Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

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:

09Oct2001 to 26Jul2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

EC Number:

269-503-2

EC Name:

Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Cas Number:

68259-00-7

Molecular formula:

C14H16N3.CH3O4S

IUPAC Name:

methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Test material form:

solid: particulate/powder

Details on test material:

See study reports for batch and purity information

Specific details on test material used for the study:

Sponsor's Identification: MIP Yellow 2982
Batch No. 028400A8AA
Date Received: September 10, 2001
Physical Description: Yellowish-orange powder with chunks
Storage Conditions: Ambient temperature

Method

Target gene:

TA98; hisD3052; uvrB; rfa; pKM101
TA100; hisG46; MvrB; rfa; pKM101
TA1535; hisG46; MvrB; rfa
TA1537; hisC3016; uvrB; rfa
WP2MvrA; trp; uvrA;

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: TA98; hisD3052; uvrB; rfa; pKM101 TA100; hisG46; MvrB; rfa; pKM101 TA1535; hisG46; MvrB; rfa TA1537; hisC3016; uvrB; rfa WP2MvrA; trp; uvrA;

Metabolic activation:

with and without

Metabolic activation system:

S9 Metabolic Activation System

Test concentrations with justification for top dose:

Dose Range finding Assay
A dose range finding study was performed using tester strains TAIOO and WP2MvrA, and ten doses of test article ranging from 6.67 to 5000 ug/plate, in the absence of S9 and in the presence of standard S9 (one plate per dose; Experiment 22733-Al; Tables 1 and 2). Inhibited growth (characterized by a decreased revertant frequency or a thinning of the background lawn) was observed in tester strain TA100 at doses >667 µg/plate with S9 and >100 µg/plate without S9, and in tester strain WP2uvrA at doses >1000 ug/plate with S9 and >667 ug/plate without S9. In addition, the test article was found to be freely soluble, at all doses evaluated with and without S9.

Based upon these results, the test article was evaluated in the initial mutagenicity assay in all five tester strains at doses of 33.3,100, 333, 1000, 3330 and 5000 ug/plate in the presence of S9 (both types), and at doses 33.3,100, 333, 1000, 2000 and 3330 µg/plate in the absence of S9 (Experiment 22733-Bl, Tables 3-5). Inhibited growth was observed in all five tester strains at the highest 2-3 dose levels with S9 (both types), and at the highest 1-4 dose levels without S9.

Vehicle / solvent:

Water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

other: 2-aminoanthracene; ICR-191;

Details on test system and experimental conditions:

Tester Strains. The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TAIOO, TAI535 and TA1537 (Ames et al., 1975) and the Escherichia coli tryptophan auxotroph WP2MvrA (Green and Muriel, 1976). The specific genotypes of the strains are shown below
TA98; hisD3052; uvrB; rfa; pKM101
TA100; hisG46; MvrB; rfa; pKM101
TA1535; hisG46; MvrB; rfa
TA1537; hisC3016; uvrB; rfa
WP2MvrA; trp; uvrA;
In addition to a mutation in the histidine or tryptophan operons, the tester strains contain two additional mutations which enhance their sensitivity to some mutagenic compounds. A mutation
Plating Procedures
These procedures were used in the dose rangefinding study and the mutagenicity assays. Each plate was labeled with a code which identified the test article, test phase, tester strain, activation condition and dose level. The S9 mixes and dilutions of the test article were prepared immediately prior to their use.
When S9 mix was required, 500 \xL of the appropriate mix was added to 13 x 100 mm glass culture tubes which had been pre-heated to 30 or 37°C ± 2°C (for reductive and standard S9 mixes, respectively). To these tubes were added 100 jiL of tester strain and 50 \iL of test or control article. If S9 mix was not required, 500 |JL of O.IM phosphate buffer was substituted for the standard S9 mix. After the required components had been added, the mixture was vortexed. Cultures treated with standard S9 or without S9 were incubated for 20 ± 2 minutes at 37 ± 2°C, while those treated with reductive S9 were incubated for 30 ± 2 minutes at 30 ± 2°C.
Following the 20- or 30-minute pre-incubation, 2 mL of molten selective top agar was then added to each tube, and the mixture was vortexed aiid overlaid onto 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 52 ± 4 hr at 37 ± 2°C. Positive control articles were plated using a 50 ML plating aliquot.
Scoring the Plates
Plates which were not evaluated immediately following the incubation period were held at 5 ± 3°C until such time that colony counting and bacterial background lawn evaluation could take place.
Bacterial Background Lawn Evaluation. The condition of the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate (except as noted below; see Protocol Deviation). Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level. Lawns were scored as normal (N), reduced (R), obscured by precipitate (O), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) also were noted.
Counting Revertant Colonies. Revertant colonies were counted by automated colony counter or by hand.

