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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The target substance Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) (Reaction product of guerbet alcohol, C24 -26, branched and cyclic with 1,2,4 -benzenetricarboxylic acid) was in the Ames test performed according to OECD 471 negative.

Additionally the source substance 1,2,4 -Benzenetricarboxylic acid, mixed decyl and octyl triesters was in the chromosome aberration test according to OECD 473 and in the mouse lymphoma test according to OECD 476 negative.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September - October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
from July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate of Compliance with Good Laboratory Practices according to Directives 2004/9/CE and 2004/10/CE, Groupe Interministeriel des Produits Chimiques, Republique Francaise, Certificat n°: 2016/48, dated 2 Nov 2016
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: delivered from Sponsor, BATCH: 05347/MA
- Expiration date of the lot/batch: June 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15 - 25°C), non hygroscopic
- Stability: stable under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO, test solutions used immediately after preparation, no further details mentioned
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data
Target gene:
his D, his C, his G, tryp E
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 microsome fraction prepared from Sprague Dawley rat liver homogenate, Aroclor 1254 induced
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix, top dose chosen according to guideline
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item is despersible in DMSO
Untreated negative controls:
yes
Remarks:
absolute negative control containing no test item; negative controls with solvents used to solubilize positive controls (Acetone, DMSO, NaCl 0.15 M)
Negative solvent / vehicle controls:
yes
Remarks:
vehicle used to solubilize test item (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
other: cis-Platinum (II) Diammine Dichloride, CAS 15663-27-1: 1 µg/plate, E. coli, without S9; 2-anthramine, CAS 613-13-8: 2 µg/plate, TA 98, 100, 1535, 1537, with S9 (plate incorporation); 1 µg/plate, TA 98, 100, 1535, 1537, with S9 (pre-incubation)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
1st assay:
- without metabolic activation: in agar (plate incorporation),
- with metabolic activation: in agar (plate incorporation)
2nd assay:
- without metbolic activation: in agar (plate incorporation),
- with metabolic activation: pre-incubation

DURATION
- Preincubation period: 30 min (at 37°C, with shaking)
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 3 plates per experimental point

DETERMINATION OF CYTOTOXICITY
- Method: Bacteriostatic activity was tested in all concentrations in strain TA 100 in a preliminary cytotoxicity experiment: percent of survival in the plates treated with test item concentrations compared to negative control colonies was measured.
Evaluation criteria:
Study was judged to be valid if the following criteria were met:
- the bacteriostatic activity of the highest concentration tested is equal of less than 75%,
- the spontaneous reversion rate of the absolute negative control complies with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent is not statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation complies with the historical values of the laboratory,
- negative and positive values don't show significant difference with the historical values of the laboratory (± 2 standard deviations).

Criteria for mutagenic activity in this assay:
The result of the test is considered as negative, if the revertant number is below three fold the number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2 (uvrA-)(pKM101) strains without and with metabolic activation.
The result of the test is considered positive if a dependent relationship concentration is obtained in one, or several of the 5 strains, without and/or with metabolic activation, a mutagenic effect being taken into account for a given dilution of test item if the number of revertant colonies is at least two fold that of spontaneous revertant colonies for TA 98, TA 100 and Escherichia coli WP2 (uvrA-)(pKM101), and three fold for TA 1535 and TA 1537.
All results must be confirmed in an independent experiment.
Statistics:
Mean values of replicates with standard deviations were calculated.
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
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
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:
valid
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
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Results of the bacteriostatic activity control testing show that neither original solution nor dilutions have bacteriostatic effect.

