Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 700-342-7 | CAS number: 1163775-81-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
1, 2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters showed no evidence of mutagenic potential in an OECD Guideline 471 AMES bacterial reverse mutation asssay involving five strains of Salmonella typhimurium, in the absence and presence of metabolic activation(±S9).
In an OECD 473 chromosomal aberration assay, 1, 2,4 -Benzenetricarboxylic acid, mixed dodecyl 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, dodecyl 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 read across study, 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 bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21.7.2015 - 2.8.2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 2013
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- N/A
- Target gene:
- N/A
- Species / strain / cell type:
- S. typhimurium TA 97
- Remarks:
- a
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium TA 102
- Species / strain / cell type:
- S. typhimurium TA 1535
- Cytokinesis block (if used):
- N/A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver S9 prepared in-house was used as the metabolic activation system
- Test concentrations with justification for top dose:
- 5, 1.5, 0.5, 0.15, 0.05 and 0.15 µL/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone (supplier: fisher Scientific, HPLC-grade, stored at RT
- Justification for choice of solvent/vehicle: the test item is soluble in acetone. - Positive controls:
- yes
- Positive control substance:
- other: 0.5 mg/L Dexon (CAS-Nr. 140-56-7) used in the tester strains TA97a, TA98 and TA102 in the absence of S9 mix (S9-)
- Remarks:
- without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: 0.2 mg/l 2-aminofluorene used in the tester strains TA97a, TA98 and TA100 in the presence of S9 mix (S9+)
- Remarks:
- with metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: 0.5 mg/ml 1, 8-Dihydroxyanthraquinone (Dorbane)
- Remarks:
- with metabolic activation
- Positive controls:
- yes
- Positive control substance:
- other: 0.02 mg/ml 2-Aminoanthracene (2-AA) used in the tester strain TA1535 in S9+
- Remarks:
- with metabolic activation
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 100 µl/plate
- Remarks:
- in each tester strain
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation)
The first experiment was done with the plate incorporation method, the second experiment to verify that the test item was negative with the preincubation method.
DURATION
- Preincubation period: for a minimum 20 min firstly (one condition in the replicate assay)
NUMBER OF REPLICATIONS: 3 (test substance), 3 (positve controls), 3 (solvent control)
DETERMINATION OF CYTOTOXICITY
The numbers of the revertant colonies had no significant decrease and the signs of the background lawn in each tester strain had no obvious difference comparing with the solvent controls, so it was considered that the test item was non-cytotoxic to all the tester strains under the conditions of this test. other: the background lawn was inspected for signs of toxicity (no further details mentioned)
- Rationale for test conditions:
- not mentioned
- Evaluation criteria:
- - Validity criteria:
The test system that meets the conditions below is considered to be valid:
1) The density of bacteria in each tester strain culture should be in the range of 0.9~9×10exp9 colony forming units (CFU)/mL;
2) The mean number of revertant colonies in all untreated controls are within the range of background data in this lab, and microscopic examination of the background lawn reveals the presence of densely packed microcolonies which form a uniform;
3) The mean number of revertant colonies in all positive controls are within the range of background data in this lab. - Statistics:
- not mentioned
- Key result
- Species / strain:
- S. typhimurium TA 97
- Remarks:
- a
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- 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
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated 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:
- 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: none
RANGE-FINDING/SCREENING STUDIES:
The preliminary test for the Bacterial Reverse Mutation Test of 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters had been performed in this lab before. In the test, according to the solubility of the test item, acetone was used as solvent. The standard plate incorporation method was performed at five dose levels, including 5, 1, 0.2,0.04 and 0.008 μL/plate, in five tester strains of TA97a, TA98, TA100, TA102 and TA1535, only with metabolic activation system. The solvent controls (Acetone, 100 μL/plate) in each tester strain were performed at the same time. The dose volumes of each dose group and solvent control group were 0.1mL/plate, in duplicate.
The results showed that there was a few of small oil droplets on the surface of the GM agar at 5 µL/plate and micro small oil droplets at 1 µL/plate before the incubation. But there was no droplet of test item found at all dose levels after the incubation. Moreover, at all doses, the numbers of the revertant colonies had no significant decrease and the signs of the background lawn in each tester strain had no obvious difference comparing with the solvent controls, so it was considered that the test item was non-cytotoxic to all the tester strains under the conditions of this test.
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 - Conclusions:
- Under the conditions of this study, the results both in the first experiment and in the validation experiment were negative. Thus, the test item, 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters is considered to be non-mutagenic in the bacterial reverse mutation assay using Salmonella typhimurium tester strains.
- Executive summary:
The results of the viable count in the two experiments showed that the density of the cultures for each tester strain was within 0.9~9×10exp9 colony forming units (CFU)/ml and were considered acceptable.
Both in the first experiment and the validation experiment, the mean number of revertant colonies in the untreated controls and positive controls were within the range of background data in this lab and the number of the solvent control was not obvious decrease compared to the corresponding untreated controls. In addition, the signs of background lawn in the solvent controls had grown as densely packed microcolonies observed with microscope. So the sensitivity of the assay and the efficacy of the S9 mix were validated.
In the first experiment, both in the absence and presence of S9 mix, a few of small oil droplets were found on the surface of GM agar at 5 µl/plate dose level and micro small oil droplets were found at 1.5µl/plate dose level before the incubation, but no oil droplet was found on the GM agar in the plate at each dose level after the incubation. In the validation experiment the same result was obtained as in the first experiment.
The study was performed to evaluate the ability of 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters to induce reverse mutations in the genome of the Salmonella typhimurium tester strains in the presence and absence of the metabolic activation system, and the method according to OECD 471. Five histidine deficient (his-) mutant tester strains of Salmonella typhimurium including TA97a, TA98, TA100, TA102 and TA1535 were treated with1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters using the standard plate incorporation method and the preincubation method at six dose levels, in triplicate, with untreated controls, solvent controls and positive controls. and both in the presence and absence of the metabolic activation system (cofactor-supplemented post-mitochondrial fraction (S9)). Based on the results of the preliminary test (initial toxicity test) that had been performed in this lab before, six dose levels in the first experiment were selected including 5, 1.5, 0.5, 0.15, 0.05 and 0.015 µL/plate, and Acetone was used as solvent. Then the validation experiment was conducted using the same dose levels and solvent as the first experiment. In the first experiment, the sign of the background lawn at all dose levels in each tester strain were no obvious difference comparing with the solvent controls in the presence and absence of S9 mix. This indicates that the test item has no obvious cytotoxicity to the tester strains at all tested dose levels. In the validation experiment, the same result was obtained as in the first experiment.
In the first experiment, the number of revertant colonies at each dose level in all tester strains was two times less than (three times in TA1535) that of the solvent controls. In the validation experiment, the same result was obtained as in the first experiment.
