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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12.12.2016 to 22.05.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In Vitro Mammalian Cell Gene Mutation Test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Xianyang Sanjing Technology Co., ltd./160612
- Expiration date of the lot/batch: June 18, 2018
- Purity: > 99.6 % w/w


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, in a cool and dry place, away from light

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: European Collection of Cell Cultures (ECACC). Lot. No.: 10H016.
- Suitability of cells: Recommended in guideline
- Number of passages if applicable: Cells underwent maximum 5 passages after thawing the original culture delivered from cell collection before using for testing.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS in incubator (5 % CO2, 37±1 °C, moistened).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes. Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. Cleansing was not performed in experiment without metabolic activation, because of bad growth of cells in HAT medium.
Metabolic activation:
with and without
Metabolic activation system:
PCB-induced rat liver S9
Test concentrations with justification for top dose:
Cytotoxicity – S9 : 0.0001, 0.0005, 0.0010 0.005, 0.01 and 0.02 mg/mL.
Mutagenicity – S9: 0.0001, 0.0005, 0.001, 0.002, 0.004 mg/mL

In the cytotoxicity test without S9, at the concentration 0.005 mg per mL, survival was 11.2 %. To ensure cytotoxicity between 10 and 20% the concentration 0.004 mg/mL was used in mutagenicity experiment without S9.

Cytotoxicity + S9: 0.0005, 0.001, 0.002, 0.003 mg/mL and 0.0075, 0.0150, 0.03, 0.06 mg/mL
Mutagenicity +S9: 0.0025, 0.005, 0.01, 0.02, and 0.04 mg/mL

In the first cytotoxicity with S9, the concentrations of 0.0005, 0.001, 0.002, 0.003 mg per mL were used, but they were completely non-toxic. The tests was repeated with concentrations of 0.075, 0.150, 0.03 and 0.06 mg/mL. 0.02 and 0.04 mg/mL were selected as the top doses for the mutagenicity test with S9 with expected cytotoxicity of 80 and 100% respectively.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle:
The test substance was soluble in DMSO up to a concentration of 133 mg per mL- a red brown solution arose. After addition to DMEM medium, precipitation occurred in all concentrations used; in lower concentrations, precipitation was visible at observation by microscope. Upon further examination, where a concentration row in DMEM was prepared starting with a concentration of 1.33 mg per mL, the first concentration with precipitation not observable with the naked eye was 0.022 mg per mL. This concentration was used as the second highest in the cytotoxicity testing. The highest concentration (the first with visible precipitation) was 0.044 mg per mL.
The test substance was also soluble in acetonitrile in ca half concentration than that in DMSO. After adding to DMEM, precipitation also occurred. As DMSO is more usual solvent with known no mutagenic effect, DMSO was used for the experiments.
Controls
Untreated negative controls:
yes
Remarks:
Complete cell culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 3 hr and 24 hr
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 9 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF CELLS EVALUATED:
Mutants: 2.2 x 10+5
Cloning efficiency: 300 cells

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency (survival)


Rationale for test conditions:
In the cytotoxicity test without S9, at the concentration 0.005 mg per mL, survival was 11.2 %. To ensure cytotoxicity between 10 and 20% the concentration 0.004 mg/mL was used in mutagenicity experiment without S9 (Table 2).

In the first cytotoxicity with S9, the concentrations of 0.0005, 0.001, 0.002, 0.003 mg per mL were used, but they were completely non-toxic. The tests was repeated with concentrations of 0.075, 0.150, 0.03 and 0.06 mg/mL. 0.02 and 0.04 mg/mL were selected as the top doses for the mutagenicity test with S9 with expected cytotoxicity of 80 and 100% respectively (Table 4).
Evaluation criteria:
Each experiment is evaluated separately using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987 (2).

The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to the negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).

There is no requirement for verification of a clearly positive or negative response.

In cases when the response is neither clearly negative nor clearly positive than a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions i.e. S9 concentration or S9 origin) could be performed.
Statistics:
For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
0.02 mg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
0.004 mg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Addition of the test substance to cultivation medium did not change pH values of treatment solutions. It slightly decreased the pH in the highest dose (not used in mutagenicity experiments). Differences approximately 0.05 pH were observed between results at the start and in the end of 3 hours period. This change has not such extent which could produce artifactual positive results. No pH adjustment was needed. (Table 6)

- Effects of osmolality: As the test substance is not soluble in DMEM, results could be taken with caution. Measuring was performed two times. Firstly, the test substance/solvent was dosed in 100 μL of DMSO. So each solution for osmolality measuring contained 9.90 mL of DMEM and 100 μL of the test substance in appropriate concentration in DMSO. Samples were very unstable, gas on thermistor released at formation of drops, which affects ofshape and size of drops. The highest change was observed between negative and solvent control. Osmolality of samples was more closely to the solvent than medium itself. The test substance is well soluble in DMSO, so volume of application form was decreased to 50 μL and the second osmolality measuring (as well as mutagenicity testing) was performed in solutions containing 9.95 mL of DMEM and 50 μL of DMSO/application form of the test substance. Such measured samples had better stability, nevertheless results were analogic (Table 7).

- Precipitation: After addition to DMEM medium, precipitation occurred in all concentrations used; in lower concentrations, precipitation was visible at observation by microscope. Upon further examination, where a concentration row in DMEM was prepared starting with a concentration of 1.33 mg per mL, the first concentration with precipitation not observable with the naked eye was 0.022 mg per mL. This concentration was used as the second highest in the cytotoxicity testing. The highest concentration (the first with visible precipitation) was 0.044 mg per mL.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
EMS 50 µL: 17.53; historical control range (95% confidence interval) is 9.89 – 17.59 mutants per 105cells;
EMS 100 µL: 36.01; historical control range (95% confidence interval) is 19.67 – 36.26 mutants per 105cells;
DMBA: 33.85 and 28.21 what is an evidence of good function of the test system. Historical control range (95% confidence interval) is 9.92 – 33.99 mutants per 105cells.

- Negative (solvent/vehicle) historical control data:
Mutation frequencies of negative control were 2.22 and 2.27 mutants per 105 plated cells. Historical control range (97.5% confidence interval) is 0.76-2.31 mutants per 105cells.
Mutation frequencies of solvent control range varied from 2.00 – 3.23 mutants per 105 plated cells. Historical control range (97.5% confidence interval) is 0.85 – 3.45 mutants per 105cells.

Applicant's summary and conclusion

Conclusions:
Under the above-described experimental design the test substance, N,N′-m-phenylenedimaleimide, was non-mutagenic in Chinese hamster V79 lung fibroblasts with and without metabolic activation.
Executive summary:

In a mammalian cell gene mutation assay (HPRT; 17-331), Chinese hamster V79 lung fibroblasts cultured in vitro were exposed to N,N′-m-phenylenedimaleimide (99.6%), in DMSO at concentrations of  0.0001, 0.0005, 0.001, 0.002, 0.004 mg/mL in the absence and 0.0025, 0.005, 0.01, 0.02, and 0.04 mg/mL in the presence of mammalian metabolic activation (PCB-induced rat liver S9).  

N,N′-m-phenylenedimaleimide was tested up to cytotoxic concentrations The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.