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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacteria reverse mutation: Key study: Test method according to the OECD Guideline 471 with GLP study. The test item PINE OIL 50% was found non-mutagenic in the Bacterial Reverse Mutation Test up to the highest tested concentration of 0.1 µL/plate under the test conditions.

In vitro mammalian chromosomal aberration: Key study: Test method according to the OECD Guideline 473 with GLP study. The test item PINE OIL 50% was found as non-clastogenic up to the concentration of 0.125 µL/mL both in the presence and absence of metabolic activation under the test conditions.

In vitro gene mutation in mammalian cells: Key study: Test methodaccording to the OECD Guideline 476 with GLP study. The test item PINE OIL 50% was found as non-mutagenic up to the concentration of 0.3125 µL/mL, both in the presence and absence of metabolic activation under the test conditions.

In vitro bacteria reverse mutation: Supporting studies: Experimental results from studies performed with the components or analogue substances terpineol, alpha terpineol, beta terpineol, cineole, d-limonene, alpha terpinene, gamma terpinene, alpha pinene, l-alpha pinene, camphene, terpinen-4 -ol, dl-borneol and estragole are available. All results obtained were negative except for the following findings from two different studies: terpineol was found weakly mutagenic in TA102 strain and camphene and beta-terpineol showed weak mutagenic activity in TA100 strain with metabolic activation. However, camphene was found negative with TA100 strain in another mutagenicity test with and without metabolic activation. Furthermore, terpineol (a mixture of different isomers included beta-terpineol) was found non-cytogenic to mammalian cells and alpha terpineol was found non-mutagenic to mammalian cells in other studies.

In vitro mammalian chromosomal aberration/sister chromatid exchange: Supporting studies: Experimental results from studies performed with components or analogue substances terpineol, cineole, d-limonene, 5-Ethylidene-2-norbornene, camphene, estragole as well as tea tree oil (a mixture of some of the components) are available. All studies were negative except for one single study with cineole which was found positive in one SCE study. However, cineole was also found negative in a chromosome aberration test. Thus, based on weight of evidence approach, the test substance is considered to be non-cytogenic to mammalian cells.

In vitro gene mutation in mammalian cells: supporting studies: Experimental results from studies performed with alpha terpineol, d-limonene and 5-Ethylidene-2-norbornene are available. All studies were negative. Based on these results, the test substance is considered to be non-mutagenic to mammalian cells.

Other studies: alpha pinene and gamma terpinene were determined non-mutagenic in an unscheduled DNA synthesis assay in rat hepatocytes. In contrast, estragole was found positive in an in vitro UDS test with rat liver cells. However, estragole was negative in an in vitro mammalian chromosome aberration test and in an in vitro bacteria reverse mutation test with and without metabolic activation and thus in an overall assessment this compound should not be considered as mutagenic according to this available information.

Link to relevant study records

Reference

Reference 1

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.

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

Target gene:

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

Species / strain / cell type:

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

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.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

2 µg/plate.

Positive control substance:

2-nitrofluorene

Remarks:

Salmonella TA 98, without metabolic activation.

Positive controls:

yes

Remarks:

1 µg/plate.

Positive control substance:

sodium azide

Remarks:

Salmonella TA 100 and TA 1535, without metabolic activation.

Positive controls:

yes

Remarks:

50 µg/plate.

Positive control substance:

9-aminoacridine

Remarks:

Salmonella TA 1537, without metabolic activation.

Positive controls:

yes

Remarks:

5 µg/plate.

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

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

Positive controls:

yes

Remarks:

4 µg/plate.

Positive control substance:

other: 2-aminoanthracene

Remarks:

All Salmonella strains, with metabolic activation.

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.

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

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

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

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 November 2018 - 13 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according OECD Guideline 473. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood from human donors
- Suitability of cells: healthy, young, non-smoking donors with no known recent exposure to genotoxic chemicals or radiation were used. Blood from individual was collected in sodium heparin vacutainer and analyzed using Advia 2120. All the parameters were within the acceptable range.
- Cell cycle length, doubling time or proliferation index: The normal cell cycle length is 12 to 15 hours.
- Sex, age and number of blood donors if applicable: 2 individual males of 27 and 28 years old were used, one for the initial cytotoxicity and the other for the chromosomal aberration test.
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Modal number of chromosomes: 46±2
- Normal (negative control) cell cycle time: 12 to 15 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin-Streptomycin)
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate

Test concentrations with justification for top dose:

0.0312, 0.0625 and 0.125 µL/mL

Based on the results of solubility, precipitation and pH tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the chromosomal aberration test at concentrations of 0.125, 0.25, 0.5, 1 and 2 µL/mL and in the same conditions of the main test. The obtained reduction in mitotic index (MI) at 0.25 µL/mL and higher concentrations was 100%. As the reduction in MI was not more than 45±5% of the negative control at 0.125 µL/mL, this value was selected as the highest concentration for the chromosomal aberration test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was conducted to determine the maximum concentration or workable suspension solution of the test item in the vehicle compatible with the test system at 200 µL/mL and the test item was miscible in dimethyl sulphoxide at 200 µL/mL. Precipitation test was conducted at 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 23 hours and 3 minutes of incubation, slight precipitation was observed at 2 µL/mL. No precipitation was observed in any other tested concentration. No change in pH was observed in any of the concentration tested. Hence, 2 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation (0.05 µg/mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (10 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: 44 to 48 hours, with phytohaemagglutinin (PHA) to induce cell division prior to exposure.
- Exposure duration: 3 h and 10 min (short term treatment, +S9 and -S9); 22 h and 9 min (long term treatment, -S9)

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After treatment, cell suspension was mixed with 3 to 4 mL of freshly prepared warm 0.56% Potassium chloride, incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 3 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2200 rpm for 10 minutes. The procedure was repeated twice by adding 3 mL of cold acetic acid: methanol fixative (1:3). Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide pre labeled with treatment/group number. The slides were air dried. Minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5% Giemsa stain for 15 minutes.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300 (150 per replication)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: For each replicate minimum of 500 cells were scored. Cytotoxicity was determined by calculating percentage reduction in mitotic index by using the formula:
(%) Reduction in MI= [(Percentage MI of VC - Percentage MI of treated)/Percentage MI of VC] ×100; VC: Vehicle Control, MI: Mitotic Index.

Evaluation criteria:

Positive result if, in any of the experimental conditions examined:
*At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
*The increase is dose-related when evaluated with an appropriate trend test.
Negative result if, in all experimental conditions examined:
*None of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
*There is no concentration-related increase when evaluated with an appropriate trend test.
*All results are inside the distribution of the historical vehicle control data.

Statistics:

Data (Percentage of cells with aberrations) was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p<0.05).

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

maximum mean % reduction in MI = 25.86 at 0.125 µL/mL (long term treatment)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A solubility test was conducted to determine the maximum concentration or workable suspension solution of the test item in the vehicle compatible with the test system at 200 µL/mL and the test item was miscible in dimethyl sulphoxide at 200 µL/mL. Precipitation test was conducted at 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Post 23 hours and 3 minutes of incubation, slight precipitation was observed at 2 µL/mL. No precipitation was observed in any other tested concentration. No change in pH was observed in any of the concentration tested. Hence, 2 µL/mL was selected as highest concentration for testing in the initial cytotoxicity test.
Initial cytotoxicity test: this test was conducted at concentrations of 0.125, 0.25, 0.5, 1 and 2 µL/mL and in the same conditions of the main test. The obtained reduction in mitotic index (MI) at 0.25 µL/mL and higher concentrations was 100%. As the reduction in MI was not more than 45±5% of the negative control at 0.125 µL/mL, this value was selected as the highest concentration for the chromosomal aberration test.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 4 below
- Negative (solvent/vehicle) historical control data: see table 4 below

The means of percentage aberrated cells observed for test item and positive controls were within the historical vehicle control and positive control ranges respectively.

Table 1. Summary of percentage mitotic index for initial cytotoxicity test

Set No.	Treatment	Concentrations (mg/mL)	Average % Mitotic Index	% reduction of Mitotic Index
Set 1 (+S9) (3 to 6 hours)	Vehicle control	0	7.60	-
	PINE OIL 50%	0.125	5.97	21.45
		0.25	0.00	100.00
		0.5	0.00	100.00
Set 2 (-S9)  (3 to 6 hours)	Vehicle control	0	7.95	-
	PINE OIL 50%	0.125	6.54	17.74
		0.25	0.00	100.00
		0.5	0.00	100.00
Set 3 (-S9) (20 to 24 hours)	Vehicle control	0	7.44	-
	PINE OIL 50%	0.125	6.09	18.15
		0.25	0.00	100.00
		0.5	0.00	100.00

Table 2. Individual data of chromosomal aberrations and mitotic index

Set No.	Treatment	Concentrations (µL/mL)	Replicate	Mitotic Index				Aberrations										Total No. of Aberrations	Total No. of Aberrations without Gaps	Total no. of Aberrant cells	Total % of Aberrated cells
								Gaps		Breaks		Exchanges		Fragments	Ring	Deletion	Dicentric
				Total No. of cells	Total No. of MP	Mitotic Index	Percentage of MI	Chromatid	Chromosome	Chromatid	Chromosome	Chromatid	Chromosome
Set 1 (+S9) (3 to6 hours)	Vehicle Control	-	R1	529	36	0.0681	6.81	-	-	-	1	-	-	1	-	-	-	2	2	1	0.67
			R2	523	34	0.0650	6.50	-	-	1	1	-	-	-	-	-	-	2	2	2	1.33
	Positive Control (Cyclophosphamide Monohydrate)	10µg/mL	R1	535	35	0.0654	6.54	2	2	6	4	1	-	7	-	1	-	23	19	15	10.00
			R2	524	31	0.0592	5.92	1	1	4	4	-	-	4	2	3	-	19	17	14	9.33
	PINE OIL 50%	0.0312	R1	518	36	0.0695	6.95	1	-	-	-	-	-	1	-	-	-	2	1	1	0.67
			R2	509	31	0.0609	6.09	-	-	-	-	-	-	1	-	-	-	1	1	1	0.67
		0.0625	R1	529	35	0.0662	6.62	-	-	-	-	-	-	1	-	1	-	2	2	2	1.33
			R2	527	32	0.0607	6.07	-	-	-	-	1	-	2	-	-	-	3	3	2	1.33
		0.125	R1	521	26	0.0499	4.99	-	-	1	1	-	-	-	-	-	-	2	2	2	1.33
			R2	525	26	0.0495	4.95	1	-	1	-	-	-	-	-	-	-	2	1	1	0.67

Set No.	Treatment	Concentrations (µL/mL)	Replicate	Mitotic Index				Aberrations										Total No. of Aberrations	Total No. of Aberrations without Gaps	Total no. of Aberrant cells	Total % of Aberrated cells
								Gaps		Breaks		Exchanges		Fragments	Ring	Deletion	Dicentric
				Total No. of cells	Total No. of MP	Mitotic Index	Percentage of MI	Chromatid	Chromosome	Chromatid	Chromosome	Chromatid	Chromosome
Set 2 (-S9) (3 to 6 hours)	Vehicle Control	-	R1	528	36	0.0682	6.82	-	-	-	-	-	-	1	-	-	-	1	1	1	0.67
			R2	526	31	0.0589	5.89	-	-	1	1	-	-	-	-	-	-	2	2	2	1.33
	Positive Control (Mitomycin C)	0.05µg/mL	R1	529	28	0.0529	5.29	2	1	6	3	-	-	5	2	-	-	19	16	14	9.33
			R2	527	33	0.0626	6.26	1	3	7	2	-	1	5	-	1	1	21	17	14	9.33
	PINE OIL 50%	0.0312	R1	518	34	0.0656	6.56	-	-	1	-	-	-	1	-	-	-	2	2	2	1.33
			R2	524	30	0.0573	5.73	-	-	1	-	-	-	-	-	1	-	2	2	2	1.33
		0.0625	R1	529	30	0.0567	5.67	-	-	-	-	-	-	1	-	-	-	1	1	1	0.67
			R2	523	34	0.0650	6.50	-	-	1	-	-	-	1	-	-	-	2	2	2	1.33
		0.125	R1	521	23	0.0441	4.41	-	-	1	1	-	-	-	-	-	-	2	2	2	1.33
			R2	529	27	0.0510	5.10	-	-	-	-	-	-	1	-	-	-	1	1	1	0.67

Set No.	Treatment	Concentrations (µL/mL)	Replicate	Mitotic Index				Aberrations										Total No. of Aberrations	Total No. of Aberrations without Gaps	Total no. of Aberrant cells	Total % of Aberrated cells
								Gaps		Breaks		Exchanges		Fragments	Ring	Deletion	Dicentric
				Total No. of cells	Total No. of MP	Mitotic Index	Percentage of MI	Chromatid	Chromosome	Chromatid	Chromosome	Chromatid	Chromosome
Set 3 (-S9) (20 to 24 hours)	Vehicle Control	-	R1	531	31	0.0584	5.84	-	-	1	1	-	-	-	-	-	-	2	2	2	1.33
			R2	523	39	0.0746	7.46	-	-	-	1	-	-	1	-	-	-	2	2	2	1.33
	Positive Control (MitomycinC)	0.05µg/mL	R1	524	34	0.0649	6.49	1	1	6	5	-	-	4	1	1	-	19	17	15	10.00
			R2	523	30	0.0574	5.74	-	2	7	5	-	1	2	-	3	-	20	18	17	11.33
	PINE OIL 50%	0.0312	R1	523	37	0.0707	7.07	-	-	1	1	-	-	-	-	-	-	2	2	2	1.33
			R2	529	32	0.0605	6.05	-	-	1	-	-	-	-	-	-	-	1	1	1	0.67
		0.0625	R1	526	32	0.0608	6.08	-	-	1	1	-	-	-	-	-	-	2	2	2	1.33
			R2	528	36	0.0682	6.82	-	-	1	-	-	-	-	-	-	-	1	1	1	0.67
		0.125	R1	528	27	0.0511	5.11	-	-	-	1	-	-	-	-	-	-	2	1	1	0.67
			R2	526	25	0.0475	4.75	-	-	1	1	-	-	-	-	-	-	2	2	2	1.33

150 metaphases were evaluated per replicate. MI: Mitotic Index, MP: Metaphase Plates, R1: Replicate 1, R2: Replicate 2, -S9: Without metabolic activation.

Table 3. Summary of chromosomal aberrations and mitotic index

Set No.	Treatment	Concentrations (µg/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 1  (+S9) (3 to 6 hours)	Vehicle Control	0	6.66	NA	2	2	1.5	1.00
	Positive Control (Cyclophosphamide monohydrate)	10 µg/mL	6.23	6.46	21	18	14.5	10.65*
	PINE OIL 50%	0.0312	6.52	2.1	1.5	1	1	0.67
		0.0625	6.35	4.65	2.5	2.5	2	1.33
		0.125	4.97	25.38	2	1.5	1.5	1.00

Set No.	Treatment	Concentrations (mg/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 2 (-S9) (3 to 6 hours)	Vehicle Control	0	6.36	NA	1.5	1.5	1.5	1.00
	Positive Control (Mitomycin-C)	0.05 µg/mL	5.78	9.12	20	16.5	14	9.33*
	PINE OIL 50%	0.0312	6.15	3.3	2	2	2	1.33
		0.0625	6.09	4.25	1.5	1.5	1.5	1.00
		0.125	4.76	25.16	1.5	1.5	1.5	1.00

Set No.	Treatment	Concentrations (µL/mL)	Mean % MI	Mean % Reduction in MI	Mean of Total Aberrations with Gaps	Mean of Total Aberrations without Gaps	Mean of Total Aberrant cells without Gaps	Mean of Percentage Aberrated Cells
Set 3 (-S9) (20 to 24 hours)	Vehicle Control	0	6.65	NA	2	2	2	1.33
	Positive Control (Mitomycin-C)	0.05 µg/mL	6.12	7.97	19.5	17.5	15	10.00*
	PINE OIL 50%	0.0312	6.56	1.35	1.5	1.5	1.5	1.00
		0.0625	6.45	3.01	1.5	1.5	1.5	1.00
		0.125	4.93	25.86	2	1.5	1.5	1.00

 MI: Mitotic Index; *: Statistically significant

Table 4. Historical data

Vehicle control - DMSO

95% Confidence level
	With S9 (3 to 6 hours)	Without S9  (3 to 6 hours)	Without S9   (20 to 24 hours)
Average	0.76	0.90	0.81
Standard deviation	0.24	0.24	0.29
Sample size	12.00	12.00	12.00
Margin of error	0.14	0.14	0.16
Upper bound	0.90	1.04	0.97
Lower boud	0.62	0.76	0.65
95% Confidence level	1.96	1.96	1.96
Max	1.00	1.33	1.30
Min	0.35	0.67	0.34

Positive control

95% Confidence level
	With S9 (3 to 6 hours)	Without S9 (3 to 6 hours)	Without S9 (20 to 24 hours)
Average	9.88	9.90	10.42
Standard Deviation	1.12	1.26	1.89
sample size	16.00	16.00	16.00
Margin of error	0.55	0.62	0.93
upper bound	10.43	10.52	11.34
lower boud	9.33	9.28	9.49
95% Confidence level	1.96	1.96	1.96
Max	12.00	13.35	16.00
Min	8.67	8.67	8.67

Conclusions:

In an in vitro chromosomal aberration test, PINE OIL 50% was found as non-clastogenic up to the concentration of 0.125 µL/mL both in the presence and absence of metabolic activation under the test conditions.

Executive summary:

The test item PINE OIL 50% was evaluated for chromosomal aberrations in human lymphocytes according to OECD Guideline 473, following the Principles of GLP. Based on the results of the solubility and precipitation tests, the test item was formulated in DMSO as vehicle and 2 µL/mL was selected as highest concentration in an initial cytotoxicity test. This test was conducted at concentrations of 0.125, 0.25, 0.5, 1 and 2 µL/mL. The obtained reduction in mitotic index (MI) at 0.25 µL/mL and higher concentrations was 100%. As the reduction in MI was not more than 45±5% of the negative control at 0.125 µL/mL, this value was selected as the highest concentration for the main test. Cultured human peripheral blood lymphocytes previously incubated for 44 -48 h with PHA in RPMI medium were treated with test item in DMSO at the concentrations of 0.0312, 0.0625 and 0.125 µL/mL. The treatment was carried out in duplicates for the short term period (3 h and 10 min) both in the presence and absence of metabolic activation (S9 mix) and for the long term period (22 h and 9 min) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at 10 µg/mL and Mytomycin-C at 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls. Colchicine was used as the metaphase-arresting substance. There was no statistically significant increase in the number of aberrated cells when compared with vehicle control at any of the concentration levels tested. The mean reduction in MI at highest dose 0.125 µL/mL was 25.16 -25.86% for all the treatments. The concurrent vehicle and positive control values were within the 95% control limits of the distribution of the laboratory’s historical control database. All acceptability criteria were met. Based on these results, the test item is considered as non-clastogenic up to the concentration of 0.125 µL/mL both in the presence and absence of metabolic activation under the presented test conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2018 - 22 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according OECD Guideline 476. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hprt (hypoxanthine-guanine-phosphoribosyl transferase) gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The derivative of the CHO-K1, CHO AA8 Cells were procured from American Type Culture Collection (ATCC)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin and streptomycin).
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate

Test concentrations with justification for top dose:

0.0390625, 0.078125, 0.15625 and 0.3125 µL/mL.

Based on the results of solubility, pH and precipitation test, an initial cytotoxicity test was conducted for the selection of test concentrations for the gene mutation test at concentrations of 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 µL/mL. The results indicated that the test item was not cytotoxic as the Relative Survival of the treated cells at the tested concentrations up to 0.3125 µL/mL was 11.11% to 12.09%, when compared with the vehicle control, both in the presence and absence of metabolic activation. Therefore 0.3125 µL/mL was selected as the highest concentration in the gene mutation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility test was conducted with distilled water and dimethyl sulphoxide to determine the maximum concentration or workable suspension of the test item in the vehicle compatible with the test system up to the maximum concentration of 500 µL/mL. The test item was miscible in DMSO at 5 µL/mL. Precipitation test was conducted and the test item showed no precipitation and no change in pH up to 5 µL/mL. Based on these results, 5 µL/mL was selected as the highest concentration for the initial cytotoxicity test.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium.

- Cell density at seeding (if applicable): approx. 2 x 10^6 cells/culture flask (Initial cytotoxicity test and Gene mutation test).

DURATION
- Exposure duration: 3 h and 15 min (with and without metabolic activation)
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 3 (cytotoxicity and cloning efficiency in non-selective medium) and 5 (cloning efficiency of mutant colonies in selective medium)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: A volume of 100 µL of vehicle/different concentrations of test item was added to tetra plate cultures to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. After treatment, medium from each flask was aspirated and monolayer was washed with DPBS. Cells were trypsinized. Trypsinization was stopped by adding culture media followed by collecting the media with cells. Tetra plate treatments were pooled into a pre labeled tube and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded and cell pellet was retained. Each treatment replicate was plated in triplicate with cell concentration of 200 cells/5 mL media in cell culture 25 cm2 flasks for estimation of parallel cytoxicity. Afterwards, the replicate cultures were sub cultured in duplicates at a density of 1×106 cells/culture flask for expression of mutant phenotype for 9 days. Finally, each replicate treatment cultures were pooled and sub cultured in quintuplicates at a density of 4x10^5 cells per group with culture media containing 6-Thioguanine and 200 cells /dish in triplicates without 6-Thioguanine for determination of cloning efficiency. 25 cm2 flasks were incubated for 8 days. Post incubation period, medium from each dish was aspirated and stained with 5% Giemsa stain, number of colonies formed were counted manually.

NUMBER OF CELLS EVALUATED: 200 x 3 per group (cytotoxicity and cloning efficiency in non-selective medium) and 4x10^5 x 5 per group (cloning efficiency of mutant colonies in selective medium)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival according to formulas included in annex 2 of OECD TG 476.

Evaluation criteria:

Positive result if, in any of the experimental conditions examined:
*At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
*The increase is dose-related when evaluated with an appropriate trend test.
*Any of the results are outside the distribution of the historical negative/vehicle control data
Negative result if, in all experimental conditions examined:
*None of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
*There is no concentration-related increase when evaluated with an appropriate trend test.
*All results are inside the distribution of the historical vehicle control data.

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups using SPSS Software version 22 at a 95% level (p<0.05) of significance.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relative survival (%)=11.63 - 39.53

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Relative survival (%)=12.64 - 40.23

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 conducted with distilled water and dimethyl sulphoxide to determine the maximum concentration or workable suspension of the test item in the vehicle compatible with the test system up to the maximum concentration of 500 µL/mL. The test item was miscible in DMSO at 5 µL/mL. Precipitation test was conducted and the test item showed no precipitation and no change in pH up to 5 µL/mL. Based on these results, 5 µL/mL was selected as the highest concentration for the initial cytotoxicity test.
Based on the results of solubility, pH and precipitation test, an initial cytotoxicity test was conducted for the selection of test concentrations for the gene mutation test at concentrations of 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 µL/mL. The results indicated that the test item was not cytotoxic as the Relative Survival of the treated cells at the tested concentrations up to 0.3125 µL/mL was 11.11% to 12.09%, when compared with the vehicle control, both in the presence and absence of metabolic activation. Therefore 0.3125 µL/mL was selected as the highest concentration in the gene 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

Data of mutant frequency observed for test item and positive controls were within the historical vehicle control and positive control ranges respectively.

Table 1. Summary of initial cytotoxicity test

Set No.	Treatment	Concentration (µL/mL)	Average Colony Count± SD	*Cloning   Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set1 +S9	Vehicle Control (DMSO)	-	169.67±5.03	0.85	0.91	-
	Pine Oil 50%	0.15625	73.33±1.53	0.37	0.17	18.68
		0.3125	71.33±3.06	0.36	0.11	12.09
		0.625	55.33±3.51	0.28	0.05	5.49
		1.25	40.67±2.08	0.20	0.02	2.20
Set2 -S9	Vehicle Cotrol (DMSO)	-	168.67±3.51	0.84	0.90	-
	Pine Oil 50%	0.15625	70.33±1.53	0.35	0.16	17.78
		0.3125	66.67±3.21	0.33	0.10	11.11
		0.625	53.00±3.00	0.27	0.05	5.56
		1.25	38.00±4.58	0.19	0.02	2.22

*Note: Cloning Efficiency = 200 cells plated for each replicate.

Adjusted CE = CE x Number of cells at the end of treatment/number of cells at the beginning of treatment.

RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.

CE = Number of colonies/Number of cells plated.

Table 2. Summary of parallel cytotoxicity test - gene mutation test

Set No.	Treatment	Concentration (µL/mL)	Average Colony Count ± SD	*Cloning Efficiency (CE)	Adjusted Cloning Efficiency (ACE)	Relative Survival (RS) (%)
Set1 +S9	Vehicle Control (DMSO)	-	170.00±2.00	0.85	0.87	-
	Pine Oil 50%	0.0390625	95.67±2.08	0.48	0.35	40.23
		0.078125	89.00±1.00	0.45	0.26	29.89
		0.15625	73.00±2.00	0.37	0.17	19.54
		0.3125	70.67±2.52	0.35	0.11	12.64
	Benzo(a)pyrene              (Positive control)	0.003	133.00±1.73	0.67	0.46	52.87
Set2 -S9	Vehicle Control (DMSO)	-	165.67±3.06	0.83	0.86	-
	Pine Oil 50%	0.0390625	88.33±2.08	0.44	0.34	39.53
		0.078125	87.00±2.65	0.44	0.25	29.07
		0.15625	72.67±1.53	0.36	0.16	18.60
		0.3125	64.67±2.52	0.32	0.10	11.63
	4 Nitroquinoline N-oxide (Positive control)	0.001	145.33±3.51	0.73	0.51	59.30

*Note: Cloning Efficiency = 200 cells plated for each replicate.

Adjusted CE = CE x Number of cells at the end of treatment/number of cells at the beginning of treatment.

RS = Adjusted CE in treated culture/Adjusted CE in the vehicle control × 100.

CE = Number of colonies/Number of cells plated.

Table 3. Summary of gene mutation test

Set No.	Treatment	Concentration (µL/mL)	*Average Colony Count ± SD	Cloning Efficiency in selective media	Cloning Efficiency in non-selective media	Total number of Mutant Colonies/ 2x10^6cells	Mutant Frequency/ 2x10^6cells
Set1 +S9	Vehicle Control (DMSO)	-	170.00±2.00	0.0000105	0.85	21	24.71
	Pine Oil 50%	0.0390625	167.67±1.53	0.0000105	0.84	21	25.00
		0.078125	172.67±3.51	0.0000110	0.86	22	25.58
		0.15625	170.67±1.53	0.0000110	0.85	22	25.88
		0.3125	173.00±3.00	0.0000115	0.87	23	26.44
	Benzo(a)pyrene (Positive control)	0.003	124.00±1.73	0.0000810	0.62	162	261.29**
Set2 -S9	Vehicle Control (DMSO)	-	172.00±2.65	0.0000110	0.86	22	25.58
	Pine Oil 50%	0.0390625	169.33±1.53	0.0000105	0.85	21	24.71
		0.078125	173.00±2.00	0.0000105	0.87	21	24.14
		0.15625	172.00±3.00	0.0000115	0.86	23	26.74
		0.3125	175.67±1.53	0.00000115	0.88	23	26.14
	4Nitroquinoline N-oxide (Positive control)	0.001	126.00±2.00	0.0000830	0.63	166	263.49**

*Note: Cloning efficiency = 200 cells plated for each replicate.  

**: Statistically significant (p˂0.05).  

Mutant Frequency = Cloning efficiency of mutant colonies in selective medium/Cloning efficiency in non-selective medium.

Table 4. Individual data of parallel cytotoxicity test and mutant phenotype

Set No.	Treatment	Concentration (µL/mL)	No. of Colonies/200 Cells			No. of Mutant Colonies/2x10^6Cells
			Replicate 1	Replicate 2	Replicate 3	Replicate 1	Replicate 2	Replicate 3	Replicate 4	Replicate 5
Set1 +S9	Vehicle control (DMSO)	-	168	172	170	3	6	4	5	3
	Pine Oil 50%	0.039062	169	166	168	4	5	3	4	5
		0.078125	173	169	176	3	4	6	4	5
		0.15625	169	171	172	6	4	3	4	5
		0.3125	170	173	176	4	5	6	3	5
	Benzo(a) pyrene (Positive control)	0.003	125	122	125	30	32	32	35	33
Set2 -S9	Vehicle control (DMSO)	-	170	175	171	3	5	5	4	5
	Pine Oil 50%	0.039062	168	169	171	3	4	2	5	7
		0.078125	171	173	175	5	5	4	3	4
		0.15625	169	172	175	5	4	4	4	6
		0.3125	177	176	174	6	4	4	5	4
	4 Nitroquinoline N-oxide (Positive control)	0.001	126	128	124	35	32	33	34	32

Table 5. Historical data

Vehicle control - DMSO

	With Metabolic Activation (3 to 6 hours)	Without  Metabolic Activation (3 to 6 hours)
Mean Data of Mutant Frequency/2x10^6Cells	24.51	25.43
Standard Deviation	2.81	1.89
Margin of Error	1.95	1.31
Upper bound	26.46	26.74
Lower bound	22.56	24.12

Positive Control - Benzo(a)pyrene and 4- Nitroquinoline N-oxide

	With Metabolic Activation (3 to 6 hours) [Benzo(a)pyrene]	Without Metabolic Activation (3 to 6 hours) [4Nitroquinoline N-oxide]
Mean Data ofMutant Frequency/2x10^6Cells	261.94	264.60
Standard Deviation	27.28	18.52
Margin of Error	17.82	12.10
Upper bound	279.76	276.70
Lower bound	244.12	252.50

Conclusions:

PINE OIL 50% was found as non-mutagenic up to the concentration of 0.3125 µL/mL, both in the presence and absence of metabolic activation under the test conditions.

Executive summary:

The test item PINE OIL 50% was evaluated for gene mutation test in CHO AA8 cells according to OECD Guideline 476, following the Principles of GLP. Based on the results of the solubility, pH and precipitation tests, the test item was formulated in DMSO as vehicle and 5 μL/mL was selected as the highest concentration in an initial cytotoxicity test. This test was conducted at concentrations of 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 µL/mL. Relative Survival values of 11.11% to 12.09% compared with the vehicle control, both in the presence and absence of metabolic activation were found at 0.3125 µL/mL which were the minimum values of Relative Survival >10% of the tested concentrations. Therefore 0.3125 µL/mL was selected as the highest concentration in the gene mutation test. The main test was conducted at the concentrations of 0.0390625, 0.078125, 0.15625 and 0.3125 µL/mL with an exposure time of 3 h and 15 min both in the presence and absence of metabolic activation and 6-Thioguanine as the selective agent. DMSO alone was tested as solvent control and Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls. Parallel cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival. There was no statistically significant increase in number of mutant colonies at any of the concentrations tested when compared with the vehicle control. All results were inside the distribution of the historical vehicle control data. Positive controls resulted in mutant frequencies, which were statistically significant when compared with the vehicle control and within the historical positive control data. Based on these results, the test item is considered as non-mutagenic at and up to the concentration of 0.3125 µL/mL, both in the presence and absence of metabolic activation under the tested conditions.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two of the recommended strains were tested)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: UTH 8414

Species / strain / cell type:

S. typhimurium, other: UTH 8413

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate (S9) from Aroclor-induced male Sprague-Dawley rats (100 µL/plate)

Test concentrations with justification for top dose:

Five concentrations, from 10 µg/plate to 500 µg/plate.
Toxicity and/or solubility determined the upper limit of the dose tested.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(With and without metabolic activation)

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Positive controls:

yes

Positive control substance:

other: Cisplatin

Details on test system and experimental conditions:

DURATION
- Exposure duration: The plates were incubated at 37 ºC for 48 h, at which time the number of colonies per plate was determined.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8414

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8413

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha pinene was tested for mutagenecity (doses: 10 -500µg/plate). The mutagenicity assay employed was that described by Maron and Ames using Salmonella typhimurium strains TAl00 and TA98, which are DNA-repair deficient, and two additional strains, UTH 8414 and UTH 8413, developed by Matney, which have full DNA repair capacity. The mutagenicity assays were carried out both with and without metabolic activation (S9). Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two of the recommended strains were tested)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: UTH 8414

Species / strain / cell type:

S. typhimurium, other: UTH 8413

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate (S9) from Aroclor-induced male Sprague-Dawley rats (100 µL/plate)

Test concentrations with justification for top dose:

Five concentrations, from 10 µg/plate to 1000 µg/plate.
Toxicity and/or solubility determined the upper limit of the dose tested.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(With and without metabolic activation)

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Positive controls:

yes

Positive control substance:

other: Cisplatin

Details on test system and experimental conditions:

DURATION
- Exposure duration: The plates were incubated at 37 ºC for 48 h, at which time the number of colonies per plate was determined.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8414

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8413

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Camphene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Camphene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Camphene was tested for mutagenecity (doses: 10 -1000µg/plate). The mutagenicity assay employed was that described by Maron and Ames using Salmonella typhimurium strains TAl00 and TA98, which are DNA-repair deficient, and two additional strains, UTH 8414 and UTH 8413, developed by Matney, which have full DNA repair capacity. The mutagenicity assays were carried out both with and without metabolic activation (S9). Camphene was not mutagenic in all strains tested with and without metabolic activation.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two of the recommended strains were tested)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: UTH 8414

Species / strain / cell type:

S. typhimurium, other: UTH 8413

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate (S9) from Aroclor-induced male Sprague-Dawley rats (100 µL/plate)

Test concentrations with justification for top dose:

three concentrations, from 10 µg/plate to 500 µg/plate.
Toxicity and/or solubility determined the upper limit of the dose tested.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(With and without metabolic activation)

Positive controls:

yes

Positive control substance:

sodium azide

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Positive controls:

yes

Positive control substance:

other: Cisplatin

Details on test system and experimental conditions:

DURATION
- Exposure duration: The plates were incubated at 37 ºC for 48 h, at which time the number of colonies per plate was determined.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8414

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: UTH 8413

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

d-limonene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

d-limonene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

d-limonene was tested for mutagenecity (doses: 10 - 500µg/plate). The mutagenicity assay employed was that described by Maron and Ames using Salmonella typhimurium strains TAl00 and TA98, which are DNA-repair deficient, and two additional strains, UTH 8414 and UTH 8413, developed by Matney, which have full DNA repair capacity. The mutagenicity assays were carried out both with and without metabolic activation (S9). D-limonene was not mutagenic in all strains tested with and without metabolic activation.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only one replicate was conducted)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Remarks:

"Spot test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Remarks:

"Quantitative test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

"Quantitative test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254 (S-9A)

Test concentrations with justification for top dose:

Spot test: 3 μmol/plate (408 μg/plate)
Quantitative test: 0.03, 0.3, 3 and 30 μmol /plate (4.08, 40.8, 408, and 4080 μg /plate)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (+S9); N-methyl-N'-nitro-N-nitrosoguanidin (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Cultures were grown in Oxoid nutrient broth No. 2. Revertants were scored on glucosenminimal salts medium supplemented with 0.05 μmol histidine and 0.05 μmol biotin. Plates used for viable counts contained 10 μmol histidine (and 0.05 μmol biotin). The experiments were carried out essentially as described by Ames.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 1

DETERMINATION OF CYTOTOXICITY
Toxicity was determined based on the absence of a background lawn of bacteria on the plates. If absence of a background lawn was found the test was repeated with a lower concentration of the substance.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(precipitates at 30 μmol/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at doses equal and higher than 3 μmol/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(precipitates at 30 μmol/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at doses equal and higher than 3 μmol/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test item precipitates at top dose of 30 μmol/plate in the quantitative test

Remarks on result:

other: spot test (rat liver S9 fraction induced by Aroclor 1254 (S-9A))

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha pinene was tested using the Ames assay for mutagenecity on Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation (S9). The test item was initially tested (spot test) at 3 μmol/plate on strains TA 98, TA 100, TA 1535 and TA 1537 both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254. As negative results were obtained, the test item was tested quantitatively at doses of 0.03, 0.3, 3 and 30 μmol /plate using TA 98 and TA 100 with and without metabolic activation using a liver fraction (S-9) from 3-methylcholanthrene. Alpha pinene was found toxic to the bacteria at doses equal and higher than 3 μmol/plate and precipitated at top dose of 30 μmol/plate. Under these test conditions, alpha pinene was found not mutagenic in all strains tested with and without metabolic activation.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only one replicate was conducted)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Remarks:

"Spot test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Remarks:

"Quantitative test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

"Quantitative test"

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254 (S-9A)

Test concentrations with justification for top dose:

Spot test: 3 μmol/plate
Quantitative test: 0.03, 0.3, 3 and 30 μmol /plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (+S9); N-methyl-N'-nitro-N-nitrosoguanidin (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Cultures were grown in Oxoid nutrient broth No. 2. Revertants were scored on glucosenminimal salts medium supplemented with 0.05 μmol histidine and 0.05 μmol biotin. Plates used for viable counts contained 10 μmol histidine (and 0.05 μmol biotin). The experiments were carried out essentially as described by Ames.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 1

DETERMINATION OF CYTOTOXICITY
Toxicity was determined based on the absence of a background lawn of bacteria on the plates. If absence of a background lawn was found the test was repeated with a lower concentration of the substance.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate (spot test)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(precipitates at 30 μmol/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at doses equal and higher than 3 μmol/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(precipitates at 30 μmol/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at doses equal and higher than 3 μmol/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: test item precipitates at top dose of 30 μmol/plate in the quantitative test

Remarks on result:

other: spot test (rat liver S9 fraction induced by Aroclor 1254 (S-9A))

Limonene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Limonene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Limonene was tested using the Ames assay for mutagenecity on Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation (S9). The test item was initially tested (spot test) at 3 μmol/plate on strains TA 98, TA 100, TA 1535 and TA 1537 both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254. As negative results were obtained, the test item was tested quantitatively at doses of 0.03, 0.3, 3 and 30 μmol /plate using TA 98 and TA 100 with and without metabolic activation using a liver fraction (S-9) from 3-methylcholanthrene. Limonene was found toxic to the bacteria at doses equal and higher than 3 μmol/plate and precipitated at top dose of 30 μmol/plate. Under these test conditions, limonene was found not mutagenic in all strains tested with and without metabolic activation.

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: TA97a

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

First set of experiments: 0, 10, 20, 35, 50, 75, 100, 200, 250, 300, 400, 500, 750, 1000, 1250, 1500, 2000, 2500 and 5000 μg/plate
Second set of experiments (complementary number of doses within the non-toxic dose interval determined in first set of experiments): 0, 5, 10, 20, 25, 35, 50, 75, 100, 200, 250, 500, 750 and 1500 μg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

(100 µL ethanol)

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (TA100/+S9 and TA1535/+S9 (1 μg/plate), TA98/+S9 (0.5 μg/plate)); 2-aminofluorene (TA97a/+S9 (10 μg/plate))

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: 100 μl of an overnight grown 100 μl of the test substance (diluted in analytical grade ethanol, Vetec™, Rio de Janeiro, Brazil), the negative (solvent) control, or the positive control (PC) and 500 μl of the sodium-phosphate buffer or the S9 mix were mixed with 2ml of top agar which was poured onto a minimal glucose plate. Plates were incubated at 37ºC for 72h in the dark and then scored for revertant his+ bacteria colonies. Each determination was made in triplicate and two independent experiments were carried out.
- Cell density at seeding: For all assays, an inoculum (200 μl) of a thawed permanent culture was added to 20ml of Nutrient Broth No. 2 and incubated at 37ºC with shaking until a concentration of approximately 1–2 x10^9 bacteria per millilitre was obtained.

DURATION
- Exposure duration: 72h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity of (-) alpha pinene to S. typhimurium strains TA97a, TA98, TA100 and TA1535 was investigated in a first set of experiments. Toxicity was apparent either as a reduction in the number of his+ revertant bacteria colonies and or as a change of auxotrophic background growth (i.e. the background lawn).
Doses at which toxicity appeared as an alteration of the background lawn are marked with an asterisk in table 1 (Any other information on results incl. Tables).
Then, a second set of experiments was conducted which included a complementary number of doses within the non-toxic dose interval determined in first set of experiments.

OTHER:
-Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox™ (Molecular Toxicology Inc., Boone, NC, USA). The S9 mixture was prepared as follows: 7.0ml of ultrapure water; 10.5ml of 200mM sodium phosphate buffer pH7.4; 0.84ml of 100mM NADP solution; 0.105ml of 1M glucose-6-phosphate; 0.42ml of 1.65M KCl + 0.4M MgCl2 salt solution; and 2.1ml of lyophilized S9 fraction reconstituted with water provided by a MilliQ™ water purification system.

Evaluation criteria:

The criteria for a positive mutagenic response, was a clear dose-dependent increase in the number of revertants within the non-toxic range (Maron and Ames, 1983).

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 100 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 100 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 100 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 500 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1500 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 500 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 200 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1000 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 250 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 750 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1. Testing of alpha-terpinene (1-isopropyl-4-methyl-1,3-cyclohexadiene) in the Salmonella/microsome assay

Dose (µg/plate)		Number of revertants (Mean ± SD)
		TA 100		TA 98		TA 97a		TA 1535
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Set 1 experiments	5000	-	-	-	43 ± 36*	-	45 ± 17*	2 ± 3*	6 ± 4*
	2500	-	-	-	65 ± 16*	-	108 ± 22*	0 ± 0*	14 ± 5*
	2000	-	104 ± 28*	13 ± 13*	-	-	-	-	-
	1500	-	-	23 ± 12*	-	-	-	-	-
	1250	-	73 ± 35*	19 ± 14*	-	-	-	-	-
	1000	-	51 ± 35*	9 ± 12*	62 ± 12	-	144 ± 17*	12 ± 1*	15 ± 6*
	750	-	-	35 ± 8*	-	-	-	-	-
	500	-	84 ± 18*	20 ± 17	59 ± 2	110 ± 16*	142 ± 5	8 ± 4*	17 ± 2
	400	-	-	-	-	79 ± 2*	-	-	-
	300	-	-	-	-	83 ± 10*	-	-	-
	250	-	-	-	67 ± 4	-	146 ± 19	-	-
	200	-	-	-	-	101 ± 8*	-	-	-
	100	102±8*	116 ± 35*	-	57 ± 4	97 ± 17	185 ± 28	16 ± 2	17 ± 4
	75	128±19	-	-	-	-	-	-	-
	50	118 ± 2	-	-	-	141 ± 6	-	-	-
	35	147 ± 19	-	-	-	-	-	-	-
	20	139 ± 8	-	-	-	-	-	-	-
	10	152 ± 36	-	-	-	-	-	-	-
	0	167 ± 10	206	43 ± 12	58 ± 8	150 ± 2	202 ± 28	18 ± 4	27 ± 5
	PC	804 ± 59	614 ± 97	173 ± 35	430 ± 15	845 ± 17	928 ± 24	506 ± 8	154 ± 4
Set 2 experiments	1500	-	-	-	50 ± 2*	-	-	-	-
	750	-	-	-	58 ± 14	-	-	-	19 ± 6*
	500	-	-	34 ± 6*	58 ± 8	-	172 ± 4	0 ± 0*	21 ± 4
	250	-	-	25 ± 5	58 ± 4	-	220 ± 8	2 ± 3*	16 ± 2
	200	-	-	-	-	107 ± 20*	-	-	-
	100	-	-	36 ± 12	56 ± 8	109 ± 13	267 ± 8	4 ± 3	19 ± 5
	75	149 ± 7	120 ± 3	-	-	138 ± 20	-	-	-
	50	133	130 ± 12	41 ± 6	60 ± 3	123 ± 16	226 ± 14	20 ± 4	26 ± 2
	35	170 ± 15	146 ± 19	-	-	-	-	-	-
	25	-	-	37 ± 2	-	141 ± 14	194 ± 4	22 ± 5	22 ± 7
	20	162 ± 12	132 ± 39	-	-	-	-	-	-
	10	170 ± 6	140 ± 1	-	-	158 ± 2	-	21 ± 5	-
	5	154 ± 15	145 ± 18	-	-	158 ± 8	-	28 ± 7	-
	0	158 ± 26	148 ± 19	37 ± 7	47 ± 1	152 ± 13	190 ± 4	30 ± 4	23 ± 5
	PC	449 ± 32	774 ± 54	88 ± 8	183 ± 22	752 ± 87	841 ± 24	696 ± 81	182 ± 10

Negative control (dose 0) = 100μl ethanol; Positive control (PC): TA100/-S9 and TA1535/-S9, SA (1μg/plate); TA100/+S9 and TA1535/+S9, 2AA (1μg/plate); TA98/-S9, 2-NF (1.5μg/plate); TA98/+S9; 2AA (0.5μg/plate); TA97a/-S9, 4-NQNO (1μg/plate); TA97a/+S9, 2AF (10μg/plate).

(-) not tested. (*) Toxicity apparent as an alteration of the background lawn. Values are means ± SD of three plates.

Conclusions:

Alpha terpinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha terpinene was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA97a and TA1535 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. The test substance was not mutagenic in all strains tested with and without metabolic activation.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: TA97a

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

First set of experiments: 0, 100, 250, 500, 750, 1000, 1250, 1500, 2000, 2500 and 5000 μg/plate
Second set of experiments (complementary number of doses within the non-toxic dose interval determined in first set of experiments): 0, 1, 5, 10, 25, 50, 100, 200, 250, 400, 500,600, 700, 750, 800, 900, 1000, 1250, 1500, 2000, 4000 μg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene (TA100/+S9 and TA1535/+S9 (1 μg/plate), TA98/+S9 (0.5 μg/plate)); 2-aminofluorene (TA97a/+S9 (10 μg/plate))

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: 100 μl of an overnight grown culture, 100 μl of the test substance (diluted in analytical grade ethanol, Vetec™, Rio de Janeiro, Brazil), the negative (solvent) control, or the positive control (PC) and 500 μl of the sodium-phosphate buffer or the S9 mix were mixed with 2ml of top agar which was poured onto a minimal glucose plate. Plates were incubated at 37ºC for 72h in the dark and then scored for revertant his+ bacteria colonies. Each determination was made in triplicate and two independent experiments were carried out.
- Cell density at seeding: For all assays, an inoculum (200 μl) of a thawed permanent culture was added to 20ml of Nutrient Broth No. 2 and incubated at 37ºC with shaking until a concentration of approximately 1–2 x10^9 bacteria per millilitre was obtained.

