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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item tested up to a cytotoxic concentration of 5000 µg/plate, without and with metabolic activation, respectively, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254, prepared according to MARON and AMES (1983) was obtained from Trinova Biochem. S9 was collected from male rats.
Test concentrations with justification for top dose:
Test cncentrations: 31.6, 100, 316, 1000, 3160 and 5000 µg of the test item per plate in the plate corporation and preincubation test, without or with metabolic activation.
Plates 3 per concentration and experiment
Experiments 2 independent experiments, each with and without metabolic activation

The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity and no test item precipitation was noted.
Hence, 5000 µg of the test item per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

Vehicle / solvent:
water
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
1st independent experiment - Plate Incorporation Method
Sterile top agar containing 0.6 % agar and 0.5% NaCl was molten on the day of the test. 10 mL of a sterile solution of 0.5 mM L-histidine HCl /0.5 mM Biotin were added to 100 mL of molten agar. 2 mL of this top agar were distributed into culture tubes held at 45 °C in a heating block. 0.1 mL of Salmonella cell suspension (containing approximately 108 viable cells in the late exponential or early stationary phase), 0.1 mL of test item solution (or 0.1 mL vehicle or 0.1 mL positive control) and 0.5 mL of S9 mix were added to these culture tubes. In the assay without metabolic activation, the S9 mix was substituted with 0.5 mL phosphate buffer mentioned above.
The test components were mixed by vortexing the soft agar for 3 sec at low speed and then poured onto a coded 27.5 mL minimal glucose agar plate (Minimal Glucose Agar medium E). To achieve a uniform distribution of the top agar on the surface of the plate, the uncovered plate was quickly tilted and rotated and then placed on a level surface with the cover on and finally allowed to harden.
Immediately, the plates were inverted and placed in a dark 37 °C incubator for 48 h and could be stored after incubation for up to 24 hours at 4 °C. The revertant colonies on the test plates and on the control plates were counted with a colony counter , and the presence of the background lawn on all plates was confirmed. A lawn that was thin compared with the lawn on the negative control plate was evidence of bacterial toxicity.
Routine examination of the background lawn of bacterial growth resulting from the trace of histidine added to the top agar could be an aid in determining the presence of toxic effects. If massive cell death has occurred, the background lawn on the test plates would have been sparse compared with control plates.
In this case more histidine would be available to the individual surviving bacteria and they would undergo more cell divisions, consequently appearing as small colonies which could be mistaken for revertants if the absence of a normal background lawn is not noted.

2nd independent experiment - Preincubation Method
The test item was preincubated with the test strain (containing approximately 108 viable cells in the late exponential or early stationary phase) and sterile buffer (0.5 mL) or the metabolic activation system (0.5 mL) for 20 minutes at 37 °C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate. 0.1 mL of the test item solution (or 0.1 mL vehicle or 0.1 mL positive control), 0.1 mL of bacteria, and 0.5 mL of S9 mix or sterile buffer, were mixed with 2 mL of overlay agar. Tubes were aerated during preincubation by using a shaker. The remaining steps were the same as described for the plate incorporation method.


Rationale for test conditions:
The test item was completely dissolved in water for injection shortly before use. The vehicle water for injection was employed as the negative control. Fresh preparations of the test item were used for the treatment in all experimental parts.
Prior to the main test two preliminary cytotoxicity tests (plate incorporation test, without and with metabolic activation) were carried out in test strain TA100.
Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures. Insolubility could have been assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. The recommended maximum test concentration for soluble non-cytotoxic test items is 5 mg/plate or 5 µL/plate. For non-cytotoxic test items that are not soluble at 5 mg/plate or 5 µL/plate, one or more concentrations tested should be insoluble in the final treatment mixture. Test items that are cytotoxic already below 5 mg/plate are tested up to a cytotoxic concentration. Interference of precipitates with the scoring should be avoided.
In the main study 6 different concentrations of the test item were tested, with half-log intervals between plates (31.6, 100, 316, 1000, 3160 and 5000 µg PU-2019-872 per plate in the plate corporation and preincubation test, without or with metabolic activation).


Evaluation criteria:
Bacteria colonies were counted employing the Biosys Biocount 5000 γ system. Print outs of the colony counts were filed with the raw data. Occurrence of test item precipitation would have been documented after visual inspection of the cultures with the unaided eye. Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a scarce background lawn.
Acceptance Criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by LPT.
The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p  0.05, U-test according to MANN and WHITNEY, see section ‎6, reference ‎3) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient (section ‎6, reference ‎3) may be applied.
Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium.
A test item for which the results do not meet the above-mentioned criteria is considered as non-mutagenic in the AMES test.

Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with metabolic activation/Preincubation test: 5000 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 3160 and 5000 µg/plate without metabolic activation/ Preincubation test: at 3160 µg/plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium 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
True negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Preincubation test: at 3160 and 5000 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test item tested up to a cytotoxic concentration of 5000 µg/plate, without and with metabolic activation, respectively, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test.

Six concentrations ranging from 31.6 to 5000 µg of the test item/plate were employed in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.

Cytotoxicity
Cytotoxicity in form of reduction of the number of revertants by more than 50% was noted in the plate incorporation test at concentrations of 3160 and 5000 µg of the test item/plate in test strain TA98 without metabolic activation and at a concentration of 5000 µg of the test item/plate in test strain TA1537 with metabolic activation. In the preincubation test, cytotoxicity in form of reduction of the number of revertants by more than 50% was noted for the following concentration(s) and test strains: at 3160 µg of the test item/plate in test strain TA98 without metabolic activation, at 3160 and 5000 µg of the test item//plate in test strain TA102 without metabolic activation, at 5000 µg of the test item/plate in test strain TA102 with metabolic activation and at 5000 µg of the test item/plate in test strain TA1537 without metabolic activation.
Mutagenicity
No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 µg/plate, in the plate incorporation test and in the preincubation test for the experiments without and with metabolic activation, respectively.
The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 5000 µg/plate, without and with metabolic activation, respectively, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test.

Executive summary:

The test item tested up to a cytotoxic concentration of 5000 µg/plate, without and with metabolic activation, respectively, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification