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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
α-Pinene is a bicyclic monounsaturated monoterpene and (-)-α-pinene is an enantiomeric form of α-pinene. Therefore, data on α-pinene can be extrapolated to (-)-α-pinene. (see read-across justification document in section 13).
Cross-reference
Reason / purpose:
read-across source

Data source

Materials and methods

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : FAB-29062016
Purity : 96.2% (sum of the two main constituents)
Name of test material (as cited in study report): alpha-pinene multiconstituent
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 28 June 2017

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no fluctuations in pH when the test item was dosed into media.
- Effects of osmolality: no fluctuations in osmolality of more than 50 mOsm at the dose levels invest
igated

- Cytotoxicity: concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 0.1, 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 µg/mL.
No precipitate was observed by eye at the end of treatment. A reduction in CBPI compared to vehicle control values, equivalent to 54.5% cytotoxicity, was obtained with Alpha-pinene multiconstituent at 27 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 13 and 27 µg/mL.

Any other information on results incl. tables

Preliminary Toxicity Test:

In all exposure conditions the highest concentration tested was 2000 µg/mL and no precipitate was observed by eye at the end of treatment.

After 3-hour treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 68.8% cytotoxicity, was obtained with alpha-pinene multiconstituent at 500 µg/mL. At higher tested concentrations, overt toxicity was observed.

After 3-hour treatment in the presence of S9-mix, no significant reduction in CBPI compared to vehicle control values was observed with alpha-pinene multiconstituent up to 250 µg/mL. At higher tested concentrations, overt toxicity was observed.

After 20-hour treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 31% cytotoxicity, was obtained with alpha-pinene multiconstituent at 125 µg/mL. At higher tested concentrations, overt toxicity was observed.

These results were used to select concentrations for the main test. 

Main Test:

3-hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 62.5, 125, 150, 175, 200, 225 and 250 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Additional 3-hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the additional main micronucleus test were 1, 10, 20, 40, 45, 50, 55, 60, 80, 100, 150, 200 and 250 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Second additional 3 -hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the second additional main micronucleus test were 1, 10, 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Third additional 3-hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the third additional main micronucleus test were 1, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45 µg/mL. No precipitate was observed by eye at the end of treatment. A reduction in CBPI compared to vehicle control values equivalent to 56.5% cytotoxicity, was obtained with Alpha-pinene multiconstituent at 33 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 27 and 33 µg/mL.

alpha-Pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle control.

Mean micronucleus induction in the vehicle control was close to the laboratory historical control limit and the data were considered acceptable for addition to the laboratories laboratory historical negative control database.

The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, within the laboratory historical positive control data, demonstrating the sensitivity of the test system

3-hour treatment in the presence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 125,250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Additional 3 -hour treatment in the presence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the additional main micronucleus test were 50, 100, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1000 µg/mL. No precipitate was observed by eye at the end of treatment. A reduction in CBPI compared to vehicle control values, equivalent to 50.6% cytotoxicity, was obtained with Alpha-pinene multiconstituent at 250 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 50, 100 and 250 µg/mL.

alpha-Pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle control.

Mean micronucleus induction in the vehicle control was within the laboratory historical control limit.

The positive control compound (cyclophosphamide) caused a statistically significant increase in the number of binucleate cells containing micronuclei, within the laboratory historical positive control data, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

20 -hour treatment in the absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 62.5, 125, 150, 175, 200, 225 and 250 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.

Additional 20-Hour Treatment in the Absence of S9 Mix:

Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 0.1, 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 µg/mL. No precipitate was observed by eye at the end of treatment. A reduction in CBPI copared to vehicle control values, equivalent to 54.5% cytotoxicity, was obtained with alpha-pinene multiconstituent at 27 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 13 and 27 µg/mL

alpha-Pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle control.

Mean micronucleus induction in the vehicle control was within the laboratory historical control limit.

The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, within the laboratory historical positive control data, demonstrating the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:
alpha-Pinene multiconstituent did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.
Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured human lymphocytes were exposed to test item, alpha-pinene multiconstituent.

Three alpha-pinene multiconstituent concentrations were assessed for determination of induction of micronuclei. The highest concentrations selected for micronucleus analysis were those which caused a reduction in cytokinesis-block proliferative index (CBPI) equivalent to 55±5% cytotoxicity. Following 3-hour treatment in the absence of S9 mix, reductions in CBPI equivalent to 56.5% cytotoxicity were obtained with alpha-pinene multiconstituent at 33 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 27 and 33 µg/mL. Following 3‑hour treatment in the presence of S9 mix, reductions in CBPI equivalent to 50.6% cytotoxicity were obtained with alpha-pinene multiconstituent at 250 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 50, 100 and 250 µg/mL. In the absence of S9 mix following 20‑hour treatment, a reduction in CBPI equivalent to 54.5% cytotoxicity was obtained with alpha-pinene multiconstituent at 27 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 13 and 27 µg/mL.

In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, alpha-pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls.

The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

It was concluded that alpha-pinene multiconstituent did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.