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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:
experimental study
Adequacy of study:
key study
Study period:
24 November 2016 until 30 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according to OECD 476

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:

Molecular Weight: 344.45 g/mol
Expiry: 31 March 2017
Storage Conditions: At room temperature
Appearance: White powder
Stability in Solvent: Not indicated by the sponsor

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/Beta-Naphtoflavone induced Rat liver S9
Test concentrations with justification for top dose:
4 hours treatment without S9 mix: 1.0; 3.1; 9.3; 27.8 (p); 83.3 (p) µg/mL
4 hours treatment with S9 mix: 1.0, 3.1; 9.3, 27.8 (p); 83.3 (p) µg/mL

p = precipitation visible to the unaided eye at the end of treatment

Vehicle / solvent:
DMSO (1.0% (v/v)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


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): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concen¬trations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).
Statistics:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.31 in the solvent control versus pH 7.34 at 250.0 µg/mL)
- Effects of osmolality: No relevant increase (445 mOsm in the solvent control versus 461mOsm at 250.0 µg/mL)
- Evaporation from medium: Not examined
- Precipitation: determined at 27.8 and 83.3 µg/mL
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation or phase separation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 2.0 µg/mL and 250 µg/mL were used. The highest concentration was based on the solubility properties of the test item in DMSO.
No relevant toxic effects were observed after 4 hours treatment up to the maximum concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred at 31.3 µg/mL and above without metabolic activation and at 15.6 µg/mL and above with metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The concentrations used in the main experiment were selected based on the data of the pre-experiment. Again, the maximum concentration was 250 µg/mL. The individual concentrations were spaced by a factor of 3.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.



COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table
   conc. µg/mL  S9 Mix

 relavtive CE I

 relative cell density  rel. adj. CE I  mutant colonies/106 cells  95% conf. interval

 Culture I

 

 

 

 

 Solvent control (DMSO)    -  100.0  100.0  100.0  40.6  1.7 - 30.2
 Positive control (EMS)  300.0  -  99.0  90.2  89.3  361.0  1.7 - 30.2  

 Test Item

 1.0  -  130.4  81.6  106.3  26.5  1.7 - 30.2
 Test Item  3.1  -  97.2  96.8 94.1   32.2  1.7 - 30.2
Test Item  9.3  -  88.6  91.1  80.7  22.1  1.7 - 30.2 
 Test Item  27.8 (p)  -  88.9  101.8  90.5  46.5  1.7 - 30.2 
 Test Item  83.3 (p)  -  117.7  59.8  70.4  21.7  1.7 - 30.2 
Test Item   250.0 (p)  -  121.2  60.4  73.1  not contiued#  
               
Solvent control (DMSO)    +  100.0  100.0  100.0  18.9  2.0 - 29.4
 Positive control (DMBA)  2.3  +  79.5  97.6  77.7  115.0  2.0 - 29.4
 Test Item  1.0  +  92.2  95.9  88.5  37.5  2.0 - 29.4 
 Test Item  3.1  +  83.0  84.0  69.7  11.4  2.0 - 29.4 
 Test Item 9.3   +  90.0 95.3   85.8 26.5   2.0 - 29.4 
 Test Item  27.8 (p)  +  89.9  89.5  80.4  19.7  2.0 - 29.4
 Test Item 83.3 (p)  + 89.3   93.4  83.4 34.4   2.0 - 29.4 
 Test Item   250.0 (p)  +  91.3  76.5  69.8  not continued#  

   conc. µg/mL  S9 Mix

 relavtive CE I

 relative cell density  rel. adj. CE I  mutant colonies/106cells  95% conf. interval

 Culture II

 

 

 

 

 Solvent control (DMSO)    -  100.0  100.0  100.0  13.3  1.7 - 30.2
 Positive control (EMS)  300.0  - 113.8  85.9  97.7  340.9 1.7 - 30.2  

 Test Item

1.0  -  94.7 105.1  99.5  12.0  1.7 - 30.2
 Test Item  3.1  -  68.2  94.9 64.8  17.3  1.7 - 30.2
Test Item  9.3  -  90.1  71.5  64.4  36.2  1.7 - 30.2 
 Test Item  27.8 (p)  -  54.9  105.1  57.6  18.1  1.7 - 30.2 
 Test Item  83.3 (p)  -  72.3  74.5 53.9   17.3  1.7 - 30.2 
Test Item   250.0 (p)  -  62.1  104.9 65.1  not contiued#  
               
Solvent control (DMSO)    +  100.0  100.0  100.0  14.0  2.0 - 29.4
 Positive control (DMBA)  2.3  +  87.1  100.4  87.4  108.7  2.0 - 29.4
 Test Item  1.0  +  90.8  107.4  97.5  16.1  2.0 - 29.4 
 Test Item  3.1  +  89.0  86.4  76.9  33.4  2.0 - 29.4 
 Test Item 9.3   +  88.1  102.3 90.2   21.9  2.0 - 29.4 
 Test Item  27.8 (p)  +  92.4  99.3  91.7  23.8  2.0 - 29.4
 Test Item 83.3 (p)  + 95.7  101.1  96.7  15.2  2.0 - 29.4 
 Test Item   250.0 (p)  +  87.3  100.7  88.0  not continued#  

CE = Cloning efficiency

P = precipitation visible to the unaided eye at the end of treatment

#    culture was not continued to avoid analysis of too many precipitating concentrations

Applicant's summary and conclusion

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.

No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiment up to the maximum concentration.

The 95% confidence interval was exceeded at several concentrations in the presence and absence of metabolic activation. A t-test run at any experimental point above the 95% confidence interval was not significant. Furthermore, the mean values of the mutation frequeny of both parallel cultures remained within the 95% confidence interval at all soluble concentrations.

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

In the main experiment with and without S9 mix the range of the solvent controls was from 13.3 up to 40.6 mutants per 106cells; the range of the groups treated with the test item was from 11.4 up to 46.5 mutants per 106cells. The highest solvent control value of 40.6 mutants per 106cells exceeded the 95% confidence interval but remained within the historical range of solvent controls. The mutation frequency of the parallel culture and the mean of both cultures (40.6 and 13.3, equal to a mean of 27.0) was fully acceptable.

EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.