Registration Dossier
Registration Dossier
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EC number: 290-754-9 | CAS number: 90218-76-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
1, 2,4 -Benzenetricarboxylic acid, mixed decyl and octyl triesters showed no evidence of mutagenic potential in an OECD Guideline 471 AMES bacterial reverse mutation asssay involving four strains of Salmonella typhimurium and one Escherichia coli strain, in the absence and presence of metabolic activation(±S9).
In an OECD 473 chromosomal aberration assay, 1, 2,4 -Benzenetricarboxylic acid, mixed decyl and octyl triesters showed no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, at any dose level of any sampling time. It was concluded that 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester did not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.
In an OECD 476 mouse lymphoma assay 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation under the test conditions
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- May 28 to August 5, 2009
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: well documented study performed according to OECD Guideline and GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- thymidine kinase locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
Minimal medium A: RPMI 1640 medium supplemented with penicillin, streptomycin sulphate, sodium pyrovate, L-glutamine, non-essential amino acids and F 68 Pluronic
Minimal medium B: same as Minimal medium A without F68 Pluronic
Complete medium (5%): Minimal medium A supplemented with 5% v/v heat-inactivated horse serum
Complete Medium (10%: Minimal medium A supplemented with 10% v/v heat-inactivated horse serum
Complete medium A (20%): Minimal medium A supplemented with 20% v/v heat-inactivated horse serum
Complete medium B (20%): Minimal medium B supplemented with 20% v/v heat-inactivated horse serum
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- other: Generation time and mutation rates (spontaneous and induced) were checked in the testing laboratory
- Metabolic activation:
- with and without
- Metabolic activation system:
- liver S9 mix of Phenobarbital - 5,6-Benzoflavone induced male Sprague Dawley rats
- Test concentrations with justification for top dose:
- With and without metabolic activation:
Toxicity test (range finding): 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/mL
1. experiment: 156, 313, 625 and 1250 and 2500 µg/mL with and without metabolic activation
2. experiment: 156, 313, 625, 1250 and 2500 µg/mL without metabolic activation; resp. 875, 1138, 1479, 1923 and 2500 µg/mL with metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solvent giving the best solubility/dispersal characteristics - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: in the absence of metabolic activation (4 h/24h exposure): methyl methanesulphonate (MMS, 5.0/10.0 µg/ml); in the presence of metabolic activation: Benzo(a)pyrene (B(a)P, 2.0 µg/mL)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
Experiment 1: 3 h (with and without metabolic activation)
Experiment 2: 24 h (without metabolic activation) and 3 h (with metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days (mutation selection assay)
SELECTION AGENT (mutation assays): trifluorothymidine (TFT), final concentration: 3.0 µg/mL in Complete medium B (20%)
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: mutation selection assay: 2000 per well; cloning efficiency assay: 1.6 cells per well (8 cells/mL)
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (Cell concentrations were adjusted to 8 cells/mL and, for each dose level, 0.2 mL was plated into 96 microtitre wells, incubated for 7 days. Wells containing viable clones were identified by the eye using background illumination and then counted.)
OTHER EXAMINTATIONS
- Colony sizing: small and large type mutants, estimated for solvent and positive controls
- Observations of pH and osmolality: the treatment solutions were measured during performance of one the main experiments - Evaluation criteria:
- Criteria for a positive result:
- the induced mutant frequency is higher than the global evaluation factor suggested for the microwell method (126 x 10E-6) at one or more doses
- there is a significant dose-relationship as indicated by the linear trend analysis
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance. - Statistics:
- Statisitical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990). The following methods were applied:
- Results of individual plates within a replicate treatment were checked for consistency by calculation of Chi-square.
- Heterogeneity factors were calculated for survival, viability and mutation. Values obtained should not exceed 10.8 times the current heterogeneity factor where 10.8 is the one-sided 0.1% level of the F-distribution with 1 and infinitive degrees of freedom.
- Overall consistency was evaluated by calculation of the ratio of the heterogeneity factors of the experiment to the current heterogeneity factor. This ratio should not exceed the one-sided 1% critical values from the F-distribution.
- The estimated heterogeneity factors of the experiment were combined with the current heterogeneity factor to define the updated estimated factors.
- Comparison of each treatment with the control: for each comparison, the ratio Di²/var(Di) was compared to the critical values for the one tailed Dunnett`s test.
- Test for linear trend: The evaluation of a linear trend in mutant frequency with the treatment dose was performed using weighted regression. The slope and its variance var(b) were calculated to form the test statistic b²/var(b), which was compared to tabulated critical values of Chi-square with 1 degree of freedom. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- Precipitation in all tested 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
- Effects of osmolality: no
- Water solubility: not soluble
- Precipitation: yes, particles in suspension and opacity were observed by adding solution at 250 mg/mL in ethanol to RPMI complete medium (10%) in a ratio 1:100 in the solubility trial, on the basis of this result a concentration of 2500 µg/mL was selcted as the highest dose level to be used in the test. Opacity, resp. slight opacity was observed at concentrations of 1250, 625 and 313 µg/mL.
- Other confounding effects: no
RANGE-FINDING/SCREENING STUDIES: No relevant toxicity was noted at any dose level tested using the short treatment time. In the absence of S9 metabolic activation, using the long treatment time, slight reduction of relative survival (RS) was noted at several concentrations without any dose relationship.
COMPARISON WITH HISTORICAL CONTROL DATA: The solvent and positive control mean mutant fractions were within the normal ranges experienced in the testing laboratory. - Conclusions:
- Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
1,2,4-Benzenetricarboxylic acid, decyl octyl ester is not mutagenic in mouse lymphoma L5178Y cells, in either the absence or the presence of S9 mix, when tested in ethanol, under the reported experimental conditions. - Executive summary:
The test item 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester was examined for mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.
The study was designed to comply with the experimental methods indicated in: Test method B.17 "in vitro mammalian cell gene mutation test" described in Council Regulation (EC) No. 440/2008 and OECD Guideline for the testing of chemicals No. 476 (adopted July 1997).
A solubility trial indicated that the maximum practicable concentration of the test item in the final test medium was 2500 µg/mL using ethanol as the solvent. On the basis of this result a preliminary cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2500 µg/mL and at a wide range of lower dose levels: 1250, 625, 313, 78.1, 39.1, 19.5 and 9.77 µg/mL. No relevant toxicity was observed at any dose level at any sampling time, in the absence and presence of S9 metabolism.
Based on the toxicity results obtained in the preliminary assay, two independent assays for mutation to 5 -trifluorothymidine resistance were performed using the following dose levels and treatment times:
Assay No. 1: 156, 313, 626, 1250 and 2500 µg/mL, 3 hours treatment with and without metabolic activation
Assay No. 2: 156, 313, 625, 1250 and 2500 µg/mL, 24 hours treatment without meatbolic activation; resp. 875, 1138, 1479, 1923 and 2500 µg/mL, 3 hours treatment with metabolic activation.
No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism.
Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.
It was concluded that 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 2009-05-21 to 2009-10-06
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- due to a technical problem, no measurement of the osmolality values of treatment media could be performed for the second main experiment
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- see above
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes: obtained from human blood from two healthy male volunteer donors (one for each experiment)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 1x (Dutch modification); 500 mL supplemented with 100 mL heat inactivated Foetal Calf Serum, 6.25 mL L-glutamine (200 mM), 1.25 mL Streptomycin sulphate 50 mg/mL Penicillin G 50,000 IU/mL
- Additional strain / cell type characteristics:
- other: stimulated to cell division in vitro by addition of the mitogen PHA (phytohaemogglutinin); cell cycle length approx. 17 hours
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 of Phenobarbital - 5,6-Benzoflavone induced male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- Range finding: 2500, 1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 µg/mL
Dose levels selected for metaphase analysis in the main tests:
First main test: 1250, 625 and 313 µg/mL (with and without S9 mix, 3 hours treatment time, 24 hours harvest time)
Second main test: 2500, 1250 and 625 µg/mL (without S9 mix, 24 hours treatment time, 24 hours harvest time) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was found to be soluble in ethanol at the concentration of 500 mg/mL. An aliquot of the stock solution in ethamol at 500 mg/mL added in the ratio of 1 : 100 to culture medium gave particles in suspension. Particles in suspension and opacity were observed when adding an aliquot at 250 mg/mL, opacity was observed by adding solutions at 125 and 6.25 mg/mL. On the basis of the results of solubility testing, the maximum dose level of 2500 µg/mL was selected for treatment, where precipitation and opacity were expected. This dose level was achieved by adding a solution of test item in ethanol at the concentration of 250 mg/mL to culture medium in the ration of 1 : 100. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol, 1% in tissue culture medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- without metabolic activation Migrated to IUCLID6: 0.75 and 0.50 µg/mL in the first main test; 0.45 and 0.30 µg/mL in the second main test
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ethanol, 1% in tissue culture medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation Migrated to IUCLID6: 18.0 and 23.0 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
48 hours after the lymphocyte cultures were initiated, they were centrifuged at 1000 rpm for 10 minutes, the culture medium was decanted and replaced with treatment medium (4.95 mL resp. 3.95 mL (in the test with S9 mix) culture medium without PHA + 0.05 mL test item or control solution + 1.00 mL S9 mix). The cultures were subsequently incubated for 3 hours at 37 °C, the medium was aspirated and the cultures were centrifuged and washed twice with PBS. Fresh medium was added and the cultures were incubated for a further period of 21 hours recovery period. Colcemid was added for the last 3 hours, leading up to harvesting after 24 hours. In the second main test the test medium was not changed (treatment for 24 hours).
DURATION
- Preincubation period: not applicable
- Exposure duration: First main test: 3 h (presence and absence of S9 mix); second main test: 24 h in the absence of S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h
SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concentration 0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa solution (3 % in tap water)
NUMBER OF REPLICATIONS: two cultures per dose level, controls and vehicle control
NUMBER OF CELLS EVALUATED: 200 metaphases per dose level (100 per replicate)
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Determination of aneupoid cells
OTHER: Measurement of pH and osmolality at the three higher dose levels in the first main test, pH measurement in the second main test at the four higher dose levels - Evaluation criteria:
- The evaluation was based on the set of results which excludes gaps.
A test item is considered to have clastogenic properties if the following criteria are all fulfilled:
- statistically significant increases in the incidence of cells bearing aberrations are observed at any dose level over the concurrent control
- the increases are reproduced in both replicate cultures and must be observed in both experiments
- the increases must exceed historical controls
- biological significance must be given - Statistics:
- Fisher's exact test was used to compare the number of cells bearing aberrations either including and excluding gaps (assumed to be Poisson ditributed) in control and treated cultures.
- Species / strain:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- lymphocytes: human peripheral blood lymphocytes
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: a slight reduction of the osmolality value was observed at the two high dose levels
- Evaporation from medium: not applicable
- Water solubility: no data
- Precipitation: In the first main test precipitation and dose-related opacity were oberved at the end of treatment at the dose levels of 2500 and 1250 µg/mL; during the performance of the second main test no precipitation was observed at the end of the treatment.
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES: No cytotoxicity up to the tested concentrations were observed.
COMPARISON WITH HISTORICAL CONTROL DATA: in the range of historical control data
ADDITIONAL INFORMATION ON CYTOTOXICITY: none - Conclusions:
- Interpretation of results (migrated information):
negative
It was concluded that 1,2,4-Benzenetricarboxylic acid, decyl octyl ester did not induce chromosomal aberration in human lymphocytes after in vitro treatment under the reported experimental conditions. - Executive summary:
The test item, 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester, was assayed for the ability to cause chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation according to Test method B.10 described in Council Regulation (EC) No. 440/2008 and OECD Guideline No. 473 (adopted July 1997).
Two independent experiments for chromosomal damage were performed. In the first experiment, the cells were treated 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 24 hours corresponding to approx. 1.5 cell cycle was used. As negative results were obtained, a second experiment was performed using the same harvest time (24 hours). A continuous treatment until harvest was used.
Both for the first and second experiments, dose levels of 2500, 1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 µg/mL were used in the absence or presence of S9 metabolism. Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. For both experiments, the dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotocity of the tets item treatments (as determined by the reduction in mitotic index.) Where no cytotoxicity occured the highest treatment level was selected as the maximum dose level for scoring.
