3-sec-[C15-18-(branched and linear)-alk-2-enyl]pyrrolidine-2,5-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames

The test article, X-16151, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains ofSalmonellatyphimurium and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 μg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 μg per plate.

Mouse Lymphoma Assay

The test article, X-16151, was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥313 μg/mL at the beginning of treatment and at concentrations ≥313 μg/mL by the end of treatment for 4-hour treatment with and without S9 and ≥156 μg/mL by the end of treatment for 24-hour treatment with S9. Relative suspension growth (RSG) was 20 and 49% at concentrations of 38.1 μg/mL (4-hour treatment with S9) and 19.5 μg/mL (4-hour treatment without S9), respectively. RSG was 0% at all higher concentrations using 4-hour treatment with and without S9. Due to excessive toxicity observed with 24-hour treatment without S9, this condition was repeated.

In the retest of preliminary toxicity assay, the concentrations tested were 0.313, 0.625, 1.25, 2.5, 5.0, 10, 15 and 20 μg/mL with 24-hour treatment without S9. No visible precipitate was observed at the beginning of treatment or end of treatment. Relative suspension growth (RSG) was 32% at concentrations of 10 μg/mL with 24-hour treatment without S9. RSG was 0% at all higher concentrations. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 10.5, 13.1, 16.4, 20.5, 25.6, 32, 40 and 50 μg/mL (4-hour treatment with S9), 8.4, 10.5, 13.1, 16.4, 20.5, 25.6, 32 and 40 μg/mL (4-hour treatment without S9) and 3.4, 4.3, 5.4, 6.7, 8.4, 10.5, 13.1 and 16.4 μg/mL (24-hour treatment without S9).

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 13.1, 16.4, 20.5, 25.6 and 32 μg/mL (4-hour treatment with S9), 8.4, 10.5, 13.1 and 16.4 μg/mL (4-hour treatment without S9) and 3.4, 4.3, 5.4, 10.5 and 13.1 μg/mL (24-hour treatment without S9) exhibited 20 to 90%, 26 to 112% and 18 to 92% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 18 to 99% (4-hour treatment with S9), 20 to 92% (4-hour treatment without S9) and 19 to 90% (24-hour treatment without S9). No increases in induced mutant frequency (IMF) ≥90 mutants/106 clonable cells were observed under any treatment condition. Statistical Analysis (Cocharan Armitage test for trend) was performed for all three conditions and there was no trend in 4 hour treatment with and without S9. There was a positive trend in the 24-hour without S9. However, it should be noted that the IMF was below 90 mutants/106 clonable cells. Thus the trend is considered to be biologically irrelevant.

These results indicate X-16151was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 November 2018 to 08 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-study according to OECD Test Guideline 471

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Lot/batch No.of test material: R17 2934STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light- Solubility of the test substance in the solvent/vehicle: The test article formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.TREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: To achieve a solution, the most concentrated dilution was vortexed for 2 minutes in the confirmatory mutagenicity assay.

Target gene:

The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes for a protein of the DNA nucleotide excision repair system, resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. The top dose was based on the guideline. Based upon initial toxicity-mutation assay results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, the dose levels tested were 33.3, 100, 333, 1000, 3333 and 5000 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO for the test article and all positive controls were diluted in dimethyl sulfoxide (DMSO) except for sodium azide, which was diluted in sterile water - Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test article and compatibility with the target cells

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Exposure duration: 48 to 72 hoursNUMBER OF REPLICATIONS: 2 in the initial-toxicity mutation assay; 3 in the confirmatory mutagenicity assayNUMBER OF CELLS EVALUATED: >/= 0.3 x 10^8 cells/plate. DETERMINATION OF CYTOTOXICITY- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.

Evaluation criteria:

The revertant colony numbers were determined for each plate (counted either manually or by automatic colony counter). The mean and standard deviation of the number of revertants per plate were calculated and reported.For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:Strains TA1535 and TA1537Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.Strains TA98, TA100 and WP2 uvrAData sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No toxicity in the initial toxicity mutation assay. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation in the confirmatory mutagenicity assay

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, X-16151 did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

The test article, X-16151, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 μg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 33.3,100, 333, 1000, 3333 and 5000 μg per plate. Precipitate was observed beginning at 3333 μg per plate with all conditions. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate X-16151 was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2018 to 17 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD Test Guideline 490

