Registration Dossier
Registration Dossier
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EC number: 201-877-4 | CAS number: 89-04-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The substance, an ester of 1,2,4 -benzenetricarboxylic acid with C8 linear side chains (TM8), has been tested in three different in vito assays.
A bacterial reverse mutation study (OECD 471) with the substance (TM8), including 4 strains of Salmonella typhimurium and one strain of Escherichia coli, with and without metabolic activation, was negative.
An in vitro chromosome aberration study (OECD 473) in human lymphocytes, conducted with the substance, with and without metabolic activiation was negative for chromosome damage.
An in vitro mutagencity study, the mouse lymphoma assay (OECD 490), with the substacne also did not induce mutation in mouse lymphoma L5178Y cells in the absence or presence of S9 metabolic activation.
In addition, a number of supporting QSAR assessments are included which demonstrate a lack of concern for mutagenic action with the substance. The results for in vitro tests for bacterial reverse mutation, chromosome aberration and mouse lymphoma assay with a close chemical analogue TM8 -10, were also negative and are also included as supporting evidence.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant study conducted according to internationally recognised test methods.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Also compliant with Guideline for Screening Mutagenicity Testing of Chemicals Japan
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- - Lot/batch No.: C-120
- Species / strain / cell type:
- E. coli WP2 uvr A
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction (mixed induction rat liver preparation)
- Test concentrations with justification for top dose:
- Without S9 mix 0 313 625 1250 2500 &5000 ug/plate
With S9 mix 0 313 625 1250 2500 & 5000 ug/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Substance is soluble in acetone. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- furylfuramide
- Remarks:
- TA100, TA98 in absence of S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA1535 in absence of S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- WP2 in absence of S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537 in absence of S9 mix
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- All strains in the presence of S9 mix
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation)
DURATION
- Preincubation period: 10h
- Exposure duration: 48h at 37 degrees C
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: plates/test: 3
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: not observed at doses of up to 5000 ug/plate with or without S9 - Evaluation criteria:
- An increase in colony count of more than 2 fold when compared with concurrent controls and/or a dose dependent increase in the colony count which is statistically significantly higher than the count for the controls.
- Statistics:
- No statistical analyses were done.
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and 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 applicable
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No data
- Water solubility: N/A
- Precipitation: No
- Other confounding effects: No
RANGE-FINDING/SCREENING STUDIES: yes range of doses tested 1.22-5000ug/plate.
COMPARISON WITH HISTORICAL CONTROL DATA:
ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity was not observed at 313-5000 ug/plate - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
Based on results under the conditions of this test the substance was not considered to be mutagenic to Salmonella typhimurium TA100, TA 1535, TA98, TA1537 & E coli WP2 uvrA. - Executive summary:
A bacterial reverse mutation assay (Ames test) has been undertaken following OECD test methods.
The substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP compliant study conducted in accordance with internationally recognised test methods
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- - Lot/batch No.: C-120
- Target gene:
- N/A
- Species / strain / cell type:
- other: Chinese hamster lung cells CHL/IU
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Inactivated calf serum in Eagle MEM
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction (mixed function induction of rat liver)
- Test concentrations with justification for top dose:
- -S9 (continuous treatment): 0, 1.3, 2.5, 5.0 mg/ml
-S9 (short term treatment): 0, 1.3, 2.5, 5.0 mg/ml
+S9 (short term treatment): 0, 1.3, 2.5, 5.0 mg/ml - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone; supplied by Wako Pure Chemical Industries Co.
- Justification for choice of solvent/vehicle: Substance is poorly soluble in water; very soluble in acetone & DMSO - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- -S9; 6 & 24 hours of exposure
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- +S9; 6 hours exposure
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Preincubation period:
- Exposure duration: For continuous treatment, cells were treated for 24 hours in the absence of S9; for short-term treatment cells were treated for 6 hours with & without S9 and cultivated with fresh media for 18 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcimid 0.1 ug/mL
STAIN (for cytogenetic assays): 3% Giemsa solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 metaphases/slide of each replicate
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No cytotoxicity up to and including 5.0 mg/mL when inhibition of cell growth determined.
