Amines, rosin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test:

In the key study (2017), Rosin amine 90 was evaluated for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria: according to the OECD TG 471, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test and Japanese Regulatory Authorities including METI, MHLW and MAFF.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the metabolizing system (10% liver S9 in standard co-factors).

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal historical range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation within normal historical ranges of the tested bacterial strains.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were initially assayed in triplicate against each tester strain, using the direct plate incorporation method.  However, the test item induced excessive toxicity to all of the Salmonella strains dosed in the absence of S9-mix and Salmonella strains TA1535 and TA1537 dosed in the presence of S9-mix.  Consequently, these bacterial strains were repeated at a later date using an amended dose range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.  E.coli strain WP2uvrA (absence and presence of S9) and Salmonella strains TA100 and TA98 (presence of S9) were tested at 1.5 to 5000 µg/plate.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method).  Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 2 (pre-incubation method).  Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the first mutation test at 15 µg/plate in the absence of S9-mix.  This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility.  Furthermore, the individual revertant counts at the statistically significant dose level was within the in-house historical untreated/vehicle control range for the tester strain and the mean maximum fold increase was only 1.4 times the concurrent vehicle control.

Chromosomal aberration test:

In the key study (2017), the test item CAS 8050-09-7 (Rosin), dissolved in THF, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations.

The highest applied concentration in this study (3500.0 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. However, in the presence of S9 mix, the highest applied concentration showed clear cytotoxic effects, but was not evaluable for cytogenetic damage. In Experiment II in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Gene mutation test:

The key study was conducted according to a method that was designed to assess the potential mutagenicity of the test material: CAS 8050-09-7 (Rosin) on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 2.5 to 40 µg/ml in the absence of metabolic activation and 10 to 80 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 2.5 to 45 µg/ml in the absence of metabolic activation, and 10 to 55 µg/ml in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2017 - 23 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Version / remarks:

The test method was designed to be compatible with the guidelines for bacterial mutagenicity
testing published by the major Japanese Regulatory Authorities including METI, MHLW and
MAFF

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Rosin amine, 90 20170116023
- Physical state/Appearance: Amber viscous liquid
- Expiration date of the lot/batch: 1 year from manufacture
- Purity test date: 100% (UVCB), No correction was required for purity allowance (UVCB product).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within four hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 5 minutes at 40 °C on the day of each experiment. No correction was required for purity allowance. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

Target gene:

Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
WP2uvrA trp-; uvrA-: base-pair substitution

TA100 his G 46; rfa-; uvrB-;R-factor

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: The Salmonella typhimurium strains have a deleted excision repair mechanism and mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base-pair substitution to tryptophan independence

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

The maximum dose level of the test item in the first experiment was initially selected as the maximum recommended dose level of 5000 µg/plate according to the OECD Guidelines for Testing of Chemicals No. 471 (1997) “Bacterial Reverse Mutation Test”. Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were initially assayed in triplicate against each tester strain, using the direct plate incorporation method.A test item film (greasy in appearance) with an associated precipitate was noted at 5000 µg/plate. However, the test item induced excessive toxicity to all of the Salmonella strains dosed in the absence of S9-mix and Salmonella strains TA1535 and TA1537 dosed in the presence of S9-mix. Consequently, these bacterial strains were repeated at a later date using an amended dose range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate. E.coli strain WP2uvrA (absence and presence of S9) and Salmonella strains TA100 and TA98 (presence of S9) were tested at 1.5 to 5000 µg/plate.

The dose range used for Experiment 2 was determined by the results of Experiment 1 (and Experiment 1 repeat) and was as follows:

E.coli strain WP2uvrA (without S9-mix) and Salmonella strains TA100 and TA98 (with
S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
All Salmonella strains (without S9-mix) and TA1535 and TA1537 (with S9-mix): 0.015,
0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate.
E.coli strain WP2uvrA (with S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.

