Trinonyl benzene-1,2,4-tricarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Trinonyl benzene-1,2,4-tricarboxylate (L9TM) did not exert mutagenic activity in a reliable reverse bacterial mutation assay (plate incorporation and pre-incubation method) with and without metabolic activation. Also no gene mutations in mammlian cells were detected when trinonyl benzene-1,2,4-tricarboxylate (L9TM) was tested in a forward gene mutation assay with chinese hamster cells (V79 cells testing for mutation at the HPRT gene locus). The submission susbtance did not produce chromosomal aberrations in peripheral human blood lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 JUN 2016 to 17 JUN 2016

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:

adopted 21. July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted 31. May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium LT2 strains TA97a, TA98, TA100, TA102 and TA1535

Additional strain / cell type characteristics:

other: Genetic characteristics of each test bacteria strain such as amino acid requirement, UV sensitivity, membrane mutation and drug resistance were verified in advance and bacteria strains possessing the required characteristics were used.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

plate incorporation test (experiments 1 a and b): 0, 50, 150, 500, 1500, and 5000 µg/plate
pre-incubation (experiment 2): 0, 156, 313, 625, 1250, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item was sufficiently soluble in DMSO, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4- Nitro-1,2-phenylene diamine (CASRN 99-56-9) without S9 mix for strains TA97a, TA98, and TA102; 2-amino-anthracene (CASRN 613-13-8) with S9 mix for strains TA97a, TA100, TA102, and TA1535

Remarks:

4- Nitro-1,2-phenylene Diamine is not a standard positive control according to OECD guideline 471 but is recommended according to Ames et al. [D.M. Maron, B.N. Ames: “Revised methods...”, Mutation Research 113 (1983) 173- 215, Elsevier Biomedical Press].

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
experiment 1: plate incorporation
experiment 2: preincubation

DURATION
- Preincubation period: only experimt 1 - 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates per concentration tested

DETERMINATION OF CYTOTOXICITY
- Method: other: Performed in experiment 1 only, analogously to the titre control (see below) with the maximum dose of test item with and without S9 mix on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1°C.
- Titer control: The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.

Rationale for test conditions:

The test material was sufficiently soluble in DMSO. Testing was performed to the maximal test concentration mentioned in the testing guideline and no relevant cytotxicity was observed. Based on this and in concordance with the previsions from the testing guideline at least four other (i.e. a total of at least 5 analysable) test concentrations were chosen.

Evaluation criteria:

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

The colonies were counted visually and the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

Key result

Species / strain:

other: S. typhimurium LT2 strains TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Trinonyl benzene-1,2,4-tricarboxylate did not exert mutagenic activity in the reverse bacterial mutation assay (AMES Test according to OECD TG471 and GLP; plate incorporation and pre-incubation method) with and without metabolic activation.

Executive summary:

Reverse mutation testing of the test material, trinonyl benzene-1,2,4 -tricarboxylate, was conducted according to OECD testing guideline 471 and GLP using 5 strains of bacteria: Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535. The test material (dissolved in DMSO) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in two experiments (experiment 1: plate incorporation method; experiment 2: pre-incubation method).

In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate. In all three experiments, the test item showed no cytotoxicity towards all bacteria strains (no reduction of background lawn and number of revertants). The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 10EXP9 bacteria/mL. All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Thus indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Since all criteria for acceptability have been met, the study is considered valid.

The test item showed no increase in the number of revertants in all bacteria strains in both experiments. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102, and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 MAY 2017 to 19 SEP 2017

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)

Version / remarks:

adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosomal aberration test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy donors
- Sex, age and number of blood donors if applicable:
Experiment I without S9: 2 female donors, 24 years old
Experiment I with S9: 2 female donors, 24 years old
Experiment II: 3 male donors, 26 years old
- Whether whole blood or separated lymphocytes were used if applicable: human peripheral blood lymphocytes in whole blood culture

Additional strain / cell type characteristics:

other: no prior chromosomal damage identified

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I without S9: 0, 0.13, 1, and 2 mg/mL
Experiment I with S9: 0, 0.5, 1, and 2 mg/mL
Experiment II: 0, 0.5, 1, and 2 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item solubility and applicability in a cell based test system

