(3S,4aS,8aS)-N-tert-butyldecahydro-3-isoquinolinecarboxamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 October 1994 to 7 December 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline compliant study with no deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

PTH_decahydroamide

IUPAC Name:

PTH_decahydroamide

Details on test material:

- Name of test material (as cited in study report): PTH-decahydroamide
- Physical state: colourless to pale yellow solid
- Analytical purity:99.8% (GC)

- Purity test date: no data
- Lot/batch No.:25927
- Expiration date of the lot/batch: 30 June 1995

- Stability under test conditions: stable for duration of study. Stable in the selected vehicle for at least 48 hours
- Storage condition of test material: room temperature in the dark

Method

Target gene:

Various chromosomal aberrations in cultured peripheral human lymphocytes

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: F10 complete culture medium - consisting of Ham's F10 mediun without thymidine and hypoxanthine, supplemented with heat inactivated foetal calf serum, L-glutamine, penicillin/streptomycin and sodum bicarbonate and heparin. Whole blood was cultured in F10 complete medium with phytohaemaglutinin
- Properly maintained: yes
Cellular source was healthy adult male volunters (average generation times were 16.6, 14.4 and 15.5 hours for volunteers aged 34, 28 and 28 respectively, for cell lines used in the preliminary study , experiment 1 and 2 respectively).
Blood samples were obtained by venapuncture, into sodium heparin, and lymphocyte cultures started wihin 4 hours of collection.
All incubations were completed at 80-95% humidity in a CO2 enriched (5%) atmosphere, in the dark at 37 degrees Centigrade

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes prepared from Aroclor 1254 induced adult male Wistar or Sprague-Dawley rats

Test concentrations with justification for top dose:

Pilot study concentrations: 10, 33, 100, 333 and 1000 ug/ml for 24 and 48 h fixation period without S-9; and for the 48 hour fixation period with S-9.

Experiment 1 without S-9 (24 h fixation): 33, 100, 178, 333, 422 and 562 ug/ml
Experiment 1 without S-9 (48 h fixation): 333, 422 and 562 ug/ml

Experiment 1 with S-9 (24 h fixation): 33, 100, 333 and 1000 ug/ml
Experiment 1 with S-9 (48 h fixation): 333 and 1000 ug/ml

Experiment 2 without S-9 (48 h fixation): 10, 33, 56, 100, 178, 333, 422 and 562 ug/ml
Experiment 2 with S-9 (48 h fixation): 33, 100, 333 and 1000 ug/ml

Concentrations selected for scoring chromosomal aberrations:
Experiment 1 without S-9 (24 h fixation): 33, 178, 422 ug/ml
Experiment 1 without S-9 (48 h fixation): 422 ug/ml

Experiment 1 with S-9 (24 h fixation): 100, 333 and 1000 ug/ml
Experiment 1 with S-9 (48 h fixation): 1000 ug/ml

Experiment 2 without S-9 (48 h fixation): 33, 178 and 422 ug/ml
Experiment 2 with S-9 (48 h fixation): 100, 333 and 1000 ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Solvent for reference substances was Hank's Balanced Salt Solution without calcium or magnesium (HBSS)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

direct acting mutagen, 0.2 ug/ml for 24hr and 0.1 ug/ml for 48 hr treatment

Migrated to IUCLID6: without S-9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

indirect acting mutagen, 15ug/ml for 3hr treatment (24 hr fixation)

Migrated to IUCLID6: with S-9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium F10 Complete culture medium

DURATION

- Exposure duration: Cytogenicity test - 24 and 48 h without S-9 and 3 h with S-9. Experiment 1 - 24 and 48 hr with and without S-9; Experiment 2 - 3 hr exposure wth 24 hour fixation, with and without S-9.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 ug/ml medium
STAIN (for cytogenetic assays): 5% Giemsa solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Mitotic index determined from metaphases in 1000 cels per culture. Chromosome aberrations determined from 100 metaphase spreads per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Assay acceptability criteria included - numbers of chromosomal aberrations should fall with laboratory background historical control range and positive controls should produce statistically significant increase in number of cells with chromosomal aberrations.

For data evalauation:
a test substance was considered a positive clastogen if it induced a dose-related statistically significant increase in the number of cells with hromosomal aberrations and a statistically significant increase in the frequency of aberrations in the absence of a clear dose relationship.

Statistics:

Control and treated groups were compared using the Chi-squared method

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Based on the results of the pilot study, various dose selections were made for the main study determinations of effects on mitotic index and induction of chromosomal aberrations. Concentrations of greater than 1000 ug/ml could not be tested due to low solubiity of the test material in the culture medium. Mitotic index was reduced at 333 ug/ml without S-9 (24 and 48 h fixation), but no clear reduction seen in the experiment with S-9.

In the main study experiments PTH-decahydroamide did not induce a statistically or biologically significant increase in the number of cells with chromosomal aberrations, either with or without the presence of metabolic activation.
The number of cells with chromosomal aberrations in the solvent controls was within the historical control background range for the laboratory. The positive controls produced significant increases in the frequency or aberrant cells.

PTH-decahydroamide was not clastogenic under the conditions of this assay.

Remarks on result:

other: strain/cell type: human peripheral lymphocytes

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

PTH-decahydroamide was not clastogenic under the conditions of this assay.

Executive summary:

In a replicated assay to determine the effects of PTH-decahydroamide on the induction of chromosomal aberrations in cultured peripheral human lymphocytes, cultures were prepared with or without metabolic activation (rat liver S-9 mix) and tested using concentrations of up to 1000 ug/ml for 24 or 48 hour fixation periods.

None of the concentrations evaluated gave a postive response under the test conditions. Vehicle and positive control cultures gave responses consistent with historical ranges and met the acceptance criteria for the assay.

PTH-decahydroamide was not clastogenic under the conditions of this assay.