Pine, Pinus sylvestris, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several key studies were available on a similar NCS:
- Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).
- In vitro SCE and micronucleus tests: negative results (Kong, 1995; Kr.2)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 January - 22 March 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation. The study was conductecd on a similar NCS, Pine dwarf oil which has a similar composition.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 10 July 2012/ signed on 30 November 2012)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan gene

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % S9; S9 fraction prepared from liver homogenates of rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg bw/day by oral route

Test concentrations with justification for top dose:

Preliminary Toxicity Test (preincubation method):
- 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix. As toxicity was observed from 5 µg/plate a second preliminary test was performed.
- 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix.

Mutation Test:
Experiment 1 & 2 (preincubation method):
- All tester strains (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5 and 15 µg/plate.
- All Salmonella tester strains (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
- E.coli strain WP2uvrA (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetonitrile
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water, dimethyl sulphoxide, dimethyl formamide at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL but was fully miscible in acetonitrile at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetonitrile was therefore selected as the vehicle.
- Formulation preparation: Test item was accurately weighed and approximate half-log dilutions prepared in acetonitrile by mixing on a vortex mixer on the day of each experiment. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetonitrile

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetonitrile

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: All strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 1987.

METHOD OF APPLICATION: Preincubation (test item, vehicle and positive controls) and plate incorporation (negative control) methods

DURATION
- Preincubation period: 20 minutes at 37 °C with shaking at approximately 130 rpm
- Incubation period: Treated plates were placed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) and incubated at 37 °C for approximately 48 h.

NUMBER OF REPLICATIONS:
-1 plate/dose for preliminary toxicity test and 3 plates/dose for treatment, negative, vehicle and positive controls in mutation test (Experiment 1 & 2).

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was determined on the basis of growth of the bacterial background lawn.

OTHER: After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies using an automated colony counter.

Evaluation criteria:

There are several criteria for determining a positive result. Any one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
- Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

PRELIMINARY TOXICITY TEST:
- In the absence of S9-mix, the test item initially exhibited toxicity to both strains of bacteria from 5 µg/plate. In the presence of S9-mix, toxicity was noted to TA 100 from 150 µg/plate and to WP2uvrA from 50 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Mutation Test: The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 5 µg/plate in the absence of S9-mix (1.5 µg/plate for TA 1535 in Experiment 2 only) and 50 µg/plate (WP2uvrA) and 150 µg/plate (all Salmonella strains) in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical negative, solvent and positive control data (2010 and 2011) and lies within range.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile.
- The culture density for each bacterial strain was also checked and considered acceptable.
- Test item formulation used in this experiment was shown to be sterile.

Table 7.6.1/3:Preliminary Toxicity Test

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500
-	TA 100	91	102	112	84	75 S	74 S	73 S	73 S	65 V
+	TA 100	86	78	86	75	83	91	83	61 S	53 V
-	WP2 uvr A	13	16	12	14	12 S	15 S	11 S	17 S	15 V
+	WP2 uvr A	25	22	17	24	31	22	21 S	16 S	14 S

 

S: sparse bacterial background lawn

V: Very weak bacterial background lawn

See the attached Document for information on tables of results - Main experiments

Conclusions:

Under the test condition, Pine dwarf oil is not mutagenic with and without metabolic activation to strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to Pine dwarf oil at the following concentrations under anaerobic conditions: 

Preliminary Toxicity Test (preincubation method):

- 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix. As toxicity was observed from 5 µg/plate a second preliminary test was performed.

- 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix.

 

Mutation Test:

Experiment 1 & 2 (preincubation method):

- All tester strains (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5 and 15 µg/plate.

- All Salmonella tester strains (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.

- E.coli strain WP2uvrA (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.

 

Metabolic activation system used in this test was10 % S9; S9 fraction prepared from liver homogenates of rats induced with Phenobarbitone/β-Naphthoflavone. Negative, vehicle and positive control groups were also included in mutagenicity tests.