Untreated negative controls:

yes

Remarks:

serum free medium RPMI 1640 or 0.9% NaCl

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: no
- Exposure duration:
Experiment I - 4 hours
Experiment II - 25 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid®

STAIN (for cytogenetic assays): 10% Giemsa

NUMBER OF REPLICATIONS: each concentration was tested in duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Each cell culture was harvested and processed separately for the preparation of chromosomes.
3 h before harvesting, Colcemid® was added to the cultures (final concentration: 0.1 μg/mL). The cells were spun down by gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were re-suspended in 12 mL hypotonic KCl solution. Then the cell suspension was allowed to stand at room temperature for 20 min. After removal of the hypotonic solution by centrifugation (10 min, 500 * g), the cell pellet was fixated with a mixture of methanol and glacial acetic acid (3:1). After fixation at 2-8°C, minimum 30 min, the cell suspension was spun down by gentle centrifugation (10 min, 500 * g), the supernatant was discarded and the cell pellet was re-suspended in fixative again. The washing procedures were repeated until the cell pellet was white.
The slides were prepared by dropping the cell suspension onto clean microscope slides.
The slides were then stained with a 10% solution of Giemsa. All slides were independently
coded before microscopic analysis.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 150 per replicate, thus in total 300 metaphases
(note for CPA (experiment I positive control with metabolic activation) only 250 metaphases were evaluated as there was severe chromosomal damage and the substance gave a clearly positive result

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (according to guideline appropriate for lymphocytes in primary cultures)

OTHER:
Evaluation was performed using Zeiss microscopes and the automatic slide scanning system “Metafer”, provided by MetaSystems, Altlussheim.

Rationale for test conditions:

Top concentration is the highest concentration to be tested according to guideline, if neither cytotoxicity nor solubility (or the substance definition) is the limiting factor. Thereof at least three lower doses were evaluated.

Evaluation criteria:

7.3.1 Acceptability
The assay is considered acceptable as it meets the following criteria:
 The concurrent solvent control is considered acceptable to be added to the historical laboratory control data.
 All experimental conditions are tested (short exposure with and without metabolic activation, extended exposure without metabolic activation) unless a positive result is achieved in any experiment.
 In each experiment, an adequate number of cells is analysable both in the controls and in at least 3 test item concentrations.
 The criteria for cell proliferation and for the selection of concentrations are fulfilled.
 The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations compared with the concurrent solvent control. The obtained values should be compatible with the historical laboratory control
data.
7.3.2 Classification
A test item is classified as non-mutagenic if:
 Neither a statistically significant nor a concentration-related increase of the number of cells with structural chromosomal aberrations in the evaluated test concentrations is observed.
 The obtained results lie within the range of the historical laboratory control data for solvent controls.

A test item is classified as mutagenic if in any of the experimental conditions:
 The number of induced structural chromosome aberrations (excluding gaps) lies above the range of the historical laboratory control data for solvent controls, at least at one concentration of the test item.
 A dose-related increase in the number of cells with structural chromosome aberrations is observed.
 A statistically significant increase of structural chromosomal aberrations is found, at least at one concentration of the test item.

Statistics:

The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher’s exact test or chi-square-test at the 5% level (p < 0.05).

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Remarks:

value was very low compared to historical controls

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human periperal blood

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: none

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see tables included as illustration
- Negative (solvent/vehicle) historical control data:see tables included as illustration

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index

Remarks on result:

other: experiment I - 4 hours exposure

Conclusions:

Trinonyl benzene-1,2,4-tricarboxylate was tested in an in vitro mammalian chromosomal aberration test according to OECD TG 473 and GLP. While experiment I - 4 h exposure either without metabolic activation system - yielded equivocal results, the same exposure time with metabolic activation yielded negative results. Also experiment II - 25 h exposure without metabolic activation system - showed no chromosomal aberrations. As the results of the 1st experiment without metabolic activation were not verified in the 2nd experiment the final assessment of the test item is negative.

