Butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo-, tetradecyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay

Key study:- Mecchi (2004) 'Salmonella-Escherichia coli/Mammalian Microsome Reverse Mutation Assay with a Confirmatory Assay with EK2003-0090' conducted in line with OECD Guideline 471. The test substance was determined to be non-genotoxic.

Chromosome aberration

Key study:- Murli (2003) 'Chromsomal Aberrations in Chinese Hamster Ovary (CHO) Cells' conducted in line with OECD Guideline 473. The test substance was determined to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2003 to 5 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

The bacterial reverse mutation assay detects point mutations, both frameshifts and/or base pair substitutions. The strains of Salmonella typhimurium and Escherichia coli used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his-) or tryptophan (trp-) dependent cells are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan), only those cells which revert to histidine (his+) or tryptophan (trp+) independence are able to form colonies. The trace amount of histidine or tryptophan in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The his+ or trp+ revertants are readily discernable as colonies against the limited background growth of the his- or trp- cells. By using several different tester strains, both base pair substitution mutations and frameshift mutations can be detected. The bacterial reverse mutation assay has been shown to be a sensitive, rapid, and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Tester Strain Genotypes:
TA98: hisD3052, uvrB, rfa and pKM101
TA100: hisG46, uvrB, rfa and pKM101
TA1535: hisG46, uvrB and rfa
TA1537: hisC3076, uvrB and rfa

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Tester Strain Genotype:
WP2uvrA: trp, uvrA and pKM101

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

10.0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In solubility testing, the test article was observed to be insoluble in deionized water at approximately 100 mg per mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene and ICR-191

Details on test system and experimental conditions:

METHOD OF APPLICATION
In agar (plate incorporation)

DURATION
- Exposure duration: After required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay solidified, plates were inverted and incubated for 52 ± 4 hours at 37 ± 2 °C.
- Fixation time (start of exposure up to fixation or harvest of cells): To ensure that cultures were harvested in late log phase, length of incubation was determined by spectrophotometric monitoring of culture density. Cultures were harvested once a predetermined density was reached which ensured that cultures had reached a density of at least 0.5 x 10ˆ9 cells per mL and had not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Cultures were removed from incubation when target density was reached and were held at >0 to 10 °C until used in the assay.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate andor by thinning or disappearance of the bacterial background lawn.

Evaluation criteria:

ASSAY EVALUATION CRITERIA
Once criteria for a valid assay had been met, responses observed in the assay were evaluated.

Tester Strains TA98, TA100, and WP2uvrA(pKM101)
For a test article to be considered positive, it must have produced at least a 2-fold increase in mean revertants per plate of at least one of these tester strains over the mean revertants per plate of its respective vehicle control.
This increase in mean number of revertants per plate must have been accompanied by a dose response to increasing concentrations of test article.

Tester Strains TA1535 and TA1537.
For a test article to be considered positive, it must have produced at least a 3-fold increase in mean revertants per plate of at least one of these tester strains over the mean revertants per plate of its respective vehicle control. This increase in mean number of revertants per plate must have been accompanied by a dose response to increasing concentrations of test article.

Statistics:

No data

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Doses tested in the mutagenicity assay were selected based on results of the dose rangefinding assay conducted on the test article using tester strains TA100 and WP2uvrA(pKM101) in the presence and absence of S9 (one plate per dose). Ten doses of test article, from 6.67 to 5000 µg per plate, were tested in Trial A1. No cytotoxicity was observed with either tester strain in the presence or absence of S9 mix, as evidenced by no dose-related decreases in the number of revertants per plate. Bacterial background lawns were observed to be normal up to 1000 µg per plate in the presence of S9 mix and up to 5000 µg per plate in the absence of S9 mix. Test article precipitate was observed on the plates at 667 µg per plate and above in the absence of S9 mix. In the presence of S9 mix, test article precipitate was observed at 3330 µg per plate and above with TA100 and at 1000 µg per plate and above with WP2uvrA(pKM101). The bacterial background lawns were obscured by precipitate at 3330 µg per plate and above in the presence of S9 mix.