Vehicle / solvent:

- Vehicle:
Identity: Dimethyl sulphoxide (DMSO)
Supplier: Fisher Scientific
CAS No.: 67-68-5
Batch number: 1690734
Purity: >99%
Expiry Date: 31 March 2022
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Remarks:

The solvent, negative and positive controls were performed in triplicate.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation up to nine dose levels
- Cell density at seeding (if applicable): All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.

DURATION: All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: in triplicate

Rationale for test conditions:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello an
Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable (within historical control ranges). T

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- not specifed

RANGE-FINDING/SCREENING STUDIES:
-

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control and negative (solvent/vehicle) historical control data: Combined historical negative and solvent control ranges for 2015 and 2016 are presented in
Appendix 1.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Remarks on result:

other: no mutagenic

Conclusions:

Rosin amine 90 was considered to be non-mutagenic under the conditions of the OECD Guidelines for Testing of Chemicals No. 471 (1997) “Bacterial Reverse Mutation Test”. It also meets the requirements of the Method B13/14 of Commission Regulation (EC) Number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial
Reverse Mutation Test and is compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and
MAFF.

Executive summary:

The purpose of the study was to evaluate Rosin amine 90 for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria: according to the OECD TG 471, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test and Japanese Regulatory Authorities including METI, MHLW and MAFF.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the metabolizing system (10% liver S9 in standard co-factors).

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal historical range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation

within normal historical ranges of the tested bacterial strains.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were initially assayed in triplicate against each tester strain, using the direct plate incorporation method.  However, the test item induced excessive toxicity to all of the Salmonella strains dosed in the absence of S9-mix and Salmonella strains TA1535 and TA1537 dosed in the presence of S9-mix.  Consequently, these bacterial strains were repeated at a later date using an amended dose range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.  E.coli strain WP2uvrA (absence and presence of S9) and Salmonella strains TA100 and TA98 (presence of S9) were tested at 1.5 to 5000 µg/plate.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method).  Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 2 (pre-incubation method).  Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the first mutation test at 15 µg/plate in the absence of S9-mix.  This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility.  Furthermore, the

individual revertant counts at the statistically significant dose level was within the in-house historical untreated/vehicle control range for the tester strain and the mean maximum fold increase was only 1.4 times the concurrent vehicle control.

Conclusion:

Rosin amine 90 was considered to be non-mutagenic under the conditions of this study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The maximum dose level used was limited by test material induced toxicity.

Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 0001423147 at 400 µg/ml for Experiment 1, and Sigma batch 0001426183 at 150 µg/ml for Experiment 2, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

Vehicle used:
Vehicle (DMSO) treatment groups were used as the vehicle controls.


Justification for choice of vehicle:
Formed a solution suitable for dosing at the required concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 2.5 to 40 µg/ml in the absence of metabolic activation and 10 to 80 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 2.5 to 45 µg/ml in the absence of metabolic activation, and 10 to 55 µg/ml in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables" section.

Statistics:

Please see "Any other information on materials and methods incl. tables" section.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 19.53 to 5000 µg/ml.
In all three of the exposure groups there were marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was very steep in all three of the exposure groups. A precipitate of the test material was observed at and above 312.5 µg/ml in all three of the exposure groups on dosing. In the subsequent mutagenicity experiments the maximum dose level was limited by toxicity.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 3 and 6). A marked reduction in viability (%V), indicative of residual toxicity, was only observed at a dose level that had been excluded from the statistical analysis due to excessive toxicity in the absence of metabolic activation (Table 3). Optimum levels of toxicity were achieved in the presence of metabolic activation (Table 6). Optimum or near optimum levels of toxicity were not achieved in the absence of metabolic activation, despite using a narrow dose interval, due to the very sharp onset of test material-induced toxicity (Table 3). However, it was considered that, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including those that achieved or exceeded the optimum level of toxicity, or in the second experiment where near optimum levels of toxicity were achieved in both the absence and presence of metabolic activation, the test material had been adequately tested. The excessive toxicity observed at and above 30 µg/ml in the absence of metabolic activation, and at and above 60 µg/ml in the in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 25 µg/ml in the absence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose level was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). No precipitate of test material was observed at any of the dose levels.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of a marked dose-related reduction in % RSG and RTG values in cultures dosed with the test material in the both the absence and presence of metabolic activation (Tables 9 and 12). There was no evidence of any significant reductions in (%V) viability, therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation (Tables 9 and 12). Near optimum levels of test material-induced toxicity were achieved in both the absence and presence of metabolic activation (Tables 9 and 12). It is considered that, due to the very steep toxicity curve of the test material, a slightly higher dose level in both the absence and presence of metabolic activation would have resulted in excessive levels of toxicity. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had no effect on the toxicity of the test material.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). No precipitate of test material was observed at any of the dose levels.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Remarks:

