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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro mutagenicity and genotoxicity assays with MGK 264 were performed.

In the first assay, MGK 264 was tested in the salmonella/mammalian microsome plate incorporation mutagenicity assay with a confirmatory assay similar to the OECD 471 guideline but predating the current version. The assay evaluates the mutagenic potential of the test article for its ability to induce back mutations at selected loci of several strains of salmonella typhimurium in the presence and absence of exogenous metabolic activation system of Arochlor-induced rat liver microsomes. The tester strains used in this study were TA 98, TA 100, TA 1535, TA 1537 and TA 1538. MGK 264 was tested at 5 dose levels along with appropriate vehicle and positive controls. Following an approximate 48 hour incubation at 37±2^oC, revertant colonies per plate were counted.

The results of the Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay with a Confirmatory Assay indicate that under the conditions of this study in the initial mutagenicity assay and a subsequent confirmatory assay, the test article MGK 264 (MBA# T5205) did not cause a positive response on any of the tester strains with or without metabolic activation by Aroclor-induced rat liver microsomes.

 

In the second assay, MGK 264 was tested twice in the chromosome aberration assay using Chinese hamster ovary cells similar to the OECD 473 guideline but predating the current version. The assay was conducted both in the absence and presence of an Aroclor-induced S-9 activation system at dose levels of 0.04, 0.02, 0.01 and 0.005 µl/ml and 0.08, 0.06, 0.04, 0.02 and 0.01 µl/ml, respectively. A delayed harvest was carried out in the absence of S-9 because of observed cell cycle delay. A statistically significant increase in the number of aberrations per cell was observed at 0.02 µl/ml in the absence of S-9 in the initial assay (p≤0.01, student's t-test); no dose response was observed and the increase was not reproduced in the confirmatory assay. No increase in structural chromosome aberrations was observed at any concentration in S-9 activated system. MGK®264 was concluded to be negative in the CHO chromosome aberration assay.

 

In the third assay, MGK 264 was tested in the mouse lymphoma mutagenesis assay in the presence and absence of Aroclor induced rat liver S-9, similar to the OECD 476 guideline but predating the current version. 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. 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. In the confirmatory assay the nonactivated cultures that were cloned were treated 0.015, 0.012, 0.009, 0.007 or 0.004 µl/ml of MGK 264 and the S-9 activated cultures that were cloned were treated with 0.059, 0.049, 0.039, 0.029 or 0.018 µl/ml. 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.

 

Based on the negative results obtained in 3 different studies, it may be concluded that MGK 264 does not have mutagenic potential.

Link to relevant study records

Reference

Reference 1

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

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.

Specific details on test material used for the study:

MGK®264, Lot Number: 3843

Target gene:

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

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

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.

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Aug to 16 Sep 1986

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)

Version / remarks:

The study was performed according to the AMES test methodologies but predated the current OECD 471 guideline

Principles of method if other than guideline:

The experimental materials, methods and procedures are based on those described by Ames, B.N., et al., Mutation Research, 31:347-364, 1975, and Maron, D.M., and B.N. Ames, Mutation Research, 113:173-215,1983.

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Specific details on test material used for the study:

Test Article ID: MGK264
Lot No.:3843

Target gene:

rfa wall mutation, uvrB and pKM101 in Salmonella typhimurium. Tester strains TA98, TA100, TA1535, TA1537 and TA1538.

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 enzymes from the livers of Male Sprague-Dawley rats, treated with Aroclor 1254 at 500 mg/kg

Test concentrations with justification for top dose:

Dose rates were determined as part of the initial phase of this study "Range-finding"
Maximum dose to be plated in the presence of metabolic activation = 3333 ug per plate.
Maximum dose to be plated in the absence of metabolic activation = 333 ug per plate.

Due to reduced toxicity compared to the range-finding study all tester strains were also repeated as below:
Maximum dose to be plated in the presence of metabolic activation =10,000 ug per plate.
Maximum dose to be plated in the absence of metabolic activation = 1,000 ug per plate.