 

In preliminary toxicity test, the test item initially exhibited toxicity to both strains of bacteria from 5 µg/plate in the absence of S9-mix. In the presence of S9-mix, toxicity was noted to TA 100 from 150 µg/plate and to WP2uvrA from 50 µg/plate. In main experiments, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 5 µg/plate in the absence of S9-mix (1.5 µg/plate for TA 1535 in Experiment 2 only) and 50 µg/plate (WP2uvrA) and 150 µg/plate (all Salmonella strains) in the presence of S9-mix.No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study. 

 

Under the test conditions, Pine dwarf oil is not considered as mutagenic in these bacterial systems.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

In an in vitro micronucleus test, cultured human lymphocytes were exposed to Pine needle extract for 48 h in the absence of metabolic activation and examined for micronucleus induction.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 (complete growth medium)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

Pine needle extract: 0.50 mg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

cyclophosphamide - 30 ng/mL

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Healthy adult peripheral blood was received from Nanjing Center blood bank, China.

PREPARATION OF CULTURE: The peripheral blood 0.3 mL was added to 4.7 mL culture medium, cultured for 24 h at 37 °C.

METHOD OF APPLICATION: In RPMI 1640 medium

DURATION
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: Triplicate

CELL HARVEST AND SLIDE PREPARATION: According to Migliore, 1989.

NUMBER OF CELLS EVALUATED: For each treated concentration, 1000 lymphocytes carrying micronuclei were scored.

Evaluation criteria:

No data

Statistics:

The data were statistically evaluated with the t-test.

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 7.6.1/2: In vitro micronucleus test results

Group	Concentration		MNF (%) Mean ± SE
	CP (ng/mL)	PNE (mg/mL)
Distilled water			1.31 ± 0.57
CP	30	-	18.26 ± 1.51*
PNE	-	0.50	1.47 ± 0.72
PNE + CP	30	0.05	15.05 ± 1.58**
PNE + CP	30	0.50	11.31 ± 2.17***
PNE + CP	30	5.00	3.80 ± 0.87**

 

CP: Cyclophosphamide; PNE: Pine needle extract

Statistical significance: * p < 0.01, compared to distilled water control

* * p < 0.01, * * * p < 0.05 compared to CP groups

PNE could decrease the micronucleus frequency (MNF) compared with the cyclophosphamide (positive group) and the PNE + CP groups. These showed that PNE could decrease the micronucleus induced by CP, and micronucleus frequency decreased markedly at the dose of 5.0 mg/mL PNE (p < 0.01). The negative correlation between PNE dose and inhibition of MNF was distinct (r = - 0.9587, p < 0.05).

Conclusions:

Under the test conditions, Pine needle extract is not considered as clastogenic or aneugenic in cultured human lymphocytes following 48 h treatments in the absence of metabolic activation.

Executive summary:

In an in vitro micronucleus test, cultured human lymphocytes were exposed to Pine needle extract (PNE) at the concentrations of 0.50 mg/mL for 48 h (continuous treatment) in the absence of metabolic activation. Negative control (distilled water) and positive controls (cyclophosphamide at 30 ng/mL) were also included in the study. Antimutagenic effect of PNE was also studied with following dose levels (PNE + CP): 0.05 mg/mL + 30 ng/mL; 0.50 mg/mL + 30 ng/mL; 5.00 mg/mL + 30 ng/mL.

No statistically significant increase in micronuclei was observed in the PNE treated group when compared with negative control. PNE could decrease the micronucleus frequency (MNF) compared with the cyclophosphamide (positive group) and the PNE + CP groups. Results showed that PNE could decrease the micronucleus induced by CP, and micronucleus frequency decreased markedly at the dose of 5.0 mg/mL PNE (p < 0.01) demonstrating the antimutagenic effect. The positive control induced statistically significant increases in the proportion of cells with micronuclei, demonstrating the sensitivity of the test system.

