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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance, Akardit, was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test with negative result.

The In Vitro Mammalian Cell Micronucleus Test assayed the test substance Akardit for mutagenicity. The results are equivocal.

Ames test-data from article/handbook. N-Methyl-N',N'-diphenylurea (CAS number 13114-72-2) was tested under for mutagenicity, in Salmonella typhimurium (TA 97, 98, 100, 1535, 1537). The test substance N-Methyl-N',N'-diphenylurea (CAS number 13114-72-2) was found nonmutagenic with/without metabolic activation.


The test substance, Akardit, was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The performed test was based on EU method B.17. Mutagenicity – In Vitro Mammalian Cell Gene Mutation Test, which is analogous to the OECD Test Guideline No. 476 (1997).

The In Vitro Mammalian Cell Micronucleus Test assayed the test substance Akardit for mutagenicity. The test was performed according to Method B.49, In Vitro Mammalian Cell Micronucleus Test, Commision Regulation (EU) No. 640/2012 and OECD Test Guideline No. 487 - In Vitro Mammalian Cell Micronucleus Test, Adopted 26th September, 2014.

These studies are GLP. Klimish score 1.

Ames test-data from article/handbook. N-Methyl-N',N'-diphenylurea (CAS number 13114-72-2) was tested under for mutagenicity, in Salmonella typhimurium (TA 97, 98, 100, 1535, 1537). Klimish score 2.
Link to relevant study records

Referenceopen allclose all

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:
16.09.-15.02.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
see any other onformation
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and mixture of cofactors.
Test concentrations with justification for top dose:
cytotoxicity: 0.0313, 0.025, 0.125, 0.25, 1.0, 2.5, 5.0 mg.mL-1 (3h treatment)mutagenicity: 0.5, 0.25, 0.125, 0.0625 mg.mL-1 (3h treatment)0.1, 0.075, 0.05, 0.025 and 0.01 mg.mL-1 (24h treatment)
Vehicle / solvent:
DMEM
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
TEST SYSTEMThe lung fibroblasts V79 from male Chinese hamster were used for testing. Frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 10H016The cells were kept at -196 ºC under liquid nitrogen. After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS in incubator (5 % CO2, 37±1 °C, moistened).Cells underwent maximum12 passages after thawing the original culture delivered from cell collection before using for test. Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants.METHOD OF APPLICATION: in mediumDURATION1st experiment: 10 days, 3h teratment with metabolic activation, 3h without metabolic activation2st experiment: 10 days, 24h teratment without metabolic activation onlyNUMBER OF REPLICATION: 2NUMEBRO OF CELLS EVALUATED: 100 000 cellsSELECTION AGENT (mutation assays): 6-thioguanine 98%, CAS No. 154-24-7, Alfa Aesar, diluted in 0,5% Na2CO3; 5 µg.mL-1 (final concentration) for selection of mutantsDETERMINATION OF CYTOTOXICITY- Method: cloning efficiency
Statistics:
Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 – 240Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutat. Res. 189, 83 - 91
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: Measuring of pH was performed for treatment solutions used in experiments without metabolic activation. The test substance hardly changed pH of cultivation medium.- Water solubility: As the test substance is insoluble in water it was dissolved in DMSO
Conclusions:
Under the above-described experimental design, the test substance, Akardit, was non-mutagenic for V79 cells with as well as without metabolic activation.
Executive summary:

The test substance,Akardit,was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The performed test was based on EU methodB.17. Mutagenicity –In Vitro Mammalian Cell Gene Mutation Test, which is analogous to the OECD Test Guideline No. 476 (1997).

V79 hamster fibroblast were used for testing.

The test substance was dissolved in dimethyl sulfoxide in concentration 25 mg.mL-1. Concentrations tested for cytotoxicity in 3-hour experiment were 0.25, 0.125, 0.0625 and 0.0313 mg.L-1and other 3 concentrations were tested as a suspension in DMEM (1.0, 2.5 and 5.0 mg.mL-1). No toxicity was observed in any dose.

