[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1991-02-06 to 1991-07-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 strains tested (instead of five)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

EC Number:

259-033-6

EC Name:

[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

Cas Number:

54229-13-9

Molecular formula:

C21H29ClN5O3.Cl

IUPAC Name:

[2-({4-[(E)-2-(2-chloro-4-nitrophenyl)diazen-1-yl]phenyl}(ethyl)amino)ethyl](2-hydroxypropyl)dimethylazanium chloride

Test material form:

solid

Method

Target gene:

Histidine locus

Species / strain

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:
The test followed the directions of Ames et al. (1973, 1975) and Maron and Ames (1983): 0.1 mL compound, 0.1 mL bacteria, 0.5 mL S9 mix or buffer, 2.0 mL soft agar, 45 °C in water bath, max. 30 sec, transfer to petri dish with solid agar

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First test: 5000, 1000, 200, 40 and 8 µg/plate;
Repeat tests: 400, 200, 100, 50 and 25 µg/plate

Vehicle / solvent:

The solvent was deionized water and for the positive controls DMSO.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: nitrofurantoin

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene diamine

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 0.1 mL TS + 0.1 mL bacteria + 0.5 mL S9 + 2 mL soft agar: 30 sec at 45 °C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 4 plates/strain/concentration

DETERMINATION OF CYTOTOXICITY
- Method: - background growth
- marked and dose-dependent reduction in mutant count compared to negative controls
- titer determination

- OTHER:
Acceptance criteria:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratory's own historical data
b) The positive controls had to show sufficient effects, as defined by the laboratory's experience
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

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 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by scientific judgement.

Statistics:

N.A.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no indication of bacteriotoxic effects of the test item at doses of up to and including 100 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only be used up to 400 µg per plate for assessment purposes. Three of the four strains concerned revealed a dose-related increase in mutant counts to well over double those of negative controls. Strains TA 100, TA 1537 and TA were affected. The findings were confirmed by a repeat test. The lowest dose at which this finding was reproducible was approximately 200 µg per plate for Salmonella typhimurium TA 100 and TA 1537, and approximately 25 µg per plate for TA 98. Positive findings were obtained with and without S9 mix, the effects being strengthened by S9 mix.

Any other information on results incl. tables

 Table 1: Summary of mean values with S9-mix from the first test

µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
30% S9-mix
0		12		107		11		32
8		12		170		12		49
40		14		185		21		101
200		10		250		77		467
1000		11		10		0		--
5000		0		0		0		0
2-AA		140		498		31		182

 

Table 2: Summary of mean values with S9-mix from repeat test

µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
30% S9-mix
0		13		165		12		44
25		16		226		26		397
50		12		213		24		185
100		14		223		58		757
200		14		245		82		802
400		9		174		25		486
2-AA		127		662		70		307

 Table 3: Summary of mean values without S9-mix from the first test

µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
0		13		91		11		24
8		9		115		10		58
40		11		141		24		196
200		10		37		--		208
1000		--		0		0		0
5000		0		0		0		0
Na-azid		641
NF				289
4-NPDA						40		57

 

Table 4: Summary of mean values without S9-mix from the repeat test

µg/plate		Strain
		TA 1535		TA 100		TA 1537		TA 98
0		12		116		11		25
25		12		201		27		157
50		10		140		22		389
100		10		113		16		303
200		7		28		0		198
400		6		13		0		--
Na-azid		824
NF				361
4-NPDA						60		58

Applicant's summary and conclusion

Conclusions:

A dose dependent increase in the mutant count at non-bacteriotoxic concentrations in comparison to the negative controls was observed after treatment with the test item in the presence and absence of metabolic activation. Therefore, the test item is considered to be mutagenic with and without S9 mix in this Salmonella/microsome test.

Executive summary:

In a reverse gene mutation assay in bacteria (equivalent to OECD 471), strains TA 1535, TA 100, TA 1537 and TA 98 of S. typhimurium were exposed to the test item (95.6 % purity) in deionized water at concentrations of 8, 40, 200, 1000 and 5000 µg/plate (first/pre-test) and at concentrations of 25, 50, 100, 200 and 400 µg/plate (repeat test) in the presence and absence of mammalian metabolic activation. 

The test item was tested up to the limit concentration (5000 µg/plate). Based on bacteriotoxic effects, dose groups above 400 µg per plate were not used for assessment purposes. The positive controls induced the appropriate responses in the corresponding strains. In Salmonella typhimurium strains TA 100, TA 1537 and TA 98 a mutagenic effect was observed at non-cytotoxic doses. In the Salmonella typhimurium strain TA 1535 no mutagenic effect was observed. Based on the result, it can be stated that the test item can be considered as a bacterial mutagen in this assay.

This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.