Under the conditions of this study, the results both in the first experiment and in the validation experiment were negative. Thus, the test item, 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters is considered to be non-mutagenic in the bacterial reverse mutation assay using Salmonella typhimurium tester strains.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2015-05-18 - 2015-06-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- one slide per cell culture was made, however, more than 100 well-spread metaphase cells could be observed on each slide, therefore the deviation did not affect the quality or integrity of the study.
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- N/A
- Species / strain / cell type:
- Chinese hamster lung (CHL/IU)
- Details on mammalian cell type (if applicable):
- Chinese Hamster Lung (CHL) cell line, purchased from Cell bank of Shanghai Institute of Cell Biology, Chinese Academy of Science, which was used in this study. CHL cell is the sensitive cell to detect the chromosome aberration.
CHL cells were maintained at 36.2~37.6ºC in the humidified atmosphere of 3.67~4.55% CO2 and the temporary deviation to the conditions was accepted because of the opening of the incubator`s door.
The culture media for daily culture of the cells was modified Eagle’s medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (10% MEM medium). - Cytokinesis block (if used):
- N/A
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 prepared in-house was used as the metabolic activation system
The S9 was prepared from the livers of the rats induced with Aroclor1254: male Sprague Dawley rats are treated by a single intraperitoneal injection of Aroclor 1254 at the dose of 500 mg/kg five days prior to the S9 preparation. The livers of the rats are taken out under asepsis conditions, homogenized in a 0.15 mol/L KCl solution (1g liver: 3ml KCl solution) and centrifuged at 11000rpm for 10 min with AllegraTM 64R Auto Freeze Centrifuge. The supernatant of S9 was stored in liquid nitrogen not more than two years. Before being used, each batch of S9 was tested again for its sterility, its protein content (not higher than 40 mg/ml), and its capability to activate known mutagens in Ames test. The S9 used in this test was prepared on Apr. 10, 2014, and rechecked during Apr.10-12, 2015. The results of the test have indicated that each index of S9 was qualified, which met the test requirements.
10% S9 mixture (S9 mix): 10% S9 mix was prepared immediately before the test and was placed in the mixture of water and ice during the test. The remaining S9 mix was discarded. - Test concentrations with justification for top dose:
- Range finding: doses: 5, 1.25, 0.31, 0.08 µg/mL
Dose levels selected for metaphase analysis in the main tests:
First main test: doses: 5, 2, 0.8, 0.32 and 0.13 µg/mL, means concentration: 1000, 400, 160, 64, 25.6 µm/l (with and without S9 mix, 3.5 hours treatment time, 24 hours harvest time)
Second main test: doses 5, 2, 0.8, 0.32 and 0.13 µg/mL, means concentration: 1000, 400, 160, 64, 25.6 µm/L (without S9 mix, 24 hours treatment time, 24 hours harvest time) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was found to be soluble in acetone. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- acetone (Fisher Scentific)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- The concurrent untreated controls, solvent controls, and positive controls were included in each treatment condition.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
400μl of the test item was weighed and put it into the asepsis pipe, then acetone was added to 1ml scale line that had been constant volume and mixed thoroughly to no oil droplet being observed by naked eyes as the working solutions which concentration was 400μl/ml (the dose was 2μL/mL). Under asepsis conditions, the working solution of other concentrations were prepared one by one via dilution in 2.5 times intervals. The highest dose (5μL/mL) did not be prepared and add the test item into the cell cultures directly. The test item and prepared working solutions were put in the asepsis pipe labeled with study No. and concentration.
As being prepared, the test item was weighed accurately and mixed with the solvent thoroughly. In addition, the test item was applied to the test system within 1h after being formulated. It is assumed that the formulation was prepared exactly, homogeneously and stable as being used, so no analysis was carried out to determine the homogeneity, concentration and stability of the test item formulation. This was an exception with regard to SEPA GLP and was reflected in the GLP compliance statement.
Cell preparation:
2×10exp5 CHL cells were seeded in the cell culture plate and cultured overnight. Duplicate cultures were included in each dose level and control. Before being treated, the cells were washed with HBSS solution, and the mixtures used during cell treatment were prepared.
Cell treatment:
The test item solution or control solution was added to the corresponding mixture.
The solution was added into each respective cell culture, and the cultures were observed by naked eyes for test item precipitation at the beginning and end of the treatment. After the treatment, the cells treated for 3.5h were washed several times with HBSS solution and cultured in 10% MEM medium, and then put into the incubator to be cultured until 24h (before cell harvest). The cells treated 24h were not washed until 24h.
Cell harvest and cytotoxicity:
As 24 hours after the beginning of cell treatment (before cell harvest), all cells were treated with Colchicine for near 2h at the final concentration of 1μg/mL, and harvested. As harvest, the cells were washed with HBSS solution and treated with 150μL of trypsin solution to break off. Then 1mL of the medium containing FBS were added to the cultures and mixed repeatedly. After all of these procedures, 100μL of the cells of each dose and control were counted using a haemocytometer under inverted Microscope and the relative increase in cell count (RICC) were calculated to evaluate the cytotoxicity as the formula below:
RICC（%） = Increase in number of cells of treated culture / Increase in number of cells of untreated control culture x 100
Increase in number of cells = number of cells as harvest - number of as cell preparation
Chromosome preparation:
The cells exposed the cells of the concentration with a RICC above 40% and two lower concentrations, and all controls were chose for chromosome preparation.
The chromosome was prepared by the procedures shown below:
Harvested cells were transferred into tubes to remove the media by centrifugation at 1000rpm for 5 minutes.
Remove the supernate, add 1mL of 75mM KCL hypotonic solution preheated at 37ºC and mix. Then incubate samples at 37ºC for 30 minutes, and centrifuge at 1000rpm for 5 minutes.
Remove the supernate, add 1mL freshly prepared Carnoy's mixture (Methanol: glacial acetic acid=3:1) slowly, mix gently with a pipette, and fix for 2 minutes.
Add 0.5ml Carnoy's mixture, mix gently with a pipette, incubate for 20 minutes, then centrifuge the samples at 1000rpm for 5 minutes.
Remove the supernate, add 1mL Carnoy's mixture, incubate at 2~8ºC for more than 12hours.
Centrifuge the samples at 1000rpm for 5 minutes, remove the supernate, and add some Carnoy's mixture to get the cell suspension.
Drop the cell suspension to clean slide, make at least 1 slides per cell culture, dry them naturally at room temperature.
Stain the dry samples with Giemsa solution (Giemsa dye liquor: Phosphate buffer=1:10(v:v)) for 30 minutes, wash them with water and dry them naturally. Then all slides were independently coded.
Microscopic analysis:
The metaphase cells with clear backgrounds and metaphase chromosome moderate contraction of mitotic cells were searched under a low power lens. Then the well-spread metaphase cells were observed and analyzed under the oil lens. 200 well-spread metaphase cells nearly equally divided among the duplicates were scored per dose and control in each treatment condition. The different types of structural chromosome aberrations were scored respectively. Gaps were scored separately. Polyploidy and endoreduplication were scored at the same time.