DURATION
- Exposure duration: 72h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity of (-) alpha pinene to S. typhimurium strains TA97a, TA98, TA100 and TA1535 was investigated in a first set of experiments. Toxicity was apparent either as a reduction in the number of his+ revertant bacteria colonies and or as a change of auxotrophic background growth (i.e. the background lawn).
Doses at which toxicity appeared as an alteration of the background lawn are marked with an asterisk in table 1 (Any other information on results incl. Tables).
Then, a second set of experiments was conducted which included a complementary number of doses within the non-toxic dose interval determined in first set of experiments.

OTHER:
-Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox™ (Molecular Toxicology Inc., Boone, NC, USA). The S9 mixture was prepared as follows: 7.0ml of ultrapure water; 10.5ml of 200mM sodium phosphate buffer pH7.4; 0.84ml of 100mM NADP solution; 0.105ml of 1M glucose-6-phosphate; 0.42ml of 1.65M KCl + 0.4M MgCl2 salt solution; and 2.1ml of lyophilized S9 fraction reconstituted with water provided by a MilliQ™ water purification system.

Evaluation criteria:

The criteria for a positive mutagenic response, was a clear dose-dependent increase in the number of revertants within the non-toxic range (Maron and Ames, 1983).

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2500 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1250 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2500 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 2000 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1250 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 100 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 5000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 5000 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1 Testing of (-)-alpha-pinene in the Salmonella/microsome assay

Dose (µg/plate)		Number of revertants (Mean ± SD)
		TA 100		TA 98		TA 97a		TA 1535
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Set 1 experiments	5000	-	-	33±8	53±6	-	124±22	10±4*	13±4*
	2500	140±8*	147±52*	35±9	46±5	-	159±11	8±2*	19±8
	2000	138±32*	162±11*	-	-	134±21*	-	-	-
	1500	144*	164±1	-	-	103±17*	-	-	-
	1250	125±23*	-	-	-	122±25*	-	-	-
	1000	-	175±34	43±9	49±8	106±6	133±20	11±5*	19±5
	750	-	-	-	-	99±3	-	-	-
	500	-	143±1	34±3	47±3	124±15	153±16	12±2*	19±4
	250	-	-	-	-	-	173±22	-	21±3
	100	-	-	33±6	51±13	-	-	16±5*	-
	0	214±6	212±25	37±6	59±4	194±8	201±11	19±1	20±1
	PC	1112±64	716±40	618±28	278±19	799±47	879±33	362±27	165±24
Set 2 experiments	4000	-	-	-	-	-	-	-	18±3
	2000	-	-	37±7	-	-	-	-	19±6
	1500	-	-	33±3	-	-	-	-	-
	1250	-	-	35±5	-	-	-	-	-
	1000	174±17	-	42±1	45±4	92±11	153±14	-	21±3
	900	190±21	165±32	-	51±6	101±5	124±2	-	-
	750	-	-	33±8	-	-	-	-	-
	800	171±8	167±19	-	39±8	106±15	111±7	-	-
	700	166±30	167±13	-	49±4	105±16	113±28	-	-
	600	165±17	156±12	-	45±5	109±3	100±4	-	-
	500	170±26	174±6	27±4	48±13	89±6	109±18	-	18±2
	400	187±3	165±16	-	39±6	110±8	-	-	-
	250	-	-	-	-	-	-	-	18±3
	100	-	-	-	-	-	-	13±4	23±4
	50	-	-	-	-	-	-	10±1	-
	25	-	-	-	-	-	-	19±2	-
	10	-	-	-	-	-	-	19±7	-
	5	-	-	-	-	-	-	19±7	-
	1	-	-	-	-	-	-	20±13	-
	0	171±17	142±21	43±2	54±3	121±26	199±37	25±2	24±3
	PC	775±69	716±73	173±35	423±25	583±27	866±114	899±44	193±13

Negative control (dose 0) = 100μl ethanol; Positive control (PC): TA100/-S9 and TA1535/-S9, SA (1μg/plate); TA100/+S9 and TA1535/+S9, 2AA (1μg/plate); TA98/-S9, 2-NF (1.5μg/plate); TA98/+S9; 2AA (0.5μg/plate); TA97a/-S9, 4-NQNO (1μg/plate); TA97a/+S9, 2AF (10μg/plate).

(-) not tested. (*) Toxicity. Values are means ± SD of three plates.

Conclusions:

(-) alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

(-) alpha pinene was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA97a and TA1535 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. (-) alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD Guideline 471 with deviations: one strain missing; no data on number of bacterial cells per culture; individual plate counts not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

one strain missing; no data on number of bacterial cells per culture; individual plate counts not reported

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Aroclor 1254-induced adult male Sprague Dawley rats (RLI) and Syrian hamsters (HLI) liver

Test concentrations with justification for top dose:

- Without S9: 0, 0.3, 1, 3, 10 and 33 µg/plate
- With S9 (RLI): 0, 33, 100, 333, 1000 and 3333 µg/plate
- With S9 (HLI): 0, 10, 33, 100, 333 and 1000 µg/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (95%)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (1 µg/plate with TA 98 and TA 100; 2.5 µg/plate with TA 1535 and TA 1537)

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol (95%)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine (5 µg/plate with TA 98), sodium azide (1 µg/plate with TA 100 and TA 1535), 9-aminoacridine (50 µg/plate with TA 1537)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): no data

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

- OTHER:
Dose-range finding experiment: Test substance was checked for toxicity to TA 100 up to a concentration of 10 mg/plate or the Iimit of solubility, both in the presence and absence of S-9 mix.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Mean plate count

 

Dose

TA100

TA 1535

TA 1537

TA 98

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0

121 ± 15.4

151 ± 11.6

150 ± 6.6

24 ± 4.2

24 ± 4.3

24 ± 5

4 ± 1.5

5 ± 1.2

5 ± 0.9

18 ± 1.2

32 ± 0.6

32 ± 2.3

0.3

132 ± 3.8

14 ± 4.3

5 ± 0.3

18 ± 1.7

1

117 ± 9.4

15 ± 0.6

3 ± 1.2

21 ± 4.7

3

131 ± 4.2

13 ± 2.1

28 ± 1.5

3 ± 0.6

17 ± 4.6

10

122 ± 7.5

136 ± 10.7

17 ± 2.3

21 ± 2.2

6 ± 1.7

10 ± 2.7

23 ± 2

31 ± 3.5

33

129 ± 4.6

153 ± 21

125 ± 4.5

0 ± 0s

31 ± 1.9

24 ± 3.3

4 ± 0.7s

5 ± 0.9

6 ± 0.7

13 ± 4.3s

39 ± 1.2

26 ± 3.0

100

143 ± 1.8

138 ± 12.5

20 ± 2.6

19 ± 4.5

7 ± 1.5

6 ± 0.9

34 ± 1.8

27 ± 5.8

333

129 ± 13.6

110 ± 9.9

24 ± 3.5

t

7 ± 3.2

6 ± 1.5

26 ± 3.1

28 ± 3.9

1000

112 ± 21.1s

105 ± 9.6s

25 ± 0.5

4 ± 1.2

6 ± 2.8s

16 ± 8.4

20 ± 2.1s

3333

133 ± 2.5

25 ± 4.4

10 ± 2.0

14 ± 8.1s

POS

410 ± 27.1

601 ± 37.7

1401 ± 53.4

406 ± 4.0

163 ± 12.2

309 ± 8.7

172 ± 18.5

193 ± 11.6

506 ± 3.4

728 ± 67.6

380 ± 16.6

1276 ± 33.1

 

Abbreviations: POS: Positive control; NA: not activated; RLI: rat liver S-9, Aroclor 1254 induced; HLI: hamster liver S-9, Aroclor 1254 induced; s: slight clearing of background lawn; t: complete clearing of background lawn

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to OECD guideline 471, strains of S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) were exposed to d-limonene in 95% ethanol with and without S9 metabolic activation [S9 fraction of Aroclor 1254-induced adult male Sprague Dawley rats (RLI) and Syrian hamsters (HLI) liver] according to the preincubation method (20 min) at the following concentrations: - Without S9: 0, 0.3, 1, 3, 10 and 33 µg/plate - With S9 (RLI): 0, 33, 100, 333, 1000 and 3333 µg/plate - With S9 (HLI): 0, 10, 33, 100, 333 and 1000 µg/plate   The positive controls induced the appropriate responses in the corresponding strains. D-limonene showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S9 mix.   Under the test conditions, d-limonene is not considered as mutagenic in this bacterial system according to the criteria of the CLP Regulation (EC) N° (1272-2008). 

Reference 12

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Data published in a peer reviewed journal. Original study report not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

25000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha pinene was tested for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Alpha pinene was not mutagenic in all strains tested with and without metabolic activation.

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

abstract

Remarks:

Data published in a peer reviewed journal. Original study report not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

150000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

d-limonene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

d-limonene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

D-limonene was tested for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. D-limonene was not mutagenic in all strains tested with and without metabolic activation.

Reference 14

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Method similar to OECD guideline 471, but only two strains were tested with metabolic activation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two strains were tested with metabolic activation)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with

Metabolic activation system:

rat liver microsome fraction, S9, prepared from Aroclor 1254-treated animals according to the procedure of Ames et al. (800 μg/plate)

Test concentrations with justification for top dose:

The amounts assayed ranged from 0.05 µl to 100 µl of the undiluted sample.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: picrolonic acid

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: a test sample of 10^8 bacterial cells, and S9 at a concentration of 800 μg protein per plate were incorporated into a tube containing top agar prepared with minimal medium (Minimal Broth Davis, Difco) and 0.05 mM histidine and 0.05 mM biotin. The top agar was then poured on a petri dish containing minimal medium supplemented with 20% glucose. After a 48-hour incubation at 37°C, each assay plate was counted and the number of spontaneous mutants for either TA98 (40) or TA100 (180) were subtracted from the total number of revertants.

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: at least 2

DETERMINATION OF CYTOTOXICITY
An additional tube of top agar was prepared as explained above and plated on nutrient agar (Difco) to examine the background lawn of bacterial growth for the presence of toxic effects.

OTHER:
-Plates containing aflatoxin B1 were also included in each experiment to confirm enzyme activation by the S9 fraction.

Evaluation criteria:

The positive response to mutagenicity with TA100 is defined as any deviation above the upper 99.9% confidence limits of the mean control value. This value (180) is the average number of spontaneous TA100 revertants observed on the control plates.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Alpha pinene was not mutagenic in all strains tested with metabolic activation.

Conclusions:

Alpha pinene was not mutagenic in all strains tested with metabolic activation.

Executive summary:

Alpha pinene was tested for mutagenecity on Salmonella typhimurium strains TA100 and TA98 with metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Alpha pinene was not mutagenic in all strains tested with metabolic activation.

Reference 15

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Method similar to OECD guideline 471, but only two strains were tested with metabolic activation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two strains were tested with metabolic activation)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with

Metabolic activation system:

rat liver microsome fraction, S9, prepared from Aroclor 1254-treated animals according to the procedure of Ames et al. (800 μg/plate)

Test concentrations with justification for top dose:

The amounts assayed ranged from 5 µl to 10 µl of the undiluted sample.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: picrolonic acid

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: a test sample of 10^8 bacterial cells, and S9 at a concentration of 800 μg protein per plate were incorporated into a tube containing top agar prepared with minimal medium (Minimal Broth Davis, Difco) and 0.05 mM histidine and 0.05 mM biotin. The top agar was then poured on a petri dish containing minimal medium supplemented with 20% glucose. After a 48-hour incubation at 37°C, each assay plate was counted and the number of spontaneous mutants for either TA98 (40) or TA100 (180) were subtracted from the total number of revertants.

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: at least 2

DETERMINATION OF CYTOTOXICITY
An additional tube of top agar was prepared as explained above and plated on nutrient agar (Difco) to examine the background lawn of bacterial growth for the presence of toxic effects.

OTHER:
-Plates containing aflatoxin B1 were also included in each experiment to confirm enzyme activation by the S9 fraction.

Evaluation criteria:

The positive response to mutagenicity with TA100 is defined as any deviation above the upper 99.9% confidence limits of the mean control value. This value (180) is the average number of spontaneous TA100 revertants observed on the control plates.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

A weak mutagenic response toward TA100 but not TA98 was observed for the test substance.

Conclusions:

Camphene was found a weak mutagenic with TA100 and but not with TA98 in a reverse mutation test with metabolic activation.

Executive summary:

Camphene was tested for mutagenecity on Salmonella typhimurium strains TA100 and TA98 with metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity.

A weak mutagenic response toward TA100 but not TA98 was observed in doses where no apparent toxicity was detected.

Reference 16

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD guideline 473 with minor deviations: no data on number of replicates; no data on karyotype stability and incubation temperature; only 2-h exposure with test substance with S9

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

no data on number of replicates; no data on karyotype stability and incubation temperature; only 2-h exposure with test substance with S9

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: Litton Bionetics Inc.
- Type and identity of media: McCoys 5A medium supplemented with antibiotics and 10% fetal calf serum
- Properly maintained: Yes; cells for experiments were thawed and grown in the medium at 37 °C using 5% CO2
- Periodically checked for Mycoplasma contamination: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)

Test concentrations with justification for top dose:

- Without S9: 0, 10, 30 or 100 µg/mL
- With S9: 0, 50, 150 or 500 µg/mL
The doses selected for the aberration trials were based on data from the SCE trials. Ten doses were selected which, generally, covered a narrower range than used in the SCE assay. The three highest doses with sufficient metaphase cells were scored for aberrations.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation (5 µg/mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (50 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 2 (+S9) or 8 (-S9) hours
- Fixation time (start of exposure up to fixation or harvest of cells): 10.5 (-S9) or 12 (+S9) hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: 100 cells/dose in vehicle control and treatment groups; 50 cells/dose in positive control groups. Cells were scored for simple (chromatid gaps and breaks, fragments, deletions, chromosome gaps and breaks, and double minutes), complex (interstitial deletions, triradials, quadriradials, rings and dicentrics) and other pulverized, polyploids, and endoreduplications) aberrations.

DETERMINATION OF CYTOTOXICITY
- Based on SCE trials (visual estimate of the confluency of each flask)

Evaluation criteria:

- If a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal;
- If a trial had significant trend and one significant dose it was judged weak positive;
- If the trial had two significant doses it was judged positive, whether or not a positive trend was obtained.
- If only one dose was significant and the increase over the control was P <0.0005 the trial was denoted weak positive*

Statistics:

- Data were evaluated for both trend and dose point increase over the solvent control.
- A binomial sampling assumption was used to evaluate an absolute increase in aberrations over the solvent control. Dose points with P values adjusted by Dunnett's method were considered significant if < 0.05, whereas a trend of P < 0.003 was significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic above 100 (-S9) and 500 (+S9) µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic above 100 (-S9) and 500 (+S9) µg/mL

Table 1: Results obtained in trial 1 (without S9)

 

Dose (µg/mL)	Cells	Percent cells with aberrations
		Total	Simple	Complex
0.0000	100	4.00	4.00	0.00
10.0000	100	2.00	2.00	0.00
30.0000	100	2.00	2.00	0.00
100.0000	100	6.00	6.00	0.00
Positive control - MMC
5.0000	50.	50.00	42.00	18.00
Trend statistic = 0.75E+00
Trend probability = 0.23E+00

 

Table 2: Results obtained in trial 1 (with S9)

 

Dose (µg/mL)	Cells	Percent cells with aberrations
		Total	Simple	Complex
0	100	4	3	1
50	100	0	0	0
150	100	4	4	0
500	100	5	5	0
Positive control - CPA
50	50	40	26	18
Trend statistic = 0.88E+00
Trend probability = 0.19E+00

Conclusions:

Under the test conditions, d-limonene is not considered as cytogenetic in CHO cells according to the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro mammalian chromosome aberration test performed similarly to OECD guideline 473, Chinese hamster Ovary (CHO) cells were exposed to d-limonene in McCoys 5A medium with and without metabolic activation [S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)] at the following concentrations: Without S9: 0, 10, 30 and 100 µg/mL, and with S9: 0, 50, 150 and 500 µg/mL. Positive controls (mitomycin C at 5 µg/mL without S9 and cyclophosphamide at 50 µg/mL with S9) induced the appropriate response. Chromosome aberrations were not induced in treatment groups over background at any tested concentrations in the presence or absence of activation system. Under the test conditions, d-limonene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 17

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CHO cells were obtained from the Oak Ridge National Laboratory with a designation CHO-K1-BH4 (subclone D1)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham`s modified F12 medium supplemented with 10% (v/v) heated inactivated fetal bovine serum, lacking in hipoxanthine. (+S9: F12 medium with 50 units/ml penicillin, 50 µg/ml streptomycin and 5% (v/v) dyalized bovine serum; -S9: without serum)
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver homogenate prepared from Arochlor 1254-induced Sprague-Dawley male rats (Microbi ological Associates, Bethesda, MD)

Test concentrations with justification for top dose:

With and without metabolic activation: 0.01, 0.02, 0.04, 0.06 and 0.08 mg/mL.
The selection of a suitable range of concentrations for testing was based upon a preliminary cytotoxicity study.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

cell culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium.
Duplicate cultures of CHO were incubated 36-48 h before treatment into 75-cm2 culture flasks. A range of test compound concentration was added and cultured for 6 h and 10 h before sampling with and without metabolic activation.

- Cell density at seeding (if applicable): 3-5 x 10^5 cells/75-cm2 culture flask.

DURATION
- Exposure duration: 6 h and 10 h

SPINDLE INHIBITOR (cytogenetic assays): Colchicine was added to culture flasks 2 h prior to cell harvesting.

STAIN (for cytogenetic assays): Dilute Geimsa solution (1:25)

NUMBER OF REPLICATIONS: 2 cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After treatment with colchicine, cells were removed from flasks by brief incubation with 0.01% trypsin and gentle agitation. They were centrifuged and suspended in 0.075 M KCl and incubated for 20-25 min at 37ºC. Cells were then centrifuged, fixed in 3 or 4 changes of Carnoy’s fixative and chromosome spreads prepared. Chromosomes were stained for 10 min in dilute Geimsa solution (1:25) and rinsed with water.

NUMBER OF CELLS EVALUATED: minimum of 50 cells/culture for each sampling time and each dosage.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was determined by inoculating 3-5 x 10^5 cells into 75-cm2 culture flasks and treating for 6-8 h with test compound in dosage ranges of 0.006-0.06 mg/mL (test 1) and 0.07-0.10 mg/mL (test 2) in the absence and presence of metabolic activation. Both growth inhibition and inhibition of mitosis (mitotic index) were performed for cytotoxicity.
- Growth inhibition: It was expressed as the relative number of surviving cells in untreated (DMSO control) compared to test item-treated cells.
- Inhibition of mitosis (mitotic index): after harvesting of cells for chromosome preparations, the mitotic inhibition was determined as the proportion of cells in metaphase.

Evaluation criteria:

The test compound was considered positive if:
a) at least one of the test concentrations exhibits a statistically significant increase in chromosome aberrations compared with the concurrent negative control,
b) the increase is dose-related,
c) any of the results are outside the range of the historical negative control

Statistics:

One-tailed Fisher Exact probability test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Other results: When present, chromatid breaks and chromosome fragment were the predominant aberration.

RANGE-FINDING/SCREENING STUDIES: A concentration of 0.08 mg/ml produced a 22.8% reduction of culture growth of CHO cells tested without S9 activation relative to control values and a 30.6% reduction of growth with metabolic activation. Excessive mitotic inhibition was produced without S9 activation above 0.08 mg/mL and with S9 activation 0.06 mg/mL produced a 70% reduction in metaphase mitoses. A concentration range of 0.01-0.07 mg/ml test compound was used without metabolic activation and 0.01-0.08 mg/ml with metabolic activation.

HISTORICAL DATA:
The concurrent negative and positive controls were within the range for historical controls.

Table 1: Summary of incidence of aberrant CHO cells in the in vitro clastogenicity study with ENB

		+S9		-S9
Material	Dosage	Cells scored	Percent of aberrant cells (mean - SD)	Cells scored	Percent of aberrant cells (mean - SD)
6 h sampling time
DMSO		100	4.0 – 5.66	100	4.0 – 2.83
ENB	0.01 mg/mL	100	1.0 – 1.-41	NE	-
ENB	0.02 mg/mL	100	5.0 – 1.41	NE	-
ENB	0.04 mg/mL	100	3.0 – 1.41	100	6.0 – 0.0
ENB	0.06 mg/mL	NE	-	100	5.0 – 4.24
ENB	0.08 mg/mL	NE	-	100	1.0 – 1.41
10 h sampling time
DMSO		100	2.0 – 0.0	100	4.0 – 2.83
ENB	0.01 mg/mL	100	2.0 – 0.0	100	5.0 – 1.41
ENB	0.02 mg/mL	100	4.0 – 0.0	100	4.0 – 0.0
ENB	0.04 mg/mL	100	3.0 – 4.24	100	4.0 – 0.0
Medium	-	50	4.0 – 0.0	50	2.0 – 0.0
TEM	1.5 µg/mL	50	36.0 – 0.3c	NE	-
CP	12.0 µg/mL	NE	-	50	26.0 – 0.0c

TEM, triethylenemelamine; CP, cyclophosphamide; NE, not evaluated

c Significant at p < 0.001 (compared to solvent control)

Conclusions:

In an in vitro chromosome aberration study, the tested material ENB was not clastogenic when tested in CHO cells in vitro with or without metabolic activation.

Executive summary:

5-ethylidene-2-norbornene (ENB) was tested in an in vitro chromosome aberration study with cultured Chinese hamster ovary CHO cells in presence and absence of metabolic activation using a method comparable to OECD guideline 473. A preliminary cytotoxicity study consisting on growth inhibition and inhibition of mitosis (mitotic index) was conducted. Based on these results, the tested concentrations were 0.01, 0.02, 0.04, 0.06 and 0.08 mg/mL both with and without metabolic activation. Duplicate cultures were incubated 36-48 h before treatment into 75-cm2 culture flasks. The test compound was added and cultured for 6 h and 10 h before sampling. There were no statistically significant or dosage-related increases in chromosome aberrations compared with the concurrent control (DMSO), with and without metabolic activation at both sampling times. When present, chromatid breaks and chromosome fragment were the predominant aberration. Both positive controls, triethylenemelamine without metabolic activation and cyclophosphamide with metabolic activation, produced highly significant increases in the numbers of aberrant cells compared to control. The concurrent negative and positive controls were within the range for historical controls. Based on these results, it can be concluded that ENB is not a clastogen.

Reference 18

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HGPRT (hypoxanthine-guanine-phosphoribosyl transferase) gene

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CHO cells were obtained from the Oak Ridge National Laboratory with a designation CHO-K1-BH4 (subclone D1)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham`s modified F12 medium supplemented with 10% (v/v) heated inactivated fetal bovine serum, lacking in hipoxanthine. (+S9: F12 medium with 50 units/ml penicillin, 50 μg/ml streptomycin and 5% (v/v) dyalized bovine serum; -S9: without serum)
For determination of mutant frecuencies, F-12-D5 medium containing 2.0 mg/ml 6-TG was used as the selective medium.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9 liver homogenate prepared from Arochlor 1254-induced Sprague-Dawley male rats (Microbiological Associates, Bethesda, MD)

Test concentrations with justification for top dose:

Without metabolic activation: 0.02, 0.04, 0.05, 0.06, 0.07 and 0.08 µL/mL.
With metabolic activation: Test 1: 0.02, 0.04, 0.05, 0.06, 0.07 and 0.10 µL/mL; test 2: 0.06, 0.07, 0.08 and 0.09 µL/mL
The selection of a suitable range of concentrations for testing was based upon a preliminary cytotoxicity study.

