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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-02-02 - 1998-02-27 (experimental phase)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Screening version of Ames test (one plate per dose and strain)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The mutagenicity evaluation was performed using a screening version (one plate per dose and strain) of the Salmonella/microsome test, also termed the Ames Test, as described by Ames et al. (1973a, 1975) and Maron and Ames (1983).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
p-tert-butylaniline
EC Number:
212-215-9
EC Name:
p-tert-butylaniline
Cas Number:
769-92-6
Molecular formula:
C10H15N
IUPAC Name:
p-tert-butylaniline

Method

Target gene:
his-
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 mix
Test concentrations with justification for top dose:
16, 50, 158, 500, 1581, 5000 µg/plate (first trial)
40, 80, 160, 320, 640, 1280 µg/plate (independent repeat)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The used solvent was selected from a priority list in the order water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether and DMF.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
other: nitrofurantoin, 4-nitro-1,2-phenylene diamine (4-NPDA)
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 OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays):
Plates with selective agar were used.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, one plate was used.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

- OTHER
- Study controls: No "untreated" negative control was set up for the used solvent, since sufficient evidence was available in the literature (e.g. Maron and Ames, 1983) and from our own experience (see Chapter 8), indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.
The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation.
- Concentrations of positive controls:
Sodium azide (NaN3) - 10µg/plate for TA 1535
nitrofurantoin (NF) - 0.2 µg/plate for TA 100
4-nitro-1,2-phenylene diamine (4-NPDA) - 10 μg/plate for TA1537, 0.5 μg/plate for TA98
Cumene - 50 μg/plate for TA102
2-Aminoanthracene (2AA) - 3 μg/plate for all strains
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 that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. Otherwise, the result is evaluated 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.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Remarks:
historical data
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Remarks:
historical data
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Remarks:
historical data
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 of p-tert.-Butylaniline at doses of up to and including 80 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only partly be used for assessment purposes up to and including 1581 µg per plate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: positive
The mutagenicity evaluation was performed using a screening version (one plate per dose and strain) of the Salmonella/microsome test, also termed the Ames Test, as described by Ames et al. (1973a, 1975) and Maron and Ames (1983). 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 this sensitivity, indications of weak mutagenic effects of p-tert.-Butylaniline could be found at assessable doses of up to 1581 µg per plate in Salmonella typhimurium TA 100. In consequence, p-tert.-Butylaniline has to be regarded as mutagenic with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.
Executive summary:

p-tert.-Butylaniline was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37°C using doses of up to and including 1280 µg per plate. Other conditions remained unchanged.

Doses up to and including 80 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 1581 µg per plate for assessment purposes.

Evidence of mutagenic activity of p-tert.-Butylaniline was seen. On Salmonella typhimurium TA 100, a weak but reproducible 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 80 µg per plate. The Salmonella/microsome test thus showed p-tert.-Butylaniline to have a weak mutagenic effect.

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

Therefore, p-tert.-Butylaniline was considered to be mutagenic with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.