Slides in the experiment conditions of exposure for 3.5 hours with and without metabolic activation were scored firstly. Because both results were negative, the slides of 24h-treatment without metabolic activation were scored.
- Rationale for test conditions:
- N/A
- Evaluation criteria:
- Under three treatment conditions, the results of the untreated controls and solvent control showed that the percentage of cells with structural chromosomal aberration was less than 5%; at the same time, the results of the positive controls showed that the percentage of cells with structural chromosomal aberration was higher than 15%, and statistically significant differences (P<0.01) were found in the cells of all positive controls as compared with the concurrent untreated controls. So the sensitivity of the assay and the efficacy of the S9 mix were validated.
positive result:
Providing that one of the acceptability criteria below are fulfilled in any of the experimental conditions examined, the result will be considered to be clearly positive:
1) at least one of the test concentrations exhibits a statistically significant increase (P＜0.05) in the number of cells with chromosome aberrations compared with the concurrent untreated control, and the increase is concentration-related；
2) at least one of the test concentrations exhibits a statistically significant increase (P＜0.05) in the number of cells with chromosome aberrations compared with the concurrent untreated control, and the increase is reproducible .
negative result:
The test item is considered to be negative if none of the above criteria is met.
equivocal result:
In case the response is neither clearly negative nor clearly positive, additional cells should be scored and the biological relevance of the result should be considered. If the result is not yet clear, a repeat experiment will be performed using modified experimental conditions. If the result in the repeat experiment is still equivocal, the test item will be conclude to be equivocal. - Statistics:
- For the duplicate cultures in each dose and control, the mean value and the standard deviation of the number of harvest cells were calculated, then the relative increase in cell count (RICC) were calculated. The percentage of cells with structural chromosomal aberration(s) excluding gaps were calculated, in addition, the number of cells with each type of structural chromosomal aberration for all doses and controls are shown separately. The number of cells with polyploidy and/or endore duplication were shown. The percentage of cells with structural chromosomal aberration of each dose and positive control were evaluated by X2 Exact test to compare with the concurrent solvent control.
- Key result
- Species / strain:
- Chinese hamster lung (CHL/IU)
- 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:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Evaporation from medium: not applicable
- Water solubility: no data
- Other confounding effects: none
RANGE-FINDING/SCREENING STUDIES: Based on the cytotoxicity results, cells at the dose with a RICC above 50% and two lower doses, and all controls in each treatment condition were selected for chromosome preparation. Then microscopic analysis was conducted for chromosomal aberrations.
COMPARISON WITH HISTORICAL CONTROL DATA: in the range of historical control data
ADDITIONAL INFORMATION ON CYTOTOXICITY: none - Conclusions:
- The results of this test under three treatment conditions were negative, thus, the test item, 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl trimesters, is not considered to cause structural chromosomal aberrations in cultured mammalian cells under the conditions of this study.
- Executive summary:
The study was performed to evaluate 1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters for its ability to cause structural chromosomal aberrations in cultured mammalian cells (Chinese Hamster Lung (CHL) cell), in three treatment conditions including exposure for 3.5 hours(h) with and without metabolic activation, and exposure for 24 hours without metabolic activation according to OECD 473.
In this test, CHL cell line was treated with 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters, in the above three treatment conditions at five exposure doses, in duplicate, with untreated controls, solvent controls (Acetone) and positive controls in each condition at the same time.
Based on the results of the preliminary test (initial toxicity test) that had been performed in this lab before, Acetone was used as solvent. Cells were exposed for 3.5 hours with and without metabolic activation (S9 mix) at the doses of 5, 2, 0.8, 0.32 and 0.13 μl/ml, respectively, and were exposed for 24 hours without metabolic activation (S9 mix) at the doses of 5, 2, 0.8, 0.32 and 0.13 μL/mL. After the exposure, all cells were harvested and counted. Then the relative increase in cell count (RICC) was calculated to evaluate the cytotoxicity of the test item. Based on the cytotoxicity results, cells at the dose with a RICC above 50% and two lower doses, and all controls in each treatment condition were selected for chromosome preparation. Then microscopic analysis was conducted for chromosomal aberrations.
Under three treatment conditions, the results of the untreated controls and solvent control showed that the percentage of cells with structural chromosomal aberration was less than 5%; at the same time, the results of the positive controls showed that the percentage of cells with structural chromosomal aberration was higher than 15%, and statistically significant differences (P<0.01) were found in the cells of all positive controls as compared with the concurrent untreated controls. So the sensitivity of the assay and the efficacy of the S9 mix were validated.
In this test, test item precipitation all kinds of oil droplets were found in the cell cultures at each exposure dose of test item at the beginning and end of three treatment conditions, but that did not affect the cell count.
The results about cytotoxicity showed that the RICC of the cells exposed for 3.5 h in the presence of S9 mix at the doses of 0.13, 0.32, 0.8, 2 and 5 μL/mL were 86%, 84%, 88%, 79% and 81%; the RICC of the cells exposed for 3.5h in the absence of S9 mix at the doses of 0.13, 0.32, 0.8, 2 and 5 μL/mL were 91%, 77%, 67%, 67% and 70%; the RICC of the cells exposed for 24h in the absence of S9 mix at the doses of 0.13, 0.32, 0.8, 2 and 5 μl/ml were 83%, 85%, 84%, 75% and 67%.
The results of the microscopic analysis showed that (gaps were excluded) the percentage of cells with structural chromosomal aberrations exposed for 3.5h in the presence of S9 mix at the doses of 0.8, 2 and 5μL/mL were 0.0%, 0.5%, 1.0%；the percentage of cells with structural chromosomal aberrations exposed for 3.5h in the absence of S9 mix at the doses of 0.8, 2 and 5μl/ml were 2.0%, 3.5%, 2.0%; the percentage of cells with structural chromosomal aberrations exposed for 24h in the absence of S9 mix at the doses of 0.8, 2 and 5μl/ml were 0.0%, 1.0%, 3.0%.
The results of statistical analysis indicated no statistically significant difference (P>0.05) in the percentage of cells with structural chromosomal aberration was found in all test item treated cells under all treatment conditions as compared with the concurrent untreated control. Also, the number of the polypoidy and endoreduplication was not obvious increased. At the same time, statistically significant differences (P<0.01) were found in the cells of all positive controls as compared with the concurrent untreated control.
The results of this test in all treatment conditions were negative, thus, it is considered that the test item, 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters could not cause structural chromosomal aberrations in cultured mammalian cells under the conditions of this study.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- 2009
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: well documented study performed according to OECD Guideline and GLP
- Justification for type of information:
- Read across justification included in Section 13
1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters is registered under REACh Regulation (EC) No. 1907/2006. The following report summarizes all available and relevant data for the read-across strategy with the substance 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters .
