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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 04 to June 23, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD guideline 473 without any deviation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Terpineol
- Physical state: Colourless to slightly amber liquid
- Storage condition of test material: Stored at 15-25 °C in the dark under nitrogen; test solutions were protected from light and used within approximately 4 hours of initial formulation
- Solubility: Miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 174.6 mg/mL. Solubility limit in culture medium was approximately 873.1-1746 µg/mL as indicated by precipitation at the higher concentration which persisted for approximately 20 hours after test article addition.

Method

Species / strain
Species / strain / cell type:
lymphocytes: cultures prepared from the pooled blood of three male donors
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2% S9 fraction of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Range-finder experiment: 5.598-1543 μg/mL (with and without S-9)
Main study:
- Experiment 1: Without S-9: 0, 350, 425 and 450 μg/mL; with S-9: 0, 300, 550 and 625 μg/mL
- Experiment 2: Without S-9: 0, 75, 200 and 225 μg/mL; with S-9: 0, 400, 550, 625 and 650 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation

Migrated to IUCLID6: 2.5 and 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

Migrated to IUCLID6: 10, 20 and 30 µg/mL
Details on test system and experimental conditions:
PREPARATION OF CULTURES: Whole blood cultures pooled from three healthy, non-smoking male volunteers were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 µg/mL gentamycin, so that the final volume following addition of S9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1 °C for approximately 48 hours and rocked continuously.

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 3 or 20 hours, 37 ± 1 ºC
- Fixation time (start of exposure up to harvest of cells): 20 or 20.75 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine, 1 µg/mL for 2 hours
STAIN (for cytogenetic assays): Giemsa (4% v/v)

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: At least 1000 cells/dose were counted in cytotoxicity test to determine the mitotic index; at least 200 metaphase cells/dose were analysed for chromosomal aberrations

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- Cells with structural aberrations including or excluding gaps, polyploidy, hyperdiploidy or endoreduplication were recorded during the study.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range were observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p ≤ 0.05)
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).

- Test article was considered as positive in this assay if all of the above criteria were met.
- Test article was considered as negative in this assay if none of the above criteria were met.
- Results which only partially satisfied the above criteria were dealt with on a case by case basis.
Statistics:
- Statistical method used was Fisher's exact test.
- Proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
- Probability values of p ≤ 0.05 were accepted as significant.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolality: No marked changes in osmolality or pH were observed at the highest concentration tested in the range-finding cytotoxicity experiment (1543 µg/mL, equivalent to 10 mM), compared to the concurrent vehicle controls.
- Solubility/Precipitation: Miscible with anhydrous analytical grade dimethyl sulphoxide (DMSO) at a concentration of at least 174.6 mg/mL. Solubility limit in culture medium was approximately 873.1-1746 µg/mL as indicated by precipitation at the higher concentration which persisted for approximately 20 hours after test article addition.

RANGE-FINDING/SCREENING STUDIES:
- In the range-finding cytotoxicity study, precipitation was observed at or above 200 μg/mL and complete cytotoxicity was seen at or above 925.8 µg/mL tested with or without S-9.
- See table 2 for more data

COMPARISON WITH HISTORICAL CONTROL DATA: Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control (normal) ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Range-finder experiment: mitotic index determinations

Treatment

Mitotic index (%)

(µg/mL)

3+17 hours, -S-9

3+17 hours, +S-9

20+0 hours, -S-9

 

