2-ethoxybenzonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Salmonella/microsome test (Ames test): positive with strain TA 1535 (+ S9 mix) and negative with strains TA 98, TA 100, TA 102 and TA 1535 (+/- S9 mix)

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:

April 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

0, 50, 158, 500, 1581, 5000 µg/plate (first test, +/-S9 mix, all strains)
0, 50, 100, 200, 400, 800, 1600 µg/plate (repeat test, +/-S9 mix, all strains)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

No solvent control was used since sufficient evidence was available in the literature and from testing laboratory experience, indicating that the solvents used had no influence on the spontaneous mutant counts of the used strains.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), cumene hydroperoxide (only TA 102), 2-aminoanthracene.

Remarks:

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Details on test system and experimental conditions:

METHOD: Standard plate test and preincubation test; each concentration including the controls was tested in triplicate.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 1537, TA 100 and TA 98 this increase should be about twice that of negative controls. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these criteria may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

not specified

Species / strain:

S. typhimurium, other: TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strong, strain-specific bacteriotoxic effect at 800 µg/plate and above

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strong, strain-specific bacteriotoxic effect at 800 µg/plate and above

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

strong, strain-specific bacteriotoxic effect at 800 µg/plate and above

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Summary of results from the Salmonella mutagenicity assay (first test) with Ethoxybenzonitril (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	9	73	12	18	170
50	7	66	9	15	185
158	4	90	8	24	172
500	4	55	7	18	128
1581	4	59	2	20	48
5000	-	0	0	-	-
Positive control	634	229	95	225	280
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	10	78	10	23	230
50	12	75	10	27	236
158	20	91	9	26	253
500	23	85	11	23	233
1581	13	64	6	18	155
5000	-	-	-	-	6
Positive control	225	1170	191	1058	429

Table 2: Summary of results from the Salmonella mutagenicity assay (repeat test) with Ethoxybenzonitril (mean values of revertants per plate)

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	6	81	7	17	226
50	5	80	6	16	218
100	7	78	6	17	239
200	8	82	6	17	226
400	9	61	5	14	183
800	6	44	4	5	131
1600	0	-	1	-	18
Positive control	536	263	125	188	419
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	10	69	8	35	284
50	12	72	7	28	295
100	15	86	7	19	270
200	22	92	6	17	227
400	21	100	5	20	244
800	21	88	4	18	210
1600	10	49	-	10	94
Positive control	114	1019	117	1172	491

 

 

Table 3: Summary of results from the Salmonella mutagenicity assay (repeated plate incorporation and pre-incubation test with TA 1535 and metabolic activation) with Ethoxybenzonitril (mean values of revertants per plate)

 

Dose (µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 1535
	plate incorporation	pre-incubation
0	9	9
50	12	12
100	20	19
200	25	27
400	31	26
800	27	18
1600	11	5
Positive control	184	259

Doses up to and including 500 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 1600 µg per plate for assessment purposes.

Evidence of mutagenic activity of Ethoxybenzonitril was seen. On Salmonella typhimurium TA 1535, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. The lowest reproducible effective dose was 100µg per plate. The Salmonella/microsome test thus showed Ethoxybenzonitril to have a mutagenic effect. However, it can be assumed that this result may be based on the effect of an impurity.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

Executive summary:

The mutagenic potential of Ethoxybenzonitril was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471. Doses up to and including 500 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 1600 µg per plate for assessment purposes. Evidence of mutagenic activity of Ethoxybenzonitril was seen. On Salmonella typhimurium TA 1535, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. The lowest reproducible effective dose was 100 µg per plate. The Salmonella/microsome test thus showed Ethoxybenzonitril to have a mutagenic effect. However, it can be assumed that this result may be based on the effect of an impurity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

The mutagenic potential of Ethoxybenzonitril was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471 (Herbold, 2000). Doses up to and including 500 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 1600 µg per plate for assessment purposes. Evidence of mutagenic activity of Ethoxybenzonitril was seen. On Salmonella typhimurium TA 1535, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was found only with S9 mix. The lowest reproducible effective dose was 100 µg per plate. The Salmonella/microsome test thus showed Ethoxybenzonitril to have a mutagenic effect. However, it can be assumed that this result may be based on the effect of an impurity.

Justification for selection of genetic toxicity endpoint
Only one study available

Justification for classification or non-classification

Based on the study results a classification according to Directive 67/548/EEC or Regulation (EC) No. 1272/2008 (CLP) is not warranted.