On the basis of all evaluated data, the similarity of the read-across substances is justified on basis of the physico-chemical properties, toxicological, ecotoxicological profiles and supported by various QSAR methods. In most cases the One-to-one approach is used to fill data gaps. There is convincing evidence that both substances have an overall common category profile. Both substances are not classified in accordance with Directive 67/548/EEC and Regulation (EC) No. 1272/2008. - Reason / purpose for cross-reference:
- read-across source
- 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
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solvent giving the best solubility/dispersal characteristics - Key result
- 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:
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 structurally related substance 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 gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Study period:
- 1987
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study with acceptable restrictions (only three Salmonella typhimurium strains tested)
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- N/A
- Target gene:
- N/A
- Species / strain / cell type:
- S. typhimurium TA 97
- Additional strain / cell type characteristics:
- other: histidine auxotrophe
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- other: histidine auxotroph
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- other: histidine auxotroph
- Cytokinesis block (if used):
- N/A
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver post-mitochondrial fraction (S9) from Arochlor-1254 induced male Wistar rats
- Test concentrations with justification for top dose:
- 0, 8, 40, 200, 1000 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: not stated - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-nitrofluorene (50 µg/plate) for TA 98; sodium azide (2 µg/plate) for TA 100; 9-aminoacridine (50 µg/plate) for TA 97
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (5 µg/plate) with all strains
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation)
DURATION
- Preincubation period: not applicable
- Exposure duration: 2 days
NUMBER OF REPLICATIONS: 3 (test substance), 5 (controls)
DETERMINATION OF CYTOTOXICITY
- Method: other: the background lawn was inspected for signs of toxicity (no further details mentioned)
OTHER:
- Colony counting: Colonies were counted electronically using a 40-10 image analyser (Analytical Measuring Systems). - Evaluation criteria:
- - Validity criteria: a) negative control within the normal range, b) clear induced increase in revertant numbers by the positive control confirming discrimination between different strains and an active S9 preparation and c) no more than 5% loss of the plates due to contamination or unforeseen event
- Evaluation criteria: the test compound is considered mutagenic if a) the assay is valid and b) if it induces a two-fold and significant increase in revertant numbers, accompanied by dose response correlations - Statistics:
- Analysis of variance (the F-test) and regression analysis were performed
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- 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:
- not examined
- Positive controls validity:
- valid
- 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:
- 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: none
RANGE-FINDING/SCREENING STUDIES: An initial toxicity range-finding experiment was carried out in TA 100 only, using final concentrations at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and positive control. From these test results the highest test dose were chosen for the main mutation assay. There were no signs of toxicity up to 5000 µg/plate either in the absence and presence of S9.
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: strain/cell type: Salmonella typhimurium
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
The test item failed to induce a two-fold increase in revertants numbers with any tester strain either in the absence or presence of S-9, and was considered to be non-mutagenic in this assay. - Executive summary:
The structurally related substance 1,2,4-Benzenetricarboxylic acid, decyl octyl ester showed no mutagnic activity neither in the presence nor in the absence of metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2016
- Reliability:
- 2 (reliable with restrictions)
- 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
- Specific details on test material used for the study:
- N/A
- Target gene:
- N/A
- 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
- Cytokinesis block (if used):
- N/A
- 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 - Rationale for test conditions:
- N/A
- 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 structurally related substance 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester, was assayed for the ability to cause chromosoaml 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
- Specific details on test material used for the study:
- N/A
- Target gene:
- N/A
- 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 - Cytokinesis block (if used):
- N/A
- 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. - Statistics:
- N/A
- 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
Table1: Results of the first experiment in the absence of S9 mix:
| Dose level (µl/plate) | Plates | Number of revertant colonies (colonies/plate) | ||||
| TA97a | TA98 | TA100 | TA102 | TA1535 | ||
| 5 | 1 2 3 |
127A 123 120 |
19A 20 22 |
117A 116 119 |
235A 240 226 |
13A 17 13 |
Mean+SD Ratio |
123 +-4 0.97 |
20 +-2 1.00 |
117 +-2 0.91 |
234 +-7 0.98 |
14 +-2 1.17 |
|
| 1.5 | 1 2 3 |
129 114 129 |
22 22 18 |
108 115 111 |
221 218 238 |
12 17 11 |
| Mean+SD | 124 +-9 0.98 |
21 +-2 1.05 |
111 +-4 0.87 |
226 +-11 0.95 |
13 +-3 1.08 |
|
| 0.5 | 1 2 3 |
123 124 117 |
21 19 19 |
113 106 104 |
215 228 238 |
15 17 12 |
| Mean+SD | 121 +-4 0.95 |
20 +-1 1.00 |
108 +-5 0.84 |
227 +-12 0.95 |
15 +-3 1.25 |
|
| 0.15 | 1 2 3 |
123 114 121 |
22 23 22 |
112 111 126 |
251 244 245 |
13 16 16 |
Mean+SD Ratio |
119 +-5 0.94 |
22 +-1 1.10 |
116 +-8 0.91 |
247 +-4 1.04 |
15 +-2 1.25 |
|
| 0.05 | 1 2 3 |
120 125 120 |
21 21 17 |
125 126 135 |
226 231 239 |
12 14 14 |
Mean+SD Ratio |
122 +-3 0.96 |
20 +-2 1.00 |
129 +-6 1.01 |
232 +-7 0.97 |
13 +-1 1.08 |
|
| 0.015 | 1 2 3 |
117 127 116 |
23 21 26 |
131 124 137 |
242 243 250 |
14 15 12 |
Mean+SD Ratio |
120 +-6 0.94 |
23 +-3 1.15 |
131 +-7 1.02 |
245 +-4 1.03 |
14 +-2 1.17 |
|
Ratio=the mean number of revertant colonies in each group/ the mean number of revertant colonies in solvent.
A-There was no significant difference in the signs of the background lawn comparing with that of the solvent control.