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Lot/batch No.of test material: R17 2934STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature, protected from light- Solubility of the test substance in the solvent/vehicle: The test article formed a clear solution in DMSO ata concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- 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 S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Due to excessive toxicity observed with 24-hour treatment without S9, this condition was repeated. In the retest of preliminary toxicity assay, the concentrations tested were 0.313, 0.625, 1.25, 2.5, 5.0, 10, 15 and 20 μg/mL with 24-hour treatment without S9.Based upon the results from retest of preliminary toxicity assay, the concentrations chosen for the definitive mutagenicity assay were 10.5, 13.1, 16.4, 20.5, 25.6, 32, 40 and 50 μg/mL (4-hour treatment with S9), 8.4, 10.5, 13.1, 16.4, 20.5, 25.6, 32 and 40 μg/mL (4-hour treatment without S9) and 3.4, 4.3, 5.4, 6.7, 8.4, 10.5, 13.1 and 16.4 μg/mL (24-hour treatment without S9).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO for hte test article. Methyl methanesulfonate (MMS) (positive control), diluted in water and used as the concurrent positive control for the mutagenicity assay, without S9. 7,12-Dimethyl-benz(a)anthracene (DMBA) (positive control) was diluted in DMSO and used as the concurrent positive control for the mutagenicity assay, with S9- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test article and compatibility with the target cells

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. DURATION- Exposure duration: Target cells were treated with the test and control articles for 4 hours with and without S9, and for 24 hours without S9.- Expression time (cells in growth medium): 2 days post-treatment- Selection time (if incubation with a selection agent): 10-11 days. SELECTION AGENT (mutation assays): trifluorothymidine. NUMBER OF REPLICATIONS: Vehicle control in duplicate cultures and test article in single cultures in the preliminary toxicity assay. Duplicate cultures for the test and control articles in the definitive assay.NUMBER OF CELLS EVALUATED: 3 x 10^6 cells/culture. DETERMINATION OF CYTOTOXICITY- Method: relative total growth

Evaluation criteria:

In evaluation of the data, increases in induced mutant frequency which occurred only at highly toxic concentrations (i.e., less than 10% total growth) were not considered biologically relevant. All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data.• A result was considered positive if a concentration-related increase in mutant frequency was observed in the treated cultures and one or more treatment conditions with 10% or greater total growth exhibited induced mutant frequencies of >/=90 mutants/10E6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average vehicle control mutant frequency was >90 mutants/10E6 clonable cells, a doubling of mutant frequency over the vehicle would also be required.• A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants/10E6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.There are some situations in which a chemical would be considered negative when there was no culture showing between 10 to 20% survival. There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/10E6) in a series of data points within 100 to 20% survival and there was at least one negative data point between 20 and 25% survival.• There was no evidence of mutagenicity (e.g. no dose response or increase in induced mutant frequencies between 45 and 89 mutants/10E6) in a series of data points between 100 to 25% survival and there was also a negative data point between 10 and 1% survival. In this case, it would be acceptable to count the TFT colonies of cultures exhibiting <10% total growth.

Statistics:

Cocharan- Armitage test for trend

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Negative for all treatment conditions

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relative total growth of the cloned cultures ranged from 18 to 99% (4-hour treatment with S9), 20 to 92% (4-hour treatment without S9) and 19 to 90% (24-hour treatment without S9)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: The test article did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose) for both preliminary toxicity assays and mutagenicity assay- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120% in the initial preliminary toxicity assay- Precipitation: Initial Preliminary toxicity assay:Visible precipitate was observed at concentrations ≥313 μg/mL at the beginning of treatment and at concentrations ≥313 μg/mL by the end of treatment for 4-hour treatment with and without S9 and ≥156 μg/mL by the end of treatment for 24-hour treatment with S9Retest of Preliminary Toxicity Assay and Mutagenicity Assay: No precipitate

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Under the conditions of the assay described in this report, X-16151 was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Executive summary:

The test article, X-16151, was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥313 μg/mL at the beginning of treatment and at concentrations ≥313 μg/mL by the end of treatment for 4-hour treatment with and without S9 and ≥156 μg/mL by the end of treatment for 24-hour treatment with S9. Relative suspension growth (RSG) was 20 and 49% at concentrations of 38.1 μg/mL (4-hour treatment with S9) and 19.5 μg/mL (4-hour treatment without S9), respectively. RSG was 0% at all higher concentrations using 4-hour treatment with and without S9. Due to excessive toxicity observed with 24-hour treatment without S9, this condition was repeated.

In the retest of preliminary toxicity assay, the concentrations tested were 0.313, 0.625, 1.25, 2.5, 5.0, 10, 15 and 20 μg/mL with 24-hour treatment without S9. No visible precipitate was observed at the beginning of treatment or end of treatment. Relative suspension growth (RSG) was 32% at concentrations of 10 μg/mL with 24-hour treatment without S9. RSG was 0% at all higher concentrations. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 10.5, 13.1, 16.4, 20.5, 25.6, 32, 40 and 50 μg/mL (4-hour treatment with S9), 8.4, 10.5, 13.1, 16.4, 20.5, 25.6, 32 and 40 μg/mL (4-hour treatment without S9) and 3.4, 4.3, 5.4, 6.7, 8.4, 10.5, 13.1 and 16.4 μg/mL (24-hour treatment without S9).

