Reaction Product of Rosin, fumarated, Tall-Oil Rosin, Ca(OH)2 and MgO

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to guideline protocol

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Rosin, fumarated

EC Number:

266-040-8

EC Name:

Rosin, fumarated

Cas Number:

65997-04-8

IUPAC Name:

Rosin, fumarated

Details on test material:

- Name of test material (as cited in study report):Rosin, fumarated
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: UVCB
- Physical state: amber crystalline solid.
- Analytical purity: normal commercial samples used
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: TWR01075-15FTOR-9.19.01
- Expiration date of the lot/batch: 19 March 2002
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:
- Storage condition of test material: ambient temperature
- Other:

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells grown as monolayers, and have a generation time of approximately 12 hrs.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes. The modal chromosome number has been determined to be 21.
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 enzymes

Test concentrations with justification for top dose:

Test 1:
Absence of S9 mix: 9 dose levels up to the maximum permitted concentration of 5000ug/ml in the absence of S9 mix
Presence of S9 mix: 10, 20, 40, 60 and 80ug/ml

Test 2:
Presence of S9 mix: 5, 10, 20, 30, 40 and 50ug/ml
Absence of S9 mix (24hr harvest): 40, 60, 80, 95, 110 and 120 ug/ml
Absence of S9 mix (48hr harvest): 20, 40, 80, 120, 160, and 200ug/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item was freely soluble in DMSO.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide at 20-50ug/ml (with S9); and methyl methanesulphonate at 10-40ug/ml (without S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. The basic medium (Ham's F-10) containing HEPES buffer, was supplemented with the antibiotic minocycline. For cell growth and treatment in the absence of S9 mix, foetal bovine serum (10% v/v) was added. the medium used for treatment in the presence of S9 mix and for washing cultures before or after treatment, was serum free.

DURATION
See treatment schedules under 'Any other information on materials and methods inc. tables'

SPINDLE INHIBITOR (cytogenetic assays): Colcemid at 0.1ug/ml for 2 hrs
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 3 slides per culture

NUMBER OF CELLS EVALUATED: A total of 100 metaphase cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: by calculating number of cells recovered per culture and compared with the number of cells (mean of 2 cultures) recovered from the vehicle control cultures.

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

OTHER:

Evaluation criteria:

Toxicity: A dose level was considered to be toxic if the cell count was reduced to less than 50% of the mean vehicle control culture values or if consistent evidence of changes to cell morphology was observed.
Clastogenicity: The results for test item and positive control treated cultures were evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures were within the 95% confidence limits for the historical negative control data. The response at a single dose was classified as significant if the percent of aberrant cells was consistently greater than the 99% confidence limits for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate. A test was positive if the response in at least one acceptable dose levels was significant by the criterion described above. At test item was positive if both Test 1 and 2 were positive, as described above or if the second test was positive after the first test gave indications of activity. These indications may be suspicious levels of aberrant cells (between 95% and 99% confidence limits). Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive.

Statistics:

estimation of confidence limits.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see below

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity was noted at 20-80ug/ml in the presence of S9 mix and at 156-5000ug/ml in the absence of S9 mix in Test 1. In Test 2, toxicity was noted in cultures treated with 50ug/ml in the presence of S9 mix. In the absence of S9 mix, toxicity was noted in cultures treated with 95-120ug/ml (24hr harvest) and 80-200ug/ml (48 hr harvest).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Cultures treated with the following dose levels were selected for assessment of chromosomal aberrations:

Test 1, Presence of S9 mix: 10, 20, and 40ug/ml

Test 1, Absence of S9 mix: 39, 78, and 156 ug/ml

Test 2, Presence of S9 mix: 30, 40 and 50 ug/ml

Test 2, Absence of S9 mix (24 hr harvest): 80, 95, and 110 ug/ml

Test 2, Absence of S9 mix (48 hr harvest): 40, 80, and 120ug/ml

All the cultures, with one exception, had levels of structural aberrations within the 95% confidence limits of the historical negative control data. The exception was seen in Test 1, presence of S9 mix, where one of the cultures treated with 40ug/ml had a suspicious level of aberrations in the aberrant cells excluding gaps parameter. The duplicate culture was within the 95% confidence limits of the historical negative control data so the suspicious response was deemed sporadic and not biologically significant.

When the extra assessment of polyploidy was carried out on the cultures treated inthe absence of S9 mix and harvested at 48 hrs there was an slight increase in the number of polyploidy cells in the cultures treated with 120ug/ml. This response was observed only at a concentration level overtly toxic to the cells.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance was not clastogenic.

Executive summary:

In this in vitro mammalian chromosomal aberration test conducted in Chinese Hamster Ovary cells according to OECD guideline, Rosin, fumarated did not show clastogenic effects under the conditions of the study, both in the presence and absence of activation system. Although the test item induced polyploidy this was at a concentration overtly toxic to the cells.