Migrated from field 'Test system'.

Please see Attached "Tables 1 to 13"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 2.5 to 40 µg/ml in the absence of metabolic activation and 10 to 80 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 2.5 to 45 µg/ml in the absence of metabolic activation, and 10 to 55 µg/ml in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD guideline 473

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000.0, 3500.0 µg/mL
Experiment II: 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000.0, 3500.0 µg/mL

Without metabolic activation:
Experiment I: 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000.0, 3500.0 µg/mL
Experiment II: 1.9, 3.4, 6.0, 10.4, 18.3, 32.0, 56.0, 98.0, 171.4, 300.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item CAS 8050-09-7 (Rosin), dissolved in THF, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphase plates per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 3500.0 µg/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests considering the solubility properties of the test item.
In Experiment I visible precipitation of the test item in the culture medium was observed at 69.6 µg/mL and above in the absence and presence of S9 mix. In Experiment II, precipitation was observed at 171.4 µg/mL and above in the absence of S9 mix and at 121.9 µg/mL and above in the presence of S9 mix at the end of treatment. No relevant increase in the osmolarity or pH value was observed (Exp. I: solvent control: 361 mOsm, pH 7.4 versus 318 mOsm and pH 7.4 at 3500.0 µg/mL; Exp. II: solvent control: 374 mOsm, pH 7.6 versus 365 mOsm and pH 7.6 at 300.0 µg/mL).
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. However, in the presence of S9 mix, the highest applied concentration showed clear cytotoxic effects, but was not evaluable for cytogenetic damage. In Experiment II in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration (44.2 % of control). In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 2.0 % aberrant cells, excluding gaps) were in accordance with the range of the solvent control values (0.0 - 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (825.0 µg/mL) or CPA (2.5 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Summary of results of the chromosomal aberration study with CAS 8050-09-7 (Rosin)

	Preparation interval	Test item concentration in µg/mL	Mitotic indices in % of control	Aberrant cells in %
				incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1	100.0	0.0	0.0	0.0
		Positive control^2	75.9	8.5	8.5^S	0.5
		22.7	96.3	1.0	0.5	0.0
		39.8	99.7	2.5	1.5	0.0
		69.6^P	81.6	1.0	0.5	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1	100.0	2.5	1.5	0.0
		Positive control^2	46.0	17.5	15.0^S	2.5
		18.3	95.7	1.0	0.5	0.0
		32.0	70.7	1.0	1.0	0.0
		56.0	44.2	0.5	0.0	0.0
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1	100.0	2.5	2.0	1.0
		Positive control^3	49.3	15.0	14.5^S	3.0
		39.8	101.3	0.5	0.5	0.0
		69.6^P	107.9	2.5	2.0	0.0
		2000.0^P	100.4	0.0	0.0	0.0
II	22 hrs	Solvent control^1	100.0	4.0	2.0	0.0
		Positive control^4	93.4	11.5	10.5^S	2.5
		39.8	121.4	2.0	1.0	0.0
		69.6	108.3	1.0	0.5	0.0
		121.9^P	104.1	0.5	0.0	0.0

 

*  Including cells carrying exchanges

^P  Precipitation occurred at the end of treatment

^S  Aberration frequency statistically significant higher than corresponding control values

¹   THF 0.5 % (v/v)

²     EMS 825.0 µg/mL

³   CPA     2.5 µg/mL

⁴   CPA     7.5 µg/mL

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

CAS 8050-09-7 (Rosin) is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or precipitating concentrations because the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, rosin is not classifiable for Germ Cell Mutagenicity according to Directive 67/548/EEC, the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Executive summary:

The test item CAS 8050-09-7 (Rosin), dissolved in THF, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations.