HISTORICAL CONTROL DATA (with number of experiments, ranges, means and standard deviation)

POSITIVE HISTORICAL CONTROL DATA
TA 1535:
- without metabolic activation, plate incorporation, Sodium Azide: n=1017, range: 190 - 1487, mean: 431.5 ± 220.2
- with metabolic activation, plate incorporation, 2-Anthramine: n=546, range: 26 - 269, mean: 190.2 ± 56.0
- with metabolic activation, pre-incubation, 2-Anthramine: n=540, range: 25 - 217, mean: 75.6 ± 37.0
TA 1537:
- without metabolic activation, plate incorporation, 9-Aminoacridine: n=1017, range: 219 - 1967, mean: 876.5 ± 448.3
- with metabolic activation, plate incorporation, 2-Anthramine: n=546, range: 24 - 170, mean: 55.1 ± 24.7
- with metabolic activation, pre-incubation, 2-Anthramine: n=540, range: 21 - 182, mean: 48.0 ± 23.7
TA 98:
- without metabolic activation, plate incorporation, 2-Nitrofluorene: n=1017, range: 187 - 1667, mean: 496.3 ± 219.9
- with metabolic activation, plate incorporation, 2-Anthramine: n=546, range: 219 - 1499, mean: 572.9 ± 222.1
- with metabolic activation, pre-incubation, 2-Anthramine: n=540, range: 174 - 1368, mean: 462.2 ± 192.3
TA 100:
- without metabolic activation, plate incorporation, Sodium Azide: n=1017, range: 381 - 1690, mean: 991.8 ± 331.2
- with metabolic activation, plate incorporation, 2-Anthramine: n=543, range: 361 - 2163, mean: 846.8 ± 359.5
- with metabolic activation, pre-incubation, 2-Anthramine: n=543, range: 309 - 1889, mean: 668.8 ± 309.4
Escherichia coli WP2 (pKM101)(uvr A-)
- without metabolic activation, plate incorporation, cis-Platinium (II) Diamine Dichloride: n=777, range: 248 - 1089, mean: 484.6 ± 168.2
- with metabolic activation, plate incorporation, Dimethyl Benzanthracene: n=402, range: 365 - 1680, mean: 686.5 ± 253.3
- with metabolic activation, pre-incubation, Dimethyl Benzanthracene: n=402, range: 281 - 1680, mean: 670.0 ± 232.0

NEGATIVE (SOLVENT/VEHICLE) HISTORICAL CONTROL DATA
TA 1535:
- without metabolic activation, plate incorporation: n=1017, range: 4 - 23, mean: 11.0 ± 3.6
- with metabolic activation, plate incorporation: n=546, range: 3 - 23, mean: 12.2 ± 4.1
- with metabolic activation, pre-incubation: n=540, range: 5 - 25, mean: 12.8 ± 4.3
TA 1537:
- without metabolic activation, plate incorporation: n=1017, range: 1 - 20, mean: 6.0 ± 2.5
- with metabolic activation, plate incorporation: n=546, range: 1 - 24, mean: 8.1 ± 3.5
- with metabolic activation, pre-incubation: n=540, range: 1 - 21, mean: 8.2 ± 3.3
TA 98:
- without metabolic activation, plate incorporation: n=1017, range: 6 - 29, mean: 16.0 ± 3.9
- with metabolic activation, plate incorporation: n=546, range: 11 - 38, mean: 23.2 ± 5.0
- with metabolic activation, pre-incubation: n=540, range: 10 - 36, mean: 23.1 ± 5.4
TA 100:
- without metabolic activation, plate incorporation: n=1017, range: 40 - 123, mean: 59.8 ± 11.8
- with metabolic activation, plate incorporation: n=543, range: 55 - 189, mean: 96.2 ± 22.3
- with metabolic activation, pre-incubation: n=540, range: 51 - 189, mean: 95.7 ± 23.9
Escherichia coli WP2 (pKM101)(uvr A-)
- without metabolic activation, plate incorporation: n=777, range: 41 - 188, mean: 83.6 ± 34.2
- with metabolic activation, plate incorporation: n=402, range: 80 - 264, mean: 156.8 ± 34.6
- with metabolic activation, pre-incubation: n=402, range: 69 - 250, mean: 158.8 ± 35.9

