6-amino-2-(2,4-dimethylphenyl)-1H-benz[de]isoquinoline-1,3(2H)-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay in the presence of metabolic activation and not mutagenic in 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:

17 July 2016 to 27 July 2016

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 forward mutation assay

Specific details on test material used for the study:

A correction factor of 1.031 was used to correct for the purity.

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.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked 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).

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.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °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 study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)
- Mutation experiment (TA1535, TA1537 and TA98): 17, 52, 164, 512, 1600 and 5000 μg/plate (absence and presence of S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Chosen following a solubility test. At a concentration of 50 mg/mL, the test material formed a suspension in dimethyl sulfoxide whereas at 16 mg/mL and lower it was fully soluble. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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 S9-mix and reported as part of the first mutation experiment.

MUTATION ASSAY
At least five different doses (increasing with approximately half-log steps) of the test material were tested in each strain both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98.

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 DMSO 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.

DETERMINATION OF CYTOTOXICITY
- Method: 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.

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 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 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: TA1537, TA98 and TA100

Metabolic activation:

with

Genotoxicity:

positive

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:

S. typhimurium, other: TA1537, TA98 and TA100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

only observed in tester strain TA100 in the absence of S9-mix at concentrations of 512 μg/plate and upwards.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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/MUTATION EXPERIMENT
- Precipitate: in the dose range finding test, precipitation of the test material was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards. At the end of the incubation period the test material precipitated on the plates at dose levels of 512 μg/plate and upwards in the absence of S9-mix and at 5000 μg/plate in the presence of S9-mix. In the first mutation experiment, precipitation of the test material on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate and at 1600 and 5000 μg/plate at the end of the incubation period.
- Toxicity: Since the test material precipitated heavily on the plates at the test substance concentration of 5000 μg/plate in tester strain TA100 (presence of S9-mix), the number of revertants at this dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants was only observed in tester strain TA100 in the absence of S9-mix at concentrations of 512 μg/plate and upwards. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix.
- Mutagenicity: No increases in the number of revertants were observed upon treatment with the test material in the absence of S9-mix.
The test material induced increases in the number of revertant colonies in the presence of S9-mix. The increases were observed in tester strain TA100 (up to 4.1-fold), in tester strain TA1537 (up to 37-fold) and in tester strain TA98 (up to 51-fold).
The test material did not induce a significant dose-related increase in the number of revertant colonies in tester strains TA1535 and WP2uvrA in the presence of S9-mix.

DISCUSSION
No increases in the number of revertants were observed upon treatment with the test material in the absence of S9-mix. In the presence of S9-mix, the test material did not induce a significant dose-related increase in the number of revertant colonies in the tester strains TA1535 and WP2uvrA. The test material induced dose related increases in three tester strains (TA1537, TA98 and TA100). The increases observed in the three tester strains were above the laboratory historical control data range, dose-related and were up to 37, 31 and 4.0-fold the concurrent controls, respectively. These increases are considered to be biologically relevant and the test material is considered to be mutagenic.
The negative and 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.

Table 1: Dose Range-finder and Mutation Experiment

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate (3 replicates)
		Base-pair Substitution Type		Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	PC DMSO 1.7 5.4 17 52 164 512 1600 5000	833 86 84 72 82 76 78 NP 56 SP 36 SP 56 n MP	803 10 - - 7 7 8 16 NP 11 SP 6 n MP	1208 21 26 32 30 26 28 NP 38 SP 32 SP 12 n MP	1302 9 - - 14 11 15 15 NP 16 SP 13 n MP	1219 5 - - 6 8 8 11 NP 13 SP 7 n MP
+	PC DMSO 1.7 5.4 17 52 164 512 1600 5000	1184 99 98 98 129 168 261 404 214 NP n HP	167 14 - - 9 11 9 21 NP 12 SP 7 n MP	411 40 40 42 51 39 42 43 33 NP 19 n MP	1119 14 - - 83 184 387 712 NP 431 SP 211 n MP	283 5 - - 16 31 70 144 NP 184 SP 80 n MP

HP= Heavy Precipitate; the number of revertant colonies could not be determined

MP = Moderate Precipitate

NP = No precipitate

SP = Slight Precipitate

n = Normal bacterial background lawn

Conclusions:

Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay in the presence of metabolic activation and not mutagenic in the Escherichia coli reverse mutation assay.

Executive summary:

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions.

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

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 512 μg/plate and upwards in the absence of S9-mix and at 5000 μg/plate in the presence of S9-mix. Since the test material precipitated heavily on the plates at the test material concentration of 5000 μg/plate in tester strain TA100 (presence of S9-mix), the number of revertants at this dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the absence of S9-mix at the concentrations of 512 μg/plate and upwards. 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 mutation assay at a concentration range of 17 to 5000 µg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. The test material precipitated on the plates at dose levels of 1600 and 5000 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.

No increases in the number of revertants were observed upon treatment with the test material in the absence of S9-mix.

In the presence of S9-mix, the test material did not induce a significant dose-related increase in the number of revertant colonies in the tester strains TA1535 and WP2uvrA. The test material induced dose related increases in three tester strains (TA1537, TA98 and TA100). The increases observed in these three tester strains were above the laboratory historical control data range, dose-related and were up to 37, 31 and 4.0-fold the concurrent controls, respectively. These increases are considered to be biologically relevant and the test material is considered to be mutagenic.

The negative and 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.

Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay in the presence of metabolic activation and not mutagenic in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of the test material to cause mutagenic effects in bacteria was assessed in accordance with the standardised guidelines OECD 471 and EU Method B.13/14 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

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 512 μg/plate and upwards in the absence of S9-mix and at 5000 μg/plate in the presence of S9-mix. Since the test material precipitated heavily on the plates at the test material concentration of 5000 μg/plate in tester strain TA100 (presence of S9-mix), the number of revertants at this dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the absence of S9-mix at the concentrations of 512 μg/plate and upwards. 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 mutation assay at a concentration range of 17 to 5000 µg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. The test material precipitated on the plates at dose levels of 1600 and 5000 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.

No increases in the number of revertants were observed upon treatment with the test material in the absence of S9-mix.

In the presence of S9-mix, the test material did not induce a significant dose-related increase in the number of revertant colonies in the tester strains TA1535 and WP2uvrA. The test material induced dose related increases in three tester strains (TA1537, TA98 and TA100). The increases observed in these three tester strains were above the laboratory historical control data range, dose-related and were up to 37, 31 and 4.0-fold the concurrent controls, respectively. These increases are considered to be biologically relevant and the test material is considered to be mutagenic.

The negative and 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.

Under the conditions of the study, it is concluded that the test material is mutagenic in the Salmonella typhimurium reverse mutation assay in the presence of metabolic activation and not mutagenic in the Escherichia coli reverse mutation assay.

Given that there were some positive responses in the assay, this must be borne in mind when selecting the appropriate studies to further investigate the genotoxic potential of the test material at the next tonnage level. Based on the results of this single in vitro study in bacteria, at this stage it is not considered appropriate to classify the material.

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.