Test Article Handling

The test article was stored at room temperature. In solubility testing, the test article was observed to be insoluble in deionized water at approximately 100 mg per mL. In dimethylsulfoxide (DMSO) the test article was observed to form a transparent, brown, non-viscous, solution at 100 mg per mL . For this reason DMSO (Acros Organics, Lot No. A017777101 for Trial 25446-Al; EM Science, Lot No. 42233 for Trial 25446-B1 and Lot No. 42317 for trials 25446-Cl and 25446-Dl) was chosen as the vehicle. In the initial mutagenicity assay (Trial 25446-Bl), the test article was observed to form a very viscous, heterogeneous suspension at 100 mg per mL. For this reason, the test article was diluted with additional DMSO to achieve a top stock concentration of 50.0 mg per mL. At a concentration of 50.0 mg per mL, the test article was observed to form a transparent, dark-orange, non-viscous solution. The test article was observed to remain in solution at all succeeding dilutions prepared for the mutagenicity assay.

Mutagenicity Assay

The mutagenicity assay results are presented in the attached Tables.

Doses tested in the mutagenicity assay with all tester strains were 10.0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate in the presence and absence of S9 mix.

In the initial mutagenicity assay, Trial B1, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any tester strain in either the presence or absence of S9 mix. A dose-related reduction in revertants per plate was observed with tester strain WP2uvrA(pKM101) in the absence of S9 mix. No other dose-related reductions in revertant count were observed. Test article precipitate was observed on the plates in the presence of S9 mix at 3330 µg per plate and above and in the absence of S9 mix at 1000 to 3330 µg per plate and above. The test article precipitate obscured the bacterial background lawns at 3330 µg per plate and above in both the presence and absence of S9 mix.

In the confirmatory mutagenicity assay, Trial C1, the mean vehicle control value for tester strain TA98 in the absence of S9 mix was not within the acceptable range for this strain as specified in the protocol. For this reason, the data generated with TA98 in the absence of S9 mix in Trial C1 were not used in the evaluation of the test article. The test article was re-tested

with tester strain TA98 in the absence of S9 mix in Trial D1. All other data generated in Trial C1 were acceptable and no positive increases in the mean number of revertants per plate were observed with any tester strain in either the presence or absence of S9 mix. A dose-related reduction in revertants per plate was observed with tester strain WP2uvrA(pKM101) in the absence of S9 mix. No other dose-related reductions in revertant count were observed. Test article precipitate was observed on the plates in the presence of S9 mix at 1000 to 3330 µg per plate and above and in the absence of S9 mix at 333 to 3330 µg per plate and above. The test article precipitate obscured the bacterial background lawns at 3330 pg per plate and above in both the presence and absence of S9 mix.

In the repeat mutagenicity assay, Trial D1, all data were acceptable and no positive increases were observed with tester strain TA98 in the absence of S9 mix. No cytotoxicity was observed as evidenced by no dose-related decreases in the mean number of revertants per plate.

Test article precipitate was observed on the plates (obscuring the bacterial background lawns) at 3330 µg per plate and above.

All criteria for a valid study were met.

Conclusions:

Interpretation of results: negative

The results of the Salmonella-Escherichia coli Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test substance did not cause positive increases in the mean number of revertants per plate with any tester strain in the presence or absence of Aroclor™ induced rat liver (S9).

Executive summary:

In a GLP compliant study conducted in line with OECD Guideline 471, butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester was evaluated for the ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at the histidine locus in the genome of several strains of Salmonella typhimunium and at the tryptophan locus of Escherichia coli tester strain WP2uvrA(pKM101).

Doses tested in the mutagenicity assay were selected based on the results of a dose range finding assay using tester strains TA100 and WP2uvrA(pKM101) and ten doses of test article ranging from 6.67 to 5000 µg per plate, one plate per dose, both in the presence and absence of S9 mix.

Tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA(pKM101). The assay was conducted in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. Doses tested with all tester strains in the initial mutagenicity assay were 10.0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate in both the presence and absence of S9 mix. Results of the initial mutagenicity assay were confirmed in an independent experiment.

The results of the Salmonella-Escherichia coli Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause positive increases in the mean number of revertants per plate with any tester strain in the presence or absence of Aroclor™ induced rat liver (S9).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2003 to 8 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO cell line was derived from an ovarian biopsy of a female Chinese hamster. The Chinese hamster ovary cells (CHO-WBL) used in this assay were from a permanent ceIl line and were originally obtained from the laboratory of Dr. S. Wolff, University of California, San Francisco. The cells were subsequently subcloned in the testing laboratory, and stock cultures stored in liquid nitrogen. The CHO-WBL subclone is a permanent cell line with an average cycle time of 12 to 14 hours and a modal chromosome number of 21.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