Table #1: Assay n°1, without metabolic activation, plate incorporation

Concentration [µg/plate] Revertant colonies / plate
TA 98 TA 100 TA 1535 TA 1537 E. coli WP2 uvrA
mean±SD R mean±SD R mean±SD R mean±SD R mean±SD R
Negative control 917.67 ± 2.08 - 67.67 ± 4.93 - 9.33 ± 3.06 - 5.00 ± 2.00 - 145.67 ± 25.50 -
Vehicle (DMSO) 16.67 ± 0.58 - 64.67 ± 2.08 - 10.67 ± 2.08 - 6.67 ± 1.53 - 153.67 ± 15.50 -
50 15.33 ± 3.21 0.92 58.33 ± 4.04 0.90 8.67 2.52 0.81 4.00 ± 0.00 0.60 115.67 ± 9.02 0.75
150 13.67 ± 2.52 0.82 57.67 ± 4.51 0.89 12.00 ± 2.65 1.13 6.67 ± 0.58 1.00 139.33 ± 19.86 0.91
500 14.00 ± 1.00 0.84 60.00 ± 3.00 0.93 9.67 ± 4.73 0.91 8.67 ± 1.53 1.30 115.33 ± 5.51 0.75
1500 13.67 ± 5.86 0.82 60.67 ± 1.53 0.94 9.33 ± 1.53 0.88 3.33 ± 2.31 0.50 142.33 ± 10.79 0.93
5000 17.33 ± 6.11 1.04 55.00 ± 4.00 0.85 9.00 ± 2.65 0.84 7.00 ± 1.00 1.05 118.33 ± 11.55 0.77
Positive control solvent 17.33 ± 3.21 - 64.00 ± 3.61 - 8.67 ± 2.08 - 9.33 ± 0.58 - 139.00 ± 25.87 -
Positive control 523.33 ± 83.48 30.19 1306.67 ± 67.83 20.42 910.67 ± 113.00 105.08 1366.33 ± 154.67 146.39 517.67 ± 40.65 3.72

Table #2: Assay n°1, with metabolic activation (10% S9 mix), plate incorporation

Concentration [µg/plate] Revertant colonies / plate
TA 98 TA 100 TA 1535 TA 1537 E. coli WP2 uvrA
mean±SD R mean±SD R mean±SD R mean±SD R mean±SD R
Negative control 29.00 ± 3.00 - 95.00 ± 7.00 - 14.33 ± 3.51 - 9.67 ± 2.52 - 209.67 ± 27.54 -
Vehicle (DMSO) 25.67 ± 2.08 - 84.67 ± 9.29 - 16.33 ± 1.53 - 10.00 ± 3.00 - 213.00 ± 28.05 -
50 25.67 ± 5.03 1.00 78.00 ± 8.54 0.92 13.67 ± 3.51 0.84 10.33 ± 4.04 1.03 211.67 ± 13.65 0.99
150 24.33 ± 2.52 0.95 79.33 ± 9.02 0.94 13.00 ± 3.46 0.80 10.00 ± 3.46 1.00 209.00 ± 16.46 0.98
500 30.00 ± 2.00 1.17 74.67 ± 1.53 0.88 15.00 ± 1.00 0.92 15.00 ± 2.65 1.50 207.33 ± 19.76 0.97
1500 25.67 ± 0.58 1.00 77.00 ± 6.24 0.91 12.33 ± 4.51 0.76 10.67 ± 2.08 1.07 205.33 ± 9.71 0.96
5000 27.33 ± 3.21 1.06 72.67 ± 2.52 0.86 13.00 ± 1.73 0.80 12.33 ± 2.52 1.23 203.67 ± 13.32 0.96
Positive control solvent 24.67 ± 6.11 - 80.33 ± 6.43 - 19.67 ± 5.13 - 10.67 ± 1.53 - 219.00 ± 17.06 -
Positive control 578.33 ± 69.57 23.45 660.00 ± 26.51 8.22 110.67 ± 24.54 5.63 57.33 ± 4.16 5.38 1002.67 ± 114.77 4.58

