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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 4 straines used
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat S9 liver
Test concentrations with justification for top dose:
8; 40; 200; 1000; 5000 Kg/plate
Vehicle / solvent:
solvent: dimethyl sulfoxide (CAS No. 67-68-5)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Details on test system and experimental conditions:
SYSTEM OF TESTING
- Metabolic activation system:
Aroclor 1254 induced rat S9 liver, obtained from Cytotest Cell Research
GmbH & Co. KG (Batches 280992 and 170593)
ADMINISTRATION:
- Dosing:
main test: 8/40/200/1000/5000 Kg/plate (+/- metabolic activation)
pre-incubation test: 8/40/200/1000/5000 Kg/plate (+/- metabolic activation)
- Number of replicates: 3
- Application: solvent dimethyl sulfoxide (CAS No. 67-68-5)
main test: 50 g/l; pre-incubation test: 100 g/l
- Positive and negative control groups and treatment:
positive, TA 98 and TA 1538: nitrofluorene
positive, TA 100 and TA 1535: sodium azide
positive, TA 1537: aminoacridine
negative: solvent + untreated controls
activity of metabolic system: aminoanthracene / TA 100
- Pre-incubation: 30 minutes at 30 +/- 1 °C
incubation ca. 96 hours at ca. 37 °C
Evaluation criteria:
mutagenic effects (i.e ratio of revertant rates treated/control >= 2) at <= 5000 Kg/plate with generally positive dose-response relationship in any strain
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENOTOXIC EFFECTS:
- With metabolic activation: None
- Without metabolic activation: None
The positive controls were functional.
PRECIPITATION CONCENTRATION: No precipitation was observed.
Conclusions:
Based on the available data from this study using four strains of Salmonella typhimurium (TA 1535, TA 1537, TA 1538, TA 98, TA 100) with and without metabolic activation. The Test Item is considered as non-mutagenic.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 October 2020 - 02 March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 30. May 2008
Deviations:
yes
Remarks:
Study was performed to fill data gaps of in vitro genotoxicity testing performed in 1993. Therefore, only the missing strain TA 102 was tested.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 26. Jun. 2020
Deviations:
yes
Remarks:
Study was performed to fill data gaps of in vitro genotoxicity testing performed in 1993. Therefore, only the missing strain TA 102 was tested.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Test concentrations with justification for top dose:
experiment 1: 5000, 1500, 500, 150 and 50 μg/plate.
experiment 2: 5000, 2500, 1250, 625, 313 and 156 μg/plate.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
other: 2-Amino-anthracene
Details on test system and experimental conditions:
7 PERFORMANCE OF THE STUDY
7.1 Culture of Bacteria
On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inoculated with one lyophilizate of Salmonella typhimurium TA102 (for detail see 6.2.2, page 12).
7.1.1 Preparations
Different media and solutions were prepared preliminary (exact production dates are docu-mented in the raw data).
On the day of the test, the bacteria culture was checked for growth visually. The incubation chambers were heated to 37 ± 1 °C. The water bath was turned to 43 ± 1 °C. The table surface was disinfected.
The S9-mix was freshly prepared and stored at 0 °C.

7.1.2 Description of the Method
Per each concentration and control, three plates with (+S9) and three plates without meta-bolic activation (-S9) were used.
The test item solutions were prepared according to chapter 6.1.4, page 11.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ± 1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
 100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control). For the positive control MMC 2.5 μL of the stock solu-tion were applied to achieve a final concentration of 0.5 μL/plate.
 500 μL S9-mix (see chapter 6.4.13, page 16 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
 100 μL bacteria suspension (see chapter 6.2.2, page 12, test system, culture of the strains)
 2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.
Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:
 100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control).
 500 μL S9-mix (see chapter 6.4.13, page 16 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
 100 μL bacteria suspension (see chapter 6.2.2, page 12, test system, culture of the strains)
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.
Rationale for test conditions:
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized (demin.) water and dimethyl sulfoxide (DMSO). The solid test item was soluble in DMSO, only.
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Evaluation criteria:
Five different analysable and non-toxic concentrations were used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A result is considered as positive if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the concentrations occurs in at least one tested strain with or without metabolic activation.
A biologically relevant increase is described as follows:
 if in the bacteria strain S. typhimurium TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
A substance is not mutagenic if it does not meet the criteria above.
If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
8.1 Experiment 1
8.1.1 Confirmation of the Criteria and Validity
The strain Salmonella typhimurium TA102 met the criterion of at least 109 bacteria/mL (cor-relating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 17, page 40 ff.). Both positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
8.1.2 Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested con-centrations.
No signs of toxicity towards the bacteria strain Salmonella typhimurium TA102 could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
8.1.3 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.

8.2 Experiment 2
8.2.1 Confirmation of the Criteria and Validity
Strain Salmonella typhimurium TA102 met the criterion of at least 109 bacteria/mL (correlating to 100 colonies/plate after dilution), and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the vehicle and negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 17, page 40 ff.). Both positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
8.2.2 Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strain Salmonella typhimurium TA102 could be observed. The bacterial background lawn was visible and not affected. The number of re-vertant colonies was not reduced.
8.2.3 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.
Conclusions:
The Test Items was tested for genotoxicity in an OECD 471 and GLP compliant study with the Salmonella typhimurium test strain TA102 with and without metabolic activation. Based on this study, the Test Item is non-mutagenic under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The substance did not show genotoxic effects in an Ames tests with Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and 1538 (50 - 5000 µg/plate), all with and without metabolic activation. In addition, a second Ames test with with Salmonella typhimurium TA 102 was performed. The test substance was determined to be non genooxic under the condistions of the test (with and without metabolic activation, 50 - 5000 µg/plate).

In the attached OECD Toolbox study (read across) for effects in V79 Chinese hamster lung cells, with and without metabolic activation, the results were:

In the first test set with no info on metabolic activation PDPI scored -0.6, ie negative for genotoxicity and cytotoxicity

In the second test set with metabolic activation PDPI scored +0.2, ie ambiguous

In the third test set without metabolic activation PDPI scored -1, ie negative for genotoxicity and cytotoxicity

In addition, the parent substances of this salt (pyromellitic acid and 2 -phenyl-2 -imidazoline) are both negative.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No effects observed. Therefore, the Test Substance must not be classified.