Reaction mass of Resin acids and Rosin acids, hydrogenated, sodium salts and sodium [1R-(1α,4aβ,10aα)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylate

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:

3 November 2009 to .... May 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Fully Guideline- and GLP-compliant, read-across from related substance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Rosin, hydrogenated and [1R-(1a,4aß,10aa)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylic acid

IUPAC Name:

Reaction mass of Rosin, hydrogenated and [1R-(1a,4aß,10aa)]-1,2,3,4,4a,9,10,10a-octahydro-7-isopropyl-1,4a-dimethylphenanthren-1-carboxylic acid

Constituent 2

Reference substance name:

Resin 835 A, parent substance to Registered substance

IUPAC Name:

Resin 835 A, parent substance to Registered substance

Details on test material:

- Name of test material (as cited in study report): Resin 835 A
- Physical state: Amber flakes
- Lot/batch No.: K49E09-01999
- Expiration date of the lot/batch: 11 August 2010
- Storage condition of test material: Room temperature, in the dark but may be used under light.

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: primary culture

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: not applicable

Metabolic activation:

with and without

Metabolic activation system:

Lyophilized Post-Mitochondrial Supernatant (Moltox, Art. No. 11-01L.2, Lot.: 2361) from male Sprague-Dawley rat livers, induced with Aroclor-1254, in 0.154M KCL

Test concentrations with justification for top dose:

Experiment A (3 hours incubation, no metabolic activation system): 0.0067, 0.02, 0.06, 0.18 mg/mL
Experiment B (20 hours incubation, no metabolic activation system): 0.0067, 0.013, 0.02, 0.04, 0.06 mg/mL
Experiment A (3 hours incubation, with metabolic activation system): 0.0067, 0.02, 0.06, 0.18 mg/mL
Experiment B (3 hours incubation, with metabolic activation system): 0.0067, 0.02, 0.06 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: On the days of the experiments, stock solutions of "Resin 835 A" were prepared in DMSO (one solution for each tested concentration). These stock solutions were then diluted with the appropriate culture medium (RPMI or - for 20 hours of incubation - complete culture medium) to achieve the intended final concentrations of the test substance (final concentration of DMSO: 1%)
- Justification for choice of solvent/vehicle: Common vehicle for non water soluble substances

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

see below for details

Migrated to IUCLID6: and cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Cultures were kept at 37 °C and 5% CO2 for ca. 48 hours before further processing
- Exposure duration: 3 hours (one experiment without and two experiments with metabolic activation) and 20 hours (one experiment without metabolic activation)
- Expression time (cells in growth medium): 15 hours for the cultures treated with test substance for 3 hours (plus 2 hours with spindle inhibitor added)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): 0.1 mL of Colcemid (Gibco BRL Life Technologies, UK, article no 15210-057, 10 µg/mL in balanced salt solution) per culture.
STAIN (for cytogenetic assays): 10 minutes with Giemsa solution (10% v/v in a buffer of 0.067 M KH2PO4 and 0.067 M Na2HPO4 x 2 H2O in deionised water), rinsed in tap-water and then in deionised water.

NUMBER OF REPLICATIONS: 2 per culture

NUMBER OF CELLS EVALUATED:
Mitotic indices: 2000 lymphocytes per culture
Chromosome aberrations: 100 metaphases per culture (i.e. 200 per concentration, apart from cultures with obviously high numbers of metaphases with aberrations)

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic indices were determined by counting a total of 2000 lymphocytes per cell culture and by recording the number of lymphocytes in any stage of mitosis. This number was then expressed as percentage of mitotic lymphocytes.

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

Evaluation criteria:

Only well spread cells with 44 to 47 chromosomes, polyploid and endoreduplicated cells were acceptable for analysis. Structural aberrations were scored according to well defined (and reported) criteria.

Statistics:

The Chi2-Test (two-tailed, p=0.05) was used for the comparison between the negative control and the "Resin 835 A" cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher’s Exact Test was used. Chi2-Test or Fisher’s Exact Test were also used for the comparison between the negative and the positive controls.

Results and discussion

Test results

Species / strain:

lymphocytes: primary cell culture

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 0.18 mg/mL

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, historical control data (on negative and positive controls) given in the report.
In experiment A without the use of a metabolic activation system the number of metaphases with gaps was slightly higher than the upper range of historical positive controls (23/100 vs. 17.2/100). This event does not affect the validity of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
see "any other information on results incl. tables"

Remarks on result:

other: strain/cell type: lymphocytes

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

For further tables see attachment.

Mitotic index

Complete cytotoxicity occurred at a test substance concentration of 0.18 mg/mL medium in the experiments with 3 hours of incubation regardless whether a metabolic activation system was used or not. Marked cytotoxicity was observed at a concentration of 0.06 mg/ml in any of the experiments (3 hours and 20 hours of incubation) regardless whether a metabolic activation system was used or not.

