Oligomerisation products of beta-pinene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Nu-Film-17

IUPAC Name:

Nu-Film-17

Constituent 2

Reference substance name:

Oligomerisation products of beta-pinene

EC Number:

701-246-8

Molecular formula:

Variable (dimer = C20-H34)

IUPAC Name:

Oligomerisation products of beta-pinene

Details on test material:

- Batch number: 154293
- Pinene oligomers content 96 wt%

Method

Target gene:

Endpoint investigated = chromosome damage

Species / strain

Species / strain / cell type:

lymphocytes: Human

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

First test concentrations: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 ug/ml.
Second test concentrations:
- Without S9 mix, 18 hour harvest: 62.5, 125, 250, 375, 500, 750 and 1000 ug/ml
- With S9 mix, 18 hour harvest: 125, 250, 500 and 1000 ug/ml
- Without S9 mix, 32 hour harvest: 15.6, 31.3, 62.5, 125, 250, 375, 500, 750 and 1000 ug/ml
- With S9 mix, 32 hour harvest: 125, 250, 500 and 1000 ug/ml

Vehicle / solvent:

Nu-Film-17 (Pinolene) was immiscible in DMSO and so was prepared directly in the tissue culture medium.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: ethyl methanesulphonate, cyclophosphamide

Details on test system and experimental conditions:

First test: Human lymphocyte cultures (0.9E06 cells/ml) that had been incubated for 48 hours were centrifuged at 200g. The cell pellets were resuspended in appropriate dosing solutions (5ml) to give the desired final concentrations of Nu-Film-17 (Pinolene) (2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250. 500 and 1000 ug/ml). Duplicate cultures in the absence of S9 mix were prepared for each concentration of the test substance and four cultures were left untreated to serve as the negative control group. For the positive control group, ethyl methansulphonate was used at concentrations of 500 and 750 ul/ml in duplicate cultures. The cultures without S9 mix were incubated for 18 hours.

The test was also performed in duplicate cultures in the presence of S9 mix. For the positive control group with S9 mix, cyclophosphamide was added to duplicate cultures at concentrations of 10 and 15 ug/ml. Three hours after dosing cultures with the S9 mix, these were centrifuged and cell pellets resuspended in fresh medium and incubated for an additional 15 hours.

Second test: Human lymphocyte cultures (1E06 cells/ml) that had been incubated for 48 hours were centrifuged at 200g. The cell pellets were resuspended in appropriate dosing solutions (5ml) to give the desired final concentrations of Nu-Film-17 (Pinolene):
- without S9 mix, 18 hour harvest: 62.5, 125, 250, 375, 500, 750 and 1000 ug/ml
- with S9 mix, 18 hour harvest: 125, 250, 500 and 1000 ug/ml
- without S9 mix, 32 hour harvest: 15.6, 31.3, 62.5, 125, 250, 375, 500, 750 and 1000 ug/ml
- with S9 mix, 32 hour harvest: 125, 250, 500 and 1000 ug/ml

Duplicate cultures were prepared and used for each concentration of the test substance and four cultures were left untreated. For the 32 hour harvest ethyl methansulphonate was used at concentrations of 250 and 500 ul/ml in duplicate cultures (without S9 mix) and cyclophosphamide was used at concentrations of 2.5 and 5 ug/ml (with S9 mix). Three hours after dosing cultures with the S9 mix, these were centrifuged and cell pellets resuspended in fresh medium and incubated for an additional 15 or 29 hours. Cultures that were treated without S9 mix were incubated for 18 or 32 hours.

Harvesting and fixation: Colchicine was added to each culture to stop mitotic activity 2 hours before harvesting. Cell suspensions were centrifuged and cell pellets treated with a hypotonic solution (20% Hanks balanced salt solution). After treatment with the hypotonic solution, cell suspensions were again centrifuged and cell pellets fixed with freshly prepared fixative (3 parts methanol:1 part glacial acetic acid). These pellets were allowed to fixed for 2 hours upon which were centrifuged and resuspended in fresh fixative. These cell suspensions were placed onto microscope slides and allowed to air-dry and then stained with Giemsa and mounted in DPX.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Only at highest concentration - not used for metaphase analysis

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity:
- First test: The mitotic indices of Nu-Film-17 (Pinolene) treated human lymphocytes can be found in table 1. A reduction in mitotic index was observed at the 500 ug/ml dose group in the absence of S9 mix. The higher dose group (1000 ug/ml) was too toxic for analysis, thus the concentrations selected for metaphase analysis were 62.5, 250 and 500 ug/ml. In the presence of S9 mix, no significant reduction in mitotic index was observed when compared to the control value, thus the doses selected for metaphase analysis were 125, 500 and 1000 ug/ml.

- Second test, 18 hour harvest: The mitotic indices of Nu-Film-17 (Pinolene) treated human lymphocytes can be found in table 1. A reduction in mitotic index was observed at the 500 ug/ml dose group in the absence of S9 mix. The higher dose group (1000 ug/ml) was too toxic for analysis, thus the concentrations selected for metaphase analysis were 62.5, 250 and 500 ug/ml. In the presence of S9 mix, no significant reduction in mitotic index was observed when compared to the control value, thus the doses selected for metaphase analysis were 125, 500 and 1000 ug/ml.

