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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-2017
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(21 Jul 1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Resin acids and Rosin acids, hydrogenated, calcium salts
EC Number:
271-402-3
EC Name:
Resin acids and Rosin acids, hydrogenated, calcium salts
Cas Number:
68554-12-1
Molecular formula:
Unspecified
IUPAC Name:
Resin acids and Rosin acids, hydrogenated, calcium salts
impurity 1
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Water
impurity 2
Chemical structure
Reference substance name:
Sodium chloride
EC Number:
231-598-3
EC Name:
Sodium chloride
Cas Number:
7647-14-5
Molecular formula:
ClNa
IUPAC Name:
sodium chloride
Test material form:
solid: particulate/powder
Details on test material:
Name in report: Resin acids and Rosin acids, hydrogenated, calcium salts
Lot No. ResinateCa1.2014
Storage conditions: refridgerator
The identity of Hydrogenated rosin, Calcium salt was confirmed with IR, UVVis and mass spectroscopy analysis
Purity: 100 % UVCB
Specific details on test material used for the study:
Water content: 1.3%
solid, off-white
storage in the refrigerator

Method

Target gene:
his+ / trp+
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
1st Experiment (Standard plate test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd Experiment (Preincubation test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
The substance is insolble in water.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer

OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Positive controls:

With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA
Evaluation criteria:
Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 100 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 1000 μg/plate onward.

Any other information on results incl. tables

Standard plate incorporation assay with metabolic activation

Strain Test group Dose (ug/plate) Mean revertants per plate standard deviation Factor Individual revertant colony counts
TA 1535 Acetone - 9.7 2.3 - 7, 11, 11
Test item 33 10.0 2.6 1.0 12, 11, 7
100 8.3 4.0 0.9 13 P, 6 P, 6 P
333 10.3 4.0 1.1 8 P, 15 P, 8 P
1000 9.0 1.0 0.9 8 P, 9 P, 10 P
2500 12.0 2.6 1.2 11 P, 15 P, 10 P
5000 7.7 2.1 0.8 7 P, 10 P, 6 P
  2-AA 2.5 293.7 52.6 30.4 303, 237, 341
TA 100 Acetone - 93.0 10.6 - 89, 85, 105
Test item 33 86.3 4.9 0.9 84, 92, 83
100 93.7 10.6 1.0 105 P, 84 P, 92 P
333 90.3 3.8 1.0 92 P, 86 P, 93 P
1000 92.7 11.4 1.0 80 P, 102 P, 96 P
2500 84.3 1.5 0.9 86 P, 84 P, 83 P
5000 70.3 9.7 0.8 68 P, 62 P, 81 P
  2-AA 2.5 1637.0 88.8 17.6 1553, 1628, 1730
TA 1537 Acetone - 7.0 1.7 - 8, 5, 8
Test item 33 5.7 1.2 0.8 5, 7, 5
100 7.3 4.7 1.0 11 P, 9 P, 2 P
333 6.0 1.0 0.9 5 P, 6 P, 7 P
1000 5.7 1.2 0.8 5 P, 5 P, 7 P
2500 5.0 2.0 0.7 5 P, 7 P, 3 P
5000 4.3 2.5 0.6 7 P, 2 P, 4 P
  2-AA 2.5 91.3 12.7 13.0 106, 85, 83
TA 98 Acetone - 25.3 4.7 - 27, 29, 20
Test item 33 23.0 6.6 0.9 24, 29, 16
100 31.0 9.2 1.2 39 P, 21 P, 33 P
333 26.7 7.1 1.1 19 P, 28 P, 33 P
1000 17.7 3.8 0.7 15 P, 22 P, 16 P
2500 16.7 4.6 0.7 22 P, 14 P, 14 P
5000 14.7 1.2 0.6 14 P, 14 P, 16 P
  2-AA 2.5 1225.0 73.8 48.4 1307, 1164, 1204
E. coli Acetone - 28.0 7.5 - 29, 35, 20
Test item 33 25.7 8.6 0.9 24, 18, 35
100 26.0 5.3 0.9 24 P, 22 P, 32 P
333 29.7 8.5 1.1 30 P, 21 P, 38 P
1000 23.3 2.1 0.8 25 P, 24 P, 21 P
2500 19.7 3.1 0.7 23 P, 19 P, 17 P
5000 17.7 3.5 0.6 18 P, 21 P, 14 P
  2-AA 60 141.3 14.5 5.0 132, 134, 158

