N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Aug to 15 December 1986

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:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

The study method is comparable to this guideline but predates it.

Principles of method if other than guideline:

Clive, D. and Spector, J.F.S. Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutation Res. 31:17-29, 1975 de Serres, et al., The Salmonella Mutagenicity Assay: Recommendations, Science 203:563-565, 1979

GLP compliance:

yes

Type of assay:

other: Assessing specific locus mutations at the TK locus in cultured L5178Y TK+/- Mouse Lymphoma cells.

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

EC Number:

204-029-1

EC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Cas Number:

113-48-4

Molecular formula:

C17H25NO2

IUPAC Name:

N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

impurity 1

Reference substance name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

Molecular formula:

C20H38N2O2

IUPAC Name:

(E)-1,4-Bis(2-ethylhexylamino)-2-butene-1,4-dione

impurity 2

Reference substance name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Molecular formula:

C12H19NO2

IUPAC Name:

1-(2-ethylhexyl)-1H-pyrrole-2,5-dione

Test material form:

liquid

Details on test material:

Purity and characterisation analysis conducted on the following sample; Supplier: McLaughlin Gormley King ; Batch Number: AB9500

Specific details on test material used for the study:

MGK®264, Lot Number: 3843

Method

Target gene:

TK locus in cultured L5178Y TK+/- Mouse Lymphoma cells.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

Based on the data derived from the toxicity test, the test article was prepared so that the highest concentration was 100% toxic. The test article was solubilized and 15 serial eighth log dilutions were carried out. This produced 16 dose levels decreasing approximately 100-fold from highest to lowest. The nonactivated cultures that were cloned had been treated with 0.018, 0.013, 0.0075, 0.0056, 0.0042, 0.0032, 0.0024, 0.0018 and 0.0013 µl/ml. The activated cultures that were cloned had been treated with 0.056, 0.042, 0.032, 0.024, 0.018, 0.013, 0.01, 0.0075, 0.0056 and 0.0042 µl/ml.

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

yes

Remarks:

Solvent vehicle for the test article was used as the negative control.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

Two concentrations of ethylmethanesulfonate (EMS) will be used as a positive control for direct acting mutagens, and two concentrations of 7,12-dimethylbenz(a)anthracene (DMBA) will be used for promutagens.

Details on test system and experimental conditions:

The plates were incubated at 37±1°c in a humidified 5% CO2 atmosphere for 10-12 days.

Rationale for test conditions:

per method described in: Clive, D. and Spector, J.F.S. Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y mouse lymphoma cells. Mutation Research 31:17-29, 1975.

Evaluation criteria:

The following criteria were used as guidelines in judging the significance of the activity of a test article in this system.

Positive - if there is a positive dose response and one or more of the three highest doses in the 10% or greater Total Growth range exhibit a mutant frequency which is two-fold greater than the background level. All data including that from cultures with less than 10% Total Growth will be used to establish the dose response relationship. The first assay and the confirmatory assay must both demonstrate a positive response to call a test article a positive mutagen.

Equivocal - if there is no dose response but any one or more of the three highest doses with 10% or greater Total Growth exhibit a two-fold increase in mutant frequency over background, or if there is a dose response but no culture exhibits a two-fold increase in mutant frequency over background. If an assay produces a positive response and the confirmatory assay produces an equivocal or negative response, then the results for the test article will be classed as having produced an equivocal response.

Negative - if there is no dose response in cultures with 10% or greater Total Growth and none of these test cultures exhibit a two-fold or greater increase in mutant frequency over background. Both assays must demonstrate a negative response for the test article to be classed as negative.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

other: Negative response in the presence of activation. Some sporadic increases in mutant frequency were seen in cultures treated in the absence of activation. There was no clear dose dependent response and no reproducible mutagenic response within a single dose

Remarks:

The response was only noted at highly toxic levels (less than 20% Total Growth). The results meet the criteria for an equivocal response.