The following dose levels were selected for scoring (100 metaphase spreads were scored for chromosomal aberrations from each culture, 200 for each experimental point):
Assay #1: 1250, 625 and 313 µg/mL with and without S9 metabolism (3 hours treatment time, 24 hours harvest time)
Assay #2: 2500, 1250 and 625 µg/mL without S9 metabolism (24 hours treatment and harvest time)
Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level of any sampling time. Statistically significant increases in the incidence of cells bearing aberrations (both including and excluding gaps) were seen following positive control treatments with Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.
It was concluded that 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester did not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Bacterial Reverse Mutation Assay
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13-04-2021 to 06-05-2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Adopted 2020
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Adopted June 2020
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Source: MOLTOX - Molecular Toxicology
Inc., Boone, North Carolina, USA - Species / strain / cell type:
- S. typhimurium TA 98
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Source: MOLTOX - Molecular Toxicology
Inc., Boone, North Carolina, USA - Species / strain / cell type:
- S. typhimurium TA 100
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Source: MOLTOX - Molecular Toxicology
Inc., Boone, North Carolina, USA - Species / strain / cell type:
- S. typhimurium TA 1537
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Source: MOLTOX - Molecular Toxicology
Inc., Boone, North Carolina, USA - Species / strain / cell type:
- E. coli WP2 uvr A
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: Surce: MOLTOX - Molecular Toxicology
Inc., Boone, North Carolina, USA - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Microbiological Laboratory of Charles River Laboratories Hungary Kft
- method of preparation of S9 mix:
Male Wistar rats (410-708 g, animals were 9-25 weeks old) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg bw/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels.
On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC.
- concentration or volume of S9 mix and S9 in the final culture medium:
S9 Mix (containing 10 % (v/v) of S9).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The sterility of the preparation was confirmed where the protein concentration of the preparation was determined by a chemical analyser at 540 nm. The mean protein concentration of the S9 fraction used were determined to be 21.1 g/L. The biological activity in the Salmonella assay of S9 was characterized in each case using the two
mutagens 2-Aminoanthracene and Benzo(a)pyrene, that require metabolic activation by microsomal
enzymes. The batches of S9 used in this study functioned appropriately.
The sterility of the preparation was confirmed. Sterilization of the salt solution for S9 mix was performed by filtration through a 0.22 μm membrane filter while sterilization of the sodium phosphate buffer was performed at 121°C in an autoclave. - Test concentrations with justification for top dose:
- A preliminary concentration range finding test was preformed where six test concentrations were prepared by successive dilutions of the stock solution, spaced by factors
of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the
background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium
TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and
10 μg/plate of the test item, in the absence and presence of metabolic activation.
Based on the results of the preliminary test, a 100 mg/mL stock solution was prepared in N,N-Dimethylformamide , which was diluted by serial dilutions in several steps to obtain the dosing formulations for lower doses. The maximum test concentration was 5000 μg/plate.
Examined concentrations in the Assay 1 and Assay 2 were 5000, 1581, 500, 158.1, 50 and
15.81 μg/plate. Examined concentrations in the Assay 3 were 5000, 1581, 500, 158.1, 50,
15.81, 5, 1.581, 0.5 and 0.1581 μg/plate. - Vehicle / solvent:
- - Solvent/ vehicle used: Dimethyl sulfoxide (DMSO) (Supplier: VWR; Batch No.: 20H054003), N,N-Dimethylformamide (DMF) (Supplier: VWR, Batch No.: 20D024011) and Distilled water (Manufacturer: MAGILAB Kft., Batch No.: 202011110,m Expiry date: 19 May 2021)
- Justification for choice of solvent/vehicle:
The solubility of the test material was examined using distilled water, and dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and acetone. The test item was insoluble (it was divided into
two phases) at 100 mg/mL concentration using distilled water and DMSO. At the same
concentration clear solubility was observed using DMF and acetone. Due to the better
biocompatibility, DMF was selected as vehicle for the study. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO, DMF and Distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-1,2-phenylenediamine
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO, DMF and Distilled water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: three
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Not stated
- Assay 1: standard plate incorporation procedure, Assay 2 and 3: standard pre-incubation procedure.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Assay 2 and 3: 20
minutes at 37ºC
- Exposure duration/duration of treatment: 48 (±1) hours
- Harvest time after the end of treatment (sampling/recovery times): Not stated
DETERMINATION OF CYTOTOXICITY
- Method: other: the background lawn was inspected for signs of toxicity (no further details mentioned) - Rationale for test conditions:
- The test conditions were determined following a preliminary compatibility test and a preliminary concentration range finding test.
- Evaluation criteria:
- Criteria for Validity
The study was considered valid if:
a)the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
b)at least five analysable concentrations are presented in all strains of the main tests.
Criteria for a Positive Response
A test item was considered mutagenic if:
(a) a concentration-related increase in the number of revertants occurs and/or;
(b) a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
(a) the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
(b) the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Criteria for a Negative Response
A test article is considered non-mutagenic if it produces neither a concentration-related
increase in the number of revertants nor a reproducible biologically relevant positive response
at any of the concentration groups, with or without metabolic activation. - Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- 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
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: not mentioned
- Precipitation: at 5000 µg/plate, this did not affect scoring of mutant colonies
- Other confounding effects: Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected
in the main tests in some other sporadic cases. However, no dose-dependence was observed in
those cases and they were below the biologically relevant threshold value. The numbers of
revertant colonies were within the historical control range in each case, so they were considered
to be within the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed
in the main tests at some non-cytotoxic concentrations. However, no background inhibition was
recorded and the mean numbers of revertant colonies were in the historical control range in all
cases, thus they were considered as biological variability of the test system.
RANGE-FINDING/SCREENING STUDIES: In the Preliminary Concentration Range Finding Test, the plate incorporation method was used.
It was performed using Salmonella typhimurium TA98 and Salmonella
typhimurium TA100 tester strains in the presence and absence of metabolic activation system with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate. The following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
The results indicated a slight precipitate was detected on the plates in the preliminary experiment in both TA98 and TA100 bacterial strains with and without metabolic activation at the concentration levels of 5000 and 2500 µg/plate.