OTHER EXAMINATIONS:
- Determination of polyploidy:Negative
- Determination of endoreplication: No data or negative if equivalent to clastogenicity - Evaluation criteria:
- A dose related increase or a reproducible increase in the number of cells with chromosome aberrations.
An increase in the number of polyploid cells.
An increase in the number of cells with endoreduplicated chromosomes - Statistics:
- No data, thought to be Fisher's Exact Test
- Species / strain:
- other: Chinese hamster lung cells (CHL/IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- 5 mg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: poor
- Precipitation: No
- Other confounding effects: None
RANGE-FINDING/SCREENING STUDIES: yes; growth inhibition test (range of concentrations: 0.0-5.0 mg/mL
COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxicity observed - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
negative
In the conditions in which this study was performed the tested substance, at doses of up to 5.0 mg/mL, did not induce chromosome aberrations with or without S9 in cultured mammalian somatic cells. - Executive summary:
The substance has been assayed for its ability to cause chromosomal damage in cultured mammalian cells following in-vitro treatment in the absence and presence of S9 metabolic activation.
The substance did not induce chromosomal aberrations in human lymphocytes after in-vitro treatment
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2017-04-13 to 2017-06-20
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- Adopted June 2016
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt Lot No. 3606217009
- Expiration date of the lot/batch: 09 Jan 2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: Soluble/miscible and stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None - Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat S-9 tissue homogenate
- Test concentrations with justification for top dose:
- 0.125, 0.250, 0.500, 1.00 and 2.00 microlitres/mL
Highest concentration examined is that indicated by the test guideline - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Commonly used vehicle for testing of poorly water soluble/miscible sustances - Untreated negative controls:
- yes
- Remarks:
- Solvent
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- S9 tissue homogenate
S9 tissue homogenate was prepared from the livers of five young male Sprague-Dawley rats which had received treatment with phenobarbital and betanaphthoflavone to induce high levels of xenobiotic
metabolising enzymes.
Preparation of test cell cultures
A cell suspension (1E6 cells/mL) in complete medium was prepared. The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed with Phosphate Buffered Saline (PBS).
Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and in the presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in PBS, cells were resuspended in 20 mL RPMI minimal medium. Cell concentrations were adjusted to 8 cells/mL using complete medium and, for each dose level, 0.2 mL was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere
(100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by eye using background illumination and then counted.
Mutation assay
Treatment of cell cultures
Experiments were performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.
Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.
In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained in this experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a long treatment time (24 hours). After washing in PBS, cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2E5 cells/mL. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
Determination of survival
Following adjustment of the cell densities, samples of the cultures were diluted to 8 cell/ml using complete medium (20%). A 0.2 mL aliquot of each diluted culture was placed into each well of two 96-well plates. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. After incubation, wells containing viable clones were identified by eye using background illumination and then counted.
Expression period
During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2E5 cells/mL.
Plating for 5-trifluorothymidine resistance
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine and an estimated 2E3 cells were plated in each well of four 96-well plates. Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified . In addition, the number of wells containing large colonies and the number containing small colonies were scored.
Plating for viability
After dilution, in complete medium (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted. - Evaluation criteria:
- The assay was considered valid if the following criteria were met:
(i) The cloning efficiencies at Day 2 in the untreated/solvent control cultures fell within the range of 65-120%.
(ii) The untreated/solvent control suspension growth over 2 days fell within the range of 8-32 (3 hours exposure or 32-180 (24 hours exposure).
(iii) The mutant frequencies in the untreated/solvent control cultures fell within the range of 50E-6 - 170E-6 viable cells.
(iv) The positive control chemicals induced a clear increase in mutant frequency above the spontaneous background with at least 40% of the induced mutant frequency reflected in the small colony mutation frequency
The assay was considered positive if:
(i) The induced mutation frequency was higher than teh global evaluation factor suggested for the assay
(ii) There was a significant dose-relationship as indicated by linear trend analysis - Statistics:
- Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The tested substance does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation.
