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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-02-10 until 2012-05-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline OECD 471 has been followed accurately and the study is GLP compliant. The study has been checked for reliability.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Guideline OECD 471 was followed.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colourless homogeneous liquid
Details on test material:
Colourless homogeneous liquid. Test article is stored at well-ventilated place in tightly closed container avoiding heat, freeze and direct sunlight.

Method

Target gene:
Mutations in the histidine operon (base pair substitution, frameshift and ochre mutation).
Species / strain
Species / strain / cell type:
other: S. typhimurium TA1535, TA97a, TA98, TA100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
S-9 Mix
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 0.03 - 3 microlitres/plate
Concentration range in the main test (without metabolic activation): 0.005 - 0.5 microlitres/plate
Vehicle / solvent:
Solvent: DMSO
Details on test system and experimental conditions:
The bacterial strains were cultured in Oxoid nutrient broth No.2. Minimal glucose agar contained agar, Vogel-Bonner minimal medium E and 40% glucose. The overlay agar contained 0.6% agar, 0.5% NaCl and 0.05mM histidine-biotine (Maron and Ames, 1983).
The cofactor supplemented post-mitochondrial fraction (S9) was employed as the metabolic activation system.
Rat liver Mammalian Liver Fraction (S9) fraction preparation was carried out using phenobarbitone and beta-naphthoflavone as inducing agents. The S9 fraction was tested for protein content, sterility and its metabolic activation before preserving in cryocans at about -196 ⁰C. For every experiment, fresh vials were removed from the cryocan, thawed and used in the preparation of S9 mix.
The S9 mix was prepared in cold condition at approximately -4⁰C, immediately prior to its use in the experiment. The microsomal enzyme reacton mixture contained for 10 mL: a) sterile analytical grade water (3.35mL), b) 0.2M phosphate buffer, pH 7.4 (5.00mL), c) 0.1M NADP (0.40mL), d) 1M glucose-6-phosphate (0.05mL), e) 0.4M MgCl₂, 1.65M KCl salt solution (0.20mL), f) rat liver S9 (1.00mL).
Evaluation criteria:
A positive response is considered if the test article induces a concentration-dependent increase and/or an increase at one or more concentrations, in revertant frequency, which is at least 2-fold (3-fold for TA1535) of that observed in the corresponding concurrent vehicle control. In addition, the response should be reproducible.
A negative response is considered if the test article does not meet the afore-mentioned criteria.
A response is considered equivocal if the test article cannot be judged to be positive or negative. Equivocal or weak positive responses may indicate the need to repeat the test, possibly with a modified protocol such as appropriate spacing of dose levels.
Statistics:
No specific statistical programme was used. The mean value and standard deviation were calculated and compared to each other (histidine revertant colonies in the treatment groups vs the control groups) for 2-fold or 3-fold increases.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4 μl/plate and 5 μl/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1 μl/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Species / strain:
other: S. typhimurium TA1535, TA97a, TA98, TA100 and TA102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
3 μl/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA1535, TA97a, TA98, TA100 and TA102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
0.5 μl/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
A preliminary test was performed in order to determine solubility, precipitation and cytotoxicity. For more details, please refere to the following information.

Remarks on result:
other: strain/cell type: preliminary test
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Exposure Concentrations

Test concentrations for this study were selected from the results obtained with solubility/precipitation test and preliminary cytotoxicity test of the test article employing only TA100, with and without the metabolic activation system.

a. Solubility I Precipitation Test

N-octyl-2-pyrrolidone was found completely soluble in DMSO at a concentration of 50 mg/ml. In absence of metabolic activation system no precipitation was observed in the final reaction mixture at the concentrations of 5 μl, 4 μl , 3 μl, 2 μl, 1 μl, 0.5 μl, 0.25 μl and 0.125 μl/plate. In presence of metabolic activation system slight precipitation was observed in the final reaction mixture at the concentrations of ~1.85 μl, corresponding to final test concentrations of 5 μl, while no precipitation was observed at all the lower concentrations of 4 μl, 3 μl, 2 μl, 1 μl, 0.5 μl, 0.25 μl and 0.125 μl/plate. Hence, 5 μl/plate were selected as highest concentration for the preliminary cytotoxicity study in the absence and presence of metabolic activation system respectively.

b. Preliminary Cytotoxicity Test

Before commencing the study, the test article was assessed for cytotoxicity to the tester bacteria using tester strain TA100. Eight concentrations viz. 5 μl, 4 μl, 3 μl, 2 μl, 1 μl, 0.5 μl, 0.25 μ1 and 0.125 μl/plate of the test article, formulated in DMSO, were tested for toxicity to bacterial cells. In the presence of metabolic activation system N-octyl-2-pyrrolidone was found to be extremely cytotoxic to the bacterial cells at the concentrations of 5 μI and 4 μl/plate. Hence, 3 μl/plate was selected as the maximum test concentration for main study in presence of metabolic activation system. N-octyl-2 -pyrrolidone was found to be extremely cytotoxic in absence of metabolic activation system to the bacterial cells over the range tested up to the concentration of 1 μl/plate. Hence, 0.5 μl/plate was selected as the maximum test concentration for the main study in absence of metabolic activation system.