A

B

MIH*

A

B

MIH*

A

B

MIH*

Vehicle

5.7

7.4

-

7.0

5.1

-

5.9

6.7

-

5.598

6.2

NT

5

6.7

NT

0

6.0

NT

5

9.330

5.6

NT

15

5.7

NT

6

6.3

NT

0

15.55

7.7

NT

0

5.1

NT

16

5.4

NT

14

25.92

4.5

NT

31

6.2

NT

0

5.3

NT

16

43.19

6.0

NT

8

7.8

NT

0

6.4

NT

0

71.99

6.2

NT

5

6.2

NT

0

6.0

NT

5

120.0

6.8

NT

0

7.1

NT

0

4.9

NT

22

200.0

6.4

NT

2P

4.0

NT

34P

4.5

NT

29P

333.3

6.1

NT

7P

4.8

NT

21P

2.2

NT

65P

555.5

0.0

NT

100P

4.0

NT

34P

0.0

NT

100P

925.8

T

NT

100P

T

NT

100P

T

NT

100P

1543

T

NT

100P

T

NT

100P

T

NT

100P

NT = Not tested; P = Precipitation observed at treatment; T = Toxic

*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

Table 3: Results summary

Treatment

Concentration (mg/mL)

Cytotoxicity (%)

% Cells with Chromosome Aberrations (Excluding Gaps)

Historical (%)#

Statistical significance

Experiment 1

3+17.75 hour -S-9

Vehiclea

-

0.50

0-3

-

 

350.0

0

0.50

 

NC

 

425.0

29

1.00

 

NC

 

450.0

50

0.50

 

NC

 

*NQO, 2.50

ND

8.00

 

p ≤ 0.001

3+17.75 hour +S-9

Vehiclea

-

0.50

0-3

-

 

300.0

0

1.00

 

NC

 

550.0

35

3.00

 

NC

 

625.0

50

5.00

 

NC

 

*CPA, 10.00

ND

25.83

 

p ≤ 0.001

Experiment 2

20+0 hour -S-9

Vehiclea

-

1.00

0-3

-

 

75.00

18

0.00

 

NC

 

200.0

34

1.50

 

NC

 

225.0

53

1.00

 

NC

 

*NQO, 5.00

ND

29.37

 

p ≤ 0.001

3+17 hour +S-9

Vehiclea

-

0.50

0-3

-

 

400.0

7

0.50

 

NC

 

550.0

34

1.50

 

NC

 

625.0

39

2.50

 

NC

 

650.0

50

1.00

 

NC

 

*CPA, 20.00

ND

42.11

 

p ≤ 0.001

a Vehicle control was DMSO

* Positive control

#95th percentile of the observed range

NC = Not calculated

ND = Not determined

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the test conditions, Terpineol-Multi is not considered as clastogenic in human lymphocytes according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to Terpineol-Multi in DMSO at concentration range of 5.598-1543 μg/mL, for 3 + 17 h (treatment + recovery) with metabolic activation (2% S-9 fraction of Aroclor 1254-induced male Sprague-Dawley rats), and for 3 + 17 h or 20 + 0 h (treatment + recovery) without metabolic activation for a preliminary cytotoxicity test (Lloyd 2010). In the main test, two experiments were performed at concentrations up to 600 µg/mL without S-9 and up to 800 µg/mL with S-9 and the following concentrations were selected for analysis: Experiment 1: Without S-9 (treatment: 3 h): 0, 350, 425 and 450 μg/mL; with S-9 (treatment: 3 h): 0, 300, 550 and 625 μg/mL. Experiment 2: Without S-9 (treatment: 20 h): 0, 75, 200 and 225 μg/mL; with S-9 (treatment: 3 h): 0, 400, 550, 625 and 650 μg/mL. Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control ranges. Positive controls (4-nitroquinoline-N-oxide at 2.5 and 5 µg/mL without S-9 and cyclophosphamide at 10, 20 and 30 µg/mL with S-9) induced the appropriate response. Treatment of cells with Terpineol-Multi in the presence or absence of S-9 in both experiments resulted in frequencies of cells with structural or numerical aberrations that were generally similar to those observed in concurrent vehicle controls for all concentrations analysed. Numbers of aberrant cells (excluding gaps) in treated cultures fell within the normal range with the exception of one culture at the highest concentration analysed with S-9 in experiment 1 (625.0 µg/mL). However, the aberration frequency (excluding gaps) in the replicate culture at 625.0 µg/mL in experiment 1 and in all other cultures analysed in experiments 1 and 2 fell within the normal range. Under the test conditions, Terpineol-Multi is not considered as clastogenic in human lymphocytes.