 

Under the test conditions, Pine needle extract is not considered as clastogenic or aneugenic in cultured human lymphocytes following 48 h treatments in the absence of metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

In an in vitro SCE test, cultured human lymphocytes were exposed to Pine needle extract for 48 h in the absence of metabolic activation and examined for SCE in lymphocytes.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 (complete growth medium)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

without

Test concentrations with justification for top dose:

0.50 mg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

cyclophosphamide - 30 ng/mL

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Healthy adult peripheral blood was received from Nanjing Center blood bank, China.

PREPARATION OF CULTURE: The peripheral blood 0.3 mL was added to 4.7 mL culture medium, cultured for 24 h at 37 °C.

METHOD OF APPLICATION: In RPMI 1640 medium

DURATION
- Exposure duration: 48 h at 37 °C

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (10^ -7 mg/L) was added for 4 h before fixation.
STAIN (for cytogenetic assays): Giemsa's method with fluorescence (Perry and Wolff, 1974)

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: The SCE frequencies (SCEF) were obtained by observing and counting 100 metaphase ceils as a single for a terminal exchange and as a double for an intercolony one.

OTHERS: To make SCE evident, BUdR (5-bromo-2-deoxyuridine) was added at final concentration of 10 µg/mL) at 24 h.

Evaluation criteria:

No data

Statistics:

The data were statistically evaluated with the t-test.

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 7.6.1/2: SCE test results

Group	Concentration		Metaphases counted	SCEs/metaphase (Mean±SE)
	CP (ng/mL)	PNE (mg/mL)
Distilled water			106	6.32 ± 0.77
CP	30	-	104	27.42 ± 2.56*
PNE	-	0.50	127	7.16 ± 0.91
PNE + CP	30	0.05	142	24.81 ± 1.97**
PNE + CP	30	0.50	149	21.04 ± 2.89**
PNE + CP	30	5.00	164	7.68 ± 1.90**

 

CP: Cyclophosphamide; PNE: Pine needle extract

Statistical significance: * p < 0.01, compared to distilled water control; * * p < 0.01, compared to CP groups.

There were significant statistical differences between the negative control group and the CP group (p < 0.01), but there was no difference between the control group and the PNE group at the concentrations tested. The SCE frequencies (SCEF) decreased in all PNE + PC groups in comparison to the CP groups, and there was a distinct negative dose-response correlation (r = -0.9265) between PNE and SCEF in the PNE + CP groups. These showed that PNE could decrease the SCEF induced by CP, and SCEF decreased markedly at the dose of 5.0 mg/mL PNE (p < 0.01).

Conclusions:

Under the test conditions, Pine needle extract did not induce sister chromatid exchanges in cultured human lymphocytes following 48 h treatments in the absence of metabolic activation.

Executive summary:

In an in vitro sister chromatid exchange test, cultured human lymphocytes were exposed to Pine needle extract at the concentrations of 0.50 mg/mL for 48 h (continuous treatment) in the absence of metabolic activation. Negative control (distilled water) and positive controls (cyclophosphamide at 30 ng/mL) were also included in the study. Antimutagenic effect of PNE was also studied with following dose levels (PNE + CP): 0.05 mg/mL + 30 ng/mL; 0.50 mg/mL + 30 ng/mL; 5.00 mg/mL + 30 ng/mL.

 

No statistically significant increases in sister chromatid exchange was observed in the Pine needle extract treated group when compared with negative control. PNE could decrease the SCE frequency compared with the cyclophosphamide (positive group) and the PNE + CP groups. Results showed that PNE could decrease the SCE frequency induced by CP, and SCE frequency decreased markedly at the dose of 5.0 mg/mL PNE (p < 0.01) demonstrating the antimutagenic effect. The positive control induced statistically significant increases in sister chromatid exchange, demonstrating the sensitivity of the test system.

