Resin acids and Rosin acids, esters with glycerol and diethylene glycol

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:

2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Resin acids and Rosin acids, esters with glycerol and diethylene glycol

EC Number:

288-327-7

EC Name:

Resin acids and Rosin acids, esters with glycerol and diethylene glycol

Cas Number:

85711-66-6

Molecular formula:

UVCB substance

IUPAC Name:

esterification product of (1R,4aR,4bR,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxylic acid; (1R,4aR,4bS,10aR)-1,4a-dimethyl-7-(propan-2-ylidene)-1,2,3,4,4a,4b,5,6,7,9,10,10a-dodecahydrophenanthrene-1-carboxylic acid; (1R,4aS,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,5,6,9,10,10a-decahydrophenanthrene-1-carboxylic acid; (1R,4aS,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylic acid and 2,2'-oxydiethanol and propane-1,2,3-triol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
3M, lot: K02007
- Purity, including information on contaminants, isomers, etc.:
no data.


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
15 to 30 °C (Room Temp)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage:
no data
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis:
no data
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
The top stock dilution of the test article was found to be non-soluble at 100 mg/mL in Saline or at 500 mg/mL in DMSO. Therefore, the test article final stock was attempted to be solubilized at a concentration at 10 mg/mL directly in THP-1 Culture Medium
- Reactivity of the test material with the incubation material used (e.g. plastic ware):
no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding):
Grinding into poweder, vortexing and sonicating.
- Preliminary purification step (if any):
- Final concentration of a dissolved solid, stock liquid or gel:
39 to 5000 µg/mL
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle):
no data.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution:
The test article stock and working dilutions were observed to be cloudy pink with white particles non-viscous suspension.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added:

Method

Target gene:

Histidine and tryptophan operons

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : phenobarbital/5,6-benzoflavone-induced rat liver S9
- method of preparation of S9 mix: The 9000 × g liver supernatant fraction (S9) from phenobarbital/5,6-benzoflavone-treated male rats (Lot No. 4442; 4475), glucose-6-phosphate and MgCl2-KCl in 0.1M phosphate buffer (Regensys “A”), and lyophilized nicotinamide adenine dinucleotide phosphate (NADP; Regensys “B”) was obtained from Molecular Toxicology, Inc. (Boone, NC). These reagents were combined to create the metabolic activation mixture (S9), maintained at 2°C to 8°C or on ice prior to use, on the day of testing.
- concentration or volume of S9 mix and S9 in the final culture medium: The S9 mixture contained 7.5% (v/v) S9.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

Test concentrations with justification for top dose:

1.0, 5.0, 10.0, 25.0, 50.0, 100, 250, 500, and 1000 µg/plate. The top dose was determined based on test article solubility and precipitation at higher concentration.

TA1537 was dosed with 0.00508, 0.0152, 0.0457, 0.137, 0.412, 1.23, 3.7, 11.1, 33.3 and 100 ug/plate in the 4th confirmatory mutagenicity assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: N, N-Dimethylformamide (DMF) at 10 mg/mL

- Justification for choice of solvent/vehicle: Test article solubility and test system compatibility.

- Justification for percentage of solvent in the final culture medium: Per OECD 471

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: Other:

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^7 and 10^9 cells per mL of culture were used
- Test substance added: in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 8.75 hours
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): Plates were scored 48 hours after the start of treatment.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8.75 hours
- Selection time (if incubation with a selective agent): 56.75 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours
- Method used: Plate incorporation
- If a selective agent is used: Histidine and tryptophan-minimal agar
- Number of cells seeded:: 10^7 and 10^9 cells per mL of culture were used

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
All 4 S. typhimurium strains used were histidine auxotrophs. The E. coli strain used was a tryptophan auxotroph. When these cells are grown on minimal medium plates containing a trace amount of histidine or tryptophan, only those cells that have reverted to histidine prototrophy (his+) or tryptophan prototrophy (trp+) are able to grow and form colonies. Because DNA replication is usually required for mutagenesis to occur, the small amount of histidine or tryptophan allows all the bacteria on the plate to undergo a few cycles of cell division. The bacteria that revert to (his+) or (trp+) are easily seen as colonies against the slight background growth. The spontaneous mutation (or reversion) frequency of each strain is relatively constant, but, when a known mutagen (positive control substance) is added to the test system, the mutation frequency is typically increased from 2 to over 100 times.

- OTHER:

Rationale for test conditions:

Per OECD 471.