No inhibitory or toxic effects of the test item was observed in the preliminary experiment in either TA98 and TA100 both examined bacterial strains with and without metabolic activation.
COMPARISON WITH HISTORICAL CONTROL DATA: yes, the mean solvent control counts fell within the normal historical range.
ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity up to 5000 µg/plate in all strains - Remarks on result:
- other: The results are presented in Any other information on results incl. tables below.
- Conclusions:
- The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and showed no mutagenic activity in the absence or presence of metabolic activation on the growth of the bacterial strains under the test conditions used in this study.
- Executive summary:
1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay with histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Plate Incorporation Method), an Assay 1 (Plate Incorporation Method), an Assay 2 (Pre-Incubation Method) and Assay 3 (Pre-Incubation Method).
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1 and Assay 2 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate. Based on the observed cytotoxicity in Assay 2, the examined concentrations in the Assay 3 were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.
The test item 1,2,4-BENZENETRICARBOXYLIC ACID, MIXED DECYL AND OCTYL TRIESTERS had no mutagenic activity in the presence or absence of a metabolic activation system on the growth of the bacterial strains under the test conditions used in this study.
Referenceopen allclose all
| Table 1a: Toxicity test in the absence of S9 mix (3h exposure) | ||||||
| Concentration µg/mL) |
Cloning efficiency | Relative Survival (%) | ||||
| 0 (Solvent control) | 0.98 | 100 | ||||
| 9.77 | 0.95 | 92 | ||||
| 19.5 | 1.18 | 100 | ||||
| 39.1 | 0.98 | 88 | ||||
| 78.1 | 1.16 | 101 | ||||
| 156 | 1.18 | 106 | ||||
| 313 | 1.23 | 104 | ||||
| 625 | 0.91 | 73 | ||||
| 1250 | 1.05 | 94 | ||||
| 2500 | 1.06 | 88 | ||||
| Table 1b: Toxicity test in the absence of S9 mix (24h exposure) | ||||||
| Concentration (µg/mL) |
Cloning efficiency | Relative Survival (%) | ||||
| 0 (Solvent control) | 1.14 | 100 | ||||
| 9.77 | 0.87 | 96 | ||||
| 19.5 | 0.72 | 81 | ||||
| 39.1 | 0.75 | 78 | ||||
| 78.1 | 0.74 | 77 | ||||
| 156 | 0.84 | 79 | ||||
| 313 | 0.76 | 60 | ||||
| 625 | 1.00 | 82 | ||||
| 1250 | 0.96 | 98 | ||||
| 2500 | 0.76 | 95 | ||||
| Table 1c: Toxicity test in the presence of S9 mix (3h exposure) | ||||||
| Concentration (µg/mL) |
Cloning efficiency | Relative Survival (%) | ||||
| 0 (Solvent control) | 1.18 | 100 | ||||
| 9.77 | 1.18 | 97 | ||||
| 19.5 | 1.03 | 99 | ||||
| 39.1 | 1.18 | 113 | ||||
| 78.1 | 1.18 | 103 | ||||
| 156 | 1.00 | 101 | ||||
| 313 | 1.20 | 85 | ||||
| 625 | 1.18 | 90 | ||||
| 1250 | 1.20 | 92 | ||||
| 2500 | 1.14 | 103 | ||||
| Table 2a: Mutation test in the absence of S9 Mix (3h exposure) (Summary of means of data) 1. Experiment | ||||||
| Concentration (µg/mL) |
Relative Total Growth (%) | Mutant fraction (x 10-6) |
Induced mutant fraction (x 10-6) | |||
| 0 (Solvent control) | 100 | 59.5 | N/A | |||
| 156* | 92 | 62.8 | 3.36 | |||
| 313* | 89 | 65.8 | 6.29 | |||
| 625* | 75 | 60.7 | 1.19 | |||
| 1250* | 83 | 57.5 | - | |||
| 2500* | 83 | 54.3 | - | |||
| MMS 10.0 (Positive Control) | 63 | 351.0 | 291.5** | |||
| N/A: not applicable | ||||||
| * precipitation ** Induced mutant frequency > global evaluation factor (GEF = 126 x 10E-6) | ||||||
| Induced mutant fraction (IMF): Mutant fraction of treatment minus mutant fraction of solvent control | ||||||
| - : IMF <= 0 | ||||||
| Table 2b: Mutation test in the presence of S9 Mix (3h exposure) (Summary of means of data) 1. Experiment | ||||||
| Concentration (µg/mL) |
Relative Total Growth (%) | Mutant fraction (x 10-6) |
Induced mutant fraction (x 10-6) | |||
| 0 (Solvent control) | 100 | 52.2 | N/A | |||
| 156* | 123 | 48.0 | - | |||
| 313* | 94 | 64.5 | 12.30 | |||
| 625* | 90 | 71.9 | 19.79 | |||
| 1250* | 96 | 67.1 | 14.89 | |||
| 2500* | 99 | 63.5 | 11.36 | |||
| B(a)P 2.00 (Positive control) | 55 | 580.5 | 528.4** | |||
| N/A: not applicable | ||||||
| * precipitation ** Induced mutant frequency > global evaluation factor (GEF = 126 x 10 E-6) | ||||||
| Induced mutant fraction (IMF): Mutant fraction of treatment minus mutant fraction of vehicle group | ||||||
| - : IMF <= 0 | ||||||
| Table 2c: Mutation test in the absence of S9 Mix (24h exposure) (Summary of means of data) 2. Experiment | ||||||
| Concentration (µg/mL) |
Relative total growth (%) | Mutant fraction (x 10-6) |
Induced mutant fraction (x 10-6) | |||
| 0 (Solvent control) | 100 | 77.2 | N/A | |||
| 156* | 86 | 60.1 | - | |||
| 313* | 82 | 73.8 | - | |||
