Ethanol, 2-[(3-nitropyrazolo[1,5-a]pyridin-2-yl)oxy]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames' test: positive in S. typhimurium TA98, negative in other strains

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 6 October 2014 to 18 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well described study performed according to OECD guideline and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

post mitochondrial supernatant prepared from the livers of phenobarbital/b-naphthoflavone-induced rats.

Test concentrations with justification for top dose:

5000, 2500, 1250, 625, 312.5, 156.25, 78.13 and 39.06 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF (N,N-Dimethylformamide)
- Justification for choice of solvent/vehicle: based on the results of a solubility test

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine (NPD), 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for the initial test; preincubation in the confirmatory test

DURATION
- Preincubation period: 20 minutes (confirmatory tests)
- Exposure duration: 48±1 hours

NUMBER OF REPLICATES: 3 per dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Criteria for a Positive Response:
The test item was considered to have shown mutagenic activity in this study if a 2-fold increase (in S. typhimurium TA98, TA100 and E. coli WP2 uvrA strains) or 3-fold increase (in S. typhimurium TA1535 and TA1537 strains) in the mean number of revertants was observed when compared to the vehicle (solvent) controls, in any strain, with an evidence of a dose-response relationship.
Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if no increase in the mean number of revertants, reaching 2-fold (for S. typhimurium TA98, TA100 and E. coli WP2 uvrA strains) or 3-fold (for S. TA1535 and TA1537 strains) of the vehicle controls value, was observed at any of the tested dose-levels.
Reference to historical data or other considerations of biological relevance might be taken into account in the evaluation of the data obtained.
Statistical method may be used as an aid in evaluating the test results.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see details below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No insolubility was detected in the main tests in any examined bacterial strains with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the Preliminary Range Finding Test, substantial increases of the revertant counts were observed in Salmonella typhimurium TA98 bacterial strain with and without metabolic activation. The calculated mutation factor values were over the relevant threshold value in this strain in several cases, dose dependence was also observed.
No precipitate was detected on the plates in the preliminary experiment in Salmonella typhimurium TA98 bacterial strain with and without metabolic activation.
No signs of inhibitory, cytotoxic effects were observed in the preliminary experiment in Salmonella typhimurium TA98 bacterial strain at any concentrations with or without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean values of revertant colony numbers of untreated, negative (vehicle/solvent) and positive control plates were in harmony with the historical control range in all strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development, reduced number of revertant colonies was also detected in some cases) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1537 strain at 5000 μg/plate concentration with and without metabolic activation.

Table 1: Summary Table of the Initial Mutation Test (Mean values of revertants)

Concentrations (µg/plate)	Salmonella typhimuriumstrains								Escherichia coli
	TA98		TA100		TA1535		TA1537		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	24.0	25.7	107.7	108.0	11.7	11.7	10.7	11.3	77.3	66.3
Distilled water control	-	-	87.3	-	13.0	-	-	-	78.0	-
DMSO control	21.3	35.7	-	97.3	-	9.7	14.7	11.3	-	68.0
DMF control	17.3	28.3	81.7	94.0	15.0	10.7	14.0	10.3	85.0	66.7
5000	486.7	510.7	116.3	108.0	9.0	9.3	16.7	12.0	55.3	71.3
2500	313.3	348.0	104.3	100.3	11.7	9.3	13.7	11.3	63.7	61.7
1250	207.3	221.3	105.3	109.0	15.7	7.0	16.3	16.0	73.3	73.7
625	126.7	133.3	102.0	103.0	18.3	13.0	15.3	15.0	76.0	71.7
312.5	64.3	96.3	83.7	103.3	15.7	12.0	16.0	15.3	66.0	67.7
156.25	47.0	56.3	92.7	105.3	14.3	11.3	15.3	12.3	74.7	69.3
78.13	26.3	43.0	85.0	98.7	13.3	12.3	13.0	15.0	78.0	71.7
39.06	23.7	40.3	93.0	85.3	16.0	12.0	10.0	10.7	110.0	77.3
NPD (4µg)	392.0	-	-	-	-	-	-	-	-	-
2AA (2µg)	-	2521.3	-	2393.3	-	190.3	-	196.7	-	-
2AA (50µg)	-	-	-	-	-	-	-	-	-	309.7
SAZ (2µg)	-	-	1093.3	-	1084.0	-	-	-	-	-
9AA (50µg)	-	-	-	-	-	-	390.7	-	-	-
MMS (2µL)	-	-	-	-	-	-	-	-	1121.3	-