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(Cell culture medium)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (20 µL/mL)

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium.
For the test without metabolic activation, 20-24 h before treatment 5x10^5 CHO cells were inoculated into 25-cm2 culture flasks containing F12-D5 medium and incubated at 37 ºC in a 5-6% CO2 atmosphere. Appropriate amounts of ENB or control materials (DMSO solvent, cell culture medium, or positive controls) were added, and the cultures were exposed for 5 h. For testing in the presence of metabolic activation, the procedure used F12 medium without serum, but containing 1.0 mL S9 activation mix per 4 mL of medium.

- Cell density at seeding (if applicable): 5 x 10^5 cells/25-cm2 culture flask.

DURATION
- Exposure duration: 5 h
- Selection time (if incubation with a selection agent): The mutant fraction was determined in duplicate cultures for each treatment group after a 9- and 12-day subculturing period. At 2- and 3-day intervals post-treatment ca. 3-5 x 10^5 cells were subcultured and incubated for 7 days before dissociation and seeding to plates in medium containing 6-thioguanine (6-TG) or without 6-TG to assess plating efficiency of the treated cell population.All cultures were incubated for an additional 6-8 days to allow cell growth.

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)


NUMBER OF REPLICATIONS: 2 cultures

NUMBER OF CELLS EVALUATED: The number of mutants per 10^6 total cells and per 10^6 viable cells were calculated

DETERMINATION OF CYTOTOXICITY
- Groth inhibition test: a preliminary test was conducted in order to select the highest dosage that produced a maximum of 80-90% cell death. It was expressed as the relative number of surviving cells in untreated (DMSO control) compared to test item-treated cells.
- Cytotoxicity, as relative survival of ENB-treated cells compared with DMSO controls, was determined 1 d after exposure ("surviving fraction"). This colony-forming potential of treated cells was used as a measure of treatment-induced cytotoxicity, using 4 plates/culture and 100 cells/plate.

Statistics:

Analysis of mutation frequencies followed the procedure for Irr and Snee (1979), employing Box-Cox transformation (Box & Cox, 1964) before parametric analysis using Student’s t-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(From 0.06 µL/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 0.1 µL/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(From 0.08 µL/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the mutagenicity test, ENB produced a dosage-related cytotoxicity to CHO cells with and without metabolic activation. The findings indicated a steep slope in the dose-response relationship between 0.06 and 0.07 µl/mL without metabolic activation, and 0.07-0.10 µl/mL with metabolic activation. In the study without metabolic activation, ENB did not produce any statistically significant or dosage-related increase in the number of mutants/10^5 clonable cells. In the test with metabolic activation, increases in the incidence of mutants were seen with only one of the duplicate cultures at each concentration. However, these increases were not statistically significant. A repeat test was therefore conducted to confirm the absence of a mutagenic effect with 0.06-0.09 µl/mL dosage range with metabolic activation. The 0.08 and 0.09 µl/mL doses were completely cytotoxic but no mutagenic effects were observed in duplicate cultures with ENB concentrations of 0.06 and 0.07 µl/mL.

Remarks on result:

other: Test 1

Conclusions:

Under test conditions, ENB did not produce any statistically significant or dosage-related increase in the number of mutants cells with and without metabolic activation.

Executive summary:

To determine the potential for 5-Ethylidene-2-norbornene (ENB) to cause forward gene mutations, a CHO cell line was used for the detection of mutations at the HGPRT (hypoxanthine-guanine-phosphoribosyl transferase) gene locus in a medium containing the purine analog 6-thioguanine (6-TG). For the test without metabolic activation, 20-24 h before treatment 5E5 CHO cells were inoculated into 25-cm2 culture flasks containing F12-D5 medium and incubated at 37 ºC in a 5-6% CO2 atmosphere. Appropriate amounts of ENB or control materials (DMSO solvent, cell culture medium, or positive controls) were added, and the cultures were exposed for 5 h. For testing in the presence of metabolic activation, the procedure used F12 medium without serum, but containing 1.0 mL S9 activation mix per 4 mL of medium. The tested concentrations were: without metabolic activation: 0.02, 0.04, 0.05, 0.06, 0.07 and 0.08 µL/mL; and with metabolic activation: 0.02, 0.04, 0.05, 0.06, 0.07 and 0.10 µL/mL (test 1) and 0.06, 0.07, 0.08 and 0.09 µL/mL (test 2). Positive controls were dimethylnitrosamine (DMN) with metabolic activation, and ethylmethanesulfonate (EMS) without metabolic activation. The mutant fraction was determined in duplicate cultures for each treatment group. A dosage-related cytotoxicity to CHO cells with and without metabolic activation was found. In the study without metabolic activation, ENB did not produce any statistically significant or dosage-related increase in the number of mutants/1E5 clonable cells. In the test 1 with metabolic activation, increases in the incidence of mutants were seen with only one of the duplicate cultures at each concentration. However, these increases were not statistically significant. Test 2 was therefore conducted to confirm the absence of a mutagenic effect. The 0.08 and 0.09 µl/mL doses were completely cytotoxic but no mutagenic effects were observed in duplicate cultures with ENB concentrations of 0.06 and 0.07 µl/mL.

Reference 19

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD Guideline 476 with deviations: no data on test material purity, source and concentration units; no data on negative/positive controls; evaluation criteria not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

no data on test material purity, source and concentration units; no data on negative/positive controls; evaluation criteria not reported

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase, TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer's medium containing 10% horse serum, antibiotics, glutamine, sodium pyruvate and Pluronic F68

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of induced rat liver supplemented with cofactors (CORE)

Test concentrations with justification for top dose:

Up to 100 µg/mL

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

no data

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: strain/cell type: -3.7.2C heterozygote

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in L5178Y cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro mammalian cell gene mutation test performed similarly to OECD Guideline 476, mouse lymphoma L5178Y TK+/- (-3.7.2C heterozygote) cells were exposed to d-limonene up to 100 µg or nL/mL in both the absence and presence of metabolic activation (S9 fraction of induced rat liver supplemented with cofactors). D-Limonene showed no substantial increases in mutant frequency up to the highest concentration tested in either presence or absence of S9 mix. Under the test conditions, d-limonene is not considered as mutagenic in L5178Y cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 20

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD Guideline 476 with minor deviations: no data on maintenance of cell cultures and absence of mycoplasma

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

no data on maintenance of cell cultures and absence of mycoplasma

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase, TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Source of cells: National Toxicology Program's (NTP) chemical repository (Radian Corporation, Austin, USA)
- Type and identity of media: Fischer’s medium used for expression and cloning; horse serum used at 20% v/v for soft agar cloning

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of male Fischer 344 rat liver induced with Aroclor-1254

Test concentrations with justification for top dose:

Without S9:
- Trial 1: 0, 10, 20, 30, 40, 50 and 60 nL/mL
- Trial 2: 0, 30, 40, 50, 60, 80 and 100 nL/mL
- Trial 3: 0, 5, 10, 20, 30, 40 and 50 nL/mL
- Trial 4: 0, 5, 10, 20, 30, 40, 50 and 60 nL/mL

With S9:
- Trail 1: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL
- Trail 2: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL
- Trail 3: 0, 30, 40, 50, 60, 80 and 100 nL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation(5 nL/mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation (2.5 µg/mL )

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 hours at 37 °C
- Expression time (cells in growth medium): 48 hours at 37 °C
- Selection time (if incubation with a selection agent): 9-12 days at 37 °C

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicate (at least)

NUMBER OF CELLS EVALUATED: 6 x 10^6 cells exposed to the test item, 3 x 10^6 cells to select mutant cells; 600 cells to determine cloning efficiency

DETERMINATION OF CYTOTOXICITY
- Method: Relative growth on Days 1 and 2, cloning efficiency and relative total growth

OTHER EXAMINATIONS:
- Colony sizing: Number of small and large mutant colonies were determined by recording TFT colony counts for increments of 0.2 on the colony size discriminator.

OTHER: Colonies were counted on an Artek 880 colony counter fitted with a 10-turn size discriminator.

Evaluation criteria:

Positive (+):
- Significant response for at least one of the three highest dose sets and a significant trend (P ≤ 0.05)

Questionable (?):
- Significant response for one of the three highest dose sets but no significant trend
- Significant trend but no significant dose set

Inconclusive (i):
- Significant response for a dose set other than one of the three highest but no significant trend
- No significant responses or trend, but the relative total growth is greater than 30% and higher toxicity can be attained

No response (=):
- No significant responses or trend, and the relative total growth is greater than 30% under conditions where a 1.5-fold increase in dose cause precipitation or where the 5 mg/mL (or 5 µL/mL) concentration limit is attained.

Negative (-):
- No significant responses or trend, and either the relative total growth is less than 30% or excessive toxicity occurs for a 1.5-fold higher dose.

Statistics:

Consistency among the mutant frequencies was analysed using chi-square test; acceptable cultures must be significant at P ≤ 0.05

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at or above 50 and 60 nL/mL (with and without S9, respectively)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Response in trials without S9:
- Trial 1: Inconclusive (i)
- Trial 2: Questionable (?)
- Trial 3 and 4: Negative (-)
- Overall response: Negative (-)

Response in trials with S9:
- Trial 1 and 2: Negative (-)
- Trial 3: Inconclusive (i)
- Overall response: Negative (-)


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Without S9: Cytotoxicity was observed in one or more replicates tested at concentration of 50 nL/mL or above
- With S9: Cytotoxicity was observed in one or more replicates tested at concentration of 60 nL/mL or above

Table 1: Chemical-induced changes in the large and small classes of mutant colonies

 

Chemical treatment	Trial	Mutant colony count and CE						Mutant frequency				Mutant frequency change
		Treatment			Solvent control			Treatment		Solvent control		Difference
		L	S	CE	L	S	CE	L	S	L	S	L	S
50 nL/mL	WO 2	48	119	98	44	53	85	16	40	17	21	-1	19
50 nL/mL	S9 2	98	143	79	72	109	115	41	60	21	32	20	28

Conclusions:

Under the test conditions, d-limonene was not considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro mammalian cell gene mutation test performed similarly to OECD guideline 476, mouse lymphoma L5178Y TK+/- cells were exposed to d-limonene in 1% ethanol in Fischer’s medium with and without metabolic activation (S9 fraction of male Fischer 344 rat liver induced with Aroclor-1254) at the following concentrations:

Without S9:

- Trial 1: 0, 10, 20, 30, 40, 50 and 60 nL/mL

- Trial 2: 0, 30, 40, 50, 60, 80 and 100 nL/mL

- Trial 3: 0, 5, 10, 20, 30, 40 and 50 nL/mL

- Trial 4: 0, 5, 10, 20, 30, 40, 50 and 60 nL/mL

With S9:

- Trial 1: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL

- Trial 2: 0, 10, 20, 30, 40, 50, 60 and 80 nL/mL

- Trial 3: 0, 30, 40, 50, 60, 80 and 100 nL/mL

 

Positive controls (methyl methanesulphonate at 5 nL/mL without S9 and 3-methylcholanthrene at 2.5 µg/mL with S9) induced the appropriate response. In experiment without S9, mutagenic responses in trials 1, 2, 3 and 4 were inconclusive, questionable, negative and negative, respectively. In experiment with S9, mutagenic responses in trials 1, 2 and 3 were negative, negative and inconclusive, respectively. Overall, d-limonene was not considered as mutagenic in either presence or absence of S9 mix. Cytotoxicity was observed in one or more replicates tested at or above 50 nL/mL.

 

Under the test conditions, d-limonene was not considered as mutagenic in mouse lymphoma L5178Y cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 21

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD guideline 479 with minor deviations: no data on number of replicates; no data on karyotype stability

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

no data on number of replicates; no data on karyotype stability

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Source: Litton Bionetics Inc.
- Type and identity of media: McCoys 5A medium supplemented with antibiotics and 10% fetal calf serum
- Properly maintained: Yes; cells for experiments were thawed and grown in the medium at 37 °C using 5% CO2
- Periodically checked for Mycoplasma contamination: Yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)

Test concentrations with justification for top dose:

Without S9:
- Trial 1: 0, 16.2, 54 or 162 µg/mL
- Trial 2: 0, 30, 50 or 100 µg/mL
- Trial 3: 0, 15, 30 or 50 µg/mL
With S9:
- Trial 1: 0, 16.2, 54 or 162 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation (0.0015 or 0.01 µg/mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (0.4 or 2.5 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 2 hours at 37 °C
- Expression time (cells in growth medium): 24 hours in presence of bromodeoxyuridine (BrdUrd): 10^-5 M
- Fixation time (start of exposure up to fixation or harvest of cells): 25-29 hours (standard harvest) or > 29 hours (delayed harvest)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.1 or 0.4 µg/mL for 2-2.5 h

NUMBER OF CELLS EVALUATED: 50 cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: Visual estimate of the confluency of each flask at the end of the treatment

Evaluation criteria:

- If a trial had a positive trend and no significant doses, or if there was no trend and only one significant dose, the trial was judged equivocal;
- If a trial had significant trend and one significant dose it was judged weak positive;
- If the trial had two significant doses it was judged positive, whether or not a positive trend was obtained.

Statistics:

- Data were evaluated for both trend and dose point increase over the solvent control.
- A trend of P < 0.005 or an individual dose with a 20% increase over the solvent control was considered significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

cytotoxic above 162 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic above 162 µg/mL

Table 1: Results obtained in trial 1 (without S9)

 

Dose (µg/mL)	Total chromosomes	Total SCE	SCE per cell
0	1050	389	7.78
16.2	1050	447	6.94
54	1051	463	9.26
162	1051	457	9.14
Positive control - MMC
0.0015	1048	701	14.02
0.0100	211	341	34.10
Trend statistic: 0.23E+01
Trend probability: 0.96E-02

 

Table 2: Results obtained in trial 2 (without S9)

 

Dose (µg/mL)	Total chromosomes	Total SCE	SCE per cell	Harvest time
0.0000	1049	366	7.32	26.50
30.0000	1049	407	8.14	26.50
50.0000	1048	405	8.10	30.50
100.0000	1041	475	9.50*	30.50
Positive control - MMC
0.0015	1046	476	9.52	26.50
0.0100	210	252	25.20	26.50
Trend statistic: 0.38E+01
Trend probability: 0.81E-04

* significant (20%) increase of SCE per chromosome over the control

 

Table 3: Results obtained in trial 3 (without S9)

 

Dose (µg/mL)	Total chromosomes	Total SCE	SCE per cell	Harvest time
0.0000	1048	345	6.90	26.50
15.0000	1049	343	6.86	26.50
30.0000	1048	349	6.98	26.50
50.0000	1046	406	8.12	30.50
Positive control - MMC
0.0015	1051	516	10.32	26.50
0.0100	209	230	23.00	26.50
Trend statistic: 0.22E+01
Trend probability: 0.15E-01

 

Table 4: Results obtained in trial 1 (with S9)

 

Dose (µg/mL)	Total chromosomes	Total SCE	SCE per cell
0.0000	1047	398	7.96
16.2000	1048	404	8.08
54.0000	1049	399	7.98
162.0000	1045	394	7.88
Positive control - CPA
0.4000	1046	620	12.40
2.5000	210	405	40.50
Trend statistic: -0.17E+00
Trend probability: 0.57E+00

Conclusions:

Under the test conditions, d-limonene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro sister chromatid exchange assay performed similarly to OECD guideline 479, Chinese hamster Ovary (CHO) cells were exposed to d-limonene in McCoys 5A medium with and without metabolic activation [S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (20 µL/mL)] at the following concentrations: Without S9: trial 1: 0, 16.2, 54 and 162 µg/mL; trial 2: 0, 30, 50 and 100 µg/mL and trial 3: 0, 15, 30 and 50 µg/mL. With S9: trial 1: 0, 16.2, 54 and 162 µg/mL. Clear increases in mean SCE/cell were induced by the positive control chemicals mitomycin C (without S-9) and cyclophosphamide (with S-9). In trial 2 (without S9), a significant linear trend and a significant increase in SCE/cell were observed at a concentration of 100 µg/mL. However, no significant increases and no significant linear trends were observed in trial 1 (with and without S9) and trial 3 (without S9). Under the test conditions, d-limonene is not considered as cytogenetic in CHO cells according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 22

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

test conducted only without metabolic activation

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

(CHO K-1)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, cloned in testing laboratory

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ham's F12 medium and humidified atmosphere with 5% CO2 at 37ºC.
The medium was supplemented with 10% fetal bovine serum, 50 IU/ml penicillin G, 50 µg/ml streptomycin sulfate and 2.5 µg/ml fungizon. Medium and all antibiotics were obtained from Flow Laboratories, Inc. (U.S.A.).

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 10, 33.3, 100, 333, 1000 µM

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium. Treatment was done with cells in the log-phase. The cells were exposed to Mitomycin C (MMC) for 21 h, and washed twice with Hanks' balanced salt solution. Treated cells were cultured in the presence or absence of tested material for 1 cell cycle.
- Cell density at seeding (if applicable): CHO K-1 cells were seeded at a density of 0.5-1.0 x 10^6 cells/100-mm dish.

DURATION
- Exposure duration: 1 cell cycle (21 h)

SPINDLE INHIBITOR (cytogenetic assays): The cells were treated with colchicine for 2 h at a final concentration of 50 µg/ml.

STAIN (for cytogenetic assays): modified Giemsa procedure (Sakanishi and Takayama, 1977)

NUMBER OF REPLICATIONS: 3 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: BrdUrd (final concentration 5 µM) was added 2 cell cycles before fixation. After addition of BrdUrd, the cultures were incubated in total darkness and all operations were performed under a red safe light. Preparations were processed using a modified Giemsa procedure (Sakanishi and Takayama, 1977) and harlequin-stained chromosomes in 50 metaphases per culture were analyzed for SCEs.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 50 metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; mitotic indices were determined and given as numbers of mitotic cells per 1000 cells. In addition, the numbers of cells which completed 2 cell cycles (M2) or less than 2 cycles (MI) were determined.

Statistics:

The SCE data were statistically analyzed using Student's t-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µM with and without MMC treatment

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Table 1. Effect of components of plant essence on MMC-induced SCEs

Component	MMC treatment (0.15 µM)	Mean frequency of SCEs
		Concentration (µM) of components
		0	10	33.3	100	333	1000
DL-camphene	+	81.7	80.9	80.5	81.9	82.0	T
	-	9.0	9.0	8.8	9.2	9.3	T

Cells treated with 0.15 µM MMC for 21 h were post-treated with dl-camphene at the indicated doses for 21 h.

Each value represents the mean of 3 independent experiments.

T, toxic; -, not tested.

Conclusions:

Camphene did not induce sister chromatid exchanges in CHO cells tested without metabolic activation.

Executive summary:

Camphene was tested on sister chromatid exchange assay in cultured Chinese hamster ovary CHO-K1 cells without metabolic activation using an method comparable to OECD guideline 479. 3 independent experiments were conducted with and without initial induction during 1 cell cycle (21 h) of Mitomycin C (MMC) and post treatment during 1 cell cycle (21 h) of tested material at concentrations of 0, 10, 33.3, 100, 333, 1000 µM. The tested material was found toxic at the high dose of 1000 μM. Under these test conditions, camphene did not induce sister chromatid exchanges in CHO cells at any of the doses tested.

Reference 23

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

test conducted only without metabolic activation

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

(CHO K-1)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, cloned in testing laboratory

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ham's F12 medium and humidified atmosphere with 5% CO2 at 37ºC.
The medium was supplemented with 10% fetal bovine serum, 50 IU/ml penicillin G, 50 µg/ml streptomycin sulfate and 2.5 µg/ml fungizon. Medium and all antibiotics were obtained from Flow Laboratories, Inc. (U.S.A.).

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 10, 33.3, 100, 333, 1000 µM

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium. Treatment was done with cells in the log-phase. The cells were exposed to Mitomycin C (MMC) for 21 h, and washed twice with Hanks' balanced salt solution. Treated cells were cultured in the presence or absence of tested material for 1 cell cycle.
- Cell density at seeding (if applicable): CHO K-1 cells were seeded at a density of 0.5-1.0 x 10^6 cells/100-mm dish.

DURATION
- Exposure duration: 1 cell cycle (21 h)

SPINDLE INHIBITOR (cytogenetic assays): The cells were treated with colchicine for 2 h at a final concentration of 50 µg/ml.

STAIN (for cytogenetic assays): modified Giemsa procedure (Sakanishi and Takayama, 1977)

NUMBER OF REPLICATIONS: 3 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: BrdUrd (final concentration 5 µM) was added 2 cell cycles before fixation. After addition of BrdUrd, the cultures were incubated in total darkness and all operations were performed under a red safe light. Preparations were processed using a modified Giemsa procedure (Sakanishi and Takayama, 1977) and harlequin-stained chromosomes in 50 metaphases per culture were analyzed for SCEs.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 50 metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; mitotic indices were determined and given as numbers of mitotic cells per 1000 cells. In addition, the numbers of cells which completed 2 cell cycles (M2) or less than 2 cycles (MI) were determined.

Statistics:

The SCE data were statistically analyzed using Student's t-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µM with and without MMC treatment

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Table 1. Effect of components of plant essence on MMC-induced SCEs

Component	MMC treatment (0.15 µM)	Mean frequency of SCEs
		Concentration (µM) of components
		0	10	33.3	100	333	1000
d-(+)-limonene	+	81.7	81.1	83.8	83.7	T
	-	9.0	8.9	9.1	9.0	T

Cells treated with 0.15 µM MMC for 21 h were post-treated with test item at the indicated doses for 21 h.

Each value represents the mean of 3 independent experiments.

T, toxic; -, not tested.

Conclusions:

d-limonene did not induce sister chromatid exchanges in CHO cells tested without metabolic activation.

Executive summary:

D-limonene was tested on sister chromatid exchange assay in cultured Chinese hamster ovary CHO-K1 cells without metabolic activation using an method comparable to OECD guideline 479. 3 independent experiments were conducted with and without initial induction during 1 cell cycle (21 h) of Mitomycin C (MMC) and post treatment during 1 cell cycle (21 h) of tested material at concentrations of 0, 10, 33.3, 100, 333, 1000 µM. The tested material was found toxic at the dose of 333 μM. Under these test conditions, d-limonene did not induce sister chromatid exchanges in CHO cells at any of the doses tested.

Reference 24

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Data published in a peer reviewed journal. Original study report not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

no data on replications

GLP compliance:

not specified

Type of assay:

other: unscheduled DNA synthesis (UDS)

Target gene:

Not applicable

Species / strain / cell type:

hepatocytes: Rat/Fischer and Sprague Dawley adult male

Metabolic activation:

without

Test concentrations with justification for top dose:

0.001, 0.003, 0.01, 0.03, 0.1, 10 μl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; Livers were perfused in situ with 0.5 mM EDTA in HEPES buffer (pH 7.2) for four minutes. Cultures of rat liver hepatocytes were incubated with the test material for 18-20 hours.

DURATION
- Exposure duration: 18-20 h

NUMBER OF CELLS EVALUATED: 75-150 cells were analyzed for each dose level.

Evaluation criteria:

UDS was measured by electronically counting nuclear grains and subtracting the average number of grains in 3 adjacent nuclear sized cytoplasmic areas.
The test was considered positive if an increase in net nuclear grain count of at least six grains per nucleus above the solvent control and/or an increase in the percent of nuclei with at least 6 net grains to more than 10% above the negative control value.

Key result

Species / strain:

hepatocytes: Rat/Fischer and Sprague Dawley adult male

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

the positive induced significant increases in the mean number of net nuclear grain counts compared to the solvent control.

