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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:
1985

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
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: Nicotinamide

Method

Target gene:
All 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 TA 1538 and TA 98
- his G 46 is a mis-sense mutation which is reverted to prototrophy by a variety of mutagens that cause base-pair substitutions.
- his C 3076 contains a frameshift which appears to have added a G/C basepair resulting in GGGG/CCCC. Tris mutation is reverted by 9-aminoacridine, ICR-191 and epoxides of polycyclic hydrocarbons.
- his D 3052 also contains a frameshift mutation with the sequence GCGCGC/GCGCGC-which is reverted with the deletion of 2 base-pairs, CG/GC. It is readily reverted by aromatic amines and derivatives.
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 hydrophobic 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
- Growth medium: Nutrient broth 8 g Difco Bacto nutrient broth, 5 g NaCl/L
- Test medium: Diluted agar (0.6 % Difco Bacto-agar, 0.6 % NaCl), autoclaved and just before use, 5 mL of sterile 1.0 mM L-histidine.HC1, 1.0 mM biotin solution was added to each 100 ml of soft agar and thoroughly mixed. This molten agar, maintained in a water bath at 45 °C (maximum)was dispensed in 2 ml volumes into small sterile tubes to which was added in order:
0.5 mL S9 mix or 0.05 M phosphate buffer, pH 7.4
0.1 mL bacteria (ca. 2.0E09 cells/mL)
0.1 mL solvent or test solution
The tube contents, which were continually cooling, were mixed and then poured in minimal medium plates obtained from Gibco Europe Limited, Paisley, Scotland. These plates contained 20 ml of 1.5 % Difco Bacto-agar in Vogel-Bonner Medium E (2) with glucose. When the soft agar had set, the plates were inverted and incubated at 37 °C for 2 days whereupon colonies were counted using a Biotran III automated counter (New Brunswick Inc., N.J., U.S.A.) at maximum sensitivity i.e. colonies of 0.1 mm or more in diameter counted. The plates were also examined for precipitates and, microscopically for microcolony growth.
- Properly maintained: yes
Additional strain / cell type characteristics:
nitroreductase deficient
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
TYPE AND IDENTITY OF MEDIA
- Growth medium: Nutrient broth 8 g Difco Bacto nutrient broth, 5 g NaCl/L
- Test medium: Diluted agar (0.6 % Difco Bacto-agar, 0.6 % NaCl), autoclaved and just before use, 5 mL of sterile 1.0 mM L-histidine.HC1, 1.0 mM biotin solution was added to each 100 ml of soft agar and thoroughly mixed. This molten agar, maintained in a water bath at 45 °C (maximum)was dispensed in 2 ml volumes into small sterile tubes to which was added in order:
0.5 mL S9 mix or 0.05 M phosphate buffer, pH 7.4
0.1 mL bacteria (ca. 2.0E09 cells/mL)
0.1 mL solvent or test solution
The tube contents, which were continually cooling, were mixed and then poured in minimal medium plates obtained from Gibco Europe Limited, Paisley, Scotland. These plates contained 20 ml of 1.5 % Difco Bacto-agar in Vogel-Bonner Medium E (2) with glucose. When the soft agar had set, the plates were inverted and incubated at 37 °C for 2 days whereupon colonies were counted using a Biotran III automated counter (New Brunswick Inc., N.J., U.S.A.) at maximum sensitivity i.e. colonies of 0.1 mm or more in diameter counted. The plates were also examined for precipitates and, microscopically for microcolony growth.
- Properly maintained: yes
Additional strain / cell type characteristics:
nitroreductase deficient
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced microsomal S-9 fraction
Test concentrations with justification for top dose:
Two independent tests were carried out, each run in triplicate.
- 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 test item was used:
33 ug, 100 ug, 333 ug, 1000 ug, 3333 ug and 10000 ug per plate
- Mutation test: 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.
- Vehicle control: Dimethylsulphoxide, 0.1 mL per plate, used as the test compound vehicle, in both the presence and the absence of S9 mix.
- Positive control: 2-Aminoanthracene, 0.5 ug per plate with all strains except strain TA 1537, was used to demonstrate activity of the S9 mix and the mutability of the bacteria. A dose of 1 ug of 2-Aminoanthracene was used for strain TA 1537, because of uncertainty as to whether this strain would give an appropriate response at the lower dose Sodium azide, 1 ug per plate, with TA 1535 and TA 100; 2-nitrofluorene, 1 ug per plate, with TA 1538 and TA 98; 9-aminoacridine, 20 ug per plate, with TA 1537. These substances served as an aid to strain identification and to demonstrate the mutability of the bacteria.
Vehicle / solvent:
- Vehicle used: Dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene
Remarks:
All controls run in triplicate with and without S-9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- In agar (plate incorporation)

DURATION
- Preincubation period: None
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): L-histidine

NUMBER OF REPLICATIONS:
- Test and control cultures in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Number of his+ revertant colonies counted per plate

QUALITY CONTROL
At the times that the experiments were conducted, 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).
Evaluation criteria:
A test was considered acceptable if for each strain:
i) the bacteria demonstrated their typical responses to crysta1 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 in the case of TA 100, x 0.5 the mean vehicle control mutant numbers per plate. 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 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
Remarks:
Highest concentration 10000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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
Remarks:
Highest concentration 10000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
- 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: The vehicle control values were within the normal ranges experienced in this laboratory and reported in the literature with these strains of S. typhimurium.
- 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 item: The results of the first test showed that the test sample did not induce 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.
The results of the second test confirmed that the test item did not induce significant increases in revertant numbers in any of the strains used in the presence or absence of S9-mix. No toxicity to the background lawn of bacteria was noted on this occasion.
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 bacteria used in this study, either in the presence or absence of S9-mix. No toxicity to the background lawn of bacteria was noted in this study.
Executive summary:

A study was carried out according to EU Method B.13/14 and OECD Guideline 471 (Bacterial Reverse Mutation Assay). The test item was tested 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 metabolising activity of the S9 mix. The test item was neither toxic nor mutagenic in any of the strains of bacteria used in this study either in the presence or absence of S9 mix.