Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid, alcohols fraction

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:

13 November 2018 - 14 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according OECD Guideline 471. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid, alcohols fraction

EC Number:

949-141-8

Molecular formula:

Not available since an UVBC substance.

IUPAC Name:

Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid, alcohols fraction

Test material form:

liquid

Method

Target gene:

Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver.

Test concentrations with justification for top dose:

0.001, 0.0032, 0.01, 0.032 and 0.1 µL/plate.

Initial cytotoxicity test: TA100 tester strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 µL /plate. There was cytotoxicity in all tested concentrations.
Follow up cytotoxicity test: Test was performed at 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 and 0.0125µL/plate. The test item showed cytotoxicity at 0.2, 0.3 and 0.4 µL/plate with lawn intensity 2+ (Thin lawn) and 0.1 µL/plate with lawn intensity 3+ (Slightly thin lawn). The test item did not result in cytotoxicity at 0.05, 0.025 and 0.0125 µL/plate with bacterial lawn 4+ intensity. Hence, 0.1 µL/plate was selected as the highest concentration for mutation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle:
A solubility test was carried out with Distilled Water and Dimethyl Sulphoxide (DMSO). The test item was found miscible in DMSO at a concentration of 50 µL/mL. Subsequently, in a precipitation test in DMSO, the test item resulted in no precipitation at and up to 5 µL/plate tested concentration.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Controls 2

Positive controls:

yes

Remarks:

2 µg/plate.

Positive control substance:

2-nitrofluorene

Remarks:

Salmonella TA 98, without metabolic activation.

Controls 3

Positive controls:

yes

Remarks:

1 µg/plate.

Positive control substance:

sodium azide

Remarks:

Salmonella TA 100 and TA 1535, without metabolic activation.

Controls 4

Positive controls:

yes

Remarks:

50 µg/plate.

Positive control substance:

9-aminoacridine

Remarks:

Salmonella TA 1537, without metabolic activation.

Controls 5

Positive controls:

yes

Remarks:

5 µg/plate.

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

E.coli WP2 uvrA (pKM 101), without metabolic activation.

Controls 6

Positive controls:

yes

Remarks:

4 µg/plate.

Positive control substance:

other: 2-aminoanthracene

Remarks:

All Salmonella strains, with metabolic activation.

Controls 7

Positive controls:

yes

Remarks:

30 µg/plate.

Positive control substance:

other: 2-aminoanthracene

Remarks:

E.coli WP2 uvrA (pKM 101), with metabolic activation.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Plate incorporation (initial mutation test): Test item, vehicle or positive control (100 µL) and the tester strains (100 µL) along with S9/phosphate buffer saline (500 µL) were mixed with 2 mL of molten soft agar and poured on to minimal glucose agar plates.

Pre-incubation (confirmatory mutation test): Test item, vehicle or positive control (100 µL) and the tester strains (100 µL) along with S9/Phosphate Buffer Saline (500 µL) were incubated in an incubator shaker at 100±5 rpm. Post incubation, the test constituents were mixed with 2 mL molten soft agar and poured on to minimal glucose agar plates.

- Cell density at seeding (if applicable): 18×108 cells/mL.

DURATION
- Preincubation period: 30 minutes (Pre-incubation test)
- Exposure duration: 65 hours and 50 minutes (Plate incorporation test) and 65 hours (Pre-incubation test)

NUMBER OF REPLICATIONS: 3 replicates per dose and controls.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Bacterial background lawn intensity
- Any supplementary information relevant to cytotoxicity:
The condition of the bacterial background lawn was evaluated for evidence of the test item toxicity using the code system as follows:
0: No lawn (Absent): Distinguished by a complete lack of any background lawn compared to solvent control plates.
1+: Very thin lawn (Extremely reduced): Distinguished by an extreme thinning of the background lawn compared to solvent control plates.
2+: Thin lawn (Moderately reduced): Distinguished by a marked thinning of the background lawn compared to solvent control plates.
3+: Slightly thin lawn (Slightly reduced): Distinguished by a noticeable thinning of the background lawn compared to solvent control plates.
4+: Thick lawn (Normal): Distinguished by a healthy background lawn comparable to solvent control plates.

