused by toxicity of the test material. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.

Mutagenicity
No greater than 2-fold increase in the number of revertants was observed upon treatment with the test material under all conditions tested.


SECOND MUTATION EXPERIMENT
Precipitate
Precipitation of the test material on the plates was observed at the start and at the end of the incubation period at concentrations of 492 μg/plate and upwards.

Toxicity
In the second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.
In tester strain TA1537 in the absence and presence of S9-mix, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test material. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.

Mutagenicity
In the second mutation assay, no biologically relevant increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

DISCUSSION
All bacterial strains showed negative responses over the entire dose-range, i.e. no biologically relevant increase in the number of revertants in two experiments.
It is noted that in the second mutation experiment, in tester strain TA100 in the presence of 10 % S9 a small but dose-related increase in revertant colonies that extended across insoluble concentrations was observed. However, as it did not achieve the criterion of twice the concurrent vehicle control and a similar effect was not apparent in the first mutation experiment (conducted with 5 % S9 mix) the findings were considered to be biologically irrelevant.
The negative control values were within the laboratory historical control data ranges.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the responses for TA1535 in the absence of S9-mix and TA1537 in the presence of S9-mix in the second experiment where results outside the laboratory historical control data ranges were observed. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the values were more than 3 times greater than the concurrent solvent control values, these deviations in the mean plate count of the positive controls had no effect on the results of the study.

Conclusions:

Under the conditions of this study the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay or the Escherichia coli reverse mutation assay.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and EU Method B13/14, under GLP conditions.

The potential of the test material to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA was determined in the presence or absence of an exogenous mammalian metabolic activation system (S9).

A correction factor of 5 was used to correct for the purity (20 %) of the test material. The vehicle of the test material was Milli-Q water.

In the dose-range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 1600 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test material was tested in the first mutation assay at a concentration range of 17 to 1600 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material precipitated on the plates at the dose level of 1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 154 to 1568 μg/plate in the absence and presence of 10 % (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 492 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The test material did not induce a biologically relevant increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control materials indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay or the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no study available

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames, Groot (2018)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and EU Method B13/14, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The potential of the test material to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA was determined in the presence or absence of an exogenous mammalian metabolic activation system (S9).

A correction factor of 5 was used to correct for the purity (20 %) of the test material. The vehicle of the test material was Milli-Q water.

In the dose-range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 1600 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test material was tested in the first mutation assay at a concentration range of 17 to 1600 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test material precipitated on the plates at the dose level of 1600 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 154 to 1568 μg/plate in the absence and presence of 10 % (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 492 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The test material did not induce a biologically relevant increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In this study, acceptable responses were obtained for the negative and strain-specific positive control materials indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Under the conditions of this study the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay or the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.