Table 2: Results of the first experiment in the presence of S9 mix
| Dose level (µl/plate) | Plates | Number of revertant colonies (colonies/plate) | ||||
| TA97a | TA98 | TA100 | TA102 | TA1535 | ||
| 5 | 1 2 3 |
150A 158 158 |
26A 22 24 |
127A 134 128 |
284A 252 276 |
20A 16 21 |
Mean+SD Ratio |
155 +-5 1.05 |
24 +-2 1.00 |
130 +-4 0.96 |
271 +-17 0.98 |
19 +-3 1.19 |
|
| 1.5 | 1 2 3 |
141 159 146 |
22 29 26 |
141 131 148 |
301 288 263 |
15 17 17 |
| Mean+SD | 149 +-9 1.01 |
26 +-4 1.08 |
140 +-9 1.04 |
284 +-19 1.03 |
16 +-1 1.00 |
|
| 0.5 | 1 2 3 |
164 149 151 |
24 27 26 |
133 138 141 |
257 289 262 |
19 21 14 |
| Mean+SD | 155 +-8 1.05 |
26 +-4 1.08 |
137 +-4 1.01 |
269 +-17 0.97 |
18 +-4 1.13 |
|
| 0.15 | 1 2 3 |
166 165 164 |
24 26 21 |
126 138 145 |
292 257 271 |
22 20 17 |
Mean+SD Ratio |
165 +-1 1.12 |
24 +-3 1.00 |
136 +-10 1.01 |
273 +-18 0.99 |
20 +-3 1.25 |
|
| 0.05 | 1 2 3 |
149 160 168 |
26 29 31 |
140 165 147 |
277 264 291 |
16 18 13 |
Mean+SD Ratio |
159 +-10 1.08 |
29 +-3 1.21 |
151 +-13 1.12 |
277 +-14 1.00 |
16 +-3 1.09 |
|
| 0.015 | 1 2 3 |
159 164 153 |
27 29 25 |
131 147 140 |
291 291 276 |
19 21 16 |
Mean+SD Ratio |
159 +-6 1.08 |
27 +-2 1.13 |
139 +-8 1.03 |
286 +-9 1.04 |
19 +-3 1.19 |
|
Notes: Ratio = the mean number of revertant colonies in each group/ the mean number of revertant colonies in solvent.
A-There was no significant difference in the signs of the background lawn comparing with that of the solvent.
Table 3: Results of the validation experiment in the absence of S9 mix
| Dose level (µl/plate) | Plates | Number of revertant colonies (colonies/plate) | ||||
| TA97a | TA98 | TA100 | TA102 | TA1535 | ||
| 5 | 1 2 3 |
134A 123 130 |
22A 21 24 |
153A 136 149 |
259A 248 253 |
19A 21 16 |
Mean+SD Ratio |
129 +-6 1.01 |
22 +-2 1.10 |
146 +-9 1.11 |
253 +-6 0.99 |
19 +-3 1.36 |
|
| 1.5 | 1 2 3 |
129 129 127 |
25 22 19 |
132 137 155 |
245 256 248 |
13 20 16 |
| Mean+SD | 128 +-1 1.00 |
22 +-3 1.10 |
141 +-12 1.08 |
250 +-6 0.98 |
16 +-4 1.14 |
|
| 0.5 | 1 2 3 |
131 136 128 |
25 23 25 |
125 134 144 |
264 262 251 |
16 17 15 |
| Mean+SD | 132 +-4 1.03 |
24 +-1 1.20 |
134 +-10 1.02 |
259 +-7 1.02 |
16 +-1 1.14 |
|
| 0.15 | 1 2 3 |
123 129 130 |
22 21 19 |
130 135 134 |
270 251 282 |
14 13 16 |
Mean+SD Ratio |
127 +-4 0.99 |
21 +-2 1.05 |
133 +-3 1.02 |
268 +-16 1.05 |
14 +-2 1.00 |
|
| 0.05 | 1 2 3 |
127 128 141 |
18 23 19 |
130 135 131 |
239 237 259 |
18 16 15 |
Mean+SD Ratio |
132 +-8 1.03 |
20 +-3 1.00 |
132 +-3 1.01 |
245 +-12 0.96 |
16 +-2 1.14 |
|
| 0.015 | 1 2 3 |
125 131 130 |
22 23 16 |
115 118 130 |
255 250 255 |
19 16 16 |
Mean+SD Ratio |
129 +-3 1.01 |
20 +-4 1.00 |
121 +-8 0.92 |
253 +-3 0.99 |
17 +-2 1.21 |
|
Notes: Ratio = the mean number of revertant colonies in each group/ the mean number of revertant colonies in solvent.
A-There was no significant difference in the signs of the background lawn comparing with that of the solvent.
Table 4: Results of the validation experiment in the presence of S9 mix
| Dose level (µl/plate) | Plates | Number of revertant colonies (colonies/plate) | ||||
| TA97a | TA98 | TA100 | TA102 | TA1535 | ||
| 5 | 1 2 3 |
155A 134 153 |
28A 26 26 |
156A 160 152 |
271A 295 272 |
34A 15 23 |
Mean+SD Ratio |
147 +-12 1.09 |
27 +-1 1.04 |
156 +-4 1.11 |
279 +-14 1.03 |
27 +-6 1.50 |
|
| 1.5 | 1 2 3 |
160 150 142 |
25 20 27 |
163 171 144 |
292 282 281 |
23 26 19 |
| Mean+SD | 151+-9 1.12 |
24 +-4 0.92 |
159 +-14 1.14 |
285 +-6 1.05 |
23 +-4 1.28 |
|
| 0.5 | 1 2 3 |
152 161 154 |
28 22 22 |
158 154 157 |
285 281 272 |
21 24 17 |
| Mean+SD | 156 +-5 1.16 |
24 +-3 0.92 |
156 +-2 1.11 |
279 +-7 1.03 |
21 +-4 1.17 |
|
| 0.15 | 1 2 3 |
150 156 156 |
27 24 28 |
158 159 168 |
276 286 274 |
22 19 19 |
Mean+SD Ratio |
154 +-3 1.14 |
26 +-2 1.00 |
162 +-6 1.16 |
279 +-6 1.03 |
20 +-2 1.11 |
|
| 0.05 | 1 2 3 |
138 130 141 |
29 27 24 |
154 140 148 |
296 297 292 |
23 19 23 |
Mean+SD Ratio |
136 +-6 1.01 |
27 +-3 1.04 |
147+-7 1.05 |
295+-3 1.09 |
22+-2 1.22 |
|
| 0.015 | 1 2 3 |
129 139 137 |
22 26 26 |
131 147 146 |
289 293 267 |
24 26 23 |
Mean+SD Ratio |
135 +-5 1.00 |
25 +-2 0.96 |
141 +-9 1.01 |
283 +-14 1.04 |
24 +-2 1.33 |
|
Notes: Ratio = the mean number of revertant colonies in each group/ the mean number of revertant colonies in solvent.
A-There was no significant difference in the signs of the background lawn comparing with that of the solvent.
Table 1: Results of the microscopic analysis for 3.5 h with S9:
| Dose level/control (µg/mL) | Cells number scored | Chromosome -break |
Chromosome- exchange |
Chromatid- break |
Chromatid- exchange |
Cells number with structural chromosomal aberration(s) |
Polyploidy | Endoreduplication | Gap | Cells percentage with structural chromosomal aberration(s) (%) |
| Untreated control | 200 | 0 | 0 | 2 | 0 | 2 | 0 | 3 | 3 | 1.0 |
| Acetone | 200 | 0 | 0 | 3 | 0 | 3 | 0 | 3 | 3 | 1.5 |
| 0.8 | 200 | 0 | 0 | 0 | 0 | 0 | 0 | 3 | 3 | 0.0 |
| 2 | 200 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | 0.5 |
| 5 | 200 | 2 | 0 | 0 | 0 | 2 | 0 | 1 | 1 | 1.0 |
| CP | 200 | 9 | 32 | 15 | 16 | 48 | 0 | 6 | 6 | 24.0* |
Note: * statistically significant difference compared with solvent control (P<0.01).