Evaluation criteria:

Positive result: there is a concentration related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system, AND this increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value in S. typhimurium strains TA98, TA100 and E. coli WP2 uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value in tester strains TA1535 and TA1537.
Equivocal response: biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non dose responsive increase that is equal to or greater than the respective threshold cited.
Negative result: neither positive nor equivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3+ (Slightly thin lawn) at 0.1 µL/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3+ (Slightly thin lawn) at 0.1 µL/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3+ (Slightly thin lawn) at 0.1 µL/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3+ (Slightly thin lawn) at 0.1 µL/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

3+ (Slightly thin lawn) at 0.1 µL/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A solubility test was carried out with Distilled Water and Dimethyl Sulphoxide (DMSO). The test item was found miscible in DMSO at a concentration of 50 µL/mL. Subsequently, in a precipitation test in DMSO, the test item resulted in no precipitation at and up to 5 µL/plate tested concentration.
Initial cytotoxicity test: TA100 tester strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 µL /plate. There was cytotoxicity in all tested concentrations.
Follow up cytotoxicity test: Test was performed at 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 and 0.0125µL/plate. The test item showed cytotoxicity at 0.2, 0.3 and 0.4 µL/plate with lawn intensity 2+ (Thin lawn) and 0.1 µL/plate with lawn intensity 3+ (Slightly thin lawn). The test item did not result in cytotoxicity at 0.05, 0.025 and 0.0125 µL/plate with bacterial lawn 4+ intensity. Hence, 0.1 µL/plate was selected as the highest concentration for mutation test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 5 below
- Negative (solvent/vehicle) historical control data: see table 5 below

The observed numbers of revertant colonies for vehicle and positive controls were within the historical control ranges except for positive control with TA1535 in the plate incorporation method with and without S9 which were slightly below the range and for positive control with E. coli in the preincubation method with S9 which were slightly above the range. These observations are considered without impact on the final conclusions of this assay.

Any other information on results incl. tables

Table1. Summary of initial cytotoxicity test - Salmonella typhimurium TA100 tester strain

Test Item Concentration (µL/plate)	No. of Revertants/plate
	With S9						Without S9
	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity
Vehicle Control	121	106	119	115	8.1	4+	108	111	102	107	4.6	4+
0.5	39	35	39	38	2.3	2+	36	36	34	35	1.2	2+
0.6	24	28	30	27	3.1	2+	26	25	21	24	2.6	2+
0.7	25	20	23	23	2.5	2+	19	22	24	22	2.5	2+
0.8	20	19	16	18	2.1	1+	18	15	18	17	1.7	1+
0.9	15	18	12	15	3.0	1+	15	17	14	15	1.5	1+
1	8	11	9	9	1.5	1+	7	10	8	8	1.5	1+
2	0	0	0	0	0.0	0	0	0	0	0	0.0	0
3	0	0	0	0	0.0	0	0	0	0	0	0.0	0
4	0	0	0	0	0.0	0	0	0	0	0	0.0	0
5	0	0	0	0	0.0	0	0	0	0	0	0.0	0

Table 2. Summary of follow up initial cytotoxicity test - Salmonella typhimurium TA100 tester strain

Test Item Concentration (µL/plate)	No. of Revertants/plate
	With S9						Without S9
	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity	R1	R2	R3	Average	±SD	Bacterial Lawn Intensity
Vehicle Control	113	103	116	111	6.8	4+	102	101	114	106	7.2	4+
0.0125	109	110	114	111	2.6	4+	106	99	113	106	7.0	4+
0.025	106	107	112	108	3.2	4+	105	100	110	105	5.0	4+
0.05	108	99	104	104	4.5	4+	102	106	100	103	3.1	4+
0.1	70	72	76	73	3.1	3+	77	75	69	74	4.2	3+
0.2	60	56	49	55	5.6	2+	61	57	55	58	3.1	2+
0.3	43	52	56	50	6.7	2+	41	46	44	44	2.5	2+
0.4	46	50	38	45	6.1	2+	42	48	39	43	2.9	2+

Lawn intensity:

4+= Thick lawns: Distinguished by a healthy (Normal) background lawn comparable to vehicle control plates.    

3+=Slightly thin lawn: Distinguished by a noticeable thinning of the background lawn compared to solvent/vehicle control plates.

2+= Thin lawn: Distinguished by a marked thinning of the background lawn compared to solvent/vehicle control plates.

1+= Very thin lawn: Distinguished by an extreme thinning of the background lawn compared to solvent/vehicle control plates.

0 = No lawn : Distinguished by a complete lack of any background lawn compared to solvent/vehicle control plates

Table 3. Summary of colony counts of revertants of trial-I. Plate Incorporation Method