| 625* | 87 | 60.1 | - | |||
| 1250* | 89 | 74.4 | - | |||
| 2500* | 102 | 78.8 | 1.22 | |||
| MMS 5.00 (Positive control) | 85 | 734.8 | 657.6** | |||
| N/A: not applicable | ||||||
| * precipitation ** Induced mutation frequency > global evaluation factor (GEF = 126 x 10 E-6) | ||||||
| Induced mutant fraction (IMF): Mutant fraction of treatment minus mutantt fraction of vehicle group | ||||||
| - : IMF <= 0 | ||||||
| Table 2b: Mutation test in the presence of S9 Mix (4h exposure) (Summary of means of data) 2. Experiment | ||||||
| Concentration (µg/mL) |
Relative total growth (%) | Mutant fraction (x 10-6) |
Induced mutant fraction (x 10-6) | |||
| 0 (Solvent control) | 100 | 56.3 | N/A | |||
| 875* | 81 | 62.6 | 6.27 | |||
| 1138* | 136 | 96.2** | 39.87** | |||
| 1479* | 90 | 54.9 | - | |||
| 1923* | 79 | 70.2 | 13.89 | |||
| 2500* | 81 | 60.8 | 4.56 | |||
| B(a)P 2.00 (Positive control) | 33 | 658.6 | 602.4*** | |||
| N/A: not applicable | ||||||
| * precipitation ** statistically significant at p<0.05 *** Induced mutant frequency > global evaluation factor (GEF = 126 x 10 E-6) | ||||||
| Induced mutant fraction (IMF): Mutant fraction of treatment minus mutantt fraction of vehicle group | ||||||
| - : IMF <= 0 | ||||||
Table #1: Summary of data obtained in chromosomal aberration test #1 (3 hours treatment time, 24 hours sampling time)
| Presence of S9 metabolism | Absence of S9 metabolism | ||||
| Treatment | Dose [µg/mL] | % cells with chromosomal aberrations excl. gaps | relative MI[%] | % cells with chromosomal aberrations excl. gaps | relative MI[%] |
| Negative control | - | 0.0 | 95 | 0.0 | 99 |
| Solvent control | 1 % Ethanol | 0.0 | 100 | 0.0 | 100 |
| Test substance | 313 | 0.0 (neg.) | 98 | 0.5 (neg.) | 95 |
| Test substance | 625 | 0.0 (neg.) | 97 | 0.5 (neg.) | 98 |
| Test substance | 1250 | 0.5 (neg.) | 103 | 0.5 (neg.) | 98 |
| Mitomycin-C | 0.50 | not tested | 12.5 (pos.***) | 65 | |
| Cyclophosphamide | 18.0 | 23.5 (pos.***) | 53 | n.d. | n.d. |
relative MI = Mitotic Index relative to solvent controls
neg. = negative aberration frequency
pos.*** = positive aberration frequency, statistically significant (p<0.001)
Table #2: Summary of data obtained in chromosmal aberration test #2 (24 hours treatment time, 24 hours sampling time)
| Presence of S9 metabolism | Absence of S9 metabolism | ||||
| Treatment | Dose [µg/mL] | % cells with chromosomal aberrations excl. gaps | relative MI[%] | % cells with chromosomal aberrations excl. gaps | relative MI[%] |
| Negative control | - | not tested | 0.0 | 132 | |
| Solvent control | 1 % Ethanol | not tested | 0.0 | 100 | |
| Test substance | 625 | not tested | 0.0 (neg.) | 66 | |
| Test substance | 1250 | not tested | 0.0 (neg.) | 66 | |
| Test substance | 2500 | not tested | 0.0 (neg.) | 74 | |
| Mitomycin-C | 0.30 | not tested | 15.0 (pos.***) | 40 | |
relative MI = Mitotic Index relative to solvent controls
neg. = negative aberration frequency
pos.*** = positive aberration frequency, statistically significant (p<0.001)
Table 2: Preliminary Concentrations Range Finding Test: Results
Concentration (µg/plate) |
Mean values of revertants/Mutation factor (MF) |
Salmonella typhimurium tester strains |
|||
TA98 |
TA100 |
||||
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
17.3 |
18.3 |
102.7 |
116.0 |
MF |
0.98 |
1.02 |
0.94 |
1.01 |
|
DMSO control |
Mean |
16.3 |
19.3 |
-- |
107.3 |
MF |
0.92 |
1.07 |
-- |
0.94 |
|
Distilled water control |
Mean |
-- |
-- |
110.7 |
-- |
MF |
-- |
-- |
1.02 |
-- |
|
DMF control |
Mean |
17.7 |
18.0 |
108.7 |
114.7 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
112.3* |
19.7* |
100.7* |
100.7* |
MF |
0.70 |
1.09 |
0.93 |
0.88 |
|
2500 |
Mean |
16.3* |
19.7* |
103.0* |
102.3 |
MF |
0.92 |
1.09 |
0.95 |
0.89 |
|
1000 |
Mean |
16.0 |
22.7 |
96.3 |
98.0 |
MF |
0.91 |
1.26 |
0.89 |
0.85 |
|
316 |
Mean |
18.3 |
20.7 |
96.7 |
98.3 |
MF |
1.04 |
1.15 |
0.89 |
0.86 |
|
100 |
Mean |
17.0 |
21.7 |
96.0 |
99.0 |
MF |
0.96 |
1.20 |
0.99 |
0.86 |
|
31.6 |
Mean |
17.7 |
20.0 |
97.3 |
100.7 |
MF |
1.00 |
1.11 |
0.90 |
0.88 |
|
10 |
Mean |
16.3 |
17.7 |
98.3 |
102.0 |
MF |
0.92 |
0.98 |
0.90 |
0.89 |
|
NDP (4µg) |
Mean |
405.3 |
-- |
-- |
-- |
MF |
24.82 |
-- |
-- |
-- |
|
2AA (2µg) |
Mean |
-- |
2413.3 |
-- |
2457.3 |
MF |
-- |
124.83 |
-- |
22.89 |
|
SAZ (2µg) |
Mean |
-- |
-- |
1105.3 |
-- |
MF |
-- |
-- |
9.99 |
-- |
|
Note:
*: Slightly precipitate
NPD: 4-nitro-1, 2-phenylene-diamine
2AA: 2-aminoanthracene
SAZ: Sodium Azide
Table 3: Summary table of Assay 1
Concentration (µg/plate) |
Mean values of revertants/Mutation factor (MF) |
Salmonella typhimurium tester strains |
Escherichia coli |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
WP2 uvrA |
|||||||
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