Alpha pinene did not cause a significant increase in UDS as measured by the mean number of net nuclear grain counts at any dose level.

Conclusions:

Alpha pinene was not mutagenic based on the results of the rat hepatocyte unscheduled DNA synthesis assay.

Executive summary:

Alpha pinene was tested on the rat hepatocyte unscheduled DNA synthesis assay following the OECD Guideline 482. Cultures of rat liver hepatocytes were incubated with the test material for 18-20 hours at concentrations of 0.001, 0.003, 0.01, 0.03, 0.1, 10 μl/ml without metabolic activation. The tested material did not cause a significant increase in the mean number of net nuclear grain counts compared to the control at any dose level. Thus, alpha pinene is considered to be negative in the unscheduled DNA synthesis assay.

Reference 25

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study performed similarly to OECD Guideline 471 with deviations: one strain missing; no data on number of bacterial cells per culture; individual plate counts not reported

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

one strain missing; no data on number of bacterial cells per culture; individual plate counts not reported

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced liver S-9 fractions obtained from male Sprague-Dawley rats and male Syrian hamsters, injected, i.p.

Test concentrations with justification for top dose:

The maximum concentration dosed was 10mg/plate unless toxicity was observed at this dose level.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [acetone; dimethyl sulphoxide; ethanol(95%); water (distilled)]
- Justification for choice of solvent/vehicle: The solvent of choice was distilled water, DMSO was used if the chemical was insoluble. Ethanol or acetone was used if the substance was not soluble or stable in DMSO. The final choice of solvent for this substance was not reported.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (1 µg/plate with TA 98 and TA 100; 2.5 µg/plate with TA 1535 and TA 1537)

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylenediamine (5 µg/plate with TA 98), sodium azide (1 µg/plate with TA 100 and TA 1535), 9-aminoacridine (50 µg/plate with TA 1537)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): no data

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

- OTHER:
Dose-range finding experiment: Test substance was checked for toxicity to TA 100 up to a concentration of 10 mg/plate or the Iimit of solubility, both in the presence and absence of S-9 mix.

Evaluation criteria:

A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not.

Statistics:

The data were evaluated using analysis based on the models presented by Margolin et al (1981).

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Conclusions:

Under the test conditions, cineole is not considered as mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to OECD guideline 471, strains of S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) were exposed to cineole with and without S9 metabolic activation [S9 fraction of Aroclor 1254-induced adult male Sprague Dawley rats and Syrian hamsters liver] according to the preincubation method (20 min). The positive controls induced the appropriate responses in the corresponding strains. Cineole showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains in either presence or absence of S9 mix. Under the test conditions, cineole is not considered as mutagenic in this bacterial system according to the criteria of the CLP Regulation (EC) N° (1272-2008). 

Reference 26

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

only 2-h exposure with test substance with S9 was performed and only 100 cells per concentration were scored.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Cell line: CHO-W-B1

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5a medium with 10 % foetal calf serum, L-glutamine and antibiotics.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix consisting of 15 µL/mL liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/mL NADP, and 4.5 mg/mL isocitric acid in serum-free medium.

Test concentrations with justification for top dose:

- Without S9: 479 µg/mL - 663 µg/mL.
- With S9: 630 µg/mL - 810 µg/mL.

- Doses were chosen for the aberration test based on a preliminary test of cell survival 24 hrs after treatment. For most tests, doses were based on observations of cell confluence and mitotic cell availability in the SCE test (reported elsewhere).

Vehicle / solvent:

- Solvents used: either water, dimethyl sulfoxide (DMSO), ethanol or acetone, in that order of preference. It is unclear which solvent was used for the test material.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Remarks:

Solvents used: either water, dimethyl sulfoxide (DMSO), ethanol or acetone, in that order of preference. It is unclear which solvent was used for the test material.

Untreated negative controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: with S9 mix: 2 hr; without S mix: throughout incubation period
- Fixation time (start of exposure up to fixation or harvest of cells): 8 to 12 hr standard (cells in first mitosis). In cases where experience suggested that mitosis was delayed by the presence of the test material, harvesting was delayed to "e.g. 18-26 hr".

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive and solvent controls.

OTHER EXAMINATIONS:
- Determination of polyploidy: Aberrations from polyploid cells not scored, but metaphases with 19-23 chromosomes were used.

- OTHER:
- All types of aberrations were recorded separately, but for data analysis they were grouped into categories of "simple" (breaks and terminal deletions), "complex" (exchanges and rearrangements), “other” (including pulverised chromosomes), and "total".

Evaluation criteria:

The analyses examined the evidence for a dose relation and absolute increase over the solvent control at each dose.

Statistics:

Linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, Margolin et al. (1983). The P values were adjusted by Dunnett's method to take into account the multiple dose comparisons. For data analysis, the "total" aberration category was used and the criterion for a positive response was that the adjusted P value be ≤ 0.05.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic above 100 (-S9) and 500 (+S9) µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation observed at concentrations ≥ 630 µg/mL.

Metabolic activation	Result	Range (µg/mL)	Least effect concentration (µg/mL)
Without S9 mix	-	479 - 663	-
With S9 mix	-	630 - 810	-

The least effective concentration tested (LEC) is the lowest dose to give a statistically significant increase (P ≤ 0.05) in aberrations. For chemicals with which the lowest dose tested gave a positive response, the LEC is preceded by "<". “-”, no LEC observed.

Conclusions:

The test substance was negative for chromosome aberrations with and without metabolic activation.

Executive summary:

In an in vitro mammalian chromosome aberration test performed similarly to OECD guideline 473, Chinese hamster Ovary (CHO) cells were exposed to 1,8 -cineole in McCoys 5a medium with and without metabolic activation [S9 fraction of livers from Aroclor 1254-induced male Sprague-Dawley rats (15 µL/mL)] at the following range concentrations: without S9: 479 - 663 µg/mL, and with S9: 630 - 810 µg/mL. Positive controls (mitomycin C without S9 and cyclophosphamide with S9) induced the appropriate response. Chromosome aberrations were not induced in treatment groups over background at any tested concentrations in the presence or absence of activation system.

Reference 27

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

No data

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Cell line: CHO-W-B1

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy's 5a medium with 10% foetal calf serum, L-glutamine and antibiotics.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix consisting of 15 μL/mL liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/mL NADP, and 4.5 mg/mL isocitric acid in serum-free medium.

Test concentrations with justification for top dose:

- Without S9: 50 μg/mL - 500 μg/mL.
- With S9: 600 μg/mL - 800 μg/mL.

Doses selected on one of two bases (unclear on which basis the concentrations of test material were determined):
-- Preliminary growth inhibition test, counting cells excluding trypan blue 24 hr after treatment. Top dose was that estimated to reduce growth by 50 %.
-- Observation of cell monolayer and confluence activity in the cultures used for analysis of SCEs. Aim to obtain results at the highest dose at which sufficient metaphase cells would be available for analysis.

Vehicle / solvent:

- Solvents used: either water, dimethyl sulfoxide (DMSO), ethanol or acetone, in that order of preference. It is unclear which solvent was used for the test material.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Remarks:

Solvents used: either water, dimethyl sulfoxide (DMSO), ethanol or acetone, in that order of pre ference. It is unclear which solvent was used for the test material.

Untreated negative controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: without metabolic activation: approx. 25 hr; with metabolic activation: approx. 2 hr. 5-Bromodeoxyuridine (BrdUrd; 10 pM) was added 2 hr after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed.
- Fixation time (start of exposure up to fixation or harvest of cells): 28 hrs. Immediately before harvesting, the cell monolayers were examined and the degree of confluence and availability of mitotic cells were noted.

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.1 µg/ml) present during the final 2-3 hr of total incubation time with 5-Bromodeoxyuridine (BrdUrd).

STAIN (for cytogenetic assays): 10 min in concentrated Hoechst 33258 (5 µg/mL in pH 6.8 buffer) and exposure to “black light” at 55 to 60 °C for approx. 5 min, then slides were stained with Giemsa.

NUMBER OF REPLICATIONS: 1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with “dilute” Hoeschst 33258 (0.5 pg/ml in Sorensen’s buffer, pH 6.8) and examined by fluorescence microscopy to assess cell cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25-26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis. Later harvests were not necessary for this test substance.

NUMBER OF CELLS EVALUATED: 50 cells per dose scored from the 3 highest doses at which sufficient M2 cells available.

Statistics:

A linear regression test (trend test) of SCEs per chromosome vs the log of the dose was used. For individual doses, absolute increases in SCEs per chromosome of 20% or more over the solvent control were considered significant.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation observed at concentrations ≥ 500 µg/mL.

The SCE test was positive only without activation at doses that induced cell cycle delay. No aberration induction was detected even after extending the incubation time without S9 to 20 hours.

Metabolic activation	Result	Range (µg/mL)	Least effective concentration (LEC) (µg/mL)
Without S9 mix	+	50 - 500	500 / 200
With S9 mix	-	600 - 800	-

The least effective concentration tested (LEC) is the lowest dose to give a 20% increase in SCEs.

Conclusions:

The test material was positive without metabolic activation but negative with metabolic activation in a sister chromatid exchange test. The SCE test was positive only without activation at doses that induced cell cycle delay. No aberration induction was detected even after extending the incubation time without S9 to 20 hours.

Executive summary:

Cloned Chinese hamster ovary cells were cultured in McCoy's 5a medium with 0 % foetal calf serum, L-glutamine and antibiotics. 5-Bromodeoxyuridine was added 2 hours after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment period was approximately 25 hours without S9 ad 2 hours with S9. The total incubation time with 5-Bromodeoxyuridine was 25 - 26 hours, with colcemid present during the final 2-3 hours. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield enough metaphase cells for analysis. Slides were stained, coded and scored.

The test material was positive without metabolic activation but negative with metabolic activation in a sister chromatid exchange test for genotoxicity. The SCE test was positive only without activation at doses that induced cell cycle delay. No aberration induction was detected even after extending the incubation time without S9 to 20 hours.

Reference 28

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(only one replicate was conducted)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254 (S-9A)

Test concentrations with justification for top dose:

3 μmol/plate (463 μg/plate)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (+S9); N-methyl-N'-nitro-N-nitrosoguanidin (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Cultures were grown in Oxoid nutrient broth No. 2. Revertants were scored on glucosenminimal salts medium supplemented with 0.05 μmol histidine and 0.05 μmol biotin. Plates used for viable counts contained 10 μmol histidine (and 0.05 μmol biotin). The experiments were carried out essentially as described by Ames.

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 1

DETERMINATION OF CYTOTOXICITY
Toxicity was determined based on the absence of a background lawn of bacteria on the plates. If absence of a background lawn was found the test was repeated with a lower concentration of the substance.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

at 3 μmol/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: rat liver S9 fraction induced by Aroclor 1254 (S-9A)

Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha terpineol was tested using the Ames assay for mutagenecity on Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation (S9). The test item was tested at 3 μmol/plate on strains TA 98, TA 100, TA 1535 and TA 1537 both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254. Under these test conditions, alpha terpineol was found not mutagenic in all strains tested with and without metabolic activation.

Reference 29

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium, other: TA97a

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

Preliminary test carried out with TA100 strain without and with addition of S9 mixture: 0, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 2000, 2500, 2750 and 3000 μg/plate
Main assay (upper limit of the dose interval tested was either the highest non-toxic dose or the lowest toxic dose determined in the preliminary assay): 0, 25, 50,100, 250, 500, 750, 1000, 1250, 1500, 2000 and 2500 μg/plate.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene (TA100/+S9 (1 µg/plate); TA98/+S9 (0.5 µg/plate)); nitro-o-phenilene-diamine (TA98/-S9 (1 µg/plate)); 2-Aminofluorene (TA97a/+S9 (10 µg/plate))

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: 2 ml of top-agar was mixed with 100 µl of an overnight grown culture of S. typhimurium, 100 µl of the test substance (diluted in ethanol analytical grade, Merck, KGaA), the negative control, or the positive control (PC) and 500 µl of the phosphate buffer or the S9 mixture. Plates were incubated at 37ºC for 72h in the dark and then scored for revertant his+ bacteria colonies. Each determination was made in triplicate and two independent experiments were carried out.
- Cell density at seeding: For all assays, an inoculum (200 μl) of a thawed permanent culture was added to 20ml of Nutrient Broth No. 2 and incubated at 37ºC with shaking until a concentration of approximately 1–2 x10^9 bacteria per millilitre was obtained.

DURATION
- Exposure duration: 72h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity of the test compound to S. typhimurium strain TA100 was investigated in the preliminary assay. Toxicity was apparent either as a reduction in the number of his+ revertant bacteria colonies and or as a change of auxotrophic background growth (i.e. the background lawn).
Doses at which toxicity appeared as an alteration of the background lawn are marked with an asterisk in tables 1 and 2 (Any other information on results incl. Tables).

OTHER:
-Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox (Molecular Toxicology, Annapolis, USA). The S9 mixture was prepared as follows: 7.0ml of ultrapure water; 10.5ml of 200mM sodium phosphate buffer pH7.4; 0.84ml of 100mM NADP solution; 0.105ml of 1M glucose-6-phosphate; 0.42ml of 1.65M KCl + 0.4M MgCl2 salt solution; and 2.1ml of lyophilized S9 fraction reconstituted with distilled water.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2000 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 2000 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2500 μg/plate)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 2500 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 1500 μg/plate)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

up to highest dose tested of 2500 μg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 1500 μg/plate)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

(up to highest dose tested of 2000 μg/plate)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 2000 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Testing of terpineol in the Salmonella/microsome assay

Dose (µg/plate)	Number of revertants (Mean ± SD)
	TA 100		TA 98		TA 97a		TA 102
	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
2500	-	141±62*	-	4/0/0+	-	-	-	-
2000	53±14*	146 ±2	-	68± 13	-	272 ±34	-	833± 57*
1500	165 ±16	212 ±28	43 ±5	60 ±10	225 ±32	248 ±12	-	1573± 191
1250	158 ±19	188 ±23	42 ±2	58 ±2	224 ±11	246 ±6	862 ±187	1727± 108
1000	176 ± 6	208 ±33	40± 8	54± 10	191 ±8	254 ±21	1004 ±488	1429± 46
750	177 ±14	196 ±1	36 ±4	69 ±10	216 ±16	246 ±12	1418 ±176	1177± 42
500	177 ±12	161 ±24	28 ±7	53 ±10	195 ±13	241 ±16	1312± 89	1116± 74
250	186± 9	-	30 ±6	-	182 ±12	-	812± 166	790± 47
100	-	-	-	-	-	-	924± 151	-
50	-	-	-	-	-	-	737± 32	-
25	-	-	-	-	-	-	611± 25	-
0	176 ±14	197 ±7	40 ±7	70 ±6	158 ±2	214 ±18	731 ±36	771± 37
PC	898 ± 51	742 ± 13	192 ± 31	312 ± 33	1098 ± 80	870 ± 140	4875 ± 1031	2024 ± 182

Dose O—Negative Control: 100 µl ethanol; PC—Positive Control: TA100/-S9, SA (0.5 µg/plate); TA100/+S9, 2AA (1 µg/plate); TA98/-S9, NPD (1 µg/plate); TA98/+S9; 2AA (0.5 µg/plate); TA97a/-S9, 4-NQNO (1 µg/plate); TA97a/+S9, 2AF (10 µg/plate); TA102/-S9, MC (0.5 µg/plate); TA102/+S9, B-alpha-P (50 µg/plate).

(-) Dose not tested.

(*) Toxicity apparent as an alteration of the background lawn.

(/+) mutant counts of individual plates.

Values are the means ±SD of three plates of one (out of two) representative experiment.

Table 2. Mutagenicity of terpineol to TA102 tester strain in the confirmatory experiment

Dose (µg/plate)	- S9	+ S9
2000	-	572 28*
1500		1562 123
1250	-	1526 219
1000	1050 38	1114 562
750	1088 32	1057 74
500	786 58	824 6
250	742 24	-
100	634 26	-
50	600 32	-
25	630 54	-
0	613 ±24	790± 120
PC	5820 ± 311	1627± 260

Dose O—negative control (100 µl ethanol); PC—positive controls, -S9 (MC: 0.5 µg/plate); +S9 (B-alpha-P: 50 µg/plate)

(*) Toxicity apparent as an alteration of the background lawn.

Values are revertant counts (mean±SD of three plates).

Conclusions:

Terpineol was found to be not mutagenic in TA 100, TA 98 and TA 97a tester strains and weakly mutagenic in TA102 tester strain with and without metabolic activation.

Executive summary:

Terpineol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA97a and TA102 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Every determination was made in triplicate and each experiment was repeated once in order to check the reproducibility of the results. In contrast to the absence of mutagenicity towards TA97a, TA98 and TA100 strains, terpineol produced a dose-related increase in the number of TA102 revertants. The maximum effects were a 2.0-fold increase in the number TA102 revertants at doses as high as 750 µg/plate in the absence of S9 mixture, and a 2.2-fold increase at doses as high as 1250 µg/plate in the presence of S9 mixture. The negative as well as the positive results were reproduced by a confirmatory experiment. In conclusion, terpineol was found to be not mutagenic in TA 100, TA 98 and TA 97a strains but weakly mutagenic in TA102 strain with and without metabolic activation.

Reference 30

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Data published in a peer reviewed journal. Original study report not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

10000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha terpineol was tested for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.

Reference 31

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Data published in a peer reviewed journal. Original study report not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

50000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Gamma terpinene was not mutagenic in all strains tested with and without metabolic activation.

Conclusions:

Gamma terpinene was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Gamma terpinene was tested for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Gamma terpinene was not mutagenic in all strains tested with and without metabolic activation.

Reference 32

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 September 2012 – 22 March 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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

Target gene:

Histidine-requiring gene in Salmonella typhimurium and tryptophan-requiring gene in Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from male Sprague-Dawley rat liver and induced by Phenobarbital (PB) and 5,6-benzoflavone (BF)

Test concentrations with justification for top dose:

Preliminary experiment: 19.5, 78.1, 313, 1250 and 5000 μg/plate.
In the preliminary test, growth inhibition was observed at 1250 μg/plate for all strains without metabolic activity. When tested with metabolic activity, growth inhibition was observed at 313 μg/plate for strains TA 98, TA 1535 and TA 1537, and at 1250 μg/plate for strains TA 100 and E. Coli.
The highest dose selected for the main experiments for each strain was the lowest dose showing growth inhibition in the preliminary test. This dose was further diluted 5 times with the ratio of 2 to obtain 6 doses.

Main tests 1 and 2 (all strains -S9 and TA 98, TA 1535 and TA 1537 +S9): 39.1, 78.1, 156, 313, 625 and 1250 μg/plate
Main tests 1 and 2 (TA 100 and E. Coli +S9): 9.77, 19.5, 39.1, 78.1, 156 and 313μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to solubility in water of 1.98 g/L, a solubility test was performed on DMSO. As a result, the test substance was dissolved in DMSO at 50 mg/mL and no reactivity such as generation of heat or gas was observed, so the test was conducted using DMSO as a solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Methoxy-6-chloro-9-[ 3-(2-chloro ethyl)-aminopropylamino] acridine 2HCL (ICR-191); 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation: 0.1 mL of solvent or positive control or test solution, 0.5 mL of S9 mix or 0.1 M phosphate buffer solution (pH 7.4) and 0.1 mL of culture solution of each strain were added to the test tube. Preincubation was carried out with shaking (80 rpm) for 20 minutes at 37ºC. After completion of the preincubation 2.0 mL of top agar were added and after stirring the mixture was uniformly layered on a minimum glucose agar plate medium. Once confirmed that the top agar layered on the minimum glucose agar plate medium was solidified, the plate was turned upside down, placed in the incubator, and incubate at 37ºC for 49 hours in preliminary test and for 48.5 h in the main tests.

- Cell density at seeding (if applicable): 1 x 10^9 cells/mL.

DURATION
- Preincubation period: 20 min.
- Exposure duration: 48.5 h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants (revertant bacteria) can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition. The presence or absence of growth inhibition was observed using a stereoscopic microscope.

Evaluation criteria:

A result was considered positive when the number of revertant colonies in the test substance treated group showed an increase of 2 times or more as compared with the number of spontaneous revertant colonies (negative control value) and a dose response and reproducibility were observed. Also, a positive result was decided when a clear dose response was not shown but an increase of more than twice the number of spontaneously reversed mutant colonies was counted and when reproducibility was confirmed in two main tests.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 625 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 625 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 313 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1250 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 625 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 313 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 625 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 313 μg/plate and above)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Results of Test 1 (-S9Mix)

Metabolic activation		Dose of test substance (μg/plate)	Number of revertants (colony count / plate)
			Base pair substitution type			Frameshift type
			TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
S9 Mix (-)		Negative control (DMSO)	100 115 120 (112±10.4)	12 13 9 (11±2.1)	29 23 25 (26±3.1)	19 13 13 (15±3.5)	8 5 7 (7±1.5)
		39.1	90 99 97 (95±4.7	5 7 5 (6±1.2)	26 31 38 (32±6.0)	15 11 13 (13±2.0)	4 5 6 (5±1.0)
		78.1	97 95 108 (100±7.0)	7 12 10 (10±2.5)	23 27 25 (25±2.0)	13 12 13 (13±0.6)	6 5 4 (5±1.0)
		156	100 103 99 (101±2.1)	5 9 5 (6 ±2.3)	36 29 42 (36±6.5)	10 12 15 (12±2.5)	8 5 11 (8±3.0)
		313	98 101 99 (99±1.5)	5 6 11 (7±3.2)	22 30 36 (29±7.0)	13 10 11 (11±1.5)	6 4 7 (6±1.5)
		625	46* 50* 47* (48±2.1)	0* 0* 0* (0±0)	27 21 27 (25±3.5)	8* 8* 8* (8±0.0)	0* 0* 0* (0±0)
		1250	0* 0* 0* (0±0)	0* 0* 0* (0±0)	0* 0* 0* (0±0)	0* 0* 0* (0±0)	0* 0* 0* (0±0)
Positive controls	S9Mix (without)	Name	AF-2	SAZ	AF-2	AF-2	ICR-191
		Dose (μg/plate)	0.01	0.5	0.01	0.1	1.0
		Number of colonies/ plate	712 690 641 (681± 36.3)	358 304 374 (345±36.7)	115 123 117 (118±4.2)	461 428 425 (438±20.0)	1993 1905 1810 (1903±91.5)

Notes:

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

SAZ: Sodium azide

ICR-191: 2-Methoxy-6-chloro-9-[ 3-(2-chloro ethyl)-aminopropylamino] acridine · 2HCL

*: Growth inhibition by the test substance was observed.

