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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 October 2015 - 20 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
EC Number:
231-034-6
EC Name:
N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)
Cas Number:
7417-99-4
Molecular formula:
C19H20N4O2
IUPAC Name:
N-[4-({4-[(aziridine-1-carbonyl)amino]phenyl}methyl)phenyl]aziridine-1-carboxamide
Test material form:
solid

Method

Target gene:
Histidine-requiring gene in Salmonella thyphimurium
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver
Test concentrations with justification for top dose:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
The test item concentrations to be applied in the main experiment were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (AppliChem Lot No. 410131997).
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
yes
Remarks:
(A. dest.)
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (10 µg/plate for TA 98 and 40 µg/plate for TA 1537 without metabolic activation); 2-Aminoanthracene (2.5 μg/plate for TA1537, TA1535, TA98 and TA100, and 10 µg/plate for TA102 with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 1 x10^9 bacteria/mL

DURATION
- Preincubation period: Samples of each tester strain were grown by culturing for 12 h at 37ºC in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10e9 cells/mL).
- Exposure duration: 48 h incubation period at 37ºC in the dark.

SELECTION AGENT (mutation assays): All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was characterised by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.





Rationale for test conditions:
According to OECD TG 471
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high, and if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
(Biologically relevant increases of revertant colony numbers observed at concentrations of 316 µg/plate and higher (-S9) and at concentrations of 2500 µg/plate (+S9))
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
(Biologically relevant increases of revertant colony numbers observed at concentrations of 31.6 µg/plate and higher (±S9))
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
(biologically relevant increases of revertant colony numbers observed at concentrations of 10 µg/plate and higher (-S9) and in all concentrations tested (+S9))
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 102
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
- Precipitation: No precipitation of the test item was observed at any concentration or tester strain used.
- Other: No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation).


RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with the tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested (3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate ) for toxicity and induction mutations with three plates each. The experimental conditions in this pre-experiment were the same as described for the main experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see tables 4 and 6 in "any other information on results incl. tables"
- Negative (solvent/vehicle) historical control data: see tables 3 and 5 in "any other information on results incl. tables"







Any other information on results incl. tables

Table 3: Historical Laboratory Control Data of the Negative Control (in 2012 - 2014) without S9 (-S9)

 

TA 98

TA 100

TA 1535

TA 1537

TA 102

Mean

22.3

95.5

10.9

7.5

230.5

SD

4.8

18.1

5.1

2.4

47.8

Min

13

61

4

2

136

Max

48

182

35

27

415

RSD [%]

21.6

18.9

46.8

31.4

20.8

n                    

1159

1281

1043

1043

684

Table 4: Historical Laboratory Control Data of the Positive Control (in 2012 - 2014) without S9 (-S9)

TA 98

TA 100

TA 1535

TA 1537

TA 102

Substance

Conc./plate  

4-NOPD

10 µg

NaN3 10 µg

NaN3 10 µg

4-NOPD

40 µg

MMS

1 µL

Mean

443.7

704.8

858.3

93.2

1733.5

SD

183.1

272.7

320.2

27.3

408.3

Min

192

132

34

30

162

Max

2213

1498

1472

273

3181

RSD [%]

41.3

38.7

37.3

29.3

23.6

n                       

1172

1285

1042

1054

688

Table 5: Historical Laboratory Control Data of the Negative Control (in 2012 - 2014) with S9 (+S9)

 

TA 98

TA 100

TA 1535

TA 1537

TA 102

Mean

29.9

103.4

9.1

7.6

290.9

SD

6.2

17.4

3.3

2.7

65.3

Min

13

68

4

3

91

Max

61

194

34

31

495

RSD [%]

20.9

16.8

36.1

35.3

22.4

n                    

1157

1286

1042

1040

683

Table 6: Historical Laboratory Control Data of the Positive Control (in 2012 - 2014) with S9 (+S9)

 

TA 98

TA 100

TA 1535

TA 1537

TA 102

Substance

Conc./plate  

2-AA 2.5 µg

2-AA 2.5 µg

2-AA 2.5 µg

2-AA 2.5 µg

2-AA

10 µg

Mean

2318.0

1839.6

100.0

218.6

663.9

SD

573.6

455.1

60.6

85.2

176.6

Min

128

169

19

28

137

Max

3606

3132

1011

489

2001

RSD [%]

24.7

24.7

60.6

39.0

26.6

n                       

1156

1284

1041

1039

688

S9: metabolic activation  

Conc.: concentration

Mean: mean of revertants/plate

Min.: minimum of revertants/plate

Max.: maximum of revertants/plate

SD: Standard Deviation

RSD: Relative Standard Deviation

n: Number of control values

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported, the test substance caused gene mutations in the tester strains TA 100, TA 1535 and TA 98. Therefore, the test substance is considered to be mutagenic in this bacterial reverse mutation assay.


Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out using Salmonella typhimurium strains TA1537, TA1535, TA98, TA100 and TA102 in the presence and absence of metabolic activation (S9 mix prepared from the livers of rats). The test item was dissolved in DMSO and diluted prior to treatment. In the initial toxicity-mutation test the bacterial cultures were exposed to test item at 8 concentrations (three plates/concentration) between 3.16 and 5000 µg/plate. No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation). In the main test, the cultures were exposed to test item at 8 concentrations (three plates/concentration) between 3.16 and 5000 µg/plate both in the absence and presence (S9 mix) of metabolic activation. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies. All validity criteria were met. Under the experimental conditions reported, the test substance caused gene mutations by base pair changes in the genome of the tester strains TA 100 and TA 1535 and frameshifts in the genome of the tester strain TA 98. Therefore, the test substance is considered to be mutagenic in this bacterial reverse mutation assay.