Executive summary:

Chromosmal aberrations of the test material, trinonyl benzene-1,2,4 -tricarboxylate, were assessed according to OECD testing guideline 473 and GLP using human peripheral blood lymphocytes in whole blood culture. The test material (dissolved in DMSO) was tested up to concentrations of 2 mg/mL in the absence and presence of S9 -mix in two independent experiments (experiment I: 4 hours exposure either with or without metabolic activation system; experiment II: 25 hours exposure without metabolic activation system).

In both independent experiments, neither haemolysis nor precipitation occurred and no relevant cytotoxicity was detected.

All positive control compounds caused distinct, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system. The solvent controls showed numbers of aberrant cells within the range of the historical data and met the acceptability criteria.

In experiment I without metabolic activation, all evaluated concentrations (2, 1 and 0.13 mg/mL) showed statistically significant increases in the proportion of aberrant cells compared with the concurrent solvent control. However, the values for DMSO itself were extremely low and therefore, the statistical significance can actually be achieved with moderate numbers of aberrant cells.

But also the mean value for the concentration 2 mg/mL lay above the historical data for the solvent control DMSO and also above the 99.7% control limit. Regarding the individual cultures, primarily one out of two replicates was responsible for the increased proportion of aberrant metaphases: the value for culture 1 was still inside the 99.7% control range of the historical data for DMSO, only the value for culture 2 lay completely out of range. The mean values for 1 mg/mL and 0.13 mg/mL lay above the historical data, but inside the 95.5% (1 mg/mL) or 99.7% control limit (0.13 mg/mL).

Yet, no clear concentration relation became evident.

In conclusion, two out of three criteria for a clear positive result are fulfilled, but there was no clear concentration relation observed, therefore, the outcome of this experimental part has by definition to be assessed as equivocal.

Neither a statistically significant nor a biologically relevant increase of structural chromosomal aberrations was observed at the evaluated concentrations in experiment I with metabolic activation. The mean values lay inside the historical data (0.5 mg/mL), inside the 95.5% control range (2 mg/mL) or inside the 99.7% control range (1 mg/mL).

Thus yielding a clear negative test result.

Neither a statistically significant nor a biologically relevant increase of structural chromosomal aberrations was observed at the evaluated concentrations in experiment II (extended exposure, only without metabolic activation). Thus yielding a clear negative test result.

Since the suspicious results of the 1st experiment without metabolic activation were not verified in the 2nd experiment without metabolic activation and extended exposure time, the outcome of the test is finally assessed as negative.

No observations were made which might cause doubts concerning the validity of the study outcome.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in human lymphocytes in vitro at the chosen test item concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 MAY 2016 to 21 AUG 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 29 July 2016

Deviations:

yes

Remarks:

In experiment Ia some cell culture plates were incubated at 0% CO2 instead of 5% CO2 for one day.Plates were further incubated with 5 % CO2 and evaluated. As there was a drastic reduction in number of colonies, the experiment was terminated and repeated.

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 30 May 2008

Deviations:

yes

Remarks:

for details see above

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro Mammalian cell Gene Mutation Test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: cells called V79-4 purchased by CLS (Eppelheim, Germany)
- Doubling time: 12-16 h
- Number of passages if applicable: 5-10 days of cultivation prior to treatment
- Modal number of chromosomes: 22


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMEM complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 from male rats treated with Aroclor 1254

Test concentrations with justification for top dose:

In the pre-test no cytotoxicity was observed in the treatments with and without metabolic activation up to the limit concentration of the test guideline.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: DMSO was chosen as vehicle, because the test item was sufficiently soluble in it and this solvent does not have any effects on the viability of the cells or the number of spontaneous mutant colonies in the tested concentration.

