Rosin, decarboxylated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Decarboxylated Rosin

Cas Number:

8050-18-8

Molecular formula:

Not availabe for UVCB substance

IUPAC Name:

Decarboxylated Rosin

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. Experiment 2 was performed on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was the same as Experiment 1 (1.5 to 5000 μg/plate). Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the urgency of result, the lack of cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates. The test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Test concentrations with justification for top dose:

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate.

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

Details on test system and experimental conditions:

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. On a regular basis (approximately monthly), batches of culture from master stocks are prepared and coded, these are then routinely tested for appropriate characteristics, viability and mutation frequency to ensure acceptability criteria is met.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 2547490 expiry date 07/2024) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Rationale for test conditions:

The test item was insoluble in both sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully soluble in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.

The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in high purity acetone by mixing on a vortex mixer. No correction for purity was required. Acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots (Maron et al., 1981). All test item preparation and dosing was performed under yellow safety lighting.

All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).

2. A reproducible increase at one or more concentrations.

3. Biological relevance against in-house historical control ranges.

4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that are statistically significant but are within the in-house historical vehicle/untreated control range are not flagged for statistical significance in the data tables.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) and viability are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation (S9-mix), are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

Experiment 1 (plate incorporation) – Table 2 and Table 3
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

A test item precipitate (greasy in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).


Experiment 2 (pre-incubation) – Table 4 and Table 5
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 μg/plate).

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
A test item precipitate (greasy in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.

No biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). One statistically significant value was noted (WP2uvrA at 50 μg/plate in the absence of metabolic activation (S9-mix)). However, as the maximum fold increase was only 1.8 times the concurrent vehicle control and the mean colony count was within the in-house historical vehicle/untreated control range for the strain, the response was considered of no biological relevance. Therefore, this value has not been highlighted in Table 4 as it did not meet the required criteria for a positive response.

Any other information on results incl. tables

Table 1 – Spontaneous Mutation Rates (Concurrent Negative Control)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA	TA98	TA1537
106	12	13	12	11
128 (117)	13 (13)	14 (15)	17 (15)	10 (13)
118	14	17	17	17
Viability – Bacterial cells 10^9 per mL
1.8	2.3	2.5	1.4	1.9

 

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA	TA98	TA1537
116	19	13	24	13
78 (102)	9 (16)	24 (18)	22 (22)	15 (14)
111	20	18	20	13
Viability – Bacterial cells 10^9 per mL
2.7	2.2	3.5	1.3	2.8

 

Table 2 – Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 18 August 2020							To: 20 August 2020
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Based-pair substitution strains								Frameshift strains
		TA 100		TA 1535			WP2uvrA			TA 98			TA 1537
	Solvent Control (Acetone)	113 115 107	(112) 4.2#	27 17 15		(20) 6.4	12 11 20		(14) 4.9	21 20 16		(19) 2.6	10 16 10		(12) 3.5
	1.5 μg	114 130 113	(119) 9.5	24 15 23		(21) 4.9	14 19 12		(15) 3.6	24 11 9		(15) 8.1	12 15 15		(14) 1.7
	5 μg	113 119 119	(117) 3.5	14 18 15		(16) 2.1	17 13 21		(17) 4.0	11 18 17		(15) 3.8	12 10 10		(11) 1.2
	15 μg	131 128 109	(123) 11.9	9 18 12		(13) 4.6	21 19 16		(19) 2.5	18 12 23		(18) 5.5	11 13 17		(14) 3.1
	50 μg	127 108 121	(119) 9.7	14 14 13		(14) 0.6	21 23 15		(20) 4.2	19 20 14		(17) 4.2	17 15 8		(13) 4.7
	150 μg	114 118 115	(116) 2.1	16 26 25		(22) 5.5	12 13 22		(16) 5.5	16 22 14		(18) 4.4	8 9 9		(9) 0.6
	500 μg	109 111 81	(100) 16.8	27 24 23		(25) 2.1	17 20 7		(15) 6.8	15 23 16		(21) 1.7	13 8 5		(9) 4.0
	1500 μg	104 113 111	(109) 4.7	22 15 16		(18) 3.8	13 21 15		(16) 4.2	20 20 23		(23) 2.1	14 14 16		(15) 12
	5000 μg	129P 116P 135P	(127) 9.7	17P 15P 16P		(16) 1.0	10P 34P 10P		(18) 13.9	24P 25P 21P		(23) 2.1	8P 10P 11P		(10) 1.5
Positive controls S9-Mix (-)	Name	ENNG		ENNG			ENNG			4NQO			9AA
	Dose level	3 μg		5 μg			2 μg			0.2 μg			80 μg
	No. of revertants	636 729 717	(694) 50.6	1045 1124 1012	(1060) 57.6		1118 964 873	(985) 123.8		138 116 121	(125) 11.5		238 766 311	(438) 286.1

