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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2009-10-21 to 2009-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Thymidine Kinase Locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: Clone 3.7.2C
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment: 39.1; 78.1; 156.3; 312.5; 625; 1250; 2500; 5000 µg/mL

Experiment I:
without S9 mix (4 hours treatment): 2.4; 4.9; 9.8; 19.5; 39.0; 58.5; 78.0 µg/mL; evaluated: 2.4; 4.9; 9.8; 39 µg/mL
with S9 mix (4 hours treatment): 4.9; 9.8; 19.5; 39.0; 78.0; 117.0; 156.0 µg/mL; evaluated: 4.9; 9.8; 19.5; 39.0; 78.0 µg/mL

Experiment II:
without metabolic activation (24 hours treatment): 2.5; 5.0; 10.0; 20.0; 40.0; 50.0; 60.0 µg/mL; evaluated: 10; 20; 40; 50; 60 µg/mL
with metabolic activation (4 hours treatment): 10.0; 20.0; 40.0; 80.0; 100.0; 110.0; 120.0 µg/mL; evaluated: 40, 80, 100, 110, 120 µg/mL



Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water (10 %)
- Justification for choice of solvent/vehicle: solubility properties
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: approx. 4000 cells per well

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
Statistics:
Linear regression analysis (least squares) using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was chosen with regard to the solubility of the test item in deionised water. Test item concentrations between 39.1 and 5000 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Strong toxic effects were observed at 78.1 µg/mL and above in the absence of metabolic activation (4 h treatment) and at 312.5 µg/mL and above in the presence of metabolic activation. Following continuous treatment (24 hours) toxic effects as described above occurred already at the lowest concentration of 39.1 µg/mL and above.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation occurred up to the maximum concentration with and without metabolic activation at both treatment intervals.
The dose range of the main experiments was limited by toxicity of the test item. The individual concentrations were generally spaced by a factor of 2.0. A closer spacing was used in the upper concentration range to cover toxic effects more closely. A very narrow dose spacing was used in the second experiment with and without metabolic activation to cover the recommended cytotoxic range of approximately 10-20% relative total growth.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment II the mutant frequency exceeded the range of the historical solvent control data at several test points without metabolic activation (both cultures) and at one test point with metabolic activation (culture I). However, the threshold described above was not reached at any test point of the second experiment and no dose dependent increase was indicated by statistical analysis.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative total growth of less than 50 % in both parallel cultures were observed in the absence of metabolic activation at 39 µg/mL in experiment I following 4 hour treatment and at 40 µg/mL and above in experiment II following 24 hours treatment. In the presence of metabolic activation toxic effects as described above occurred at 100 µg/mL and above in experiment II. No reproducible cytotoxic effects were noted in the first experiment with metabolic activation. The recommended toxic range of approximately 10-20 % RTG was covered in the second experiment with and without metabolic activation.
The isolated minor reduction of the relative total growth to 43.5 % in the first culture of experiment I with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table
      relative mutant   relative mutant  
  conc. µg S9 total colonies/   total colonies/  
  per mL mix growth 106cells threshold growth 106cells threshold
Column 1 2 3 4 5 6 7 8
Experiment I / 4 h treatment     culture I culture II
Solv. control with water - 100.0 147 273 100.0 141 267
Pos. control with MMS  19.5 -  32.4 424 273  23.7 403 267
Test item   2.4 - 131.7 122 273  67.5 209 267
Test item   4.9 - 122.7 134 273 129.3 139 267
Test item   9.8 - 103.9 137 273 126.0 104 267
Test item  19.5 -  43.7 275 273  96.9  96 267
Test item  39.0 -  31.1 187 273  39.3 181 267
Test item  58.5 - culture was not continued# culture was not continued#
Test item  78.0 - culture was not continued# culture was not continued#
       
