Resin acids and Rosin acids, manganese 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:

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, manganese salts

EC Number:

232-716-6

EC Name:

Resin acids and Rosin acids, manganese salts

Cas Number:

9008-34-8

Molecular formula:

Unspecified

IUPAC Name:

(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,4aS,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylic acid dimanganese

Test material form:

solid

Details on test material:

Batch 20120706
Purity 83%
Other components: Manganese sulfate and sodium sulfate
Water 2.3%
Physical state: solid, beige
storage: in the refrigerator
Stability; guaranteed until Dec 31, 2018

Specific details on test material used for the study:

purity: 83% UVCB
physical state: solid, beige
Other components: water, inorganic salts

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; 3200 and 6400 μg/plate
2nd Experiment (Preincubation test with and without S9 mix): 0; 33; 100; 333; 1000; 3200 and 6400 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

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

Key result

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:

other: A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward.

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 333 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 333 μg/plate onward.

Any other information on results incl. tables

Table 1: Standard plate test without metabolic activation

Strain	Test group	Dose	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
		(μg/plate)
TA 1535	Acetone	-	8.3	1.2	-	7, 9, 9
	Test item	33	9.3	2.1	1.1	7, 10, 11
		100	10.7	2.5	1.3	8, 11, 13
		333	10.3	1.5	1.2	9 P, 10 P, 12 P
		1000	7.3	0.6	0.9	8 P, 7 P, 7 P
		3200	8.7	2.5	1.0	11 P, 9 P, 6 P
		6400	8.0	2.6	1.0	7 P, 6 P, 11 P
	MNNG	5.0	5389.3	255.2	646.7	5428, 5623, 5117
TA 100	Acetone	-	89.3	13.7	-	83, 105, 80
	Test item	33	101.3	5.7	1.1	106, 103, 95
		100	101.0	14.7	1.1	104, 85, 114
		333	96.0	7.8	1.1	92 P, 105 P, 91 P
		1000	89.7	8.1	1.0	99 P, 86 P, 84 P
		3200	94.3	1.2	1.1	95 P, 93 P, 95 P
		6400	74.7	4.0	0.8	74 P, 71 P, 79 P
	MNNG	5.0	3395.0	185.3	38.0	3504, 3181, 3500
TA 1537	Acetone	-	5.0	1.0	-	6, 5, 4
	Test item	33	3.7	1.2	0.7	3, 5, 3
		100	5.7	0.6	1.1	6, 6, 5
		333	5.3	2.1	1.1	3 P, 6 P, 7 P
		1000	4.7	1.5	0.9	6 P, 5 P, 3 P
		3200	3.3	0.6	0.7	3 P, 3 P, 4 P
		6400	3.0	2.0	0.6	5 P, 1 P, 3 P
	AAC	100	673.0	127.9	134.6	684, 540, 795
TA 98	Acetone	-	17.7	6.5	-	18, 11, 24
	Test item	33	17.7	0.6	1.0	17, 18, 18
		100	16.7	1.5	0.9	15, 17, 18
		333	20.7	2.5	1.2	21 P, 23 P, 18 P
		1000	12.0	1.0	0.7	11 P, 13 P, 12 P
		3200	12.3	1.5	0.7	11 P, 14 P, 12 P
		6400	9.7	1.2	0.5	9 P, 9 P, 11 P
	NOPD	10	1294.3	54.0	73.3	1326, 1325, 1232
E.coli	Acetone	-	25.3	1.5	-	27, 25, 24
	Test item	33	26.7	1.5	1.1	25, 28, 27
		100	24.3	4.6	1.0	27, 27, 19
		333	24.0	1.7	0.9	25 P, 22 P, 25 P
		1000	20.7	2.1	0.8	20 P, 23 P, 19 P
		3200	21.0	7.2	0.8	23 P, 13 P, 27 P
		6400	21.7	4.5	0.9	17 P, 22 P, 26 P
	4-NQO	5	904.0	13.1	35.7	910, 913, 889

P = precipitation

Table 2: Standard plate test with metabolic activation

Strain	Test group	Dose	Mean revertants per plate	Standard deviation	Factor	Individual revertant colony counts
TA 1535	Acetone	-	6.3	1.2	-	5, 7, 7
	Test item	33	6.0	1.7	0.9	5, 5, 8
		100	7.7	2.3	1.2	9, 9, 5
		333	5.7	2.3	0.9	7 P, 7 P, 3 P
		1000	6.7	1.5	1.1	8 P, 7 P, 5 P
		3200	5.3	1.5	0.8	7 P, 4 P, 5 P
		6400	6.0	1.0	0.9	5 P, 6 P, 7 P
	2-AA	2.5	296.0	25.2	46.7	267, 309, 312
TA 100	Acetone	-	97.7	3.2	-	100, 99, 94
	Test item	33	91.3	9.5	0.9	88, 102, 84
		100	99.7	4.2	1.0	101, 103, 95
		333	101.0	7.5	1.0	109 P, 100 P, 94 P
		1000	111.7	11.6	1.1	106 P, 104 P, 125 P
		3200	76.3	7.5	0.8	84 P, 69 P, 76 P
		6400	78.3	11.0	0.8	71 P, 73 P, 91 P
	2-AA	2.5	2603.7	206.1	26.7	2814, 2595, 2402
TA 1537	Acetone	-	10.0	3.6	-	13, 11, 6
	Test item	33	9.0	1.7	0.9	11, 8, 8
		100	10.7	4.2	1.1	6, 14, 12
		333	8.3	2.5	0.8	8 P, 11 P, 6 P
		1000	8.3	3.5	0.8	8 P, 5 P, 12 P
		3200	5.7	1.5	0.6	6 P, 4 P, 7 P
		6400	3.3	0.6	0.3	3 P, 4 P, 3 P
	2-AA	2.5	216.0	32.2	21.6	190, 252, 206
TA 98	Acetone	-	24.0	5.6	-	29, 25, 18
	Test item	33	27.7	2.9	1.2	31, 26, 26
		100	27.7	4.7	1.2	24, 33, 26
		333	21.0	7.5	0.9	14 P, 20 P, 29 P
		1000	15.7	1.2	0.7	15 P, 17 P, 15 P
		3200	12.3	3.2	0.5	10 P, 16 P, 11 P
		6400	13.3	1.2	0.6	12 P, 14 P, 14 P
	2-AA	2.5	1781.0	188.5	74.2	1615, 1742, 1986
E.coli	Acetone	-	27.3	4.5	-	32, 23, 27
	Test item	33	28.7	2.9	1.0	27, 32, 27
		100	25.0	5.3	0.9	19, 29, 27
		333	24.0	7.0	0.9	21 P, 19 P, 32 P
		1000	22.7	5.0	0.8	22 P, 18 P, 28 P
		3200	19.7	3.5	0.7	20 P, 16 P, 23 P
		6400	14.0	2.0	0.5	14 P, 12 P, 16 P
	2-AA	60	93.3	21.1	3.4	71, 96, 113

P = Precipitation

Applicant's summary and conclusion

Conclusions:

The substance was not mutagenic in the Ames test.

Executive summary:

In a key bacterial reverse mutation assay, the test material (CAS# 9008-34-8; 83%) was tested on Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli strain WP2 at concentrations of 0; 33; 100; 333; 1000; 3200 and 6400 μg/plate in the presence and absence of metabolic activation (±S9).

The test material was not found to be mutagenic in the Ames test.