Table 2: Summary Table of the Confirmatory Mutation Test (Mean values of revertants)

Concentrations (µg/plate)	Salmonella typhimuriumstrains								Escherichia coli
	TA98		TA100		TA1535		TA1537		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Untreated control	17.0	26.0	100.3	104.0	12.0	7.3	4.0	11.7	30.7	41.7
Distilled water control	-	-	98.0	-	14.0	-	-	-	31.3	-
DMSO control	21.0	27.3	-	94.3	-	11.0	3.7	7.0	-	31.7
DMF control	24.3	32.7	91.0	100.0	18.7	9.0	6.3	7.0	27.0	32.7
5000	488.0	341.3	102.3	116.0	8.7	10.3	4.7	0.3	28.7	29.7
2500	774.7	335.3	106.3	104.3	11.0	11.7	9.3	4.3	38.7	43.0
1250	214.7	272.0	88.3	99.7	12.0	11.0	7.7	14.3	49.3	52.7
625	157.0	183.7	103.7	97.3	15.0	9.7	9.7	12.7	48.0	59.3
312.5	89.7	104.3	101.3	111.3	17.3	7.7	7.7	8.7	51.0	58.7
156.25	52.7	71.7	103.3	105.7	12.3	11.0	7.0	7.0	52.3	56.3
78.13	34.3	47.0	93.3	100.3	19.0	9.3	8.0	9.7	53.0	60.7
39.06	29.7	48.7	105.0	108.3	12.7	8.0	10.7	9.7	44.0	44.7
NPD (4µg)	362.3	-	-	-	-	-	-	-	-	-
2AA (2µg)	-	2328.0	-	2406.7	-	315.7	-	193.3	-	-
2AA (50µg)	-	-	-	-	-	-	-	-	-	247.7
SAZ (2µg)	-	-	1194.7	-	1198.7	-	-	-	-	-
9AA (50µg)	-	-	-	-	-	-	385.3	-	-	-
MMS (2µL)	-	-	-	-	-	-	-	-	896.0	-

Conclusions:

Interpretation of results (migrated information):
positive without and without metabolic activation (in S. typhimurium TA 98)
negative all other strains with and without metabolic activation

R0056895A had mutagenic activity in Salmonella typhimurium TA98 strain in the presence and absence of metabolic activation system. However, no mutagenic activity of the test item was observed in the other four experimental strains under the test conditions of this study. Based on these data, the overall result is positive.

Executive summary:

R005895A was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay, based on OECD Guidelines for Testing of Chemicals, No.471 and Commission Regulation (EC) No. 440/2008, B.13/14. with Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA, in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats. R0058407A was tested up to 5000 µg/plate in DMF.

R0056895A had mutagenic activity in Salmonella typhimurium TA98 strain in the presence and absence of metabolic activation system. However, no mutagenic activity of the test item was observed in the other four experimental strains under the test conditions of this study.

Based on these data, the overall result is positive.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Additional information from genetic toxicity in vitro:

R005895A was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay, based on OECD Guidelines for Testing of Chemicals, No.471 and Commission Regulation (EC) No. 440/2008, B.13/14. with Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA, in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/b-naphthoflavone-induced rats. R0058407A was tested up to 5000 µg/plate in DMF.

R0056895A had mutagenic activity in Salmonella typhimurium TA98 strain in the presence and absence of metabolic activation system. However, no mutagenic activity of the test item was observed in the other four experimental strains under the test conditions of this study.

Based on these data, the overall result is positive.

Justification for selection of genetic toxicity endpoint
Only one study, well described and performed according to OECD guideline and GLP

Justification for classification or non-classification

A positive result was observed in one strain of S. typhimurium in the Ames' test. However, this result is insufficient for classification. Additional information as in-vitro mammalian assays could be useful for classification purpose.