First mutagenicity experiment (3 hour treatment) with the test substance followed then. Four concentrations of test substance dissolved in dimethyl sulfoxide were used-0.0625,0.125, 0.25 and 0.5 mg.mL-1. Each concentration was tested in two independent runs. Experiments were performed with as well as without metabolic activation.

Results of the first experiment were negative, so the modification of experiment was performed with extended treatment time of 24 hours and lower concentrations considering toxicity of the test substance shown in 24 hours cytotoxicity experiment (0.01, 0.025, 0.05, 0.075 and 0.1 mg.mL-1). The test was performed without metabolic activation only; each concentration was tested in replicate. Also the second experiment gives no evidence of the mutagenicity of test substance.

In the arrangement given above, the test substance, Akardit, was non-mutagenic for V79 cells without as well as with metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03.02.-26.05.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
yes
Remarks:
The second experiment without metabolic activation with 24 hours exposure was repeated, because of poor quality of slides. Therefore the study proceeded longer that it was planned. This deviation has no impact to study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
mammalian cell line, other: peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
The human peripheral blood lymphocytes used for testing were obtained from healthy non smoking donors (up to 35 years of age). Peripheral blood (heparinized) is taken from donors in certified medical laboratory (MeDiLa) in the morning and as soon as possible transported into the test facility.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and mixture of cofactors.
Test concentrations with justification for top dose:
In the first experiment without and with metabolic activation (S9-mix) the concentrations 250, 500 and 1000 μg/mL were used for analysis of genotoxic effect. In the second experiment (prolonged exposition without activation) the concentrations 62.5, 125, 250 and 500 μg/mL were used for analysis of genotoxic effect.
Untreated negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: Colchicine, Cyclophosphamide monohydrate
Details on test system and experimental conditions:
Principle of test is the detection of binucleated cells with micronuclei, which are induced by the test substance in human peripheral blood lymphocytes. Lymphocytes are cultured in growth medium and test substance is added to them. Cell cycle is then stopped by cytochalasin B, cultures are sampled and microscopic preparations are prepared. Preparations are then analysed by microscope. Genotoxicity is indicated by increased incidence of binucleated cells with micronuclei.Experiments with and without metabolic activation with short treatment (3 hours) are done at first. If both experiments with the short treatments are negative or equivocal, subsequently, extended exposure treatment without metabolic activation is performed.
Key result
Species / strain:
mammalian cell line, other: peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:Actual results of negative and positive controls fulfilled the acceptability criteria - the actual values did not exceed limits of historical controls in our laboratoryResults overviewResults of experiments are summarized in the tables (cytotoxicity) and (genotoxicity). The tables contain the dose applied per culture in g/mL (concentrations of test substance were applied to cultures at a volume of 50 l), amount of S9 per culture in l, number of mononucleated, binucleated and multinucleated cells, CBPI index and % cytotoxicity, numbers of binucleated cells with micronuclei and average numbers of binucleated cells with micronuclei in 1000 cells, number of micronuclei and average number of micronuclei in 1000 cells, parameter Mt / Mc , i.e. ratio of number of binucleated cells with micronuclei at tested dose (Mt) to number of binucleated cells with micronuclei at negative control (Mc, untreated control or S9-mix). Numbers of binucleated cells with micronuclei in negative and positive controls were compared with historical controls in our laboratory. See (Table 7 and Table 8). Values of negative and positive controls in this study are within the ranges of historical data, so that test system responds adequately and the experiment is acceptable.The cytotoxic effect was characterized as % of cytotoxicity. Results for the cytotoxic effect are given in the Table 1, Table 2 and Table 3Test concentrations 5000 and 2500 μg/mL show the cytotoxicity higher than 55±5 %. On the basis of this result, the concentration of 1000 μg/mL was selected as the highest concentration to be used for the analysis of genotoxic effect. The genotoxic effect characterized by numbers of binucleated cells with micronuclei is presented in Tables 4 – 6 The results from the first experiment with and without metabolic activation with short exposure (see Tab. 4 and 5) showed the dependence between dose and effect, however the ratio of number of binucleated cells with micronuclei at tested dose to number of binucleated cells with micronuclei in negative control (Mt/Mc) was lower than<2. The first experiments gave negative results, so the second experiment without metabolic activation had to be done with extended exposure (23 hours) in the presence of cytochalasin B (see Tab. 6). Results discussionBoth experiments with the short treatments at the doses 250-1000 μg/mL and in the experiment with the long treatment at the doses 125-500 μg/mL showed trend in Mt/Mc ratio. But at no evaluated concentration the doubling in the number of binucleated cells with micronuclei occurred. Numbers of binucleated cells with micronuclei in trated cultures versus numbers of binucleated cells with micronuclei in negative controls were lower than 2 (Mt/M<2) at all doses.Under the conditions described above the effect of test substance is not clearly positive or negative. The results are equivocal.
Remarks on result:
other: The result are equivocal.

