Reaction mass of methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate and methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

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:

28 March 2000 - 23 June 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Column 1 of REACH Annex VII informs on the standard mutagenicity information requirements, for substances produced or imported in quantities >1 tpa. The Bacterial Reverse Mutation Test (OECD 471) detects gain of function point mutations and frameshifts in vitro, and is required to fulfil Annex VII information requirements.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Molecular formula:

C17H21NO2

IUPAC Name:

Methyl 2-[[(Z)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Constituent 2

Reference substance name:

Methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Molecular formula:

C17H21NO2

IUPAC Name:

Methyl 2-[[(E)-(2,4-dimethylcyclohex-3-en-1-ylidene)methyl]amino]benzoate

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures mutation in the histidine operon. Gain of function his- to his+ mutation is induced by chemicals which cause base changes or frameshift mutations in the genome of this organism. S. typhimurium strains TA98 and TA1537 primarily respond to frameshift mutations at the histidine gene locus his D 3052 and his C 3076, respectively. Strains TA100, TA102 and TA1535 respond to base-pair substitutions in the his G 46, his G 428 and his G 46 locus.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: rfa

Remarks:

loss of the outer lipopolysaccharide barrier

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homgenate (S9)

Test concentrations with justification for top dose:

15, 50, 150, 500, 1500 and 5000 µg per plate. In the presence and absence of S9-mix the test item was bacteriotoxic towards strain TA102 at 5000 μg/plate. Precipitation of the test compound an the plates was observed at 1500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

CELLS USED
- Source of cells: Dr. Bruce N. Ames, University of California, Berkeley, California, U.S.A.
- Suitability of cells: All tester strains except the strain TA102 contain a uvrB- mutation, which causes loss of the excision repair system, and greatly increases the sensitivity of the test for detecting of many mutagens.
- Methods for maintenance in cell culture if applicable: Master plates are used as a source of bacteria for inoculating the overnight cultures and are always kept at 4°C and discarded alter one month or sooner if the number of spontaneous revertants per plate falls out of the normal range specific for a strain. The tester strain cultures used in this study were grown in Oxoid nutrient broth No.2 (2.5%) on a shaker at 37°C for 11-12 hours to a density of 1E9 to 3E9 cells per mL. They were then kept at 4°C. Only fresh cultures are used for mutagenicity assays.

MEDIA USED
- Type and identity of media: Agar, 1.5%; Difco bacto nutrient broth, 0.8%; NaCl, 0.5%
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

Number of spontaneous revertant colonies (his+ revertants)

Statistics:

Estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level was calculated using a X2-test (Mohn & Ellenberger, 1977).

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Bacteriotoxic at 5000 µg/plate

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound an the plates was observed at 1500 and 5000 µg/plate

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: TA1535 (-S9: 31±11, range 13-58; +S9: 19±6, range 9-27), TA1537 (-S9: 11±4, range 5-20; +S9: 16±5, range 9-29), TA98 (-S9: 30±8, range 18-47; +S9: 40±10, range 21-63), TA100 (-S9: 150±35, range 89-226; +S9: 139±38, range 88-238), TA102 (-S9: 280±37, range 218-359; +S9: 306±45, range 237-400)
- Negative (solvent/vehicle) historical control data: TA1535 (-S9: 30±11, range 10-62; +S9: 18±6, range 9-28), TA1537 (-S9: 11±4, range 6-21; +S9: 16±5, range 9-29), TA98 (-S9: 29±8, range 19-50; +S9: 38±9, range 23-62), TA100 (-S9: 138±29, range 94-205; +S9: 133±38, range 79-222), TA102 (-S9: 269±38, range 163-337; +S9: 298±46, range 207-404)

Applicant's summary and conclusion

Conclusions:

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. The Bacterial Reverse Mutation Test (OECD 471, EU B.13/14) detects gain of function point mutations and frameshifts in vitro, and is required to fulfil REACH Annex VII information requirements. Under the test conditions, the test item did not present any mutagenic properties in the Bacterial Reverse Mutation Test. Conducted according to the aforementioned guidelines and GLP, the Ames Test passed all validity criteria and was considered to be reliable without restriction (Klimisch 1).

Executive summary:

An Ames test was conducted on the test item using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 (King 2000). The test was performed by the plate incorporation assay at test item concentrations of 15, 50, 150, 500, 1500 and 5000 µg/plate in the presence of S9 and 50, 150, 500, 1500 and 5000 µg/plate in the absence of S9, alongside negative, solvent and positive controls. The test item was incubated for 48 to 72 hours and mutagenicity was assessed based on the number of revertant colonies. The test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolising system. In the presence and absence of S9-mix the test item was bacteriotoxic towards the strain TA102 at a concentration of 5000 µg/plate. The study is reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD Guideline 471.