The highest applied concentration in this study (3500.0 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. The chosen treatment concentrations and the rationale for the dose selection are reported in Table1. The evaluated experimental points and the results are summarised in Table2.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. However, in the presence of S9 mix, the highest applied concentration showed clear cytotoxic effects, but was not evaluable for cytogenetic damage. In Experiment II in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

The maximum dose level used was limited by test material induced toxicity.

Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 0001423147 at 400 µg/ml for Experiment 1, and Sigma batch 0001426183 at 150 µg/ml for Experiment 2, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.

Vehicle / solvent:

Vehicle used:
Vehicle (DMSO) treatment groups were used as the vehicle controls.


Justification for choice of vehicle:
Formed a solution suitable for dosing at the required concentration.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 2.5 to 40 µg/ml in the absence of metabolic activation and 10 to 80 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 2.5 to 45 µg/ml in the absence of metabolic activation, and 10 to 55 µg/ml in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables" section.

Statistics:

Please see "Any other information on materials and methods incl. tables" section.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

non-mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS

Preliminary Toxicity Test

The dose range of the test material used in the preliminary toxicity test was 19.53 to 5000 µg/ml.
In all three of the exposure groups there were marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was very steep in all three of the exposure groups. A precipitate of the test material was observed at and above 312.5 µg/ml in all three of the exposure groups on dosing. In the subsequent mutagenicity experiments the maximum dose level was limited by toxicity.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 3 and 6). A marked reduction in viability (%V), indicative of residual toxicity, was only observed at a dose level that had been excluded from the statistical analysis due to excessive toxicity in the absence of metabolic activation (Table 3). Optimum levels of toxicity were achieved in the presence of metabolic activation (Table 6). Optimum or near optimum levels of toxicity were not achieved in the absence of metabolic activation, despite using a narrow dose interval, due to the very sharp onset of test material-induced toxicity (Table 3). However, it was considered that, with no evidence of any toxicologically significant increases in mutant frequency at any of the dose levels, including those that achieved or exceeded the optimum level of toxicity, or in the second experiment where near optimum levels of toxicity were achieved in both the absence and presence of metabolic activation, the test material had been adequately tested. The excessive toxicity observed at and above 30 µg/ml in the absence of metabolic activation, and at and above 60 µg/ml in the in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 25 µg/ml in the absence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose level was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). No precipitate of test material was observed at any of the dose levels.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of a marked dose-related reduction in % RSG and RTG values in cultures dosed with the test material in the both the absence and presence of metabolic activation (Tables 9 and 12). There was no evidence of any significant reductions in (%V) viability, therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation (Tables 9 and 12). Near optimum levels of test material-induced toxicity were achieved in both the absence and presence of metabolic activation (Tables 9 and 12). It is considered that, due to the very steep toxicity curve of the test material, a slightly higher dose level in both the absence and presence of metabolic activation would have resulted in excessive levels of toxicity. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had no effect on the toxicity of the test material.
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). No precipitate of test material was observed at any of the dose levels.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Remarks on result:

other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Remarks:

Migrated from field 'Test system'.

Please see Attached "Tables 1 to 13"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 2.5 to 40 µg/ml in the absence of metabolic activation and 10 to 80 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 2.5 to 45 µg/ml in the absence of metabolic activation, and 10 to 55 µg/ml in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD guideline 473

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000.0, 3500.0 µg/mL
Experiment II: 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000.0, 3500.0 µg/mL

