Resin acids and Rosin acids, aluminum 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, aluminum salts

EC Number:

263-075-0

EC Name:

Resin acids and Rosin acids, aluminum salts

Cas Number:

61789-65-9

Molecular formula:

Unspecified

IUPAC Name:

Resin acids and Rosin acids, aluminum salts

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

Test material form:

solid

Details on test material:

Name of test substance: Resin acids and Rosin acids, aluminium salts
Batch identification: V11794/4M
Identity: confirmed
Purity: The calculated purity based on the analyzed carbon content was found to be 94%.
The calculated purity based on the analyzed aluminium content was found to be 100%.
Physical state, appearance: solid, off-white
Storage conditions: refrigerator

Specific details on test material used for the study:

Resin acids and Rosin acids, aluminium salts
Purity: 94% based on total carbon content and 100% based on Aluminium content
solid, off-white
storage in the refrigerator
manufactured in 2013

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: DMSO
The substance is insoluble 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 1000 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was
occasionally observed in the standard plate test depending on the strain and test conditions at a concentration of 5400 μg/plate.
In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions from about
2700 μg/plate onward.

Any other information on results incl. tables

Standard plate incorporation assay without metabolic activation

Strain	Test group	Dose (ug/plate)	Mean revertants per plate	standard deviation	Factor	Individual revertant colony counts
TA 1535	DMSO	-	9.3	2.3	-	8, 8, 12
	Test item	33	9.0	4.6	1.0	10, 13, 4
		100	8.7	4.0	0.9	8, 5, 13
		333	10.0	2.6	1.1	9, 8, 13
		1000	14.3	3.8	1.5	10 P, 17 P, 16 P
		2700	9.3	1.5	1.0	11 P, 9 P, 8 P
		5400	6.3	1.5	0.7	5 P, 6 P, 8 P
	MNNG	5.0	5325.7	123.0	570.6	5354, 5191, 5432
TA 100	DMSO	-	83.7	11.4	-	71, 87, 93
	Test item	33	92.3	9.3	1.1	103, 86, 88
		100	82.3	11.0	1.0	76, 76, 95
		333	101.0	3.6	1.2	105, 100, 98
		1000	111.7	23.1	1.3	102 P, 138 P, 95 P
		2700	89.0	11.0	1.1	100 P, 78 P, 89 P
		5400	93.7	12.7	1.1	89 P, 84 P, 108 P
	MNNG	5.0	3863.0	45.2	46.2	3872, 3814, 3903
TA 1537	DMSO	-	7.0	2.0	-	5, 9, 7
	Test item	33	10.7	2.1	1.5	13, 10, 9
		100	11.0	4.4	1.6	6, 14, 13
		333	7.0	4.6	1.0	12, 6, 3
		1000	7.3	2.1	1.0	9 P, 5 P, 8 P
		2700	6.0	1.0	0.9	5 P, 6 P, 7 P
		5400	4.0	1.7	0.6	3 P, 6 P, 3 P
	AAC	100	771.3	128.7	110.2	918, 677, 719
TA 98	DMSO	-	11.7	2.5	-	12, 9, 14
	Test item	33	15.0	2.6	1.3	17, 12, 16
		100	17.0	1.7	1.5	16, 16, 19
		333	15.7	0.6	1.3	16, 15, 16
		1000	16.0	5.0	1.4	11 P, 16 P, 21 P
		2700	12.3	5.1	1.1	18 P, 8 P, 11 P
		5400	10.0	3.6	0.9	6 P, 11 P, 13 P
	NOPD	10	1047.7	48.2	89.8	1076, 1075, 992
E. coli	DMSO	-	23.7	4.2	-	25, 19, 27
	Test item	33	28.3	3.8	1.2	30, 31, 24
		100	21.7	3.2	0.9	18, 23, 24
		333	22.3	4.0	0.9	20, 27, 20
		1000	18.0	3.6	0.8	17 P, 15 P, 22 P
		2700	18.0	4.0	0.8	22 P, 14 P, 18 P
		5400	15.3	7.6	0.6	24 P, 12 P, 10 P
	4-NQO	5	596.0	8.5	25.2	587, 597, 604

P = precipitation

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	DMSO	-	9.3	2.9	-	11, 6, 11
	Test item	33	8.0	1.0	0.9	7, 8, 9
		100	14.7	1.2	1.6	14, 16, 14
		333	11.3	2.3	1.2	10, 14, 10
		1000	14.3	4.9	1.5	20 P, 11 P, 12 P
		2700	6.7	0.6	0.7	6 P, 7 P, 7 P
		5400	6.7	2.5	0.7	7 P, 4 P, 9 P
	2-AA	2.5	279.0	14.9	29.9	285, 290, 262
TA 100	DMSO	-	89.3	4.5	-	89, 94, 85
	Test item	33	82.7	5.1	0.9	77, 87, 84
		100	102.7	13.1	1.1	89, 104, 115
		333	97.7	15.0	1.1	115, 89, 89
		1000	103.3	4.9	1.2	109 P, 101 P, 100 P
		2700	107.7	7.4	1.2	116 P, 102 P, 105 P
		5400	80.0	6.2	0.9	87 P, 75 P, 78 P
	2-AA	2.5	2133.3	566.4	23.9	2594, 1501, 2305
TA 1537	DMSO	-	9.3	2.3	-	12, 8, 8
	Test item	33	6.3	0.6	0.7	6, 6, 7
		100	9.0	1.7	1.0	8, 11, 8
		333	5.7	2.1	0.6	4, 8, 5
		1000	8.3	1.2	0.9	9 P, 7 P, 9 P
		2700	7.0	2.0	0.8	7 P, 5 P, 9 P
		5400	4.7	1.5	0.5	5 P, 6 P, 3 P
	2-AA	2.5	131.3	31.9	14.1	116, 168, 110
TA 98	DMSO	-	23.0	4.0	-	19, 27, 23
	Test item	33	21.7	2.5	0.9	22, 19, 24
		100	27.7	2.5	1.2	30, 25, 28
		333	28.0	5.6	1.2	33, 29, 22
		1000	23.3	5.8	1.0	20 P, 20 P, 30 P
		2700	16.0	3.6	0.7	19 P, 12 P, 17 P
		5400	6.3	4.9	0.3	4 P, 3 P, 12 P
	2-AA	2.5	1347.0	69.2	58.6	1371, 1269, 1401
E. coli	DMSO	-	27.3	5.5	-	31, 30, 21
	Test item	33	27.0	14.9	1.0	44, 16, 21
		100	25.3	5.1	0.9	31, 24, 21
		333	30.7	6.4	1.1	26, 28, 38
		1000	21.7	2.1	0.8	21 P, 20 P, 24 P
		2700	24.0	4.6	0.9	19 P, 28 P, 25 P
		5400	16.0	2.6	0.6	19 P, 15 P, 14 P
	2-AA	60	132.0	13.5	4.8	146, 131, 119

P = precipitation

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in bacteria.

Executive summary:

In a key bacterial reverse mutation assay, the test material (CAS# 61789-65-9; purity 94%) 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 5000μg/plate in a DMSO 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.