Experiment without a metabolic activation system, 3 hours of incubation:

Test substance concentration (mg/mL)		0.0067		0.02		0.06		0.18
Mitotic index (% of respective negative control)		97.7		74.1		51.1		0.0

Bold figures: These concentrations were analysed

Experiment without a metabolic activation system, 20 hours of incubation:

Test substance concentration (mg/mL)		0.0067		0.013		0.02		0.04		0.06
Mitotic index (% of respective negative control)		49.4		92.4		88.8		58.8		43.5

Bold figures: These concentrations were analysed

Experiments with a metabolic activation system, 3 hours of incubation:

Test substance concentration (mg/mL)		0.0067		0.0067		0.2		0.2		0.6		0.6		0.18
Mitotic index (% of respective negative control)		91.4		42.5		118.3		65.5		84.8		35.8		0.0

Bold figures: These concentrations were analysed

Numerical aberrations

In both experiments with a metabolic activation system the number of metaphases with less than 46 chromosomes was statistically significantly higher at the highest test substance concentration of 0.06 mg/mL compared to the concurrent negative controls, which is regarded as a random event without biological relevance.

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any other experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

 

Gaps

No statistically significant increases in the number of gaps were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Structural aberrations

No marked or statistically significant increases in the number of metaphases with structural aberrations were noted in any of the experiments performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not. All figures were within the range of historical negative controls.

 

Positive controls

The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.However, in experiment A without the use of a metabolic activation system the number of metaphases with gaps was slightly higher than the upper range of historical positive controls (23/100 vs. 17.2/100).This event does not affect the validity of the study.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro.
Therefore, "Resin 835 A" is considered to be non-clastogenic in this chromosome aberration test when tested up to precipitating concentrations in the absence and presence of metabolic activation.

Executive summary:

Objective

The study was performed to determine possible mutagenic properties of "Resin 835 A" by means of anin vitromammalian chromosome aberration test in human lymphocytes, according to Regulation (EC) No 440/2008 method B.10. and to OECD Guideline 473.

 

Methods

A total of four experiments was performed and analysed: two of them without and two with the use of a metabolic activation system (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution). A concentration range between 0.0067 and 0.18 mg test substance per mL medium was tested. 0.18 mg test substance per mL medium was the highest technically feasible concentration, leading to precipitation of the test substance visible by the unaided eye.

Primary lymphocyte cultures were established from whole blood freshly obtained from two female donors. After 48 hours of incubation, "Resin 835 A" was added. In all experiments with the use of a metabolic activation system and in one experiment without addition of a metabolic activation system the test substance was washed out three hours later and the cultures were cultivated for another 17 hours. In one experiment without a metabolic activation system the cultures were treated with the test substance for 20 hours. In all experiments Colcemid was added two hours before the end of the cultivation period, and then cells were fixed and slides prepared.

The test substance was dissolved in DMSO and then added to RPMI medium for the 3h treatments or to complete culture medium for the 20h treatments (final concentration of DMSO: 1%). For each concentration of the test substance two cultures were established. One negative control (appropriate medium containing 1% DMSO) and one positive control (methanesulfonic acid methyl ester, MMS, for cultures without metabolic activation system and cyclophosphamide, CP, for cultures with a metabolic activation system) were set up concurrently in each experiment.

The concentrations of "Resin 835 A" in the experiments performed were between 0.0067 and 0.18 mg/L.

In general, apart from cultures with obviously high numbers of metaphases with aberrations, 100 metaphases per culture (i.e. 200 per concentration) were analysed for structural and numerical chromosomal aberrations. The slides were coded before analysis. The mitotic indices were calculated from 2000 lymphocytes per culture for an assessment of cytotoxicity.

 

Results

Cytotoxicity

Complete cytotoxicity occurred at a test substance concentration of 0.18 mg/mL mediumin the experiments with 3 hours of incubation regardless whether a metabolic activation system was used or not.Marked cytotoxicity was observed at a concentration of 0.06 mg/ml in any of the experiments (3 hours and 20 hours of incubation) regardless whether a metabolic activation system was used or not.

 

Numerical aberrations

In both experiments with a metabolic activation system the number of metaphases with less than 46 chromosomes was statistically significantly higher at the highest test substance concentration of 0.06 mg/mL compared to the concurrent negative controls, which is regarded as a random event without biological relevance.

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any other experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Gaps

No statistically significant increases in the number of gaps were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

 

Structural aberrations

No marked or statistically significant increases in the number of metaphases with structural aberrations were noted in any of the experiments performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not. All figures were within the range of historical negative controls.

 

Positive controls

The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.

 

Conclusion

"Resin 835 A" caused complete cytotoxicity at a test substance concentration of 0.18 mg/mL mediumin the experiments with 3 hours of incubation regardless whether a metabolic activation system was used or not. At a concentration of 0.06 mg/ml severe cytotoxicity (35.8% - 84.8%) occurred in any oft the experiments (3 hours and 20 hours of incubation) regardless whether a metabolic activation system was used or not.The highest concentration used (0.18 mg/mL) caused precipitation of the test substance, visible even to the unaided eye.

Statistically significantly higher numbers of metaphases with less than 46 chromosomes in both experiments with a metabolic activation system (treatment length of 3 hours) at a test substance concentration of 0.06 mg/mL are regarded as a random event without biological relevance.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro.

Therefore, "Resin 835 A" is considered to be non-clastogenicin this chromosome aberration test when tested up to precipitating concentrations in the absence and presence of metabolic activation.