32.9, 47.1 ,67.2, 96.0, 137, 196, 280 and 400 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: For the assay without S9, 1 day after culture initiation, cells were incubated at 37 ± 2 °C with the test article at predetermined concentrations, vehicle and positive controls for ~17.8 hours. The cultures were then washed with buffered saIine. For the assay with S9, 1 day after culture initiation, cultures were incubated at 37 ± 2 °C for ~3 hours in the presence of the test article at predetennined concentrations, vehicle and positive controls, and the S9 reaction mixture. The cultures were then washed with buffered saline.
- Expression time (cells in growth medium): Cultures were initiated by seeding approximately 0.9 x 10ˆ6 cells per 75 cm2 flask into a sufficient volume of culture medium so that the final volume was 10 mL in the assay without metabolic activation after the addition of the test article in its chosen vehicle or was 10 mL in the assay with metabolic activation after the addition of the test article in its chosen vehicle and the S9 mix.
- Selection time (if incubation with a selection agent): The cells were re-fed with complete McCoy’s 5a medium with 0.1 µg/mL Colcemid® and incubated for 2 ± 0.5 hours. The cultures were then harvested.
- Fixation time (start of exposure up to fixation or harvest of cells): In the confirmatory aberrations assay without and with S9, ~20-hour harvests were conducted. This harvest time corresponds to 1.5 times a cell cycle time of approximately 13 hours (Galloway et al., 1994).

SELECTION AGENT (mutation assays): Colcemid®

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Percent polyploidy was analyzed by evaluating at least 100 metaphases, if available, and tabulated.
- Determination of endoreplication: Percent endoreduplication was analyzed by evaluating at least 100 metaphases, if available, and tabulated.

Evaluation criteria:

ASSAY EVALUATION CRITERIA
After completion of microscopic analysis, data were decoded. The following factors are taken into account in evaluation of the test article data:
- The number and percentages of aberrant cells excluding gaps (-g).
- The number and percentages of aberrant cells including gaps (+g).
- Evidence of a dose-response relationship.
The experimental unit is the cell, and therefore the percentage of cells with structural aberrations was the basis for evaluation.

EVALUATION OF A POSITIVE RESPONSE
A test article was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when p ≤ 0.01) in the number of cells with chromosomal aberrations is observed at one or more concentrations. The linear trend test evaluated the dose responsiveness. A dose-response should be observed if a significant increase was seen at one or more concentrations.

EVALUATION OF A NEGATIVE RESPONSE
A test article was considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of ceIls with chromosomal aberrations at any of the concentrations.

EQUIVOCAL EVALUATION
Although most assays give clearly positive or negative results, in rare cases the data set would preclude making a definitive judgment about the activity of
the test article. Results might remain equivocal or questionable regardless of the number of times the assay is repeated.

Although the evaluation criteria provided here normally are sufficient, the Study Director might use additional considerations to obtain a final evaluation of the test article based upon the Study Director’s scientific judgment.

Statistics:

Statistical analysis employed a Cochran-Armitage test for linear trend and Fisher's Exact Test (Thakur et aI., 1985) to compare the percentage of cells with aberrations in treated cells to the results obtained for the vehicle controls.
Statistical analysis was also performed for cells exhibiting numerical aberrations (polyploidy and endoreduplication) in order to indicate significant (p ≤ 0.01) increases in these events as indicators of possible induction of numerical aberrations; however, the test articles were evaluated only for structural aberrations and not for numerical aberrations by this protocol.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES

Initial Chromosomal Aberrations Assay
- Without metabolic activation: No reductions were observed in the mitotic indices of the cultures treated with 137, 196, 280 and 400 µg/mL as compared with the vehicle control cultures. ChromosomaI aberrations were analyzed from the cultures treated with 96.0, 137, 196, and 400 µg/mL. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.

- With metabolic activation: Slight reductions of 0, 0, 6 and 10 % were observed in the mitotic index of the cultures treated with 137, 196, 280 and 400 µg/mL as compared with the vehicle control cultures. Chromosomal aberrations were analyzed from the cultures treated with 96.0, 137, 196 and 400 µg/mL. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.

Based on the results from the initial assay, the confirmatory chromosomal aberrations assay was conducted at doses of 6.25, 12.5, 25.0, 50.0, 100, 150, 225, 300, and 400 µg/mL without metabolic activation and 50.0, 100, 150, 225, 300 and 400 µg/mL with metabolic activation. Treatment periods were for ~18 and ~3 hours without and with metabolic activation, respectively, and the cultures were harvested ~20 hours from the initiation of treatment.