 

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

# Standard deviation

 

 

 

Table 3 – Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 18 August 2020							To: 20 August 2020
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Based-pair substitution strains								Frameshift strains
		TA 100		TA 1535			WP2uvrA			TA 98			TA 1537
	Solvent Control (Acetone)	117 140 131	(129) 11.6#	11 14 20		(15) 4.6	26 22 30		(26) 4.0	36 24 22		(27) 7.6	13 9 22		(15) 6.7
	1.5 μg	114 84 126	(108) 21.6	15 12 12		(13) 1.2	17 17 25		(20) 4.6	23 32 17		(24) 7.5	10 25 14		(16) 7.8
	5 μg	106 107 98	(104) 4.9	12 11 16		(13) 2.6	20 10 10		(13) 5.8	32 20 22		(25) 6.4	14 17 12		(14) 2.5
	15 μg	106 118 126	(117) 10.1	11 19 15		(15) 4.0	17 20 28		(22) 5.7	20 25 24		(23) 2.6	9 10 15		(11) 3.2
	50 μg	115 132 143	(130) 14.1	14 17 20		(17) 3.0	26 20 22		(23) 3.1	8 18 23		(16) 7.6	6 10 12		(9) 3.1
	150 μg	125 102 116	(114) 11.6	11 11 8		(10) 1.7	23 22 10		(18) 7.2	24 22 19		(22) 2.5	6 11 15		(11) 4.5
	500 μg	105 131 112	(116) 13.5	15 17 14		(15) 1.5	26 14 24		(21) 6.4	13 16 21		(17) 4.0	8 9 12		(10) 2.1
	1500 μg	101 114 98	(104) 8.5	14 21 12		(16) 4.7	12 28 30		(23) 9.9	17 26 21		(21) 4.5	10 12 16		(13) 3.1
	5000 μg	118P 108P 103P	(110) 7.6	16P 15P 16P		(16) 0.6	26P 32P 23P		(27) 4.6	29P 29P 32P		(30) 1.7	7P 11P 11P		(10) 2.3
Positive controls S9-Mix (-)	Name	2AA		2AA			2AA			BP			2AA
	Dose level	1 μg		2 μg			10 μg			5 μg			2 μg
	No. of revertants	1877 2070 2102	(2016) 121.7	316 372 318	(335) 31.8		103 104 105	(104) 1.0		97 95 109	(100) 7.6		267 334 364	(322) 49.7

 

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

P Test item precipitate

# Standard deviation

 

 

Table 4 – Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 24 August 2020							To: 27 August 2020
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Based-pair substitution strains								Frameshift strains
		TA 100		TA 1535			WP2uvrA			TA 98			TA 1537
	Solvent Control (Acetone)	112 119 124	(118) 6.0#	15 23 12		(17) 5.7	15 16 17		(16) 1.0	29 23 27		(26) 3.1	9 7 12		(9) 2.5
	1.5 μg	120 134 129	(128) 7.1	14 16 15		(15) 1.0	19 19 23		(20) 2.3	20 22 19		(20) 1.5	12 8 9		(10) 2.1
	5 μg	122 138 118	(126) 10.6	17 12 14		(14) 2.5	22 21 22		(22) 0.6	27 23 18		(23) 4.5	11 10 10		(10) 0.6
	15 μg	147 111 105	(121) 22.7	9 19 15		(14) 5.0	18 24 19		(20) 3.2	23 26 24		(24) 1.5	7 8 10		(8) 1.5
	50 μg	117 130 130	(126) 7.5	17 10 16		(14) 3.8	21 31 34		(29) 6.8	28 25 20		(24) 4.0	9 7 10		(9) 1.5
	150 μg	122 122 125	(123) 1.7	13 20 10		(14) 5.1	21 13 20		(18) 4.4	30 31 25		(29) 3.2	12 4 9		(8) 4.0
	500 μg	126 124 120	(123) 3.1	20 27 16		(21) 5.6	15 29 18		(21) 7.4	17 23 25		(22) 4.2	8 7 9		(8) 1.0
	1500 μg	109 114 120	(114) 5.5	13 20 10		(18) 1.2	22 18 19		(20) 2.1	18 26 26		(23) 4.6	9 5 7		(7) 2.0
	5000 μg	122P 104P 106P	(111) 9.9	20 27 16		(15) 0.6	32P 23P 17P		(24) 7.5	34P 32P 21P		(29) 7.0	10P 6P 9P		(8) 2.1
Positive controls S9-Mix (-)	Name	ENNG		ENNG			ENNG			4NQO			9AA
	Dose level	3 μg		5 μg			2 μg			0.2 μg			80 μg
	No. of revertants	807 718 722	(749) 50.3	141 205 225	(190) 43.9		749 478 698	(642) 144.0		261 177 194	(211) 44.4		537 456 368	(454) 84.5