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 13.1, 16.4, 20.5, 25.6 and 32 μg/mL (4-hour treatment with S9), 8.4, 10.5, 13.1 and 16.4 μg/mL (4-hour treatment without S9) and 3.4, 4.3, 5.4, 10.5 and 13.1 μg/mL (24-hour treatment without S9) exhibited 20 to 90%, 26 to 112% and 18 to 92% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 18 to 99% (4-hour treatment with S9), 20 to 92% (4-hour treatment without S9) and 19 to 90% (24-hour treatment without S9). No increases in induced mutant frequency (IMF) ≥90 mutants/106 clonable cells were observed under any treatment condition. Statistical Analysis (Cocharan Armitage test for trend) was performed for all three conditions and there was no trend in 4 hour treatment with and without S9. There was a positive trend in the 24-hour without S9. However, it should be noted that the IMF was below 90 mutants/106 clonable cells. Thus the trend is considered to be biologically irrelevant.

These results indicate X-16151was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo Micronucleus, Flaunders & Durward (2009)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 474, EU Method B12, USA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines, under GLP conditions.

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 800 mg/kg and with 400 and 200 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups, each of 7 mice, were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 400 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ataxia and ptosis.

Marked statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 800 mg/kg test material dose groups when compared to their concurrent control groups and, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Under the conditions of this study, the test material was considered to be non-genotoxic.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 April 2009 to 16 July 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USA EPA, TSCA and FIFRA guidelines

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese METI/MHLW guidelines for testing of new chemical substances

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

other: Hsd: ICR (CD-1)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately five to eight weeks old
- Weight at study initiation: 23 to 29 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: The animals were housed in groups of up to seven, by sex, in solid-floor polypropylene cages with wood-flake bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 – 25 °C
- Humidity: 30 – 70 %
- Air changes: approximately fifteen changes per hour
- Photoperiod: the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Amount of vehicle: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was freshly prepared as required as a solution at the appropriate concentration in arachis oil.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Once

Post exposure period:

24 or 48 hours

Dose / conc.:

800 mg/kg bw/day (nominal)

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main test: 7 males per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Cyclophosphamide
- For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water
- Justification for choice of positive control(s): Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test.
- Route of administration: oral
- Doses / concentrations: 50 mg/kg

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the micronucleus test. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. The range-finding toxicity test was also used to determine if the main test was to be performed using both sexes or males only. Groups of mice were dosed as follows: 2000 mg/kg (oral) and 100, 1600, 1200 and 800 mg/kg (intraperitoneal). All animals were dosed once only at the appropriate dose level by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing. Animals were observed within the time periods of 0.5, 1 and 2 hours after dosing and subsequently once daily for up to two days. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
- Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration; therefore, this was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 800 mg/kg, was selected for use in the main test, with 400 and 200 mg/kg as the lower dose levels.

TREATMENT AND SAMPLING TIMES:
- One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test material at 800 mg/kg was killed after 48 hours.
- In addition, three further groups of mice were included in the test; two groups (each of seven mice) were dosed via the intraperitoneal route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing.
- All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.

DETAILS OF SLIDE PREPARATION:
- Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS:
- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micro nucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
- The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
- A positive mutagenic response was demonstrated when a statistically significant, dose­responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
- If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.
- A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

- All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part Ill (1989). The data was analysed following a √(x + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 800 – 2000 mg/kg
- Clinical signs of toxicity in test animals: No evidence of toxicity was observed in animals dosed with test material via the oral route and, therefore systemic absorption could not be confirmed using this dose route. In animals dosed with the test material via the intraperitoneal route premature deaths occurred at and above 1200 mg/kg, and clinical signs were observed at and above 800 mg/kg as follows: Hunched posture, ptosis, ataxia, pilo-erection, lethargy, pallor of the extremities, hypothermia, decreased respiratory rate, splayed gait and laboured respiration. The test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration; therefore, this was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 800 mg/kg, was selected for use in the main test, with 400 and 200 mg/kg as the lower dose levels.