Table #3: Assay n°2, without metabolic activation, plate incorporation

Concentration [µg/plate] Revertant colonies / plate
TA 98 TA 100 TA 1535 TA 1537 E. coli WP2 uvrA
mean±SD R mean±SD R mean±SD R mean±SD R mean±SD R
Negative control 17.33 ± 3.51 - 92.00 ± 11.14 - 7.67 ± 0.58 - 9.00 ± 2.65 - 136.33 ± 5.13 -
Vehicle (DMSO) 19.00 ± 2.00 - 83.33 ± 5.51 - 10.33 ± 2.31 - 9.00 ± 1.00 - 142.67 ± 3.21 -
50 16.67 ± 2.08 0.88 75.33 ± 5.03 0.90 12.33 ± 0.58 1.19 7.33 ± 3.21 0.81 128.33 ± 9.29 0.90
150 14.33 ± 0.58 0.75 72.33 ± 3.06 0.87 11.00 ± 1.00 1.06 11.67 ± 3.21 1.30 133.00 ± 7.21 0.93
500 17.67 ± 1.53 0.93 77.00 ± 5.57 0.92 9.67 ± 3.06 0.94 5.00 ± 2.08 0.59 134.00 ± 11.36 0.94
1500 19.33 ± 3.51 1.02 74.67 ± 2.52 0.90 10.33 ± 3.51 1.00 7.00 ± 3.21 0.81 127.00 ± 4.36 0.89
5000 19.67 ± 1.53 1.04 65.00 ± 6.00 0.78 9.33 ± 3.21 0.90 8.67 ± 3.06 0.96 133.00 ± 9.54 0.93
Positive control solvent 20.33 ± 4.16 - 88.00 ± 6.56 - 10.00 ± 2.00 - 7.67 ± 1.15 - 145.33 ± 22.55 -
Positive control 456.00 ± 34.37 22.43 1252.33 ± 110.01 14.23 957.33 ± 44.50 95.73 906.33 ± 17.01 118.22 556.00 ± 22.87 3.83

Table #4: Assay n° 2, with metabolic activation (10% S9 mix), pre-incubation

Concentration [µg/plate] Revertant colonies / plate
TA 98 TA 100 TA 1535 TA 1537 E. coli WP2 uvrA
mean±SD R mean±SD R mean±SD R mean±SD R mean±SD R
Negative control 25.33 ± 1.53 - 89.67 ± 15.14 - 11.00 ± 4.00 - 11.33 ± 0.58 - 213.67 ± 20.74 -
Vehicle (DMSO) 30.67 ± 1.15 - 102.00 ± 7.55 - 12.67 ± 5.51 - 9.00 ± 2.00 - 221.33 ± 6.66 -
50 28.00 ± 6.24 0.91 92.33 ± 5.13 0.91 11.00 ± 1.73 0.87 13.33 ± 2.31 1.48 205.33 ± 2.08 0.93
150 28.33 ± 4.73 0.92 86.33 ± 8.02 0.85 10.00 ± 4.57 0.79 10.33 ± 1.53 1.15 202.67 ± 6.66 0.92
500 30.67 ± 6.11 1.00 87.33 ± 5.13 0.86 11.33 ± 3.21 0.89 13.00 ± 3.00 1.44 196.00 ± 8.89 0.89
1500 28.33 ± 2.08 0.92 88.67 ± 12.74 0.87 12.33 ± 4.93 0.97 10.33 ± 2.08 1.15 199.00 ± 6.24 0.90
5000 27.67 ± 5.86 0.90 85.67 ± 2.08 0.84 15.67 ± 4.93 1.24 13.33 ± 2.52 1.48 199.33 ± 6.03 0.90
Positive control solvent 26.67 ± 4.62 - 92.67 ± 1.53 - 11.33 ± 5.86 - 10.67 ± 6.43 - 216.33 ± 19.86 -
Positive control 238.00 ± 56.79 8.93 310.00 ± 18.52 3.35 183.67 ± 15.01 16.21 48.33 ± 5.03 4.53 763.67 ± 62.94 3.53