Treatment	Test Concentration   (µL/plate)			No. of Revertants (Mean of 3 Plates)
				With S9						Without S9
				Salmonella typhimurium				E.coli WP2 uvrA (pKM 101)		Salmonella typhimurium				E.coli WP2 uvrA (pKM 101)
				TA 98	TA 100	TA 1535	TA 1537			TA 98	TA 100	TA 1535	TA 1537
Vehicle Control	100 µL of Dimethyl Sulphoxide		Mean	25.0	109.0	21.0	9.3	167.0		23.0	104.7	19.7	8.3	169.7
			±SD	2.0	6.6	3.0	2.5	9.6		2.6	4.2	2.5	1.5	10.7
	Lawn Intensity			4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
PINE OIL 50%	0.001	Mean		23.7	110.3	21.0	8.7	167.0		21.7	103.0	20.0	7.0	160.0
		±SD		2.5	6.1	2.0	2.5	6.2		1.5	5.0	2.0	1.0	3.0
	Fold Increase			0.9	1.0	1.0	0.9	1.0		0.9	1.0	1.0	0.8	0.9
	Lawn Intensity			4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.0032	Mean		24.0	108.7	20.3	9.3	166.7		21.7	107.3	19.7	8.3	158.0
		±SD		1.7	8.3	2.5	3.8	5.1		2.9	3.2	2.1	2.1	8.5
	Fold Increase			1.0	1.0	1.0	1.0	1.0		0.9	1.0	1.0	1.0	0.9
	Lawn Intensity			4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.01	Mean		22.0	103.3	20.3	6.7	155.0		21.0	100.0	19.0	6.7	152.7
		±SD		2.0	5.5	1.5	0.6	5.0		2.0	5.0	1.7	2.1	5.7
	Fold Increase			0.9	0.9	1.0	0.7	0.9		0.9	1.0	1.0	0.8	0.9
	Lawn Intensity			4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.032	Mean		18.7	88.7	18.0	6.0	148.0		17.7	88.3	16.7	5.3	144.3
		±SD		2.1	1.5	1.0	1.0	4.6		1.5	3.5	0.6	0.6	4.0
	Fold Increase			0.7	0.8	0.9	0.6	0.9		0.8	0.8	0.8	0.6	0.9
	Lawn Intensity			4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.1	Mean		14.7	68.7	15.0	4.0	126.0		14.0	68.3	14.7	4.3	125.7
		±SD		0.6	5.1	1.0	1.0	6.6		1.0	3.5	0.6	0.6	5.9
	Fold Increase			0.6	0.6	0.7	0.4	0.8		0.6	0.7	0.7	0.5	0.7
	Lawn Intensity			3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Control	100 µL of respective Positive Control		Mean	364.0	417.0	111.0	106.3	404.7		359.3	403.0	106.3	103.3	407.3
			±SD	6.6	7.5	17.1	5.7	15.2		9.5	9.5	7.4	6.7	10.1
	Fold Increase			14.6	3.8	5.3	11.4	2.4		15.6	3.9	5.4	12.4	2.4
	Lawn Intensity			4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Table 4. Summary of colony counts of revertants of trial-II. Preincubation Method

Treatment	Test Concentration   (µL/plate)		No. of Revertants (Mean of 3 Plates)
			With S9						Without S9
			Salmonella typhimurium				E.coli WP2 uvrA (pKM 101)		Salmonella typhimurium				E.coli WP2 uvrA (pKM 101)
			TA 98	TA 100	TA 1535	TA 1537			TA 98	TA 100	TA 1535	TA 1537
Vehicle Control	100 µL of Dimethyl Sulphoxide	Mean	26.7	113.7	22.7	8.0	170.7		22.7	108.0	20.7	7.7	165.3
		±SD	1.2	5.9	3.5	2.0	6.0		2.5	7.5	3.1	3.1	6.7
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
PINE OIL 50%	0.001	Mean	24.0	105.0	21.0	8.0	174.0		21.0	109.0	21.0	7.3	169.0
		±SD	2.6	4.4	3.6	3.0	5.6		2.6	3.6	2.0	1.5	5.6
	Fold Increase		0.9	0.9	0.9	1.0	1.0		0.9	1.0	1.0	1.0	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.0032	Mean	24.3	115.3	21.3	7.0	172.3		22.3	108.3	20.3	6.7	161.0
		±SD	0.6	7.4	2.3	1.0	6.7		3.2	8.1	3.1	2.1	8.0
	Fold Increase		0.9	1.0	0.9	0.9	1.0		1.0	1.0	1.0	0.9	1.0
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.01	Mean	22.7	108.3	22.0	7.7	156.3		21.3	99.7	20.7	7.0	153.3
		±SD	2.5	2.1	3.6	3.8	6.5		2.1	12.4	3.5	2.0	4.5
	Fold Increase		0.9	1.0	1.0	1.0	0.9		0.9	0.9	1.0	0.9	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.032	Mean	20.7	92.0	18.3	5.7	158.0		19.0	89.3	16.7	6.0	147.3
		±SD	2.9	6.6	1.5	0.6	14.8		2.6	4.5	1.2	1.0	5.7
	Fold Increase		0.8	0.8	0.8	0.7	0.9		0.8	0.8	0.8	0.8	0.9
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+
	0.1	Mean	16.7	68.7	15.7	4.3	128.7		16.0	68.3	15.3	3.7	123.7
		±SD	0.6	6.5	0.6	0.6	2.5		1.0	1.5	0.6	0.6	7.1
	Fold Increase		0.6	0.6	0.7	0.5	0.8		0.7	0.6	0.7	0.5	0.7
	Lawn Intensity		3+	3+	3+	3+	3+		3+	3+	3+	3+	3+
Positive Control	100 µL of respective Positive Control	Mean	357.7	413.7	127.7	107.3	413.3		355.3	405.7	115.7	103.0	406.0
		±SD	9.6	10.7	8.5	14.3	4.5		5.0	9.5	7.0	11.8	8.5
	Fold Increase		13.4	3.6	5.6	13.4	2.4		15.7	3.8	5.6	13.4	2.5
	Lawn Intensity		4+	4+	4+	4+	4+		4+	4+	4+	4+	4+

