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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:
24 March to 31 March, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
study conducted on the analogue substance; the read across justification is detailed in section 13. The Reliability of the Source Study is 1.
Justification for type of information:
The complete read-across justification is detailed in section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
-Toxicity test: 5000, 1580, 500, 158 and 50.0 μg/plate
- Main test: 5000, 2500, 1250, 625 and 313 µg/plate.
On the basis of the results obtained in the preliminary toxicity test, 5000 µg/plate was selected as the top concentration for the Main Assay.
Vehicle / solvent:
Sterile water for injection and Dimethylsulfoxide (DMSO)
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
without metabolic activation (S9)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation system (S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
-Nutrient Broth: Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5 % in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
-Nutrient Agar: Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
-Minimal Agar: Minimal medium agar was prepared as 1.5 % Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
-Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10 mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 mL).

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assay. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatment was performed both in the absence and presence of S9 metabolism (Rat Mixed Induction) using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

SOLUBILITY
Solubility of the test item was evaluated in a preliminary trial using distilled water.

INCUBATION AND SCORING
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, plates were immediately scored by counting the number of revertant colonies and analysing the background lawn on each plate.

Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as: y = a +bx
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
SOLUBILITY
A clear solution was obtained after 30 minute sonication at 37 °C at the concentration of 50.0 mg/ml. This permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

TOXICITY TEST
No precipitation of the test item was observed at the end of the incubation period at any concentration tested, in the absence or presence of S9 metabolic activation. Plates treated with the test item showed a dose dependent brown color of the agar, which did not interfere with the scoring of colonies or the evaluation of thinning of the background lawn.
No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.
Large increases in revertant numbers were observed both in the absence and presence of S9 metabolic activation with TA1537, TA98 and TA100 tester strains. In the presence of S9 metabolism, increases in revertant numbers were also observed for TA1535 tester strain.

MAIN TEST
No toxicity was observed at any dose level, with any tester strain, in the absence or presence of rat liver S9 metabolic activation. Plates treated with the test item presented a dose dependent brown color of the agar, which did not interfere with the scoring of colonies.
The test item induced large increases in the number of revertant colonies with TA1537, TA98 and TA100 tester strains, both in the absence and presence of S9 metabolism. A moderate increase (2.9-fold) was also observed with TA1535 tester strain in the presence of S9 metabolism.
These increases were dose-related and the observed revertant numbers fell out the historical control range.
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions.
Marked increases in revertant numbers were obtained in the test following treatment with the positive control items, indicating that the assay system was functioning correctly.
Since a clear positive response was observed, no further experiment was undertaken.

VALIDITY CRITERIA
The study was accepted as valid:
- Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.
- The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain.
- No plates were lost through contamination or cracking.

EVALUATION CRITERIA
The test item induced more than two-fold increases in the number of revertant colonies with TA98, TA100 and TA1537 tester strains, both in the absence and presence of S9 metabolism and with TA1535 in its presence. A statistically significant dose-effect relationship was indicated. Since a clear positive response was observed, no further experiment was undertaken.

Applicant's summary and conclusion

Conclusions:
The test item induces reverse mutation in bacteria both in the absence and presence of S9 metabolism, under the reported experimental conditions.
Executive summary:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The test was performed according to the OECD Guideline 471 (1997) and the EU method B.13/14 of EC 440/2008. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in sterile water for injection. The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals. On the basis of toxicity test results, in the Main Assay using the plate incorporation method, the test item was assayed at five dose levels from 313 to 5000 µg/plate. Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.

The test item induced dose-related and large increases in the number of revertant colonies in TA1537, TA98 and TA100 tester strains both in the absence and presence of S9 metabolism. Positive increases in the number of revertant colonies (2.9-fold) were also observed with TA1535 tester strain in the presence of S9 metabolism. All the validity criteria were met. Since a clear positive response was observed, no further experiment was undertaken. It is concluded that the test item induces reverse mutation in bacteria both in the absence and presence of S9 metabolism, under the reported experimental conditions.