Remarks on result:

mutagenic potential (based on QSAR/QSPR prediction)

Confirmatory Chromosomal Aberrations Assay

Without metabolic activation

No reductions were observed in the mitotic indices of the cultures treated with 100, 150, 225, 300 and 400 µg/mL, respectively, as compared with the vehicle control cultures (attached Table 12). Chromosomal aberrations were analyzed from the cultures treated with 25.0, 50.0, 100 and 150 µg/mL (attached Table 13). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.

With metabolic activation

Reductions of 0, 1 , 7, 19 and 66 % were observed in the mitotic index of the cultures treated with 100, 150, 225, 300 and 400 µg/mL as compared with the vehicle control cultures (attached Table 15). Chromosomal aberrations were analyzed from the cultures treated with 50.0, 100, 225 and 400 µg/mL (attached Table 16). No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.

Under nonactivation conditions, the sensitivity of the cell cultures for induction of chromosomal aberrations is shown by the increased frequency of aberrations in the cells exposed to mitomycin C, the positive control agent. The test article is considered negative for inducing chromosomal aberrations, polyploidy, or endoreduplication without metabolic activation. The successful activation by the metabolic system is illustrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive control agent. The test article was considered negative for inducing chromosomal aberrations, polyploidy, or endoreduplication with metabolic activation.

Conclusions:

Interpretation of results: negative

Under the conditions of the test, the test substance was considered to be negative for inducing chromosomal aberrations in CHO cells with and without metabolic activation.

Executive summary:

In a GLP compliant study conducted in line with OECD Guideline 473, butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester was evaluated for the ability to induce chromosomal aberrations in cultured Chinese Hamster Ovary (CHO) cells with and without an exogenous metabolic activation system. The test substance was formulated in dimethylsulfoxide (DMSO) for the assay. The highest concentration tested in the initial assay was 400 µg/mL, which was above the solubility limit of the test article after dosing in culture medium. All dosing was achieved using dosing volumes of 1 % (10 µL/mL) and the vehicle control cultures were treated with 10.0 µL/mL of DMSO.

In the initial chromosomal aberrations assay, the treatment period was for ~3 hours with and without metabolic activation and cultures were harvested ~20 hours from the initiation of treatment. Concentrations of 1.36, 1.94, 2.77, 3.95, 5.65, 8.07, 11.5, 16.5, 23.5, 33.6, 48.0, 68.6, 98.0, 140 and 200 µg/mL were tested with and without metabolic activation. Due to the lack of toxicity at the dose levels tested and the lack of precipitate at the time of wash or harvest of these cultures, the assay was repeated testing concentrations of 32.9, 47.1, 67.2, 96.0, 137, 196, 280 and 400 µg/mL with and without metabolic activation. Cultures treated with concentrations of 96.0, 137, 196 and 400 µg/mL without and with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.

In a confirmatory chromosomal aberrations assay, the treatment period was for ~17.8 hours without metabolic activation and ~3 hours with metabolic activation and the cultures were harvested ~20 hours from the initiation of treatment. Concentrations of 6.25, 12.5, 25.0, 50.0, 100, 150, 225, 300 and 400 µg/mL without metaboIic activation and 50.0, 100, 150, 225, 300 and 400 µg/mL with metabolic activation were tested. Cultures treated with concentrations of 25.0, 50.0, 100, and 150 µg/mL without metabolic activation and 50.0, 100, 225 and 400 µg/mL with metabolic activation were analyzed for chromosomal aberrations. No significant increase in cells with chromosomal aberrations, polyploidy, or endoreduplication was observed in the cultures analyzed.

The test substance was considered negative for inducing chromosomal aberrations in CHO cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay

The key study (Mecchi, 2004) was performed in compliance with GLP and to OECD Guideline 471 with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard, in line with an accepted, standardised guideline and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch et al. (1997). The test substance was determined to be non-genotoxic.

Chromosome aberration

The key study (Murli, 2003) was performed in compliance with GLP and to OECD Guideline 473 with a sufficient level of detail to assess the quality of the presented data. The study was performed to a good standard, in line with an accepted, standardised guideline and was assigned a reliability score of 1 using the principles for assessing data quality as set out in Klimisch et al. (1997). The test substance was determined to be non-genotoxic.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.