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Toxicological information

Genetic toxicity: in vitro

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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:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Reliable without restriction; study was conducted by methods similar to OECD Guideline 471.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Guideline states E. coli WP2 uvrA, E. coli WP2 uvrA (pKM101), or S. typhimurium TA 102 should be one of the strains used in standard testing. In this study, S. typhimurium 1538 was used as 5th tester strain. This does not impact the quality of the study.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) terephthalate
EC Number:
229-176-9
EC Name:
Bis(2-ethylhexyl) terephthalate
Cas Number:
6422-86-2
Molecular formula:
C24H38O4
IUPAC Name:
bis(2-ethylhexyl) terephthalate
Constituent 2
Reference substance name:
Reference substance 001
Cas Number:
6422-86-2
Details on test material:
-Test material (as cited in study report): Di(2-ethylhexyl) terephthalate (DEHT)
-Synonyms: Kodaflex® DOTP, dioctyl terephthalate
-Supplier: Eastman Chemical Products, Kingsport, TN
-Purity: 98.4% as determined by gas chromatography with mass spectroscopy. The impurities were identified as 0.6% methyl-2-ethylhexyl terephthalate and 1% unidentifiable impurities.

Method

Target gene:
His (-)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The tester strains were obtained from Dr. Bruce Ames (University of California, Berkeley, CA)
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
The tester strain was obtained from Dr. Bruce Ames (University of California, Berkeley, CA)
Metabolic activation:
with and without
Metabolic activation system:
Induced Rat Liver S9 (no further details regarding preparation of the metabolic activation system were provided in the publication).
Test concentrations with justification for top dose:
Screening Study:
10 doses from 0.32 to 10000 µg/plate

Mutagenicity Study:
1.0, 10, 100, 1000, 10000 µg/plate
Vehicle / solvent:
ethanol
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-n'-nitro-n-nitrosoguanidine
Remarks:
N-methyl-n'-nitro-n-nitrosoguanidine was used with tester strains TA 100 and TA 1535 at dose concentrations of 0.75 and 2.0 µg/plate, respectively, without metabolic activation.
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
9-Aminoacridine was used with tester strain TA 1537 at a dose concentration of 75 µg/plate without metabolic activation.
Positive controls:
yes
Positive control substance:
other: Picrolonic acid
Remarks:
Picrolonic acid was used with tester strain TA 1538 at a dose concentration of 200 µg/plate without metabolic activation.
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
ICR-191 was used with tester strain TA 98 at a dose concentration of 3.0 µg/plate without metabolic activation.
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
2-Aminoanthracene was used with tester strains TA 1535, TA 1537, and TA 1538 at 2.5 µg/plate and TA 98 and TA 100 at 0.6 µg/plate with metabolic activation.
Details on test system and experimental conditions:
Screening Assay:
The screening study used tester strain TA 100 and half-log dose intervals of the test substance from 0.32 µg/plate to 10,000 µg/plate. Tester strain TA 100 was used because of its high spontaneous reversion rate. Spontaneous revertant numbers were counted and plotted against the dose of the test substance, producing a survival curve for the his+ genotype. The three highest doses designated for the mutagenicity assay were those which produced 10, 50, and 90% survival and the lowest two doses were 1/10 and 1/100 of the amount giving 90% survival in the toxicity assay. In this case, where toxicity was absent, doses from 10,000 µg/plate to 1.0 µg/plate were tested.

Mutagenicity Assay:
The mutagenicity assay was performed by mixing approximately 10^8 cells from an overnight tester strain growth culture with a known amount of the test substance, the induced rat liver S9 mixture (when required), and top agar containing a minimal amount of histidine. The resulting mixture was poured onto the surface of a sterile Petri dish containing 25 mL of solidified bottom agar and incubated for 48 hours at 37 °C. Revertant colonies were counted using a Biotran automated colony counter. Vehicle control plates contained no added test chemical and positive control plates containing appropriate amounts of chemicals known to be active were also performed with each tester strain. All platings were done in triplicate.
Evaluation criteria:
A response was considered to be positive if there was a dose-dependent increase in revertants per plate resulting in
(1) at least a doubling of the background reversion rate for strains TA 98 or TA 100, or
(2) at least a tripling of the background reversion rates for strains TA 1535, TA 1537, or TA 1538.
Statistics:
Statistical analyses were not needed due to the absence of an increase in the number of revertant colonies at any dose level beyond that seen with the positive control.

Results and discussion

Test resultsopen allclose all
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:
other: No cytotoxicity was observed at concentrations up to 10,000 micrograms/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: No cytotoxicity was observed at concentrations up to 10,000 micrograms/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The results of the experiment conducted on the test substance with and without metabolic activity were all negative at dose concentrations up to 10,000 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
It is concluded that, under the conditions of this test, di (2-ethylhexyl) terephthalate showed no evidence of mutagenic activity in this bacterial system with and without S9-induced metabolic activation at dose concentrations up to 10,000 µg/plate.

Based on the absence of genotoxic or mutagenic effects in this study with and without metabolic activation, di (2-ethylhexyl) terephthalate is not classified for “Germ Cell Mutagenicity” according to GHS.
Executive summary:

In an Ames reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 of S. typhimurium were exposed to di (2-ethylhexyl) terephthalate in ethanol at concentrations of 1.0, 10, 100, 1000, or 10000 µg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method. Under the conditions of the test, di (2-ethylhexyl) terephthalate did not induce gene mutations by base pair changes or frameshifts, while positive control substances induced appropriate responses. Di (2-ethylhexyl) terephthalate was considered to be non-mutagenic in this study. 

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