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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Four in vitro mutagenicity tests are performed. All tests show no evidence of genetic toxicity in vitro. No classification according to CLP regulation (EC) No 1272/2008 is needed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07.11.2014 to 01.07.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his D 3052 (strain TA98#), his G 46 (strain TA100#, TA1535# ), his G 428 (strain TA102#), his C 3076 (strain TA1537#)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: Histidine deficiency
Metabolic activation:
with and without
Metabolic activation system:
S9 - mix
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 3160 or 5000 μg Di-(iso)-pentyl terephthalate (DPT) per plate
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
10 µg / plate
Positive control substance:
sodium azide
Remarks:
TA 1535 and TA 100 without metabolic activation in highly purified water
Positive controls:
yes
Remarks:
10 µg / plate
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without metabolic activation in DMSO
Positive controls:
yes
Remarks:
100 µg / plate
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without metabolic activation in ethanol, abs.
Positive controls:
yes
Remarks:
10 µg / plate
Positive control substance:
mitomycin C
Remarks:
TA 102 without metabolic activation in highly purified water
Positive controls:
yes
Remarks:
10 µg / plate
Positive control substance:
benzo(a)pyrene
Remarks:
TA 98, TA 102 and TA 1537 with metabolic activation in DMSO
Positive controls:
yes
Remarks:
2 µg / plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 100 and TA 1535 with metabolic activation in DMSO
Untreated negative controls:
yes
Remarks:
The vehicle dimethylsulfoxide (DMSO) was used as negative reference item (all test strains).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (First expermient: plate incorporation and Second experiment: preincubation)
- Cell density at seeding (if applicable): 10^8 viable cells in the late exponential or early stationary phase

DURATION
- Preincubation period: The test item was preincubated with the test strain (containing approximately 108 viable cells in the late exponential or early stationary phase) and sterile buffer (0.5 mL) or the metabolic activation system (0.5 mL) for 20 minutes at 37°C prior to mixing with the overlay agar and pouring onto the surface of a minimal agar plate.
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

DETERMINATION OF CYTOTOXICITY
- Method: colony reduction
- Any supplementary information relevant to cytotoxicity: Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is significantly increased compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

The range of spontaneous reversion frequencies per plate is based on Kirkland :
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20

The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment.

The results of the negative and positive control cultures have to be within the range of the historical data generated by LPT.
Statistics:
None reported
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA102, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 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
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
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 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
Conclusions:
In conclusion, under the present test conditions Di-(iso)-pentyl terephthalate (OPT)
tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the
Salmonella typhimurium strains TA98, TA 100, TA 102, TA 1535 and TA 1537
neither in the plate incorporation test nor in the preincubation test each carried out
without and with metabolic activation.
Executive summary:

The potential of Di-(iso)-pentyl terephthalate (DPT) to induce gene mutation was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. Di-(iso)-pentyl terephthalate (DPT) was completely dissolved in dimethylsulfoxide (DMSO). The vehicle DMSO served as the negative control.

Preliminary test

Di-(iso)-pentyl terephthalate (DPT) was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 μg Di-(iso)-pentyl terephthalate (DPT)/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 μg/plate. Hence, 5000 μg Di-(iso)-pentyl terephthalate (DPT)/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations, 31.6, 100, 316, 1000, 3160 or 5000 μg Di-(iso)-pentyl terephthalate (DPT)/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 μg Di-(iso)-pentyl terephthalate (DPT)/plate in any test strain.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for Di-(iso)-pentyl terephthalate (DPT), tested up to a concentration of 5000 μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01.09.2014 to 01.07.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: In vitro mammalian chromosomal aberration test
Species / strain / cell type:
other: human lymphocyte cultures
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human peripheral blood was obtained by venipuncture
- Sex, age and number of blood donors if applicable: young (age: between 18 and 35 years) non-smoking, healthy donors known to be without any medication
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
mammalian metabolic activation system
Vehicle / solvent:
dimethylsulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Positive control for the study in the absence of metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Positive control for the study in the presence of metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
Two independent experiments without (24 and 4-hour exposure) and one experiment with metabolic activation (4-hour exposure) were carried out.

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid®

STAIN (for cytogenetic assays):
Giemsa stain


METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The tubes were centrifuged, the fixative removed and the cell pellet resuspended in a few drops of 60% acetic acid. Single drops of the cell suspension were spread on clean, grease-free glass slides on a hot plate (approx. 50°C) and the slides were left to air-dry. Two slides were prepared per culture, stained for 30 minutes in Giemsa stain (1:10 in WEISE's buffer11 pH 6.8), washed in buffer and left to air-dry. Permanent slides were made using CONSUL MOUNT mountant after clearing in xylene.


NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
For each treatment and culture 150 metaphases were examined.

NUMBER OF CELLS EVALUATED:
To examine the toxicity of the test item, 1000 cells were scored and the mitotic index was calculated as the percentage of cells in metaphase.
Evaluation criteria:
Observed aberrations were noted and scored according to J. R. K. SAVAGE (1975).
In addition, the total number of gaps was recorded for each culture, but excluded from evaluation.
Metaphases which differed from the normal diploid complement (46) were excluded from evaluation. However, test item-related variations of the normal chromosome number were noted (polyploidy / endoreduplication).

Statistics:
The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p  0.05) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, Statistical evaluation of mutagenicity test data, 1989).
Key result
Species / strain:
other: human lymphocyte cultures
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the present test conditions, Di-(iso)-pentyl-terephthalate (DPT) tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation,
revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.
Executive summary:

Tests without metabolic activation (4- and 24-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Di-(iso)-pentyl-terephthalate (DPT) at concentrations ranging from 62.5 to 500 or 15.6 to 250 μg/mL medium (4- and 24-hour exposure) in the absence of metabolic activation ranged from 0.3% to 2.1%. These results are within the range of the historical control data (0 - 4%). The vehicle control cultures had a mean of 0.7% cells with aberrations (excluding gaps), and the positive control cultures had a significantly increased mean frequency of cells with aberrations of 10.3% (4-hour exposure) or 11.3 (24-hour exposure), which was in line with the historical control range. Therefore, the test is considered to be valid.

Test with metabolic activation (4-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Di-(iso)-pentyl-terephthalate (DPT) at concentrations from 125 to 1000 J.lg/ml medium in the presence of metabolic activation ranged from 0.3% to 1.0%. These results were within the range of the historical control data (0 - 4%). The vehicle control cultures had a mean of 1.0% cells with aberrations (excluding gaps), and the positive control cultures had a significantly increased mean frequency of cells with aberrations of 1 0.0%, which was in line with the historical control range. Therefore, the test is considered to be valid.

No test item-related polyploidy or endoreduplication was noted in the experiments without or with metabolic activation.

The range of incidence of cells with chromosomal aberrations (excluding gaps) of the solvent controls and positive controls Mitomycin C and cyclophosphamide, without and with metabolic activation, for experiments of the years 2013 to 2014 (most recent background data) is shown below:

   % of cells with aberations excluding gaps#         
   Without metabolic activation    With metabolic activation     
   Solvent controls  Positiv controls (Mitomycin C)   Solvent controls  Positive control (cyclophosphamide)
mean  0.7 10.5  0.7  10.7
 SD  0.8  2.3  0.8  2.3
 range  0.0 - 4.0  7.0 - 19.0  0.0 - 4.0  8.0 - 21.0

# - data obtained from analysis of 100 meta phases in 22 experiments performed during 2013 to 2014; the control data of this study are not included in the background data.

SD - standard deviation

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12.01.2015 to 01.07.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
other: human peripheral lymphocytes
Details on mammalian cell type (if applicable):
Human peripheral blood was obtained by venipuncture from young (approximately 18 – 35 years of age), healthy, non-smoking male or female individuals with no known recent exposures to genotoxic chemicals or radiation.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
CytoB (Cytochalasin B)
Metabolic activation:
with and without
Metabolic activation system:
Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254, prepared according to MARON and AMES (1983) was purchased from Trinova Biochem . S9 was collected from male rats.
Test concentrations with justification for top dose:
In the preliminary experiment without and with metabolic activation concentrations of 0.078, 0.156, 0.313, 0.625, 1.25, 2.5 and 5 µL Di-(iso)-pentyl terephthalate (DPT)/mL medium were employed.
Cytotoxicity was noted at the concentrations of 0.625 µL (4-hour exposure) or 0.313 µL (20-hour exposure) test item/mL medium and higher. Hence, 0.625 µL test item/mL were employed as top concentration for the experiments with 4-hour exposure (with and without metabolic activation) or 0.313 µL test item/mL in the experiment with 20-hour exposure (without metabolic activation).
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.2 μg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: colchicine
Remarks:
0.02 μg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
20 μg/mL
Details on test system and experimental conditions:
At least 500 cells per replicate cell culture (two cultures per concentration in the main study, one culture per concentration in the preliminary test) were scored and classified as mononucleates, binucleates or multinucleates to estimate the proliferation index as a measure of toxicity. The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI).
Thus, an RI of 53% means that, compared to the numbers of cells that have divided to form binucleate and multinucleate cells in the control culture, only 53% of this number divided in the treated culture, i.e. 47% cytostasis.
All slides, including those of the solvent controls, were independently coded before the microscopic analysis.
The micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration (at least 1000 binucleated cells per culture; two cultures per concentration). If substantially fewer than 1000 binucleate cells per culture are available for scoring at each concentration, and if a significant increase in micronuclei is not detected, the test would be repeated using more cells, or at less toxic concentrations, whichever is appropriate. Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.
Statistics:
Only the frequencies of binucleate cells with micronuclei (independent of the number of micronuclei per cell) were used in the evaluation of micronucleus induction. Concurrent measures of cytotoxicity and/or cytostasis for all treated and vehicle control cultures were determined. Individual culture data were provided.
If a test item induces a concentration-related increase or a statistical significant and reproducible increase in the number of cells containing micronuclei, it is classified as a positive result.
Consideration of whether the observed values are within or outside of the historical control range can provide guidance when evaluating the biological significance of the response.
The assessment was carried out by a comparison of the samples with the positive and the vehicle control, using a chi-square test corrected for continuity according to YATES (COLQUHOUN, 1971[1]) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989: Statistical evaluation of mutagenicity data).
Key result
Species / strain:
lymphocytes: human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the present test conditions, Di-(iso)-pentyl terephthalate (OPT) tested up to cytotoxic concentrations in the absence and
in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed
no indications of chromosomal damage in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant
chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively.
Therefore, the test is considered valid.
Executive summary:

