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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in Bacteria- Not mutagenic to Salmonella typhimurium or Saccharomyces cerevisiae (OECD TG 471)

In-vitro gene mutation in Bacteria- Not mutagenic to Salmonella typhimurium, mutagenic to Escherichia coli without S9 (OECD TG 471)

In-vitro gene mutation in mammalian cells- Not mutagenic in L5178Y mouse lymphoma cells (OECD TG 490)

Link to relevant study records

Reference

Reference 1

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

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

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

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 / 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

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 April 1997 - 13 May 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: batch# CRU 96269
- Expiration date of the lot/batch: not specified
- Purity test date: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: test substance remained in solution throughout duration of assay
- Solubility and stability of the test substance in the solvent/vehicle: test substance was soluble in vehicle up to 100 mg/mL and formed a clear, yellow solution
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no reactivity with vehicle was reported

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 thepKM101 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.

The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene. Tester strain WP2uvrA (pKM101) has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base substitution mutations.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: rfa wall mutation; pKM101 plasmid

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA deletion

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 Rat Liver S9 homogenate

Test concentrations with justification for top dose:

100, 250, 500, 1000, 2500, 5000 ug/plate
Cytotoxicity was observed in the dose rangefinding study and the highest concentration used in the mutagenicity assay was a dose which gave a reduction of revertants per plate and/or a thinning of the bacterial background lawn.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:test substance was soluble in DMSO up to 100 mg/mL

Untreated negative controls:

yes

Remarks:

historical average of spontaneous revertants strain TA98

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: 2-amino anthracene

Untreated negative controls:

yes

Remarks:

historical average of spontaneous revertants Strain TA100

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-aminoanthracene

Untreated negative controls:

yes

Remarks:

historical average of spontaneous revertants Strain TA1535

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-aminoanthracene

Untreated negative controls:

yes

Remarks:

Historical average of spontaneous revertants Strain TA1537

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; ICR-191

Untreated negative controls:

yes

Remarks:

Historical average of spontaneous revertants Strain WP2uvrA

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): > or = to 0.5 x 10^9 bacteria per mL

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 ± 8 hours

NUMBER OF REPLICATIONS: 3 replicates per dose

DETERMINATION OF CYTOTOXICITY
- Method: Thinning or dissapearance in 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:

The following criteria were used to determine a positive response:
1. Tester Strains TA98, TA100, and WP2uvrA: 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.
2. Tester Strains TA1535 and TA1537: for a test article to be considered positive, it had to produce at leas a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the 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:

The mean revertants per plate and and the standard deviation were calculated.

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

Untreated negative 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative 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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Main Assay

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 positive in Escherichia coli.

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 Escherichia coli, in both the presence and absence of an S9 metabolic activation system.  Two separate bacterial mutation tests were performed. The doses were: 100, 250, 500, 1000, 2500, 5000 ug/plate. In the initial mutagenicity assay, all data were acceptable and positive increases in the number of revertants per plate were observed with tester strain WP2uvrA both in the presence (2.8-fold) and absence (2.2-fold) of S9 mix. No positive increases were observed with any of the remaining tester strain/activation condition combinations. In the confirmatory assay, all data were acceptable and a 3.3 fold positive increase in the number of revertants per plate was observed with tester strain WP2uvrA in the absence of S9 mix. The increase in revertants per plate observed in WP2uvrA with S9 mix was not reproducible. Under the conditions in this study, Hydrazine Carboximidamide 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was mutagenic Escherichia coli.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 July 1997 - 08 October 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

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

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test article was stored at room temperature, protected from light, and the vehicle of choice was DMSO.
- Stability under test conditions: The test article was prepared in DMSO at 100-times the highest desired concentration. Lower lOOx stocks were prepared by serial dilution with DMSO. The test article remained in solution in culture medium up to about 100 ug/ml.
- Solubility and stability of the test substance in the solvent/vehicle: Test substance was soluble in DMSO up to 3 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The test article remained in solution in culture medium up to about 100 ug/ml.

