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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance, Pivalic Acid, was tested in an OECD 471 Bacterial Reverse Mutation Assay, an OECD 473 In Vitro Mammalian Cell Gene Mutation Test in L5178Y/TK+/- Mouse Lymphoma cells and an OECD 473 In Vitro Chromosome Aberration assay in Chinese hamster ovary (CHO) cells in the presence and absence of an exogenous metabolic activation system. Water was used as the vehicle for all three studies.

All criteria for a valid study were met as described in the protocols. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, Pivalic Acid 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. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Under the conditions of the assay described in the report, Pivalic Acid 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.

Under the conditions of the assay described in the report, Pivalic Acid was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosomal aberration test using CHO cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identification: Pivalic Acid
Batch No.: PH5D0530
CAS No.: 75-98-9
Purity: 99.5%
Molecular Weight: 102.1317 g/mol
Description: Clear colorless solid
Storage Conditions: Room temperature, protected from light
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Test concentrations with justification for top dose:
50.0, 150, 500, 1500 and 5000 μg per plate
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.
Evaluation criteria:
The following criteria must be met for the mutagenicity assay to be considered valid:
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.
All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60.
BioReliance Study No. AE59KE.502REACH.BTL 14
To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.
The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control.
A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At least a moderate reduction in the background lawn (background code 3, 4 or 5).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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, Pivalic Acid 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. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.
Executive summary:

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, Pivalic Acid 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. The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: L5178Y/TK+/- Mouse Lymphoma Assay
Specific details on test material used for the study:
Identification: Pivalic Acid
Lot No.: PH5D0530
CAS No.: 75-98-9
Purity: 99.5% (per Certificate of Analysis)
Molecular Weight: 102.1317 g/mol
Description: Clear colorless solid
Target gene:
L5178Y/TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y/TK+/- mouse lymphoma cells are heterozygous at the normally diploid thymidine kinase (TK) locus. L5178Y/TK+/- cells, clone 3.7.2C, were received from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC. Each batch of frozen cells was tested and found to be free of mycoplasma contamination. This test system has been demonstrated to be sensitive to the mutagenic activity of a variety of chemicals.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
Pivalic Acid was evaluated at concentrations of 3.99, 7.98, 16.0, 31.9, 63.8, 128, 255, 511 and 1021 μg/mL. No visible precipitate was observed at the beginning or end of treatment.
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
methylmethanesulfonate
Evaluation criteria:
The average spontaneous mutant frequency of the vehicle control cultures must be within 35 to 140 TFT-resistant mutants/106 surviving cells. Low spontaneous mutant frequencies, i.e., 20 to 34 mutants/106 surviving cells, were considered acceptable if small colony recovery was demonstrated (Mitchell et al., 1997). The average cloning efficiency of the vehicle controls must be between 65 and 120% and the total suspension growth between 8 to 32 for the 4-hour exposure and 20 to 180 for the 24-hour exposure (Moore et al., 2002, 2006, and 2007).
The mutant frequency for at least one dose of each positive control must meet the criteria for a positive response. The mutant frequency for at least one dose of one of the positive controls must induce an increase in small colony mutants according to the following criteria: Induced Mutant Frequency (IMF) positive control ≥300 x 10-6 mutants with 40% small colonies or small colony IMF for positive control ≥150 x 10-6 (Moore et al., 2002; 2006).
Cultures treated with a minimum of four concentrations of test substance must be evaluated and their mutant frequencies reported. Results may be accepted, with justification, when only three concentrations of test substance are evaluated and otherwise meet the other criteria for a valid test. The highest test substance concentration must produce 80 to 90% toxicity unless limited by solubility or the maximum required concentration. In the case of a test substance with a steep toxicity curve (no concentrations with 10 to 20% survival), the results may be considered acceptable if a concentration spacing of ≤2-fold is used and the highest concentration tested showed <20% survival or total kill (Sofuni et al., 1997).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Under the conditions of the assay described in this report, Pivalic Acid 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 substance, Pivalic Acid, 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. Water was used as the vehicle. In the preliminary toxicity assay, the concentrations tested were 3.99, 7.98, 16.0, 31.9, 63.8, 128, 255, 511 and 1021 μg/mL. The maximum concentration evaluated approximated the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Relative suspension growth (RSG) was 98, 99 and 103% at a concentration of 1021 μg/mL for the 4-hour treatments with and without S9 and the 24-hour treatment without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 63.8, 128, 255, 511 and 1021 μg/mL for all treatment conditions. In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at all concentrations exhibited 100 to 113% (4-hour treatment with S9), 78 to 105% (4-hour treatment without S9) and 66 to 109% (24-hour treatment without S9) RSG, and were cloned. Relative total growth of the cloned cultures ranged from 82 to 110% (4-hour treatment with S9), 75 to 89% (4-hour treatment without S9) and 60 to 102% (24-hour treatment without S9). One concentration (5 μg/mL) of the positive control in the 24-hour treatment without S9 was lost prior to cloning due to dilution error. No increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed under any treatment condition. These results indicate Pivalic Acid was 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:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In Vitro Mammalian Chromosomal Aberration Assay in Chinese Hamster Ovary (CHO) Cells
Specific details on test material used for the study:
Identification: Pivalic Acid
Batch No.: PH5D0530
Purity: 99.5% (per Certificate of Analysis)
Molecular Weight: 102.1317 g/mol
Description: Clear colorless solid
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Non-activated: 100, 250, 500, 750, 1020
S9-activated: 100, 250, 500, 750, 900, 1020
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 15. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 hours with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens (Preston et al., 1981).
Evaluation criteria:
Vehicle Controls
The frequency of cells with structural chromosomal aberrations should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are not extreme outliers (indicative of experimental or human error).
Positive Controls
The frequency of cells with structural chromosomal aberrations must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).
Test Conditions
The test substance must be tested using a 4-hour treatment with and without S9, as well as a 20-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.
Analyzable Concentrations
At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored may be reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Initial

 Treatment Condition  Treatment Time Highest Evaluated Dose (μg/mL) Cell Growth Inhibition (%) Mitotic Inhibition (%)   
    Non-activated  4 hr  1020  -12  17
 20 hr  1020  29  32
 S9-activated  4 hr  750  56  14

Retest

 Treatment Condition  Treatment Time Highest Evaluated Dose (μg/mL) Cell Growth Inhibition (%) Mitotic Inhibition (%)   
 S9-activated  4 hr  1020  54  29
Conclusions:
Under the conditions of the assay described in this report, Pivalic Acid was concluded to be negative for the induction of structural and numerical chromosomal aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosomal aberration test using CHO cells.
Executive summary:

The test substance, Pivalic Acid, was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster ovary (CHO) cells in both the absence and presence of an of an exogenous metabolic activation system. Water was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.102 to 1020 μg/mL (10 mM), which was the limit dose for this assay. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was not observed at any dose in the non-activated 4 and 20-hour exposure groups. Cytotoxicity was observed at 1020 μg/mL in the S9-activated 4-hour exposure group. Based upon these results, the doses chosen for the chromosomal aberration assay ranged from 100 to 1020 μg/mL for all three exposure groups. In the initial chromosomal aberration assay, cytotoxicity (55 ± 5% reduction in cell growth index relative to the vehicle control), was not observed at any dose in the non-activated 4 and 20-hour exposure groups. Cytotoxicity was observed at doses ≥ 750 μg/mL in the S9-activated 4-hour exposure group. The doses selected for evaluation of chromosomal aberrations were 250, 500, and 1020 μg/mL for the non-activated 4 and 20-hour exposure groups; and 250, 500, and 750 μg/mL for the S9-activated 4-hour exposure group. In the non-activated 4-hour exposure group, no significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). In the S9-activated 4-hour exposure group, no significant or dose-dependent increases in structural aberrations were observed (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). No significant increase in numerical (polyploid or endoreduplicated cells) aberrations was observed (p > 0.05; Fisher’s Exact test); however, the Cochran-Armitage test was positive for a dose response (p ≤ 0.05). In the non-activated 20-hour exposure group, a statistically significant increase in structural aberrations was observed at 1020 μg/mL (p ≤ 0.01; Fisher’s Exact). Although the Cochran-Armitage test was negative for a dose response (p > 0.05), the induction of structural aberrations (2.3%) was slightly outside the historical 95% control limit of 0.00% - 2.21%. Therefore, the Study Director has concluded that the statistically significant increase in structural aberrations was considered to be biologically irrelevant. No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed at any dose (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). In the S9-activated 4-hour exposure group, the induction of numerical aberrations in solvent control was outside the historical 95% control limit. Therefore, the chromosome aberration assay was repeated at doses ranging from 100 to 1020 μg/mL in the S9-activated 4-hour exposure group. In the repeat assay, cytotoxicity (55 ± 5% reduction in cell growth index relative to the vehicle control), was observed at 1020 μg/mL in the S9-activated 4-hour exposure group. BioReliance Study No. AE59KE.331REACH.BTL 8 The doses selected for evaluation of chromosomal aberrations were 250, 750, and 1020 μg/mL. No significant or dose-dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in the S9-activated 4-hour exposure group (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). These results indicate Pivalic Acid was negative for the induction of structural and numerical chromosomal aberrations in the presence and absence of the exogenous metabolic activation system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Performed studies were all negative for genotoxicity.