||
Untreated control |
Mean |
15.0 |
17.0 |
88.0 |
92.7 |
13.7 |
13.3 |
16.7 |
16.7 |
57.7 |
59.0 |
MF |
1.00 |
0.96 |
1.03 |
1.00 |
1.00 |
0.98 |
1.00 |
1.00 |
0.98 |
1.03 |
|
DMSO control |
Mean |
16.0 |
17.0 |
-- |
93.7 |
-- |
14.0 |
17.3 |
17.0 |
-- |
59.0 |
MF |
1.07 |
0.96 |
-- |
1.01 |
-- |
1.02 |
1.04 |
1.02 |
-- |
1.03 |
|
Distilled water control |
Mean |
-- |
-- |
88.7 |
-- |
12.7 |
-- |
-- |
-- |
56.3 |
-- |
MF |
-- |
-- |
1.04 |
-- |
0.93 |
-- |
-- |
-- |
0.95 |
-- |
|
DMF control |
Mean |
15.0 |
17.7 |
85.7 |
93.0 |
13.7 |
13.7 |
16.7 |
16.7 |
59.0 |
57.3 |
MF |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
|
5000 |
Mean |
16.0* |
17.3* |
84.0* |
88.7* |
14.0* |
14.0* |
14.3* |
16.3* |
56.3* |
59.3* |
MF |
1.07 |
0.98 |
0.98 |
0.95 |
1.02 |
1.02 |
0.86 |
0.98 |
0.95 |
1.03 |
|
1581 |
Mean |
15.7 |
17.7 |
83.0 |
91.3 |
14.0 |
13.0 |
15.0 |
16.7 |
58.7 |
59.7 |
MF |
1.04 |
1.00 |
0.97 |
0.98 |
1.02 |
0.95 |
0.90 |
1.00 |
0.99 |
1.04 |
|
500 |
Mean |
15.3 |
17.0 |
84.3 |
92.7 |
13.7 |
13.3 |
15.0 |
16.3 |
58.3 |
58.3 |
MF |
1.02 |
0.96 |
0.98 |
1.00 |
1.00 |
0.98 |
0.90 |
0.98 |
0.99 |
1.02 |
|
158.1 |
Mean |
16.7 |
16.7 |
87.0 |
94.3 |
14.7 |
13.3 |
15.3 |
16.7 |
58.0 |
59.0 |
MF |
1.11 |
0.94 |
1.02 |
1.01 |
1.07 |
0.98 |
0.92 |
1.00 |
0.98 |
1.03 |
|
50 |
Mean |
17.0 |
17.7 |
82.3 |
91.3 |
14.0 |
12.7 |
14.3 |
15.7 |
58.0 |
57.0 |
MF |
1.13 |
1.00 |
0.96 |
0.98 |
1.02 |
0.93 |
0.86 |
0.94 |
0.98 |
0.99 |
|
15.81 |
Mean |
16.0 |
17.0 |
84.7 |
95.7 |
13.7 |
14.3 |
15.0 |
16.0 |
59.7 |
58.0 |
MF |
1.07 |
0.96 |
0.99 |
1.03 |
1.00 |
1.05 |
0.90 |
0.96 |
1.01 |
1.01 |
|
NDP (4µg) |
Mean |
453.3 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
MF |
28.33 |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
|
2AA (2µg) |
Mean |
-- |
2385.3 |
-- |
2448.0 |
-- |
207.7 |
-- |
205.0 |
-- |
-- |
MF |
-- |
140.31 |
-- |
26.14 |
-- |
14.83 |
-- |
12.06 |
-- |
-- |
|
2AA (50µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
249.0 |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
4.22 |
|
SAZ (2µg) |
Mean |
-- |
-- |
1170.7 |
-- |
1142.7 |
-- |
-- |
-- |
-- |
-- |
MF |
-- |
-- |
13.20 |
-- |
90.21 |
-- |
-- |
-- |
-- |
-- |
|
9AA (2µg) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
438.7 |
-- |
-- |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
25.31 |
-- |
-- |
-- |
|
MSS (2µL) |
Mean |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
976.0 |
-- |
MF |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
-- |
17.33 |
-- |
|
Note:
*: Slightly precipitate
2AA: 2-aminoanthracene
NPD: 4-nitro-1, 2-phenylene-diamine
SAZ: Sodium Azide
9AA: 9-aminoacridine
MMS: Methyl methanesulfonate
Table 4: Summary results of Assay 2 and Assay 3
| Concentrations (µg/plate) | Mean values of revertants / Mutation factor (MF) | Salmonella typhimurium tester strains | Escherichia coli | ||||||||
| TA98 | TA100 | TA1535 | TA1537 | WP2 uvrA | |||||||
| -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | -S9 | +S9 | ||
| Untreated control | Mean | 15.7 | 14.3 | 85.3 | 87.3 | 10.3 | 12.3 | 12.3 | 11.3 | 56.3 | 57.0 |
| MF | 0.98 | 0.96 | 1.12 | 1.05 | 1.07 | 1.00 | 1.06 | 1.06 | 1.01 | 1.00 | |
| DMSO control | Mean | 14.3 | 15.0 | -- | 85.7 | -- | 11.7 | 11.7 | 10.7 | -- | 56.3 |
| MF | 0.90 | 1.00 | -- | 1.03 | -- | 0.95 | 1.00 | 1.00 | -- | 0.99 | |
| Distilled water control | Mean | -- | -- | 78.3 | -- | 11.7 | -- | -- | -- | 56.0 | -- |
| MF | -- | -- | 1.03 | -- | 1.21 | -- | -- | -- | 1.01 | -- | |
| DMF control | Mean | 16.0 | 15.0 | 76.0 | 83.0 | 9.7 | 12.3 | 11.7 | 10.7 | 55.7 | 57.0 |
| MF | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | |
| 5000 | Mean | 15.7*# | 16.3* | 72.7* | 81.0* | 14.3*# | 11.7* | 13.0*# | 10.0* | 56.7* | 56.7* |
| MF | 0.98 | 1.09 | 0.96 | 0.98 | 1.48 | 0.95 | 1.11 | 0.94 | 1.02 | 0.99 | |
| 1581 | Mean | 15.0# | 16.3 | 75.3 | 84.3 | 11.0# | 12.3 | 13.3# | 9.3 | 56.0 | 56.7 |
| MF | 0.94 | 1.09 | 0.99 | 1.02 | 1.14 | 1.00 | 1.14 | 0.88 | 1.01 | 0.99 | |
| 500 | Mean | 14.3# | 16.3 | 73.7 | 81.7 | 11.0## | 12.3 | 13.7# | 9.0 | 55.7 | 57.3 |
| MF | 0.90 | 1.09 | 0.97 | 0.98 | 1.14 | 1.00 | 1.17 | 0.84 | 1.00 | 1.01 | |
| 158.1 | Mean | 16.3# | 15.7 | 74.0 | 83.3 | 11.3 | 12.3 | 12.7# | 10.3 | 56.3 | 56.0 |
| MF | 1.02 | 1.04 | 0.97 | 1.00 | 1.17 | 1.00 | 1.09 | 0.97 | 1.01 | 0.98 | |
| 50 | Mean | 15.0## | 15.3 | 75.0 | 83.7 | 11.7 | 12.0 | 13.3## | 10.7 | 54.7 | 57.3 |
| MF | 0.94 | 1.02 | 0.99 | 1.01 | 1.21 | 0.97 | 1.14 | 1.00 | 0.98 | 1.01 | |
| 15.81 | Mean | 15.3 | 14.7 | 74.7 | 75.3 | 10.7 | 11.7 | 13.0 | 9.3 | 57.7 | 57.0 |
| MF | 0.96 | 0.98 | 0.98 | 0.91 | 1.10 | 0.95 | 1.11 | 0.88 | 1.04 | 1.00 | |
| 5 | Mean | 16.7 | -- | -- | -- | 11.0 | -- | 13.0 | -- | -- | -- |