- Executive summary:
The substance has been 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 substance does not induce mutation in mouse lymphoma L5178Y cells after in vitro treatment in the absence or presence of S9 metabolic activation.
Referenceopen allclose all
Table 1 - Results of reverse mutation test I
With (+) or |
Dose (µg/plate) |
Number of revertants Mean (±SD) |
||||
without(-) |
Base-pair substitution type |
Frameshift type |
||||
S9 mix |
TA100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
|
|
0 |
105 (±3) |
15 (±5) |
34 (±4) |
23 (±4) |
9 (±2) |
|
313 |
106 (±6) |
17 (±4) |
33 (±7) |
23 (±4) |
10 (±2) |
S9 mix |
625 |
113 (±7) |
20 (±5) |
38 (±2) |
25 (±4) |
8 (±1) |
(-) |
1250 |
124 (±16) |
19 (±3) |
38 (±6) |
28 (±5) |
10 (±1) |
|
2500 |
123 (±12) |
21 (±2) |
36 (±6) |
26 (±2) |
8 (±2) |
|
5000 |
116 (±9) |
23 (±1) |
36 (±3) |
25 (±8) |
8 (±3) |
|
0 |
107 (±8) |
16 (±1) |
29 (±5) |
35 (±3) |
11 (±3) |
|
313 |
114 (±8) |
17 (±2) |
35 (±2) |
31 (±3) |
13 (±3) |
S9 mix |
625 |
115 (±12) |
20 (±2) |
42 (±2) |
35 (±2) |
11 (±1) |
(+) |
1250 |
116 (±10) |
20 (±1) |
43 (±5) |
39 (±3) |
12 (±0) |
|
2500 |
125 (±18) |
21 (±3) |
34 (±3) |
36 (±3) |
11 (±1) |
|
5000 |
131 (±24) |
21 (±3) |
37 (±3) |
39 (±3) |
12 (±2) |
+ve control |
Chemical |
AF-2 |
SA |
ENNG |
AF-2 |
9-AA |
S9 mix(-) |
Doseµg/plate |
0.01 |
0.5 |
2 |
0.1 |
80 |
|
Colonies/plate |
514 (±14) |
441 (±30) |
833 (± 42) |
549 (±40) |
313 (±6) |
+ve control |
Chemical |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
S9 mix(+) |
Doseµg/plate |
1 |
2 |
10 |
0.5 |
2 |
|
Colonies/plate |
1301 (±82) |
228 (±3) |
1092 (±28) |
461 (±10) |
189 (±13) |
AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; SA = sodium azide; ENNG = N-ethyl-n’-nitro-N-nitrosoguanidine; 9-AA = 9-aminoacridine; 2 -AA = 2 -aminoanthracene
Table 2 - Results of reverse mutation test II
With (+) or |
Dose (µg/plate) |
Number of revertants Mean (±SD) |
||||
without(-) |
Base-pair substitution type |
Frameshift type |
||||
S9 mix |
TA100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
|
|
0 |
151 (±5) |
11 (±2) |
33 (±2) |
20 (±3) |
8 (±2) |
|
313 |
130 (±6) |
10 (±1) |
41 (±5) |
18 (±2) |
8 (±1) |
S9 mix |
625 |
135 (±3) |
10 (±1) |
39 (±2) |
21 (±5) |
7 (±2) |
(-) |
1250 |
135 (±13) |
10 (±3) |
39 (±7) |
17 (±2) |
9 (±3) |
|
2500 |
142 (±6) |
10 (±1) |
42 (±1) |
19 (±2) |
11 (±1) |
|
5000 |
140 (±20) |
9 (±1) |
41 (±1) |
18 (±1) |
8 (±1) |
|
0 |
111 (±6) |
10 (±1) |
40 (±3) |
28 (±2) |
17 (±2) |
|
313 |
135 (±13) |
10 (±2) |
48 (±3) |
26 (±3) |
13 (±2) |
S9 mix |
625 |
151 (±17) |
10 (±1) |
45 (±8) |
29 (±5) |
16 (±3) |
(+) |
1250 |
135 (±24) |
12 (±1) |
45 (±7) |
30 (±4) |
13 (±4) |
|
2500 |
146 (±8) |
10 (±1) |
41 (±4) |
31 (±10) |
16 (±4) |
|
5000 |
140 (±5) |
12 (±2) |
39 (±2) |
29 (±3) |
13 (±3) |
+ve control |
Chemical |
AF-2 |
SA |
ENNG |
AF-2 |
9-AA |
S9 mix(-) |
Doseµg/plate |
0.01 |
0.5 |
2 |
0.1 |
80 |
|
Colonies/plate |
633 (±27) |