Untreated negative controls:

yes

Remarks:

culture media only

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration:
Experiment I - 4 hours
Experiment II - 24 hours
- Expression time (cells in growth medium): total expression time at least 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 6- Thiosguanine (6-TG)

NUMBER OF REPLICATIONS: duplicate cultures per concentration level


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; :
- Any supplementary information relevant to cytotoxicity: In the pre-test, 7 concentrations of the test item were used and tested with and without metabolic activation (0, 0.03, 0.06, 0.13, 0.25, 0.5, 1, and 2 mg/mL). The exposure time was 4 hours.
500 cells were exposed to each concentration of the test item for 4 hours with and without metabolic activation (duplicate cultures per concentration level). Following treatment, the cells were washed two times with PBS Dulbecco (2.5 % HS). After an expression period of 7 d the cells were stained with methylene blue. Afterwards the colonies were counted and the absolute and the relative cloning efficiency were calculated

Rationale for test conditions:

Top concentration is the highest concentration to be tested according to guideline, if neither cytotoxicity nor solubility (or the substance definition) is the limiting factor. A total of at least four concentrations have to be evaluated (in addition to controls).

Evaluation criteria:

Acceptability
The gene mutation assay is considered acceptable if it meets the following criteria:
1. the mutant frequency found in the solvent controls falls within the laboratory historical control data range
2. the positive control substances must produce a significant increase (p < 0.05) in mutant frequency and lies in the range of the historical data.
3. two experimental conditions (+S9 and -S9) are tested.
4. adequate number of cells (spontaneous MF is 5 - 20 * 10-6) and concentrations (minimum of 4) are analysable.
5. appropriate concentration selection is given

Classification
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
 at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
 the increase of the mutant frequency is concentration-related
 any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
 none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
 there is no concentration-related increase of the mutant frequency
 all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
However, in a case by case evaluation this decision depends on the level of the corresponding
solvent control data (e.g. low spontaneous mutation rate within the laboratories historical control data range, a concentration-related increase of the mutations within this range has to be discussed; check variability of the mutation rates of solvent controls).

Statistics:

The number of mutant cells in each treatment group was compared with the solvent control. Statistical significance was tested with a fourfold chi-squared test calculating a test variable which is approximately chi-squared distributed (with one degree of freedom).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Experiment I: 4 hours exposure

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Experiment II: 24 hours exposure

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES:
During treatment small oily droplets were visible at the concentrations 2 mg/mL - 0.25 mg/mL. No cytotoxicity was observed in the treatments with and without metabolic activation.

Conclusions:

Under the experimental conditions of this study Oxsoft L9TM (Trinonyl benzene-1,2,4-tricarboxylate) did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
Therefore, the test item Oxsoft L9TM is considered to be “non-mutagenic under the conditions of the HPRT assay”.

Executive summary:

In this experimental study the potential of the test item Oxsoft L9TM to induce mutations at the HPRT locus on chromosome X Chinese Hamster V79 cells was investigated. The test was conducted according to OECD testing guideline 476 and GLP.

The assay was performed in two independent valid experiments, using two parallel cultures each (replicates). In experiment I, 6 concentrations of the test item were used and tested with and without metabolic activation. The exposure time was 4 hours The following nominal concentrations of the test item were investigated in experiments I with and without metabolic activation: 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.13 mg/mL and 0.06 mg/mL

In experiment II, again 6 concentrations of the test item were used and tested without metabolic activation. The exposure time was 24 hours. The following nominal concentrations of the test item were investigated in experiment II without metabolic activation: 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.13 mg/mL and 0.06 mg/mL

In all experiments very small oily drops were visible at the concentrations 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL during treatment.

Ethylmethane sulfonate (experiment I: 300 μg/mL, experiment II: 150 μg/mL) and 7,12 - dimethylbenz(a)anthracene (1.5 μg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies but without reduction of the viability.

In both experiments of this study (with and without S9 mix) the mutant colony numbers/10EXP6 cells of the solvent controls were in the range of the historical data.

No cytotoxic effects were observed in none of the test item concentrations in experiment I and II. Therefore, all concentrations were analysable for mutagenicity.

In experiment I without metabolic activation, some of the test item concentrations induced a statistically significant increase in mutant fequency in comparison to the solvent control, however all those values are still within the range of the spontaneous mutant frequency (5 - 20 * 10EXP-6) or of the historical data of the solvent control. Therefore, no biologically relevant and reproducible increase in mutant colony numbers/10EXP6 cells was observed in the main experiments up to the maximum concentration. All mutant frequencies remained well within the historical range of solvent control. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency but did not reveal a linear regression. Therefore, all criteria for a clearly negative result are fulfilled.