Evaluation criteria:

Criteria for a positive response:
The test substance is considered positive for mutagenicity if it induces an increase of mean revertants per concentration with increasing test substance concentration. The increases should be at least 2 times the vehicle control substance background frequency for strains with high spontaneous levels (i.e., TA100) and 3 times for those with low spontaneous levels (TA98, TA1535, TA1537, and WP2 uvrA). These increases should be seen in at least 2 or more successive concentrations or the response should be repeatable at a single concentration.

Criteria for a negative response:
The test substance was considered to be negative for inducing mutagenicity if it did not induce a response which fulfills the criteria for a positive response.

Statistics:

For each concentration level and for each condition, the mean revertant count and standard deviation (SD) were determined.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was noted at 50 ug/plate and higher concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was noted at 25 ug/plate and higher concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation was noted at 50 ug/plate and higher concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitates were observed in initial assays with TA1537 at concentrations of 5.0 ug/plate and greater. No precipitation was observed in the 4th confirmatory assay with TA1537 with dosing from 0.00508 ug/plate to 100 ug/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitates were observed at 10 ug/plate and higher concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: Not expected based - test article is a solid.
- Water solubility: <0.43 mg/L (See IUCLID section 4.8)
- Precipitation and time of the determination: See 'Results and Discussion' section for precipitating concentrations. Observations for precipitation was made at plate scoring.

RANGE-FINDING/SCREENING STUDIES (if applicable): The test article was tested at 1.0, 5.0, 10, 50, 100, 500, 1000, and 5000 µg/plate using the plate incorporation method. Precipitates were observed at ≥ 50 µg/plate in both strains without metabolic activation; at 100 µg/plate in strain TA100 with metabolic activation; at ≥ 500 µg/plate in both strains with metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or number of revertant colonies) was not observed in any strain with or without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : All vehicle control and positive control data was within historical ranges for the reported results.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Ames test:
- Signs of toxicity : No cytotoxicity was observed (measured via background lawn reduction)

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, rosin acid derivatives are negative with and without metabolic activation (S9) in the bacterial reverse mutation assay (Ames assay).

Executive summary:

Rosin acid derivative was tested in a bacterial reverse mutation assay (Ames assay) according to OECD 471. The study was conducted in compliance with OECD GLP. Rosin acid derivative was prepared in N, N-dimethylformamide (DMF) and administered to S. typhimurium strains TA98, TA100, TA1535, and TA1537 and E. coli strain WP2 uvrA at concentrations up to 5000 ug/plate for the range-finding assay and 1000 ug/plate for the mutagenicity and repeat mutagenicity assays. Mutagenicity testing was performed in triplicate at each concentration with and without a phenobarbital/5,6 -benzoflavone-induced rat liver S9 metabolic activation system.

In the range-finding assay, rosin acid derivatives were tested at 1.0, 5.0, 10, 50, 100, 500, 1000, and 5000 µg/plate using the plate incorporation method. Precipitates were observed at ≥ 50 µg/plate in both strains without metabolic activation; at 100 µg/plate in strain TA100 with metabolic activation; at ≥ 500 µg/plate in both strains with metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or number of revertant colonies) was not observed in any strain with or without metabolic activation.

In the mutagenicity assay, rosin acid derivatives were tested at 1.0, 5.0, 10.0, 25.0, 50.0, 100, 250, 500, and 1000 µg/plate using the plate incorporation method. precipitates were observed at ≥ 10.0 µg/plate in strain WP2 uvrA without
metabolic activation; at ≥ 25.0 µg/plate in strain TA100 without metabolic activation; at ≥ 50.0 µg/plate in strains TA98 and TA1535 without metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was not observed in any strain without metabolic activation. In the repeat mutagenicity assay, rosin acid derivatives were tested at 1.0, 5.0, 10.0, 25.0, 50.0, 100, 250, 500, and 1000 µg/plate using the plate incorporation method. Precipitates were observed at ≥ 25.0 µg/plate in strains TA100, TA1535, and WP2 uvrA with metabolic activation; at ≥ 50.0 µg/plate in strain TA98 with metabolic activation. Cytotoxicity (i.e., reduction in the background lawn and/or mean number of revertant colonies) was not observed in any strain with metabolic activation. Due to equivocal results with the strain TA1537 and precipitation issues in the testing media, several repeat assays were required with this strain. In the final confirmatory assay with TA1537, rosin acid derivatives were tested at 0.00508, 0.0152, 0.0457, 0.137, 0.412, 1.23, 3.70, 11.1, 33.3, and 100 μg/plate. No precipitates or cytotoxicity were observed. In the two definitive mutagenicity assays, criteria for a negative response were met for TA1537.

Based on the results of the study, rosin acid derivatives are negative with and without metabolic activation (S9) in the bacterial reverse mutation assay (Ames assay).