Target gene:

The cell line used in this test measures forward mutations in a reporter gene, specifically the endogeneous thymidine kinase gene contained in the L5178Y TK+/--3.7.2C mouse lymphoma cell line (generally called L5178Y). The autosomal and heterozygous nature of the thymidine kinase gene enables the detection of viable colonies whose cells are deficient in the enzyme thymidine kinase following mutation from TK+/- to TK-/-. This deficiency can result from genetic events affecting the TK gene including both gene mutations (point mutations, frame-shift mutations, small deletions, etc.) and results in a slow growing phenotype. Slow growing mutants have suffered genetic damage that involves putative growth regulating gene(s) near the TK locus which results in prolonged doubling times and the formation of late appearing or small colonies.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

Clone 3.7.2C

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Stock cultures were obtained from Dr. Donald Clive
- Suitability of cells: The mouse TK locus is located on the distal end of chromosome 11. The L5178Y TK+/- -3.7.2C cell line has mutations in both p53 alleles and produces mutant-p53 protein. The p53 status of the TK+/--3.7.2C cell line is likely responsible for the ability of the test to detect large-scale damage
- Cell cycle length, doubling time or proliferation index:
- Methods for maintenance in cell culture if applicable: All laboratory cultures were maintained in logarithmic growth by serial subculturing for up to 4 months and were then replaced by cells from the frozen stock. To reduce the frequency of spontaneous TK"'" mutants prior to use in the mutation assay, cell cultures were exposed to conditions that selected against the TK"'" phenotype and then returned to normal growth medium for three to eight days
- Modal number of chromosomes: 40
- Number of passages if applicable: not specified

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The medium used for this study was RPMI 1640 (Amacher et a l , 1980; Clive et a l , 1987) supplemented with horse serum (10% by volume), Pluronic® F68, L-glutamine, sodium pyruvate, penicillin and streptomycin. Treatment medium was Fischer's medium with the same media supplements used in the culture medium except that the horse senun concentration was reduced to 5% by volume. Cloning medium consisted of the RPMI 1640 growth medium with up to 20% horse serum, without Pluronic® F68 and with the addition of 0.24% BBL® agar to achieve a
semisolid state. Selection medium was cloning medium that contained 3 ug/ml of TFT (Clive et a l , 1987).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver homogenate

Test concentrations with justification for top dose:

Dose range finding assay: 1.97, 3.93, 7.85, 15.7, 31.3, 62.5, 125, 250, 500, 1000 ug/mL

The test article was noncytotoxic to moderately cytotoxic without metabolic activation from 1.97 ug/ml to 62.5 ug/ml. The 125 pg/ml treatment was highly cytotoxic and higher concentrations were lethal. In the presence of metabolic activation, the test article was noncytotoxic to moderately cytotoxic from 1.97 pg/ml to 125 pg/ml and the 250 pg/ml treatment was highly cytotoxic. Higher concentrations were lethal.

Main Assay:
Two nonactivation mutation assays were performed.
Trial 1- 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 175, 200, 225, 250 and 300 pg/ml were initiated and treatments from 175 ug/ml to 300 ug/ml were terminated because of excessive cytotoxicity.
Trial 2- 12.5, 25.0, 50.0, 75.0, 100, 125, 150,175, 200, 225 and 250 ug/ml were initiated and treatments above 175 ug/ml were terminated because of excessive cytotoxicities

Two trials of the activation assay were performed.
Trial 1- 25.0, 50.0, 75.0, 100, 125,150, 200, 225, 250, 300, 350 and 400 ug/ml but doses below 200 ug/ml were terminated because a sufficient number of doses were available for analysis.
Trial 2- 50.0, 100, 150, 200, 250, 300, 350, 400, 450 and 500 ug/ml were initiated and doses at 50.0 pg/ml, 150 pg/ml and 250 pg/ml were terminated in order to leave six doses with a good range of cytotoxicity for analysis

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test substance was soluble in DMSO up to 3 mg/mL

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9 homogenate

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

With S9 homogenate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 6 x 10^6 cells

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 - 14 days

SELECTION AGENT (mutation assays): Triflurothymidine (TFT)

NUMBER OF REPLICATIONS: The assay conditions consisted of vehicle controls in triplicate, two positive controls and eleven or twelve different test article dose levels using one culture per dose level.