- Second test, 32 hour harvest: The mitotic indices of Nu-Film-17 (Pinolene) treated human lymphocytes can be found in table 1. A reduction in mitotic index was observed at the 250 and 375 ug/ml dose groups in the absence of S9 mix, thus the concentrations selected for metaphase analysis were 31.3, 125 and 250 ug/ml. In the presence of S9 mix, a slight reduction in mitotic index was observed in the 1000 ug/ml dose group when compared to the control value. The doses selected for metaphase analysis were 125, 500 and 1000 ug/ml.

Metaphase analysis:

- First test: See table 2 for metaphasis analysis data. No statistically significant increases of chromosome aberrations were observed at any concentration either in the presence or absence of S9 mix. The positive control groups (with ethyl methanesulphonate or cyclophosphamide) caused statistically significant increases of aberrant cells.

- Second test, 18 hour harvest: See table 2 for metaphasis analysis data. No statistically significant increases of chromosome aberrations were observed at any concentration either in the presence or absence of S9 mix. The positive control groups (with ethyl methanesulphonate or cyclophosphamide) caused statistically significant increases of aberrant cells.

- Second test, 32 hour harvest: See table 2 for metaphasis analysis data. There were no statistically significant increases in aberrant cells in any dose group in the presence of S9 mix. However, in the absence of S9 mix there was a statistically significant increase in chromosomal damage in the 250 ug/ml dose group, but this was associated with high cytotoxicity and was not considered to be of biological significance.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Mitotic indices

	Test substance concentration (ug/ml)	S9 mix	Mean mitotic index (%)	Relative mitotic index (%)
First test, 18 hour harvest	Untreated	-	3.6	100
	2	-	4.0	111
	3.9	-	4.5	125
	7.8	-	4.0	111
	15.6	-	4.3	119
	31.3	-	4.3	119
	62.5	-	3.3	92
	125	-	4.4	122
	250	-	4.3	119
	500	-	1.8	50
	1000	-	0.3	8
	Untreated	+	3.8	100
	2	+	3.6	95
	3.9	+	3.0	79
	7.8	+	3.0	79
	15.6	+	4.5	118
	31.3	+	4.0	105
	62.5	+	4.2	111
	125	+	4.7	124
	250	+	4.0	105
	500	+	3.7	97
	1000	+	4.9	129
Second test, 18 hour harvest	Untreated	-	4.0	100
	62.5	-	3.3	83
	125	-	2.9	73
	250	-	2.7	68
	375	-	2.0	50
	500	-	1.8	45
	750	-	0.3	8
	1000	-	0.0	0
	Untreated	+	2.6	100
	125	+	2.5	96
	250	+	2.2	85
	500	+	2.3	88
	1000	+	2.2	85
Second test, 32 hour harvest	Untreated	-	4.8	100
	15.6	-	4.2	88
	31.3	-	3.0	63
	62.5	-	3.3	69
	125	-	2.8	58
	250	-	1.6	33
	375	-	1.6	33
	500	-	0.5	10
	750	-	0.1	2
	1000	-	0.0	0
	Untreated	+	3.6	100
	125	+	3.9	108
	250	+	3.1	86
	500	+	3.5	97
	1000	+	2.2	61

+ = Presence of S9 mix

- = Absence of S9 mix

Table 2. Metaphase analysis

Test	Test material	Concentration	S9 mix	Number of aberrant cells
				Mean % (Excluding gaps)	Mean % (Including gaps)
First test	Untreated	-	-	1.25	1.25
	Nu-Film-17 (Pinolene)	62.5	-	1.00	1.00
		250	-	1.00	1.00
		500	-	2.50	2.50
	Ethyl methanesulphonate	500	-	19.00*	19.5*
	Untreated	-	+	1.25	1.25
	Nu-Film-17 (Pinolene)	125	+	3.50	3.50
		500	+	3.00	3.50
		1000	+	3.00	3.50
	Cyclophosphamide	15	+	16.5*	16.5*
Second test, 18 hour harvest	Untreated		-	2.50	2.50
	Nu-Film-17 (Pinolene)	32.5	-	1.50	1.50
		250	-	1.00	1.00
		500	-	4.50	4.50
	Ethyl methanesulphonate	500	-	18.5*	18.5*
	Untreated	-	+	0.50	0.50
	Nu-Film-17 (Pinolene)	125	+	0.00	0.00
		500	+	0.00	0.50
		1000	+	0.50	1.00
	Cyclophosphamide	10	+	13.0*	13.0*
Second test, 32 hour harvest	Untreated	-	-	1.50	1.50
	Nu-Film-17 (Pinolene)	31.3	-	0.50	0.50
		125	-	4.50	4.50
		250	-	5.5*	5.5*
	Ethyl methanesulphonate	500	-	32.5*	32.5*
	Untreated		+	1.50	1.50
	Nu-Film-17 (Pinolene)	125	+	2.00	2.00
		500	+	1.00	1.00
		1000	+	1.50	1.50
	Cyclophosphamide	5	+	24.7*	25.2*

* Statistically significant

+ = Presence of S9 mix

- = Absence of S9 mix

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, no evidence of clastogenic activity was detected.