| MF | 1.04 | -- | -- | -- | 1.14 | -- | 1.11 | -- | -- | -- | |
| 1.581 | Mean | 17.7 | -- | -- | -- | 10.7 | -- | 12.7 | -- | -- | -- |
| MF | 1.10 | -- | -- | -- | 1.10 | -- | 1.09 | -- | -- | -- | |
| 0.5 | Mean | 15.3 | -- | -- | -- | 11.3 | -- | 13.3 | -- | -- | -- |
| MF | 0.96 | -- | -- | -- | 1.17 | -- | 1.14 | -- | -- | -- | |
| 0.1581 | Mean | 15.7 | -- | -- | -- | 12.0 | -- | 13.3 | -- | -- | -- |
| MF | 0.98 | -- | -- | -- | 1.24 | -- | 1.14 | -- | -- | -- | |
| NPD (4µg) | Mean | 412.0 | -- | -- | -- | -- | -- | -- | -- | -- | -- |
| MF | 28.74 | -- | -- | -- | -- | -- | -- | -- | -- | -- | |
| 2AA (2µg) | Mean | -- | 2458.7 | -- | 2478.7 | -- | 233.3 | -- | 212.0 | -- | -- |
| MF | -- | 163.91 | -- | 28.93 | -- | 20.00 | -- | 19.88 | -- | -- | |
| 2AA (50µg) | Mean | -- | -- | -- | -- | -- | -- | -- | -- | -- | 243.0 |
| MF | -- | -- | -- | -- | -- | -- | -- | -- | -- | 4.31 | |
| SAZ (2µg) | Mean | -- | -- | 1062.7 | -- | 1052.0 | -- | -- | -- | -- | -- |
| MF | -- | -- | 13.57 | -- | 90.17 | -- | -- | -- | -- | -- | |
| 9AA (50µg) | Mean | -- | -- | -- | -- | -- | -- | 430.7 | -- | -- | -- |
| MF | -- | -- | -- | -- | -- | -- | 36.91 | -- | -- | -- | |
| MMS (2µL) | Mean | -- | -- | -- | -- | -- | -- | -- | -- | 1022.7 | -- |
| MF | -- | -- | -- | -- | -- | -- | -- | -- | 18.26 | -- | |
Note:
*: Slightly precipitate
#: Reduced background lawn
##: Slightly reduced background lawn
Table 5. Historical Control data (Period of 2015 -2020)
Untreated control data |
||||||||||
|
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
20.9 |
98.3 |
13.2 |
8.8 |
40.6 |
25.6 |
104.6 |
12.3 |
9.9 |
44.1 |
St. dev. |
4.4 |
12.7 |
3.5 |
3.2 |
9.4 |
5.8 |
13.2 |
3.2 |
3.6 |
9.4 |
Range |
11-50 |
67-152 |
1-33 |
2-26 |
14-77 |
13-54 |
67-152 |
3-39 |
1-29 |
16-89 |
n |
1296 |
1297 |
1302 |
1314 |
1302 |
1311 |
1314 |
1314 |
1323 |
1299 |
DMSO control data |
||||||||||
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
20.5 |
95.4 |
13.0 |
8.4 |
39.6 |
24.9 |
101.0 |
11.8 |
9.4 |
43.0 |
St. dev. |
4.2 |
12.1 |
3.5 |
3.1 |
9.7 |
5.4 |
13.5 |
3.1 |
3.5 |
9.5 |
Range |
7-41 |
60-145 |
3-34 |
1-27 |
12-75 |
11-50 |
53-165 |
2-33 |
1-29 |
9-76 |
n |
1428 |
1419 |
1425 |
1449 |
1425 |
1443 |
1443 |
1449 |
1455 |
1431 |
Distilled water control data |
||||||||||
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
21.5 |
97.5 |
13.2 |
9.3 |
41.5 |
25.9 |
103.1 |
12.1 |
10.4 |
44.6 |
St. dev. |
4.4 |
12.5 |
3.3 |
3.4 |
9.1 |
5.6 |
13.6 |
3.1 |
3.7 |
9.0 |
Range |
13-37 |
58-150 |
2-32 |
3-20 |
17-72 |
15-45 |
59-164 |
3-34 |
3-24 |
13-76 |
n |
288 |
1332 |
1332 |
303 |
1341 |
291 |
1314 |
1326 |
300 |
1320 |
DMF control data |
||||||||||
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
19.6 |
93.5 |
12.9 |
9.2 |
42.0 |
23.8 |
96.2 |
11.9 |
10.5 |
43.3 |
St. dev. |
3.9 |
13.3 |
3.1 |
3.4 |
10.8 |
5.5 |
13.2 |
3.0 |
3.6 |
10.9 |
Range |
11-33 |
57-121 |
6-24 |
2-18 |
16-70 |
11-37 |
68-143 |
3-21 |
3-19 |
18-72 |
n |
114 |
114 |
114 |
117 |
111 |
114 |
114 |
114 |
114 |
111 |
Acetone control data |
||||||||||
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
20.9 |
97.1 |
12.9 |
8.2 |
40.1 |
25.5 |
102.2 |
11.5 |
9.1 |
44.1 |
St. dev. |
4.3 |
10.0 |
3.6 |
2.8 |
8.5 |
5.6 |
11.3 |
3.0 |
3.2 |
8.7 |
Range |
11-35 |
63-126 |
6-32 |
2-17 |
21-63 |
16-44 |
66-132 |
4-19 |
1-19 |
20-70 |
n |
219 |
222 |
222 |
225 |
219 |
219 |
222 |
225 |
225 |
219 |
Positive reference control data |
||||||||||
without metabolic activation (-S9 Mix) |
with metabolic activation (+S9 Mix) |
|||||||||
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
TA98 |
TA100 |
TA1535 |
TA1537 |
E. coli |
|
Mean |
395.9 |
1130.9 |
1138.8 |
413.3 |
1020.6 |
2391.6 |
2408.0 |
219.0 |
214.0 |
239.5 |
St. dev. |
99.4 |
82.5 |
102.0 |
34.0 |
114.4 |
146.2 |
115.7 |
32.1 |
22.3 |
35.6 |
Range |
182-2336 |
536-1480 |
568-2004 |
208-629 |
488-2496 |
312-2736 |
1116-3104 |
101-418 |
147-424 |
127-384 |
n |
1296 |
1296 |
1302 |
1314 |
1305 |
1311 |
1317 |
1317 |
1323 |
1299 |
Note: n: number of cases
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In Vitro Genetic Mutation in Bacterial Cells
In a key OECD 471 bacterial reverse mutation test (Orosz, I; Klimisch score 1) 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters was tested in DMSO for potential mutagenic activity using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
A preliminary compatibility test, a preliminary concentration range finding test (Plate Incorporation Method) and three main mutagenticity assays (Assay 1 -Plate Incorporation Method; Assay 2 -Pre-Incubation Method and Assay 3 -Pre-Incubation Method) were conducted in the study.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the preliminary experiment, the examined test concentrations in the Assay 1 and Assay 2 were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate. Based on the observed cytotoxicity in Assay 2, the examined concentrations in the Assay 3 were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.