 

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA 9-Aminoacridine

P Test item precipitate

# Standard deviation

 

 

Table 5 – Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 18 August 2020							To: 20 August 2020
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Based-pair substitution strains								Frameshift strains
		TA 100		TA 1535			WP2uvrA			TA 98			TA 1537
	Solvent Control (Acetone)	135 134 117	(129) 10.1#	9 16 23		(16) 7.0	24 27 27		(26) 1.7	39 38 25		(34) 7.8	11 20 10		(14) 5.5
	1.5 μg	153 133 114	(133) 19.5	10 13 15		(13) 2.5	24 32 17		(24) 7.5	30 51 37		(39) 10.7	9 10 15		(11) 3.2
	5 μg	130 139 144	(138) 7.1	9 12 12		(11) 1.7	23 23 25		(24) 1.2	29 23 29		(27) 3.5	15 14 10		(13) 2.6
	15 μg	133 135 123	(130) 6.4	7 11 8		(9) 2.1	22 30 26		(26) 4.0	36 25 55		(39) 15.2	9 13 11		(11) 2.0
	50 μg	115 108 135	(119) 14.0	21 8 12		(14) 6.7	32 20 34		(29) 7.6	51 26 26		(34) 14.4	14 13 4		(10) 5.5
	150 μg	130 137 134	(134) 3.5	9 18 21		(16) 6.7	25 26 20		(24) 3.2	29 24 21		(25) 4.0	10 17 13		(13) 3.5
	500 μg	145 145 133	(141) 6.9	11 15 25		(16) 6.2	33 22 33		(29) 6.4	25 25 24		(25) 0.6	10 7 3		(7) 3.5
	1500 μg	157 141 120	(139) 18.6	11 5 8		(17) 7.2	23 19 27		(23) 4.0	26 37 35		(33) 5.9	14 12 21		(16) 3.5
	5000 μg	125P 119P 121P	(122) 3.1	18P 23P 16P		(8) 3.0	21P 27P 27P		(25) 3.5	23P 25P 36P		(28) 7.0	12P 8P 12P		(11) 2.3
Positive controls S9-Mix (-)	Name	2AA		2AA			2AA			BP			2AA
	Dose level	1 μg		2 μg			10 μg			5 μg			2 μg
	No. of revertants	1803 1698 1593	(1698) 105.0	278 298 310	(295) 16.2		104 106 127	(112) 12.7		151 116 116	(128) 20.2		238 240 271	(250) 18.5

 

BP Benzo(a)pyrene

2AA 2-Aminoanthracene

P Test item precipitate

# Standard deviation

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item AA-948-61 (Decarboxylated Rosin, CAS no. 8050-18-8) did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test AA-948-61 (Decarboxylated Rosin, CAS no. 8050-18-8) was considered to be non-mutagenic.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. Experiment 2 was performed on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was the same as Experiment 1 (1.5 to 5000 μg/plate). Eight test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the urgency of result, the lack of cytotoxicity noted in Experiment 1 and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item AA-948-61 (Decarboxylated Rosin, CAS no. 8050-18-8) did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test AA-948-61 (Decarboxylated Rosin, CAS no. 8050-18-8) was considered to be non-mutagenic.