5-chloro-2-(2,4-dichlorophenoxy)aniline

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-chloro-2-(2,4-dichlorophenoxy)aniline

EC Number:

260-480-4

EC Name:

5-chloro-2-(2,4-dichlorophenoxy)aniline

Cas Number:

56966-52-0

Molecular formula:

C12H8Cl3NO

IUPAC Name:

5-chloro-2-(2,4-dichlorophenoxy)aniline

Details on test material:

- Name of test material (as cited in study report): FAT 80'023/G (TADE)
- Physical state: solid, grey to green
- Analytical purity: > 98 %
- Lot/batch No.: ICMDJuli 1995
- Expiration date of the lot/batch: August 31, 2000
- Stability under test conditions: In solvent: 48 hours in H2O, polyethylene glycol, and CMC
- Storage condition of test material: room temperature

Method

Target gene:

his

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced liver S9-mix from 8 - 12 weks old male rats

Test concentrations with justification for top dose:

10.0; 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate

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 for the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

for TA 1535, TA 100, without metabolic activation

Positive control substance:

sodium azide

Remarks:

Purity: at least 99 %, Dissolved in: aqua dest., Concentration: 10 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

TA 1537, TA 98, without metabolic activation

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Purity: at least 99.9 %, Dissolved in: DMSO, Concentration: 10 µg/plate

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

TA 1535, TA 1537, TA 98, TA 100, with metabolic activation

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

Purity: at least 97.5 %, Dissolved in: DMSO, Concentration: 2.5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, plate incorporation test in experiment I and I A and preincubation test in experiment II

DURATION
- Preincubation period: 60 min
- Selection time (if incubation with a selection agent): 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, reduction in the number of revertants,

POSITIVE CONTROLS
- The stability of the positive control substances in solution was unknown but a mutagenic response in the expected range is sufficient evidence of biological stability. The dilutions of the stock solutions were prepared on the day of the experiment and used immediately.

OTHER:
- The test article precipitated from 2500.0 µg/plate up to 5000.0 µg/plate in the overlay agar. The undissolved particles of the test article had no influence on the data recording.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates. Range of spontaneous reversion frequencies: TA 1535: 10 - 29; TA 1537: 5 - 28; TA 98: 15 -57; TA 100: 77 - 189

- A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
- A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

Statistics:

A significant response is described as follows:
- A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537.
- Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

other: S. Typhimurium TA 100 and TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Since overlapping mutagenic and toxic effects occurred in strain TA 1535 in the presence of metabolic activation an additional experiment was carried out as plate incorporation test using strain TA 1535 with S9 mix to verify the data (I A).
- Toxic effects, evidenced by a reduction in the number of revertants, occurred in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) with and without metabolic activation in all strains used.
- At 5000.0 µg/plate an exceptionally strong background growth was observed in strain TA 1535 without metabolic activation in the first experiment. This effect might indicate a high number of mutant bacteria which could not form real colonies due to overlapping toxic effects as stated in the original report.
- Strain TA 1535: Substantial increases in revertant colony numbers were observed following treatment with FAT 80'023/G (TADE) at the higher concentrations with S9 mix in experiment I, I A, and II with as well as without S9 mix.
- Strain TA 1537: Substantial increases in revertant colony numbers were observed at the highest concentration with S9 mix in experiment II. Nevertheless in the concentration of 1000.0 µg/plate and 2500 µg/plate strongly reduced revertant number points to toxicity.
A summary of the results in detail see the attached file.

Remarks on result:

other: other: S. typhimurium TA 1535

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Plate Incorporation test (10 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	31	1.0	no	negative
	yes	35	1.2	no	negative
TA 1537	no	13	1.0	no	negative
	yes	16	1.3	no	negative
TA 1535 exp I exp IA	no	15	1.3	no	negative
	yes	13	9.5	yes	positive
	yes	13	6.7	yes	positive
TA 100	no	116	1.0	no	negative
	yes	129	1.0	no	negative

Preincubation test (10 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	27	1.0	no	negative
	yes	37	1.2	no	negative
TA 1537	no	18	0.7	no	negative
	yes	24	9.6	no	positive
TA 1535	no	43	5.0	no	positive
	yes	29	4.4	yes	positive
TA 100	no	145	0.9	no	negative
	yes	162	1.0	no	negative

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Based on the study results, the substance is considered mutagenic in Salmonella typhimurium TA 1535 and TA 1537.

Executive summary:

It can be stated that during the mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and possibly frameshifts in the genome ofthe strains TA 1535 and TA 1537. Therefore, FAT 80'023/G (TADE) is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.