Rationale for test conditions:

The bacterial reverse mutation assay detects point mutations, both frameshifts and/or base pair substitutions. The strains of Salmonella typhimurium and Escherichia coli used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his-) or tryptophan (trp-) dependant cells, are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan) only those cells which revert to histidine (his+) or tryptophan (trp+) independence are able to form colonies. The trace amount of histidine or tryptophan in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The his+ or trp+ revertants are readily discernible as colonies against the limited background growth of the his- or trp- cells. By utilizing several different tester strains, both base pair substitution mutations and frameshift mutations can be detected.

The bacterial reverse mutation assay has been shown to be a sensitive, rapid and accurate
indicator of the mutagenic activity of many materials including a wide range of chemical classes.

Evaluation criteria:

Assay Evaluation Criteria
Once the criteria for a valid assay had been met, responses observed in the assay were evaluated.

Tester Strains TA98, TA100 and WP2avrA. For a test article to be considered positive, it had to produce at least a 2-fold 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. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Tester Strains TA1535 and TA1537. For a test article to be considered positive, it had to produce at least a 3-fold 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. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Statistics:

N/A

Results and discussion

Test results

Test results 1

Key result

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed during range finding study

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 4

Key result

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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed dring range finding study

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay: Preincubation Method with a Confirmatory Assay indicate that, under the conditions of this study, the test item did not cause a positive increase in revertant frequencies in any tester strain with or without standard or reductive S9.

Executive summary:

The objective of this study was to evaluate the test article and/or its metabolites for their ability to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2MvrA. Evaluation of the test article was performed in the absence of exogenous metabolic activation, as well as in the presence of standard (Aroclor 1254-TM-induced male Sprague-Dawley rat liver) and reductive (uninduced male Golden Syrian hamster liver with FMN) metabolic activation systems (S9s).

 

A dose range finding study was performed using tester strains TA100 and WP2MvrA, and ten doses of test article ranging from 6.67 to 5000 µg/plate, in the absence of S9 and in the presence of standard S9 (one plate per dose). Inhibited growth (characterized by a decreased revertant frequency or a thinning of the background lawn) was observed in tester strain TA100 at doses >667 µg/plate with S9 and >100 µg/plate without S9, and in tester strain WP2uvrA at doses >1000 µg/plate with S9 and >667 µg/plate without S9. In addition, the test article was found to be freely soluble, at all doses evaluated with and without S9.

 

Based upon these results, the test article was evaluated in the initial mutagenicity assay in all five tester strains at doses of 33.3, 100, 333,1000, 3330 and 5000 µg/plate in the presence of S9 (both types) and at doses 33.3, 100, 333,1000, 2000 and 3330 µg/plate in the absence of S9. Inhibited growth was observed in all five tester strains at the highest 2-3 dose levels with S9 (both types), and at the highest 1-4 dose levels without S9. The test article again was found to be freely soluble at all doses evaluated with and without S9. Increases in revertant frequencies, to approximately 2.1- to 3.1-fold control values, were observed in tester strains WP2uvrA and TA1537, respectively, in the presence of reductive S9. However, these increases did not appear to be dose dependent, and those observed in tester strain WP2uvrA were within acceptable vehicle control ranges. Revertant frequencies for all other tester strain/S9 combinations approximated or were less than those observed in the concurrent negative control cultures.

 

The test article was re-evaluated in an independent confirmatory assay under identical conditions (except that a dose of 2000 µg/plate was added with both S9s). Inhibited growth again was observed in all five tester strains at the highest 2-4 dose levels with and without S9 (both types), and the test article again was found to be freely soluble at all doses evaluated with and without S9. Revertant frequencies for all doses of MIP Yellow 2982, in all tester strain/S9 combinations, approximated negative control values. All positive and negative control values in both assays were within acceptable ranges, and all criteria for a valid study were met.

 

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay: Preincubation Method with a Confirmatory Assay indicate that, under the conditions of this study, the test item did not cause a positive increase in revertant frequencies in any tester strain with or without standard or reductive S9.