2-Propenenitrile, reaction products with 2,2,4(or 2,4,4)-trimethyl-1,6-hexanediamine

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:

from 2017-09-19 to 2017-11-14

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998-08

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use

Version / remarks:

2012-06

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenenitrile, reaction products with 2,2,4(or 2,4,4)-trimethyl-1,6-hexanediamine

EC Number:

292-059-6

EC Name:

2-Propenenitrile, reaction products with 2,2,4(or 2,4,4)-trimethyl-1,6-hexanediamine

Cas Number:

90530-20-4

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

3-[(3,5,5-trimethylhexyl)amino]propanenitrile; 3-[(6-amino-2,2,4-trimethylhexyl)amino]propanenitrile; 3-[(6-amino-2,4,4-trimethylhexyl)amino]propanenitrile; 3-[(6-amino-3,3,5-trimethylhexyl)amino]propanenitrile

Specific details on test material used for the study:

Batch No.: D1608206

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Method

Target gene:

his/trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

uvrA

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

16.0, 50.0, 160.0, 500.0, 1600.0, 5000.0 µg/plate, recommended maximum test concentration

Vehicle / solvent:

- Solvent used: ultrapure water (ASTM Type 1)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

without S9 mix, 4 µg/plate for TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix, 2 µg/plate for TA100 and TA 1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix, 50 µg/plate for TA1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

witout S9 mix, 2 µL/plate for E.coli WP2 uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

with S9 mix, 2 µg/plate for all TA strains, 50 µg/plate for E.coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: n agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: colony and background lawn development

Evaluation criteria:

Evaluation of Experimental Data
The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye), the mean values, standard deviations and the mutation rates were calculated.

Mutation Rate = (Mean revertants at the test item (or control*) treatments) / mean revertants of vehicle control
* untreated, vehicle or positive control

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control. According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Initial Mutation Test (Plate Incorporation Test)

Concentrations (μg/plate)	Salmonella typhimuriumtester strains																Escherichia coli WP2 uvrA
	TA98				TA100				TA1535				TA1537
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean Values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	19.7	1.09	26.0	1.26	87.7	1.00	91.7	1.01	9.0	0.79	13.3	0.95	8.3	0.93	9.0	1.13	32.7	0.91	46.7	0.85
DMSO Control	16.0	1.00	21.3	1.00	–	–	94.3	1.00	–	–	14.7	1.00	6.0	1.00	7.3	1.00	–	–	43.7	1.00
Ultrapure Water Control	18.0	1.00	20.7	1.00	87.3	1.00	90.7	1.00	11.3	1.00	14.0	1.00	9.0	1.00	8.0	1.00	36.0	1.00	54.7	1.00
5000	21.3	1.19	24.7	1.19	83.3	0.95	107.0	1.18	10.7	0.94	12.3	0.88	9.3	1.04	9.3	1.17	49.7	1.38	48.7	0.89
1600	22.0	1.22	24.7	1.19	82.0	0.94	108.0	1.19	9.7	0.85	14.3	1.02	6.7	0.74	6.0	0.75	44.3	1.23	54.0	0.99
500	27.7	1.54	24.0	1.16	89.0	1.02	103.7	1.14	10.3	0.91	11.0	0.79	8.0	0.89	9.0	1.13	40.7	1.13	51.0	0.93
160	25.0	1.39	28.3	1.37	92.3	1.06	102.0	1.13	8.7	0.76	11.0	0.79	8.3	0.93	9.0	1.13	46.3	1.29	49.3	0.90
50	28.7	1.59	24.0	1.16	86.7	0.99	104.0	1.15	10.7	0.94	14.3	1.02	9.3	1.04	11.7	1.46	32.7	0.91	44.7	0.82
16	23.0	1.28	31.0	1.50	93.0	1.06	98.0	1.08	9.3	0.82	13.3	0.95	9.3	1.04	8.7	1.08	32.3	0.90	50.0	0.91
NPD (4 μg)	357.3	22.33	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 μg)	–	–	–	–	933.3	10.69	–	–	533.3	47.06	–	–	–	–	–	–	–	–	–	–
9AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	758.0	126.33	–	–	–	–	–	–
MMS (2 μL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	821.3	22.81	–	–
2AA (2 μg)	–	–	1828.0	85.69	–	–	1493.3	15.83	–	–	237.3	16.18	–	–	153.3	20.91	–	–	–	–
2AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	193.7	4.44

Table 2: Summary Table of the Results of the Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (μg/plate)	Salmonella typhimuriumtester strains																Escherichia coli WP2 uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean Values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	27.3	1.08	29.0	1.19	92.7	1.06	92.0	0.88	11.0	1.00	11.0	0.92	10.3	1.19	9.0	1.13	17.0	0.46	39.3	0.84
DMSO Control	23.0	1.00	22.7	1.00	–	–	94.3	1.00	–	–	13.0	1.00	7.3	1.00	6.3	1.00	–	–	38.7	1.00
Ultrapure Water Control	25.3	1.00	24.3	1.00	87.3	1.00	104.0	1.00	11.0	1.00	12.0	1.00	8.7	1.00	8.0	1.00	36.7	1.00	46.7	1.00
5000	5.3	0.21	19.7	0.81	24.3	0.28	52.3	0.50	0.0	0.00	4.3	0.36	1.3	0.15	6.3	0.79	14.7	0.40	26.7	0.57
1600	31.7	1.25	26.7	1.10	92.0	1.05	100.3	0.96	8.0	0.73	9.0	0.75	9.0	1.04	12.7	1.58	35.3	0.96	44.3	0.95
500	32.7	1.29	24.3	1.00	78.3	0.90	98.3	0.95	12.7	1.15	11.3	0.94	12.3	1.42	8.7	1.08	33.7	0.92	43.3	0.93
160	26.0	1.03	34.3	1.41	75.3	0.86	93.7	0.90	10.0	0.91	10.3	0.86	8.0	0.92	7.3	0.92	35.0	0.95	39.7	0.85
50	21.3	0.84	30.3	1.25	77.0	0.88	97.7	0.94	11.0	1.00	9.3	0.78	9.7	1.12	8.3	1.04	27.7	0.75	39.3	0.84
16	24.0	0.95	28.3	1.16	85.3	0.98	99.3	0.96	12.0	1.09	13.0	1.08	7.3	0.85	9.7	1.21	28.0	0.76	39.7	0.85
NPD (4 μg)	253.3	11.01	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 μg)	–	–	–	–	797.3	9.13	–	–	509.3	46.30	–	–	–	–	–	–	–	–	–	–
9AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	528.0	72.00	–	–	–	–	–	–
MMS (2 μL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	741.3	20.22	–	–
2AA (2 μg)	–	–	1532.0	67.59	–	–	2826.7	29.96	–	–	164.0	12.62	–	–	121.7	19.21	–	–	–	–
2AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	243.7	6.30

Applicant's summary and conclusion

Conclusions:

In an bacterial reverese mutation assay (AMES) according to OECD Guideline 471, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used.

Executive summary:

The mutagenic potential of the test item was determined in an in vitro bacterial reverse mutation assay (AMES) according to OECD Guideline 471. Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations (16, 50, 160, 500, 1600, 5000 µg/plate), including the controls, were tested in triplicate (positive and negative controls were run concurrently). nagative, vehicle and positive controls were valid. No cytotoxicity was observed up to the max. concentration. No precipitation was observed throughout the study. No substantial increases were observed in revertant colony numbers of any of the five tester strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.