 

Under the test conditions, Pine needle extract did not induce sister chromatid exchanges in cultured human lymphocytes following 48 h treatments in the absence of metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

See RAAF document.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

PRELIMINARY TOXICITY TEST:
- In the absence of S9-mix, the test item initially exhibited toxicity to both strains of bacteria from 5 µg/plate. In the presence of S9-mix, toxicity was noted to TA 100 from 150 µg/plate and to WP2uvrA from 50 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Mutation Test: The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 5 µg/plate in the absence of S9-mix (1.5 µg/plate for TA 1535 in Experiment 2 only) and 50 µg/plate (WP2uvrA) and 150 µg/plate (all Salmonella strains) in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical negative, solvent and positive control data (2010 and 2011) and lies within range.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile.
- The culture density for each bacterial strain was also checked and considered acceptable.
- Test item formulation used in this experiment was shown to be sterile.

Table 7.6.1/3:Preliminary Toxicity Test

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500
-	TA 100	91	102	112	84	75 S	74 S	73 S	73 S	65 V
+	TA 100	86	78	86	75	83	91	83	61 S	53 V
-	WP2 uvr A	13	16	12	14	12 S	15 S	11 S	17 S	15 V
+	WP2 uvr A	25	22	17	24	31	22	21 S	16 S	14 S

 

S: sparse bacterial background lawn

V: Very weak bacterial background lawn

See the attached Document for information on tables of results - Main experiments

Conclusions:

Based on read-across approach, the target substance is not mutagenic with and without metabolic activation to strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to Pine dwarf oil at the following concentrations under anaerobic conditions: 

Preliminary Toxicity Test (preincubation method):

- 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix. As toxicity was observed from 5 µg/plate a second preliminary test was performed.

- 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix.

 

Mutation Test:

Experiment 1 & 2 (preincubation method):

- All tester strains (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5 and 15 µg/plate.

- All Salmonella tester strains (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.

- E.coli strain WP2uvrA (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.

 

Metabolic activation system used in this test was10 % S9; S9 fraction prepared from liver homogenates of rats induced with Phenobarbitone/β-Naphthoflavone. Negative, vehicle and positive control groups were also included in mutagenicity tests.

 

In preliminary toxicity test, the test item initially exhibited toxicity to both strains of bacteria from 5 µg/plate in the absence of S9-mix. In the presence of S9-mix, toxicity was noted to TA 100 from 150 µg/plate and to WP2uvrA from 50 µg/plate. In main experiments, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 5 µg/plate in the absence of S9-mix (1.5 µg/plate for TA 1535 in Experiment 2 only) and 50 µg/plate (WP2uvrA) and 150 µg/plate (all Salmonella strains) in the presence of S9-mix.No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study. 

 

Based on read-across approach, the target substance is not mutagenic with and without metabolic activation to strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

See RAAF document.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 7.6.1/2: In vitro micronucleus test results

Group	Concentration		MNF (%) Mean ± SE
	CP (ng/mL)	PNE (mg/mL)
Distilled water			1.31 ± 0.57
CP	30	-	18.26 ± 1.51*
PNE	-	0.50	1.47 ± 0.72
PNE + CP	30	0.05	15.05 ± 1.58**
PNE + CP	30	0.50	11.31 ± 2.17***
PNE + CP	30	5.00	3.80 ± 0.87**

 

CP: Cyclophosphamide; PNE: Pine needle extract

Statistical significance: * p < 0.01, compared to distilled water control

* * p < 0.01, * * * p < 0.05 compared to CP groups

PNE could decrease the micronucleus frequency (MNF) compared with the cyclophosphamide (positive group) and the PNE + CP groups. These showed that PNE could decrease the micronucleus induced by CP, and micronucleus frequency decreased markedly at the dose of 5.0 mg/mL PNE (p < 0.01). The negative correlation between PNE dose and inhibition of MNF was distinct (r = - 0.9587, p < 0.05).

Conclusions:

Based on the read-across approach, the target substance is not considered as clastogenic or aneugenic in cultured human lymphocytes following 48 h treatments in the absence of metabolic activation.