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY:
- Method: In the pre-experiment: determination of titre (the test item was considered non-toxic, if the qu otient titre/toxicity is below 2), in the main experiments: evaluation of background lawn, reduction in numb er of revertants in comparison to negativ/solvents control

OTHER EXAMINATIONS:
- Visual counting of mutant colonies, mean values and standard deviations were calculated.
- Quality control of bacterial strains: genotype confirmation for each batch of bacteria before stock culture preparation: all bacterial strains were tested for crystal violet sensitivity and spontaneous revertants, TA98 and TA100 were tested for resistance to Ampicillin; furthermore the following examinations were performed: determination of titre, sterility control and positive control.

Evaluation criteria:

For a test article to be considered positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase in the mean number of revertants per plate musy be accompanied by a dose response to increasing concentrations of the test article. In those cases revertants per plate is less than three-fold, the response must be reproducible.

Statistics:

For all replicate platings, the mean number of revertants per plate was calculated and the standard deviation around the mean was also calculated.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

other: No positive response observed

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

other: No positive response observed

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

other: No positive response observed

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

other: No positive response observed

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

not specified

Genotoxicity:

other: No positive response observed

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The results of the Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay with a Confirmatory Assay indicate that under the conditions of this study in the initial mutagenicity assay and a subsequent confirmatory assay, McLaughlin Gormley King Company's test article MGK®264 (MBA# T5205) did not cause a positive response on any of the tester strains with or without metabolic activation by Aroclor induced rat liver microsomes.

Executive summary:

MGK 264 was tested in the salmonella/mammalian microsome plate incorporation mutagenicity assay with a confirmatory assay. The assay evaluates the mutagenic potential of the test article for its ability to induce back mutations at selected loci of several strains of salmonella typhimurium in the presence and absence of exogenous metabolic activation system of arochlor induced rat liver microsomes. The tester strains used in this study were TA 98, TA 100, TA 1535, TA 1537 and TA 1538. MGK 264 was tested at 5 dose levels along with appropriate vehicle and positive controls. Following an approximate 48 hour incubation at 37 +-2 degree celcius, revertant colonies per plate were counted.

The results of the Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay with a Confirmatory Assay indicate that under the conditions of this study in the initial mutagenicity assay and a subsequent confirmatory assay, the test article MGK®264 (MBA# T5205) did not cause a positive response on any of the tester strains with or without metabolic activation by Aroclor induced rat liver microsomes.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 1986 to 14 January 1987

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Similar to previous versions of this guideline. The study predates the current guideline which has had several modifications.

Principles of method if other than guideline:

'The test article, MGK 264, was tested twice in the chromosome aberration assay using Chinese hamster ovary cells. The assay was conducted both in the absence and presence of an Aroclor-induced S-9 activation system at dose levels of 0.04, 0.02, 0.01 and 0.005 ul/ml and 0.08, 0.06, 0.04, 0.02 and 0.01 ul/ml, respectively.

GLP compliance:

yes

Type of assay:

other: In vitro mammalian: chromosome aberration assay using Chinese hamster ovary cells

Specific details on test material used for the study:

MGK 264, Lot No. 3843

Target gene:

The CHO-K cell line is a proline auxotroph with a modal chromosome number of 20 and a population doubling time of 10-14 hours. CHO-K1 cells were obtained from the American Type Culture Collection (repository number CCL 61), Rockville, MD. This system has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1 cells were obtained from the American Type Culture Collection (repository number CCL 61), Rockville, MD.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 activation system

Test concentrations with justification for top dose:

Dose levels for the chromosome aberration assay were selected following a preliminary toxicity test based upon cell proliferation after treatment relative to the solvent control. CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 1.0 to 0.0001 µl/ml in the presence and absence of an S-9 reaction mixture. The test article was soluble in solvent; however some precipitation occurred when added to aqueous treatment medium at final concentrations of 1.0 and 0.3 µl/ml. Cell growth was severely inhibited at levels of 0.1 µl/ml and greater in both test systems and moderately inhibited at 0.03 µl/ml in the absence of S-9. Based upon these findings, dose levels of 0.04, 0.02, 0.01 and 0.005 µl/ml in the absence of exogenous metabolic activation and 0.08, 0.06, 0.04, 0.02 and 0.01 µl/ml in the presence of an S-9 activation system were selected for further study.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Remarks:

The positive and negative controls fulfilled the requirements for a valid test.

