1H-Isoindole-1,3(2H)-dione, 2-[(2R)-2-hydroxy-3-[[4-(3-oxo-4-morpholinyl)phenyl]amino]propyl]-

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:

Feb 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1H-Isoindole-1,3(2H)-dione, 2-[(2R)-2-hydroxy-3-[[4-(3-oxo-4-morpholinyl)phenyl]amino]propyl]-

IUPAC Name:

1H-Isoindole-1,3(2H)-dione, 2-[(2R)-2-hydroxy-3-[[4-(3-oxo-4-morpholinyl)phenyl]amino]propyl]-

Constituent 2

Reference substance name:

2-[(2R)-2-hydroxy-3-{[4-(3-oxomorpholin-4-yl)phenyl]amino}propyl]-1H-isoindole-1,3(2H)-dione

EC Number:

610-200-5

Cas Number:

446292-07-5

Molecular formula:

C21 H21 N3 O5

IUPAC Name:

2-[(2R)-2-hydroxy-3-{[4-(3-oxomorpholin-4-yl)phenyl]amino}propyl]-1H-isoindole-1,3(2H)-dione

Specific details on test material used for the study:

purity > 98.2%

Method

Target gene:

histidin gene locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Hydroxyaminophthalimid: 50-5000 µg/plate (repeat tests in TA 1535 and TA 100: 100-3200 µg/plate)
Sodium-azide: 10 µg/plate (TA 1535)
Nitrofurantoin: 0.2 µg/plate (TA 100)
4-Nitro-1,2-phenylene diamine: 10 µg/plate (TA 1537), 0.5 µg/plate (TA 98)
Mitomycin C: 0.2 µg/plate (TA 102)
2-Aminoanthracene: 3 µg/plate

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

No solvent control was used since sufficient evidence was available in the literature and from testing laboratory experience, indicating that the solvents used had no influence on the spontaneous mutant counts of the used strains.

Positive controls:

yes

Positive control substance:

other: Sodium-azide, Nitrofurantoin, 4-Nitro-1,2-phenylene diamine, Mitomycin C, 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): E+06 dilution
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA 1535 and TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

lowest reproducible effective dose for TA 1535: 200 µg/plate; lowest reproducible effective dose for TA 100: 1600 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium, other: TA 1537, TA 98 and TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

There was no indication of a bacteriotoxic effect of Hydroxyaminophthalimid up to and including 3200 µg/plate. No inhibition of growth was noted as well. At 3200 µg/plate, the substance started to precipitate, so that at 5000 µg/plate no further evaluation was possible.

The

Applicant's summary and conclusion

Conclusions:

based on positive results in strains TA1535 and TA100 the test item has mutagenic potential

Executive summary:

Hydroxyaminophthalimid was investigated using the Salmonella/microsome plate incorporation test for point-mutagenic effects in doses up to and including 5000 µg/plate on the five histidine-auxotrophic Salmonella typhimurium LT2 strains TA 1535, TA 100, TA 1537, TA 98 and TA 102.

Doses up to and including 3200 µg/plate did not cause any bacteriotoxic effects. No inhibition of growth was noted as well. Substance precipitation occurred at the dose of 3200 µg/plate and above. Therefore, the test was no longer interpretable at 5000 µg/plate.

Evidence of mutagenic activity of Hydroxyaminophthalimid was seen. On Salmonella typhimurium TA 1535 and TA 100, a biologically relevant increase was found in the mutant count compared to the corresponding negative control. Positive response was only found without S9 mix. The lowest reproducible effective dose was 200 µg/plate for S. typhimurium TA 1535 and 1600 µg/plate for TA 100. The Salmonella/microsome test thus showed Hydroxyaminophthalimid to have a mutagenic effect.