485 (±8) |
929 (±39) |
531 (±53) |
436 (±15) |
+ve control |
Chemical |
2-AA |
2-AA |
2-AA |
2-AA |
2-AA |
S9 mix(+) |
Doseµg/plate |
1 |
2 |
10 |
0.5 |
2 |
|
Colonies/plate |
1230 (±38) |
250 (±2) |
1305 (±68) |
510 (±19) |
190 (±27) |
AF-2 = 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; SA = sodium azide; ENNG = N-ethyl-n’-nitro-N-nitrosoguanidine; 9-AA = 9-aminoacridine; 2 -AA = 2 -aminoanthracene
Table 1 - Chromosome analysis of cells following short-term treatment with and without S9 mix
Group |
Concentration (μg/mL) |
S9 |
Exposure Time (hours) |
Cell |
No. of cells analysed |
No. of structural aberrations |
Number of cells with aberrations (%) |
Polyploidy |
||||||
mix |
growth index |
gap |
ctb |
cte |
csb |
cse |
frg |
Total |
(%) |
|||||
Solvent control |
0 |
- |
6 |
100 |
200 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 (0.5) |
0.0 |
|
1250 |
- |
6 |
107 |
200 |
0 |
1 |
0 |
0 |
0 |
0 |
1 |
1 (0.5) |
0.0 |
Test substance |
2500 |
- |
6 |
111 |
200 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
1 (0.5) |
0.0 |
|
5000 |
- |
6 |
107 |
200 |
0 |
1 |
0 |
2 |
0 |
0 |
3 |
3 (1.5) |
0.0 |
MMC |
0.1 |
- |
6 |
113 |
200 |
4 |
17 |
76 |
1 |
2 |
0 |
96 |
91 (45.5) |
0.0 |
Solvent control |
0 |
+ |
6 |
100 |
200 |
1 |
1 |
1 |
2 |
0 |
0 |
4 |
3 (1.5) |
0.0 |
TOTM |
1250 |
+ |
6 |
105 |
200 |
0 |
2 |
0 |
1 |
2 |
0 |
5 |
5 (2.5) |
0.0 |
T0TM |
2500 |
+ |
6 |
103 |
200 |
0 |
0 |
0 |
1 |
1 |
0 |
2 |
2 (1.0) |
0.0 |
TOTM |
5000 |
+ |
6 |
94 |
200 |
0 |
1 |
1 |
0 |
0 |
0 |
2 |
2 (1.0) |
0.0 |
BP |
20 |
+ |
6 |
133 |
200 |
2 |
14 |
144 |
0 |
2 |
0 |
160 |
147 (73.5) |
0.0 |
Abbreviations:
gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring etc.),frg: fragment,MMC: mitomycin C,BP: benzo(a)pyrene
Table 2 - Chromosome analysis of cells following continuous treatment without S9 mix
Group |
Concentration (μg/mL) |
S9 |
Exposure Time (hours) |
Cell |
No. of cells analysed |
No. of structural aberrations |
Number of cells with aberrations (%) |
Polyploidy |
||||||
mix |
growth index |
gap |
ctb |
cte |
csb |
cse |
frg |
Total |
(%) |
|||||
Solvent control |
0 |
- |
24 |
100 |
200 |
1 |
0 |
1 |
1 |
0 |
0 |
2 |
2 (1.0) |
0.0 |
|
1250 |
- |
24 |
98 |
200 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
0 (0.0) |
0.0 |
Test substance |
2500 |
- |
24 |
104 |
200 |
0 |
0 |
1 |
1 |
0 |
0 |
2 |
2 (1.0) |
0.0 |
|
5000 |
- |
24 |
107 |
200 |
2 |
1 |
0 |
0 |
0 |
0 |
1 |
1 (0.5) |
0.0 |
MMC |
0.03 |
- |
24 |
103 |
200 |
3 |
20 |
38 |
2 |
2 |
0 |
62 |
61 (30.5) |
0.0 |
Abbreviations:
gap: chromatid gap and chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange (dicentric and ring etc.),frg: fragment,MMC: mitomycin C
In Mutation assay I (3 hours exposure), slight precipitation was noted upon addition of the highest concentration of the test item to the cultures both in the absence and presence of S9 metabolism. No precipitation was observed in Mutation assay II (24 hours exposure).