Table 1: Evaluation of cytotoxic effect without metabolic activation-3h exposure

- MA I

Culture No.

Treatment/Test substance concentration

Number of MNC

Number of BNC

Number of MTNC

CBPI

Cytotoxicity (%)

3

DMSO

210

677

113

1.90

-

10

250 μg/mL

211

694

95

1.88

2.1

11

500 μg/mL

169

737

94

1.93

-2.4

12

1000 μg/mL

315

633

52

1.74

18.4

13

2500 μg/mL

845

140

15

1.17

81.2

14

5000 μg/mL

760

212

28

1.27

70.3

15

Colchicine

720

237

43

1.32

64.2

1

UTC

247

649

104

1.86

5.1

 


 

 

Table 2: Evaluation of cytotoxic effect with metabolic activation (S9-mix) -3h exposure

+MA I

Culture No.

Treatment/Test substance concentration

Number of MNC

Number of BNC

Number of MTNC

CBPI

Cytotoxicity (%)

4

DMSO + S9-mix

171

705

124

1.95

-

5

250mg/mL +

S9-mix

139

758

103

1.96

-1.2

6

500mg/mL +

S9-mix

279

616

105

1.83

13.3

7

1000mg/mL +

S9-mix

348

591

61

1.71

25.2

8

2500mg/mL +

S9-mix

843

120

37

1.19

79.6

9

5000mg/mL +

S9-mix

685

265

50

1.37

61.7

16

Cyclophosphamide + S9-mix

266

690

44

1.78

18.4

1

UTC

247

649

104

1.86

10.1

3

DMSO

210

677

113

1.90

5.2

2

S9-mix

276

573

151

1.88

8.2

 


 

Table 3: Evaluation of cytotoxic effect without metabolic activation -24h exposure

- MA III

Culture No.

Treatment/Test substance concentration

Number of MNC

Number of BNC

Number of MTNC

CBPI

Cytotoxicity (%)

2

DMSO

501

421

93

1.60

-

3

62.5 μg/mL

569

382

77

1.52

12.8

4

125 μg/mL

641

313

46

1.41

32.3

5

250 μg/mL

640

309

53

1.41

30.7

6

500 μg/mL

741

213

46

1.31

49.0

7

1000 μg/mL

913

90

3

1.10

84.0

8

Colchicine

616

288

102

1.49

18.2

1

UTC

531

368

106

1.58

3.5

 

Table No. 4: Evaluation of genotoxicity without metabolic activation-3h exposure

Culture No.

Treatment/Test substance concentration

Number of binucleated cells with MN

Number of MN

Average number of BN cells with MN per 1000 binucleated cells

Average number of MN per 1000 binucleated cells

Mt/Mc

3

DMSO

11

11

5.5

5.5

-

10

250mg/mL

10

11

5

5.5

0.91

11

500mg/mL

13

13

6.5

6.5

1.18

12

1000mg/mL

16

19

8

9.5

1.45

1

UTC

16

20

8

10

1.45

15

Colchicine

129

167

64.5

83.5

11.73

 


 

Table No. 5:Evaluation of genotoxicity with metabolic activation (S9-mix) -3h exposure

Culture No.

