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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the described experimental design, the test substance, Propatyl nitrate, was non mutagenic for the Salmonella typhimurium TA 100, TA98, TA 1537 and E. coli in experiment with as well as without metabolic activation and Salmonella typhimurium TA 1535 without metabolic activation.The test substance was slightly mutagenic for Salmonella typhimurium TA 1535 with metabolic activation.

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:
17.10.2017 – 12.01.2018
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:
Council Regulation (EC) No.440/2008. Published in O.J. L 142, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
gene for histidine or tryptophan synthesis
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
supernatant of rat liver and a mixture of cofactors
Test concentrations with justification for top dose:
(3), 10, 30, 100, 300, 1000 μg per plate
Selection of doses/toxicity:
The test substance was soluble in dimethyl sufoxide - it was dissolved up to the highest recommended concentration 5000 µg per 0.1 mL. The maximum concentration was slightly clouded.
For toxicity experiment the highest concentration 5 mg per mL was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for toxicity in strain TA 100 without metabolic activation.
Precipitation occurred in top agar strating from 500 µg per plate. The test substance in background occurred in Petri dishes from 1000 µg per plate.
No toxicity was observed in any dose. The concentration of 1000 µg per plate was used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.
In the first mutagenicity experiments cytotoxicity (diminution of background, bacterial colonies in background) occurred in Salmonella typhimurium TA 98 experiment without metabolic activation. No signs of cytotoxicity occurred in the other experiments. The test substance in background in the highest concentration made evaluation somewhat difficult.
The same concentration range was used in the second mutagenicity experiments. In experiments with metabolic activation 50 instead 30 µL S9 were used for better metabolic conversion of the test substance.
In one case (strain Salmonella typhimurium TA 1535), the third mutagenicity experiment was performed for clarification of results. The test was performed with pre-incubation and dose range 3-1000 µg per plate. Precipitation occurred from 300 µg per tube in pre-incubation mixture and in 1000 µg per plate in top agar.
Vehicle / solvent:
dimethyl sulfoxide (DMSO), Merck Lot. No. K48069252 644, exp. 10/2018
- Justification for choice of solvent/vehicle: solubility of the substance
Untreated negative controls:
yes
Remarks:
negative controls - 0.1 mL of DMSO
Remarks:
Untreated controls - no solvent
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
(AS)
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
(NPD)
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Remarks:
(2-AF)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
(2-AA)
Positive controls:
yes
Positive control substance:
other: N-methyl-N´-nitro-N-nitrosoguanidine
Remarks:
(MNNG)
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride monohydrate
Remarks:
(9-AAc)
Details on test system and experimental conditions:
The bacterial tester strains
Salmonella typhimurium
TA 1535 (CCM 3814, lot. No. 2101200916917),
TA 98 (CCM 3811, lot No. 01022001220053),
TA 100 (CCM 3812, lot No. 0102201220054) and
TA 1537 (CCM 3815, lot No. 2101200916918)
as well as
Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732),
were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno.
Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2uvrA detects cross-linking mutagens


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

NUMBER OF REPLICATIONS: 2 series ( 3 for TA1335)

DETERMINATION OF CYTOTOXICITY
- Method: decrease of number of revertants or diminution of bacterial background

Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (see below). After this rule the result is positive, if a reproducible doseresponse effect occurs and/or a doubling of the ratio Rt/Rc is reached.
Statistics:
For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods:
Dunkel V. C.. Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation. Elsevier North-Holland Biomedical Press. 231 - 417
Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutat. Res. 189. 83 - 91
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
slightly mutagenic
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of DMSO. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance was soluble in dimethyl sufoxide - it was dissolved up to the highest recommended concentration 5000 µg per 0.1 mL. The maximum concentration was slightly clouded.
For toxicity experiment the highest concentration 5 mg per mL was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for toxicity in strain TA 100 without metabolic activation.
Precipitation occurred in top agar strating from 500 µg per plate. The test substance in background occurred in Petri dishes from 1000 µg per plate.
No toxicity was observed in any dose. The concentration of 1000 µg per plate was used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.

In preliminary cytotoxicity test, no toxicity occurred in any concentration.
In mutagenicity experiments, cytotoxicity (diminution of background, bacterial colonies in background) occurred in Salmonella typhimurium TA 98 in the first as well as in the second experiment without metabolic activation. No signs of cytotoxicity occurred in the other experiments including pre-incubation experiment.
Conclusions:
Under the above-described experimental design, the test substance, Propatyl nitrate, was non mutagenic for the Salmonella typhimurium TA 100, TA98, TA 1537 and E. coli in experiment with as well as without metabolic activation and Salmonella typhimurium TA 1535 without metabolic activation.
The test substance was slightly mutagenic for Salmonella typhimurium TA 1535 with metabolic activation.
Executive summary:

The test substance, Propatyl nitrate, was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

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 substance was dispersed in dimethylsulfoxide and assayed in doses of (3)10 - 1000 μg per plate, which were applied to plates in volume of 0.1 or 0.05 mL.

The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μL or 100 per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 10 – 1000 μg per plate.

In the second mutagenicity experiments concentrations were not changed but in experiments with metabolic activation volume of S9 was increased to 50 μL.

The third experiment with as well as without metabolic activation and 30 minutes of pre-incubation at 37±1°C and shaking was performed due to suspicion of mutagenicity based on the second experiment.

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, the test substance, Propatyl nitrate, was non mutagenic for the Salmonella typhimurium TA 98, TA 100, TA 1537 and E. coli with as well as without metabolic activation and Salmonella typhimurium TA 1535 without metabolic activation.

The test substance increased the number of revertants in experiments with metabolic activation in Salmonella typhimurium TA 1535 above critical limit. It appears in second a it was confirmed in third experiment. Therefore, the test substance is regarded as slightly mutagenic for this bacterial strain.

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification