Hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2-undecanone

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:

14 March 1978 - 26 April 1978

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Remarks:

Study predates GLP

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2-undecanone

EC Number:

270-151-7

EC Name:

Hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2-undecanone

Cas Number:

68411-85-8

Molecular formula:

C19H32N4O2

IUPAC Name:

2-{[(6-oxocyclohexa-2,4-dien-1-ylidene)methyl]amino}guanidine; undecan-2-one

Test material form:

liquid: viscous

Specific details on test material used for the study:

- Source and lot/batch No. of test material: Mobil Chemical Company (Edison, NJ) Lot # 1086
- Expiration date of the lot/batch: not specified
- Purity test date: not specified

Method

Target gene:

Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98 and TA100 also contain the pKM101 plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions. TA1538 is reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens.

Saccharomyces cerevisiae test strains can be used to assess mitotic recombination within genes. This event is most frequently non-reciprocal and is called gene conversion. Gene conversion is assayed by the production of prototrophic revertants produced in an auxotrophic heteroallelic strain carrying two different defective alleles of the same gene. The D4 strain for the detection of mitotic gene conversion carries heteroallelic genes AD2 (ade 2-2, ade 2-1) and TRP5 (trp 5-12, trp 5-27). A genotoxic chemical may produce prototrophic colonies carrying one wild type allele which allows for growth on selective medium lacking either tryptophane or adenine as a result of mitotic gene conversion.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa mutation, uvrB deletion, pKM101 plasmid

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: rfa mutation, uvrB deletion

Species / strain 3

Species / strain / cell type:

Saccharomyces cerevisiae

Remarks:

D4

Additional strain / cell type characteristics:

other: heteroallelic deficiency ade2 and trp5

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver homogenate

Test concentrations with justification for top dose:

0.01, 0.1, 1.0, 5.0, 10.0 uL/plate
Test substance exhibited cytotoxicity in all strains at 5 and 10.0 uL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test substance was soluble and formed a very ligth amber solution in DMSO at up to 200 uL/mL

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 2-aminoanthracene

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-aminoanthracene

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: 2-aminoanthracene

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: 2-aminoanthracene

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 2-aminoanthracene

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10^8

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 hours (Salmonella typhimurium); 3-5 days (Saccharomyces cerevisiae)

NUMBER OF REPLICATIONS: 2 replicate plates per dose

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertants per plate
The revertant colonies will be counted manually and the plates will be examined for bacterial background lawn. The condition of the bacterial background lawn will be evaluated for evidence of test substance toxicity. Evidence of toxicity will be scored relative to the vehicle control plate and recorded along with the revertant count for that plate. Toxicity will be evaluated as a decrease in the number of revertant colonies per plate and/or a thinning or disappearance of
the bacterial background lawn.

Rationale for test conditions:

The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens.

Evaluation criteria:

1. Strains TA1535, TA1537 and TA1538: If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.
2. Strains TA98, TA100, and D4: If the solvent control value is within the normal range, a chemical that produces a positive dose response over three concentrations with the lowest increase equal to twice the solvent control value for strain TA100 and twice - three times the solvent control for strains TA98 and D4 is considered to be mutagenic.
3. Reproducibility: If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test date loses significance.

Statistics:

Mean revertants per plate were counted and standard deviations were established.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

Saccharomyces cerevisiae

Remarks:

Strain D4

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The in vitro bacterial gene mutation test to assess the genotoxicity of Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was negative in Salmonella typhimurium and Saccharomyces cerevisiae.

Executive summary:

Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was examined for its potential to induce mutations in Salmonella typhimurium and Saccharomyces cerevisiae, in both the presence and absence of an S9 metabolic activation system. The doses were: 0.01, 0.10, 1.0, 5.0, and 10.0 uL/plate). In the mutagenicity assay, all data were acceptable and no significant increase in the number of revertants per plate were observed with any test strain in any tester strain/activation condition combinations. Under the conditions in this study, Hydrazine Carboximidamide 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was not mutagenic to Salmonella typhimurium or Saccharomyces cerevisiae.