ethyl N2-dodecanoyl-.sc.l.sc.-argininate hydrochloride

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:

From January 18, 1995 to August 24, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-630-6

EC Name:

-

Cas Number:

60372-77-2

Molecular formula:

Hill formula: C20H41N4O3Cl

IUPAC Name:

ethyl N2-dodecanoyl-l-argininate hydrochloride

Test material form:

liquid

Details on test material:

- Purity: 25% LAE
- Physical state: Transparent to slightly opalescent liquid
- Lot/batch No.: 3
- Expiration date of the lot/batch: for 6 months from 1995-03-24
- Stability under test conditions: c.a. 4ºC in the dark

Method

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 rats

Test concentrations with justification for top dose:

TEST 1: Preliminary test
Concentration range (with metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Concentration range (without metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate

TEST 2: Mutation test
Concentration range (with metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
Concentration range (without metabolic activation): 5, 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

Solvent: Water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

Without methabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

TA1535; TA100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

other: 2-Aminoanthracene

Remarks:

All strains

Details on test system and experimental conditions:

METHOD OF APPLICATION:

TEST 1: Preliminary test: in agar (plate incorporation)
An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S-9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solution was added, followed immediately by 2 ml of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. A single petri dish was used for each dose level. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period the appearance of the background bacterial lawn was examined. Revertant colonies were counted using a Seescan Automatic Colony Counter.

NUMBER OF REPLICATIONS: 3


TEST 2: MAIN TEST: in agar (plate incorporation)
An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S-9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solution was added, followed immediately by 2 ml of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S-9 mix or phosphate buffer (0 /tg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period revertant colonies were counted using a Seescan Automatic Colony Counter.


NUMBER OF REPLICATIONS: 3

Evaluation criteria:

The mean number of revertant colonies for each treatment group was compared with those obtained for the solvent/vehicle control groups. The mutagenic activity of a test substance was assessed by applying the following criteria:
a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, the test substance will be considered to show evidence of mutagenic activity in this test system. No statistical analysis will be performed.
b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent/vehicle controls in either mutation test, the test substance will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.
c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs a) and b), even after the additional testing outlined in the mutation test procedure, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at a maximum exposure to LAE at 5000 and 500 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Concentrations of test substance up to 5000 µg/plate were tested in the first range-finder test.
The range-finder test was repeated using lower concentrations, up to 150 µg/plate, because of observed toxicity. Both tests were standard plate incorporation assays.
A second main test was conducted at concentrations up to 150 µg/plate. This test involved a pre-incubation stage.

RANGE-FINDING/SCREENING STUDIES:
The revertant colony counts obtained in the preliminary toxicity test was observed. The test substance was toxic at the highest concentration towards tester strains TA 98 and TA 100, and towards TA 1537 in the presence of S-9 mix only. Toxicity was also seen towards TA 98 at 500 µg/plate in the absence of S-9 mix. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests followed by a further five dose levels.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Following treatment with the test substance in the first mutation test, toxicity was observed towards all the strains between 5000 and 500 µg/plate, except TA 1535 at 500 µg/plate in the presence of S-9 mix. Therefore the range of dose levels selected was changed, and the highest concentration was reduced to 500 µg/plate.

Applicant's summary and conclusion

Conclusions:

The in vitro assessment of the mutagenic potential of the test substance in a bacterial system performed according to OECD Guideline 471 of Genetic Toxicology on Bacterial Reverse Mutation Test. The test substance showed no evidence of mutagenic activity observed in the Bacterial reverse Mutation Test with and without metabolic activation tested for strains of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100).

Executive summary:

This report describes a study designed to assess the mutagenic potential of the test substance in a bacterial system. The study was conducted in compliance with the following guidelines: OECD Guidelines for the Testing of Chemicals (1997) Genetic Toxicology: Bacterial Reverse Mutation Test, Guideline 471.

In this in vitro assessment of the mutagenic potential of the test substance, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test substance, diluted in water which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary toxicity test with dose levels of up to 5000 µg/plate toxicity was observed at the highest concentration towards TA 98 and TA 100, and towards TA 1537 in the presence of S-9 mix only. Toxicity was also seen towards TA 98 at 500 µg/plate in the absence of S-9 mix. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50, 15, 5 µg/plate.

Following treatment with the test substance in the first mutation test, toxicity was observed towards all the strains between 5000 and 500 µg/plate, except TA 1535 at 500/µg/plate in the presence of S-9 mix. Therefore the range of dose levels selected was changed, and the highest concentration was reduced to 500 µg/plate.

Following treatment with the test substance in the second mutation test, toxicity was observed towards all the strains at 500 µg/plate, except TA 1535 in the presence of S-9 mix.

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with the test substance at any dose level, in the presence or absence of S-9 mix, in either mutation test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparation.

It is concluded that, when tested in water, the test substance was not mutagenic in this bacterial system.