Executive summary:

In an in vitro micronucleus test, cultured human lymphocytes were exposed to Pine needle extract (PNE) at the concentrations of 0.50 mg/mL for 48 h (continuous treatment) in the absence of metabolic activation. Negative control (distilled water) and positive controls (cyclophosphamide at 30 ng/mL) were also included in the study. Antimutagenic effect of PNE was also studied with following dose levels (PNE + CP): 0.05 mg/mL + 30 ng/mL; 0.50 mg/mL + 30 ng/mL; 5.00 mg/mL + 30 ng/mL.

No statistically significant increase in micronuclei was observed in the PNE treated group when compared with negative control. PNE could decrease the micronucleus frequency (MNF) compared with the cyclophosphamide (positive group) and the PNE + CP groups. Results showed that PNE could decrease the micronucleus induced by CP, and micronucleus frequency decreased markedly at the dose of 5.0 mg/mL PNE (p < 0.01) demonstrating the antimutagenic effect. The positive control induced statistically significant increases in the proportion of cells with micronuclei, demonstrating the sensitivity of the test system.

 

Based on the read-across approach, the target substance is not considered as clastogenic or aneugenic in cultured human lymphocytes following 48 h treatments in the absence of metabolic activation.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

See RAAF document.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to other study

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 7.6.1/2: SCE test results

Group	Concentration		Metaphases counted	SCEs/metaphase (Mean±SE)
	CP (ng/mL)	PNE (mg/mL)
Distilled water			106	6.32 ± 0.77
CP	30	-	104	27.42 ± 2.56*
PNE	-	0.50	127	7.16 ± 0.91
PNE + CP	30	0.05	142	24.81 ± 1.97**
PNE + CP	30	0.50	149	21.04 ± 2.89**
PNE + CP	30	5.00	164	7.68 ± 1.90**

 

CP: Cyclophosphamide; PNE: Pine needle extract

Statistical significance: * p < 0.01, compared to distilled water control; * * p < 0.01, compared to CP groups.

There were significant statistical differences between the negative control group and the CP group (p < 0.01), but there was no difference between the control group and the PNE group at the concentrations tested. The SCE frequencies (SCEF) decreased in all PNE + PC groups in comparison to the CP groups, and there was a distinct negative dose-response correlation (r = -0.9265) between PNE and SCEF in the PNE + CP groups. These showed that PNE could decrease the SCEF induced by CP, and SCEF decreased markedly at the dose of 5.0 mg/mL PNE (p < 0.01).

Conclusions:

Based on the read-across approach, the target substance did not induce sister chromatid exchanges in cultured human lymphocytes in the absence of metabolic activation.

Executive summary:

In an in vitro sister chromatid exchange test, cultured human lymphocytes were exposed to Pine needle extract at the concentrations of 0.50 mg/mL for 48 h (continuous treatment) in the absence of metabolic activation. Negative control (distilled water) and positive controls (cyclophosphamide at 30 ng/mL) were also included in the study. Antimutagenic effect of PNE was also studied with following dose levels (PNE + CP): 0.05 mg/mL + 30 ng/mL; 0.50 mg/mL + 30 ng/mL; 5.00 mg/mL + 30 ng/mL.

 

No statistically significant increases in sister chromatid exchange was observed in the Pine needle extract treated group when compared with negative control. PNE could decrease the SCE frequency compared with the cyclophosphamide (positive group) and the PNE + CP groups. Results showed that PNE could decrease the SCE frequency induced by CP, and SCE frequency decreased markedly at the dose of 5.0 mg/mL PNE (p < 0.01) demonstrating the antimutagenic effect. The positive control induced statistically significant increases in sister chromatid exchange, demonstrating the sensitivity of the test system.

