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Genetic toxicity in vitro

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In vitro bacteria reverse mutation: Weight of evidence: (+) alpha pinene was not mutagenic in all strains tested with and without metabolic activation. Furthermore, some experimental studies were performed with the analogue substance alpha pinene. All study results were negative. Based on these results, the read-across approach was applied and d-alpha pinene is considered negative under test conditions.

In vitro gene mutation in mammalian cells: Weight of evidence: Experimental results from studies performed with the analogue substances alpha pinene and 5-Ethylidene-2-norbornene. Both study results were negative. Based on these results, the read-across approach was applied and d-alpha pinene is considered to be non-mutagenic under test conditions.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
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, 400, 500, 600, 700, 800, 900, 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, 75, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 and 1000 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
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:
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 500 μg/plate and above)
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 1000 μg/plate)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 500 μg/plate and above
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 5000 μg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 μg/plate and above
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 1000 μg/plate)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 400 μg/plate and above)
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 5000 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 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:
valid
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 and above)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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

-

-

-

0 ± 0*

-

0 ± 0

0 ± 0

0 ± 0

2500

2 ± 1*

144 ± 12*

-

0± 0*

-

11 ± 14

0 ± 0

0 ± 0*

2000

2 ± 2*

141 ± 4*

-

-

-

-

-

-

1500

4 ± 6*

144*

-

-

-

-

-

-

1250

15 ± 21

-

-

-

-

-

-

-

1000

-

-

31 ± 12*

48 ± 6

168 ± 16*

181 ± 3

0 ± 0

13 ± 4*

900

-

-

23 ± 9*

-

212 ± 68*

-

-

-

800

-

-

24 ± 3*

-

225 ± 33*

-

-

-

700

-

-

28 ± 2*

-

272 ± 28*

-

-

-

600

-

-

26 ± 2*

-

177 ± 37

-

-

-

500

-

138 ± 2*

41 ± 12

48 ± 10

170 ± 41*

200 ± 21

0 ± 0*

22 ± 8

400

-

-

38 ± 5

-

150 ± 29*

-

-

-

250

-

-

-

-

-

174 ± 16

-

22 ± 3

100

-

-

-

52 ± 4

-

-

0 ± 0*

-

0

214 ± 6

166 ± 22

39 ± 6

59 ± 4

121 ± 26

201 ± 11

19 ± 1

20 ± 1

PC

1112 ± 64

716 ± 40

337 ± 39

278 ± 19

583 ± 27

879 ± 33

362 ± 27

165 ± 24

Set 2 experiments-

1000

293 ± 22

-

-

50 ± 13

-

136 ± 8

-

17 ± 4*

900

210 ± 8

174 ± 18

-

48 ± 4

-

109 ± 13

-

-

800

212 ± 12

200 ± 35

-

44 ± 8

-

127 ± 16

-

-

750

-

-

-

-

-

-

-

17 ± 4*

700

248 ± 42

178 ± 24

-

45 ± 6

-

143 ± 18

-

-

600

233 ± 6

150 ± 29

-

42 ± 1

-

136 ± 13

-

-

500

224 ± 6

158 ± 12

32 ± 8*

42 ± 8

181 ± 6*

126 ± 13

-

16 ± 1

400

227 ± 6

160 ± 12

29 ± 3

49 ± 8

160 ± 7

-

-

-

300

224 ± 8

-

34 ± 10

-

135 ± 32

-

-

-

250

-

-

-

-

-

-

-

22 ± 6

200

-

-

33 ± 2

-

112 ± 9

-

-

-

100

-

-

32 ± 8

-

141 ± 23

-

-

18 ± 4

75

-

-

-

-

-

-

3 ± 2*

-

50

-

-

34 ± 8

-

115 ± 2

-

11 ± 1

-

25

-

-

-

-

-

-

20 ± 2

24 ± 2

10

-

-

-

-

-

-

24 ± 1

-

5

-

-

-

-

-

-

24 ± 1

-

1

-

-

-

-

-

-

23 ± 4

-

0

171 ± 17

142 ± 21

37 ± 6

54 ± 3

128 ± 15

199 ± 37

25 ± 2

24 ± 3

PC

775 ± 69

716 ± 73

312 ± 20

423 ± 25

825 ± 115

866 ± 114

889 ± 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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Method similar to OECD guideline 471, but only two strains were tested with metabolic activation, no data on test item doses tested, no data on results with controls.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(Only two strains were tested with metabolic activation, no data on test item doses tested, no data on results with controls)
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:
No data
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:
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
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 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.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance d-alpha pinene is an enantiomeric form of the analogue substance alpha pinene, therefore they share the same functional groups and also have comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
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
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in bacteria

Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be not mutagenic in all strains tested with and without metabolic activation.

Conclusions:
Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be 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. Based on these results, d-alpha pinene was applied and the reaction mass is considered to be not mutagenic in the Ames test for the strains tested with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance d-alpha pinene is an enantiomeric form of the analogue substance alpha pinene, therefore they share the same functional groups and also have comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
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
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in the spot test (rat liver S9 fraction induced by Aroclor 1254 (S-9A))

Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be not mutagenic in all strains tested with and without metabolic activation.

Conclusions:
Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be 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. Based on these results, the read-across was applied and d-alpha pinene is considered to be not mutagenic in the Ames test for the strains tested with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance d-alpha pinene is an enantiomeric form of the analogue substance alpha pinene, therefore they share the same functional groups and also have comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
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
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in bacteria

Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be not mutagenic in all strains tested with and without metabolic activation.

Conclusions:
Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be 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. Based on these results, the read-across was applied and d-alpha pinene is considered to be not mutagenic in the Ames test for the strains tested with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance d-alpha pinene is an enantiomeric form of the analogue substance alpha pinene, therefore they share the same functional groups and also have comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
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
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
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in bacteria

Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be not mutagenic in all strains tested with metabolic activation.

Conclusions:
Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be 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. Based on these results, the read-across was applied and d-alpha pinene is considered to be not mutagenic in the Ames test for the strains tested with metabolic activation.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance d-alpha pinene is an enantiomeric form of the analogue substance alpha pinene, therefore they share the same functional groups and also have comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
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.
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in a unscheduled DNA synthesis test.

Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be not mutagenic in the rat hepatocyte unscheduled DNA synthesis assay.

Conclusions:
Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is considered to be not mutagenic in 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. Based on these results, the read-across approach was applied and d-alpha pinene is determined to be negative in the unscheduled DNA synthesis assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance 5-Ethylidene-2-norbornene (ENB) which shares the same functional groups with the target substance d-alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
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
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in the gene mutation assay.
Conclusions:
Based on read-across approach from the analogue ENB, d-alpha pinene is considered to be not mutagenic in the mammalian cell gene mutation assay.
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. Based on these results, the read-across approach was applied and d-alpha pinene is determined to be negative in the mammalian cell gene mutation assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Key study: Based on experimental results with analogue substance alpha pinene, read-across approach was applied and d-alpha pinene is determined to be not mutagenic in the Erythrocyte Micronucleus Test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
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 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
- aclimatation 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

5

1.4 ± 0.40

0.6426

3.10 ± 0.20

P=0.742e

Female

Air

0

5

1.4 ± 0.19

2.40 ± 0.19

Alpha pinene

25

5

2.1 ± 0.43

0.1182

2.16 ± 0.26

50

5

1.8 ± 0.25

0.2396

2.16 ± 0.20

100

5

1.7 ± 0.44

0.2949

2.74 ± 0.36

200

5

1.7 ± 0.30

0.2949

2.06 ± 0.29

400

5

1.1 ± 0.19

0.7259

2.16 ± 0.06

P=0.899

a Study was performed at ILS, Inc. The detailed protocol is presented by MacGregor et al. (1990). NCE=normochromatic erythrocyte; PCE=polychromatic erythrocyte

b Mean ± standard error

c Pairwise comparison with the chamber control group, significant at P≤0.005

d Chamber control

e Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test; significant at P≤0.025

Conclusions:
Alpha-Pinene was not mutagenic in the mouse peripheral blood micronucleus test
Executive summary:

In a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, alpha pinene was administered through inhalation to groups of B6C3F1 mice (5/sex/dose) at dose levels of 0, 25, 50, 100, 200 or 400 ppm; 5 days/week for 14 weeks. At the end of the study, peripheral blood samples were obtained from mice. Smear slides were air-dried, fixed, stained with fluorescent DNA-specific stain (acridine orange) and scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined.