Solvent and positive control cultures were included in each mutation experiment and mutant frequencies in the solvent control cultures fell within the normal range. Both positive control items resulted in an increase in the small colony mutant frequency above that seen in the solvent control.
The cloning efficiencies at Day 2 in the negative control cultures fell within the range of 65 -120%. The control suspension growth over 2 days fell within the range of 8 - 32 for 3 hour treatments and 32 - 180 for 24 hour treatment.
Mild toxicity was observed at the intermediate dose levels in the 3 hour treatment series in the absence of S9 metabolic activation. No relevant toxicity was observed at any concentration tested, with the remaining treatment conditions.
In all experimental conditions examined, no increase in mutation frequency above the concurrent negative control value exceeded the Global Evaluation Factor.
In Main Assay I (3 hours exposure), in the absence of S9 metabolic activation, higher than usual heterogeneity was observed between replicate cultures for mutation at 1.00 µL/mL, therefore this concentration
has been excluded from the statistical analysis. This finding did not affect the validity of the test.
No statistically significant dose-effect relationship was observed at any treatment time, in the absence or presence of S9 metabolism.
The test substance did not have any obvious effect on the osmolality or pH of the treatment medium.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
All three in vitro methods show a negative response with the test substance, therefore in vivo testing at 7.6.2 is not regarded as necessary.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: gene mutation
- Data waiving:
- study technically not feasible
- Justification for data waiving:
- other:
- Justification for type of information:
- No indication of a postive result for 3 in vitro studies; bacterial reverse mutation (OECD 471), in vitro chromosome aberration (OECD 473), or in vitro gene mutation assay (OECD 490) with or without metabolic activation
- Reason / purpose for cross-reference:
- data waiving: supporting information
- Reason / purpose for cross-reference:
- data waiving: supporting information
- Reason / purpose for cross-reference:
- data waiving: supporting information
Reference
Additional information
The substance, an ester of 1,2,4 -benzenetricarboxylic acid with C8 linear side chains, has been tested in:
a bacterial reverse mutation assays (Ames test) which showed the substance not to induce reverse mutation in Salmonella typhimurium or Escherichia coli;
an in-vitro chromosome aberration test in cultured human lymphocytes which showed the substance not to induce chromosomal aberrations;
in an in vitro mutagenisis assay with mouse lymphoma L5178Y cells, which was also negative.
(Q)SAR results by OECD toolbox and Danish (Q)SAR database indicates that the substance is not predicted to exhibit mutagenic activity.
A structurally related substance, an ester of 1,2,4 -benzenetricarboxylic acid with mixed C8 and C10 linear side chains, has been examined in the bacterial reverse mutation test, chromosome aberration test and also in an assay for the induction of 5‑trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment. The structurally related substance was negative in all these in vitro tests too.
REACH Regulation 1907/2006 (Annex VIII, 8.4 Column 2) states that appropriate in-vivo mutagenicity studies should be considered in those cases of a positive result in any of the in vitro genotoxicity studies. In vitro investigations with the substance and two structurally similar substances were negative and in vivo studies are therefore regarded as inappropriate and not in line with current concerns regarding animal welfare and the use of animals in scientific experiments.
Short description of key information:
Genetic toxicity in-vitro: Negative
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
Non-classification is justified on the basis of negative findings in a number of in-vitro tests on the substance and a structurally similar substance for gene mutation / mutagenicity.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