( ): The inside shows the average value and standard deviation of three plates

Table 2: Results of Test 1 (+S9Mix)

Metabolic activation		Dose of test substance (μg/plate)	Number of revertants (colony count / plate)
			Base pair substitution type			Frameshift type
			TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
S9 Mix (+)		Negative control (DMSO)	114 123 138 (125±12.1)	8 9 7 (8±1.0)	30 30 33 (31±1.7)	22 27 26 (25±2.6)	6 7 10 (8±2.1)
		9.77	NT	11 10 7 (9±2.1)	NT	20 20 19 (20±0.6)	8 7 10 (8±1.5)
		19.5	NT	9 8 9 (9±0.6)	NT	18 21 26 (22±4.0)	8 6 9 (8±1.5)
		39.1	116 121 110 (116±5.5)	5 11 10 (9±3.2)	22 31 38 (30±8.0)	24 23 20 (22±2.1)	5 8 8 (7±1.7)
		78.1	116 100 109 (108±8.0)	11 10 8 (10±1.5)	33 24 29 (29±4.5)	21 20 24 (22±2.1)	9 11 7 (9±2.0)
		156	110 91 111 (104±11.3)	8 11 8 (9±1.7)	26 25 23 (25±1.5)	17 18 23 (19±3.2)	7 6 8 (7±1.0)
		313	123 112 105 (113±9.1)	5* 7* 7* (6±1.2)	31 27 39 (32±6.1)	22* 24* 18* (21±3.1)	5* 7* 8* (7±1.5)
		625	90* 91* 103* (95±7.2)	NT	21 34 23 (26±7.0)	NT	NT
		1250	0* 0* 0* (0±0.0)	NT	0* 0* 0* (0±0.0)	NT	NT
Positive controls	S9Mix (with)	Name	B[α] P	2AA	2AA	B[α] P	B[α] P
		Dose (μg/plate)	5.0	2.0	10.0	5.0	5.0
		Number of colonies/ plate	782 731 725 (746 ± 31. 3)	359 288 344 (330±37.4)	556 717 633 (635±80.8)	338 337 376 (350±22.2)	58 55 77 (63±11.9)

Notes:

B[α] P: Benzo [α] pyrene

2AA: 2-Aminoanthracene

*: Growth inhibition by the test substance was observed.

NT: not tested

( ): The inside shows the average value and standard deviation of three plates.

Table 3: Results of Test 2 (-S9Mix)

Metabolic activation		Dose of test substance (μg/plate)	Number of revertants (colony count / plate)
			Base pair substitution type			Frameshift type
			TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
S9 Mix (-)		Negative control (DMSO)	103 104 116 (108±7.2)	10 7 10 (9±1.7)	38 38 41 (39±1.7)	24 21 19 (21±2.5)	7 10 8 (8±1.5)
		39.1	85 91 112 (96±14.2)	10 13 12 (12±1.5)	39 36 33 (36±3.0)	22 20 20 (21±1.2)	6 7 10 (8±2.1)
		78.1	92 88 95 (92±3.5)	10 10 12 (11±1.2)	39 49 38 (42±6.1)	21 22 16 (20±3.2)	8 8 8 (8±0.0)
		156	116 97 111 (108±9.8)	7 10 7 (8±1.7)	44 44 28 (39±9.2)	18 21 20 (20±1.5)	15 10 11 (12±2.6)
		313	104 117 112 (111±6.6)	6 10 8 (8±2.0)	41 38 47 (42±4.6)	19 13 18 (17±3.2)	10 10 9 (10±0.6)
		625	53* 44* 69* (55±12.7)	2* 5* 3* (3±1.5)	34 31 30 (32±2.1)	10* 11* 10* (10±0.6)	0* 0* 0* (0±0)
		1250	0* 0* 0* (0±0)	0* 0* 0* (0±0)	0* 0* 0* (0±0)	0* 0* 0* (0±0)	0* 0* 0* (0±0)
Positive controls	S9Mix (without)	Name	AF-2	SAZ	AF-2	AF-2	ICR-191
		Dose (μg/plate)	0.01	0.5	0.01	0.1	1.0
		Number of colonies/ plate	637 692 650 (660± 28.7)	293 309 353 (318±31.1)	118 102 106 (109±8.3)	439 428 474 (447±24.0)	1548 1594 1489 (1544±52.6)

Notes:

AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

SAZ: Sodium azide

ICR-191: 2-Methoxy-6-chloro-9-[ 3-(2-chloro ethyl)-aminopropylamino] acridine · 2HC1

*: Growth inhibition by the test substance was observed.

( ): The inside shows the average value and standard deviation of three plates.

Table 4: Results of Test 2 (+S9Mix)

Metabolic activation		Dose of test substance (μg/plate)	Number of revertants (colony count / plate)
			Base pair substitution type			Frameshift type
			TA 100	TA 1535	WP2uvrA	TA 98	TA 1537
S9 Mix (+)		Negative control (DMSO)	120 108 114 (114±6.0)	14 10 11 (12±2.1)	42 44 30 (39±7.6)	27 27 28 (27±0.6)	13 10 10 (11±1.7)
		9.77	NT	10 7 10 (9±1.7)	NT	28 25 22 (25±3.0)	10 7 13 (10±3.0)
		19.5	NT	7 10 6 (8±2.1)	NT	29 27 24 (27±2.5)	9 7 8 (8±1.0)
		39.1	105 119 100 (108±9.8)	11 11 10 (11±0.6)	42 47 43 (44±2.6)	25 30 27 (27±2.5)	10 10 13 (11±1.7)
		78.1	125 114 116 (118±5.9)	8 10 13 (10±2.5)	51 40 39 (43±6.7)	31 23 29 (28±4.2)	7 6 7 (7±0.6)
		156	106 122 123 (117±9.5)	8 9 6 (8±1.5)	54 42 53 (50±6.7)	30 29 29 (29±0.6)	7 11 10 (9±2.1)
		313	119 133 114 (122±9.8)	8* 10* 12* (10±2.0)	39 37 41 (39±2.0)	26* 27* 23* (25±2.1)	8* 7* 7 (7±0.6)
		625	90* 104* 75* (90±14.5)	NT	38 38 47 (41±5.2)	NT	NT
		1250	0* 0* 0* (0±0.0)	NT	0* 0* 0* (0±0.0)	NT	NT
Positive controls	S9Mix (with)	Name	B[α] P	2AA	2AA	B[α] P	B[α] P
		Dose (μg/plate)	5.0	2.0	10.0	5.0	5.0
		Number of colonies/ plate	727 771 804 (767 ± 38.6)	379 339 352 (357±20.4)	852 841 890 (861±25.7)	288 266 253 (269±17.7)	79 82 93 (85±7.4)

Notes:

B[α] P: Benzo [α] pyrene

2AA: 2-Aminoanthracene

*: Growth inhibition by the test substance was observed.

NT: not tested

( ): The inside shows the average value and standard deviation of three plates.

Conclusions:

Terpineol did not show any mutagenic effect in bacteria under test conditions with and without metabolic activation.

Executive summary:

Terpineol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, 1535 and TA1537 and Escherichia coli strain WP2 uvrA with and without metabolic activation (S9). The experiment was performed according to OECD guideline 471 with GLP. Based on growth inhibition examined in a preliminary test up to a maximum dose of 5000μg/plate, tested concentrations in the main test were: 39.1, 78.1, 156, 313, 625 and 1250μg/plate for all strains without metabolic activationand for strains TA 98, TA 1535 and TA 1537 with metabolic activation, and 9.77, 19.5,39.1, 78.1, 156 and 313μg/plate for strains TA 100 and E. Coli WP2 with metabolic activation. Two main tests were performed in triplicate using preincubation method and DMSO as solvent. Negative and positive controls were within background data of the test laboratory. Under test conditions, terpineol was found not mutagenic in all strains tested with and without metabolic activation.

Reference 33

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 homogenate prepared from male Sprague-Dawley rats and Syrian golden hamsters induced with Aroclor 1254.

Test concentrations with justification for top dose:

0, 10, 33, 100, 333 and 1000 μg/plate.
These doses were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels, one plate per dose, were tested in both the presence and the absence of induced hamster S9. As some toxicity was observed at highest doses, a total maximum dose of 1 mg per plate was used.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: For testing in the absence of S9 mix, 100 μL of the tester strain and 50 μL of the solvent or test chemical were added to 2.5 mL of molten selective top agar at 45 ± 2 ºC. When S9 was used, 0.5 mL of S9 mix, 50 μL of tester strain, and 50 μL of solvent or test chemical were added to 2.0 mL of molten selective top agar at 45 ± 2 ºC. After it was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were incubated for 48 h at 37 ± 2 ºC.
- Cell density at seeding: Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of 1-2x10e9 cells/mL.

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
A preliminary dose range-finding study using strain TA100 (both with and without metabolic activation) was performed.

Evaluation criteria:

For the test substance to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence
and the absence of induced hamster S9. As some toxicity was observed at the highest doses, a total maximum dose of 1 mg of test chemical per plate was used in the main study.

Table 3 Salmonella Test Data

Chemical Name

CAS #

Dose

TA98

TA100

TA1535

TA1537

TA1538

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

alpha-Terpineol

Negative

98-55-5

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

DMSO

13 ± 4

20 ± 3

23 ± 4

87 ± 5

97 ± 2

124 ± 7

19 ± 7

14 ± 4

14 ± 2

14 ± 4

6 ± 1

10 ± 1

10 ± 4

21 ± 6

14 ± 6

10ug

20 ± 5

26 ± 3

24 ± 7

132 ± 7

118 ± 4

133 ± 9

24 ± 8

19 ± 4

22 ± 11

13 ± 1

11 ± 4

11 ± 2

20 ± 5

18 ± 2

23 ± 13

33ug

25 ± 1

25 ± 4

29 ± 5

129 ± 14

106 ± 10

129 ± 4

22 ± 2

12 ± 5

22 ± 6

11 ± 1

13 ± 2

12 ± 3

15 ± 4

17 ± 1

25 ± 8

100ug

17 ± 5

30 ± 5

28 ± 1

120 ± 11

113 ± 15

131 ± 15

29 ± 5

21 ± 4

22 ± 2

11 ± 1

12 ± 2

17 ± 3

16 ± 4

30 ± 1

28 ± 6

333ug

16 ± 3

31 ± 1

31 ± 7

130 ± 10

117 ± 10

111 ± 21

30 ± 4

18 ± 5

6 ± 4

10 ± 6

19 ± 4

13 ± 3

17 ± 6

23 ± 4

24 ± 4

1000ug

20 ± 3

31 ± 6

40 ± 4

111 ± 30

102 ± 20

127 ± 11

28 ± 3

15 ± 5

19 ± 4

9 ± 2

15 ± 2

13 ± 3

19 ± 7

21 ± 1

28 ± 1

Positive 1

142 ± 18

435 ± 77

578 ± 43

451 ± 51

535 ± 48

576 ± 57

488 ± 24

173 ± 14

208 ± 19

223 ± 129

206 ± 58

403 ± 38

406 ± 37

754 ± 12

773 ± 118

Positive 2

201 ± 13

953 ± 208

1516 ± 68

575 ± 67

1068 ± 121

1372 ± 77

Conclusions:

alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.

Executive summary:

Alpha terpineol was tested for mutagenecity on Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation (S9 mix prepared from both rat and hamster liver). The experiment was performed using the Ames Salmonella assay for mutagenicity. In a preliminary dose range-finding study ten dose levels of the test substance, one plate per dose, were tested in both the presence and the absence of induced hamster S9 using strain TA100. As some toxicity was observed, a maximum dose of 1 mg per plate was used. Based on this preliminary study, the selected doses for the main study were 0, 10, 33, 100, 333 and 1000 μg/plate. DMSO was used as solvent and tested as solvent control. Under these experimental conditions, the test substance was found not mutagenic in all strains tested with and without metabolic activation.

Reference 34

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

4 October, 2012 - 22 March, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster Lung Fibroblast (CHL /IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human Science Research Resource Bank
- Suitability of cells: Properties of cryopreserved cells were periodically checked (culture mode, cell doubling time within 15 hours 20 hours, average number of chromosomes, contamination of mycoplasma, etc.).
- Cell cycle length, doubling time or proliferation index: cell doubling time within 15 to 20 hours.
- Number of passages if applicable: The cell passage number used was 19 passages in the cell proliferation inhibition test, 25 passages in the short treatment method for the chromosomal aberration test, and 8 passages in the continuous treatment method.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimum Essential Medium (MEM) supplemented with 10% (v/v) heated inactivated bovine serum. Using a carbonic acid gas incubator, cultivation was carried out under conditions of 5% CO2, 37ºC and high humidity. Passage was done every 1-4 days.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes.

Metabolic activation:

with and without

Metabolic activation system:

Co-factor-supplemented S9 from Sprague-Dawley male rats induced with a combination of phenobarbital (PB) and 5, 6-benzoflavone (BF)

Test concentrations with justification for top dose:

Cell growth inhibition test: 1600, 800, 400, 200, 100, 50.0, 25.0, 12.5 and 0 (negative) μg/mL.
Chromosome aberration test:
short-time treatment (+S9): 500, 400, 300, 200, 100 and 0 (negative) μg/mL
short-time treatment (-S9) and continuous treatment (24 h and 48 h): 400, 300, 200, 100 and 0 (negative) μg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium.

- Cell density at seeding (if applicable): 2 x 10^4 cells per plate

DURATION
- Exposure duration: 6 h (short time treatment); 24 h and 48 h (continuous treatment)

SPINDLE INHIBITOR (cytogenetic assays): 0.1 mL of colcemid (10 μg / mL solution) was added about 2 hours before the end of the culture for preparing specimens for chromosome observation.

STAIN (for cytogenetic assays): 2% Giemsa solution for about 15 minutes

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After treatment, cells were incubated with 0.25% trypsin, suspended in 0.075 M KCl and incubated for about 15 minutes. Cells were then centrifuged and fixed with methyl alcohol: Acetic acid = 3:1 solution. Finally, the cells were stained with 2% Giemsa solution for about 15 minutes to prepare chromosome specimens.

NUMBER OF CELLS EVALUATED: 200 cells per group

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 cells analysed per group.

DETERMINATION OF CYTOTOXICITY
- Method: cell growth inhibition test
This assay was conducted in duplicate for short-term treatment group (6 h) with and without metabolic activation and for continuous treatment group (24 h and 48 h) without metabolic activation at doses of 1600, 800, 400, 200, 100, 50.0, 25.0, 12.5 and 0 (negative control) μg/mL. The cell-growth ratio was determined against the negative control regarded as 100% growth. Condition of cells was observed at the end of treatment. Color of medium was observed immediately after addition of the test solutions. Precipitates/crystals were observed immediately after addition of the test solutions and at the end of treatment. The 50% cell proliferation inhibiting concentration of the test substance was determined for every treatment group.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The evaluation of results was performed according to the following criteria:
If incidence of chromosomal aberrations is less than 5% the test is considered negative
If incidence of chromosomal aberrations is more than 5% and less than 10% the test is considered as false positive.
If incidence of chromosomal aberrations is more than 10% the test is considered positive.

For the overall assessment the structural chromosomal aberrations excluding gaps were used. Also, a dose dependency or reproducibility was considered for the final assessment.

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Lung Fibroblast (CHL /IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see tables 1-4 on "Any other information on results incl. tables"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Immediately after addition of the test solution, precipitation was observed at doses of 400 μg/mL or more in all treatment groups. At the end of treatment, precipitation was observed at 500 μg/mL(+S9) and 400 μg/mL(-S9) in the short-time treatment group. In the continuous treatment group no precipitation was observed at the end of treatment.
- Other confounding effects: No change in color tone of the culture solution was observed after addition of the test solution.


RANGE-FINDING/SCREENING STUDIES:
In the preliminary study, immediately after addition of the test solution, precipitation was observed at doses of 400 μg/mL or more in all treatment methods. No change in color tone of the culture solution was observed.
At the end of the treatment, precipitation was observed at a dose of 400 μg mL or more for metabolic activation in the short-time treatment group and at a dose of 800 μg/mL or more by non-metabolic activation and continuous treatment group. The 50% cell-growth inhibition concentration (approximate value) was calculated to be 449 μg/mL in the short-time treatment group (+S9), 295 μg/mL in the short-time treatment group (-S9), 276 μg/mL in the 24-hour continuous treatment group and 242 μg/mL in the 48-hour continuous treatment group.

Table 1: Chromosome aberration in cultured Chinese hamster (CHL/IU) cells treated with Tepineol [Short-term treatment:+S9 mix]

Time (h)	S9 mix	Conc. Test article (µg/mL)	Number of cells with structural chromosome aberration (%)										Cell growth ratio (%)	Number of cells with numerical chromosome growth aberration (%)
			Cells observed	ctb	cte	csb	cse	other	TA(%)	g	TAG(%)	Judgement		Cells observed	Polyploid cells	Other	Total (%)	Judgement
6-18	+	NC	100	0	0	0	0	0	0	0	0	-	100	100	0	0	0	-
			100	0	0	0	0	0	0	0	0		109	100	0	0	0
			200	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		(100)	200	0 (0.0)	0 (0.0)	0 (0.0)
		100	100	0	0	0	0	0	0	2	2	-	89	100	1	0	1	-
			100	0	0	0	0	0	0	0	0		79	100	1	0	1
			200	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (1.0)	2 (1.0)		(80)	200	2 (1.0)	0 (0.0)	2 (1.0)
		200	100	0	1	0	0	0	1	0	1	-	69	100	1	0	1	-
			100	0	0	0	0	0	0	0	0		69	100	0	0	0
			200	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	1 (0.5)		(66)	200	1 (0.5)	0 (0.0)	1 (0.5)
		300	100	0	0	0	0	0	0	0	0	-	69	100	0	0	0	-
			100	1	0	0	0	0	1	1	2		69	100	0	0	0
			200	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	1 (0.5)	2 (1.0)		(66)	200	0 (0.0)	0 (0.0)	0 (0.0)
		400	100	0	0	0	0	0	0	0	0	-	79	100	0	0	0	-
			100	0	0	0	0	0	0	0	0		69	100	0	0	0
			200	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		(71)	200	0 (0.0)	0 (0.0)	0 (0.0)
		500	100	0	1	0	0	0	1	0	1	-	49	100	0	0	0	-
			100	0	0	0	0	0	0	0	0		49	100	0	0	0
			200	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	1 (0.5)		(47)	200	0 (0.0)	0 (0.0)	0 (0.0)
		PC	100	4	24	0	0	0	28	0	28	+	89	100	0	0	0	-
			100	3	22	0	0	0	25	0	25		99	100	0	0	0
			200	7 (3.5)	46 (23.0)	0 (0.0)	0 (0.0)	0 (0.0)	53 (26.5)	0 (0.0)	53 (26.5)		(90)	200	0 (0.0)	0 (0.0)	0 (0.0)

g: chromatid or chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange,

other: including fragmentation

TA: total number of cells with aberration excluding gap, TAG: total number of cells with aberration including gap.

NC: Negative control (DMSO)

PC: Positive control (cyclophosphamide, 14 μg/mL)

Each value in parenthesis on cell- growth ratio (%) data showed mean of cell-growth ratio against the negative control.

Table 2: Chromosome aberration in cultured Chinese hamster (CHL/IU) cells treated with Tepineol [Short-term treatment:-S9 mix]

Time (h)	S9 mix	Conc. Test article (µg/mL)	Number of cells with structural chromosome aberration (%)										Cell growth ratio (%)	Number of cells with numerical chromosome growth aberration (%)
			Cells observed	ctb	cte	csb	cse	other	TA(%)	g	TAG(%)	Judgement		Cells observed	Polyploid cells	Other	Total (%)	Judgement
6-18	-	NC	100	0	1	0	0	0	1	0	1	-	100	100	2	0	2	-
			100	0	0	0	0	0	0	0	0		99	100	0	0	0
			200	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	1 (0.5)		(100)	200	2 (1.0)	0 (0.0)	2 (1.0)
		100	100	0	0	0	0	0	0	0	0	-	74	100	0	0	0	-
			100	0	0	0	0	0	0	0	0		87	100	0	0	0
			200	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		(81)	200	0 (0.0)	0 (0.0)	0 (0.0)
		200	100	1	0	0	0	0	1	0	1	-	49	100	0	0	0	-
			100	0	0	0	0	0	0	0	0		49	100	0	0	0
			200	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	1 (0.5)		(49)	200	0 (0.0)	0 (0.0)	0 (0.0)
		300	100	0	0	0	0	0	0	0	0	-	49	100	0	0	0	-
			100	0	0	0	0	0	0	0	0		74	100	1	0	1
			200	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		(62)	200	1 (0.5)	0 (0.0)	1 (0.5)
		400	100	0	0	0	0	0	0	0	0	-	24	100	0	0	0	-
			100	0	0	0	0	0	0	0	0		24	100	0	0	0
			200	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		(24)	200	0 (0.0)	0 (0.0)	0 (0.0)
		PC	100	3	28	0	0	0	31	0	31	+	112	100	0	0	0	-
			100	4	32	0	0	0	36	1	37		112	100	0	0	0
			200	7 (3.5)	60 (30.0)	0 (0.0)	0 (0.0)	0 (0.0)	67 (33.5)	1 (0.5)	68 (34.0)		(113)	200	0 (0.0)	0 (0.0)	0 (0.0)

g: chromatid or chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange,

other: including fragmentation

TA: total number of cells with aberration excluding gap, TAG: total number of cells with aberration including gap.

NC: Negative control (DMSO)

PC: Positive control (mitomycin C, 0.075 μg/mL)

Each value in parenthesis on cell- growth ratio (%) data showed mean of cell-growth ratio against the negative control.

Table 3: Chromosome aberration in cultured Chinese hamster (CHL/IU) cells treated with Tepineol [Continuous treatment: 24 h]

Time (h)	S9 mix	Conc. Test article (µg/mL)	Number of cells with structural chromosome aberration (%)										Cell growth ratio (%)	Number of cells with numerical chromosome growth aberration (%)
			Cells observed	ctb	cte	csb	cse	other	TA(%)	g	TAG(%)	Judgement		Cells observed	Polyploid cells	Other	Total (%)	Judgement
24-0	-	NC	100	0	0	0	0	0	0	0	0	-	100	100	1	0	1	-
			100	2	0	0	0	0	2	0	2		100	100	0	0	0
			200	2 (1.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (1.0)	0 (0.0)	2 (1.0)		(100)	200	1 (0.5)	0 (0.0)	1 (0.5)
		100	100	0	0	0	0	0	0	1	1	-	100	100	1	0	1	-
			100	0	0	0	0	0	0	0	0		100	100	1	0	1
			200	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	1 (0.5)		(100)	200	2 (1.0)	0 (0.0)	2 (1.0)
		200	100	1	0	0	0	0	1	0	1	-	83	100	2	0	2	-
			100	0	0	0	0	0	0	0	0		83	100	1	0	1
			200	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	1 (0.5)		(83)	200	3 (1.5)	0 (0.0)	3 (1.5)
		300	100	0	1	0	0	0	1	0	1	-	49	100	1	0	1	-
			100	0	2	0	0	0	2	0	2		49	100	2	0	2
			200	0 (0.0)	3 (1.5)	0 (0.0)	0 (0.0)	0 (0.0)	3 (1.5)	0 (0.0)	3 (1.5)		(49)	200	3 (1.5)	0 (0.0)	3 (1.5)
		400	100	0	0	0	0	0	0	0	0	-	33	100	1	0	1	-
			100	0	0	0	0	0	0	0	0		33	100	0	0	0
			200	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		(33)	200	1 (0.5)	0 (0.0)	1 (0.5)
		PC	100	14	24	0	0	0	38	0	38	+	100	100	0	0	0	-
			100	11	24	0	0	0	35	0	35		116	100	0	0	0
			200	25 (12.5)	48 (24.0)	0 (0.0)	0 (0.0)	0 (0.0)	73 (36.5)	0 (0.0)	73 (36.5)		(108)	200	0 (0.0)	0 (0.0)	0 (0.0)

g: chromatid or chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange,

other: including fragmentation

TA: total number of cells with aberration excluding gap, TAG: total number of cells with aberration including gap.

NC: Negative control (DMSO)

PC: Positive control (mitomycin C, 0.050 μg/mL)

Each value in parenthesis on cell- growth ratio (%) data showed mean of cell-growth ratio against the negative control.