Treatment/Test substance concentration

Number of binucleated cells with MN

Number of MN

Average number of BN cells with MN per 1000 binucleated cells

Average number of MN per 1000 binucleated cells

Mt/Mc

4

DMSO + S9-mix

12

15

6

7.5

-

5

250 mg/mL +

 S9-mix

10

10

5

5

0.83

6

500mg/mL +

S9-mix

10

10

5

5

0.83

7

1000mg/mL +

 S9-mix

13

13

6.5

6.5

1.08

1

UTC

16

20

8

10

1.33

3

DMSO

11

11

5.5

5.5

0.92

2

S9-mix

16

16

8

8

1.33

16

Cyclophosphamide + S9-mix

28

37

14

18.5

2.33

 


 

Table No. 6: Evaluation of genotoxicity without metabolic activation- 23h exposure

Culture No.

Treatment/Test substance concentration

Number of binucleated cells with MN

Number of MN

Average number of binucleated cells with MN per 1000 binucleated cells

Average number of MN per 1000 binucleated cells

Mt/Mc

2

DMSO

45

52

22.5

26

-

3

62.5mg/mL

46

54

23

27

1.02

4

125mg/mL

38

46

19

23

0.84

5

250mg/mL

46

47

23

23.5

1.02

6

500mg/mL

69

92

34.5

46

1.53

1

UTC

54

61

27

30.5

1.20

8

Colchicine

100

115

50

57.5

2.22

 

Conclusions:
Under the test condition used, the results of mutagenicity testing of test substance Akardit are not clearly positive or negative. The results are equivocal. Another testing should be usefull.
Executive summary:

The In Vitro Mammalian Cell Micronucleus Test assayed the test substance Akardit for mutagenicity. The test was performed according to Method B.49, InVitro Mammalian Cell Micronucleus Test, Commision Regulation (EU) No. 640/2012 and OECD Test Guideline No. 487 -In Vitro Mammalian Cell Micronucleus Test, Adopted 26thSeptember, 2014.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was disolved in DMSO and assayed in concentrations of 62.5‑5000  µg/mL, which were applied to cultures in volume of 50 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the test condition used, the results of mutagenicity testing of test substance Akardit are not clearly positive or negative. The results are equivocal. Another testing should be usefull.

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:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Maron DM, Ames BN (1983): Revised methods for the Salmonella mutagenicity test. Mutat Res 113:173-215
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Target gene:
gene for synthesis of histidine
Species / strain / cell type:
S. typhimurium, other: TA 97, 98, 100, 1535, 1537
Additional strain / cell type characteristics:
other: histidine dependent strain
Metabolic activation:
with and without
Metabolic activation system:
Livers S-9 fraction ( male Spraque-Dawley rat, male Syrian hamster livers Aroclor 1254 (10% and 30%)
Test concentrations with justification for top dose:
Dose: 33, 100, 333, 1000, 3333, 6666, 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, destilled water, 95% ethanol, acetone
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
Test system: Cultures were grown at 37°C overnight, with shakingPositive control without metabolic activation: sodium azide (TA1535, 100)9-aminoacridine (TA97, 1537)4-nitro-o-phenylenediamine (TA98)Positive control with metabolic activation:2-aminoanthracene (all strains)METHOD OF APPLICATION: in agar (plate incorporation), preincubationDURATION- Incubation period: 20 min- Exposure duration: 2 days- Temperature: 37°C- without shakingNUMBER OF REPLICATIONS: 3DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other:
Evaluation criteria:
Evaluations were made at both the individual trial and overall chemical levels.Individual trials were judged mutagenic (+), weakly mutagenic (+W), questionable (?), or nonmutagenic (-) depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible dose-related response over the solvent control, under a single metabolic activationcondition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increase in his+ revertants did not meet the criteria for a "W+" response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet criteria for a mutagenic or questionable response.
Key result
Species / strain:
S. typhimurium, other: TA 97, 98, 100, 1535, 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
The test substance N-Methyl-N',N'-diphenylurea (CAS number 13114-72-2) was found nonmutagenic with/without metabolic activation.
Executive summary:

N-Methyl-N',N'-diphenylurea (CAS number 13114-72-2) was tested under for mutagenicity, in Salmonella typhimurium (TA 97, 98, 100, 1535, 1537). The test was conducted using a preincubation protocol with and without metabolic activation. The results and data from this test are presented.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The In vitro tests are not sufficient for classification.