Tests without metabolic activation (4- and 20-hour exposure):

The micronucleus frequencies of cultures treated with the concentrations of 0.078, 0.156, 0.313 or 0.625 μL Di-(iso)-pentyl terephthalate (DPT)/mL medium (4-hour exposure) or 0.039, 0.078, 0.156 or 0.313 μL Di-(iso)-pentyl terephthalate (DPT)/mL medium (20-hour exposure) in the absence of metabolic activation ranged from 2.0 to 5.5 micronuclei per 1000 binucleated cells. There was no dose related increase in micronuclei up to the top concentrations of 0.625 or 0.313 μL Di-(iso)-pentyl terephthalate (DPT)/mL medium. The micronuclei frequency was within the historical control range of the untreated and vehicle control. Vehicle controls should give reproducibly low and consistent micronuclei frequencies. In this test the following frequencies were observed: vehicle control: 2.5 or 1.5 micronuclei per 1 000 binucleated cells for the 4-hour and 20-hour exposure, respectively. The vehicle results were within the historical control ranges.

Test with metabolic activation (4-hour exposure):

The micronucleus frequencies of cultures treated with the concentrations of 0.078, 0.156, 0.313 or 0.625 fll Di-(iso)-pentyl terephthalate (DPT)/ml medium (4-h exposure) in the presence of metabolic activation ranged from 2.5 to 4.5 micronuclei per 1 000 binucleated cells. There was no dose related increase in micronuclei up to the top concentration of 0.625 fll Di-(iso)-pentyl terephthalate (DPT)/ml medium. The micronuclei frequency was within the historical control range of the untreated and vehicle control. Vehicle controls should give reproducibly low and consistent micronuclei frequencies. In this test a mean frequency of 2.5 micronuclei per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-4
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Clone: 3.7.2C
Supplier: ATCC (American Type Culture Collection),
0801 University Blvd., Manassas, VA
20110-2209, USA
Additional strain / cell type characteristics:
other: THeterozygous at hymidine kinase locus +/-
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The following test concentrations were tested in a preliminary cytotoxicity study: 10.0, 31.6, 100, 316, 1000, and 2000 µg Di(iso)-pentyl terephthalate (DPT)/mL medium.
Based on the results of the preliminary study the following concentrations were employed in the mutagenicity tests:
3.13, 6.25, 12.5, 25, and 50 µg for the experiments without metabolic activation
31.3, 62.5, 125, 250, and 500 µg for the experiments with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10^5 cells/mL

DURATION
Assay without metabolic activation:
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): At the end of the expression period, a minimum of 4 concentration levels plus positive and negative controls were selected for the determination of survival (plating efficiency) and 5-trifluoro-thymidine (TFT) resistance. After 1 week incubation, the number of viable clones was recorded.

Assay with metabolic activation:
-Exposure duration: 3 hours

SELECTION AGENT (mutation assays): 5-trifluoro-thymidine (TFT)

NUMBER OF CELLS EVALUATED: The mutant frequency is expressed as mutants/10^6 viable cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity:
Cytotoxicity is defined as the Relative Total Growth (RTG) which includes the Relative Suspension Growth (RSG) during the 2 day expression period and the Relative Plating Efficiency (RPE) obtained at the time of mutant selection.