Solv. control with water + 100.0 130 256 100.0 141 267
Pos. control with CPA   3.0 +  61.5 232 256  90.0 250 267
Pos. control with CPA   4.5  +   44.8 320 256  54.6 348 267
Test item   4.9  +   66.2 164 256 137.6  99 267
Test item   9.8  +  104.3 140 256 134.1 146 267
Test item  19.5  +   57.7 202 256 125.6 134 267
Test item  39.0  +  111.4 105 256 147.5  83 267
Test item  78.0  +   43.5 149 256 108.2 154 267
Test item  117.0  +  culture was not continued# culture was not continued#
Test item  156.0  +  culture was not continued# culture was not continued#
Experiment II / 24 h treatment     culture I culture II
Solv. control with water - 100.0 158 284 100.0 153 279
Pos. control with MMS  13.0 -  19.2 569 284  25.0 573 279
Test item   2.5 - culture was not continued## culture was not continued##
Test item

  5.0

- culture was not continued## culture was not continued##
Test item  10.0 -  65.9 217 284  78.7 264 279
Test item  20.0 -  44.3 208 284  71.1 230 279
Test item  40.0 -  33.1 173 284  45.8 208 279
Test item  50.0 -  38.5 182 284  29.8 214 279
Test item  60.0 -  20.7 209 284  16.2 241 279
Experiment II / 4 h treatment     culture I culture II
Solv. control with water + 100.0 164 290 100.0 145 271
Pos. control with CPA   3.0 +  52.2 355 290  25.0 312 271
Pos. control with CPA   4.5 +  23.6 385 290  23.3 407 271
Test item  10.0 + culture was not continued## culture was not continued##
Test item  20.0 + culture was not continued## culture was not continued##
Test item  40.0 +  75.5 171 290 102.9 189 271
Test item  80.0 +  40.2 210 290  61.6 159 271
Test item  100.0 +  47.1 133 290  49.0 149 271
Test item  110.0 +  32.6 183 290  42.0 167 271
Test item  120.0 +  22.0 203 290  29.0 201 271

Threshold = number of mutant colonies per 106cells of each solvent control plus 126

#      culture was not continued due to exceedingly severe cytotoxic effects
##
    culture was not continued since a minimum of only four analysable concentrations is required

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

The study was performed to investigate the potential of C8 -18 AAPB to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix:                     2.4; 4.9; 9.8; 19.5; and 39.0 µg/mL
with S9 mix:                         4.9; 9.8; 19.5; 39.0; and 78.0 µg/mL

Experiment II:

without S9 mix:                                10; 20; 40; 50; and 60 µg/mL

with S9 mix:                               40; 80; 100; 110; and 120 µg/mL

Relevant cytotoxic effects indicated by a relative total growth of less than 50 % in both parallel cultures were observed in the absence of metabolic activation at 39 µg/mL in experiment I following 4 hour treatment and at 40 µg/mL and above in experiment II following 24 hours treatment. In the presence of metabolic activation toxic effects as described above occurred at 100 µg/mL and above in experiment II. No reproducible cytotoxic effects were noted in the first experiment with metabolic activation. The recommended toxic range of approximately 10-20 % RTG was covered in the second experiment with and without metabolic activation.

The isolated minor reduction of the relative total growth to 43.5 % in the first culture of experiment I with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation. The mutation frequency did not reproducibly reach or exceed the threshold of 126 above the mutation frequency of the corresponding solvent control in any of the experimental parts. An isolated increase exceeding the threshold was noted in the first culture of experiment I without metabolic activation at 19.5 µg/mL. However, this increase was judged as irrelevant fluctuation since it was not reproduced in the parallel culture under identical experimental conditions. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. In experiment II the mutant frequency exceeded the range of the historical solvent control data at several test points without metabolic activation (both cultures) and at one test point with metabolic activation (culture I). However, the threshold described above was not reached at any test point of the second experiment and no dose dependent increase was indicated by statistical analysis.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTATâ11statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups.

In this study the range of the solvent controls was from 130 up to 164 mutant colonies per 106cells; the range of the groups treated with the test item was from 83 up to 275 mutant colonies per 106cells. The solvent controls remained within the range of the historical data.

Methylmethanesulfonate (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and cyclophosphamide (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at at least one of the concentrations of the controls.

There was no concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guidelines Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation Test“ and Commission Regulation (EC) No. 440/2008 B.17: ”Mutagenicity –In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008 for in vitro mutagenicity (mammalian forward gene mutation) data.