SD = Standard deviation; R = Number of revertant colonies in the presence of the test item/ Number of revertant colonies in the absence of the test item

Conclusions:
Doses (50, 150, 500, 1500 and 5000 µg/plate) performed from solution of the test item did not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA-)(pKM101) without, or with metabolic activation, according to the OECD Guideline n°471.
Executive summary:

A bacterial reverse mutation test using Salmonella typhimurium his- and Escherichia coli WP2 (uvrA-)(pKM101) was performed according to OECD Guideline n°471 in compliance with Good Laboratory Practices (GLP).

Solutions obtained from the test item have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2 (uvrA-)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out. For assay n°1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9 -mix 10% (v/v)). For assay n°2, various concentrations were put in contact with test strains in absence and presence of a metabolic activation system (S9 -mix 10%(v/v)). For the two assays, negative and postive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There was no siginificant difference between the number of spontaneous reversions, the number of reversions obtained in the prositive controls (without and with metabolic activation), and the mean of corresponding experimental "historical" values obtained in the laboratory. These results validated the two tests. There was no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (50, 150, 500, 1500, and 5000 µg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and in Escherichia coli WP2 (uvrA-)(pKM101). The test item was judged to be non-mutagenic in this test system under the described conditions.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
from 2009-05-21 to 2009-10-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Justification for type of information:
Please see Analogue Approach
Reason / purpose:
read-across source
Qualifier:
according to
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:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
see above
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: obtained from human blood from two healthy male volunteer donors (one for each experiment)
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 1x (Dutch modification); 500 mL supplemented with 100 mL heat inactivated Foetal Calf Serum, 6.25 mL L-glutamine (200 mM), 1.25 mL Streptomycin sulphate 50 mg/mL Penicillin G 50,000 IU/mL
Additional strain / cell type characteristics:
other: stimulated to cell division in vitro by addition of the mitogen PHA (phytohaemogglutinin); cell cycle length approx. 17 hours
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 of Phenobarbital - 5,6-Benzoflavone induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Range finding: 2500, 1250, 625, 313, 156, 78.1, 39.1, 19.5 and 9.77 µg/mL
Dose levels selected for metaphase analysis in the main tests:
First main test: 1250, 625 and 313 µg/mL (with and without S9 mix, 3 hours treatment time, 24 hours harvest time)
Second main test: 2500, 1250 and 625 µg/mL (without S9 mix, 24 hours treatment time, 24 hours harvest time)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was found to be soluble in ethanol at the concentration of 500 mg/mL. An aliquot of the stock solution in ethamol at 500 mg/mL added in the ratio of 1 : 100 to culture medium gave particles in suspension. Particles in suspension and opacity were observed when adding an aliquot at 250 mg/mL, opacity was observed by adding solutions at 125 and 6.25 mg/mL. On the basis of the results of solubility testing, the maximum dose level of 2500 µg/mL was selected for treatment, where precipitation and opacity were expected. This dose level was achieved by adding a solution of test item in ethanol at the concentration of 250 mg/mL to culture medium in the ration of 1 : 100.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol, 1% in tissue culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.75 and 0.50 µg/mL in the first main test; 0.45 and 0.30 µg/mL in the second main test
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol, 1% in tissue culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 18.0 and 23.0 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
48 hours after the lymphocyte cultures were initiated, they were centrifuged at 1000 rpm for 10 minutes, the culture medium was decanted and replaced with treatment medium (4.95 mL resp. 3.95 mL (in the test with S9 mix) culture medium without PHA + 0.05 mL test item or control solution + 1.00 mL S9 mix). The cultures were subsequently incubated for 3 hours at 37 °C, the medium was aspirated and the cultures were centrifuged and washed twice with PBS. Fresh medium was added and the cultures were incubated for a further period of 21 hours recovery period. Colcemid was added for the last 3 hours, leading up to harvesting after 24 hours. In the second main test the test medium was not changed (treatment for 24 hours).