Without metabolic activation:
Experiment I: 22.7, 39.8, 69.6, 121.9, 213.2, 373.2, 653.1, 1142.9, 2000.0, 3500.0 µg/mL
Experiment II: 1.9, 3.4, 6.0, 10.4, 18.3, 32.0, 56.0, 98.0, 171.4, 300.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: THF
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 100 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item CAS 8050-09-7 (Rosin), dissolved in THF, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours (Exp. I & II) after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 100 metaphase plates per culture were scored for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 3500.0 µg/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests considering the solubility properties of the test item.
In Experiment I visible precipitation of the test item in the culture medium was observed at 69.6 µg/mL and above in the absence and presence of S9 mix. In Experiment II, precipitation was observed at 171.4 µg/mL and above in the absence of S9 mix and at 121.9 µg/mL and above in the presence of S9 mix at the end of treatment. No relevant increase in the osmolarity or pH value was observed (Exp. I: solvent control: 361 mOsm, pH 7.4 versus 318 mOsm and pH 7.4 at 3500.0 µg/mL; Exp. II: solvent control: 374 mOsm, pH 7.6 versus 365 mOsm and pH 7.6 at 300.0 µg/mL).
In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. However, in the presence of S9 mix, the highest applied concentration showed clear cytotoxic effects, but was not evaluable for cytogenetic damage. In Experiment II in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration (44.2 % of control). In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 2.0 % aberrant cells, excluding gaps) were in accordance with the range of the solvent control values (0.0 - 2.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (825.0 µg/mL) or CPA (2.5 or 7.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Summary of results of the chromosomal aberration study with CAS 8050-09-7 (Rosin)

	Preparation interval	Test item concentration in µg/mL	Mitotic indices in % of control	Aberrant cells in %
				incl. gaps*	excl. gaps*	carrying exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control^1	100.0	0.0	0.0	0.0
		Positive control^2	75.9	8.5	8.5^S	0.5
		22.7	96.3	1.0	0.5	0.0
		39.8	99.7	2.5	1.5	0.0
		69.6^P	81.6	1.0	0.5	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control^1	100.0	2.5	1.5	0.0
		Positive control^2	46.0	17.5	15.0^S	2.5
		18.3	95.7	1.0	0.5	0.0
		32.0	70.7	1.0	1.0	0.0
		56.0	44.2	0.5	0.0	0.0
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control^1	100.0	2.5	2.0	1.0
		Positive control^3	49.3	15.0	14.5^S	3.0
		39.8	101.3	0.5	0.5	0.0
		69.6^P	107.9	2.5	2.0	0.0
		2000.0^P	100.4	0.0	0.0	0.0
II	22 hrs	Solvent control^1	100.0	4.0	2.0	0.0
		Positive control^4	93.4	11.5	10.5^S	2.5
		39.8	121.4	2.0	1.0	0.0
		69.6	108.3	1.0	0.5	0.0
		121.9^P	104.1	0.5	0.0	0.0

 

*  Including cells carrying exchanges

^P  Precipitation occurred at the end of treatment

^S  Aberration frequency statistically significant higher than corresponding control values

¹   THF 0.5 % (v/v)

²     EMS 825.0 µg/mL

³   CPA     2.5 µg/mL

⁴   CPA     7.5 µg/mL

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

CAS 8050-09-7 (Rosin) is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or precipitating concentrations because the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, rosin is not classifiable for Germ Cell Mutagenicity according to Directive 67/548/EEC, the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Executive summary:

The test item CAS 8050-09-7 (Rosin), dissolved in THF, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I & II
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations.

The highest applied concentration in this study (3500.0 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473. The chosen treatment concentrations and the rationale for the dose selection are reported in Table1. The evaluated experimental points and the results are summarised in Table2.

In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. However, in the presence of S9 mix, the highest applied concentration showed clear cytotoxic effects, but was not evaluable for cytogenetic damage. In Experiment II in the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentration. In the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Rosin amine 90 was considered to be non-mutagenic in the AMES test.

The test material, CAS 8050-09-7 (Rosin), read-across analogue was considered to be non-mutagenic to L5178Y cells. No evidence of an increase in polyploid metaphases as well induction of structural chromosomal aberrations in human lymphocytes in vitro were noticed after treatment with the test item: CAS 8050-09-7 (Rosin).

Rosin amine 90 is not classified according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 or UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).