In conclusion, it can be stated that under the experimental conditions reported, the test material did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

No single study was selected since, all available data (covering the data requirements) were negative.

Additional information

Reverse mutation testing of the test material, trinonyl benzene-1,2,4 -tricarboxylate, was conducted according to OECD testing guideline 471 and GLP using 5 strains of bacteria: Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535. The test material (dissolved in DMSO) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in two experiments (experiment 1: plate incorporation method; experiment 2: pre-incubation method).

All negative and nearly all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate. The test item showed no increase in the number of revertants in all bacteria strains in both experiments. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102, and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study (LAUS GmbH, 2016, AMES, RL1).

Chromosmal aberrations of the test material, trinonyl benzene-1,2,4 -tricarboxylate, were assessed according to OECD testing guideline 473 and GLP using human peripheral blood lymphocytes in whole blood culture. The test material (dissolved in DMSO) was tested up to concentrations of 2 mg/mL in the absence and presence of S9 -mix in two independent experiments (experiment I: 4 hours exposure either with or without metabolic activation system; experiment II: 25 hours exposure without metabolic activation system).

In experiment I without metabolic activation, all evaluated concentrations (2, 1 and 0.13 mg/mL) showed statistically significant increases in the proportion of aberrant cells compared with the concurrent solvent control. However, the values for DMSO itself were extremely low and therefore, the statistical significance can actually be achieved with moderate numbers of aberrant cells.

But also the mean value for the concentration 2 mg/mL lay above the historical data for the solvent control DMSO and also above the 99.7% control limit. Regarding the individual cultures, primarily one out of two replicates was responsible for the increased proportion of aberrant metaphases: the value for culture 1 was still inside the 99.7% control range of the historical data for DMSO, only the value for culture 2 lay completely out of range. The mean values for 1 mg/mL and 0.13 mg/mL lay above the historical data, but inside the 95.5% (1 mg/mL) or 99.7% control limit (0.13 mg/mL).

Yet, no clear concentration relation became evident.

In conclusion, two out of three criteria for a clear positive result are fulfilled, but there was no clear concentration relation observed, therefore, the outcome of this experimental part has by definition to be assessed as equivocal.

Neither a statistically significant nor a biologically relevant increase of structural chromosomal aberrations was observed at the evaluated concentrations in experiment I with metabolic activation. The mean values lay inside the historical data (0.5 mg/mL), inside the 95.5% control range (2 mg/mL) or inside the 99.7% control range (1 mg/mL).

Thus yielding a clear negative test result.

Neither a statistically significant nor a biologically relevant increase of structural chromosomal aberrations was observed at the evaluated concentrations in experiment II (extended exposure, only without metabolic activation). Thus yielding a clear negative test result.

Since the suspicious results of the 1st experiment without metabolic activation were not verified in the 2nd experiment without metabolic activation and extended exposure time, the outcome of the test is finally assessed as negative.

In both independent experiments, neither haemolysis nor precipitation occurred and no relevant cytotoxicity was detected, and all positive control compounds caused distinct, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system. The solvent controls showed numbers of aberrant cells within the range of the historical data and met the acceptability criteria.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in human lymphocytes in vitro at the chosen test item concentrations (LAUS GmbH, 2017, CA, RL1).

To adress gene mutation in mammalian cells in an experimental study the potential of test item trinonyl benzene-1,2,4 -tricarboxylate

to induce mutations at the HPRT locus on chromosome X Chinese Hamster V79 cells was investigated. The test was conducted according to OECD testing guideline 476 and GLP.

The assay was performed in two independent valid experiments, using two parallel cultures each (replicates). In experiment I, 6 concentrations of the test item were used and tested with and without metabolic activation. The exposure time was 4 hours The following nominal concentrations of the test item were investigated in experiments I with and without metabolic activation: 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.13 mg/mL and 0.06 mg/mL

In experiment II, again 6 concentrations of the test item were used and tested without metabolic activation. The exposure time was 24 hours. The following nominal concentrations of the test item were investigated in experiment II without metabolic activation: 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.13 mg/mL and 0.06 mg/mL

In all experiments very small oily drops were visible at the concentrations 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL during treatment.