NUMBER OF CELLS EVALUATED: A total of 3 x 106 cells from each selected tube was suspended in selection medium in soft agar to recover mutants.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The test article was noncytotoxic to moderately cytotoxic without metabolic activation from 1.97 ug/ml to 62.5 ug/ml.
- Any supplementary information relevant to cytotoxicity: Concentrations were chosen to cover a toxicity range from 10% to 20% survival to no apparent effect on growth compared to the vehicle control, or if little or no toxicity was observed and solubility was maintained, the mutation experiment was initiated with a maximum concentration of 5 mg/ml or, if precipitation of the test article occurred in the culture medium, the maximum applied dose was at leas twice the solubility limit in culture medium.

Rationale for test conditions:

The assay procedure used was based on that reported by Clive and Spector (1975), Clive, et al. (1979), Amacher et al. (1980) and Clive et al. (1987). Although
doses were selected to initiate a mutation experiment, the objective was to carry at least six doses through the entire assay. This procedure compensated for normal variations in cellular toxicity and helped to ensure the choice of at least four doses over a wide range of cytotoxicities.

Evaluation criteria:

Controls: The average absolute cloning efficiency of the vehicle controls should be between 60% and 130%. Average suspension growth of the vehicle controls for two days should be an 8.0-fold increase over the original cell numbers. The background mutant frequency (average of vehicle control values) is calculated separately for concurrent activation and nonactivation assays and should be 30 x 10"6 to 120 x 10"6.

High Dose: For test articles with weak/no mutagenic activity, relative growth should be 10% or 20% of the average vehicle control or reach the maximum applied concentrations given in the evaluation criteria. This requirement was waived if the concentration of the highest assayed dose was at least 75 % of a higher, excessively cytotoxic dose level. There is no maximum toxicity requirement for test articles which clearly show mutagenic activity.

Number of Doses: Relative cloning efficiency is 10% or greater and the total number of viable clones exceeds about 60. Minimum of four analyzed cultures is considered necessary to accept a single assay for evaluation of the test article.

Positive Criteria: The test article is evaluated as positive if dose-dependent increases of 2-fold or greater in mutant frequency are obtained over the concurrent background mutant frequency. It is desirable to obtain this relationship for at least three doses, but this goal depends on the dose steps chosen for the assay and toxicity at which mutagenic activity appears.

Negative Criteria: The test article was evaluated as negative in a single trial if a 2-fold increase in mutant frequency was not observed for (1) a range of doses that extended to toxicities causing 10-20% relative total growth, or (2) for relatively nontoxic test articles, a range of doses extending to the maximum concentration of 5 mg/ml, or (3) a range of doses that extended to a level approximately twice the solubility limit in culture medium, or (4) the increase(s) are not repeatable in a confirmatory trial.

Statistics:

The mutant selection data includes the total mutant colonies (i.e. total number of mutant colony counts obtained from 3 x 106 cells sampled from one culture, seeded into selective medium and divided among three culture dishes) and the total viable colonies (i.e. the total number of colony counts obtained from 600 cells sampled from one culture, seeded into nonselective medium and divided among three culture dishes). These were used to calculate the mutant frequency for each culture. The ratio of cells seeded for mutant selection to cells seeded for cloning efficiency was 0.5 x 104. Therefore, the mutant frequency was: (total mutant colonies/total viable colonies) x 2 x 10"4. Mutant frequency was given in units of 10"6. Also included in this section was the cloning efficiency of vehicle and positive controls, calculated as (total number of viable colonies/600) x 100%.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: no evaporation observed
- Water solubility: Test substance was not slublel in water; Test substance was soluble in DMSO up to 3 mg/mL
- Precipitation: The test article remained in solution in culture medium up to about 100 pg/ml.

RANGE-FINDING/SCREENING STUDIES: The test article was noncytotoxic to moderately cytotoxic without metabolic activation from 1.97 (xg/ml to 62.5 pg/ml. The 125 pg/ml treatment was highly cytotoxic and higher concentrations were lethal. In the presence of metabolic activation, the test article was noncytotoxic to moderately cytotoxic from 1.97 pg/ml to 125 pg/ml and the 250 pg/ml treatment was highly cytotoxic.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The measurement of the cytotoxicity of each treatment was the relative suspension growth of the cells over the two-day expression period multiplied by the relative cloning efficiency at the time of selection.

The average cloning efficiencies for the vehicle controls varied from 107.8% and 95.2% without activation to 97.1% and 88.1% with S9 metabolic activation which demonstrated acceptable cloning conditions for the assays. The positive control cultures, MMS (nonactivation) and MCA (activation) induced large increases in mutant frequency that were greatly in excess of the minimum criteria.