Table 5. Historical data

Plate Incorporation Method
Metabolic Activation		With Metabolic Activation						Without Metabolic Activation
Tester strain		Salmonella typhimurium				E.coli uvrA            (pKM 101)		Salmonella typhimurium				E.coli uvrA            (pKM 101)
		TA 98	TA 100	TA 1535	TA 1537			TA 98	TA 100	TA 1535	TA 1537
Vehicle Control (DMSO)	Mean	21.3	103.4	21.2	9.2	169.8		21.2	102.8	21.2	9.4	171.1
	±SD	3.2	5.5	2.2	1.8	7.6		2.7	4.7	2.3	1.9	6.8
	Min	16	96	13	6	156		15	91	15	7	157
	Max	40	130	26	14	191		32	126	27	15	191
Positive Control	Mean	401.7	383.6	140.1	119.2	383.5		396.6	379.7	141.1	120.1	383.1
	±SD	23.4	16.9	8.0	6.6	9.8		27.4	18.3	9.9	7.3	10.4
	Min	256	246	118	100	320		290	270	110	98	371
	Max	440	419	182	149	421		430	446	190	158	416
Preincubation Method
Metabolic Activation		With Metabolic Activation						Without Metabolic Activation
Tester strain		Salmonella typhimurium				E.coli uvrA            (pKM 101)		Salmonella typhimurium				E.coli uvrA            (pKM 101)
		TA 98	TA 100	TA 1535	TA 1537			TA 98	TA 100	TA 1535	TA 1537
Vehicle Control (DMSO)	Mean	21.2	102.9	20.9	9.4	171.7		20.9	102.1	21.1	9.6	171.3
	±SD	2.8	4.6	2.2	2.0	7.2		2.8	4.3	2.3	2.6	6.8
	Min	16	89	14	4	153		15	88	16	6	152
	Max	32	128	28	17	198		32	129	28	23	198
Positive Control	Mean	402.5	383.9	140.6	118.6	382.9		398.3	381.3	141.3	119.5	382.8
	±SD	18.5	16.1	9.3	6.3	10.4		24.5	16.2	11.0	6.4	10.3
	Min	290	293	103	99	330		281	312	110	97	321
	Max	429	425	187	151	412		428	450	200	173	418

Applicant's summary and conclusion

Conclusions:

The test item PINE OIL 50% is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.1 µL/plate under the test conditions.

Executive summary:

The test item PINE OIL 50% was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471, following the Principles of GLP. The experiments were carried out in triplicate using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA (pKM101) in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). Based on the results of the solubility test, the test item was formulated in DMSO as vehicle. In an initial cytotoxicity test, TA100 tester strain was exposed to vehicle control, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 and 5 µL /plate. There was cytotoxicity in all tested concentrations, thus a follow up cytotoxicity test was performed at 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 and 0.0125 µL/plate. Based on the results of these cytotoxicity tests, concentrations of 0.001, 0.0032, 0.01, 0.032 and 0.1 µL/plate were finally selected for testing in the mutation test. Vehicle control and appropriate positive controls (2-nitrofluorene, sodium azide and 9-Aminoacridine, 4-nitroquinoline N-oxide for trials “without metabolic activation” and 2-Aminoanthracene for trials “with metabolic activation”) were tested simultaneously. Two trials were carried out for this study in triplicate. Trial I was carried out as plate incorporation method and Trial II as pre incubation method. Based on the experimental results obtained, the mean numbers of revertant colonies at the tested concentrations were comparable to those of the vehicle control, in both the trials, in the presence and absence of metabolic activation. There was no appreciable increase in number of revertant colonies at any of the tested concentrations in both the trials. The mean values of revertant colonies of the negative (vehicle) control plates were within the historical control range. The number of revertant colonies in the positive controls resulted in 2.4 to 15.7 fold increase under identical conditions. Based on the results of the study, it is concluded that the test item is “non-mutagenic” in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.1 µL/plate under the test conditions.