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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Ames Test Rat lymphocyte chromosome aberration assay In vitro mammalian mutation assay: Mouse Lymphoma In vivo mouse peripheral blood cell micronucleus assay
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Standard Guideline study conducted according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella strains = Histidine Auxotrophs
E. coli strains = Tryptophan auxotrophs
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:
microsomal enzymes prepared from AroclorÖ-induced rat liver (S9)
Test concentrations with justification for top dose:
10.0, 33.3, 100, 333, 1000, 2500, and 5000 μg per plate in both the presence and absence of S9 mix
Vehicle / solvent:
Di-methylsulfoxide (DMSO)
Justification: In solubility testing with deionized water, the test article was observed to form a heterogeneous, white, opaque suspension
at 100 mg per mL and a homogeneous, white, opaque suspension at 50 mg per mL. In solubility testing with dimethylsulfoxide (DMSO), the test article was observed to form a colorless, transparent solution at 99.6 mg per mL. Based on these results, DMSO (Acros Organics, Lot No.
04934ME, expiration date January 2009, and Sigma-Aldrich, Lot Nos. 00347TE, expiration date February 2009 and 00148CH, expiration date March 2009) was selected as the vehicle for this study. At 100 mg per mL, which was the most concentrated stock dilution prepared for testing,
the test article was observed to form a colorless, transparent, non-viscous solution. The test article remained in solution at all succeeding dilutions prepared.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation method

Rangefinding Study
The growth inhibitory effect (cytotoxicity) of the test article to the test system was determined in order to allow the selection of appropriate concentrations to be tested in the mutagenicity assay. Design. The rangefinding study was performed using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix. Seven concentrations of test article were tested at two plates per concentration. The test article was checked for cytotoxicity up to a maximum concentration of 5000 μg per plate.

Rationale.
The cytotoxicity of the test article observed on tester strain TA100 is generally representative of that observed on the other Salmonella typhimurium tester strains and because of the comparatively high number of spontaneous revertants per plate observed with this strain, gradations of cytotoxicity can be readily discerned from routine experimental variation. The Escherichia coli tester strain WP2uvrA does not possess the rfa wall mutation that the Salmonella typhimurium strains have and thus, a different range of cytotoxicity may be observed. Also, the cytotoxicity induced by a test article in the presence of S9 mix may vary greatly from that observed in the absence of S9 mix. Therefore, this would require that different test article concentration ranges be tested in the mutagenicity assay based on the presence or absence of the microsomal enzymes.

Evaluation of the Rangefinding Study.
Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.

Selection of the Maximum Concentration for the Mutagenicity Assay.
Since indications of cytotoxicity were observed at the higher concentrations in the rangefinding study, the highest concentration of test article used in the mutagenicity assay was the same as that tested in the rangefinding study.

Mutagenicity Assay

Design.
The assay was performed using tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA both in the presence and absence of S9 mix along with appropriate vehicle control and positive controls. Results of the initial mutagenicity assay were confirmed in an independent experiment.

Frequency and Route of Administration.
Tester strains were exposed to the test article via the preincubation modification of the Ames Test originally described by Yahagi et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the preincubation methodology, S9 mix (or phosphate buffer, where appropriate), tester strain, and test article were preincubated prior to addition of molten agar. The agar and preincubation reaction mixture were mixed and then overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. Test article, vehicle controls, and positive controls were plated in triplicate.

Plating Procedures
These procedures were used in both the rangefinding study and the mutagenicity assay. Each plate was labeled with a code that identified the test article, test phase, tester strain, activation condition and concentration. S9 mix was prepared immediately prior to use. When S9 mix was required, 500 μL of S9 mix was added to 13 x 100 mm glass culture tubes, which had been pre-heated to 37 ± 1°C. To these tubes were added 100 μL of tester strain and 50 μL of vehicle control article or test article. When S9 mix was not required, 500 μL of 0.1M phosphate buffer was substituted for S9 mix. After required components had been added, the mixture was vortexed and allowed to incubate for 20 ± 2 minutes at 37 ± 1°C. Two mL of molten selective top agar was then added to each tube, and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay solidified, plates were inverted and incubated for 52 ± 4 hours at 37 ± 1°C. Positive control articles were plated using a 50 μL plating aliquot.

