N-(2-chloroethyl)-N-ethylaniline

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-01-31 - 1993-06-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as set out in EEC Directive 84/449/EEC, method B.14

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

as of 1983-05-26

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: New and Revised Health Effects Test Guidelines October 1984. HG - Gene Muta - S. typhimurium

Version / remarks:

October 1984

Deviations:

not specified

Principles of method if other than guideline:

The study was performed according to OECD 471 as adopted in 1983, so only four strains (instead of five strains including S. typhimurium TA 102) were used, which was first foreseen in the revised version of 1997. Consequently, the study does not show any deviations from the guideline as adopted in 1983, but from the most recent guideline.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-chloroethyl)-N-ethylaniline

EC Number:

245-082-0

EC Name:

N-(2-chloroethyl)-N-ethylaniline

Cas Number:

22564-43-8

Molecular formula:

C11H16ClN

IUPAC Name:

N-(2-chloroethyl)-N-ethylaniline

Test material form:

liquid

Method

Target gene:

his-

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: deep rough; reduced ability to repair damage from UV light

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 mix

Test concentrations with justification for top dose:

0, 8, 40, 200, 1000, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (test item); DMSO (positive controls)
- Justification for choice of solvent/vehicle: The used solvent was selected from a priority list in the order water, methanol, ethanol, acetone, DMSO, DMF and ethylene glycol dimethylether (EGDE).

Controls

Untreated negative controls:

yes

Remarks:

vehicle

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: nitrofurantoin; 4-nitro-1,2-phenylene diamine, 2-aminoanthracene

Remarks:

sodium azide 10 µg/plate (only TA1535), nitrofurantoin 0.2 µg/plate (only TA100), 4-nitro-1,2-phenylene diamine 10 µg/plate (only TA1537) / 0.5 µg/plate (only TA98), 2-aminoanthracene 3 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 30 sec
- Exposure duration: 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): his-negative agar

NUMBER OF REPLICATIONS: 4 plates per strain, dose, ± S9

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity of the substance was assessed in three ways. First background growth on the plates for mutant determination was inspected. If a reduction in background growth was observed, this indication for toxicity was indicated in the tables by the letter "B" after the mutant count. A single "B" without any numerical value for a mutant count represents four plates with reduced background growth at a given concentration. (The same applies to the symbols "C", "V", "P", "N" or "%", which may also appear in the tables.) Secondly, a toxic effect of the substance was assumed when the mutant count per plate was reduced significantly and in a dose-dependent manner as compared to the corresponding negative control. The third criterion was the bacteria titer. Total bacterial counts were taken on two plates with S9 mix for each concentration studied. If a test was performed only without S9 mix, however, the bacterial count was taken on plates without S9 mix.

Evaluation criteria:

A test is defined as being positive if a reproducible and dose-related increase of mutant colony numbers becomes apparent for at least one strain. For TA 1535, TA 100 and TA 98 mutant colony numbers should increase by a factor of two or more over negative control numbers, while at least a three-fold increase should be apparent for TA 1537. Otherwise, the result is judged as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: TA 1535 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: TA 1537 and TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was no indication of a bacteriotoxic effect at doses of up to and including 40 µg per plate. The total bacteria counts consistently produced results in the range of the negative controls, or differed only insignificantly. No growth inhibition was observed. Higher doses revealed a weak bacteriotoxic effect. Nevertheless, doses up to 2000 µg per plate could still be used for assessment purposes.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: positive
Testing for mutagenicity in bacteria was performed according to OECD guideline 471 as adopted 1983-05-26. Although not being conducted to recent guidelines, the test was conducted scientifically reasonable with negligible deficiencies. Also, the testing was sufficiently documented, positive and negative controls gave the appropriate response. Hence, the results can be considered as sufficiently reliable to assess the mutagenic potential of the test substance in bacteria. Due to the sensitivity, clear indication of mutagenic effects for the test substance could be found at assessable doses up to 2000 µg per plate for the two strains TA 1535 and TA 100. In consequence, the test substance is considered to be mutagenic under the conditions of this test.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471), histidine-auxotrophic strains TA1535, TA100, TA1537 and TA98 of S. typhimurium were exposed to the test substance in ethanolat concentrations of 0, 8, 40, 200, 1000, 5000 µg/plate in the presence and absence of mammalian metabolic activation (induced S9) via plate co-incubation.

The positive controls induced the appropriate responses in the corresponding strains.

Doses of up to 40 µg of test substance per plate did not induce bacteriotoxic effect in the Salmonella/microsome test: Total bacteria counts remained unchanged and no growth inhibition was observed. the substance revealed weak bacteriotoxic effects at higher doses but doses up to 2000 µg per plate could still be used for assessment purposes.

Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility), revealed clear, biologically relevant variations from the respective negative controls for TA 1535 and TA 100. These were regarded as mutagenic effects of the test substance. Since the lowest reproducible effective dose was in the low dose range, and a mutagenic effect was noted both in Salmonella typhimurium TA 1535 and TA 100, the Salmonella/microsome test showed the test substance to be a strong and definite mutagen.

In spite of the low doses used, positive controls increased the mutant counts significantly over negative control levels which demonstrated the system's high sensitivity.

Due to the sensitivity, clear indication of mutagenic effects for the test substance could be found at assessable doses up to 2000 µg per plate for the two strains TA 1535 and TA 100.

This study is classified as acceptable; it satisfies the requirement for Test Guideline OECD 471 (as adopted 1983-05-26) for in vitro mutagenicity (bacterial reverse gene mutation) data with minor deviations from the recent Guideline. According to REACH Annex VII column 2, further mutagenicity studies shall be considered in case of a positive result. However, further tests can be omitted as "de-risking" in mammalian cells / in vivo is not necessary, because the substance is only registered as an intermediate under strictly controlled conditions, where exposure is controlled anyway.