Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 July to 11 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 471 Guideline without any deviation.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
08 April 2015
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Robertet S.A. / 2493442
- Appearance: Yellow Liquid/May cristallize
- Expiration date of the lot/batch: 29 April 2017

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Fridge (2-8 °C), protected from air and light

Method

Target gene:
Histidine gene for Salmonella typhimurium
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
0.74 % final concentration in the treatment; S9 fraction produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg bw intraperitoneally.
Test concentrations with justification for top dose:
Experiment 1a: 50, 150, 500, 1500 and 5000 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Experiment 1b: 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method
Experiment 2: 8, 16, 31, 63, 125, 250 and 500 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using pre-incubation method
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in DMSO. DMSO was chosen as vehicle, because the test item was sufficiently soluble in it, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Test substance preparation:
- On the day of the start of experiment 1a, a stock solution containing 50 g/L of the test item in DMSO was prepared. The test item solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for experiment 1a: 5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
- On the day of the start of experiment 1b, a stock solution containing 50 g/L of the test item in DMSO was prepared. The test item solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for experiment 1b: 1500 μg/plate, 500 μg/plate, 150 μg/plate, 50 μg/plate, 15 μg/plate, 5 μg/plate and 1.5 μg/plate.
- On the day of the start of experiment 2, a stock solution containing 5 g/L of the test item in DMSO was prepared. The test item solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for experiment 2: 500 μg/plate, 250 μg/plate, 125 μg/plate, 63 μg/plate, 31 μg/plate, 16 μg/plate and 8 μg/plate.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-Nitro-1,2-phenylene Diamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Amino-Anthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem GmbH (batch of the bacteria strains: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilisates in the fridge at 2-8 °C.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 37 ±1 °C for 20 min
- Exposure duration: 37 ±1 °C for 48 h

NUMBER OF REPLICATIONS:
- Experiment 1a and 1b (Range-finding test): 3 plates/dose
- Experiment 2: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.

OTHERS:
- After incubation for 48 h, the revertants were counted and the numbers for each plate were recorded. The colonies were counted visually, the numbers were recorded.
Evaluation criteria:
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Statistics:
None

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate.

MAIN TEST:
Experiment 1a
Solubility and Toxicity: The test item showed no precipitates on the plates in all tested concentrations. The bacterial background lawn was not reduced at any of the concentrations, except at the bacteria strain TA100 with metabolic activation at the highest concentration, but a relevant decrease in the number of revertants was observed in all bacteria strains in the two highest concentrations (5000 and 1500 μg/plate). A relevant decrease of revertants was also observed in the bacteria strain TA1535 without metabolic activation in the third concentration (500 μg/plate). The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation.
Mutagenicity: No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test item is stated as not mutagenic under the test conditions. Due to toxicity in this experiment, a further experiment was performed.

Experiment 1b
Solubility and Toxicity: The test item showed no precipitates on the plates in all tested concentrations. The bacterial background lawn was reduced at the following concentrations: 1500 μg/plate in strain TA97a and TA98 with and without metabolic activation. A relevant decrease in the number of revertants was observed in all bacteria strains in the highest concentration (1500 μg/plate). The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation.
Mutagenicity: No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. The concentration 15 μg/plate showed an increase in the number of revertants in the bacteria strain TA98 with metabolic activation. This can be seen as uncritical, because this is an outlier. On the base of the experiments 1a and 1b, a further experiment was performed.

Experiment 2
Solubility and Toxicity: The test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
Mutagenicity: No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
History of the spontaneous revertants and positive controls of the performed experiments with these strains up to 13 July 2016 is stated in comparison with the experiments performed within this study.
- Positive historical control data:
Without metabolic activation: 389-1152 (616 ± 187); 112-793 (420 ± 154); 223-984 (536 ± 169); 491-2157 (1083 ± 403); 55-484 (244 ± 103) in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, respectively
With metabolic activation: 241-1181 (537 ± 157); 39-217 (78 ± 37); 325-1371 (654 ± 186); 408-6083 (1234 ± 885); 45-284 (108 ± 49) in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, respectively
- Negative (water) historical control data:
Without metabolic activation: 61-144 (99 ± 20); 6-35 (15 ± 6); 62-141 (97 ± 15); 120-389 (266 ± 64); 6-30 (16 ± 6) in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, respectively
With metabolic activation: 63-138 (100 ± 17); 11-41 (18 ± 6); 66-141 (99 ± 15); 125-432 (283 ± 77); 7-30 (16 ± 5) in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, respectively
- Vehicle (DMSO) historical control data:
Without metabolic activation: 58-135 (99 ± 19); 7-46 (14 ± 6); 60-136 (95 ± 17); 149-381 (265 ± 59); 8-33 (16 ± 6) in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, respectively
With metabolic activation: 70-144 (106 ± 16); 10-36 (17 ± 5); 63-199 (96 ± 22); 129-365 (272 ± 59); 6-29 (16 ± 6) in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, respectively

OTHERS
- Confirmation of genotype was performed for each batch of lyophilized bacteria before stock culture preparation. The last performance showed no abnormalities
- Confirmation of the Criteria and Validity: All strains met the criterion of at least 10^9 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges.

Any other information on results incl. tables

Acceptance criteria:

In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate.

In experiment 1a, the test item caused cytotoxicity towards all bacteria strains in the two highest concentrations (5000 and 1500 μg/plate). A relevant decrease of revertants was also observed in the bacteria strain TA1535 without metabolic activation in the third concentration (500 μg/plate).

In experiment 1b, the test item caused cytotoxicity towards all bacteria strains only in the highest concentration (1500 μg/plate).

In the second experiment no cytotoxicity was observed.

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.

Nearly all of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.

Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that test item is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system. 

Experiment 1a: 50, 150, 500, 1500 and 5000 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method

Experiment 1b: 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using plate incorporation method

Experiment 2: 8, 16, 31, 63, 125, 250 and 500 μg/plate in TA 97a, TA 98, TA 100, TA 102 and TA 1535 strains, with and without S9 mix using pre-incubation method

 

Negative, vehicle and positive control groups were also included in mutagenicity tests. 

 

The sterility control and the determination of the titre did not show any inconsistencies. The determined values for the spontaneous revertants of the negative controls (vehicle) were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation. Therefore, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate.

 

In Experiment 1a, the bacterial background lawn was not reduced at any of the concentrations, except at the bacteria strain TA100 with metabolic activation at the highest concentration, but a relevant decrease in the number of revertants was observed in all bacteria strains in the two highest concentrations (5000 and 1500 μg/plate). A relevant decrease of revertants was also observed in the bacteria strain TA1535 without metabolic activation in the third concentration (500 μg/plate). The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations caused a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Due to toxicity in this experiment, a further experiment (Experiment 1b) was performed.

 

In Experiment 1b, the bacterial background lawn was not reduced at any of the concentrations, except at the bacteria strains TA97a and TA98 with and without metabolic activation at the highest concentration, but a relevant decrease in the number of revertants was observed in all bacteria strains only in the highest concentration (1500 μg/plate). The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations caused a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. The concentration 15 μg/plate showed an increase in the number of revertants in the bacteria strain TA98 with metabolic activation. This can be seen as uncritical, because this is an outlier.

 

Due to the experiments 1a and 1b, test item was tested up to concentrations of 500 μg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strain using the pre-incubation method (experiment 2). Test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that test item is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.