Scoring the Plates
Plates not evaluated immediately following the incubation period were held at >0°C to 10°C until colony counting and bacterial background lawn evaluation could take place. Bacterial Background Lawn Evaluation. Condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored
relative to the vehicle control plate and was recorded along with the revertant counts for all platesat that concentration. Lawns were scored as normal (N), reduced (R), obscured by precipitate (O), macroscopic precipitate present (P), absent (A), contaminated (C) as needed.

Counting Revertant Colonies.
Revertant colonies were counted either by automated colony counter or by hand.

Assay Acceptance Criteria
Before assay data were evaluated, criteria for a valid assay had to be met. The following criteria were used to determine a valid assay:

Tester Strain Integrity
rfa Wall Mutation.
Demonstrating presence of the rfa wall mutation, Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet.

pKM101 Plasmid.
Demonstrating presence of the pKM101 plasmid, cultures of tester strains TA98 and TA100 exhibited resistance to ampicillin. Characteristic Number of Spontaneous Revertants. Demonstrating the requirement for histidine (Salmonella typhimurium) or tryptophan (Escherichia coli), tester strain cultures exhibited a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions.

Acceptable ranges for mean vehicle controls were as follows:
TA98 8 - 60
TA100 60 - 240
TA1535 4 - 45
TA1537 2 - 25
WP2uvrA 5 - 40

Tester Strain Culture Density.
Demonstrating that appropriate numbers of bacteria were plated, density of tester strain cultures was greater than or equal to 1.0 x 109 bacteria per mL and/or had reached a target density demonstrated to produce cultures with at least 1.0 x 109 bacteria per mL.

Positive Control Values in the Absence of S9 Mix.
Demonstrating that tester strains were capable of identifying a mutagen, the mean value of a positive control for a respective tester strain exhibited at least a 3-fold increase over the mean value of the vehicle control for that strain.

Positive Control Values in the Presence of S9 Mix (S9 Mix Integrity).
Demonstrating that S9 mix was capable of metabolizing a promutagen to its mutagenic form(s), the mean value of the positive control for a respective tester strain in the presence of the S9 mix exhibited at least a 3-fold increase over the mean value of the vehicle control for that strain. An acceptable positive control in the presence of S9 mix for a specific strain was evaluated as having demonstrated both integrity of the S9 mix and ability of the tester strain to detect a mutagen.

Cytotoxicity.
A minimum of three non-toxic concentrations was required to evaluate assay data. Cytotoxicity was detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn. Thinning of the bacterial background lawn not accompanied by a reduction in the number of revertants per plate was not evaluated as an indication of cytotoxicity.
Evaluation criteria:
Once criteria for a valid assay had been met, responses observed in the assay were evaluated.

Tester Strain TA100.
For a test article to be considered positive, it had to produce at least a 2-fold concentration-related and reproducible increase in the mean revertants per plate over the mean revertants per plate of the appropriate vehicle control. A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.

Tester Strains TA98, TA1535, TA1537, and WP2uvrA.
For a test article to be considered positive, it had to produce at least a 3-fold concentration-related and reproducible increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
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:
cytotoxicity
Remarks:
Cytotoxicity was observed with tester strain TA100 in the presence and absence of S9 mix at ≥3330 μg per plate. Cytotoxicity was observed with strain WP2uvrA in the presence of S9 mix at 5000 μg per plate, and in the absence of S9 mix at ≥3330 μg plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Rangefinding Study

Concentrations tested in the mutagenicity assay were selected based on the results of the rangefinding study conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with two plates per concentration. Seven concentrations of test article, from 10.0 to 5000 μg per plate, were tested in Trial 29063-A1. The results of Trial 29063-A1 are presented in Table 1 and Table 2. Cytotoxicity was observed with tester strain TA100 in the presence and absence of S9 mix at ≥3330 μg per plate as evidenced by concentration-related decreases in the number of revertants per plate and reduced bacterial background lawns. Cytotoxicity was observed with tester strain WP2uvrA in the presence of S9 mix at 5000 μg per plate, and in the absence of S9 mix at ≥3330 μg per plate, as evidenced by concentration-related decreases in the number of revertants per plate and reduced or absent bacterial background lawns.