Conclusions:

The in-vitro gene mutation test to assess the genotoxicity of Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was negative in the absence or presence of metabolic activation in in the mouse L5178Y cell line. This finding does not warrant classification of Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone as genotoxic under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Executive summary:

Two nonactivation mutation assays were performed with the test article. In Trial 1 (Table 2), twelve treatments at 12.5, 25.0, 50.0, 75.0, 100, 125, 150, 175, 200, 225, 250 and 300 ug/ml were initiated and treatments from 175 ug/ml to 300 ug/ml were terminated because of excessive cytotoxicity. The 12.5 ug/ml treatment was also terminated because sufficient doses were available for analysis. The remaining six treatments induced a wide range of cytotoxicity (138.4% to 23.4% relative growths). In order for a culture to be evaluated as mutagenic in Trial 1 of the nonactivation assay, a mutant frequency greater than 134.0 x 10^6 was required. This threshold value was equal to twice the average mutant frequency of the concurrent vehicle controls. None of the test article treated cultures induced this level of mutant action. A confirmatory assay was initiated.

In Trial 2 of the nonactivation assay, eleven treatments at 12.5, 25.0, 50.0, 75.0, 100, 125, 150,175, 200, 225 and 250 ug/ml were initiated and treatments above 175 ug/ml were terminated because of excessive cytotoxicities. The two lowest concentrations were also terminated leaving six dose levels. These remaining six treatments induced a wide range of toxic action (74.4% to 11.6% relative growths). The minimum criterion for a positive response in Trial 2 of the nonactivation assay was 94.2 x 10^6. None of the assayed treatments induced this level of mutant action and no dose related trend was observed. The test article was therefore considered nonmutagenic without activation in this assay.

Two trials of the activation assay were performed. In Trial 1, cultures were dosed with the test article at 25.0, 50.0, 75.0, 100, 125,150, 200, 225, 250, 300, 350 and 400 ug/ml but doses below 200 ug/ml were terminated because a sufficient number of doses were available for analysis. The remaining six doses were weakly cytotoxic to moderately cytotoxic (66.3% to 33.6% relative growth). In order for a culture to be evaluated as mutagenic in this assay, a mutant frequency of greater than178.2 x 10^6 was required. None of the treatments induced a mutant frequency that exceeded the minimum criterion. The test article was considered nonmutagenic in this trial.

Another trial was initiated to confirm these results. In the Trial 2 of the activation assay, ten treatments at 50.0, 100, 150, 200, 250, 300, 350, 400, 450 and 500 ug/ml were initiated and doses at 50.0 ug/ml, 150 ug/ml and 250 ug/ml were terminated in order to leave six doses with a good range of cytotoxicity for analysis. The selected doses induced weak cytotoxicity to high cytotoxicity (83.2% to 17.4% relative growth). In order for a dose to be considered positive in this assay, a mutant frequency exceeding 136.4 x 10^6 was required. Again, none of the analyzed treatments induced a mutant frequency that exceeded the minimum criterion. The results of the two activation mutation trials were therefore evaluated as negative for inducing forward mutations at the TK locus in mouse lymphoma cell.

Under the conditions in this study, Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was not mutagenic in the absence or presence of metabolic activation in L5178Y cells. These findings do not warrant classification of Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone as genotoxic under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP). 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was examined for its potential to induce mutations in Salmonella typhimurium bacterial cells, and Saccharomyces cerevisiae yeast cells, in both the presence and absence of an S9 metabolic activation system. The test substance did not induce a statistically significant increase in the number of revertant cells at any of the doses chosen with or without metabolic activation. In a subsequent assay, Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was examined for its potential to induce mutations in Salmonella typhimurium and Escherichia coli, in both the presence and absence of an S9 metabolic activation system. No positive increases were observed with any Salmonella typhimurium tester strain/activation condition combination. A reproducible positive increase in the number of revertants was observed for Escherichia coli without S9 mix. The in-vitro gene mutation test in mammalian cells was conducted to provide a definitive conclusion on mutagenicity. No mutagenicity was observed in the absence or presence of metabolic activation in the mouse L5178Y cell line. Taken together, these findings do not warrant the classification of Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone as a genotoxin under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).