Details on test system and experimental conditions:

For each of the chromosome aberration assays, CHO cells were seeded at approximately 5 x 10^5 cells/25 cm2 flask and were incubated at 37±1°C in a humidified atmosphere of 5±1% CO2 in air for 16-24 hours. Treatment was carried out by refeeding duplicate flasks with 5 ml complete medium for the non-activation study or with 5 ml S-9 reaction mixture for the activated study to which was added 50 ul of dosing solution of test or control article in solvent or solvent alone. An untreated control consisting of cells in complete medium or S-9 reaction mixture was also included.

In the non-activation study, the cells were exposed for 14 hours at 37±1°C in a humidified atmosphere of 5±1% CO2 in air. Two hours prior to the
scheduled cell harvest, the treatment medium was removed in order to avoid interference with cell collection and fixation and the cells were washed with PBS and refed with growth medium containing 0.1 ug/ml of colcemid. In the 8-9 activated study, the cells were exposed for 2 hours at 37±1°C in a humidified atmosphere of 5±1% CO2 in air. After the exposure period, the treatment medium was removed, the cells were washed with PBS, refed with complete medium and returned to the incubator for an additional 6 hours. At this time, colcemid was added to duplicate flasks for each treatment condition at a final concentration of 0.1 ug/ml.
Two hours after the addition of Colcemid, metaphase cells were harvested for both the activated and non-activated studies by mitotic shake-off. The cells were collected by centrifugation at approximately 800 rpm for 5 minutes. The cell pellet was resuspended in 2-4 ml 0.075 M KCl and allowed to stand at room temperature for 5 minutes. The cells were collected by centrifugation, the supernatant was aspirated and the cells were fixed with two washes with approximately 2 ml Carnoy’s fixative and were stored overnight or longer in Carnoy's fixative at approximately 4°C.

Evaluation criteria:

To prepare slides, the fixed cells were centrifuged at approximately 250 x g for 5 minutes, the supernatant fluid decanted, and the cells were resuspended to opalescence in Carnoy’s fixative. One-2 drops of cell suspension were dropped onto the center of a moist glass slide and allowed to air dry overnight. Slides were identified by the study number, date prepared and the treatment condition. The dried slides were stained with 5% Giemsa, air dried and permanently mounted.

Slides were coded with random numbers and scored without regard to treatment group. Metaphase cells with 20±2 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 100 metaphase spreads from each dose level (50 per duplicate flask) were examined and scored for chromatid and chromosome gaps and breaks, chromatid and acentric fragments, chromatid deletions, dicentrics, rings, triradials, quadriradials, complex rearrangements , pulverized chromosome (s) and cells and severly damaged cells (>10 aberrations). The XY coordinates for each cell with chromosomal aberrations was recorded using a calibrated microscope stage. Each slide was also scanned under medium power or oil immersion and the number of polyploid metaphases and metaphases with endoreduplication were counted in a total of 100 metaphase cells.

Statistics:

The cytotoxic effects of treatment are expressed relative to the solvent treated control (relative cell growth). The number and types of aberrations found are presented for each treatment group. The percentage of structurally damaged cells in the total population of cells examined was calculated for each group. Chromatid and chromosome gaps are presented in the data but not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell. Statistical analysis of the frequency of structural aberrations per cell was performed. using the Student's t-test. The t-test was used to compare pairwise the number of aberrations per cell of each treatment group with that of the solvent control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

MGK® 264 did not reproducibly induce structural chromosome aberrations in CHO cells when tested in the presence and absence of an exogenous metabolic activation system and is concluded to be negative in this test system.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity (rangeing from slight to toxic) observed at >=0.02 ul/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The activity of MGK®264 in the induction of structural chromosome aberrations in CHO cells when treated in the absence of an exogenous source of metabolic activation - initial assay.