Table 2: Results of the microscopic analysis for 3h without S9
| Dose level/control (µg/mL) | Cells number scored | Chromosome -break |
Chromosome- exchange |
Chromatid- break |
Chromatid- exchange |
Cells number with structural chromosomal aberration(s) |
Polyploidy | Endoreduplication | Gap | Cells percentage with structural chromosomal aberration(s) (%) |
| Untreated control | 200 | 2 | 0 | 0 | 0 | 2 | 0 | 0 | 1 | 1.0 |
| Acetone | 200 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 2 | 0.5 |
| 0.8 | 200 | 2 | 0 | 2 | 0 | 4 | 0 | 0 | 1 | 2.0 |
| 2 | 200 | 2 | 0 | 5 | 0 | 7 | 0 | 0 | 3 | 3.5 |
| 5 | 200 | 1 | 0 | 3 | 0 | 4 | 0 | 0 | 2 | 2.0 |
| EMS | 200 | 8 | 13 | 11 | 6 | 35 | 0 | 0 | 10 | 17.5* |
Note: * statistically significant difference compared with solvent control (P<0.01).
Table 3: Results of the microscopic analysis for 24h without S9
| Dose level/control (µg/mL) | Cells number scored | Chromosome -break |
Chromosome- exchange |
Chromatid- break |
Chromatid- exchange |
Cells number with structural chromosomal aberration(s) |
Polyploidy | Endoreduplication | Gap | Cells percentage with structural chromosomal aberration(s) (%) |
| Untreated control | 200 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 1 | 0.5 |
| Acetone | 200 | 0 | 0 | 1 | 0 | 1 | 1 | 0 | 1 | 0.5 |
| 0.8 | 200 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 2 | 0.0 |
| 2 | 200 | 0 | 0 | 2 | 0 | 2 | 3 | 0 | 2 | 1.0 |
| 5 | 200 | 1 | 0 | 5 | 0 | 6 | 1 | 0 | 2 | 3.0 |
| EMS | 200 | 15 | 5 | 21 | 0 | 38 | 0 | 0 | 9 | 19.0* |
Note: * statistically significant difference compared with solvent control (P<0.01).
| 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: Number of revertants per plate (mean of 3 (test substance) resp. 5 (control) plates)
|
[Strain TA 97] |
[Strain TA 98] |
[Strain TA 100] |
||||||
Conc. |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
- MA |
+ MA |
Cytotoxic |
0* |
85.4 |
157.4 |
no |
19.8 |
23.0 |
no |
90.2 |
126.6 |
no |
8 |
76.7 |
143.7 |
no |
15.7 |
22.7 |
no |
82.3 |
125.3 |
no |
40 |
83.0 |
137.7 |
no |
24.3 |
33.0 |
no |
97.0 |
140.0 |
no |
200 |
80.0 |
159.0 |
no |
22.3 |
29.7 |
no |
93.0 |
136.7 |
no |
1000 |
79.7 |
154.3 |
no |
19.0 |
28.7 |
no |
93.0 |
126.7 |
no |
5000 |
86.7** |
140.3** |
no |
15.7** |
32.7** |
no |
95.0** |
120.7** |
no |
Positive control |
484.6 |
520.6 |
no |
1224.2 |
940.8 |
no |
536.0 |
675.8 |
no |
*solvent control with DMSO ** precipitate observed
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)
Genetic toxicity in vivo
Description of key information
In an OECD 474 in vivo micronucleus assay, 1, 2, 4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters did not induce an increase of the incidence of micronucleated PCE in mice.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30th of July 2015 - 7th of September 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Specific details on test material used for the study:
- N/A
- Species:
- mouse
- Strain:
- ICR
- Details on species / strain selection:
- N/A
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Beijing Vital River Laboratory animal technology Co., Ltd.
- Initial age at start of acclimatisation: 49-62 days
- Age at start of treatment: 55-68 days
- Weight at study initiation: 29.95-34.61 g (males)
- Assigned to test groups randomly: yes
- Fasting period before study: not mentioned
- Housing: 1 animal per cage (plastic cages (L29.0×W18.0×H16.0cm) lied on shelf (L167.0×W70.0×H171.0cm) in the SPF grade barrier system (room D124). There were 7 cages per layer, and 5 layers per rack)
- Bedding: Corn cob bedding was supplied by Beijing Keao Xieli Feed Co., Ltd. (Batch No.: 15049811)
- Diet (e.g. ad libitum): ad libitum (SPF rodent growth and breeding feed bought from Beijing Keao Xieli Feed Co., Ltd (Product License No: SCXK (jing) 2012-0001, Batch No. was 15033113)
- Water (e.g. ad libitum): ad libitum (drinking water, purified by HT-R01000 purity system)
- Acclimation period: adequate duration, not specified
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8-24.4
- Humidity (%): 54 - 70
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: relatively non-toxic to the animals, result of solubility test
- Amount of vehicle: 0.2 mL/10 g bw
- Lot/batch no. (if required): MKBF8603V - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
4.000g/4.000g (the first administration/ the second administration) of the test item were weighed using the electronic balance and mixed with vehicle to the certain volume (40mL) as the concentration of 100mg/ml, then the suspension were continuously mixed round with homogenate machine for one minute at least. The test suspension of other concentrations were prepared by dilution by factor 2.
All test suspensions were prepared just before on the day of each administration. When prepared, the test item was weighed accurately and mixed with the vehicle thoroughly, in addition, the test item was applied to the test system within two hours after being formulated. It is assumed that the formulation was prepared exactly, homogeneously and was stable during the test. So no analysis was carried out to determine the homogeneity, concentration and stability of the test item formulation.
The order of administration was 0, 500mg/kg, 1000mg/kg, 2000mg/kg and CP (cyclophosphamide monohydrate = positive control) group. The accuracy of injector used was 0.02mL and the max volume was 1mL. Dosing formulations and vehicle were stirred on the magnetic stirrer at least 5 minutes prior to dosing and continuously stirred during dosing. At the first administration, academic dosing volume was calculated based on the body weight as grouping, and the corresponding volume based on the accuracy of injector was dosed to the animal. At the last administration, animals were weighed firstly, then academic volume was calculated and corresponding volume based on the accuracy of injector was dosed to the animal. All data during the treatment were recorded accurately. - Duration of treatment / exposure:
- N/A
- Frequency of treatment:
- twice with the test item with an interval approximately 24 hours
- Post exposure period:
- none
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 7 (6 (3 males and 3 females) in pre-experiment)
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): not mentioned
- Supplier: Sigma-Aldrich
- Batch no.: SLBG4216V
- Doses / concentrations: 50 mg/kg bw, single dose (2.5 mg/mL)
In this test, cyclophosphamide monohydrate (CP) was used as positive control item at the dose level of 50mg/kg (the concentration was 2.5mg/mL). 0.025g of CP was weighed and prepared in 10mL water as the positive control solution. The CP solution was prepared on the day of each treatment. - Tissues and cell types examined:
- N/A
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Prior to the start of this study, a preliminary test had been performed to determine the maximum tolerated dose (MTD) in this lab. The test item came from the same supplier (Batch No.: 04698/MA; Content:＞98%). Based on the result of the preliminary test, if the test item did not produce toxicity at 2000 mg/kg/body weight/day, the highest dose for an administration period should be 2000 mg/kg/body weight/day in this micronucleus test. However, if the test item does cause toxicity, the maximum tolerated dose (MTD) should be the highest dose administered in this micronucleus test.
