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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1984

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Inveresk Research International
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material: Nicotinic acid

Method

Target gene:
Five strains of Salmonella typhimurium were used:
S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
They were obtained in 1976 from Professor B.N. Ames, Department of Biochemistry. University of California, Berkeley, CA., U.S.A., and stored in liquid nitrogen since that time until used.
All these strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. Three mutations in the histidine operon are involved:
his G 46 in TA 1535 and TA 100
his C 3076 in TA 1537
his D 3052 in TA 1538 and TA 98
All 5 strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharide coat of the bacterial cell surface. This deletion increases cell permeability to more hydro phobic substances and, furthermore, greatly decreases the pathogenicity of these organisms.
The second deletion through uvrB, renders the organisms incapable of DNA excision repair and thus more susceptible to mutagenicity. These 2 deletions include the nitrate reductase (chl) and biotin (bio) genes also. Differences between TA 1535 and TA 1538, on the one hand, and the corresponding TA 100 and TA 98 strains on the other hand, are due to a plasmid the latter pair contains. A plasmid, R-Utrecht, was originally shown to increase the sensitivity of the his G 46 mutation in S. typhimurium to methyl methanesulphonate and trimethyl phosphate. The particular R-factor in TA 100 and TA 98 carried resistance to ampicillin. It is not yet clear why the presence of this particular R-factor should increase the sensitivity of strains TA 1535 and TA 1538 to the mutagenicity of certain chemicals. The involvement of an error-prone repair mechanism has been postulated.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel-Bonner Medium E agar plates with 2% glucose obtained from Gibco Europe limited, Paisley, Scotland.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
- Type and identity of media: Vogel-Bonner Medium E agar plates with 2% glucose obtained from Gibco Europe limited, Paisley, Scotland.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver preparation and co-factors (S9 mix)
Test concentrations with justification for top dose:
TOXICITY TEST
A toxicity test using strain TA 100 only was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests. One plate of each of the following concentrations of P0076D was used: 33 ug, 100 ug, 333 ug, 1000 ug, 3333 ug and 10000 ug per plate.

MUTATION ASSAY
Two independent mutation tests were conducted using 5 bacterial strains (TA 1535, TA 1537, TA 1538, TA 98 and TA 100). The dose levels used in both of these experiments and selected on the basis of the results of the toxicity test, were 33 ug, 100 ug, 333 ug, 1000 ug, 3333 ug and 10000 ug per plate. Triplicate plates were prepared for each bacterial strain and dose level in both the presence and absence of S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulphoxide (DMSO) at 0.1 mL per plate, exception sodium azide dissolved in sterile, distilled water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
without and with S9
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
1st test: all strains at 0.5 ug per plate with S9; 2nd test: TA 1537 at 1.0 ug per plate with S9
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
1st and 2nd test: TA 1535 and TA 100 at 1 ug per plate
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
1st and 2nd test: TA 1538 and TA 98 at 1 ug per plate
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
1st and 2nd test: TA 1537 at 20 ug per plate
Details on test system and experimental conditions:
METHOD OF APPLICATION
- plate incorporation

DURATION
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays)
- Histidine auxotrophs. Further, each strain was tested for its resistance to ampicillin (indicating the presence of pKM101) and sensitivity to crystal violet (indicating persistence of the rfa mutation).

NUMBER OF REPLICATIONS
- Two independent mutation tests were conducted. Triplicate plates were prepared for each bacterial strain and dose level in both the presence and absence of S9 mix.

DETERMINATION OF CYTOTOXICITY
- A toxicity test using strain TA 100 only was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests.
Evaluation criteria:
A test was considered acceptable if for each strain:
i) the bacteria demonstrated their typical responses to crystal violet and ampicillin.
ii) at least 2 of the vehicle control plates were within the following ranges: TA 1535, 1-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and TA 1538, 10-35.
iii) on at least 2 of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or for TA 100, x 1.5 the mean vehicle control mutant numbers per plate.
If the mean colony count on the vehicle control plate was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates.
iv) no toxicity or contamination was observed in at least 4 dose levels.
v) no more than one dose level was discarded before the highest significant mean colony number was achieved.

Where these criteria were met, a significant mutagenic response was recorded if there was:
i) for S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolizing enzymes where mutagens require metabolic activation by the liver.
iii) a reproducible effect in independent tests.

Results and discussion

Test resultsopen allclose all
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TOXICITY TEST
The sample was not toxic to the background lawn of bacteria either in the presence or absence of S9 mix when tested to a highest dose level of 10000 ug per plate.

MUTATION TESTS
Quality Control
All strains of S. typhimurium were sensitive to crystal violet, whereas only the plasmid-containing strains, TA 98 and TA 100, were resistant to ampicillin. These results are consistent with the known properties of these bacteria.
Vehicle Control Groups
The vehicle control values were within the normal ranges experienced in this laboratory and reported in the literature with these strains of S. typhimurium, except in one instance as detailed below (see Test Rejection).
Positive Control Groups
The results obtained with all positive controls in both tests were within the normal ranges expected for each bacterial strain and metabolic activation state.
Test Rejection
All tests were acceptable according to the study criteria except:
Test No 1: Strain TA 1538 without S9 Mix
Reason for Rejection: Inadequate vehicle control values

The results of the first test on the test item showed that the test substance did not induce significant increases in revertant numbers over vehicle control values in any strain of bacteria either in the presence or absence of S9 mix. No toxicity to the background lawn of bacteria was noted in this test.
The results of the second test confirmed that the test item did not give significant increases in revertant numbers in any of the strains used either in the presence or absence of S9 mix. No toxicity to the background lawn of bacteria was noted in this test.
Inadequate vehicle control values were obtained in the first test with strain TA 1538 in the absence of S9 mix, so a repeat test was performed with this strain. The results of this repeat test showed that the test item did not give significant increases in revertant numbers in strain TA 1538 in the absence of S9 mix. The test substance was not toxic to the bacteria in this repeat test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item was not mutagenic in any of the 5 strains of S. typhimurium used either in the presence or absence of S9 mix. No toxicity to the background lawn of bacteria was recorded in this study.
Executive summary:

A study according to EU Method B.13/14 and OECD Guideline 471 (Bacterial Reverse Mutation Assay) was carried out. The test item was assessed for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations ranging from 33 ug to 10000 ug per plate.

The tests were conducted on agar plates in the presence and absence of an Aroclor 1254-induced rat liver preparation and co-factors (S9 mix) required for mixed function oxidase activity. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolizing activity of the S9 mix.

No toxicity to the bacteria was observed in any strain of bacteria. The test item was not mutagenic in any of the bacterial strains used in this study.