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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

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
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)
Controlsopen allclose all
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

Results and discussion

Test results
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:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

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.