Mutagenicity Assay

The preincubation mutagenicity assay results are presented in Data Tables 3 through 7. These data were generated in Trials 29063-B1, 29063-C1, and 29063-D1. Data are presented as individual plate counts (Table 3, Table 5, and Table 7) and as mean revertants per plate ± standard deviation (Table 4, Table 6, and Table 7) for each test article and control group. The tester strains used in the preincubation mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with seven concentrations of the test article in presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per concentration. The concentrations tested in the initial mutagenicity assay were selected based on the results of the rangefinding study. Concentrations tested in the mutagenicity assay with all tester strains were 10.0, 33.3, 100, 333, 1000, 2500, and 5000 μg per plate in both the presence and absence of S9 mix.

In the initial mutagenicity assay (Trial 29063-B1, Table 3 and Table 4), all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. In the confirmatory mutagenicity assay (Trial 29063-C1, Table 5 and Table 6), all data generated with tester strains TA98, TA100, TA1535, and WP2uvrA were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of these tester strains in either the presence or absence of S9 mix. However, the mean vehicle control value for TA1537 in the presence of S9 mix was not within the acceptable range for this tester strain. For this reason, the data generated with TA1537 in both the presence and absence of S9 mix in Trial 29063-C1 were not used to evaluate the mutagenicity of the test article. The test article was retested with tester strain TA1537 in the presence and absence of S9 mix in Trial 29063-D1.

In the repeat confirmatory mutagenicity assay (Trial 29063-D1, Table 7), all data were acceptable and no positive increases in the mean number of revertants per plate were observed with tester strain TA1537 in either the presence or absence of S9 mix.

All criteria for a valid study were met.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Tables 1 to 7 referred to above and the historical control data are attached in the document below.

Conclusions:
Interpretation of results (migrated information):
negative

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay with octanolamine indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).
Executive summary:

The objective of this study was to evaluate the test article, octanolamine and/or its metabolites, for the ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at 1) the histidine locus in the genome of several strains of Salmonella typhimurium and at 2) the tryptophan locus of Escherichia coli strain WP2uvrA. This assay satisfied the following guidelines: U.S. EPA (1998), EEC (2000), and OECD (1997).

The concentrations tested in the mutagenicity assay were selected based on the results of a rangefinding study using tester strains TA100 and WP2uvrA and seven concentrations of test article ranging from 10.0 to 5000 μg per plate, two plates per concentration, both in the presence and absence of S9 mix.

The tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted with seven concentrations of test article in the presence and absence of S9 mix, along with concurrent vehicle control and positive controls using three plates per concentration. The concentrations tested in the mutagenicity assay were 10.0, 33.3, 100, 333, 1000, 2500, and 5000 μg per plate in both the presence and absence of S9 mix for all the tester strains. The results of the initial mutagenicity assay were confirmed in an independent experiment.

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay with octanolamine indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).

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

Additional information

Additional information from genetic toxicity in vitro:

Based on the studies available, this substance does not appear to be mutagenic or capable of causing chromosome aberrations (in vitro and in vivo) under the conditions of the assays in the presence or absence of metabolic activation.


Justification for selection of genetic toxicity endpoint
All studies are guideline OECD studies conducted according to GLP.
All studies were negative, but only possible to assign 1 study to the endpoint summary.

Justification for classification or non-classification

The data available do not suggest that this substance possesses genotoxic or mutagenic properties. Thus no classification or further in vivo testing is warranted.