Table 4: Chromosome aberration in cultured Chinese hamster (CHL/IU) cells treated with Tepineol [Continuous treatment: 48 h]

Time (h)	S9 mix	Conc. Test article (µg/mL)	Number of cells with structural chromosome aberration (%)										Cell growth ratio (%)	Number of cells with numerical chromosome growth aberration (%)
			Cells observed	ctb	cte	csb	cse	other	TA(%)	g	TAG(%)	Judgement		Cells observed	Polyploid cells	Other	Total (%)	Judgement
48-0	-	NC	100	0	0	0	0	0	0	0	0	-	100	100	0	0	0	-
			100	0	1	0	0	0	1	0	1		99	100	0	0	0
			200	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	1 (0.5)		(100)	200	0 (0.0)	0 (0.0)	0 (0.0)
		100	100	0	0	0	0	0	0	0	0	-	92	100	1	0	1	-
			100	0	0	0	0	0	0	0	0		92	100	0	0	0
			200	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)		(92)	200	1 (0.5)	0 (0.0)	1 (0.5)
		200	100	0	1	0	0	0	1	0	1	-	76	100	0	0	0	-
			100	0	0	0	0	0	0	0	0		76	100	0	0	0
			200	0 (0.0)	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	1 (0.5)		(76)	200	0 (0.0)	0 (0.0)	0 (0.0)
		300	100	1	0	0	0	0	1	0	1	-	46	100	0	0	0	-
			100	0	0	0	0	0	0	0	0		53	100	1	0	1
			200	1 (0.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (0.5)	0 (0.0)	1 (0.5)		(50)	200	1 (0.5)	0 (0.0)	1 (0.5)
		400	100	0	1	0	0	0	1	1	2	-	23	100	0	0	0	-
			100	0	1	0	0	0	1	0	1		30	100	0	0	0
			200	0 (0.0)	2 (1.0)	0 (0.0)	0 (0.0)	0 (0.0)	2 (1.0)	1 (0.5)	3 (1.5)		(27)	200	0 (0.0)	0 (0.0)	0 (0.0)
		PC	100	13	56	0	0	0	69	0	69	+	107	100	3	0	3	-
			100	5	68	0	0	0	73	0	73		115	100	3	0	3
			200	18 (9.0)	124 (62.0)	0 (0.0)	0 (0.0)	0 (0.0)	142 (71.0)	0 (0.0)	142 (71.0)		(112)	200	6 (3.0)	0 (0.0)	6 (3.0)

g: chromatid or chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange,

other: including fragmentation

TA: total number of cells with aberration excluding gap, TAG: total number of cells with aberration including gap.

NC: Negative control (DMSO)

PC: Positive control (mitomycin C, 0.050 μg/mL)

Each value in parenthesis on cell- growth ratio (%) data showed mean of cell-growth ratio against the negative control.

Conclusions:

In an in vitro chromosome aberration study, terpineol was found not clastogenic when tested in CHL/IU cells in vitro with or without metabolic activation.

Executive summary:

Terpineol was tested in an in vitro chromosome aberration study with cultured Chinese hamster lung CHL/IU cells in presence and absence of metabolic activation using a method according to OECD guideline 473. Based on a preliminary cytotoxicity study consisting on growth inhibition the tested concentrations were 500, 400, 300, 200, 100 and 0 (negative) μg/mL in the short-time treatment (+S9) and 400, 300, 200, 100 and 0 (negative) μg/mL in the short-time treatment (-S9) and continuous treatment (24 h and 48 h). Duplicate cultures were exposed to the test compound for 6 h (short time treatment) and for 24 h and 48 h (continuous treatment) before sampling. There were no statistically significant or dosage-related increases in both structural and numerical chromosome aberrations compared with the concurrent control (DMSO), with and without metabolic activation at both short-term and continuous treatment. Both positive controls, cyclophosphamide with metabolic activation and mitomycin C without metabolic activation, produced highly significant increases in the numbers of aberrant cells compared to control. From the above results, it was concluded that terpineol does not induce chromosome structural abnormality and chromosome number abnormality under the test conditions.

Reference 35

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine Kinase Gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Donald Clive, Burroughs Wellcome Co. (Research Triangle Park, NC).
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Fischer’s medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI).

Metabolic activation:

with and without

Metabolic activation system:

liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

- S9 mix: 0.14, 0.2, 0.26, 0.32 and 0.38 µL/mL
+S9 mix: 0.17, 0.27, 0.36, 0.46 and 0.56 µL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not specified.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

( DMSO)

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium.
- Cell density at seeding (if applicable): Cells in the cultures were adjusted to 3x10e5/mL at 24 h intervals. They were then cloned (1x10e6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium.

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium, to allow expression of the TK- mutation ): 48h
- Selection time (plating for -trifluorothymidine (TFT) resistance): 10-12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT) (final concentration, 3 µg /mL)

NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.

NUMBER OF CELLS EVALUATED: 1.2 x 10e7 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: The toxicity was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats. Cells at a concentration of 6 x 10e5/mL (6 x10e6 cells total) were exposed for 4 h to a range of concentrations from 0.0005 to 10000 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1ºC for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent control. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.

Evaluation criteria:

Mutant frequencies were expressed as mutants per 10e6 surviving cells.
A positive result was interpreted following two different methods:
1. Using a doubling of the mutant frequency over the concurrent solvent-treated control value, together with evidence of a dose-related increase (cited as old evaluation in the report).
2. if a concentration-related increase in mutant frequency is observed and one or more dose levels with 10% or greater total growth exhibit mutant frequencies of ≥100 mutants per 10e6 clonable cells over the background level (cited as new evaluation in the report).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

27-157% RTG

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

19-112% RTG

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 4 Mouse Lymphoma Test Data

		Non-Activated Cultures					S9-Activated Cultures
Chemical Name	Solvent	Dose (ug/mL)	Average TFT	Average VC	Mut Freq	RTG	Dose (ug/mL)	Average TFT	Average VC	Mut Freq	RTG
alpha-Terpineol	DMSO	0.14 to 0.56 ul/mL					ul/mL
		0,14	78	190	0,8	92	0,17	73	203	0,7	112
			72	191	0,8	81	0,27	80	224	0,7	111
		0,2	87	391	0,4	157		91	194	0,9	86
		0,26	66	147	0,9	55	0,36		175		52
			74	192	0,8	75		86	238	0,7	89
		0,32	82	173	0,9	53	0,46	79	182	0,9	62
			67	152	0,9	46		86	204	0,8	46
		0,38	71	161	0,9	27	0,56	68	247	0,6	19
			71	180	0,8	29			196		23
		Solvent	73	192	0,8		Solvent	62	193	0,6
		Positive	104	13	16,1	1	Positive	170	58	5,9	109

Conclusions:

The test substance was found negative in the Mouse Lymphoma Assay in the presense and in the absence of metabolic activation.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD test guideline 490) to test the potential to cause gene mutation. Treatment was performed for 4 hours with and without metabolic activation (±S9 mix). DMSO was used as solvent. The test item was examined previously in a cytotoxicity Test. Based on these results, the test item concentrations in the mutation assay ranged from 0.14 to 0.56 µL/mL. The experiments were performed using appropriate negative (vehicle) and positive control samples. Under conditions of the assay, the test substance was found negative in the presense and in the absence of metabolic activation.

Reference 36

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Data published in a peer reviewed journal. Original study report not available.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

yes

Remarks:

no data on replications

GLP compliance:

not specified

Type of assay:

other: unscheduled DNA synthesis (UDS)

Target gene:

Not applicable

Species / strain / cell type:

hepatocytes: Rat/Fischer and Sprague Dawley adult male

Metabolic activation:

without

Test concentrations with justification for top dose:

Up to 30 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; Livers were perfused in situ with 0.5 mM EDTA in HEPES buffer (pH 7.2) for four minutes. Cultures of rat liver hepatocytes were incubated with the test material for 18-20 hours.

DURATION
- Exposure duration: 18-20 h

NUMBER OF CELLS EVALUATED: 75-150 cells were analyzed for each dose level.

Evaluation criteria:

UDS was measured by electronically counting nuclear grains and subtracting the average number of grains in 3 adjacent nuclear sized cytoplasmic areas.
The test was considered positive if an increase in net nuclear grain count of at least six grains per nucleus above the solvent control and/or an increase in the percent of nuclei with at least 6 net grains to more than 10% above the negative control value.

Key result

Species / strain:

hepatocytes: Rat/Fischer and Sprague Dawley adult male

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks:

the positive induced significant increases in the mean number of net nuclear grain counts compared to the solvent control.

Gamma terpinene did not cause a significant increase in UDS as measured by the mean number of net nuclear grain counts at any dose level.

Conclusions:

Gamma terpinene was not mutagenic based on the results of the rat hepatocyte unscheduled DNA synthesis assay.

Executive summary:

Gamma terpinene was tested on the rat hepatocyte unscheduled DNA synthesis assay following the OECD Guideline 482. Cultures of rat liver hepatocytes were incubated with the test material for 18-20 hours at concentrations of up to 30 μg/ml without metabolic activation. The tested material did not cause a significant increase in the mean number of net nuclear grain counts compared to the control at any dose level. Thus, gamma terpinene is considered to be negative in the unscheduled DNA synthesis assay.

Reference 37

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The standard plate incorporation as described by Ames et al. (1975) and modified by Batzinger et al. (1978) and Babish et al. (1983) was used in this research.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Assay 1: Strains TA 97, TA 98 and TA 100 with preincubation time of 20 min both in the presence and absence of 0.5 ml of S9 mix.
Assay 2: Strain TA 100 with S9 activation and incorporation of another sample preincubated for 60 min.

DURATION
- Preincubation period: 20 min

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

Evaluation according to "MR = mean revertants per plate / mean spontaneous revertants per plate" with the following criteria:
Positive: MR greater than 2

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Conclusions:

No evidence of mutagenicity was observed at test conditions for Borneol.

Executive summary:

A bacterial reverse mutation test (Ames test) was performed on test substance Borneol using Salmonella typhimurium strains TA 97, TA 98 and TA 100 with and without S9 mix metabolic activation and 20 min standard preincubation time. Dimethylsulfoxide (DMSO) was used as solvent at a concentration of 1 mg/ml. Positive and negative/solvent controls were included. No evidence of mutagenicity was observed at test conditions.

Reference 38

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: No GLP. Equivalent or similar to OECD 471. Documentation insufficient for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

up to 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation method

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Under the conditions of the study, DL-Borneol was found to be not mutagenic with or without metabolic activation in any of the tested strains.

Executive summary:

The mutagenic potential of DL-Borneol was assessed in an Ames study following experimental outlines similar to those in OECD Guideline 471 using the plate incorporation method. Salmonella

typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with DL-Borneol at concentrations up to 5000 μg/plate in the presence and absence of metabolic activation (S9 mix). Under the conditions of the study, DL-Borneol is considered not mutagenic in bacteria.

Reference 39

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: No data on GLP. Test method not reliable for full assessment of genetic toxicity in bacteria.

Principles of method if other than guideline:

A mutation test in Escherichia coli WP 2 uvrA (trp-) was performed in Borneol and some other synthetic flavoring agents widely used in foodstuffs.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

not specified

Test concentrations with justification for top dose:

Test concentration range: 0.4 to 3.2 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Evaluation criteria:

Evaluation in mutation test according to "ratio = maximal revertants / spontaneous revertants" with the following criteria:
Positive: when ratio >=2
Negative: when ratio < 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

The results were as follows:

Reagents	Dose range (mg/plate)	Ratio	Conclusion
Borneol	0.4-3.2	0.9	-

Conclusions:

No evidence of mutagenicity was observed at test conditions for Borneol.

Executive summary:

No mutagenic activity was observed for Borneol in the test mutation using E. Coli WP2 uvrA within a concentration range for the test substance of 0.4 -3.2 mg/plate.

Reference 40

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Method similar to OECD guideline 471, but only two strains were tested with metabolic activation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(Only two strains were tested with metabolic activation)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with

Metabolic activation system:

rat liver microsome fraction, S9, prepared from Aroclor 1254-treated animals according to the procedure of Ames et al. (800 μg/plate)

Test concentrations with justification for top dose:

The amounts assayed ranged from 5 µl to 10 µl of the undiluted sample.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: picrolonic acid

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Direct plate incorporation method: a test sample of 10^8 bacterial cells, and S9 at a concentration of 800 μg protein per plate were incorporated into a tube containing top agar prepared with minimal medium (Minimal Broth Davis, Difco) and 0.05 mM histidine and 0.05 mM biotin. The top agar was then poured on a petri dish containing minimal medium supplemented with 20% glucose. After a 48-hour incubation at 37°C, each assay plate was counted and the number of spontaneous mutants for either TA98 (40) or TA100 (180) were subtracted from the total number of revertants.

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: at least 2

DETERMINATION OF CYTOTOXICITY
An additional tube of top agar was prepared as explained above and plated on nutrient agar (Difco) to examine the background lawn of bacterial growth for the presence of toxic effects.

OTHER:
-Plates containing aflatoxin B1 were also included in each experiment to confirm enzyme activation by the S9 fraction.

Evaluation criteria:

The positive response to mutagenicity with TA100 is defined as any deviation above the upper 99.9% confidence limits of the mean control value. This value (180) is the average number of spontaneous TA100 revertants observed on the control plates.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

A weak mutagenic response toward TA100 but not TA98 was observed for the test substance.

Conclusions:

Beta-terpineol was found a weak mutagenic with TA100 and but not with TA98 in a reverse mutation test with metabolic activation.

Executive summary:

Beta-terpineol was tested for mutagenecity on Salmonella typhimurium strains TA100 and TA98 with metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity.

A weak mutagenic response toward TA100 but not TA98 was observed in doses where no apparent toxicity was detected.

Reference 41

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed according to OECD guideline 473.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

only tested without metabolic activation

GLP compliance:

not specified

Remarks:

(no data reported)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Peripheral venous human blood
- Suitability of cells: Current study was approved by the Ethics Committee of the Universidade Estadual de Maringá, Maringá PR Brazil.
- Sex, age and number of blood donors if applicable: two healthy males and one female aged 25 years, non-smoking, non-alcoholic, not under drug therapy and with no recent history of exposure to mutagens.
- Whether whole blood or separated lymphocytes were used if applicable: yes, separated by centrifugation at 1100 rpm for 5 min.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium, supplemented with 15% fetal calf serum, 1% L-glutamine 200 mM and 2% phytohemagglutinin.
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

95 μg/ml, 182 μg/ml and 365 μg/ml.
4 concentrations of TTO (95 μg/ml, 182 μg/ml, 365 μg/ml and 548 μg/ml) were evaluated by the mitotic index (MI). At 548 μg/ml a cytotoxicity of approx. 80% was obtained when compared to the negative control. Thus, the TTO concentrations selected were 95 μg/ml, 182 μg/ml and 365 μg/m which produced cytotoxicity ranging approximately 9–40% when compared to the negative control.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.1 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium; The lymphocyte cultures were incubated at 37ºC, 5.0% CO2 for 72 h. At 48 h incubation, TTO and mitomycin C were added to each culture individually.

DURATION
- Preincubation period: 48 h
- Exposure duration: 24 h

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After 72 h incubation, the cells from the culture were treated with a hypotonic solution (75 mM KCl) for 20 min at 37ºC and fixed with a cold solution of methanol:glacial acetic acid (3:1 v/v). The cells were fixed with two changes of fixatives. Slides were prepared for microscopic analysis by dripping 3–4 drops of the pre-fixed lymphocyte suspension from a distance of 30 cm, dried for 5 days at 22ºC and stained with 5% Giemsa (pH 6.8 Sorensen's buffer).

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 400 well-spread metaphases (200 metaphases per dose and per replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: mitotic index
- Any supplementary information relevant to cytotoxicity: level of cytotoxicity was determined by the reduction in mitotic index (MI) when compared to that in negative control, with the highest oil concentration producing 50–60% cytotoxicity. MI was calculated by the number of dividing cells/total number of the cells x 100 (Chandrasekaran et al., 2009).

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Results were judged as follows:
negative if the frequency of chromosomal aberrations was <5%; inconclusive if the frequency of chromosomal aberrations was ≥5% but <10%; and positive if the frequency of chromosomal aberrations was ≥10% (Maenosono et al., 2009).

Statistics:

Results were analyzed statistically (Z-test, p <0.05).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Effect of TTO on the mitotic index (MI) in human lymphocytes.

TTO concentrations(μg/ml)	MI ± SD (%)
0	3.3 ± 0.948
95	2.9 ± 0.360
182	3.0 ± 0.665
365	2.2 ± 0.450
548	0.7 ± 0.776 *
positive control (a)	2.2 ± 0.608

a Positive control: mitomycin C (0.1 µg/ml).

* Significantly different from negative control (Kruskal–Wallis test, p< 0.05).

Table 2. Chromosome analysis of human lymphocytes treated with TTO

Compounds	Concentrations (μg/ml)	Frequency of chromosomal aberrations (%)
		Cog	Ceg	Cag	Cob	Ceb	Cab	Frag	Rear	Endo	Total (a)	Judge (b)
None	0	0	0	0	0	0	0	0	0	0	0	-
Mytomycin C	0.1	0.25	1	1	5.25	2.75	10.5	0	0	0.5	19 *	+
TTO	95	0	0.25	0.5	0	0	0	0	0	0.75	0.75	-
	182	0	0.75	0.5	0.25	0	0.75	0	0	0.75	1.75	-
	365	0	0	0.25	0.25	0	1	0	0	0.25	1.5	-

Cog: chromosome gap; Ceg: centromeric gap; Cag: chromatid gap; Cob: chromosome break; Ceb: centromeric break; Cab: chromatid break; Frag: fragment; Rear: rearrangement; Endo: endorreduplication. Four hundred metaphases were scored for each treatment.

a Cog, Ceg and Cag, according to OECD guideline no. 473 (1997), are classified as achromatic lesions and therefore should not be included in the total chromosomal aberrations.

b According to Maenosono et al. (2009).

* Significantly different from negative control (Z-test, p < 0.05).

Conclusions:

Under the test conditions, the test substance was found negative in a chromosomal aberration test using human blood lymphocytes.

Executive summary:

In an in vitro mammalian chromosomal aberration test performed accordingly with OECD guideline 473, human blood lymphocytes cells were exposed to the test substance tea tree oil (TTO) composed of the following main components as identified by GC/MS and NMR: Terpinen-4-ol (42.8%), γ-terpinene (20.4%), p-cymene (9.6%), α-terpinene (7.9%), 1,8-cineole (3%), α-terpineol (2,8%) and α-pinene (2.4%). Based on the cytotoxicity observed in a mitotic index (MI), the concentrations used in the test were 95, 182 and 365 μg/ml. Untreated culture was used as negative control and mitomycin C at 0.1 µg/mL was used as positive control. None of the tested TTO concentrations caused significant differences in the frequencies of both structural and numerical (polyploidy and endorreduplication) chromosomal aberrations when compared to those of the negative control. Thus, it was concluded that TTO is not genotoxic in in vitro mammalian cells.

Reference 42

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 3 different strains were tested

GLP compliance:

not specified

Remarks:

This information was not reported

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: uvrB and rfa mutated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction

Test concentrations with justification for top dose:

0, 5, 15, 50, 150, 500, 1500, and 5000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: in a previous study (Gomes-Carneiro et al., 1998) using ethanol as the solvent it was considered that the ethanol might exert some oxidising properties on terpinen-4-ol and change the chemical structure.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: 1,8-dihydroxyanthraquinone; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In agar (plate incorporation): 0.1 ml of each concentration of test material was added to 2.0 ml of molten top agar at 45ºC containing 0.1 mL in nutrient broth of the appropriate tester, and 0.5 ml of phosphate buffer or the S9 mixture (Ames, 1973). This was
mixed and poured onto a Vogel–Bonner agar plate.

- Cell density at seeding: approximately 1–9.9 x10^9 bacteria per mL.

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 2 (treated groups) and 3 (positive and negative controls)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The background ‘lawn’ was examined microscopically for toxicity and precipitation.
- Any supplementary information relevant to cytotoxicity: Toxicity was apparent either as a reduction of the number of revertants, and/or as an alteration of the auxotrophic background growth (i.e. background lawn) (Gomes-Carneiro et al., 1998).

Statistics:

Statistics were not used to evaluate this study.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1500-5000 μg/mL)

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1500-5000 μg/mL)

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1500-5000 μg/mL)

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Terpinen-4-ol was found to be not mutagenic in TA 100, TA 98 and TA102 tester strains with and without metabolic activation.

Executive summary:

Terpinen-4 -ol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98 and TA102 with and without metabolic activation (S9) following the Ames test. The experiment was performed in duplicate using the plate incorporation method with the test material diluted in acetone at doses of 0 (untreated control), 5, 15, 50, 150, 500, 1500, and 5000 μg/mL. Negative control and positive controls were tested in triplicate and all showed the expected responses.Toxicity was observed at the higher dose levels of 1500–5000 μg/mL. The test material did not induce any increase in the number of revertants over negative control values obtained for any of the strains TA102, TA100 and TA98 either in the presence or absence of metabolic activation. Thus, terpine-4-ol can be considered as non-mutagenic in the bacterial reverse assay for the tester strains used.

Reference 43

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium and tryptophan-requiring gene in Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from male Sprague-Dawley rat liver

Test concentrations with justification for top dose:

0, 30, 60, 120 and 300 μg/plate.
The highest dose used showed killing on bacteria based on previous examination with dissecting microscope.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-aminoanthracene (2-AA):

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation method (-S9) and preincubation method described by Yahagi et al. (1975). (+S9).

DURATION
- Exposure duration: 48-72 h (Not specified for the test substance)

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants (revertant bacteria) can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3-5 (Not specified for the test substance)

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition. The presence or absence of growth inhibition was observed using a dissecting microscope.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 300 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 300 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 300 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 300 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 300 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 300 μg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Results of mutagenicity tests on safrole and related compounds in S. typhimurium and in E. coli WITHOUT S9

Sample	Dose a ( µg/plate)	Revertants/plate (mean ± SD)
		Salmonella typhimurium strains					Escherichia coli
		TA100	TA1535	TA98	TA1537	TA1538	WP2 uvra
DMSO	50 µL	99 ± 6	8 ± 2	24 ± 4	4 ± 2	15± 3	55 ± 6
Positive control	b	769± 54	266 ± 34	660 ± 30	822± 88	301 ± 33	452 ± 68
Estragole	30	104± 5	7 ± 4	25 ± 6	5 ± 2	19 ± 2	68 ± 9
	60	95± 9	6 ± 3	23 ± 4	4 ± 3	14 ± 1	45 ± 3
	120	91± 10	11 ± 4	27 ± 7	4 ± 3	17 ± 5	54 ± 11
	300	109± 8	6 ± 4	25 ± 4	5 ± 1	14± 4	63 ± 13

a) All test compounds showed killing on bacteria at the highest doses used (examined with a dissecting microscope).

b) TA100 (AF-2, 0.02); TA1535 (Na N3, 0.5); TA98 (AF-2, 0.1); TA1537 (9-AAc, 80); TA1538 (2 -NF, 2); WP2 uvrA (AF-2, 0.01).