 

Based on the read-across approach, the target substance did not induce sister chromatid exchanges in cultured human lymphocytes in the absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

By read-across approach:

- In vivo micronucleus test: negative results (Kong, 1995; Kr.2)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Read across from structural analogue

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Principles of method if other than guideline:

In an in vivo micronucleus test, mice injected intraperitoneally with Pine needle extract and animals sacrificed for examination of micronucleated polychromatic erythrocytes.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Balb/c

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal Breeding Center of Nanjing University, Nanjing, China.
- Weight at study initiation: 18-22 g (at receipt)

Route of administration:

intraperitoneal

Duration of treatment / exposure:

30 h after test item administration

Frequency of treatment:

Single

Post exposure period:

None

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

10 males/dose

Control animals:

yes

Positive control(s):

- Positive control: Cyclophosphamide: 30 mg/kg bw
- Route of administration: Intraperitoneal

Tissues and cell types examined:

Micronucleus frequency in polychromatic erythrocytes (PCE) was determined by the routine method of Schmid (1973). 1000 PCEs in each animal were examined for micronucleus.

Evaluation criteria:

No data

Statistics:

The data were statistically evaluated with the t-test.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 7.6.2/2: In vivo micronucleus test results

Group	Concentration		MNF (%)	PCE (%)
	CP (mg/kg bw)	PNE (mg/kg bw)
Distilled water			2.18 ± 0.58	52.90 ± 4.71
CP	30	-	36.68 ± 1.56*	40.74 ± 4.42*
PNE	-	2000	2.38 ± 1.18	53.95 ± 4.50
PNE + CP	30	500	27.31 ± 1.29*	51.13 ± 4.21*
PNE + CP	30	1000	18.40 ± 2.73*	47.58 ± 3.25***
PNE + CP	30	2000	8.88 ± 1.38*	42.50 ± 4.32***
PNE + CP	30	4000	10.53 ± 2.42*	40.40 ± 3.96***

 

CP: Cyclophosphamide; PNE: Pine needle extract

* p < 0.01, compared to distilled water control; * * p < 0.01, * * * p < 0.05, compared to CP groups.

There was a significant negative correlation (r = -0.9782, p < 0.05) between PNE dose and MNF in the PNE-treated groups in comparison to the CP positive group up to 2000 mg/kg bw of PNE. However, when the dose of PNE reached 4000 mg/kg bw, it exhibited some increase of MNF compared with the 2000 mg/kg bw group. This suggested that PNE inhibited the MNF in certain concentration ranges.

Conclusions:

Under the test conditions, Pine needle extract did not induce micronuclei in mice.

Executive summary:

In an in vivo micronucleus test, ten male mice were injected intraperitoneally with Pine needle extract at the dose level of 2000 mg/kg bw and animals were sacrificed 30 h after the test item administration. Negative control (distilled water) and positive controls (cyclophosphamide at 30 mg/kg) were also included in the study. Antimutagenic effect of PNE was also studied with following dose levels at 10 males/group: (PNE + CP): 500 mg/kg bw + 30 mg/kg bw; 1000 mg/kg bw + 30 mg/kg bw; 2000 mg/kg bw + 30 mg/kg bw; 4000 mg/kg bw + 30 mg/kg bw.

Test material did not induce micronucleated PCE when compared with negative control. PNE could decrease the micronucleus frequency (MNF) compared with the cyclophosphamide (positive group) and the PNE + CP groups. Results showed that PNE could decrease the micronucleus induced by CP, and micronucleus frequency decreased markedly at the dose of 2000 mg/kg bw PNE (p < 0.01) demonstrating the antimutagenic effect. Positive control induced a statistically significant increase in micronucleated polychromatic erythrocytes indicating the validity of the study.