OTHER EXAMINATIONS:
- Mutant frequency
-Analysis of mutant colony size
Rationale for test conditions:
Thymidine kinase (TK) is a cellular enzyme that allows cells to salvage thymidine from the surrounding medium for use in DNA synthesis. If the thymidine analogue 5 trifluoro-thymidine (TFT) is included in the growth medium, the analogue will be phosphorylated via the TK pathway and will cause cell death by inhibiting DNA synthesis. Cells, which are heterozy¬gous at the TK locus (TK +/-), may undergo a single-step forward mutation to the TK-/- genotype in which little or no TK activity remains. Such mutants are as viable as the heterozygotes in normal medium because DNA synthesis may still proceed by de novo synthetic pathways that do not involve thymidine as an intermediate. The basis for selection of the TK / mutants is the lack of any ability to utilize toxic analogues of thymidine, which enable only the TK / mutants to grow in the presence of TFT. Cells, which grow to form colonies in the presence of TFT, are therefore assumed to have mutated, either spontaneously or induced by the test item, to the TK-/- genotype.
Two types of mutated cells can be distinguished, large, normal-growing colonies and small, slow-growing colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions or deletions), whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations. Thus, in this system, gene mutations within the tk gene and chromosomal events involving the gene may be detected and distinguished.
Evaluation criteria:
For the MLA, significant work on biological relevance and criteria for a positive response has been conducted by The Mouse Lymphoma Expert Workgroup of the IWGT (M.M. Moore et al., 2006). Therefore, the interpretation of test chemical results is based on those recommendations:
To define positive and negative results and to assure that the increased MF is biologically relevant instead of a statistical analysis (generally used for other tests), the interpretation relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated as the Global Evaluation Factor (GEF). The GEF (126 x 10-6) is based on the analysis of the distribution of the negative control MF data from participating laboratories (M.M. Moore et al., 2006).
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
In cases when the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result the data is evaluated by expert judgement and/or further investigations.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Cytotoxicity was noted at concentrations of 25 and 50 µg/mL in the absence of S9 (3- or 24-hour exposure). No signs of cytotoxicity were noted in the experiments with S9 up to the highest concentration of 500 µg DPT /mL medium.
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/ osmolarity: No relevant changes in pH or osmolality of the test item formulations at concentrations of 10.0 to 2000 µg/mL medium compared to the negative control were noted.
- Precipitation: In the main study with metabolic activation, test item precipitation was noted at concentrations of 250 and 500 µg DPT/mL medium.

RANGE-FINDING/SCREENING STUDIES:
A preliminary study was conducted to establish the highest concentration for the main study. This study was performed without and with metabolic activation. A wide range of test item concentrations of 10.0, 31.6, 100, 316, 1000, and 2000 µg Di(iso)-pentyl terephthalate (DPT)/mL medium were tested for cytotoxicity. In two preliminary experiments without and with metabolic activation, test item precipitation was noted at concentrations of 316 µg DPT/mL medium and higher. Cytotoxicity (decreased survival) was noted starting at a concentration of 31.6 µg/mL in the absence of metabolic activation (24-hour exposure) and starting at 316 µg/mL in the presence of metabolic activation (3 hour exposure). No changes in pH or osmolality were noted in the test item formulations compared to the control.

Conclusions:
Under the present test conditions, Di(iso)-pentyl terephthalate (DPT), tested up to concentrations that led to cytotoxicity or test item precipitation, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions, the positive controls exerted potent mutagenic effects and demonstrated the sensitivity of the test system and conditions.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, Di(iso)-pentyl terephthalate (DPT) also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that Di(iso)-pentyl terephthalate (DPT), tested up to concentrations that led to cytotoxicity or test item precipitation, neither induced mutations nor had any chromosomal aberration potential.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Mouse Lymphoma forward mutation assay (in vitro) according to OECD TG 490:

Under the present test conditions, Di(iso)-pentyl terephthalate (DPT), tested up to concentrations that led tocytotoxicity ortest item precipitation, in two independent experiments was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, DPT also did not exhibit clastogenic potential at the concentration-range investigated. These findings indicate that Di(iso)-pentyl terephthalate (DPT), tested up to concentrations that led to cytotoxicity or test item precipitation, neither induced mutations nor had any chromosomal aberration potential.

Reverse mutation assay (in vitro) according to OECD TG 471:

Under the present test conditions Di-(iso)-pentyl terephthalate (DPT) tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

In vitro assessment of the clastogenic activity in cultered human peripheral lymphocytes according to OECD 473:

Di-(iso)-pentyl-terephthalate (DPT) tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation, revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

In vitro micronucleus test in cultured human peripheral lymphocytes according to OECD 487

Di-(iso)-pentyl terephthalate (DPT) tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Justification for classification or non-classification

No classification for genetic toxicity is indicated according to the classification, labeling, and packaging (CLP) regulation (EC) No 1272/2008.