Under the conditions of the test method 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters did not inducte a two-fold increase in revertant numbers with any tester strain either in the absence or presences of metabolic activation (+/-S9) and is therefore considered non-mutagenic in this assay.
In Vitro Mammalian Cell Chromosome Aberration Assay
In a key Guideline OECD 473 in vitro chromosome aberration assay (Ciliutti, P. Klimisch Score =1), the potential of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters was assayed for the ability to cause chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation.
Two independent experiments for chromosomal damage were performed. In the first experiment, the cells were treated 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 24 hours corresponding to approx. 1.5 cell cycle was used. As negative results were obtained, a second experiment was performed using the same harvest time (24 hours). A continuous treatment until harvest was used.
Both for the first and second experiments, dose levels of 2500, 1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 µg/mL were used in the absence or presence of S9 metabolism. Each experiment included appropriate negative and positive controls. Two cell cultures were prepared at each test point. For both experiments, the dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotocity of the tets item treatments (as determined by the reduction in mitotic index.) Where no cytotoxicity occured the highest treatment level was selected as the maximum dose level for scoring.
The following dose levels were selected for scoring (100 metaphase spreads were scored for chromosomal aberrations from each culture, 200 for each experimental point):
Assay #1: 1250, 625 and 313 µg/mL with and without S9 metabolism (3 hours treatment time, 24 hours harvest time)
Assay #2: 2500, 1250 and 625 µg/mL without S9 metabolism (24 hours treatment and harvest time)
Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations, including or excluding gaps, was observed at any dose level of any sampling time.
It was concluded that 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester did not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.
In Vitro Genetic Mutation in Mammalian Cells
In a key in vitro mammalian cell gene mutation assay (Salvador, M.; Klimisch Score = 1) 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester was examined for mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.
A solubility trial indicated that the maximum practicable concentration of the test item in the final test medium was 2500 µg/mL using ethanol as the solvent. On the basis of this result a preliminary cytotoxicity assay was performed. Both in the absence and presence of S9 metabolic activation, the test item was assayed at a maximum dose level of 2500 µg/mL and at a wide range of lower dose levels: 1250, 625, 313, 78.1, 39.1, 19.5 and 9.77 µg/mL. No relevant toxicity was observed at any dose level at any sampling time, in the absence and presence of S9 metabolism.
Based on the toxicity results obtained in the preliminary assay, two independent assays for mutation to 5 -trifluorothymidine resistance were performed using the following dose levels and treatment times:
Assay No. 1: 156, 313, 626, 1250 and 2500 µg/mL, 3 hours treatment with and without metabolic activation
Assay No. 2: 156, 313, 625, 1250 and 2500 µg/mL, 24 hours treatment without metabolic activation; resp. 875, 1138, 1479, 1923 and 2500 µg/mL, 3 hours treatment with metabolic activation.
No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism.
It was concluded that 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Justification for classification or non-classification
1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters showed no evidence of mutagenic potential in OECD Guideline 471 Ames bacterial test with strains of Salmonella typhimurium and Escherichia coli, with and without metabolic activation (+/-S9). The test material did not induce any chromosomal damage in cultured human lymphocytes, following in vitro treatment in the absence and presence of S9 metabolic activation (OECD 476) and was not mutagenic at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation (OECD 476).
Overall, according to the CLP Regulation (EC) No 1272/2008, 1, 2,4 -Benzenetricarboxylic acid, mixed decyl and octyl triesters is not classified as a mutagen.
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