Table 2: Results of mutagenicity tests on safrole and related compounds in S. typhimurium and in E. coli WITH S9

Sample	Dose a ( µg/plate)	Revertants/plate (mean ± SD)
		Salmonella typhimurium strains					Escherichia coli
		TA100	TA1535	TA98	TA1537	TA1538	WP2 uvra
DMSO	50 µL	106 ± 9	10 ± 3	32 ± 4	7 ± 2	21± 4	59 ± 5
Positive control	b	431 ± 92	158 ± 45	180 ± 7	125 ± 37	154 ± 17	753 ± 95
Estragole	30	124 ± 1	12 ± 4	28 ± 4	8 ± 0	20 ± 2	54 ± 8
	60	107 ± 14	8 ± 0	38 ± 2	7 ± 4	18 ± 1	60 ± 6
	120	119 ± 9	9 ± 2	34 ± 3	8 ± 0	21 ± 7	53 ± 12
	300	91 ± 6	7 ± 2	20 ± 8	7 ± 4	13 ± 3	57 ± 9

a) All test compounds showed killing on bacteria at the highest doses used (examined with a dissecting microscope).

b) TA100 (BP, 5); TA1535 (2-AA, 5); TA98 (BP, 5); TA1537 (2-AA, 5); TA1538 (2-AA, 2); WP2 uvrA (2-AA, 80).

Conclusions:

Estragole did not show any mutagenic effect in bacteria under test conditions with and without metabolic activation.

Executive summary:

Estragole was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, 1535, TA1537, TA1538 and Escherichia coli strain WP2 uvrA with and without metabolic activation (S9). The experiment was performed according to Ames method. Based on growth inhibition examined in a preliminary test, the concentrations used in the main test were: 0, 30, 60, 120 and 300 μg/plate for all strains with and without metabolic activation. The assay without S9 was performed by the plate incorporation method and the assay with S9 was conducted by the pre-incubation method described by Yahagi et al. (1975). DMSO was used as solvent. Negative (solvent) and positive controls were within background data of the test laboratory. Under test conditions, estragole was found not mutagenic in all strains tested with and without metabolic activation.

Reference 44

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Remarks:

(no data reported)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Modal number of chromosomes: ± 21 chromosomes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

1% Aroclor-induced rat liver S9 mix and primary rat hepatocytes.

Test concentrations with justification for top dose:

0 (negative and solvent controls), 1e-3, 3.16e-4, 1e-4, 3.16e-5 and 1e-5 mol/L.
The top concentration was chosen because of a visible tiny oily drop on top of the treatment medium a couple of minutes after preparation of the mixture.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: Cultures were harvested 18 h (approx. 1.5 cell cycle) after beginning of treatment with a continuous treatment without external metabolism and a 2-h treatment in the presence of S9 mix.
The co-culture experiment with hepatocytes and V79 cells was carried out via seeding hepatocytes on to V79 cells that had been seeded 24 h before (Müller et al., 1992). Cells were treated continuously throughout the whole co-culture period of 18 h.

DURATION
- Exposure duration: 2 h (+S9) and 18 h (-S9)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were arrested in mitosis following a 2-h incubation with colcemid (80 ng/ml). Mitotic cells were shaken off and standard techniques for metaphase preparation were used. The cells were stained with Giemsa (5% in phosphate buffer at pH 6.88).

NUMBER OF CELLS EVALUATED: 200 per dose

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100 metaphases per dose and per replicate

Evaluation criteria:

Chromosomal aberrations were scored as described previously (Müller et al.,1992): Chromatid-type aberrations (chromatid breaks or fragments, chromatid exchanges) and chromosome-type aberrations (chromosome breaks or fragments, dicentrics, and rings) were scored. Metaphases showing multiple aberrations
were recorded seperately. Gaps and isogaps (achromatic lesions not greater than the width of a chromatid) were scored but were not included in the calculation of damaged cells.

Statistics:

Differences between single control and treatment groups were evaluated with chi-square distribution. Differences between all control and treatment groups were analyzed with the non-parametric H-test (Kruskal and Wallis, 1952), a one-way analysis of variance for independent data and different sample sizes. P-values below 0.01 were considered to indicate significant differences.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1. Chromosomal aberrations in V79 cells after treatment with estragole in the absence and in the presence of external metabolisation

Treatment [mol/l]	cells scored	Aberrations per 100 metaphases						Aberrant metaphases  excl. Gaps
		G	B'	RB'	B''	dic/ring	MA	n	%	(SE)
(a) No external metabolism, harvest time: 18 h, continuous treatment
0, control	200	4.0	4.5	0.0	1.0	0.0	0.0	10	5.0	(1.5)
DMSO, control	200	5.0	4.0	0.0	0.0	0.0	0.0	6	3.0	(1.2)
MMC 10-7	200	5.5	4.0	7.0	6.0	0.0	0.5	31	15.5*	(2.6)
estragole 10 -3	200	3.5	5.5	1.0	1.0	0.0	0.0	13	6.5	(1.7)
estragole 3.16 × 10 -4	200	1.5	1.0	0.0	0.5	0.0	0.0	3	1.5	(0.9)
estragole 10 -4	200	2.5	0.0	0.0	0.0	0.0	0.0	0	0.0	(0.0)
estragole 3.16 x 10 -5	200	2.0	4.5	0.0	0.5	0.0	0.0	9	4.5	(1.5)
estragole 10 -5	200	2.0	5.5	0.5	1.5	0.0	0.0	14	7.0	(1.8)
(b) Presence of 1% Aroclor-induced rat liver S9 mix, harvest time: 18 h, 2-h treatment
0, control	200	2.0	1.0	0.0	0.5	1.0	0.0	5	2.5	(1.1)
DMSO, control	200	3.0	0.0	0.0	1.0	0.0	0.0	2	1.0	(0.7)
CP 3.16 x 10 -5	200	5.5	18.0	21.5	6.5	0.0	0.5	51	25.5*	(3.1)
CP 10 -5	200	4.0	0.5	0.0	1.0	0.0	0.0	3	1.5	(0.9)
estragole 10 -3	200	2.0	2.5	0.0	0.5	0.0	0.0	6	3.0	(1.2)
estragole 3.16 × 10 -4	200	1.0	0.0	0.5	0.5	0.0	0.0	2	1.0	(0.7)
estragole 10 -4	200	5.5	1.5	0.0	0.5	0.0	0.0	4	2.0	(1.0)
estragole 3.16 x 10 -5	200	3.0	1.5	0.0	0.0	0.0	0.0	2	1.0	(0.7)
estragole 10 -5	200	2.5	1.0	0.0	1.5	0.0	0.0	4	2.0	(1.0)
(c) Presence of primary rats hepatocytes, harvest time: 18 h, continuous treatment
0, control	200	0.5	1.0	0.0	1.0	0.5	0.0	5	2.5	(1.1)
DMSO, control	200	1.0	1.0	0.0	0.0	0.0	0.0	2	1.0	(0.7)
CP 10 -4	150	3.5	25.0	17.5	22.5	0.5	2.5	109	54.5*	(4.1)
CP 3.16 x 10 -5	200	2.5	13.5	10.5	12.5	0.0	0.0	49	24.5*	(3.0)
estragole 10 -3	200	0.5	0.5	1.0	1.0	0.0	0.0	3	1.5	(0.9)
estragole 3.16 × 10 -4	200	3.0	0.5	0.0	1.0	0.5	0.0	4	2.0	(1.0)
estragole 10 -4	200	0.0	2.0	0.0	3.5	0.5	0.0	9	4.5	(1.5)
estragole 3.16 x 10 -5	200	0.5	1.5	0.0	1.0	0.0	0.0	5	2.5	(1.1)
estragole 10 -5	200	0.5	0.5	0.5	3.0	0.0	0.0	8	4.0	(1.4)

MMC, mitomycin C; CP, cyclophosphamide.

* Significant difference vs. DMSO control at p < 0.01.

Conclusions:

Under the test conditions, the test substance was found negative in a chromosomal aberration test using V79 cells.

Executive summary:

In an in vitro mammalian chromosomal aberration test performed similarly with OECD guideline 473, V79 cells were exposed to estragole dissolved in DMSO either via direct treatment, with rat liver S9 mix or with rat hepatocytes as metabolic activation systems. The concentrations used in the test were 0 (negative and solvent controls), 1e-3, 3.16e-4, 1e-4, 3.16e-5 and 1e-5  mol/L in each experiment. The top concentration was chosen because of a visible tiny oily drop on top of the treatment medium a couple of minutes after preparation of the mixture. Mitomycin C and cyclophosphamide were used as positive controls in appropriate concentrations. The experiments were run with two cultures in parallel. Estragole was not capable of inducing chromosomal aberrations in V79 cells in either the direct treatment or S9 mix or primary hepatocytes used as external metabolisation systems. Although chromosomal aberrations were slightly elevated at some concentrations in the experiments without S9 mix and in the presence of hepatocytes, there was no concentration-effect relationship and no significant difference from the concurrent negative controls. The positive control had a pronounced effect. Thus, estragole was found negative in the chromosomal aberration test.

Reference 45

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

GLP compliance:

not specified

Remarks:

(this information was not reported)

Type of assay:

other: unscheduled DNA synthesis (UDS)

Target gene:

Not applicable

Species / strain / cell type:

hepatocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: primary hepatocytes were isolated from 8-10-week-old Wistar rats
with the two-step collagenase perfusion technique as previously reported (Müller et al.,1994).
- Suitability of cells: Viability of the cells as determined by trypan blue exclusion exceeded 75%.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: WE medium supplemented with 2 mM L-glutamine, 0.1 mg/ml streptomycin, 100 U/ml penicillin and 10% foetal calf serum (Biochrom, Berlin). Non attached cells were removed after 2 h.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

0 (solvent control), 1e-5, 1e-4, 1e-3 and 1e-2 mol/L.
The top concentration was chosen because of a visible tiny oily drop on top of the treatment medium a couple of minutes after preparation of the mixture.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; WE medium supplemented with 2 mM L-glutamine, 0.1 mg/ml streptomycin, 100 U/ml penicillin and 10% foetal calf serum (Biochrom, Berlin). Non attached cells were removed after 2 h. Treatment medium with dissolved test compound and supplemented with 5 µC/ml tritiated thymidine was added after removal of non attached cells.

- Cell density at seeding (if applicable): 2 x 105 viable hepatocytes were seeded onto 25-mm round collagen-coated Thermanox coverslips (Nunc, Wiesbaden) in 35-mm 6-well dishes (Nunc, Wiesbaden).

DURATION
- Exposure duration: 18 h

NUMBER OF REPLICATIONS: 2 experiments with 3 cultures each.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Coverslips were washed, fixed, dried, mounted, film-coated (K5 emulsion, Ilford, Cheshire, UK), developed and stained as described in Müller, K., P. Kasper and L. Müller (1994)

NUMBER OF CELLS EVALUATED: 50 hepatocytes per slide from 3 different parallel cultures per concentration, i.e. 150 cells per experiment per concentration.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxic effects were qualified by determination of necrotic cells.

Evaluation criteria:

Grains were counted with a microscope combined with an Artek counter (Model 982b). Net grain values were obtained by subtracting the mean of three cytoplasm grain counts from the nuclear grain counts. A response was considered to be positive when the average net grain count exceeded the concurrent control level by five or more grains as described in Müller, K., P. Kasper and L. Müller (1994).

Key result

Species / strain:

hepatocytes:

Metabolic activation:

not applicable

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(at 1e-2 M)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

the positive induced significant increase in the mean number of net nuclear grain counts compared to the solvent control.

Remarks on result:

other: see details of results in the graph attached below.

Conclusions:

Estragole induced a positive response in an in vitro rat hepatocyte unscheduled DNA synthesis assay.

Executive summary:

Estragole was tested in an in vitro rat hepatocyte unscheduled DNA synthesis assay following a method similar to the OECD Guideline 482. Primary hepatocytes were isolated from 8-10-week-old Wistar rats. In each of 2 independent experiments 3 different parallell cultures of hepatocytes were incubated with the test material dissolved in DMSO for 18 hours at concentrations of 0 (solvent control), 1e-5, 1e-4, 1e-3 and 1e-2 mol/L. 50 hepatocytes per culture (150 cells per experiment) were evaluated for UDS per concentration. A response was considered positive when the average net grain count exceeded the concurrent control level by 5 or more grains. The test material induced a marked and dose-dependent response in the test. An effect was found even with the lowest concentration tested, i.e. 1e-5 M. A concentration of 1e-2 M was toxic to the cells. Positive control induced a expected response which was considered valid. Based on these results, estragole was demostrated to be an UDS inducer in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo: Supporting studies: Several experimental results from studies performed with the main components d-limonene, alpha pinene and estragole are available. All study results were negative except for one UDS study with estragole in which it was found positive in rat liver cells. However, estragole was negative in an in vitro mammalian chromosome aberration test and in an in vitro bacteria reverse mutation test with and without metabolic activation and thus in an overall assessment this compound should not be considered as mutagenic according to this available information.

Based on these results, no genotoxicity is predicted for the test substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Comet assay.

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Method: Comet assay (Sasaki et al., 1997; Sasaki et al., 1999; Sasaki et al., 2000; Tsuda et al., 2000)

GLP compliance:

no

Type of assay:

mammalian comet assay

Species:

mouse

Strain:

other: ddY

Sex:

male

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Once

Post exposure period:

No data

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

- Treatment groups: 4 males
- Vehicle control and untreated control groups: 12 males

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Tissues and cell types examined:

Stomach, colon, liver, kidney, urinary, bladder, lung, brain and bone marrow

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
A preliminary range-finding test was conducted using 4-5 male mice/dose to determine the LD50 value.
Animals were observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment.

METHOD OF ANALYSIS:
Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in Comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, 4 male ddY mice were administered a single oral dose of d-limonene in olive oil by gavage at dose levels of 2000 mg/kg bw. Animals were then observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment. Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal. A preliminary range-finding test was also conducted using 4-5 male mice/dose to determine the LD50 value. No death, morbidity or distinctive clinical and microscopic signs were observed. D-limonene did not induced DNA damage in the studied organs. Under the test conditions, d-limonene is not considered as mutagenic in Comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Comet assay

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Method: Comet assay (Tice et al., 2000).

GLP compliance:

no

Type of assay:

mammalian comet assay

Species:

rat

Strain:

other: OFA Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Saint-Germain-sur-l’Arbresle, France)
- Age at study initiation: 5-6 weeks
- Assigned to test groups randomly: Yes
- Housing: Housed in groups of 2-3 in polypropylene cages
- Diet (e.g. ad libitum): Commercial pellets (SAFE, Augy, France), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 15%
- Air changes (per hour): 20/hour
- Photoperiod (hours dark / hours light): 20 hours dark / 20 hours light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.1% CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test solutions were prepared with 0.1% CMC.

Duration of treatment / exposure:

3-6 or 22-26 hours

Frequency of treatment:

Once

Post exposure period:

No

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

In 0.5% CMC.

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

In 0.5% CMC.

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

In 0.5% CMC.

No. of animals per sex per dose:

- Vehicle control and treatment groups: Four males
- Positive control group: Three males

Control animals:

yes, concurrent vehicle

Positive control(s):

Streptozotocin
- Justification for choice of positive control(s): Known renal epigenetic carcinogen
- Route of administration: Intravenous
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

Kidney cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary range-finding test was conducted using 4 male rats/dose and animals were observed at least 2 days for mortality and clinical signs of toxicity. Maximum tolerated dose (MTD) determined in the preliminary test was selected as the highest dose for the main study.

TREATMENT AND SAMPLING TIMES: After an exposure period of 3-6 or 22-26 hours, treated animals were sacrificed and kidney cells were isolated by specific enzymatic method (Bruggeman et al., 1989). Cytotoxicity was determined on a small sample of each isolated cell suspension following the trypan blue vital dye-exclusion technique.

DETAILS OF SLIDE PREPARATION: Slides (16/dose/expression period) with the cell suspensions (3 × 10^4 cells), embedded in a layer of 0.5% of low melting-point agarose, were immersed in a lysing solution for at least 1 hour at +4 °C in the dark and then run in a horizontal gel electrophoresis unit for 20 min at 0-4 °C by applying an electric current of 0.7 V/cm (25 V/300 mA). After electrophoresis, the slides were neutralized with 0.4 M Tris (pH 7.5) and the DNA was exposed for 5 min to absolute ethanol in order to preserve all the Comet assay slides.

METHOD OF ANALYSIS: Prepared slides were stained with propidium iodide (20 µg/mL distilled water; 25 µL/slide) and scanned using a fluorescent microscope (Leica Microscopy and Scientific Instruments Group, Switzerland), connected through a gated CCD camera to Comet Image Analysis System version 4.0 software (Kinetic Imaging Ltd., UK), to determine mean Olive Tail Moment (OTM) median value in 150 cells per animal (Tice et al., 2000).

Evaluation criteria:

- Olive Tail Moment (OTM) preconised by Olive (1990) was used to evaluate DNA damage.
- OTM, expressed in arbitrary units, is calculated by multiplying the percent of DNA (fluorescence) in the tail by the length of the tail in µm. The tail length is measured between the edge of Comet head and the end of the Comet tail.

Statistics:

- Kruskall-Wallis test was used to display a possible dose–effect relationship.
- Statistical significance of differences in the median values between each group versus the control was determined with the non-parametric Mann-Whitney U-test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- No deaths, morbidity, or distinctive clinical signs were observed after any of the treatments.
- Viability, using the trypan-blue exclusion method, was >70% for each cell suspension in all control and treated groups up to the MTD.
- See table 1

Table 1: DNA damage measured by the Comet assay in isolated rat kidney cells 3–6 or 22–26 hours after a single administration of d-limonene at dose levels of 1000 and 2000 mg/kg bw

 

Sampling time (h)	Group	Dose (mg/kg)	OTM
3-6	d-limonene	0	1.76
		1000	1.81
		2000	1.35
	Streptozotocin	20	41.1***
22-26	d-limonene	0	1.87
		1000	1.91
		2000	2.21
	Streptozotocin	20	40.8***

Significant difference (Mann–Whitney U-test) as compared with the vehicle control; ***p < 0.001.

OTM: mean Olive Tail Moment median value

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in Comet assay on isolated kidney cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, groups of 4 OFA Sprague-Dawley male rats were administered a single oral dose of d-limonene in 0.5% CMC by gavage at dose levels of 0, 1000 and 2000 mg/kg bw. After an exposure period of 3-6 or 22-26 hours, treated animals were sacrificed and the kidney cells were isolated and the prepared slides were scanned to determine mean Olive Tail Moment (OTM) median value in 150 cells per animal using the method described by Tice et al (2000). A preliminary range-finding test has also been conducted using 4 males rats/dose and animals were observed at least 2 days for any clinical signs of toxicity and any mortalities in order to determine the maximum tolerated dose (MTD). Positive control (streptozotocin, 20 mg/kg bw) caused a clear increase in the mean OTM median value. D-limonene showed no substantial increase in the mean OTM median value. Under the test conditions, d-limonene is not considered as mutagenic in Comet assay on isolated kidney cells and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Comet assay.

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Method: Comet assay (Sasaki et al., 1997; Sasaki et al., 1999; Sasaki et al., 2000; Tsuda et al., 2000)

GLP compliance:

no

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Once

Post exposure period:

No data

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

- Treatment groups: 4 males
- Vehicle control and untreated control groups: 12 males

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Tissues and cell types examined:

Stomach, colon, liver, kidney, urinary, bladder, lung, brain and bone marrow

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
A preliminary range-finding test was conducted using 4-5 male rats/dose to determine the LD50 value.
Animals were observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment.

METHOD OF ANALYSIS:
Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in Comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, 4 male Wistar rats were administered a single oral dose of d-limonene in olive oil by gavage at dose levels of 2000 mg/kg bw. Animals were then observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment. Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal. A preliminary range-finding test was also conducted using 4-5 male rats/dose to determine the LD50 value. No death, morbidity or distinctive clinical and microscopic signs were observed. D-limonene did not induced DNA damage in the studied organs. Under the test conditions, d-limonene is not considered as mutagenic in Comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

no positive controls were tested

GLP compliance:

yes

Remarks:

in compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 22.5-23 g (male) and 19.3-19-7 g (female)
- Housing: individually. Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); rotated weekly; cageboard: Untreated paper cage pan liner (Shepherd Specialty Papers, Kalamazoo, MI), changed daily
- Diet (e.g. ad libitum): NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods)
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- Acclimation period: Animals were quarantined for 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Room fluorescent light: 12 hours/day
- Chamber air changes: 15 ± 2/hour

Route of administration:

inhalation: vapour

Vehicle:

- Vehicle(s)/solvent(s) used: no data

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was held in an 8-gallon stainless-steel chemical reservoir. Test item was pumped into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the test item vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
- Temperature, humidity, pressure in air chamber: 72 ± 3ºF; 50% ± 15%.
- Air change rate: 15 air changes per hour
- Method of particle size determination: A condensation particle detector (Model 3022A, TSI, Inc., St. Paul, MN) was used with and without animals in the exposure chambers. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.

TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatograph. Samples were analyzed using GC/FID to measure the stability and purity of test item in the generation and delivery system. To assess whether impurities or degradation products coeluted with test item or the solvent, a second GC/FID analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities.
- Samples taken from breathing zone: yes.

Duration of treatment / exposure:

14 weeks; 6 hours plus T90 (10 minutes) per day

Frequency of treatment:

five times per week, weekdays only

Dose / conc.:

0 ppm

Dose / conc.:

25 ppm

Remarks:

(0.14 mg/L)

Dose / conc.:

50 ppm

Remarks:

(0.28 mg/L)

Dose / conc.:

100 ppm

Remarks:

(0.56 mg/L)

Dose / conc.:

200 ppm

Remarks:

(1.13 mg/L)

Dose / conc.:

400 ppm

Remarks:

(2.26 mg/L)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Positive control(s):

none

Tissues and cell types examined:

Peripheral blood samples

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
2-week preliminary study were conducted to determine the highest administrable non lethal dose level. 5 mice per sex and per dose were exposed to 0, 100, 200, 400, 800, and 1,600 ppm test item.

TREATMENT AND SAMPLING TIMES:
At the end of the 3-month toxicity study, peripheral blood samples were obtained from male and female mice. Smears were immediately prepared and fixed in absolute methanol.

DETAILS OF SLIDE PREPARATION:
Slides were air-dried, fixed and stained with a fluorescent DNA-specific stain (acridine orange).

METHOD OF ANALYSIS:
Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) in each of five animals per exposure group. In addition, the percentage of polychromatic erythrocytes (PCEs) among a population of 1000 erythrocytes was scored for each exposure group as a measure of bone marrow toxicity.

Evaluation criteria:

In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction was preferably based on reproducibly positive trials. Ultimately, the final call was determined by the scientific staff after considering the results of statistical analyses, the reproducibility of any effects observed, and the magnitudes of those effects.

Statistics:

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one-tail Cochran-Armitage trend test, followed by pairwise comparisons between each exposed group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): See table 1
- Ratio of PCE/NCE (for Micronucleus assay): See table 1

Table 1: Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with alpha Pinene by Inhalation for 3 Months a

	Concentration (ppm)	Number of Mice with Erythrocytes Scored	Micronucleated NCEs/1,000 NCEs b	P Value c	PCEs b (%)
Male
Aird	0	5	1.6 ± 0.33		2.50 ± 0.39
Alpha pinene	25	5	1.8 ± 0.30	0.3657	2.34 ± 0.19
	50	5	1.9 ± 0.53	0.3059	2.20 ± 0.26
	100	5	2.1 ± 0.43	0.2053	2.88 ± 0.31
	200	5	1.9 ± 0.29	0.3059	2.74 ± 0.19
	400