Benzoic acid, 4-(acetylamino)-5-iodo-2-methoxy-, methyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: Micronucleus test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 07 July 2011 - Experimental completion date: 30 August 2011 (Report approval date: 17 October 2011)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test procedures are according to international guidelines and described in sufficient detail, but are not GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487 dated 22 July 2010

Principles of method if other than guideline:

The study was further conducted according to IWGT recommendations. Micronuclei detected in the cytoplasm of interphase cells, result from either chromosome breakage leading to acentric fragments (clastogenicity) or impairment of the structure and/or function of the mitotic apparatus leading to lagging chromosomes (aneugenicity).

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Iodomesac

IUPAC Name:

Iodomesac

Constituent 2

Reference substance name:

methyl 4-(acetylamino)-5-iodo-2-methoxybenzoate

IUPAC Name:

methyl 4-(acetylamino)-5-iodo-2-methoxybenzoate

Constituent 3

Reference substance name:

methyl 4-acetamido-5-iodo-2-methoxybenzoate

EC Number:

606-440-5

Cas Number:

201214-53-1

Molecular formula:

C11H12INO4

IUPAC Name:

methyl 4-acetamido-5-iodo-2-methoxybenzoate

Details on test material:

Test article identification: PE1091 (iodomesac)
Test article batch number: DS00014
Certificate of analysis: dated 14 April 2011
Expiration date: 7 April 2012
Conversion factor: Not applicable
A certificate of analysis is included in the report.

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

With metabolic activation (3-hour treatment and 21-hour recovery):
Range of concentrations tested in the culture medium: from 2.5 to 600 µg/mL.
Marked precipitate was noted from 300 µg/mL in the culture medium; 200 µg/mL (-7% of cytotoxicity) was retained as top concentration for scoring, considered as the lowest precipitating concentration in the culture medium based on the observations performed throughout the study (see second test and third test without metabolic activation). The evaluated concentrations were 80, 100 and 200 µg/mL.

Without metabolic activation (28-hour treatment without recovery):

First test:
Range of concentrations tested in the culture medium: from 2.5 to 600 µg/mL. Marked precipitate was noted from 300 µg/mL in the culture medium.
As the required cytotoxicity was not reached, a second test was performed with another range of concentrations. No results are presented.

Second test:
Range of concentrations tested in the culture medium: from 100 to 250 µg/mL. Precipitate was noted from 190 µg/mL in the culture medium.
The evaluated concentrations were 140, 170 and 180 µg/mL. The top concentration retained for scoring 180 µg/mL (50% of cytotoxicity) corresponded to the required cytotoxicity (50% to 60%).

Third test
Range of concentrations tested in the culture medium: from 100 to 250 µg/mL. Precipitate was noted from 180 µg/mL in the culture medium.
The evaluated concentrations were 130, 140 and 150 µg/mL. The top concentration retained for scoring 150 µg/mL (57% of cytotoxicity) corresponded to the
required cytotoxicity (50% to 60%).

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Group without metabolic activation

Migrated to IUCLID6: Without S9-mix

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Group with metabolic activation

Migrated to IUCLID6: With S9-mix

Details on test system and experimental conditions:

L5178Y cells were treated with the test article in 96-well microtiter plates for 3 hours with metabolic activation and for 28 hours without metabolic activation. Without metabolic activation the cells were harvested directly after the treatment time and with metabolic activation 21 hours (recovery time) after the end of the treatment. Cells were then fixed and stained with Giemsa, then scored manually.
The cytotoxicity of the test article was evaluated by the population doubling (PD), expressed as a percentage of the negative control (relative population doubling). Formula: PD = (log (C f /C 0 )) / log 2
C f = final cell count (24 hours after the start of treatment with metabolic activation or 28 hours without metabolic activation)
C 0 = initial cell count

Evaluation criteria:

Experiment acceptance criteria:
The experiment is considered valid when all the following criteria are fulfilled:
• the population doubling of the negative control is higher or equal to 1.5 but not higher than 2.2;
• the number of micronucleated cells per 1000 cells in the concurrent solvent control is lower than or equal to the acceptable upper value for historical solvent control data (99% percentile of our historical negative control data):
- 7 for 28-hour treatment without S9-mix;
- 11 for 3-hour treatment with S9-mix.
• the positive controls induce a marked increase in the number of micronucleated cells;
• at least three analyzable concentrations are scored;
• the highest concentration evaluated corresponds either to 5000 µg/mL (or 10 mM), or to the solubility limit in the solvent, or to the lowest precipitating concentration in the culture medium or to 50% to 60% of cell cytotoxicity.
Test evaluation criteria:
The ability of the test article to induce structural/numerical chromosome damage is evaluated by the increase in the number of micronucleated mononucleated cells, scored out of 1000 mononucleated cells.
A test article is considered to induce a positive response in the in vitro micronucleus test if the two criteria listed below are fulfilled for at least one concentration:
• the test article induces a statistically significant increase in the number of micronucleated cells for a given concentration;
• for this concentration, this value is strictly above positive threshold value (99% percentile of our historical negative control data):
- 7 for 28-hour treatment without S9-mix
- 11 for 3-hour treatment with S9-mix.
When none of the above criteria are fulfilled, the test article is considered negative.

Statistics:

Statistical analysis is performed using the pairwise Fisher exact test with Bonferroni-Holm correction.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

An increase (>7) in the number of micronucleated cells was observed from 170 µg/mL with a statistical significance.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cell death of 50 % or higher at and above 180 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

An increase (>7) in the number of micronucleated cells was observed from 140 µg/mL with a statistical significance at 150 µg/mL.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

57% of cytotoxicity at the top concentration retained for scoring 150 µg/mL.

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation

Under the experimental conditions of this study, PE1091 (iodomesac) was found positive in the exploratory in vitro micronucleus test in mouse lymphoma cells in the absence of metabolic activation. It was found negative in the presence of metabolic activation.