Reaction mass of isomers of disodium amino-(3-carboxylatopropanamido)hexanoate and trisodium 2,6-bis(3-carboxylatopropanamido)hexanoate

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:

9 October 2017 - 23 November 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Version / remarks:

August 1998

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:

June 2012

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:

Reaction mass of isomers of disodium amino-(3-carboxylatopropanamido)hexanoate and trisodium 2,6-bis(3-carboxylatopropanamido)hexanoate

EC Number:

947-940-6

Cas Number:

1917323-94-4

Molecular formula:

C14H21N2Na3O8 and C10H16N2Na2O5

IUPAC Name:

Reaction mass of isomers of disodium amino-(3-carboxylatopropanamido)hexanoate and trisodium 2,6-bis(3-carboxylatopropanamido)hexanoate

Test material form:

liquid

Remarks:

highly viscous, semi-solid mass

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100)and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from the Phenobarbital and β-Naphthoflavone-induced rat liver

Test concentrations with justification for top dose:

Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).
The following concentrations were examined: ±S9 Mix: 5000, 1600, 500, 160, 50 and 16 μg/plate in Experiment I (Plate incorporation method) and Experiment II (pre-incubation method).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water, Dimethyl sulfoxide (DMSO)

- Justification for choice of solvent/vehicle: Test item was completely dissolved in ultrapure water. In the study two vehicle control groups were used depending on the solubility of the test item and the solubility of positive control chemicals.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: NPD (4-Nitro-1,2-phenylenediamine)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each.

Evaluation criteria:

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 TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and E.coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
Conditions for the Validity of the Test:
The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The E.coli uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 10E9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).
A dose level is considered toxic if:
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs

Statistics:

none

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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 2

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 3

Key result

Species / strain:

S. typhimurium TA 98

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 4

Key result

Species / strain:

S. typhimurium TA 100

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:

E. coli WP2 uvr A

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
- Water solubility: Test item was completely dissolved in ultrapure water.
- Precipitation: No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.
In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

HISTORICAL CONTROL DATA (Please refer to "Any other information on results incl.tables")
In the Initial Mutation Test and Confirmatory Mutation Test most of the obtained higher revertant colony numbers (higher than the revertant colony numbers of the vehicle control) remained within the corresponding historical control data ranges.

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Range Finding Test

Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	18.3	1.04	24.3	0.80	101.3	1.05	108.0	1.12
DMSO Control	16.0	1.00	27.7	1.00	–	–	93.0	1.00
Ultrapure Water Control	17.7	1.00	30.3	1.00	96.3	1.00	96.7	1.00
5000	25.0	1.42	24.7	0.81	82.3	0.85	118.0	1.22
1600	23.3	1.32	24.0	0.79	79.3	0.82	106.0	1.10
500	26.0	1.47	25.7	0.85	91.3	0.95	106.3	1.10
160	20.3	1.15	53.0	1.75	79.7	0.83	117.0	1.21
50	24.3	1.38	24.3	0.80	90.3	0.94	113.0	1.17
16	19.0	1.08	26.0	0.86	88.3	0.92	111.7	1.16
5	19.0	1.08	29.0	0.96	98.0	1.02	123.7	1.28
NPD (4mg)	257.7	16.10	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1353.3	14.05	–	–
2AA (2mg)	–	–	1341.3	48.48	–	–	1212.0	13.03

MR:Mutation Rate

NPD:4-Nitro-1,2-phenylenediamine

SAZ:Sodium azid

2AA:2-aminoanthracene

 Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and the DMSO was applied as vehicle for positive control substances: NPD and 2AA. The mutation rate of the test item, SAZ and untreated control is given referring to the ultrapure water; the mutation rate of NPD and 2AA is given referring to DMSO.

 

Table 2: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-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	25.3	1.07	27.0	0.98	87.3	0.99	115.0	1.13	12.3	1.03	12.0	1.00	8.7	1.04	9.3	1.47	34.0	0.94	43.3	0.86
DMSO Control	20.3	1.00	26.0	1.00	–	–	94.7	1.00	–	–	12.0	1.00	6.0	1.00	9.3	1.00	–	–	40.0	1.00
Ultrapure Water Control	23.7	1.00	27.7	1.00	88.3	1.00	101.3	1.00	12.0	1.00	12.0	1.00	8.3	1.00	6.3	1.00	36.0	1.00	50.7	1.00
5000	31.3	1.32	30.3	1.10	95.3	1.08	101.0	1.00	14.3	1.19	10.3	0.86	10.3	1.24	7.7	1.21	45.7	1.27	59.0	1.16
1600	22.3	0.94	28.3	1.02	100.3	1.14	80.7	0.80	12.7	1.06	10.0	0.83	9.3	1.12	7.7	1.21	46.0	1.28	56.0	1.11
500	29.3	1.24	25.0	0.90	89.3	1.01	80.0	0.79	11.7	0.97	10.7	0.89	6.7	0.80	6.3	1.00	36.7	1.02	39.3	0.78
160	23.0	0.97	29.3	1.06	89.7	1.02	88.7	0.88	15.0	1.25	11.7	0.97	7.0	0.84	7.0	1.11	36.7	1.02	47.0	0.93
50	26.3	1.11	35.3	1.28	93.7	1.06	92.7	0.91	11.0	0.92	11.7	0.97	7.7	0.92	6.3	1.00	39.0	1.08	59.0	1.16
16	23.7	1.00	27.3	0.99	93.0	1.05	93.7	0.92	10.0	0.83	11.7	0.97	8.3	1.00	7.7	1.21	40.3	1.12	44.7	0.88
NPD (4mg)	464.3	22.84	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1658.7	18.78	–	–	1717.3	143.11	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	1052.7	175.44	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	919.3	25.54	–	–
2AA (2mg)	–	–	2664.0	102.46	–	–	2954.7	31.21	–	–	258.0	21.50	–	–	239.0	25.61	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	179.0	4.48

