Amines, rosin

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

Amines, rosin

EC Number:

263-139-8

EC Name:

Amines, rosin

Cas Number:

61790-47-4

Molecular formula:

C19H31N1

IUPAC Name:

1-[(1R,4aR,10aS)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl]methanamine; 1-[1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthren-1-yl]methanamine

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Rosin Amine 90
- Physical state: liquid
- Colour: pale amber
- Viscosity: high to extreme

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:

Method

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

Species / strain 1

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 2

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.

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Applicant's summary and conclusion

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.