Ethylmethane sulfonate (experiment I: 300 μg/mL, experiment II: 150 μg/mL) and 7,12 - dimethylbenz(a)anthracene (1.5 μg/mL) were used as positive controls and showed a distinct

increase in induced total mutant colonies but without reduction of the viability.

In both experiments of this study (with and without S9 mix) the mutant colony numbers/10EXP6 cells of the solvent controls were in the range of the historical data.

No cytotoxic effects were observed in none of the test item concentrations in experiment I and II. Therefore, all concentrations were analysable for mutagenicity.

In experiment I without metabolic activation, some of the test item concentrations induced a statistically significant increase in mutant fequency in comparison to the solvent control, however all those values are still within the range of the spontaneous mutant frequency (5 - 20 * 10EXP-6) or of the historical data of the solvent control. Therefore, no biologically relevant and reproducible increase in mutant colony numbers/10EXP6 cells was observed in the main experiments up to the maximum concentration. All mutant frequencies remained well within the historical range of solvent control. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequency but did not reveal a linear regression. Therefore, all criteria for a clearly negative result are fulfilled.

In conclusion, it can be stated that under the experimental conditions reported, the test material did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation (LAUS GmbH, 2017, HPRT, RL1).

The following adequate and reliable data for a structural anologues (i.e. C8TM) is reported as supporting information.

Reverse mutation testing of 1,2, 4 benzenetricarboxlyic acid, trioctyl ester was conducted similar to OECD testing guideline 471 using 5 strains of bacteria: Salmonella typhimurium TA100, TA1535, TA98, TA1537, and Escherichia coli WP uvrA.

Pre-testing was performed with 7 doses at 0, 1.22, 4.88, 19.5, 78.1, 313, 1250, and 5000 μg/plate, and no increase in the revertant colony count or bacteria growth inhibition was observed either with or without S9 mix. Based on these results, the main test was performed with ( phenobarbital and 5,6-benzoflavone induced rat liver S9 mix) and without metabolic activation at concentrations of 0, 313, 625, 1250, 2500, and 5000 µg/plate using the preincubation method.

As a result of 2 rounds of testing (in triplicate), the test substance did not show an increase in the revertant colony count greater than two times that of the value of the negative (solvent) control either with or without S9 mix, for any of the bacteria strains.

From the above result it was concluded that 1,2, 4 benzenetricarboxlyic acid, trioctyl ester was non-mutagenic (negative) in reverse mutation testing using bacteria. The appropriate reference mutagenes showed distinct positive mutagenic effects (MoHW, 2001, Ames, RL1).

Induction of chromosome aberration by 1,2, 4 benzenetricarboxylic acid, trioctyl ester was tested by conducting anin vitrochromosome aberration study using mammalian cultured cells (GLP study, equivalent to OECD testing guideline 473). Cells were exposed to 0, 1250, 2500, and 5000 µg/mL. No cytotoxic effects were seen up to the highest concentration tested. Short-term conditions included treatment for up to 6 hours with or without S9 mix, with subsequent culture in media only, preparation of chromosomes was performed 24 hours after test beginn. Under continuous treatment conditions cells were treated for up to 24 hours with the test material without S9 mix following immediate chromosome fixation.

Results showed that under the treatment conditions for both short-term and continuous treatment, the frequency of cells with chromosomal aberrations was less than 5%, thus indicating a negative test result.

Negative and positive controls produced expected values for cells with chromosomal aberration, showing that the tests were technically sound.

Accordingly, it was concluded that 1,2, 4 benzenetricarboxylic acid, trioctyl ester did not induce chromosomal aberrations in CHL/IU cells (MoHW, 2001, Chromosome aberration, RL1).

Justification for classification or non-classification

Based on the negative results obtained in all tests for genotoxicity performed with the the submission substance (L9TM) and the structural analogue (C8TM) of the submission substance, it is concluded that no classification is required for germ cell mutagenicity according to Regulation (EC) No 1272/2008.