RESULTS OF DEFINITIVE STUDY
- There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 400 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ataxia and ptosis.
- Evaluation of Bone Marrow Slides: Marked statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 800 mg/kg test material dose groups when compared to their concurrent control groups and, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. There were no statistically significant increases in the frequency of micronucleated PCE in any of the test material dose groups when compared to their concurrent vehicle control groups. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

 Table 1: Micronucleus Test - Summary of Group Mean Data

Treatment Group	Number of PCE With Micronuclei per 2000 PCE		PCE/NCE Ratio
	Group Mean	SD	Group Mean	SD
Vehicle Control 10 mL/kg (48-hour Sampling Time)	2.0	2.1	0.81	0.18
Vehicle Control 10 mL/kg (24-hour Sampling Time)	1.7	1.4	0.80	0.16
Positive Control 50 mg/kg (24 -hour Sampling Time)	29.0***	9.1	0.64	0.25
Test Material 800 mg/kg (48 -hour Sampling Time)	1.0	1.2	0.49**	0.19
Test Material 800 mg/kg (24 -hour Sampling Time)	0.7	0.8	0.52**	0.18
Test Material 400 mg/kg (24 -hour Sampling Time)	1.6	1.5	0.71	0.21
Test Material 200 mg/kg (24 -hour Sampling Time)	0.9	1.2	0.88	0.36

PCE = Polychromatic erythrocytes, NCE = Normochromatic erythrocytes, SD = Standard deviation, ** = P <0.01, *** = P <0.001

Conclusions:

Under the conditions of this study, the test material was considered to be non-genotoxic.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 474, EU Method B12, USA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines, under GLP conditions.

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 800 mg/kg and with 400 and 200 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups, each of 7 mice, were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 400 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ataxia and ptosis.

Marked statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 800 mg/kg test material dose groups when compared to their concurrent control groups and, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Under the conditions of this study, the test material was considered to be non-genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames

The test article, X-16151, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains ofSalmonellatyphimurium and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 μg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 μg per plate.

Mouse Lymphoma Assay

The test article, X-16151, was evaluated for its ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥313 μg/mL at the beginning of treatment and at concentrations ≥313 μg/mL by the end of treatment for 4-hour treatment with and without S9 and ≥156 μg/mL by the end of treatment for 24-hour treatment with S9. Relative suspension growth (RSG) was 20 and 49% at concentrations of 38.1 μg/mL (4-hour treatment with S9) and 19.5 μg/mL (4-hour treatment without S9), respectively. RSG was 0% at all higher concentrations using 4-hour treatment with and without S9. Due to excessive toxicity observed with 24-hour treatment without S9, this condition was repeated.

In the retest of preliminary toxicity assay, the concentrations tested were 0.313, 0.625, 1.25, 2.5, 5.0, 10, 15 and 20 μg/mL with 24-hour treatment without S9. No visible precipitate was observed at the beginning of treatment or end of treatment. Relative suspension growth (RSG) was 32% at concentrations of 10 μg/mL with 24-hour treatment without S9. RSG was 0% at all higher concentrations. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 10.5, 13.1, 16.4, 20.5, 25.6, 32, 40 and 50 μg/mL (4-hour treatment with S9), 8.4, 10.5, 13.1, 16.4, 20.5, 25.6, 32 and 40 μg/mL (4-hour treatment without S9) and 3.4, 4.3, 5.4, 6.7, 8.4, 10.5, 13.1 and 16.4 μg/mL (24-hour treatment without S9).

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 13.1, 16.4, 20.5, 25.6 and 32 μg/mL (4-hour treatment with S9), 8.4, 10.5, 13.1 and 16.4 μg/mL (4-hour treatment without S9) and 3.4, 4.3, 5.4, 10.5 and 13.1 μg/mL (24-hour treatment without S9) exhibited 20 to 90%, 26 to 112% and 18 to 92% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 18 to 99% (4-hour treatment with S9), 20 to 92% (4-hour treatment without S9) and 19 to 90% (24-hour treatment without S9). No increases in induced mutant frequency (IMF) ≥90 mutants/106 clonable cells were observed under any treatment condition. Statistical Analysis (Cocharan Armitage test for trend) was performed for all three conditions and there was no trend in 4 hour treatment with and without S9. There was a positive trend in the 24-hour without S9. However, it should be noted that the IMF was below 90 mutants/106 clonable cells. Thus the trend is considered to be biologically irrelevant.

These results indicate X-16151was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

In vivo Micronucleus, Flaunders & Durward (2009)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 474, EU Method B12, USA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 800 mg/kg and with 400 and 200 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups, each of 7 mice, were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 400 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ataxia and ptosis.

Marked statistically significant decreases in the PCE/NCE ratio were observed in the 24 and 48-hour 800 mg/kg test material dose groups when compared to their concurrent control groups and, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Under the conditions of this study, the test material was considered to be non-genotoxic.