(2S)-2-amino-3-[3,5-diiodo-4-(4-methoxyphenoxy)phenyl]propanoic 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

Study period:

9 January 2019 - 30 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-amino-3-[3,5-diiodo-4-(4-methoxyphenoxy)phenyl]propanoic acid

EC Number:

619-013-3

Cas Number:

94345-95-6

Molecular formula:

C16H15I2NO4

IUPAC Name:

(2S)-2-amino-3-[3,5-diiodo-4-(4-methoxyphenoxy)phenyl]propanoic acid

Test material form:

solid: particulate/powder

Details on test material:

Name: O-METHYL-L-DIIODOTHYRONIN
Batch No.: B488840
Manufacturing date: 25 September 2018
Expiry date: 24 September 2020
Storage condition: at room temperature, protected from light

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsomal enzyme activation mixture (rat liver extract, S9 fraction).

Test concentrations with justification for top dose:

5 to 5000 μg/plate

Vehicle / solvent:

In the study two vehicle control groups were used according to the solubility of the test item and the solubility of positive control reference items.
Dimethyl sulfoxide (DMSO) for Test Item, NPD, 9AA and 2AA;
Ultrapure water for SAZ and MMS.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, NPD; 2-aminoanthracene, 2AA;

Details on test system and experimental conditions:

The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 1.38 x 10^9 cells per mL.
The plates were incubated at 37 °C for 48 hours and the growth of the bacterial background and the density of revertant colonies was determined.
The test substance was tested without as well as with an external metabolising system (S9-mix). The results were verified by a second, independent experiment. Three plates were used per concentration and per strain for the test substance; 6 for the negative control.

Evaluation criteria:

The colony numbers on the controls (untreated, vehicle, positive) and the test item plates were determined (counted manually, evaluated by unaided eye), the mean values and appropriate standard deviations and mutation rates were calculated.
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 is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic 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:

cytotoxicity

Remarks:

The lowest concentration level where signs of cytotoxicity were noticed was 50 μg/plate

Vehicle 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:

cytotoxicity

Remarks:

The lowest concentration level where signs of cytotoxicity were noticed was 50 μg/plate

Vehicle 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:

cytotoxicity

Remarks:

The lowest concentration level where signs of cytotoxicity were noticed was 50 μg/plate

Vehicle 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:

cytotoxicity

Remarks:

The lowest concentration level where signs of cytotoxicity were noticed was 50 μg/plate

Vehicle 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

Positive controls validity:

valid

Additional information on results:

The used strains of Salmonella typhimurium showed the expected genetic properties and were sensitive against several mutagenic chemicals.

All positive control substances increased the mutation frequency to more than the threshold values. As 2-aminoanthracene, 1,8-dihydroxy-anthraquinone and 7,12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the results of these substances demonstrate also the efficiency of the metabolising system.

Solubility
No precipitation of the test substance was seen in any of the concentration groups.

Toxicity
In the preliminary test with strain TA100 a reduced number of revertants was obtained at 5000 µg/plate. In the main test, reduced numbers of revertant colonies were observed in all strains in the higher concentrations. The bacterial background lawn was only affected in strain TA97a in the first experiment. Metabolic activation did not significantly change the toxicity.

Mutagenicity:
No increase in the number of mutants was observed in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

the test item O-METHYL-L-DIIODOTHYRONIN has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item O-METHYL-L-DIIODOTHYRONIN was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (±S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and Confirmatory Mutation Tests (Pre-Incubation Tests).

As the evaluation of the Confirmatory Mutation Test in the case of Salmonella typhimurium strains was not possible (for an unforeseen technical reason), the Confirmatory Mutation Test (Pre-Incubation Test) was repeated, completed with these strains with the same conditions and concentration levels as the originally proposed procedure.

Based on the results of the Solubility Test and the Concentration Range Finding Test the test item was dissolved in dimethyl sulfoxide (DMSO). Due to the limited solubility of the test item, lower stock solution concentration and consequently higher treatment volumes (0.2 mL) were applied in the test item treatments. According to the referred OECD 471 guideline [6] the proposed, usual treatment volumes vary between 0.05 mL and 0.1 mL. This higher treatment volume did not dilute significantly the top agar or change its composition. The applicability of higher treatment volume was based on the testing laboratory’s experience and referred literature data [12].

At the concentration choice the solubility and the cytotoxicity of the test item (obtained in the Concentration Range Finding Test) were taken into consideration and based on the recommendations of OECD 471 guideline [6] the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:

±S9: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.

Following the plate incorporation and pre-incubation procedures precipitate was noticed in the examined strains at the concentrations of 5000 and 1600 μg/plate (±S9). The obtained precipitate did not influence, disturb the scoring in any case.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development were not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.

The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and all of the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

No biologically relevant increases in revertant colony numbers were observed in revertant colony numbers of any of the five test strains following treatment with O-METHYL-L-DIIODOTHYRONIN 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 show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.