Reaction mass of (8R)-(11S)-11-[(4-hydroxyphenyl)methyl]-8-alkyl-polyoxo-1-phenyl-polyaza-2-oxadodecane and (8R)-(11R)-11-[(4-hydroxyphenyl)methyl]-8-alkyl-polyoxo-1-phenyl-polyaza-2-oxadodecane

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:

02 June 2009 – 16 June 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with OECD, EC, US EPA, and other international test guidelines, and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-1, Agricultural Production Bureau, November 24, 2000.

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444. ICH(1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The study report included a current certificate of GLP compliance for the test facility, issued by the MHRA.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: Off-white solid
- Storage condition of test material: aprox. -20 degrees in the dark.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

First test: 5, 15, 50, 150, 500, 1500, and 5000 µg/plate
Second test: 50, 150, 500, 1500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The sponsor indicated that the test substance was soluble in DMSO.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

2 µg/plate for TA100 and TA1535

Migrated to IUCLID6: In the absence of S9 Mix

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate for TA1537

Migrated to IUCLID6: In the absence of S9 Mix

Controls 3

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2 µg/plate for TA98

Migrated to IUCLID6: In the absence of S9 Mix

Controls 4

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2 µg/plate for WP2 uvrA (pKM101)

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

In the presence of S9-mix. 5 µg/plate for TA100 and TA1535; 10 µg/plate for WP2 uvrA (pKM101)

Controls 6

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for TA98 and TA1537.

Migrated to IUCLID6: In the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (first test); in agar with preincubation (second test)


DURATION
- Preincubation period: 30 minutes at 37°C (second test only).
- Exposure duration: 72 hours
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):


NUMBER OF REPLICATIONS: Three plates per concentration per strain


NUMBER OF CELLS EVALUATED:


DETERMINATION OF CYTOTOXICITY
- Method: other: reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn.


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Evaluation criteria:

The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls
were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains
TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit
mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of
mutagenic activity in this test system. No statistical analysis is performed.

Statistics:

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be
subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be
considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the
vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal
response if no clear results can be obtained.

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:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: No evidence of toxicity was obtained following exposure to the test substance in the first test. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.


COMPARISON WITH HISTORICAL CONTROL DATA: The mean revertant colony counts for the vehicle controls were within or close to the 99%
confidence limits of the current historical control range of the laboratory

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

A bacterial reverse mutation test was performed by Huntingdon Life Sciences, UK, to assess the potential of the test substance to cause gene mutation. The study was conducted to OECD, EC, US EPA, and other National and International guidelines, and was performed in compliance with GLP. In each of two tests, no evidence of cytotoxicity was observed, and no increase in the number of revertant colonies relative to the controls were seen. It was concluded that the test substance showed no evidence of mutagenic activity in the bacterial system under the test conditions employed.