2-hydroxyethyliminodi(acetic acid)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxyethyliminodi(acetic acid)

EC Number:

202-263-9

EC Name:

2-hydroxyethyliminodi(acetic acid)

Cas Number:

93-62-9

Molecular formula:

C6H11NO5

IUPAC Name:

2-hydroxyethyliminodi(acetic acid)

Test material form:

solid: crystalline

Method

Target gene:

uvrB-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

dimethyl formamide

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

The five strains of bacteria used, and their mutations, are defined above.
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the
histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to
larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the
strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers
ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA
repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would
normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• Syngenta CTL, Alderley Edge, as frozen vials, on 20 March 2007.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1758279 10/20) and incubated at 37 °C for
approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item
(1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing
0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls
were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was
assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the
molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were
viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method

As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test
methodology from plate incorporation to pre-incubation.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were
incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in
triplicate.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added
to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this
experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were
viewed microscopically for evidence of thinning (toxicity). Several manual counts were required, predominantly due to revertant colonies spreading slightly, thus distorting the actual
plate count.

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls).
Acceptable ranges are
presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix.
All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels. There should be no evidence of excessive contamination.

Evaluation criteria:

Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and
Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of
this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix
used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of
the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and
consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose
level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test item precipitate was observed on the plates at
any of the doses tested in either the presence or absence of S9-mix. There were no toxicologically significant increases in the frequency of revertant colonies
recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no
toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic
activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA100 revertant colony frequency were observed in the first mutation test at
150 μg/plate (presence of S9-mix) and in the second mutation test at 5000 μg/plate (absence of S9-mix). These increases were considered to be of no biological relevance because there
was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house
historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.
The vehicle (dimethyl formamide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases
in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Applicant's summary and conclusion

Conclusions:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with Hydroxyethyl Iminoacetic Acid using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with Hydroxyethyl Iminoacetic Acid using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Test material was considered to be non-mutagenic under the conditions of this test.