N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay/Ames test: negative (OECD 471; GLP)

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:

2017-08-01 to 2017-08-16

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:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-09-14

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Target gene:

TA98: his D3052
TA100 and TA1535: his G 46
TA1537: his C 3076
E. coli WP2 uvrA: trp-

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (containing approx. 10 % v/v S9): sodium-ortho-phosphate buffer (100 mM, pH 7.4); NADP (4 mM); glucose-6-phosphate (5 mM); MgCl2 (8 mM); KCl (33 mM)

Test concentrations with justification for top dose:

Pre-experiment/Experiment 1: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Experiment 2: 3, 10, 33, 100, 333, 1000, and 2500 µg/plate (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (without metabolic activation) & 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Pre-experiment/Experiment 1: in agar (plate incorporation); Experiment 2: preincubation

EXPERIMENTAL PERFROMANCE
Experiment 1:
- the following materials were mixed in a test tube and poured onto the selective agar plates: 100 μL test solution at each dose level (solvent or positive control), 500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 μL bacteria suspension, and 2000 μL overlay agar
- after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Experiment 2:
- 100 μL test solution (solvent or positive control), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes.
- after pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube.
- mixture was poured on minimal agar plates.
- after solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

After incubation, revertant colonies were counted. Due to precipitation of the test item the colonies were partly counted manually.

In parallel to each test a sterile control of the test item was performed. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. This pre-experiment was conducted as plate incorporation test.
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Rationale for test conditions:

In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since toxic effects and precipitation of the test item were observed in experiment I, seven concentrations were tested in experiment II. 2500 µg/plate were chosen as maximal concentration.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

A statistical analysis of the data is not mandatory.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I in strain TA 1537 with metabolic activation from 2500 to 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in experiment I and from 100 to 2500 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in experiment I and from 333 to 2500 µg/plate in experiment II. The undissolved particles had no influence on the data recording.

CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred only in experiment I in strain TA 1537 with metabolic activation from 2500 to 5000 µg/plate. In the remaining test groups no toxic effects were observed neither with nor without metabolic activation.

EXPERIMENT I AND EXPERIMENT II
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with N-[2-(4-oxo-4H-3,1-benzoxazin-2-yl)phenyl]naphthalene-2-sulphonamide at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Please also refer for results to the field "Attached background material" below

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Please refer to the field "Any other information on results incl. tables" below.

Table 1: Historical data

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
	Solvent control	12	2.5	6	25	12	2.5	7	26
TA 1535	Untreated control	12	3.1	6	28	12	2.9	7	26
	Positive control	1130	143.1	334	1816	388	58.2	176	668
	Solvent control	10	2.2	6	19	13	3.5	7	30
TA1537	Untreated control	11	2.7	5	21	14	4.0	7	31
	Positive control	82	12.7	43	157	191	60.8	83	434
	Solvent control	25	4.4	13	43	34	6.2	15	58
TA 98	Untreated control	27	4.9	12	43	37	6.5	11	57
	Positive control	378	73.7	211	627	3949	771.8	360	6586
	Solvent control	156	26.0	78	209	148	32.3	73	208
TA 100	Untreated control	176	23.6	79	217	172	25.4	85	218
	Positive control	1966	293.2	498	2767	3798	830.4	536	6076
	Solvent control	41	5.6	27	63	50	6.8	28	72
WP2uvrA	Untreated control	42	5.8	30	63	52	6.8	36	88
	Positive control	798	362.7	319	4732	378	112.6	167	1265

Mean = mean value of revertants/plate
SD = standard deviation

Min = minimal value/Max = maximal value

Conclusions:

The substance tested non-mutagenic under the conditions of the study.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance should not be considered to have a mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

The substance was not observed to be mutagenic in a reliable bacterial reverse mutation assay (OECD 471).

Justification for classification or non-classification

Genetic toxicity in vitro

The substance should not be considered to have a mutagenic potential based on a bacterial reverse mutation assay (OECD 471 (1997)). The substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.