Resin acids and Rosin acids, hydrogenated, calcium salts

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:

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

Test material information

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:

calcium bis((1R,4aR,4bS,8aR,10aR)-1,4a-dimethyl-7-(propan-2-yl)-tetradecahydrophenanthrene-1-carboxylate)

impurity 1

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

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.