DURATION
- Preincubation period: not applicable
- Exposure duration: First main test: 3 h (presence and absence of S9 mix); second main test: 24 h in the absence of S9 mix
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h


SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concentration 0.2 µg/mL)
STAIN (for cytogenetic assays): Giemsa solution (3 % in tap water)


NUMBER OF REPLICATIONS: two cultures per dose level, controls and vehicle control


NUMBER OF CELLS EVALUATED: 200 metaphases per dose level (100 per replicate)


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Determination of aneupoid cells


OTHER: Measurement of pH and osmolality at the three higher dose levels in the first main test, pH measurement in the second main test at the four higher dose levels
Evaluation criteria:
The evaluation was based on the set of results which excludes gaps.
A test item is considered to have clastogenic properties if the following criteria are all fulfilled:
- statistically significant increases in the incidence of cells bearing aberrations are observed at any dose level over the concurrent control
- the increases are reproduced in both replicate cultures and must be observed in both experiments
- the increases must exceed historical controls
- biological significance must be given
Statistics:
Fisher's exact test was used to compare the number of cells bearing aberrations either including and excluding gaps (assumed to be Poisson ditributed) in control and treated cultures.
Key result
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
Key result
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

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)

Conclusions:
It was concluded that the source substance 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 source 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 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:
May 28 to August 5, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please see Analogue Approach
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
Minimal medium A: RPMI 1640 medium supplemented with penicillin, streptomycin sulphate, sodium pyrovate, L-glutamine, non-essential amino acids and F 68 Pluronic
Minimal medium B: same as Minimal medium A without F68 Pluronic
Complete medium (5%): Minimal medium A supplemented with 5% v/v heat-inactivated horse serum
Complete Medium (10%: Minimal medium A supplemented with 10% v/v heat-inactivated horse serum
Complete medium A (20%): Minimal medium A supplemented with 20% v/v heat-inactivated horse serum
Complete medium B (20%): Minimal medium B supplemented with 20% v/v heat-inactivated horse serum
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Generation time and mutation rates (spontaneous and induced) were checked in the testing laboratory
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix of Phenobarbital - 5,6-Benzoflavone induced male Sprague Dawley rats
Test concentrations with justification for top dose:
With and without metabolic activation:
Toxicity test (range finding): 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250 and 2500 µg/mL
1. experiment: 156, 313, 625 and 1250 and 2500 µg/mL with and without metabolic activation
2. experiment: 156, 313, 625, 1250 and 2500 µg/mL without metabolic activation; resp. 875, 1138, 1479, 1923 and 2500 µg/mL with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solvent giving the best solubility/dispersal characteristics
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: in the absence of metabolic activation (4 h/24h exposure): methyl methanesulphonate (MMS, 5.0/10.0 µg/ml); in the presence of metabolic activation: Benzo(a)pyrene (B(a)P, 2.0 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 3 h (with and without metabolic activation)
Experiment 2: 24 h (without metabolic activation) and 3 h (with metabolic activation)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 14 days (mutation selection assay)

SELECTION AGENT (mutation assays): trifluorothymidine (TFT), final concentration: 3.0 µg/mL in Complete medium B (20%)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: mutation selection assay: 2000 per well; cloning efficiency assay: 1.6 cells per well (8 cells/mL)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (Cell concentrations were adjusted to 8 cells/mL and, for each dose level, 0.2 mL was plated into 96 microtitre wells, incubated for 7 days. Wells containing viable clones were identified by the eye using background illumination and then counted.)