Standard plate incorporation test without metabolic activation

Strain Test group Dose (ug/plate) Mean revertants per plate standard deviation Factor Individual revertant colony counts
TA 1535 Acetone - 8.0 1.7 - 6, 9, 9
Test item 33 10.0 1.7 1.3 12, 9, 9
100 8.7 2.3 1.1 10 P, 6 P, 10 P
333 12.0 1.0 1.5 13 P, 12 P, 11 P
1000 9.7 1.2 1.2 11 P, 9 P, 9 P
2500 6.3 0.6 0.8 6 P, 6 P, 7 P
5000 7.0 1.0 0.9 8 P, 7 P, 6 P
  MNNG 5.0 3818.3 106.8 477.3 3920, 3707, 3828
TA 100 Acetone - 83.7 3.5 - 84, 80, 87
Test item 33 96.0 10.4 1.1 84, 103, 101
100 89.3 9.7 1.1 87 P, 100 P, 81 P
333 82.3 5.0 1.0 77 P, 87 P, 83 P
1000 78.3 7.8 0.9 72 P, 87 P, 76 P
2500 86.7 8.4 1.0 92 P, 91 P, 77 P
5000 88.7 13.4 1.1 83 P, 104 P, 79 P
  MNNG 5.0 3511.3 90.4 42.0 3560, 3567, 3407
TA 1537 Acetone - 6.7 1.2 - 6, 8, 6
Test item 33 9.3 3.5 1.4 9, 6, 13
100 5.0 1.0 0.8 5 P, 6 P, 4 P
333 6.0 1.0 0.9 5 P, 6 P, 7 P
1000 5.7 0.6 0.9 6 P, 6 P, 5 P
2500 4.3 0.6 0.7 4 P, 5 P, 4 P
5000 3.7 2.1 0.6 3 P, 6 P, 2 P
  AAC 100 791.3 108.6 118.7 757, 704, 913
TA 98 Acetone - 18.7 7.2 - 15, 27, 14
Test item 33 19.0 7.0 1.0 27, 16, 14
100 19.3 2.3 1.0 18 P, 22 P, 18 P
333 21.0 0.0 1.1 21 P, 21 P, 21 P
1000 14.3 2.9 0.8 11 P, 16 P, 16 P
2500 15.3 4.7 0.8 17 P, 19 P, 10 P
5000 13.0 5.0 0.7 8 P, 18 P, 13 P
  NOPD 10 1128.3 85.9 60.4 1149, 1034, 1202
E. coli Acetone - 24.0 5.2 - 30, 21, 21
Test item 33 23.0 6.2 1.0 30, 18, 21
100 28.7 4.5 1.2 29 P, 33 P, 24 P
333 27.3 4.9 1.1 25 P, 33 P, 24 P
1000 17.0 2.6 0.7 19 P, 14 P, 18 P
2500 16.7 7.2 0.7 12 P, 25 P, 13 P
5000 14.3 1.5 0.6 14 P, 13 P, 16 P
  4-NQO 5 635.7 33.3 26.5 666, 641, 600
P = Precipitation

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in bacteria.
Executive summary:

In a key Guideline (OECD 471) bacterial reverse mutation assay, the test material (CAS# 68554-12-1; purity 99.6%) was tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli strain WP2 at concentrations of 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate in an acetone vehicle in the presence and absence of metabolic activation (±S9). Solvent controls were used in the study along with positive controls 2 -aminoanthracene (2 -AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4 -nitro-o-phenylenediamine (NOPD), 9 -aminoacridine (AAC), 4 -nitroquinoline-N-oxide (4 -NQO).

The test material was not mutagenic under the experimental conditions of this Ames test.