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

McLaughlin Gormley King Company's test article MGK® 264, Lot Number 3843 (MBA #T5205) was tested in the L5178Y TK+/- mouse lymphoma mutagenesis assay in the presence and absence of Aroclor induced rat liver S-9. Two mutagenesis assays were conducted. In the first assay, the nonactivated cultures that were cloned had been treated with a range of test article concentrations from 0.018 µl/ml to 0.0013 µl/ml. These concentrations produced a range in Total Growth of 11% to 102%. One non activated culture that was cloned exhibited a mutant frequency which was twice the mean mutant frequency of the solvent controls. The Total Growth of this culture was 11%. There was no dose-dependent response in the treated cultures. The S-9 activated cultures that were cloned had been treated with a range of test article concentrations from 0.056 µl/ml to 0.0042 µl/ml. These concentrations produced a range in Total Growth of 22% to 160%. None of the S-9 activated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls.

 

In the second assay, the non activated cultures that were cloned had been treated with a range of test article concentrations from 0.015 µl/ml to 0.004 µl/ml test article. These concentrations produced a range in Total Growth of 3% to 19%. Three nonactivated cultures with greater than 10% Total Growth exhibited mutant frequencies which were twice the mean mutant frequency of the solvent controls. There was no clear dose-dependent response in the treated cultures and no reproducibility in response within a concentration. None of the S-9 activated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls.

Applicant's summary and conclusion

Conclusions:

The results from both assays indicate that the test article produced a negative response in the presence of exogenous metabolic activation. Some sporadic increases in mutant frequency were noted in cultures treated in the absence of metabolic activation. However, there was no clear dose dependent response and no reproducible mutagenic response within a single dose. The response was only noted at highly toxic levels (less than 20% Total Growth). The results meet the criteria for an equivocal response, as stated in the protocol, however the responses observed in the assays are unlikely to be a significant biological response and may be due to biological variation (c.f. the solvent control mutant frequency in the results for the nonactivated positive control), or epigenetic responses due to toxicity.

Executive summary:

McLaughlin Gormley King Company's test article MGK® 264, Lot Number 3843 (MBA #T5205) was tested in the L5178Y TK+/- mouse lymphoma mutagenesis assay in the presence and absence of Aroclor induced rat liver S-9. Two mutagenesis assays were conducted. In the first assay, the nonactivated cultures that were cloned had been treated with a range of test article concentrations from 0.018 µl/ml to 0.0013 µl/ml. These concentrations produced a range in Total Growth of 11% to 102%. One nonactivated culture that was cloned exhibited a mutant frequency which was twice the mean mutant frequency of the solvent controls. The Total Growth of this culture was 11%. There was no dose-dependent response in the treated cultures. The S-9 activated cultures that were cloned had been treated with a range of test article concentrations from 0.056 µl/ml to 0.0042 µl/ml. These concentrations produced a range in Total Growth of 22% to 160%. None of the S-9 activated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls.

 

In the second assay, the nonactivated cultures that were cloned had been treated with a range of test article concentrations from 0.015 µl/ml to 0.004 µl/ml test article. These concentrations produced a range in Total Growth of 3% to 19%. Three nonactivated cultures with greater than 10% Total Growth exhibited mutant frequencies which were twice the mean mutant frequency of the solvent controls. There was no clear dose-dependent response in the treated cultures and no reproducibility in response within a concentration. None of the S-9 activated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls.

 

The results from both assays indicate that the test article produced a negative response in the presence of exogenous metabolic activation. Some sporadic increases in mutant frequency were noted in cultures treated in the absence of metabolic activation. However, there was no clear dose dependent response and no reproducible mutagenic response within a single dose. The response was only noted at highly toxic levels (less than 20% Total Growth). The results meet the criteria for an equivocal response, as stated in the protocol, however the responses observed in the assays are unlikely to be a significant biological response and may be due to biological variation (c.f. the solvent control mutant frequency in the results for the nonactivated positive control), or epigenetic responses due to toxicity.