No increase in the frequency of micronucleated erythrocytes and no significant changes in the percentages of polychromatic erythrocytes were observed in peripheral blood samples in male or female B6C3F1 mice administered alpha-pinene.

Therefore, alpha-pinene was not mutagenic in the mouse peripheral blood micronucleus test.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance d-alpha pinene is an enantiomeric form of the analogue substance alpha pinene, therefore they share the same functional groups and also have comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: read-across from an analogue determined to be not mutagenic in the Erythrocyte Micronucleus Test
Conclusions:
Based on read-across approach from the analogue alpha-pinene, d-alpha pinene is determined to be not mutagenic in the Erythrocyte Micronucleus Test.
Executive summary:

In a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, alpha pinene was administered through inhalation to groups of B6C3F1 mice (5/sex/dose) at dose levels of 0, 25, 50, 100, 200 or 400 ppm; 5 days/week for 14 weeks. At the end of the study, peripheral blood samples were obtained from mice. Smear slides were air-dried, fixed, stained with fluorescent DNA-specific stain (acridine orange) and scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined.

No increase in the frequency of micronucleated erythrocytes and no significant changes in the percentages of polychromatic erythrocytes were observed in peripheral blood samples in male or female B6C3F1 mice administered alpha-pinene. Therefore, alpha-pinene was not mutagenic in the mouse peripheral blood micronucleus test. Based on these results, the read-across approach was applied and d-alpha pinene is determined to be not mutagenic in the Erythrocyte Micronucleus Test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro gene mutation in bacteria: Weight of evidence:

(+) 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.

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. Based on these results, the read-across was applied and d-alpha pinene is considered to be not mutagenic in the Ames test for the strains tested with and without metabolic activation.

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. Based on these results, the read-across was applied and d-alpha pinene is considered to be not mutagenic in the Ames test for the strains tested with metabolic activation.

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. Based on these results, the read-across was applied and d-alpha pinene is considered to be not mutagenic in the Ames test for the strains tested with and without metabolic activation.

Alpha pinenewas 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 pinenewas not mutagenic in all strains tested with and without metabolic activation.Based on these results, d-alpha pinene was applied and the reaction mass is considered to be not mutagenic in the Ames test for the strains tested with and without metabolic activation.

In vitro gene mutation in mammalian cells. Weight of evidence:

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. Based on these results, the read-across approach was applied and d-alpha pinene is determined to be negative in the unscheduled DNA synthesis assay.

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-guaninephosphoribosyl transferase) gene locus in a medium containing the purine analog 6-thioguanine (6-TG). 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. Based on these results, the read-across approach was applied and d-alpha pinene is determined to be negative in the mammalian cell gene mutation assay.

In vivo genetic toxicity. Key study:

In a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, alpha pinene was administered through inhalation to groups of B6C3F1 mice (5/sex/dose) at dose levels of 0, 25, 50, 100, 200 or 400 ppm; 5 days/week for 14 weeks. At the end of the study, peripheral blood samples were obtained from mice. Smear slides were air-dried, fixed, stained with fluorescent DNA-specific stain (acridine orange) and scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) per animal. In addition, the percentage of polychromatic erythrocytes (PCEs) in a population of 1000 erythrocytes was determined.

No increase in the frequency of micronucleated erythrocytes and no significant changes in the percentages of polychromatic erythrocytes were observed in peripheral blood samples in male or female B6C3F1 mice administered alpha-pinene. Therefore, alpha-pinene was not mutagenic in the mouse peripheral blood micronucleus test. Based on these results, the read-across approach was applied and d-alpha pinene is determined to be not mutagenic in the Erythrocyte Micronucleus Test.

Justification for classification or non-classification

Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.