OTHER EXAMINTATIONS
- Colony sizing: small and large type mutants, estimated for solvent and positive controls
- Observations of pH and osmolality: the treatment solutions were measured during performance of one the main experiments
Evaluation criteria:
Criteria for a positive result:
- the induced mutant frequency is higher than the global evaluation factor suggested for the microwell method (126 x 10E-6) at one or more doses
- there is a significant dose-relationship as indicated by the linear trend analysis
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statisitical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990). The following methods were applied:
- Results of individual plates within a replicate treatment were checked for consistency by calculation of Chi-square.
- Heterogeneity factors were calculated for survival, viability and mutation. Values obtained should not exceed 10.8 times the current heterogeneity factor where 10.8 is the one-sided 0.1% level of the F-distribution with 1 and infinitive degrees of freedom.
- Overall consistency was evaluated by calculation of the ratio of the heterogeneity factors of the experiment to the current heterogeneity factor. This ratio should not exceed the one-sided 1% critical values from the F-distribution.
- The estimated heterogeneity factors of the experiment were combined with the current heterogeneity factor to define the updated estimated factors.
- Comparison of each treatment with the control: for each comparison, the ratio Di²/var(Di) was compared to the critical values for the one tailed Dunnett`s test.
- Test for linear trend: The evaluation of a linear trend in mutant frequency with the treatment dose was performed using weighted regression. The slope and its variance var(b) were calculated to form the test statistic b²/var(b), which was compared to tabulated critical values of Chi-square with 1 degree of freedom.
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.
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
Conclusions:
The source substance 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 source 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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A bacterial reverse mutation test using Salmonella typhimurium his- and Escherichia coli WP2 (uvrA-)(pKM101) was performed with the target substance Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) (Reaction product of guerbet alcohol, C24 -26, branched and cyclic with 1,2,4 -benzenetricarboxylic acid) according to OECD Guideline n°471 in compliance with Good Laboratory Practices (GLP).

Solutions obtained from the test item have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2 (uvrA-)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out. For assay n°1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9 -mix 10% (v/v)). For assay n°2, various concentrations were put in contact with test strains in absence and presence of a metabolic activation system (S9 -mix 10%(v/v)). For the two assays, negative and postive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There was no siginificant difference between the number of spontaneous reversions, the number of reversions obtained in the prositive controls (without and with metabolic activation), and the mean of corresponding experimental "historical" values obtained in the laboratory. These results validated the two tests. There was no evidence of any increase in the number of revertant colonies in the presence of the various concentrations of the test item (50, 150, 500, 1500, and 5000 µg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and in Escherichia coli WP2 (uvrA-)(pKM101). The test item was judged to be non-mutagenic in this test system under the described conditions.

The source substance 1,2,4 -Benzenetricarboxylic acid, mixed decyl and octyl triesters was tested for mutation in three histidine requiring strains (TA97, TA98 and TA100) of Salmonella typhimurium both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S9) according to OECD guideline 471. The substance failed to induce a two-fold increase in revertant numbers with any tester strain either in the absence or presence of S9, so was considered non-mutagenic in this assy.

The source substance 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 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. 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. Also in a second chromosome aberration test the test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. Therefore also in the second test according to OECD 473 the test item can be considered to be non-clastogenic to human lymphocytes in vitro.

The source substance 1,2,4 -Benzenetricarboxylic acid, mixed decyl and octyl triesters

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). 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. 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.

All available data clearly demonstrate the lack of genotoxic effects.


Justification for classification or non-classification

Data from various tests with the target substance and the structurally similar source substance clearly demonstrate that the material is not genotoxic and therefore the target substance Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) (Reaction product of guerbet alcohol, C24 -26, branched and cyclic with 1,2,4 -benzenetricarboxylic acid) does not need to be classified.