2-Cyclopentene-1-carboxylic acid, 5-(dimethylphenylsilyl)-2-(hydroxymethyl)-, (1R,2R)-2-amino-1,3-dihydroxy-1-(4-nitrophenyl)propyl ester, (1R,5S)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2000 to 04 June 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in 2000 according to OECD Method 471 and EU Annex V test B14 and in accordance with GLP. Study material is well characterized.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-910-2

EC Name:

-

Cas Number:

1388152-30-4

Molecular formula:

Hill formula: C24 H32 N2 O7 Si CAS formula: C15 H20 O3 Si . C9 H12 N2 O4

IUPAC Name:

(1R,2R)-1,3-Dihydroxy-1-(4-nitrophenyl)propyl-2-ammonium (1R, 5S)-5-(dimethylphenylsilyl)-2-(hydroxymethyl)cyclopent-2-ene-1-carboxylate

Details on test material:

Test material is a white solid which was received at testing laboratory on 17 April 2000 and stored at room temperature in the dark.

Method

Target gene:

four histidine-requiring strains and one tryptophan-requiring strain

Species / strain

Species / strain / cell type:

bacteria, other: Salmonella typhimurium: TA1535, TA1537, TA98, TA100 Escherichia coli: WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/B-napthoflavone rat liver homogenate

Test concentrations with justification for top dose:

Preliminary toxicity (Range finding) experiment carried out at concentrations of 0., 0.15. 0.5, 1.5, 5, 15 ,50, 150, 500, 1500 and 5000 ug/plate . For experiment 1 concentrations (with & without metabolic activation) used: 50, 150, 500, 1500 and 5000 µg/plate . For experiment 2 an additional intermdiate dose of 3000 ug/plate was included for strains TA100 and TA 1535.

Vehicle / solvent:

Solvent: Dimethyl sulphoxide

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

vehicle controls used in parallel with the test material

Controls 2

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene at 1 ug/plate for TA100, 2 ug/plate for TA1535 &TA1537 and 10 ug/plate for WP2uvrA with metabolic activation.

Controls 3

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 5 ug/plate for TA98 with metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: 3 ug/plate for TA 100, 5 ug/plate for TA1535 and 2 ug/plate for WP2uvrA

Controls 5

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 80 ug/plate for TA1537

Controls 6

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 0.2 ug/plate for TA98

Details on test system and experimental conditions:

A toxicity range finding experiment was conducted with strain TA100 and WP2uvrA at ten concentrations plus negative (vehicle) controls in absence and in the presence of metabolic activation S-9. No evidence of toxicity was observed following this treatment and formulation and S9 mix were shown to be sterile. This test was acceptable and no evidence of toxicity was observed following this treatment. For experiment 1 five concentrations were assayed in triplicate against each tester strain with and without metabolic activation. Experiment 2 was performed using the same methodology but one additional intermediate dose concentration was added to TA100 and TA1535 to enhance the test material dose-response after statistically significant increases in revertant colony frequency had been observed in experiment 1. No evidence of toxicity was observed following this treatment. Negative and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates were all acceptable

Evaluation criteria:

Evaluation criteria: Acceptance criteria for validity of assay : the mean negative control counts fell within normal ranges, the positive control chemical induced clear increases in revertant numbers confirming discrimination between different strains and an active S-9 preparation, and no more than 5 % of the plates were lost through contamination or some other unforeseen event. Evaluation criteria: test article would be considered mutagenic if: the assay was valid, Dunnett's test gave significant response and the data set(s) showed a significant dose correlation and the positive responses described above were reproducible in atleast one strain of bacteria.

Statistics:

Mean and standard deviation of the plate counts for each treatment were determined. The Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked using linear regression analysis.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

other: preliminary test

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

other: preliminary test

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 5000 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

other: statistically significant increase but non-reproducible

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
All positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 mix were validated.

The test substance caused no visible reduction in the growth of the bacterial lawn at any dose level. The test substance was, therefore, tested up to the maximum recommended dose of 5000 ug/plate. No test substance precipitate was observed on the plates at any doses tested in either the presence or absence of S9 mix.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

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

Statistically significant, dose-related and reproducible increases in revertant colony frequency were observed in tester strains TA100 and TA1535, both with and without S9, at the upper dose levels of the test substance. The dose- responsiveness of the test substance was confirmed in the second experiment after the inclusion of an intermediate dose level. Statistically significant increases were also observed in TA98, although the responses in this instance were non-reproducible. The test substance was considered to be mutagenic under the conditions of this test.

Executive summary:

The study is considered valid and assigned a reliability rating of 2.

Ames Test, in vitro: Statistically significant, dose-related and reproducible increases in revertant colony frequency were observed in tester strains TA100 and TA1535, both with and without S9, at the upper dose levels of the test substance. The dose-responsiveness of the test substance was confirmed in the second experiment after the inclusion of an intermediate dose level. The test substance was considered to be mutagenic under the conditions of this test.