MR:Mutation Rate; NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA: 9-Aminoacridine;MMS: Methyl methanesulfonate;2AA: 2-aminoanthracene  

Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

 

Table 3: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-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	25.3	1.00	37.3	1.53	97.7	1.12	106.0	1.02	10.7	0.97	11.7	0.97	7.3	0.85	12.0	1.50	52.0	1.42	51.3	1.10
1600	23.7	0.93	31.0	1.27	84.0	0.96	110.0	1.06	9.7	0.88	12.3	1.03	9.0	1.04	11.7	1.46	42.3	1.15	48.3	1.04
500	26.0	1.03	37.3	1.53	82.3	0.94	98.7	0.95	10.3	0.94	12.3	1.03	8.0	0.92	10.0	1.25	35.3	0.96	43.7	0.94
160	23.7	0.93	27.3	1.12	79.3	0.91	100.3	0.96	11.0	1.00	13.7	1.14	7.7	0.88	9.3	1.17	40.7	1.11	43.7	0.94
50	27.7	1.09	33.3	1.37	77.7	0.89	94.3	0.91	11.3	1.03	10.0	0.83	9.0	1.04	10.3	1.29	40.3	1.10	39.7	0.85
16	26.0	1.03	31.0	1.27	88.0	1.01	103.3	0.99	9.7	0.88	12.3	1.03	9.3	1.08	11.0	1.38	35.0	0.95	47.0	1.01
NPD (4mg)	253.3	11.01	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	797.3	9.13	–	–	509.3	46.30	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	528.0	72.00	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	741.3	20.22	–	–
2AA (2mg)	–	–	1532.0	67.59	–	–	2826.7	29.96	–	–	164.0	12.62	–	–	121.7	19.21	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	243.7	6.30

MR:Mutation Rate; NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA: 9-Aminoacridine;MMS: Methyl methanesulfonate;2AA: 2-aminoanthracene

Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

 

Table 4: Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.0	105.0	10.5	8.1	25.4
		SD	3.7	25.7	1.4	2.3	5.2
		Minimum	9	66	3	2	11
		Maximum	39	155	23	19	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.5	117.1	11.8	9.0	33.9
		SD	4.3	18.1	1.4	1.9	5.2
		Minimum	12	75	4	2	17
		Maximum	46	166	23	20	56
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	20.4	100.1	10.3	7.9	24.7
		SD	3.6	24.8	1.3	2.4	4.6
		Minimum	10	64	3	2	11
		Maximum	38	147	23	20	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	26.5	113.8	11.8	8.8	33.7
		SD	4.1	18.3	1.5	1.9	5.0
		Minimum	15	71	3	3	16
		Maximum	47	162	25	20	57
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.9	104.7	10.5	7.6	26.1
		SD	3.7	25.9	1.5	2.2	5.5
		Minimum	12	68	3	2	12
		Maximum	35	154	24	16	48
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.4	117.3	11.4	8.7	34.9
		SD	4.0	18.5	1.3	2.2	4.9
		Minimum	15	83	4	3	18
		Maximum	43	167	22	16	57
			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	260.1	977.2	847.3	478.6	724.5
		SD	31.8	150.6	126.3	104.5	65.0
		Minimum	123	521	359	110	320
		Maximum	664	1970	1855	1601	1313
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1222.7	1436.4	164.1	147.0	257.7
		SD	274.9	318.3	33.1	20.1	72.5
		Minimum	386	583	85	69	140
		Maximum	2676	2988	498	399	477

 

                           SD: Standard deviation

Applicant's summary and conclusion

Conclusions:

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

A bacterial reverse mutation assay according OECD TG 471, EU method B.13/14 and EPA OTS 789.5100 was performed to investigate the mutagenic potential in two independent experiments, in a plate incorporation test (Initial Mutation Test) and in a pre-incubation test (Confirmatory Mutation Test).

In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated. The test item was dissolved in ultrapure water. In the Initial and Confirmatory Mutation Tests the following concentrations were examined: ±S9 Mix: 5000, 1600, 500, 160, 50 and 16 μg/plate.

Five bacterial strains were used to investigate the mutagenic potential in two independent experiments. Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

No substantial increases were observed in revertant colony numbers of any of the five test 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. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

The positive controls showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

In the performed experiments inhibitory effect of the test item (decreased number of revertant colony numbers and/or affected background lawn development) was not observed in any case.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.