Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 September 2002 to 05 November 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch no: AAQ0195UN

Method

Target gene:
his G 46 in TA 1535 and TA 100
his C 3076 in TA 1537
his D 3052 in TA 1538 and TA 98
E.coli WP2uvrA/pKMl01 ochre mutation
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 Fraction
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 Fraction
Test concentrations with justification for top dose:
First test (range-finding)
BMS 482204-03 (MSA salt) was added to cultures of the five tester strains at seven concentrations separated by ca half-log10 intervals. The highest concentration of BMS 482204-03 (MSA salt) tested was SO mg/ml in the chosen solvent, which provided a final concentration of 5000 µg/plate.

Second Test
Top concenetration was 5000 µg/plate
Vehicle:
Purified water
Controls
Negative controls:
yes
Remarks:
Purified water
Solvent controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
furylfuramide
other: 2-Aminoanthracene
Details on test system and conditions:
First test (range-finding)
BMS 482204-03 (MSA salt) was added to cultures of the five tester strains at seven concentrations separated by ca half-log10 intervals. The highest concentration of BMS 482204-03 (MSA salt) tested was SO mg/ml in the chosen solvent, which provided a final concentration of 5000 µg/plate. This is the standard limit concentration recommended in the regulatory guidelines this assay follows. The negative control was the chosen solvent, water. The appropriate positive controls were also included.

Aliquots of 0.1 ml of the test dilution, positive control or negative control were placed in glass vessels. S9 mix (O.S ml) or 0.1 M pH 7.4 phosphate buffer (O.S ml) was added, followed by 0.1 ml of a 10 hour bacterial culture and 2 ml of agar containing histidine (O.S mM), biotin (O.S mM) and tryptophan (O.S mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 ml minimal agar. Each Petri dish was individually labelled with a unique code corresponding to a sheet, identifying the contents of the dish. Three Petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of BMS 482204-03 (MSA salt), S9 mix and sodium phosphate buffer. All plates were incubated at
37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined
and revertant colonies counted using a Domino automated colony counter.

Any toxic effects of BMS 482204-03 (MSA salt) would be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration normally used in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at the top concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating dose levels should be obtained, unless otherwise justified by the Study Director.

Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The top concentration chosen was again 5000 µg/plate, but only five concentrations were used.

Third test
As a statistically significant response was obtained in the second test, a third test was conducted in strain WP2uvrA/pKMIOI (CM891) only. The test procedure was exactly the same as was used in the second test.
Evaluation criteria:
For a test to be considered valid, the mean of the solvent/vehicle control revertant colony numbers for each strain should lie within the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range will be maintained as a rolling record over a maximum of five years. Also, the positive control compounds must cause at least a doubling of mean revertant colony numbers over the negative control.

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA 1535 and TAJ 537) the concurrent solvent/vehicle controls, with some evidence of a positive dose-response relationship, it will be considered to exhibit mutagenic activity in this test system. No statistical analysis will be performed.

If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed,

If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al ( 1989) and will usually be Dunnett' s test followed, if appropriate, by trend analysis. Biological significance should always be considered along with statistical significance. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
increases did not meet all the criteria for a positive response, but analysis of the data using Dunnett's test indicated that the counts obtained at the highest concentration were significantly different from the control counts
Cytotoxicity:
no
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

The results obtained with BMS 482204-03 (MSA salt) and positive control compounds are presented in Tables I to 11.  The mean values quoted have been corrected to the nearest whole number. The absence of colonies on sterility check plates confirmed the absence of microbial contamination. The total colony counts  on nutrient agar plates (see Tables) confirmed the viability and high cell density of the cultures of the individual organisms.

The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory.  Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

First TEST (RANGE-FINDING)

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to BMS 482204-03 (MSA salt) at any concentration in either the presence or absence of S9 mix. No visible thinning of the background lawn of non-revertant cells was obtained following exposure to BMS 482204-03 (MSA salt).  A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.

SECOND TEST

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to BMS 482204-03 at any concentration in either the presence or absence of S9 mix.  Small increases (reaching 1.6 x control counts) were seen in strain WP2uvrA/pKM101 (CM891) in the presence of S9 mix.  These increases did not meet all the criteria for a positive rnsponse, but analysis of the data using Dunnett's test indicated that the counts obtained at the highest concentration were significantly different from the control counts.

No visible thinning of the background lawn of non-revertant cells was obtained following exposure to BMS 482204-03

THIRD TEST 

Small increases in reversion to prototrophy (reaching 1.3 x control counts) were again seen in strain WP2uvrA/pKM101 (CM891) in the presence of S9 mix. These increases did not meet all the criteria for a positive response, but analysis of the data using Dunnett's test indicated that the counts obtained at the highest concentration were significantly different from the control counts. 

No visible thinning of the background lawn of non-revertant cells was obtained following exposure to BMS 482204-03 

Applicant's summary and conclusion

Conclusions:
It is concluded that, under the test conditions employed, BMS 482204-03 (MSA salt) did not exhibit mutagenic activity in this bacterial system.
Executive summary:

In this in vitro assessment of the mutagenic  potential of BMS 482204-03 (MSA  salt),  histidine dependent auxotrophic mutants of Salmonella typhimurium, strains  TAl535, TAl537,  TA98  and TAIOO, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKMIOI (CM891), were exposed  to BMS 482204-03 (MSA salt) diluted in water.   Water was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-treated rats (S9 mix).  The first (range-finding) test was a standard plate incorporation assay; the second involved a pre-incubation stage.  A third test, in strain WP2uvrA/pKM101 (CM891), was also performed in order to confirm the results of the second test.

Concentrations of BMS 482204-03 (MSA salt) up to 5000 µg/plate were tested.  This is the standard limit  concentration recommended in  the  regulatory guidelines  that this  assay  follows.    Other concentrations used were a series of ca half-logj., dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test.

Small increases in reversion to prototrophy (reaching only 1.6 x and 1.3 x controls  in tests 2 and 3 respectively) were seen in strain WP2uvrAlpKMIOI (CM891) in the presence of 89 mix following pre­ incubation. Although analysis of the data using Dunnett's test indicated that the counts obtained at the highest concentration were significantly different from the control counts in both tests, these results did not meet all the criteria for a positive response.

No evidence of mutagenic activity was seen at any concentration of BMS 482204-03 (MSA salt) in any other strain in either mutation test.  The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, BMS 482204-03 (MSA salt) did not exhibit mutagenic activity in this bacterial system.