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Toxicological information

Genetic toxicity: in vitro

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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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Remarks:
Study predates GLP
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 / strainopen allclose all
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 / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: rfa mutation, uvrB deletion
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
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: 2-aminoanthracene
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
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
other: 2-aminoanthracene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
other: 2-aminoanthracene
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: 2-aminoanthracene
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 resultsopen allclose all
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
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
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
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
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
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.