hexyl nitrite

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13/03/2020-24/04/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

No deviations from study plan or test guidelines/methods occurred during the study.

GLP compliance:

yes (incl. QA statement)

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 number of test material: B2120
- Expiration date of the lot/batch: 03/2035

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In cool and dry place (fridge), The test item has been stored in the original package - a glass vial, in refrigerator, in the dark.
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: soluble in DMSO up to the highest concentration recommended in OECD TG 471 what is 5000 μg per plate (0.1 mL).

Method

Target gene:

A reverse mutation test - In either Salmonella typhimurium or Escherichia coli detects mutation in amino-acid requiring strain (histidine or tryptophan, respectively) to produce a strain independent of an outside supply of amino-acid.

Base pair substitution mutagens - Are agents that cause a base change in DNA. In a reversion test this change may occur at the site of the original mutation, or at a second site in the bacterial genome.

Frameshift mutagens - Are agents that cause the addition or deletion of one or more base pairs in the DNA, thus changing the reading frame in the RNA.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : prepared in-house, Lot 18.09.2017/I, exp. 30/06/2020, Lot 9.12.2019/I, exp. 9.12.2021
- method of preparation of S9 mix : prepared according to the methods described by Maron and Ames (Maron D. M., Ames B. N. (1983): Revised methods for the Salmonella mutagenicity test. Mutat. Res. 113, 173 - 215)
- concentration or volume of S9 mix and S9 in the final culture medium: Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 20, or 30 L S9 (the respective concentration of S9 in the S9mix was 3.8 or 5.7 %). In experiments without metabolic activation only buffer was added to the top agar.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: the test substance was insoluble in water (two phases)

- Justification for percentage of solvent in the final culture medium: soluble in DMSO up to the highest concentration recommended in OECD TG 471 what is 5000 μg per plate (0.1 mL).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (NPD); 2-aminofluorene (2-AF); 2-aminoanthracene (2-AA); N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate plating was used in cytotoxicity tesing at each concentration level, triplicate plating was used in plate incorporation test and experiments with pre-incubation at each dose level.
- Number of independent experiments: two cytotoxicity experiments, two types of mutagenicity experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 30 min
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Petri dishes were incubated for 48-72 h at 37+-1 °C
- Method used: agar, Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 20, or 30 L S9 (the respective concentration of S9 in the S9mix was 3.8 or 5.7 %). In experiments without metabolic activation only buffer was added to the top agar.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: the number of revertant colonies on plates was counted manually with exception of positive controls, which were counted by a colony counter ProtoCol3.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: number of revertants per plate, changes in background

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Method: number of revertants per plate

Evaluation criteria:

The main criterion used for the evaluation of reversion results was a modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). Per this rule, the result is positive if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached (Rt – number of revertants at tested dose, Rc – number of revertants of the solvent control).
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD TG 471, the biological relevance is the criterion for the interpretation of results, and a statistical evaluation of the results is not necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

True negative controls validity:

valid

Remarks:

Negative controls contain no solvent, solvent controls contain 0.1 or 0.05 mL of solvent (DMSO). All control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. Actual numbers were in ranges of historical numbers.

Positive controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

True negative controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

Positive controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

True negative controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

Positive controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

True negative controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

Positive controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

True negative controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

Positive controls validity:

valid

Remarks:

All the control numbers were compared with historical ranges of mutant frequencies obtained in laboratory. The actual numbers were in ranges of the historical numbers.

Applicant's summary and conclusion

Conclusions:

Under the above-described experimental design, the test item, Hexyl Nitrite, was found to be non mutagenic for all the used S. typhimurium and Escherichia coli WP2 uvrA with and without metabolic activation.

Executive summary:

The test item,Hexyl Nitrite,was assayed for mutagenicity using the Bacterial Reverse Mutation Test. The test was performed according to the OECD Test Guideline No. 471 in agreement with the EU Method B.13/14.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used.

The test item was soluble in dimethyl sulfoxide (DMSO) at the maximum recommended concentration 5000 μg per plate. A cytotoxicity experiment was performed first, with a concentration range of 10-5000 μg per plate in Salmonella typhimurium TA100 without metabolic activation. Based on the results, the concentrations used in the first mutagenicity experiments (plate incorporation) were 3, 10, 30, 100 and 300 μg per plate.

No cytotoxicity and only a few increased numbers of revertants in S. typhimurium TA 100 and TA 1535 were observed in the first mutagenicity experiments, so the results of first mutagenicity experiment shave been evaluated as non-mutagenic.

In case of negative results the OECD TG 471 requires either reasonable justification of no further testing or the confirmation of negative experiment with another test with changes in experiment arrangement. So we perform the second mutagenicity experiment with preincubation. These experiments were started with its own (second)cytotoxicity test with pre-incubation with a concentration range of 1.5-300 μg per plate in Salmonella typhimurium TA100 without metabolic activation.

 Based on the results, the concentrations used in the second mutagenicity experiments were 5, 10, 25, 50 and 100 μg per plate.

Both mutagenicity experiments were performed with and without metabolic activation using Delor 106-induced rat liver S9. The second mutagenicity experiment was performed with pre-incubation (30 minutes at 37±1°C and shaking).

 The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

 

In the arrangement given above, thetest item,Hexyl Nitrite,wasnon-mutagenic for all the used indicator strains in experiments with and without metabolic activation.

Change of experimental conditions performed in the second experiments had no influence on effect detected.