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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restriction; study was conducted according to OECD Guideline 471 and GLPs

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
No guideline deviations; slight change in the study plan did not impact the quality or integrity of the study.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Test Substance:
-Identity: Dibutyl terephthalate
-Batch No.: X29455-190-1
-Aggregate state at room temperature: Liquid
-Color: Colourless
-Purity: 98.6 %
-Solubility in water: Unsoluble (0.06 mg/L)
-Stability in solvent: Not indicated
-Storage: At room temperature
-Expiration Date: July 31, 2007

Method

Target gene:
His (-), Trp (-)
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 bacterial strains TA 1535, TA 1537, TA 98, and TA 100 were obtained from Trinova Biochem GmbH (Gießen, Germany).
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
WP2 uvr A was obtained from Trinova Biochem GmbH (Gießen, Germany).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ β-Naphthoflavone induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate
Vehicle / solvent:
DMSO (Merck, Darmstadt, Germany; purity > 99 %)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Sodium azide (SERVA, Heidelberg, Germany; purity = 99%) was used with tester strains TA 1535 and TA 100 at a concentration of 10 µg/plate without metabolic activation.
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
4-nitro-o-phenylene-diamine (Sigma, Deisenhofen, Germany; purity > 99.9%) was used with tester strains TA 1537 and TA 98 at a concentration of 10 µg/plate (TA 98) and 50 µg/plate (TA 1537) without metabolic activation.
Positive control substance:
methylmethanesulfonate
Remarks:
Methylmethanesulfonate (Merck-Schuchardt, Hohenbrunn, Germany; purity > 99.0%) was used with tester strain WP2 uvr A at a concentration of 3 µL/plate without metabolic activation.
Positive control substance:
other: 2-aminoanthracene
Remarks:
2-aminoanthracene (Sigma, Deisenhofen, Germany; purity = 97.5%) was used at a concentration of 2.5 µg/plate (TA 98, TA 100, TA 1535, TA 1537) and 10 µg/plate (WP2 uvr A) with metabolic activation.
Details on test system and experimental conditions:
Study Dates:
-Experimental Starting Date: September 01, 2006
-Experimental Completion Date: September 13, 2006

Preparation of S9:
S9 was prepared from 8-12 week old male Wistar Hanlbm rats (weight approx. 220-320 g), induced by administration of 80 mg/kg bw Phenobarbital (i.p.) and β-Naphthoflavone (p.o.) each on three consecutive days. Liver homogenates were prepared 24 hours after the last treatment. Liver homogenate was diluted with KCl solution followed by centrifugation at 9000 g. Aliquots of supernatant were frozen and stored at -80 °C. The protein concentration in the S9 preparation was 31.7 mg/mL (lot no. R280706) in both experiments.

S9 Mix:
The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP, in 100 mM sodium-ortho-phosphate buffer, pH 7.4.

Pre-Experiment/Experiment I:
The dose rangefinding study used the plate incorporation method and was performed with eight dose concentrations up to 5000 µg/plate on all tester strains in the presence and absence of metabolic activation. If no relevant toxic effects are observed, the pre-experiment is reported as Experiment I.

Experiment II:
The tester strains were exposed to six dose concentrations of the test substance via the pre-incubation test.

Plating Procedure:
For each strain and dose level including the controls, three plates were used. For the plate incorporation method, the following materials were mixed in a test tube and poured onto the selective agar plates: 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control); 500 µL S9 mix (for test with metabolic activation), or S9 mix substitution buffer (for test without metabolic activation); 100 µL bacteria preculture suspension; and 2000 µL overlay agar. The bacterial preculture suspension was prepared using the method described in "other information on material and methods".

In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on selective agar plates.

After solidification, the plates were incubated upside down for at least 48 hours at 37°C in the dark.

Plate Scoring and Counting:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk, UK) with a software program. The counter was connected to an IBM AT compatible PC with printer to print out the individual values and the means from the plates for each concentration together with standard deviations; the enhancement factors, as compared to spontaneous reversion rates, were also printed. Due to precipitation of the test material, some plates were counted manually.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of 2X (strains TA 98, TA 100, and WP2 uvr A) or 3X (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant only if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, if the colony counts remain within the historical range of negative and solvent controls, such an increase is not considered biologically relevant.
Statistics:
No statistical analysis was performed on this study. Statistical analysis of data collected in this type of study is not mandatory according to OECD Guideline 471.

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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. An isolated reduction in the number of revertants was observed at 100 µg/plate in strain TA 1537 with metabolic activation in experiment I. This result was considered to be spurious.

No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment with dibutyl terephthalate with and without metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It is concluded that, under the conditions of this test, dibutyl terephthalate showed no evidence of mutagenic activity in this bacterial system with and without metabolic activation.

Based on the absence of genotoxic or mutagenic effects in this study with and without metabolic activation, the substance is not classified for germ cell mutagenicity according to GHS.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535 and TA 1537 of S. typhimurium and strain WP2 uvr A of E. coli were exposed to dibutyl terephthalate in DMSO at concentrations up to 5000 µg/plate in the presence and absence of mammalian metabolic activation using the pre-incubation method and the plate incorporation method. Under the conditions of the test, dibutyl terephthalate did not induce gene mutations by base pair changes or frameshifts, while positive control substances induced appropriate responses. Dibutyl terephthalate was considered to be non-mutagenic in this study.