Experimental Procedure

Maximum dose of 3 μl and 0.5 μl/plate, both in presence and absence of metabolic activation system, which was found to be moderately cytotoxic to TA100 in the preliminary cytotoxicity, was selected as the highest dose level in the main study. In presence of metabolic activation system four lower dose levels viz. 0.9 μl, 0.3 μl, 0.09 μl and 0.03 μl/plate and in absence of metabolic activation system 0.15 μl, 0.05 μl, 0.015 μl and 0.005 μl/plate being selected for the main assay. Triplicate plating was performed at each dose level for an adequate evaluation of the variation.

Bacterial Lawn

In the absence of metabolic activation, slight reduction in the bacterial background lawn was observed at the highest concentration of 0.5 μl/plate in the tester strain TA1535 while no reduction in the bacterial background lawn was observed in the tester strains viz. TA97a, TA98, TA100 and TA102. In the presence of metabolic activation system, extreme reduction was observed at the highest concentration of 3 μl/plate in the tester strains TA97a, TA98, TA102 while moderate reduction in the bacterial lawn was observed at the concentration of 0.9 μl/plate in the tester strain TA100 metabolic activation system. In case of the tester strain TA1535, slight reduction in the bacterial background lawn was observed at the highest concentration of 0.5 μl/plate in absence of metabolic activation system while extreme reduction in the bacterial background lawn was observed at the concentrations of 3 μ1 and 0.9 μl/plate in presence of metabolic activation system.

Revertant Frequencies in Vehicle Control Groups

For the tester strains TA97a, TA98, TA100, TA102 and TA1535, the plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data at INTOX.

Revertant Frequencies in Treatment Groups

Results of this test indicated that the frequencies of histidine revertant colonies of all the tester strain were found to be comparable over the range tested of N-octyl-2-pyrrolidone both in presence and absence of metabolic activation system, during both the experiments, when I compared with the vehicle control group, as per the criteria employed for evaluation of mutagenic potential.

Revertant Frequencies in Positive Control Groups

Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation. Frequencies of histidine revertant colonies observed in the positive control plates for all tester strains, viz. TA1535, TA97a, TA98, TA100 and TA102, with and without the presence of a metabolic activation system, were significantly higher than those observed in the respective vehicle control groups, as per the criteria employed for evaluation of mutagenic potential.

Evaluation of Results

Since the observed frequencies of histidine revertant colonies of Salmonella typhimurium tester strains TA1535, TA97a, TA98, TA100 and TA102 exposed at all test concentrations of N-octyl-2 -pyrrolidone, with and without metabolic activation system, did not revealed any reproducible, dose-dependent or 2-fold (3-fold for TA1535) increases as compared to the respective vehicle control groups according to the criteria of evaluation of findings of this Ames Test, it is concluded that, N-octyl-2-pyrrolidone is non-mutagenic in Salmonella typhimurium tester strains.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The substance N-(n-octyl)-2-pyrrolidinone (NOP) is non-mutagenic in Salmonella triphimurium Reverse Mutation Test (Ames test).
Executive summary:

Salmonella typhimurium, Reverse Mutation Assay of N-octyl-2 -pyrrolidone was carried out in compliance with the OECD Guideline No. 471.

N-octyl-2-pyrrolidone was evaluated in the Ames/Salmonella Pre-incubation Assay to determine its ability to induce reverse mutation at selected histidine loci in five tester strains of Salmonella typhimurium viz. TA1535, TA97a, TA98, TA100 and TA102 in the presence and absence of metabolic activation system (S9). Based upon the preliminary tests conducted to assess the solubility/precipitation and cytotoxicity of N-octyl-2-pyrrolidone, the tester strains were exposed to the test article in triplicate cultures at the doses of 3 μl, 0.9 μl, 0.3 μl, 0.09 μ1 and 0.03μl/plate with metabolic activation system and 0.5 μl, 0.15 μl, 0.05 μl, 0.015 μl and 0.005 μl/plate for without metabolic activation system (S9). Liver S9, induced in rats by phenobarbitone with Pnaphthoflavone, was used for this purpose.

DMSO was used as a vehicle. The exposed bacteria were plated onto minimal glucose agar medium supplemented with L-histidine. The plates were incubated at 37 ± 1 οC for 48-72 hours after which the histidine revertant colonies were counted and their frequency was compared with that in vehicle control groups. Concurrent positive control groups were also included in the experiment, as specified by the test guideline.

Results of this test indicated that the frequencies of histidine revertant colonies of all the tester strain were found to be comparable over the range tested of N-octyl-2-pyrrolidone both in presence and absence of metabolic activation system, when compared with the vehicle control group, as per the criteria employed for evaluation of mutagenic potential.

Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be comparable with the range reported in the literature. Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation.

It is concluded that, under the conditions of this study, N-octyl-2-pyrrolidone is nonmutagenic in Salmonella typhimurium strains.