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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:
from 30th April to 14th may 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with international recognized guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: crystalline powder
Details on test material:
Name:DICYCLOHEXYLPHTHALATE
Batch:031-12
Expiry date:27-Jan-2013
Received from: COIM S.p.A.
Date received: 07-Mar-2012
Amount received:100 g
Description by the Sponsor:white crystalline powder
Description at first use:white powder
Storage at RTC :room temperature

Method

Target gene:
not applicable
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver microsomes (S9)
Test concentrations with justification for top dose:
Preliminary test:
The test item was test at 50, 158, 500, 1580 and 5000 µg/plate

Main test ( I experiment, without preincubation):
313, 625, 1250, 2500, 5000 µg/plate

Main test (II experiment with preincubation):
156, 313, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535, TA 100 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537, without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98 and TA 100, WP2 uvr A.
Details on test system and experimental conditions:
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.
Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation
The second experiment was performed using a pre-incubation method.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:
Regression analysis.
i)The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as:

y = a + bx
where y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).

ii)The regression line does not include the untreated control data, but includes the solvent control data.

iii)Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

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
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item DICYCLOHEXYLPHTHALATE was assayed in the toxicity test at a maximum dose level of 5000 g/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.

Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration. No toxicity was observed with any tester strain at any dose level, in the presence or absence of S9 metabolism.
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 the test item DICYCLOHEXYLPHTHALATE does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.
Executive summary:

The test item DICYCLOHEXYLPHTHALATE was examined for the ability to induce gene mutations in tester strains ofSalmonella typhimuriumandEscherichia coli,as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

The test item DICYCLOHEXYLPHTHALATE was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.

In Main Assay I, using the plate incorporation method, the test item was assayed at the maximum dose level of 5000µg/plate and at four lower dose levels spaced by two-fold dilutions: 2500, 1250, 625 and 313 µg/plate. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations. As no increases in revertant numbers were observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the dose levels of 5000, 2500, 1250, 625, 313 and 156 µg/plate. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item DICYCLOHEXYLPHTHALATE does not induce reverse mutation inSalmonella typhimuriumorEscherichia coliin the absence or presence of S9 metabolism, under the reported experimental conditions.