1,2,4-Benzenetricarboxylic acid, mixed lauryl and octyl triesters

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

tris(mixed dodecyl and octyl)benzene-1,2,4-tricarboxylate

EC Number:

700-342-7

Cas Number:

1163775-81-2

Molecular formula:

C14H47O6 to C18H55O6

IUPAC Name:

tris(mixed dodecyl and octyl)benzene-1,2,4-tricarboxylate

Constituent 2

Reference substance name:

1,2,4-Benzenetricarboylic acid, mixed dodecyl and octyl triesters

IUPAC Name:

1,2,4-Benzenetricarboylic acid, mixed dodecyl and octyl triesters

Constituent 3

Reference substance name:

Linplast 812 TM

IUPAC Name:

Linplast 812 TM

Details on test material:

Smiles notation (if other than submission substance): O=C(c1ccc(cc1C(=O)OCCCCCCCC)C(=O)OCCCCCCCC)OCCCCCCCCCCCC

Specific details on test material used for the study:

N/A

Method

Target gene:

N/A

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

Positive controls:

yes

Positive control substance:

other: 0.5 mg/ml 1, 8-Dihydroxyanthraquinone (Dorbane)

Remarks:

with metabolic activation

Controls 4

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

Controls 5

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.