In the preliminary test, the same species animals with the micronucleus test were used. Before administration, based on the result of solubility of the test item and designed dose, the test item was prepared to suspend in corn oil. Because corn oil was used as vehicle, the route of administration by gavage was used. In the preliminary test, two dose levels were set, include: 2000 and 1000 mg/kg/body weight/day. There were three males and three females in each group. The animals were dosed twice with an interval of 24 hours. The dose volume of 0.2ml/10g bw by gavage. After the second administration, all the animals continued to be observed one day. The results indicated that all animals had no obvious toxic symptoms or were death during the administration of the test item.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling of peripheral blood was carried out on all animals (7 per dose group)
DETAILS OF SLIDE PREPARATION:
All animals were sacrificed by cervical dislocation nearly 20 hours after the second administration. Bone marrow from sternum was harvested and mixed with a drop of fetal calf serum on a side of a slide. Then the slides were smeared. After air-dried, the slides were fixed in methanol for nearly 10 minutes, and stained with Giemsa’s solution for nearly 30 minutes, flushed and air dried.
METHOD OF ANALYSIS:
The slides were analysed by the manual method using microscopy. All slides were coded randomly so the manual scorer was unaware of the treatment condition. 2000 polychromatic erythrocytes (PCE) were scored per animal for the number of micronucleated polychromatic erythrocytes (MNPCE) and the incidence of micronuclei was shown in permillage. At the same time, the PCE in 200 red blood cells (RBC) were scored and the ratio between PCE and RBC was calculated for each animal.
Observed under immersion objective, both PCE and NCE (normochromatic erythrocytes) are anucleate cells. After Giemsa staining, PCE shows bice and NCE shows light jacinth.
Identification of micronuclei:
Circle is the main form, and oval, ring, hemicycle is found occasionally. Outline of micronuclei is clear and flat. The staining of Micronuclei should be same as the nucleus of an adjacent nucleated cell.
Usually, the upper limit is half of a diameter of an erythrocyte. PCE with two or more micronuclei is counted as one MNPCE. - Evaluation criteria:
- If the incidences of MNPCE in the treated groups show statistically significant increase (P<0.01) compared to the values of the NG and the increase is dose-related or very clear in one dose level, the results are deemed as positive.
The result is evaluated as negative if none of the above criteria is met.
Where small significant increase in the incidences of MNPCE in the treated groups occurs, additional cells may be scored and the biological relevance of the result will be considered. Results of this type will be reported as equivocal if the results maintain unclear. - Statistics:
- Before each administration and sampling, the mean and standard deviation of the body weight in each group was calculated, and plotted graphically respectively; the ratio between PCE and RBC of each animal was calculated, and the mean and standard deviation of the ratios in each group were calculated at the same time (the ratio in the treated groups should not be less than 20% of NG (control). The incidence of MNPCE of each animal was calculated, and the mean and standard deviation in each group were calculated at the same time, then the data were evaluated with two tailed t-test method in Excel to observe if there were statistical significant differences in the treated groups and CP group (positive control) as compared to the value of NG.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw and 1000 mg/kg bw
- Clinical signs of toxicity in test animals: none observed
- Evidence of cytotoxicity in tissue analyzed: no tissue analyzed
- Rationale for exposure: 2000 mg/kg was chosen as MTD
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically relevant increase of micronuclei was found, nor were any significant differences between the negative control groups, dose groups and the historical control data found.
- Ratio of PCE/NCE (for Micronucleus assay): No significant difference between the negative control groups, dose groups and the historical control data found.
- Appropriateness of dose levels and route: Both, MTD and oral administration, were appropriate to investigate the genotoxic potential of the test item. The positive control groups showed a significantly increased micronucleus frequency.
- Statistical evaluation: No statistically significant differences between the negative control groups and the dose group were found. - Conclusions:
- Under the conditions of this study, the results were negative, so the study suggested that 1, 2, 4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters did not induce an increase of the incidence of micronucleated PCE in mice.
- Executive summary:
This study was conducted to detect the possibility that 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters could induce the increase of the incidence of micronucleated polychromatic erythrocytes (PCE) in mice, in order to provide genotoxicity related data for the test item according to OECD 474. This study was conducted in ICR mice. All animals were SPF grade. According to the related information about the test item and the results of the preliminary test, in the micronucleus test, the animals were treated at 2000, 1000 and 500mg/kg. Negative control group (NG, corn oil) and positive control (Cyclophosphamide, CP, 50mg/kg) group were performed at the same time with the same method. All animals were randomly grouped based on body weight with 7 male animals in each group. Mice were administered the test item orally by gavage twice, with a 24 hours interval between doses. All animals were sacrificed by cervical dislocation nearly 20 hours after the last administration. Bone marrow from sternum was harvested. Bone marrow smear was prepared and analysed with microscope. The data were evaluated with t-test (two-tail) in Excel.
During the administration, no obvious decrease in the body weights of the mice was found in all designed dose groups as compared with the NG. No animal exhibited obvious toxic symptoms and death during the administration.
The frequencies of micronucleated PCE were 2.3±0.6‰ in the negative control group, 2.2±0.6‰ in the 500mg/kg group, 2.3±0.7‰ in the1000 mg/kg group, 1.8±0.9‰ in the 2000mg/kg group and25.9±3.8‰ in the CP group. The results of t-test indicated that statistically no significant difference (P>0.05) in the incidence of micronucleated PCE was found in the treated groups as compared with NG. At the same time, a statistically significant difference (P<0.01) in the incidence of micronucleated PCE was found in the CP group as compared with NG. Furthermore, the PCE/RBC ratios in all treated groups and positive control group were more than twenty percent of the ratio in NG.
Under the conditions of this study, the results were negative, so the study suggested that 1, 2, 4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters did not induce an increase of the incidence of micronucleated PCE in mice.
