N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.