The test article was soluble in treatment medium at all levels tested. At the time of harvest, dose levels of 0.04 and 0.02 µl/ml were observed to be slightly toxic to toxic upon microscopic examination of the cell monolayer. Due to low cell yield, only 68 cells were scorable in the untreated control, 22 cells at 0.04 µl/ml and 53 cells at 0.02 µl/ml. The number of structural aberrations per cell was significantly increased above that of the solvent control at 0.02 µl/ml (P≤0.01, Student's t-test). All other concentrations were not significantly increased relative to the solvent control (p>0.05, Student's t-test). T'EM induced 2.11 aberrations per cell which was statistically increased above the untreated control (p≤0.01, Student's t-test).

 

The activity of MGK®264 in the induction of structural chromosome aberrations in CHO cells when treated in the presence of an S-9 reaction mixture - initial assay.

The test article was soluble in treatment medium at all levels tested. At the time of harvest, dose levels of 0.08, 0.06 and 0.04 µl/ml were observed to be slightly toxic to toxic upon microscopic examination of the cell monolayer. Due to toxicity, no scorable metaphases were located at 0.08 and 0.06 µl/ml and only 3 scorable metaphases were located at 0.04 µl/ml. The data at 0.04 µl/ml was not analyzed due to insufficient numbers. The number of structural aberrations per cell in the test article-treated groups was not statistically increased above that of the solvent control (p>0.05, Student's t-test). CP induced 1.58 aberrations per cell which was statistically increased above the untreated control (p≤0.01, Student's t-test).

 

The activity of MGK®264 in the induction of structural chromosome aberrations in CHO cells when treated in the absence of an exogenous source of metabolic activation - confirmatory assay.

The test article was soluble in treatment medium at all levels tested. At the time of harvest, dose levels of 0.04 and 0.02 µl/ml were observed to be slightly to moderately toxic upon microscopic examination of the cell monolayer. The number of structural aberrations per cell was not significantly increased above that of the solvent control at any of the concentrations tested. TEM induced 0.73 aberrations per cell which was statistically increased above the untreated control (p≤0.01, Student's t-test).

 

The activity of MGK®264 in the induction of structural chromosome aberrations in CHO cells when treated in the presence of an S-9 reaction mixture - confirmatory assay.

The confirmatory S-9 activated assay was performed twice due to technical difficulties in slide preparation; only the results of the valid assay are presented in this report. The test article was soluble in treatment medium at all levels tested. At the time of harvest, dose levels of 0.08 and 0.06 µl/ml were observed to be toxic upon microscopic examination of the cell monolayer.

The number of structural aberrations per cell in the test article-treated groups was not statistically increased above that of the solvent control (p>0.05, Student's t-test). CP induced 0.61 aberrations per cell which was statistically increased above the untreated control (p≤0.05, Student's t-test).

Conclusions:

Under the conditions of the assay with confirmatory study as described in this report, MGK 264 did not reproducibly induce structural chromosome aberrations in CHO cells when tested in the presence and absence of an exogenous metabolic activation system and is concluded to be negative in this test system.

Executive summary:

The test article, MGK®264, was tested twice in the chromosome aberration assay using Chinese hamster ovary cells. The assay was conducted both in the absence and presence of an Aroclor-induced S-9 activation system at dose levels of 0.04, 0.02, 0.01 and 0.005 µl/ml and 0.08, 0.06, 0.04, 0.02 and 0.01 µl/ml, respectively. A delayed harvest was carried out in the absence of S-9 because of observed cell cycle delay. A statistically significant increase in the number of aberrations per cell was observed at 0.02 µl/ml in the absence of S-9 in the initial assay (p≤0.01, student's t-test); no dose response was observed and the increase was not reproduced in the confirmatory assay. No increase in structural chromosome aberrations was observed at any concentration in S-9 activated system. MGK®264 was concluded to be negative in the CHO chromosome aberration assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

On the basis of a negative OECD 471 Ames test, a negative OECD 473 chromosome aberration study and a negative OECD 476 mouse lymphoma assay, it may be concluded that MGK 264 does not require to be classified for mutagenicity.