Reference
Table 1: Statistics results of microscopic analysis
| Group (mg/kg) | Animal number | Number of PCE | MNPCE/PCE (‰) Mean±SD |
P value | PCE/RBC Mean±SD |
| 0 | 7 | 14000 | 2.3±0.6 | - | 0.55±0.03 |
| 500 | 7 | 14000 | 2.2±0.6 | > 0.05 | 0.59#±0.07 |
| 1000 | 7 | 14000 | 2.3±0.7 | > 0.05 | 0.58#±0.04 |
| 2000 | 7 | 14000 | 1.8±0.9 | > 0.05 | 0.54#±0.08 |
| CP | 7 | 14000 | 25.9±3.8 | <0.01* | 0.47#±0.04 |
Note: * statistically significant difference compared to negative control (P<0.01);
# the ratio was more than 20 percent of the ratio in the negative control group.
During the test, the change of animal body weights was determined, there was no obvious decrease as compared with the concurrent NG during the administration.
The clinical observation results showed that all animals had no toxic symptom and death.
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 (Wei, 2015; Klimisch score 1) 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters was tested for potential mutagenic activity using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA97a, TA98, TA100, TA1535 and TA1537 using the standard plate incorporation method and the preincubation method at six dose levels, in triplicate, with untreated controls, solvent controls and positive controls. and both in the presence and absence of the metabolic activation system (cofactor-supplemented post-mitochondrial fraction (S9)). Based on the results of the preliminary test (initial toxicity test) that had been performed in this lab before, six dose levels in the first experiment were selected including 5, 1.5, 0.5, 0.15, 0.05 and 0.015 µL/plate, and acetone was used as solvent. The validation experiment was conducted using the same dose levels and solvent as the first experiment. In the first experiment, the sign of the background lawn at all dose levels in each tester strain were no obvious difference comparing with the solvent controls in the presence and absence of S9 mix. This indicates that the test item has no obvious cytotoxicity to the tester strains at all tested dose levels. In the validation experiment, the same result was obtained as in the first experiment. Under the conditions of this study, the results both in the first experiment and in the validation experiment were negative. Thus, the test item, 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters is considered to be non-mutagenic in the bacterial reverse mutation assay using Salmonella typhimurium tester strains.
In a key read across 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 (Wei, 2015); the potential of 1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters was assayed for its ability to cause structural chromosomal aberrations in cultured mammalian cells (Chinese Hamster Lung (CHL) cell), in three treatment conditions including exposure for 3.5 hours with and without metabolic activation, and exposure for 24 hours without metabolic activation.
Two independent experiments for chromosomal damage were performed. In the first experiment, the cells were treated for 3.5 hours with and without metabolic activation (S9 mix) at the doses of 5, 2, 0.8, 0.32 and 0.13 μl/ml, respectively. The second experiment the cells were exposed for 24 hours without metabolic activation (S9 mix) at the doses of 5, 2, 0.8, 0.32 and 0.13 μL/mL. After the exposure, all cells were harvested and counted. Then the relative increase in cell count (RICC) was calculated to evaluate the cytotoxicity of the test item. Based on the cytotoxicity results, cells at the dose with a RICC above 50% and two lower doses, and all controls in each treatment condition were selected for chromosome preparation. Then microscopic analysis was conducted for chromosomal aberrations. Each experiment included appropriate negative and positive controls.
The cytotoxicity results showed that the RICC of the cells exposed for 3.5 h in the presence of S9 mix at the doses of 0.13, 0.32, 0.8, 2 and 5 μL/mL were 86%, 84%, 88%, 79% and 81%; the RICC of the cells exposed for 3.5h in the absence of S9 mix at the doses of 0.13, 0.32, 0.8, 2 and 5 μL/mL were 91%, 77%, 67%, 67% and 70%; the RICC of the cells exposed for 24h in the absence of S9 mix at the doses of 0.13, 0.32, 0.8, 2 and 5 μl/ml were 83%, 85%, 84%, 75% and 67%.
There were no statistically significant difference (P>0.05) in the percentage of cells with structural chromosomal aberration in all test item treated cells under all treatment conditions as compared with the concurrent untreated control. Also, the number of the polypoidy and endoreduplication was not obvious increased.
The results of this test in all treatment conditions were negative, thus, it is considered that the test item, 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters could not cause structural chromosomal aberrations in cultured mammalian cells under the conditions of this study.
In a read-across 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 read across 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.
In vivo Mammalian Micronucleus assay (OECD 474)
In a key mouse micronucleus study; OECD 474 (Wei, 2015) 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters was examined to understand if it could induce an increased incidence of micronucleated polychromatic erythrocytes (PCE) in ICR mice. Groups of 7 male mice were treated on two occasions with 2000, 1000 and 500mg/kg test material via oral gavage, with a 24 hour interval between doses. Negative control group (corn oil) and positive control (Cyclophosphamide, 50mg/kg) group were also included in the study design. All animals were sacrificed by cervical dislocation nearly 20 hours after the last administration. Bone marrow from sternum was harvested. Bone marrow smear was prepared and analysed under a microscope. The data were evaluated with t-test (two-tail).
There was no obvious decrease in the body weights of the mice in any dose groups when compared with control and no animal exhibited any toxic symptoms or death during the administration.
The frequencies of micronucleated PCE were 2.3±0.6‰ in the negative control group, 2.2±0.6‰ in the 500mg/kg group, 2.3±0.7‰ in the1000 mg/kg group, 1.8±0.9‰ in the 2000mg/kg group and25.9±3.8‰ in the CP group. The results of t-test indicated that statistically no significant difference (P>0.05) in the incidence of micronucleated PCE when compared with control. A statistically significant difference (P<0.01) was observed in the incidence of micronucleated PCE in the positive control group. Furthermore, the PCE/RBC ratios in all treated groups and positive control group were more than twenty percent of the ratio in NG.
Under the conditions of this study, 1, 2, 4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters did not induce an increase of the incidence of micronucleated PCE in mice.
All available data clearly demonstrate the lack of genotoxic effects.
Short description of key information:
1,2,4 -benzenetricarboxylic acid, mixed dodecyl and octyl triesters did not cause genetic toxicity.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters showed no evidence of mutagenic potential in OECD Guideline 471 Ames bacterial test with strains of Salmonella typhimurium with and without metabolic activation (+/-S9). The test material did not cause structural chromosomal aberrations in cultured mammalian cells following in vitro treatment in the absence and presence of S9 metabolic activation (OECD 473) and in an in vivo mouse micronucleus study (OECD 474) did not induce an increase of the incidence of micronucleated PCE in mice
Supporting data from 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters also showed no evidence of mutagenic potential in an OECD Guideline 471 5 strain Ames bacterial test (Salmonella typhimurium and Escherichia coli) with and without metabolic activation (+/-S9); it 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). 1, 2,4 -Benzenetricarboxylic acid, mixed decyl and octyl triesters is not classified as a mutagen.
Overall, according to the CLP Regulation (EC) No 1272/2008, 1, 2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters is not classified as a mutagen.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