 

Under the test conditions, Pine needle extract did not induce micronuclei in mice.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Read across from structural analogue

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to other study

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Table 7.6.2/2: In vivo micronucleus test results

Group	Concentration		MNF (%)	PCE (%)
	CP (mg/kg bw)	PNE (mg/kg bw)
Distilled water			2.18 ± 0.58	52.90 ± 4.71
CP	30	-	36.68 ± 1.56*	40.74 ± 4.42*
PNE	-	2000	2.38 ± 1.18	53.95 ± 4.50
PNE + CP	30	500	27.31 ± 1.29*	51.13 ± 4.21*
PNE + CP	30	1000	18.40 ± 2.73*	47.58 ± 3.25***
PNE + CP	30	2000	8.88 ± 1.38*	42.50 ± 4.32***
PNE + CP	30	4000	10.53 ± 2.42*	40.40 ± 3.96***

 

CP: Cyclophosphamide; PNE: Pine needle extract

* p < 0.01, compared to distilled water control; * * p < 0.01, * * * p < 0.05, compared to CP groups.

There was a significant negative correlation (r = -0.9782, p < 0.05) between PNE dose and MNF in the PNE-treated groups in comparison to the CP positive group up to 2000 mg/kg bw of PNE. However, when the dose of PNE reached 4000 mg/kg bw, it exhibited some increase of MNF compared with the 2000 mg/kg bw group. This suggested that PNE inhibited the MNF in certain concentration ranges.

Conclusions:

Based on the read-across approach, the target substance did not induce micronuclei in mice.

Executive summary:

In an in vivo micronucleus test, ten male mice were injected intraperitoneally with Pine needle extract at the dose level of 2000 mg/kg bw and animals were sacrificed 30 h after the test item administration. Negative control (distilled water) and positive controls (cyclophosphamide at 30 mg/kg) were also included in the study. Antimutagenic effect of PNE was also studied with following dose levels at 10 males/group: (PNE + CP): 500 mg/kg bw + 30 mg/kg bw; 1000 mg/kg bw + 30 mg/kg bw; 2000 mg/kg bw + 30 mg/kg bw; 4000 mg/kg bw + 30 mg/kg bw.

Test material did not induce micronucleated PCE when compared with negative control. PNE could decrease the micronucleus frequency (MNF) compared with the cyclophosphamide (positive group) and the PNE + CP groups. Results showed that PNE could decrease the micronucleus induced by CP, and micronucleus frequency decreased markedly at the dose of 2000 mg/kg bw PNE (p < 0.01) demonstrating the antimutagenic effect. Positive control induced a statistically significant increase in micronucleated polychromatic erythrocytes indicating the validity of the study.

 

Based on the read-across approach, the target substance did not induce micronuclei in mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of genotoxicity test (tested substance: pine dwarf oil)

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Thompson, 2013	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA-	-S9 +S9	Up to cytotoxic concentration	-S9 : non mutagenic +S9 : non mutagenic
2Kong, 1995	in vitro SCE (Kr. 4)	Chromosome aberration	human lymphocytes	-S9	0.5 mg/mL	negative (+ antimutagenic effects on cyclophosphamide)
3 Kong, 1995	in vitro micronucleus (Kr. 4)	Chromosome aberration	human lymphocytes	-S9	0.5 mg/mL	negative (+ antimutagenic effects on cyclophosphamide)
4Kong, 1995	in vivo micronucleus (Kr.4)	Chromosome aberration	Mouse	N/A	2000 mg/kg bw	negative (+ antimutagenic effects on cyclophosphamide)

 

 

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with Pine dwarf oil. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. Pine dwarf oil does not induce gene mutations in bacteria under the test conditions whereas the positive control chemical (with and without metabolic activation) induced significant increase of colonies. Pine dwarf oil is therefore considered as non-mutagenic according to the Ames test.

Published data was also available on Pine dwarf oil which showed that this oil didi not induced chromosome aberration in vitro and in vivo. According to this article Pine dwarf oil has even antimutagenic effect on cyclophosphamide.

The composition of Pine dwarf oil is very similar compared to and cover the one of Pine scotch. Therefore it can be considered that Pine scotch is not genotoxic (read-across approach).

Justification for classification or non-classification

Harmonized classification:

Pine scotch oil has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the data available, Pine scotch oil is not classified as genotoxic according to CLP and UN GHS criteria.