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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
Study period:
January 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: modified OECD 471 protocol (Ames II), non-GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Principles of method if other than guideline:
Liquid fluctuation Ames II test with and without liver S9 mix from induced male Wistar rats.
The test method is used to evaluate the mutagenic potential based on the ability to induce point mutations in selected loci of several strains of Salmonella typhimurium in a modified version of the Ames test (OECD 471), designated Ames II Assay (microtiter version), both with and without the addition of a metabolizing system (S9 mix) obtained from liver from induced rats.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Solid, yellow
- Storage condition of test material: Room temperature

Method

Target gene:
his
Species / strain
Species / strain / cell type:
other: TA 98, TA Mix (TA 7001 - TA 7006)
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix
Test concentrations with justification for top dose:
O; 4; 20; 100; 500; 2500 and 5000 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (with S9) and 2-nitrofluorene plus 4-nitroquinoline- N-oxide (without S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Bacteria from overnight cultures showing an optical density of at least 2.0 (measured at a wavelength of 600 nm) were used. 5 ml of the overnight cultures were added to tubes containing 25 ml Ames II Exposure medium and were gently mixed. After thorough pipetting bactiera suspension, test substance (or vehicle or control) and S9 mix (if applicable) were added. The 24-well plates were incubated at 37°C with shaking at 250 rpm for about 90 minutes. After this incubation period, 2.8 ml Ames II Reversion indicator medium (containing bromocresol purple) was pipetted to each well of the 24-well plate. The contents of each well of the 24-well plates were distributed in 50 μl aliquots over 48 wells of a 348-well Revertant Colony Selection plate (RCSP). The plates were sealed in plastic bags and incubated at 37°C in the dark. After 48 hours incubation each 48-well section of the RCSP were scored and the number of positive wells (yellow= high number of his+ revertants) were counted.

NUMBER OF REPLICATIONS: 48 wells in triplicate plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: beckground lawn, decrease in the number of yellow wells
Evaluation criteria:
Evaluation was performed by the following comparisons/calculation:
- An increase in the mean number of positive wells in dose groups was compared to the mean value of the concurrent negative control (Evaluation factor 1 F).
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of the actual experiment (Evaluation factor 2 F). The baseline was derived from the mean spontaneous revertant number plus the value of standard deviation (mean+ SD) from the distribution of spontaneous data.
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of an experimental run (Evaluation factor 3 F). A run consists of a variable number of experiments generally testing different test substances together each using the same vehicle control. This leads to an accumulation of replicates for negative controls which was used to calculate the mean spontaneous reversion number for each run.
A test substance is considered mutagenic in this test system if more than a doubling of Evaluation factor 3 F is observed in at least one test group. This finding should be dose-dependent and/or reproducible.

Results and discussion

Test results
Species / strain:
other: TA 98, TA Mix (TA 7001 - TA 7006)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
An increase in the number of positive wells (his+ revertants) was not observed either without S9 mix or after the addition of a metabolizing system.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No bacteriotoxic effect (clearing of the background lawn, decrease in the number of yellow wells) was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 500 μg/ml and above
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

SUMMARY OF RESULTS

TA98 without S9 mix

DOSE [µg/ml] REM RCSP 1 RCSP 2 RCSP 3 MEAN MEANC SD 1F 2F 3F
DMSO   0 0 1 0.3 1.0 0.47 1.0 0.7 0.5
4 1 0 1 0.7 0.47 0.7 0.5 0.3
20 0 1 0 0.3 0.47 0.3 0.2 0.2
100 2 1 1 1.3 0.47 1.3 0.9 0.6
500 p 3 4 2 3.0 0.82 3.0 2.0 1.4
2500 p 1 1 2 1.3 0.47 1.3 0.9 0.6
5000 p 3 1 2 2.0 0.82 2.0 1.4 1.0
4-NQO+2-NF 43 41 47 43.7 2.49 43.7 29.7 20.9

TA98 with S9 mix

DOSE [µg/ml] REM RCSP 1 RCSP 2 RCSP 3 MEAN MEANC SD 1F 2F 3F
DMSO 0 1 1 0.7 1.0 0.47 1.0 0.7 0.4
4 3 1 1 1.7 0.94 1.7 1.1 0.6
20 0 2 3 1.7 1.25 1.7 1.1 0.6
100 0 5 1 2.0 2.16 2.0 1.4 0.8
500 p 5 3 1 3.0 1.63 3.0 2.0 1.1
2500 p 5 1 1 2.3 1.89 2.3 1.6 0.9
5000 p 4 1 2 2.3 1.25 2.3 1.6 0.9
2-AA 48 48 48 48.0 0.00 48.0 32.6 18.4

TA Mix without S9

DOSE [µg/ml] REM RCSP 1 RCSP 2 RCSP 3 MEAN MEANC SD 1F 2F 3F
DMSO 1 1 0 0.7 1.0 0.47 1.0 0.7 0.8
4 0 0 0 0.0 0.00 0.0 0.0 0.0
20 1 0 2 1.0 0.82 1.0 0.7 0.8
100 0 0 0 0.0 0.00 0.0 0.0 0.0
500 p 0 0 1 0.3 0.47 0.3 0.2 0.3
2500 p 1 0 0 0.3 0.47 0.3 0.2 0.3
5000 p 0 1 0 0.3 0.47 0.3 0.2 0.3
4-NQO+2-NF 46 47 46 46.3 0.47 46.30 31.50 37.4

TA Mix with S9

DOSE [µg/ml] REM RCSP 1 RCSP 2 RCSP 3 MEAN MEANC SD 1F 2F 3F
DMSO   0 1 2 1 1 0.82 1.0 0.6 0.8
4 0 0 0 0.0 0.00 0.0 0.0 0.0
20 1 0 0 0.3 0.47 0.3 0.2 0.3
100 2 2 1 1.7 0.47 1.7 0.9 1.3
500 p 0 1 0 0.3 0.47 0.3 0.2 0.3
2500 p 0 0 0 0.0 0.00 0.0 0.0 0.0
5000 p 2 0 0 0.7 0.94 0.7 0.4 0.5
2-AA 19 20 23 20.7 1.70 20.7 11.4 15.5

REM = Remarks

P = Precipitation

RCSP = Revertant Colony Selection Plate (384-Well)

SD = Standard Deviation

MEAN = Mean of Replicates

MEANC = Mean corrected : < 1 = 1

F = Factor

1F = Based on Meanc

2F = Baseline Data I Based on Meanc + SD

3F = Baseline Data I Based on Mean + SD of a Run

4-NQO + 2-NF = 4-nitroquinoline: 5 μg/ml + 2-nitroflourene 100 μg/ml

2-AA = 2-aminoanthracene: 125 μg/ml

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is not a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation.