Registration Dossier
Registration Dossier
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EC number: 813-120-0 | CAS number: 1262967-45-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Reverse gene mutation assay: Negative (non-mutagenic); OECD 471; May, K. (2011)
Mammalian cell cytogenicity: Negative (non-clastogenic); OECD 473; Pritchard, L. (2011)
Mammalian cell gene mutation: Negative (non-mutagenic); OECD 476; Yamakage, K. (2017)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 01 Spetember 2011 - 02 November 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study was conducted in accordance with International guidelines and GLP. All guideline validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Commission Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: JMAFF, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-1, Agricultural Production Bureau
- Version / remarks:
- 25 November 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark and under nitrogen.
- Stability under test conditions: Assumed stable.
- Solubility and stability of the test substance in the solvent/vehicle: Diluted in acetone at a top concentration of 50 mg/mL (used to prepare plates at 5000 µg/plate)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material): Diluted in acetone.
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A
OTHER SPECIFICS: No - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver
- Test concentrations with justification for top dose:
- Seven concentrations up to 5000 µg/plate i.e. 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: test item was not soluble in DMSO and acetone was preferred over ethanol. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- other: 2-aminoanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): Not reported
DURATION
- Preincubation period: N/A
- Exposure duration: 2-3 days
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A
SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A
NUMBER OF REPLICATIONS: 3
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A
NUMBER OF CELLS EVALUATED: N/A
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A
DETERMINATION OF CYTOTOXICITY
- Method: thinning/ absence of bacteria lawn
OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A
- OTHER: N/A - Rationale for test conditions:
- The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted.
- Evaluation criteria:
- For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values.
2. Any observed response was reproducible under the same treatment conditions.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met. - Statistics:
- The statistical significance of results was analysed using Dunnett’s test to aid in the evaluation of potential positive responses.
- 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 examined
- 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 examined
- 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 examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- 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 examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- 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 examined
- Positive controls validity:
- valid
- Conclusions:
- The test item was considered to be non-mutagenic under the conditions of the test.
- Executive summary:
OECD 471 (2011) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 (S. typhimurium) and WP2uvrA (pKM101) (Escherichia coli) were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid in acetone at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in a screening experiment followed by concentrations of 50, 150, 1500 and 5000 µg/plate in a secondary experiment. Both tests were conducted in the presence and absence of S9 mix. The S9 mix content was increased from 10 % v/v to 20 % v/v in the secondary test.
The test item was tested up to the standard limit concentration recommended in the regulatory guidelines i.e. 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains.
There was no evidence of cytotoxicity or induced mutant colonies (over background levels) in any of the test item treated colonies in either of the two tests.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 July - 02 November 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Commission Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Japanese Ministry of Health and Welfare. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notification No. 1604
- Version / remarks:
- November 1999
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.
- Version / remarks:
- 1996
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
- Version / remarks:
- 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark and under nitrogen.
- Stability under test conditions: Assumed stable.
- Solubility and stability of the test substance in the solvent/vehicle: Diluted in acetone at a top concentration of 500 mg/mL (used to prepare plates at 5000 µg/mL)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: At 5000 µg/mL a fluctuation in osmolality of more than 50 mOsm/kg was observed when compared to the solvent control. No fluctuation in pH of more than 1.0 unit was observed when compared with the solvent control.
Following serial dilution to lower concentrations of 4500 and 4000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed at 4500 μg/mL when compared with the solvent control. However, at 4000 μg/mL, no fluctuation in osmolality of more than 50 mOsm/kg was observed. No pH fluctuation of more than 1.0 unit was observed at either concentration when compared with the solvent control.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system.
In this case, the highest final concentration used for subsequent testing was 4000 μg/mL.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material): Diluted in acetone.
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A
OTHER SPECIFICS: No - Species / strain / cell type:
- lymphocytes: Human blood was collected aseptically from two healthy, non-smoking male donors,
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Human blood was collected aseptically from two healthy, non-smoking male donors.
- Suitability of cells: Not reported
- Cell cycle length, doubling time or proliferation index: Not reported
- Sex, age and number of blood donors if applicable: Not reported
- Whether whole blood or separated lymphocytes were used if applicable: Not reported
- Number of passages if applicable: Not reported
- Methods for maintenance in cell culture if applicable: Blood was pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM glutamine. Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37 °C in a 5 % CO2 atmosphere for approximately 48 hours. The cultures were gently shaken daily to resuspend the cells.
- Modal number of chromosomes: Not reported
- Normal (negative control) cell cycle time: Not reported
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: See above
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not reported
- Periodically checked for karyotype stability: Not reported
- Periodically 'cleansed' against high spontaneous background: Not reported - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix; obtained from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver
- Test concentrations with justification for top dose:
- 40.31, 67.18, 111.97, 186.62, 311.04, 518.4, 864, 1440, 2400 and 4000 μg/mL.
At 5000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed when compared to the solvent control. Following serial dilution to lower concentrations of 4500 and 4000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed at 4500 μg/mL when compared with the solvent control. However, at 4000 μg/mL, no fluctuation in osmolality of more than 50 mOsm/kg was observed.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system. In this case, the highest final concentration used for subsequent testing was 4000 μg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid was found to be miscible in acetone at 500 mg/mL (1M) - 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:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Not reported
DURATION
- Preincubation period: N/A
- Exposure duration: 3 h
- Expression time (cells in growth medium): 18 h
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): 10 mins
SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): Colcemid® (at 0.1 µg/mL)
STAIN (for cytogenetic assays): 10% Giemsa, prepared in buffered water (pH 6.8)
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37 °C. After a 10 minute period of incubation at 37 °C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear. The pellets were resuspended, then centrifuged at 500 g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10 % Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at approximately 4 °C until slide analysis was completed.
NUMBER OF CELLS EVALUATED: The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures, or cultures where there were no signs of cytotoxicity.
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): One hundred metaphase figures were examined from each culture. Chromosome aberrations were scored according to the classification of the ISCN (1985).
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:
OTHER EXAMINATIONS:
- Determination of polyploidy: Polyploid cells were noted when seen using a low power objective and examined at a magnification of x1000 using an oil immersion objective.
- Determination of endoreplication: Endoreduplicated cells were noted when seen using a low power objective and examined at a magnification of x1000 using an oil immersion objective.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A
- OTHER: In a second test a 21 hour continuous treatment was used in the absence of S9 mix. In the presence of S9 mix, a 3 hour treatment was used, as in the first test. However, to modify study parameters the final concentration of S9 mix was increased from 2% v/v to 5% v/v. - Evaluation criteria:
- An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
The increases exceed the solvent control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration. - Statistics:
- The number of aberrant metaphase cells in each test substance group was compared with the solvent control value using the one-tailed Fisher exact test (Fisher 1973).
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.
D20s (the minimum concentration (mg/mL) at which aberrations were found in 20 % of metaphases) were estimated using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002). - Key result
- Species / strain:
- lymphocytes: human
- 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 examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuation in pH of more than 1.0 unit was observed when compared with the solvent control.
- Effects of osmolality: At 5000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed when compared to the solvent control. Following serial dilution to lower concentrations of 4500 and 4000 μg/mL, a fluctuation in osmolality of more than 50 mOsm/kg was observed at 4500 μg/mL when compared with the solvent control. However, at 4000 μg/mL, no fluctuation in osmolality of more than 50 mOsm/kg was observed.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system. In this case, the highest final concentration used for subsequent testing was 4000 μg/mL.
- Evaporation from medium: No
- Water solubility: Not soluble. Acetone used as a vehicle
- Precipitation: Not observed
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES: In the absence of S9 mix following 3 hour treatment,the test item caused a reduction in the mitotic index to 68 % of the solvent control value at 4000 μg/mL. The concentrations selected for metaphase analysis were 40.31, 864 and 4000 μg/mL. In the presence of S9 mix (2 % v/v final concentration) following 3 hour treatment, the test item caused a reduction in the mitotic index to 86 % of the solvent control value at 4000 μg/mL. The concentrations selected for metaphase analysis were 186.62, 864 and 4000 μg/mL. In both the absence and the presence of S9 mix, the test item caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: N/A
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Within laboratory historical control range (99 % CI).
- Negative (solvent/vehicle) historical control data: All mean values for the solvent control (acetone), and alltest item treatment concentrations were within laboratory historical control range, when taken at the 99% confidence limit.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Mitotic indices of cultured human lymphocytes treated with Test item were compared with solvent control values.
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]: No - Conclusions:
- It is concluded that the test substance Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
- Executive summary:
OECD 473 (2011) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at concentrations of 40.31, 67.18, 111.97, 186.62, 311.04, 518.4, 864, 1440, 2400 and 4000 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested with and without metabolic activation at test item concentrations of 25, 100, 250, 500, 100, 1500, 200, 2500, 3000, 3500 and 4000 µg/mL and 100, 250, 500, 1000, 2000, 3000 and 4000 µg/mL, respectively. The S9 mix concentration in the second test was raised from 2 to 5 % v/v as a modification parameter.
4000 µg/mL was determined as the highest concentration that did not induce unacceptable fluctuations in osmolarity in a screening test. This concentration was assigned as the top concentration in both the first and secondary tests.
Both the solvent control and positive controls induced the appropriate responses and were with laboratory historical ranges with 99 % CI.
There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 March - 09 June 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Study was conducted in accordance with International guidleines and in accordance with GLP. All guideline validity criteria were met.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark and under nitrogen.
- Stability under test conditions: Assumed stable.
- Solubility and stability of the test substance in the solvent/vehicle: Diluted in acetone at a top concentration of 50 mg/mL (used to prepare plates at 5000 µg/plate)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material): Diluted in acetone.
TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A
OTHER SPECIFICS: No - Target gene:
- Hprt
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: V79 cells originated from Chinese hamster lung cells obtained from Health Science Reserch Resources Bank.
- Cell cycle length, doubling time or proliferation index: Doubling time = 13 hours
- Sex, age and number of blood donors if applicable: N/A
- Whether whole blood or separated lymphocytes were used if applicable: Not reported
- Number of passages if applicable: 6 for preliminary testing and 10 for main test.
- Methods for maintenance in cell culture if applicable: Cultured in Eagle's Minimum Medium (MEM) supplemented with 10 vol % fetal calf serum in a hhumidified CO2 incubator (5 % CO2, 37 ºC).
- Modal number of chromosomes: 22
- Normal (negative control) cell cycle time: 13 hours
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No
- Periodically checked for karyotype stability: No
- Periodically 'cleansed' against high spontaneous background: No - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Prelim: 0.0078 - 0.25 mg/mL
Main: 0.031, 0.063, 0.13 and 0.25 mg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Soluble at 25 mg/mL - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 300 x 10E4
DURATION
- Preincubation period: 4 h
- Exposure duration: 4 and 24 h
- Expression time (cells in growth medium): 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days
STAIN (for cytogenetic assays): Crystal violet (0.1 % w/v)
NUMBER OF REPLICATIONS: 2 - Evaluation criteria:
- Positive result if;
1. Mean MF's in treatment groups are 3 times higher than control values.
2. The mean MF's in the treatment groups are over ranges of negative (all data) in the historical control data. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Under the conditions of this test Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid did not induce gene mutation in the Hprt locus in V79 cells.
- Executive summary:
OECD 476 (2017) - In a mammalian cell gene mutation assay (HPRT locus of V79 cells), Chinese hamster lung cells cultured in vitro were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at concentrations of 0.031, 0.063, 0.13 and 0.25 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix) for an exposure period of 4 hours.
The test item was tested up to solubility limits which were defined in a screening test. There was no evidence of cytotoxicity or induced mutant colonies over background levels at any of the concentrations tested.
The positive controls induced the appropriate response.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.
Referenceopen allclose all
Table 1 Test 1 (in the presence of S9 mix)
Strain |
Concentration (µg/plate) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
TA98 |
Solvent control |
37.3 |
3.1 |
|
38, 40, 34 |
5 |
44.3 |
2.1 |
1.2 |
45, 46, 42 |
|
15 |
33.0 |
5.3 |
0.9 |
35, 27, 37 |
|
50 |
38.7 |
0.6 |
1.0 |
38, 39, 39 |
|
150 |
42.3 |
8.3 |
1.1 |
45, 49, 33 |
|
500 |
37.3 |
6.5 |
1.0 |
37, 31, 44 |
|
1500 |
42.0 |
3.6 |
1.1 |
43, 38, 45 |
|
5000 |
40.0 |
3.0 |
1.1 |
43, 40, 37 |
|
TA100 |
Solvent control |
154.0 |
20.0 |
|
137, 149, 176 |
5 |
138.0 |
12.5 |
0.9 |
134, 152, 128 |
|
15 |
149.0 |
8.7 |
1.0 |
159, 144, 144 |
|
50 |
141.0 |
15.1 |
0.9 |
143, 125, 155 |
|
150 |
153.7 |
27.2 |
1.0 |
123, 163, 175 |
|
500 |
155.0 |
9.2 |
1.0 |
153, 165, 147 |
|
1500 |
151.3 |
7.8 |
1.0 |
145, 160, 149 |
|
5000 |
138.3 |
5.9 |
0.9 |
145, 136, 134 |
|
TA1535 |
Solvent control |
21.7 |
1.5 |
|
23, 23, 22 |
5 |
18.7 |
1.2 |
0.9 |
20, 18, 18 |
|
15 |
19.3 |
1.2 |
0.9 |
20, 20, 18 |
|
50 |
25.0 |
1.7 |
1.2 |
24, 24, 27 |
|
150 |
23.3 |
1.2 |
1.1 |
22, 24, 24 |
|
500 |
20.3 |
4.9 |
0.9 |
26, 18, 17 |
|
1500 |
21.3 |
4.2 |
1.0 |
18, 20, 26 |
|
5000 |
18.7 |
2.3 |
0.9 |
20, 20, 16 |
|
TA1537 |
Solvent control |
12.0 |
1.0 |
|
12, 13, 11 |
5 |
11.7 |
1.5 |
1.0 |
13, 10, 12 |
|
15 |
11.3 |
1.5 |
0.9 |
11, 13, 10 |
|
50 |
9.7 |
2.5 |
0.8 |
10, 7, 12 |
|
150 |
9.7 |
2.3 |
0.8 |
11, 11, 7 |
|
500 |
13.0 |
2.0 |
1.1 |
13, 15, 11 |
|
1500 |
12.7 |
2.9 |
1.1 |
11, 16, 11 |
|
5000 |
7.7 |
2.1 |
0.6 |
6, 10, 7 |
|
WP2 uvrA (pKM101) |
Solvent control |
139.0 |
7.9 |
|
142, 130, 145 |
5 |
157.7 |
21.9 |
1.1 |
145, 145, 183 |
|
15 |
156.7 |
9.1 |
1.1 |
147, 165, 158 |
|
50 |
134.7 |
31.0 |
1.0 |
165, 136, 103 |
|
150 |
158.7 |
10.0 |
1.1 |
149, 169, 158 |
|
500 |
157.7 |
16.3 |
1.1 |
165, 139, 169 |
|
1500 |
146.0 |
15.6 |
1.1 |
154, 128, 156 |
|
5000 |
128.0 |
15.6 |
0.9 |
110, 138, 136 |
|
TA98 |
2a |
313.0 |
31.7 |
8.4 |
280, 343, 317 |
TA100 |
2b |
902.0 |
138.7 |
5.9 |
787, 1056, 863 |
TA1535 |
2b |
509.3 |
22.2 |
23.5 |
535, 497, 496 |
TA1537 |
50c |
648.3 |
69.9 |
54.0 |
725, 632, 588 |
WP2 uvrA (pKM101) |
2d |
2259.0 |
200.3 |
16.3 |
2280, 2448, 2049 |
a2-nitrofluorene
bsodium azide
c9-aminoacridine
d4-nitroquinoline-1-oxide
Table 2 Test 1 (in the absence of S9 mix)
Strain |
Concentration (µg/plate) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
TA98 |
Solvent control |
52.3 |
1.2 |
|
53, 51, 53 |
5 |
58.3 |
8.7 |
1.1 |
68, 51, 56 |
|
15 |
52.3 |
3.1 |
1.0 |
53, 49, 55 |
|
50 |
45.7 |
6.4 |
0.9 |
42, 53, 42 |
|
150 |
47.3 |
2.3 |
0.9 |
46, 50, 46 |
|
500 |
49.7 |
4.2 |
0.9 |
53, 45, 51 |
|
1500 |
41.0 |
7.5 |
0.8 |
40, 49, 34 |
|
5000 |
41.0 |
6.1 |
0.8 |
44, 34, 45 |
|
TA100 |
Solvent control |
164.7 |
10.1 |
|
170, 153, 171 |
5 |
162.0 |
17.6 |
1.0 |
155, 182, 149 |
|
15 |
154.3 |
5.0 |
0.9 |
149, 155, 159 |
|
50 |
156.0 |
13.0 |
0.9 |
171, 148, 149 |
|
150 |
159.3 |
18.5 |
1.0 |
138, 171, 169 |
|
500 |
148.7 |
8.1 |
0.9 |
145, 158, 143 |
|
1500 |
146.3 |
8.4 |
0.9 |
156, 141, 142 |
|
5000 |
148.3 |
12.7 |
0.9 |
163, 141, 141 |
|
TA1535 |
Solvent control |
19.3 |
2.3 |
|
18, 18, 22 |
5 |
22.0 |
3.5 |
1.1 |
20, 26, 20 |
|
15 |
22.7 |
0.6 |
1.2 |
23, 22, 23 |
|
50 |
17.3 |
1.2 |
0.9 |
18, 18, 16 |
|
150 |
14.0 |
1.7 |
0.7 |
13, 13, 16 |
|
500 |
17.7 |
2.9 |
0.9 |
21, 16, 16 |
|
1500 |
17.7 |
2.1 |
0.9 |
16, 20, 17 |
|
5000 |
15.0 |
2.0 |
0.8 |
17, 13, 15 |
|
TA1537 |
Solvent control |
38.0 |
4.6 |
|
42, 39, 33 |
5 |
33.0 |
6.0 |
0.9 |
27, 39, 33 |
|
15 |
32.0 |
7.2 |
0.8 |
38, 24, 34 |
|
50 |
31.7 |
5.5 |
0.8 |
26, 32, 37 |
|
150 |
29.7 |
3.2 |
0.8 |
26, 32, 31 |
|
500 |
22.7 |
2.3 |
0.6 |
24, 24, 20 |
|
1500 |
24.0 |
1.7 |
0.6 |
23, 26, 23 |
|
5000 |
27.0 |
3.5 |
0.7 |
29, 29, 23 |
|
WP2 uvrA (pKM101) |
Solvent control |
159.7 |
10.5 |
|
160, 149, 170 |
5 |
154.7 |
19.1 |
1.0 |
175, 137, 152 |
|
15 |
146.3 |
14.0 |
0.9 |
160, 132, 147 |
|
50 |
158.0 |
11.3 |
1.0 |
145, 165, 164 |
|
150 |
142.3 |
19.6 |
0.9 |
137, 164, 126 |
|
500 |
139.3 |
11.9 |
0.9 |
143, 149, 126 |
|
1500 |
142.7 |
15.1 |
0.9 |
160, 136, 132 |
|
5000 |
133.0 |
5.0 |
0.8 |
138, 128, 133 |
|
TA98 |
5a |
219.7 |
29.2 |
4.2 |
236, 186, 237 |
TA100 |
5b |
2259.7 |
405.0 |
13.7 |
1858, 2668, 2253 |
TA1535 |
5b |
302.0 |
4.6 |
15.6 |
297, 306, 303 |
TA1537 |
5a |
241.7 |
17.0 |
6.4 |
241, 259, 225 |
WP2 uvrA (pKM101) |
10b |
1332.7 |
61.1 |
8.3 |
1398, 1323, 1277 |
abenzo(a)pyrene
b2-aminoanthracene
Table 3 Test 2 (in the presence of S9 mix)
Strain |
Concentration (µg/plate) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
TA98 |
Solvent control |
36.7 |
4.6 |
|
42, 34, 34 |
50 |
35.7 |
2.9 |
1.0 |
39, 34, 34 |
|
150 |
36.7 |
8.5 |
1.0 |
45, 37, 28 |
|
500 |
38.3 |
10.8 |
1.0 |
46, 43, 26 |
|
1500 |
41.3 |
5.7 |
1.1 |
46, 43, 35 |
|
5000 |
38.7 |
5.0 |
1.1 |
44, 38, 34 |
|
TA100 |
Solvent control |
123.3 |
16.3 |
|
120, 109, 141 |
50 |
138.7 |
16.2 |
1.1 |
120, 148, 148 |
|
150 |
129.0 |
6.1 |
1.0 |
136, 125, 126 |
|
500 |
117.7 |
8.1 |
1.0 |
109, 119, 125 |
|
1500 |
118.7 |
6.5 |
1.0 |
112, 125, 120 |
|
5000 |
122.7 |
3.1 |
1.0 |
126, 122, 120 |
|
TA1535 |
Solvent control |
22.7 |
0.6 |
|
22, 23, 23 |
50 |
26.0 |
2.0 |
1.1 |
24, 26, 28 |
|
150 |
23.0 |
1.0 |
1.0 |
23, 22, 24 |
|
500 |
22.3 |
7.0 |
1.0 |
15, 23, 29 |
|
1500 |
20.7 |
1.2 |
0.9 |
20, 22, 20 |
|
5000 |
20.7 |
1.2 |
0.9 |
20, 20, 20 |
|
TA1537 |
Solvent control |
16.7 |
5.0 |
|
22, 16, 12 |
50 |
15.3 |
2.1 |
0.9 |
16, 13, 17 |
|
150 |
19.7 |
1.5 |
1.2 |
18, 21, 20 |
|
500 |
14.0 |
1.7 |
0.8 |
13, 16, 13 |
|
1500 |
18.3 |
1.5 |
1.1 |
17, 18, 20 |
|
5000 |
14.7 |
1.5 |
0.9 |
15, 16, 13 |
|
WP2 uvrA (pKM101) |
Solvent control |
145.7 |
16.4 |
|
152, 127, 158 |
50 |
156.3 |
21.6 |
1.1 |
141, 181, 147 |
|
150 |
152.7 |
9.1 |
1.0 |
161, 154, 143 |
|
500 |
152.0 |
6.2 |
1.0 |
159, 150, 147 |
|
1500 |
140.0 |
8.7 |
1.0 |
145, 145, 130 |
|
5000 |
145.3 |
13.6 |
1.0 |
150, 156, 130 |
|
TA98 |
2a |
404.7 |
29.7 |
11.0 |
438, 395, 381 |
TA100 |
2b |
1015.3 |
103.6 |
8.2 |
1125, 1002, 919 |
TA1535 |
2b |
701.3 |
24.0 |
30.9 |
729, 689, 686 |
TA1537 |
50c |
501.7 |
20.6 |
30.1 |
500, 523, 482 |
WP2 uvrA (pKM101) |
2d |
1816.7 |
181.5 |
12.5 |
1782, 2013, 1655 |
a2-nitrofluorene
bsodium azide
c9-aminoacridine
d4-nitroquinoline-1-oxide
Table 4 Test 2 (in the absence of S9 mix)
Strain |
Concentration (µg/plate) |
Mean revertants per plate |
SD |
Fold increase relative to vehicle |
Individual revertant colony counts |
TA98 |
Solvent control |
47.0 |
6.2 |
|
42, 54, 45 |
50 |
51.0 |
9.5 |
1.1 |
62, 46, 45 |
|
150 |
43.7 |
1.5 |
0.9 |
45, 44, 42 |
|
500 |
51.7 |
11.2 |
1.1 |
64, 42, 49 |
|
1500 |
46.3 |
10.0 |
1.0 |
50, 35, 54 |
|
5000 |
44.7 |
4.6 |
1.0 |
50, 42, 42 |
|
TA100 |
Solvent control |
133.7 |
21.0 |
|
150, 110, 141 |
50 |
136.0 |
4.4 |
1.0 |
131, 139, 138 |
|
150 |
141.7 |
12.3 |
1.1 |
152, 145, 128 |
|
500 |
148.3 |
9.9 |
1.1 |
155, 137, 153 |
|
1500 |
140.7 |
13.6 |
1.1 |
128, 139, 155 |
|
5000 |
133.7 |
6.8 |
1.0 |
139, 136, 126 |
|
TA1535 |
Solvent control |
25.0 |
7.2 |
|
17, 31, 29 |
50 |
20.7 |
5.0 |
0.8 |
26, 20, 16 |
|
150 |
21.3 |
4.6 |
0.9 |
16, 24, 24 |
|
500 |
22.0 |
5.3 |
0.9 |
28, 18, 20 |
|
1500 |
19.0 |
2.6 |
0.8 |
17, 22, 18 |
|
5000 |
19.3 |
2.9 |
0.8 |
21, 21, 16 |
|
TA1537 |
Solvent control |
30.0 |
6.9 |
|
26, 26, 38 |
50 |
28.3 |
4.0 |
0.9 |
26, 33, 26 |
|
150 |
28.3 |
2.3 |
0.9 |
27, 27, 31 |
|
500 |
28.3 |
3.2 |
0.9 |
32, 27, 26 |
|
1500 |
31.3 |
6.8 |
1.0 |
39, 29, 26 |
|
5000 |
27.0 |
1.7 |
0.9 |
26, 29, 26 |
|
WP2 uvrA (pKM101) |
Solvent control |
156.0 |
14.4 |
|
172, 152, 144 |
50 |
165.0 |
11.3 |
1.1 |
159, 158, 178 |
|
150 |
158.3 |
15.5 |
1.0 |
158, 174, 143 |
|
500 |
154.7 |
4.5 |
1.0 |
155, 159, 150 |
|
1500 |
157.7 |
4.6 |
1.0 |
155, 163, 155 |
|
5000 |
152.7 |
4.2 |
1.0 |
156, 154, 148 |
|
TA98 |
5a |
380.7 |
36.9 |
8.1 |
409, 394, 339 |
TA100 |
5b |
2087.7 |
206.8 |
15.6 |
1864, 2272, 2127 |
TA1535 |
5b |
243.7 |
68.6 |
9.7 |
169, 304, 258 |
TA1537 |
5a |
236.0 |
14.5 |
7.9 |
222, 251, 235 |
WP2 uvrA (pKM101) |
10b |
1179.3 |
60.6 |
7.6 |
1117, 1238, 1183 |
abenzo(a)pyrene
b2-aminoanthracene
Table 1 First test summary of results
Exposure period (h) |
S9 mix (v/v) |
Test concentration (µg/mL) |
Cells with aberrations (excluding gaps) |
Cells with aberrations (including gaps) |
Relative mitotic index (%) |
Polyploidy mean incidence (%) |
||||
Individual values (%) |
Mean (%) |
Individual values (%) |
Mean (%) |
|||||||
3 |
- |
Solvent control |
2.0 |
2.0 |
2.0 |
4.0 |
2.0 |
3.0 |
100 |
0.5 |
40.31 |
0.0 |
2.0 |
1.0 |
0.0 |
3.0 |
1.5 |
93 |
0.0 |
||
864 |
0.0 |
1.0 |
0.5 |
0.0 |
2.0 |
1.0 |
85 |
1.5 |
||
4000 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
68 |
0.0 |
||
0.2a |
14.0 |
13.0 |
13.5*** |
15.0 |
15.0 |
15.0*** |
- |
0.0 |
||
|
||||||||||
3 |
+ (2 %) |
Solvent control |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
100 |
0.0 |
186.62 |
1.0 |
0.0 |
0.5 |
1.0 |
2.0 |
1.5 |
106 |
1.0 |
||
864 |
3.0 |
1.0 |
2.0 |
3.0 |
3.0 |
3.0 |
83 |
0.5 |
||
4000 |
0.0 |
1.0 |
0.5 |
0.0 |
1.0 |
0.5 |
86 |
0.5 |
||
5b |
19.0 |
17.0 |
18.0*** |
21.0 |
18.0 |
19.5*** |
- |
0.5 |
||
amitomycin C
bcyclophosphamide
*** p <0.001 (one-tailed Fisher’s exact test)
Table 2 Second test summary of results
Exposure period (h) |
S9 mix (v/v) |
Test concentration (µg/mL) |
Cells with aberrations (excluding gaps) |
Cells with aberrations (including gaps) |
Relative mitotic index (%) |
Polyploidy mean incidence (%) |
||||
Individual values (%) |
Mean (%) |
Individual values (%) |
Mean (%) |
|||||||
3 |
- |
Solvent control |
1.0 |
3.0 |
2.0 |
5.0 |
4.0 |
4.5 |
100 |
1.5 |
3000 |
1.0 |
3.0 |
2.0 |
3.0 |
4.0 |
3.5 |
101 |
1.0 |
||
3500 |
3.0 |
2.0 |
2.5 |
4.0 |
4.0 |
4.0 |
90 |
0.5 |
||
4000 |
2.0 |
2.0 |
2.0 |
2.0 |
3.0 |
2.5 |
112 |
0.0 |
||
0.1a |
14.0 |
20.0 |
17.0*** |
16.0 |
22.0 |
19.0*** |
- |
1.0 |
||
|
||||||||||
3 |
+ (5 %) |
Solvent control |
0.0 |
0.0 |
0.0 |
1.0 |
0.0 |
0.5 |
100 |
1.0 |
2000 |
0.0 |
1.0 |
0.5 |
0.0 |
1.0 |
0.5 |
104 |
2.0 |
||
3000 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
110 |
0.5 |
||
4000 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
100 |
0.0 |
||
5b |
10.0 |
9.0 |
9.5*** |
10.0 |
10.0 |
10.0*** |
- |
0.0 |
||
amitomycin C
bcyclophosphamide
*** p <0.001 (one-tailed Fisher’s exact test)
Table 1 Mutation frequency on Hprt gene in V79 cells treated with test item for 4 hours without S9 mix (mean values)
Concentration (mg/mL) |
Cell confluency |
Cytotoxicity |
Mutation |
|||||||
Cell confluency OD %/ dish |
% of control |
Relative cell numbers |
Cytotoxicity (colonies/well) |
Relative cloning efficiency (%) |
Relative survival (%) |
Mutant colonies/ well |
Relative cloning efficiency (%) |
Mean number of resistant colonies |
Mutation frequency (x106) |
|
Solvent control |
98.8 |
100.0 |
100.0 |
119.2 |
100.0 |
100.0 |
106.5 |
100.0 |
0.6 |
5.8 |
0.031 |
99.2 |
100.3 |
102.3 |
119.5 |
100.3 |
102.6 |
93.5 |
87.7 |
0.6 |
6.0 |
0.063 |
99.5 |
100.7 |
104.6 |
111.2 |
93.3 |
97.6 |
94.7 |
89.3 |
0.3 |
3.4 |
0.13 |
98.8 |
100.0 |
105.7 |
111.2 |
93.3 |
98.5 |
98.5 |
92.7 |
0.7 |
6.6 |
0.25 |
98.5 |
99.7 |
107.8 |
103.0 |
86.4 |
93.1 |
88.7 |
83.5 |
0.5 |
5.0 |
1a |
94.0 |
95.1 |
107.9 |
84.8 |
71.2 |
76.8 |
80.2 |
75.5 |
94.6 |
1179.2 |
apositive control (ethyl methanesulfonate)
Table 2 Mutation frequency on Hprt gene in V79 cells treated with test item for 4 hours with S9 mix (mean values)
Concentration (mg/mL) |
Cell confluency |
Cytotoxicity |
Mutation |
|||||||
Cell confluency OD %/ dish |
% of control |
Relative cell numbers |
Cytotoxicity (colonies/well) |
Relative cloning efficiency (%) |
Relative survival (%) |
Mutant colonies/ well |
Relative cloning efficiency (%) |
Mean number of resistant colonies |
Mutation frequency (x106) |
|
Solvent control |
100.3 |
100.0 |
100.0 |
109.7 |
100.0 |
100.0 |
104.8 |
100.0 |
0.2 |
1.7 |
0.031 |
98.8 |
98.5 |
106.7 |
102.8 |
93.8 |
100.0 |
107.3 |
103.5 |
1.0 |
9.4 |
0.063 |
99.3 |
99.0 |
103.8 |
105.5 |
96.2 |
99.8 |
114.0 |
109.4 |
0.4 |
3.5 |
0.13 |
98.3 |
98.0 |
103.0 |
118.2 |
107.8 |
111.1 |
107.0 |
102.5 |
0.5 |
4.5 |
0.25 |
97.7 |
97.3 |
101.9 |
110.0 |
100.4 |
102.3 |
118.0 |
115.3 |
0.4 |
3.3 |
1a |
96.8 |
96.5 |
103.8 |
98.5 |
89.9 |
93.3 |
102.8 |
100.9 |
41.0 |
107.0 |
apositive control (N-nitrosodimethylamine)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Mammalian somatic cell: Negative (non-clastogenic); OECD 474; Barfield, W. (2011)
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18th July - 24 Aug 2011
- 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:
- OJ L 142/240.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
- Version / remarks:
- EPA 712-C-98-226.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- November 24, 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian erythrocyte micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: JA01YX10
- Expiration date of the lot/batch: April 2012
- Purity test date: 95.8%
RADIOLABELLING INFORMATION (if applicable): N/A
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ca 20°C), in the dark under nitrogen.
- Stability under test conditions: Not stated
- Solubility and stability of the test substance in the solvent/vehicle: Not stated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not stated
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspensions of the test substance were prepared in Corn oil obtained from Sigma batch number MKBF6012V.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: 10 mL/kg/day
- Final preparation of a solid: N/A
FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A - Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
TEST ANIMALS - Source: Charles River UK Limited, Margate, Kent, England, animals
- Age at study initiation: Males and females ca 35 days old. Males and females ca 40 days
- Weight at study initiation: Males weighed between 30.0 g to 30.4 g. Females weighed between 22.4 g to 25.5 g
- Assigned to test groups randomly: yes
- Fasting period before study: Not stated
- Housing: Each group was kept, with the sexes separated, in cages and maintained in a controlled environment
- Diet (e.g. ad libitum): Nestlets
- Water (e.g. ad libitum): tap water ad libitum.
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours per day
IN-LIFE DATES: From: To: 27 July 2011 to 25 August 2011- Route of administration:
- oral: gavage
- Vehicle:
- Corn oil obtained from Sigma batch number MKBF6012V
- Details on exposure:
- Preliminary toxicity test:
A dose of 2000 mg/kg/day (200 mg/mL) was administered to 4 animals (2/sex) on two consecutive occasions approximately 24 hours apart followed by 48 hours observation.
The micronucleus test:
Dose levels of 500, 1000 and 2000 mg/kg/day were used once to 5 groups of animals once followed by 24hour observation - Duration of treatment / exposure:
- 24 hours
- Frequency of treatment:
- Once
- Post exposure period:
- Preliminary toxicity test:
48 hours observation.
The micronucleus test:
24hour observation - Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Vehicle
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Remarks:
- Test item
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Remarks:
- Test item
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- Remarks:
- Test item
- Dose / conc.:
- 12 mg/kg bw/day (nominal)
- Remarks:
- Mitomycin C
- No. of animals per sex per dose:
- 6 animals per group and 5 for positive control
- Control animals:
- yes
- Positive control(s):
- Mitomycin C
- Tissues and cell types examined:
- Bone marrow
- Details of tissue and slide preparation:
- TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):Femurs were cleaned of all excess tissue and blood and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal were flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by aspiration.
FIXATION AND SLIDE STAINING:
1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry
2 Rinsed in purified water
3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
4 Washed in purified water for 5 minutes
5 Rinsed in cold tap water for 2 minutes
6 Stored at room temperature until required
7 Immediately prior to scoring, slides are wet mounted with coverslips using purified water
DETAILS OF SLIDE PREPARATION:
The resulting cell suspensions were centrifuged at 1000 rpm (150 × g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides
METHOD OF ANALYSIS:
Fluorescence microscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei.
OTHER:
The proportion of polychromatic erythrocytes was assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes was recorded. - Evaluation criteria:
- The following criteria were applied for assessment of assay acceptability:
1. Each treated and control group should include at least 5 analysable animals.
2. Vehicle control values for micronucleated polychromatic erythrocytes must be consistent
with the laboratory historical vehicle control data.
3. Positive controls must show clear unequivocal positive responses. - Statistics:
- The data were received in an Excel document and analysed using SAS 9.1.3 (SAS Institute Inc., 2002) (Jonckheere's and Wilcoxon tests) and StatXact 3 (Cytel 1995) (Linear-by-Linear and Permutation tests).
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- No mortalities were observed throughout the duration of the micronucleus test. No clinical signs of toxicity were noted for the vehicle control, positive control and Test item group animals over the duration of the test. Some small incidences of bodyweight loss in all dose groups, including controls, were recorded.
- Conclusions:
- Under the condition of the study, it is concluded that Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid is not clastogenic.
- Executive summary:
OECD 474 (2011) - In a study designed to assess the potential induction of micronuclei by Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid in bone marrow cells of CD1 mice, the animal were treated with the test item by oral garage on two occasions approximately 24hr apart.
The preliminary toxicity test demonstrated that a dose of 2000 mg/kg/day, (the standard limit dose for the micronucleus test) administered on two consecutive occasions approximately 24 hours apart was tolerated. Therefore the main study was conducted at dose levels of 500, 1000 and 2000 mg/kg/day using a dose volume of 10 mL/kg/day.
The negative control group received the vehicle corn oil (dose volume of 10 mL/kg/day) and the positive control group received Mitomycin C at 12 mg/kg using a dose volume of 20 mL/kg.
Bone marrow smears were obtained from all test groups 24 hours after administration. One smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.
The test item did not induce any statistically significant increases in the frequency of micronucleated polychromatic erythrocytes or decreases in the proportion of polychromatic erythrocytes were observed in all tested concentrations. Both control control groups produced results within historical control data.
It is concluded that under the condition of the study, the test it is not considered clastogenic.
Reference
Table 3 Summary of results and statistical analysis
Sampling time after 2nddose |
Treatment |
Concentration (mg/mL) |
Proportion of PCE (%) # |
Incidence MPCE (mean) # |
24 Hours
|
Vehicle |
- |
51.4 |
1.3 |
Test item |
500 |
47.0 |
1.7 |
|
Test item |
1000 |
50.0 |
1.8 |
|
Test item |
2000 |
53.2 |
1.8 |
|
Mitomycin Ca |
12 |
51.3 |
53.6** |
Vehicle = Corn Oil
PCE = Polychromatic erythrocytes
MPCE = Number of micronucleated polychromatic erythrocytes observed per 2000 polychromatic erythrocytes examined
a = Positive control dosed once only approximately 24 hours prior to termination at a dose volume of 20 mL/kg/day.
# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table
Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):
** p < 0.01 (significant), otherwise p > 0.01 (not significant)
Table 4 Results for individual animals - 24 hour sampling time.
Treatment (mg/kg/day) |
AnimalNO |
Proportion PCE (%) |
MPCE
|
PCE
|
NCE
|
MNCE
|
Vehicle |
401 402 403 404 405 406
|
50.6 53.2 54.5 49.2 48.3 52.7 |
1 2 3 0 1 1 |
515 535 548 500 495 537 |
503 471 457 516 529 482 |
0 0 0 0 0 0 |
Test item (500) |
411 451 452 414 415 416 |
41.5 45.7 53.6 47.9 45.8 47.7 |
1 1 0 1 5 2 |
417 457 540 484 467 490 |
588 544 467 526 553 537 |
0 0 0 0 0 0 |
Test item (1000) |
421 422 423 424 425 426 |
51.1 50.4 49.7 50.1 55.8 43.0 |
1 2 2 1 2 3 |
514 514 506 510 564 435 |
492 505 512 507 446 576 |
0 0 0 0 0 0 |
Test item (2000) |
431 432 433 434 435 436 |
59.6 59.4 44.5 49.8 55.1 50.6 |
3 2 3 1 1 1 |
600 602 452 519 572 513 |
407 411 563 524 467 501 |
0 0 0 0 0 0 |
Mitomycin Ca(12) |
441 442 443 444 445 |
51.5 50.8 52.5 48.6 51.2 |
58 60 35 40 75 |
540 508 528 488 516 |
470 492 477 516 491 |
0 0 0 0 0
|
Vehicle = Corn Oil
PCE = Polychromatic erythrocytes
MPCE = Number of micronucleated polychromatic erythrocytes observed per 2000 polychromatic erythrocytes examined
NCE = Total number of normochromatic erythrocytes examined for micronuclei
MNCE: Number of micronucleated normochromatic erythrocytes observed
a = Positive control dosed once only approximately 24 hours prior to termination at a dose volume of 20 mL/kg/day.
Table 5 Body weight - Preliminary Toxicity Test
Treatment (mg/kg/day)
|
Animal Number
|
Bodyweights (g)
|
|||||||
Day after arrival
|
Day 1 |
Day 2 |
Day 3 |
||||||
Individual
|
Mean
|
Individual
|
Mean
|
Individual
|
Mean
|
Individual
|
Mean
|
||
Test item (2000)
|
M -21 |
30.0 |
30.2 |
34.3
|
34.5 |
33.6# |
33.8 |
35.3 |
34.7 |
M-22 |
30.4 |
34.6
|
34.0# |
34.1 |
|||||
F -27 |
22.4 |
24.0 |
24.2
|
24.6 |
23.6# |
24.4 |
23.7 |
24.7 |
|
F- 28 |
25.5 |
25.0 |
25.2
|
25.7 |
|||||
# = Denotes weight loss from previous weighing
M Male
F Female
Table 6 Body weight - Micronucleus Test
Treatment (mg/kg/day)
|
Animal Number
|
Bodyweights (g)
|
|||||||
Day after arrival
|
Day 1 |
Day 2 |
Day 3 |
||||||
Individual
|
Mean
|
Individual
|
Mean
|
Individual
|
Mean
|
Individual
|
Mean
|
||
Vehicle |
401 402 403 404 405 406 |
29.8 32.0 29.6 29.5 30.7 31.4 |
30.5 |
31.8 32.2 34.1 32.7 31.8 33.6 |
32.7 |
31.8 34.0 33.4# 33.2 31.9 33.1#
|
32.9 |
31.9 34.7 32.7# 32.7# 31.6# 33.8
|
32.9 |
Test item (500)
|
411 451 452 414 415 416 |
30.9 31.1 31.3 29.9 28.0 29.5 |
30.1 |
33.0 33.2 34.7 31.3 31.0 30.6 |
32.3 |
34.5 33.1# 34.8 31.6 31.5 31.3
|
32.8 |
35.3 33.5 35.0 32.6 32.5 31.8 |
33.5 |
Test item (1000)
|
421 422 423 424 425 426 |
30.5 28.8 33.2 31.8 31.2 30.1 |
30.9 |
32.8 31.6 35.6 34.4 34.4 32.4 |
33.6 |
32.8 31.9 35.2# 35.0 34.4# 32.6
|
33.7 |
33.0 32.4 35.8 35.0 34.2# 32.7
|
33.9 |
Test item (2000) |
431 432 433 434 435 436 |
31.1 30.3 31.2 32.1 30.6 29.6 |
30.8 |
33.3 33.2 33.7 35.1 32.2 32.0 |
33.3 |
33.0# 33.0# 33.7 35.2 31.8# 32.3
|
33.2 |
32.5# 33.5 34.0 35.1# 32.4 32.7
|
33.4 |
Mitomycin Ca(12)
|
441 442 443 444 445 |
30.4 30.5 31.1 32.0 30.5 |
30.9 |
- - - - - |
- |
31.2 31.7 33.5 32.7 35.5 |
32.9 |
31.8 31.5# 33.9 32.8 35.8 |
33.2 |
# = Denotes weight loss from previous weighing
M Male
F Female
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Mode of Action Analysis / Human Relevance Framework
Not applicable as there were no adverse effects observed.
Additional information
Mutagenicity (in vitro)
OECD 471 (2011) - In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA1537 (S. typhimurium) and WP2uvrA (pKM101) (Escherichia coli) were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid in acetone at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in a screening experiment followed by concentrations of 50, 150, 1500 and 5000 µg/plate in a secondary experiment. Both tests were conducted in the presence and absence of S9 mix. The S9 mix content was increased from 10 % v/v to 20 % v/v in the secondary test.
The test item was tested up to the standard limit concentration recommended in the regulatory guidelines i.e. 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains.
There was no evidence of cytotoxicity or induced mutant colonies (over background levels) in any of the test item treated colonies in either of the two tests.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
OECD 473 (2011) - In a mammalian cell cytogenetics assay (in vitro chromosome aberration, OECD 473), primary lymphocyte cultures were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at concentrations of 40.31, 67.18, 111.97, 186.62, 311.04, 518.4, 864, 1440, 2400 and 4000 µg/mL for 3 h with and without metabolic activation. Additionally, a continuous exposure of 24 h was tested with and without metabolic activation at test item concentrations of 25, 100, 250, 500, 100, 1500, 200, 2500, 3000, 3500 and 4000 µg/mL and 100, 250, 500, 1000, 2000, 3000 and 4000 µg/mL, respectively. The S9 mix concentration in the second test was raised from 2 to 5 % v/v as a modification parameter.
4000 µg/mL was determined as the highest concentration that did not induce unacceptable fluctuations in osmolarity in a screening test. This concentration was assigned as the top concentration in both the first and secondary tests.
Both the solvent control and positive controls induced the appropriate responses and were with laboratory historical ranges with 99 % CI.
There was no evidence (or a concentration related positive response) of chromosome aberration induction over background values at the highest tested concentration.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro Mammalian Chromosome Aberration Test (OECD 473) in human lymphocyte cells.
OECD 476 (2017) - In a mammalian cell gene mutation assay (HPRT locus of V79 cells), Chinese hamster lung cells cultured in vitro were exposed to Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at concentrations of 0.031, 0.063, 0.13 and 0.25 µg/mL in the absence and presence of mammalian metabolic activation (S9-mix) for an exposure period of 4 hours.
The test item was tested up to solubility limits which were defined in a screening test. There was no evidence of cytotoxicity or induced mutant colonies over background levels at any of the concentrations tested.
The positive controls induced the appropriate response.
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.
Mutagenicity (in vivo)
OECD 474 (2011) - In a study designed to assess the potential induction of micronuclei by Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid in bone marrow cells of CD1 mice, the animal were treated with the test item by oral garage on two occasions approximately 24 hr apart.
The preliminary toxicity test demonstrated that a dose of 2000 mg/kg/day, (the standard limit dose for the micronucleus test) administered on two consecutive occasions approximately 24 hours apart was tolerated. Therefore the main study was conducted at dose levels of 500, 1000 and 2000 mg/kg/day using a dose volume of 10 mL/kg/day.
The negative control group received the vehicle corn oil (dose volume of 10 mL/kg/day) and the positive control group received Mitomycin C at 12 mg/kg using a dose volume of 20 mL/kg.
Bone marrow smears were obtained from all test groups 24 hours after administration. One smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.
The test item did not induce any statistically significant increases in the frequency of micronucleated polychromatic erythrocytes or decreases in the proportion of polychromatic erythrocytes were observed in all tested concentrations. Both control control groups produced results within historical control data.
It is concluded that under the condition of the study, the test it is not considered clastogenic.
Justification for classification or non-classification
The substance does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008 (CLP).
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