R-AMINR

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Preceeds OECD guideline

Deviations:

no

Remarks:

Test missing the strains with AT base pair at primary reversion site.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

419-800-1

EC Name:

-

Cas Number:

112101-81-2

Molecular formula:

C10H16N2O3S

IUPAC Name:

(R)-5-(2-Aminopropyl)-2-methoxybenzenesulfonamide

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Batches were checked for strain characteristics of histidine dependence, rfa character and resistance to ampicillin (TA98 and TA100).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
The S9 liver fraction was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, USA. Batches were stored frozen and thawed just prior to incorporation into the top agar. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens and CYP450 catalysed enzyme activities.

Test concentrations with justification for top dose:

Range-finder and Mutation experiment 1: 8, 40, 200, 1000 and 5000 µg/plate.
Mutation experiment 2: 312.5, 625, 1250, 2500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: standard as per OECD guideline

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Range-finder: The test substance was tested in TA100. Triplicate plates without and with S9 mix were used. Negative and positive controls were included in quintuplicate and triplicate respectively without and with S9 mix. These platings were achieved by the following sequence of additions to 2.5 mL molten agar at 46°C: 0.1 mL bacterial culture, 0.1 mL test article solution or control and 0.5 mL 10% S9 mix or buffer solution. This was followed by rapid mixing and pouring on to Minimal Davis agar plates. When set, the plates were inverted and incubated at 37°C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn and where possible revertant colonies were counted.

Main experiment: The test substance was tested in S. typhimurium strains TA98, TA100, TA1535 and TA1537 using triplicate plates with and without S9. Negative (solvent) controls wre included in both assays, in quintuplicate with and without S9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S9. The activity of the S9 mix used ine ach experiment was confirmed by AAN treatments (in triplicate) of at least 1 strain in the presence of S9. Platings were achieved as described for the range finder.

As the results of the first experiment were negative, treatments in the presence of S9 in experiment 2 included a pre-incubation step, where the quantities of test article or control solution, bacteria and S9 mixwere mixed together and incubated for 1 hour at 37°C, before tha ddition of 2.5 mL molten agar at 46°C. Plating of these treatments then proceeded as for the normal plate-incorporation rpocedure. This was performed to help increase the sensitivity of the assay.

Colony counting:
Colonies were counted electronically using a Seescan Colony Counter and the background lawn inspected for signs of toxicity.

Rationale for test conditions:

Standard as per OECD guideline

Evaluation criteria:

Treatment of data: Individual plate counts from both experiments were recorded seperately and the mean and SD of the plate counts for each treatment were determined.

The assay was considered valid if the following criteria were met:
1) The mean negative control counts fell within the normal ranges.
2) The positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains and an active S9 preparation.
3) No more than 5% of the plates were lost through contamination or some other unforeseen event.

The test article was considered to be mutagenic if:
1) The assay was valid.
2) Dunnett's test gave a significant response (P 3) The positive responses described were reproducible.

Statistics:

The m-statistic was calculated to check that the data were Poisson-distributed and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity at 5000 µg/plate (+/- S9)

Remarks:

Slight thinning of background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity at 5000 µg/plate (+S9)

Remarks:

Slight thinning of background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity at 5000 µg/plate (+S9)

Remarks:

Slight thinning of background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity at 5000 µg/plate (+/- S9)

Remarks:

Slight thinning of background lawn

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Toxicity and dose selection:
The range-finder was carried out with strain TA100 only at concentrations of 8, 40, 200, 1000 and 5000 µg/plate with negative and positive controls. No evidence of toxicity was observed, so the results were considered acceptable as mutagenicity data for this strain. Treatments of the remaining strains were performed using the same doses. Evidence of toxicity in the form of a thinning of teh background lawn was observed following 5000 µg/plate treatments with strain TA1537 ain the absence and presence of S9, and of strain TA98 in the presence of S9.

As evidence of toxicity was observed in 2 strains and solely at the maximum dose, this dose was retained as the maximum test dose for all the second experiment treatments (this dose being the best estimate of the limit of toxicity). A narrowed dose range was employed in order to more closely investigate those doses of the substance most likely to induce mutation. In addition, all experiment 2 treatments in the presence of S9 incorporate a pre-incubation step. Evidence of toxicity (in the form of a slight thinning of the background lawn) was observed in all strains at 5000 µg/plate in the presence of S9 and in TA98 and TA1537 at 5000 µg/plate in the absence of S9.

Mutation: The mean solvent controls fell within the normal historical ranges and the positive control chemicals all induced large increases in revertant numbers in the appropriate strains and that less than 5% of plates were lost, leaving adequate numbers of plates at all treatments. The study was accepted as valid.

No test substance treatment of any of the test strains in the absence or presence of S9 producted an increase in revertant numbers that was statistically significant at the 1% level. There was, therefore, no evidence of mutagenic activity of the substance in this study.

Applicant's summary and conclusion

Conclusions:

The test substance was unable to induce mutations in 4 strains of S. typhimurium when tested at concentrations up to 5000 µg/plate both in the absence and presence of a metabolic activation system.