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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1995-06-14 to 1995-08-30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The reliability of the original study used in Read Across is 1. Justification for Read Across is given in Section 13 of IUCLID.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Nutrient medium: 8 g Merck Nutrient Broth and 5 g NaCl per liter,
Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal agar was used as selective agar,
Overlay Agar: 6.0 g Merck Agar Agar, 6.0 g NaCl, 10.5 mg L-Histidine, 12.2 mg Biotin per liter.
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen:
Experiment I: 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate
Experiment II: 33.3, 66.6, 100, 166.6, 333.3 and 1000 µg/plate
Experiment IIa (strains TA 1535 and TA 1537): 3.3, 10.0, 33.3, 66.6, 100.0 and 166.6 µg/plate
Vehicle / solvent:
On the day of the experiment, the test article was dissolved in aqua bidest. The solvent was chosen because of its solubility properties.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9, TA 1535 and TA 100, 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-NOPD
Remarks:
without S9, TA 1537 and TA 98, 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9, all strains, 2.5 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C.

NUMBER OF REPLICATIONS: 3 plates/strain/dose level

DETERMINATION OF CYTOTOXICITY: toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

ACCEPTED CONDITIONS FOR EVALUATION:
- corresponding background growth on both negative and test plates
- normal range of spontaneous reversion rates


Rationale for test conditions:
N.A.
Evaluation criteria:
A test article is considered positive if either a dose related and/or reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test article producing neither a dose related nor reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A significant response is described as follows: a test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strain TA 100 or thrice on TA 1535, TA 1537, and TA 98. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.
Statistics:
According to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Distinct toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. All plates incubated with the test article showed reduced background growth starting at concentrations as low as 33.3 µg/plate in some of the strains. Substantial increases in revertant colony numbers were observed following treatment with the test item with and without metabolic activation in all strains used. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Any other information on results incl. tables

Table 1: Summary of Results without S9 mix
Revertants/plate
mean from three plates
Concentration
 µg/plate
TA 1535 TA 1537 TA 98 TA 100 TA 1535 TA 1537
I II I II I II I II IIa Iia
Negative Control 17 17 5 13 44 30 141 125 16 10
Solvent Control 15 14 7 14 43 41 149 126 15 11
Positive Control 360 843 37 47 279 146 870 767 537 50
3.3 / / / / / / / / 19 8
10 / / / / / / / / 43 15
33.3 21 34 16 30 35 53 718 775 28 27
66.6 / 17 / 31 / 173 / 1083 9 12
100 7 9 26 23 72 161 303 1086 6 20
166.6 / 6 / 12 / 92 / 245 6 12
333.3 0 3 6 19 54 195 895 272 / /
1000 0 3 0 3 38 127 230 42 / /
2500 0 / 0 / 26 / 76 / / /
5000 10 / 0 / 30 / 65 / / /

/= not performed

Table 2: Summary of Results with S9 mix
Revertants/plate
mean from three plates
Concentration
 µg/plate
TA 1535 TA 1537 TA 98 TA 100 TA 1535 TA 1537
I II I II I II I II IIa Iia
Negative Control 16 19 6 21 48 43 147 149 12 16
Solvent Control 20 16 7 18 50 46 155 168 15 19
Positive Control 221 372 261 149 629 849 899 1209 206 126
3.3 / / / / / / / / 17 19
10 / / / / / / / / 20 21
33.3 47 70 26 35 51 158 192 209 74 38
66.6 / 63 / 37 / 129 / 318 17 39
100 12 13 15 38 39 176 308 428 12 41
166.6 / 7 / 13 / 280 / 620 8 17
333.3 0 4 7 11 52 284 472 444 / /
1000 0 1 0 10 44 311 293 164 / /
2500 0 / 0 / 23 / 176 / / /
5000 0 / 0 / 26 / 181 / / /

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100. Therefore, the test item is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to the test item in water at concentrations of 33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate (experiment I) and at concentrations of 33.3, 66.6, 100, 166.6, 333.3 and 1000 µg/plate (experiment II) in the presence and absence of mammalian metabolic activation. Strains TA 1535 and TA 1537 were additionally tested in at concentrations of 3.3, 10, 33.3, 66.6, 100 and 166.6 µg/plate (experiment IIa). The test item was tested up to the limit concentration (5000 µg/plate). Due to overlapping toxic effects, the number of revertants was reduced at the higher concentrations in the strains TA 1535, TA 1537, and TA 100 in experiments I, II and IIa.

The positive controls induced the appropriate responses in the corresponding strains. Substantial increases in revertant colony numbers were observed following treatment with the test item with and without metabolic activation in all strains used.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains TA 1535, TA 1537, TA 98, and TA 100. Therefore, the test item is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay. This study is classified as acceptable. The study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.