N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)Glycine

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:

2022-01-11 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

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-(N-{2-[bis(carboxymethyl)amino]ethyl}nonanamido)acetic acid

EC Number:

853-587-8

Cas Number:

199387-97-8

Molecular formula:

C17 H30 N2 O7

IUPAC Name:

2-(N-{2-[bis(carboxymethyl)amino]ethyl}nonanamido)acetic acid

Test material form:

solid: particulate/powder

Details on test material:

- Test material form: white to off-white crystalline powder
- Batch No.: LFND3B1003
- Storage conditions: 2 - 8 °C
- Expiry date: 01 August 2025

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: LFND3B1003
- Purity: 100% (HPLC)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in a cool, dry place, protected from light and heat

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was dissolved in DMSO and diluted prior to treatment.

Method

Target gene:

histidine operon (S. typhimurium strains TA 98, TA 100, TA 1535 and TA 1537) and tryptophane operon (E.coli strain WP2 uvrA (pKM101))

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 liver microsomal fraction was prepared at Eurofins Munich and obtained from Trinova Biochem GmbH, Gießen, Germany. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins Munich) and male Sprague Dawley rats were induced with phenobarbital/β-naphthoflavone (Trinova).
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL (Eurofins Munich) and 5 mL (Trinova) and stored at ≤ -75 °C.
The protein concentration in the S9 preparation of Eurofins Munich was 35.0 mg/mL (Lot: 191121), and in the S9 preparation of Trinova, 35.6 mg/mL (Lot: 4449). The protein concentration of Lot No.: 4449 was adjusted to 30 mg/mL. Trinova S9 preparation was used in the pre-experiment and Experiment I, while Eurofins S9 preparation was used in Experiment II.

- method of preparation of S9 mix:
The S9 mix preparation was performed according to Ames et al.
100 mM of ice-cold sodium orthophosphate-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts
During the experiment the S9 mix is stored on ice.

- concentration or volume of S9 mix and S9 in the final culture medium: 500 µL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):

The following quality control determinations were performed:
• Eurofins Munich-prepared S9 Homogenate:
Quality control determinations performed by Eurofins Munich:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
• Trinova Biochem GmbH-prepared S9 Homogenate:
Quality control determinations performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)

Test concentrations with justification for top dose:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. The maximum concentration was selected based on the toxicity of the test item. The concentration range covered two logarithmic decades. Three independent experiments were performed at the following concentrations:
Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Experiment II: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA100, TA1537 and E. coli WP2 uvrA (pKM101); TA98 and TA1535 [with metabolic activation])
3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (TA98 and TA1535 [without metabolic activation])
Experiment III: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA1535 [without metabolic activation])

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

A. dest

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

A. dest

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine (CAS 99-56-9)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

A. dest

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

A. dest

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (CAS 613-13-8)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approx. 10^9 cells/mL
- Test substance added: Experiment 1: in agar (plate incorporation); Experiment 2&3: Pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
- Any supplementary information relevant to cytotoxicity: Cytotoxicity can be detected 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.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The colonies were either counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH) or using a Sorcerer Colony Counter (Perceptive Instruments). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA1535 and TA1537 may have been counted manually.

Evaluation criteria:

Criteria of Validity
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM101))
- the negative control plates (A. dest.) with and without S9 mix are within the historical control data range (mean values of the spontaneous reversion frequency)
- corresponding background growth on both negative control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable

Evaluation of Mutagenicity
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 TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to the 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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Experiment II at concentrations of 31.6 µg/plate and higher (without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True 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:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Experiment II at concentrations of 31.6 µg/plate and higher (without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not specified
- Data on osmolality: not specified
- Possibility of evaporation from medium: not specified
- Water solubility: not specified
- Precipitation and time of the determination: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation) and in experiment III (without metabolic activation).
- Definition of acceptable cells for analysis:

RANGE-FINDING/SCREENING STUDIES (if applicable):
The toxicity of the test item was determined with tester strains TA98 and TA100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test). The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment

STUDY RESULTS
- Concurrent vehicle negative and positive control data

Ames test:
- Signs of toxicity: In experiment I, 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 experiment II, toxic effects of the test item were observed in tester trains TA98 and TA1535 at concentrations of 31.6 µg/plate and higher (without metabolic activation).
The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment II in tester strain TA100 at a concentrations of 31.6 and 316 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of the background lawn.
In experiment III, no toxic effects of the test item were noted in tester strain TA1535 up to the highest dose group evaluated (without metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment III in tester strain TA1535 at concentrations of 0.316, 3.16 and 10 µg/plate (without metabolic activation) were regarded as not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of the background lawn.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)glycine at any concentration level, neither in the presence nor absence of metabolic activation in the experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data).
- Detailed information on historical control data for the negative and positive control can be found in section "Any other information on materials & methods incl. tables”

Applicant's summary and conclusion

Conclusions:

Under the conditions of this bacterial reverse mutation assay, the test item N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)glycine did not cause an increase in the frequency of revertants both with and without metabolic activation in any of the test strains under investigation (S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA (pKM101)). Therefore, N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)glycine is considered to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria (according to OECD 471), strains of S. typhimurium (TA 98, TA 100, TA 1535, TA 1537) and E. coli (WP2 uvrA (pKM101)) were exposed to N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)glycine (100% purity) in DMSO in the presence and absence of mammalian metabolic activation (S9-mix). Experiment 1 was conducted according to the plate-incorporation test method, while in Experiment 2&3 the pre-incubation method was applied. The following concentrations of the test item were prepared and used in the experiments:

Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA100, TA1537 and E. coli WP2 uvrA (pKM101); TA98 and TA1535 [with metabolic activation]) 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate (TA98 and TA1535 [without metabolic activation])

Experiment III: 0.316, 1.0, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA1535 [without metabolic activation])

The positive controls induced the appropriate responses in the corresponding strains. The response of the negative controls was within the historical control data range of the laboratory.

There was no evidence or a concentration-related positive response of induced mutant colonies over background for the test item.

Based on the results of this study N-[2-[bis(carboxymethyl)amino]ethyl]-N-(1-oxononyl)glycine is considered to be non-mutagenic in the bacterial reverse mutation assay.

This study is classified as acceptable as it satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.