Cypress, Cupressus sempervirens, ext.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A weight of evidence strategy was followed to complete the endpoint requirements. Negative Ames and UDS tests performed on delta-3-carene and alpha-pinene, the main components of the Cypress oil.

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:

weight of evidence

Study period:

From April 16 to May 24, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD guideline 471 without any deviation.

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), obtained from Molecular Toxicology Incorporated, USA

Test concentrations with justification for top dose:

- Range-finder experiment: 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without S-9);
- Experiment 1: 0.064, 0.32, 1.6, 8, 40, 200 and 1000 µg/plate, (with and without S-9);
- Experiment 2: 7.813, 15.63, 31.25, 62.5, 125, 250 and 500 µg/plate (strain TA102 in the absence of S-9 and strains TA 100 and TA 1535 in the presence of S-9);
0.9766, 1.953, 3.906, 7.813, 15.63, 31.25 and 62.5 µg/plate (strains TA 98, TA 100, TA 1535 and TA 1537 in the absence of S-9 and strains TA 98, TA 1537 and TA 102 in the presence of S-9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Solubility of delta-3-carene in DMSO = 100 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation method; as the results of Experiment 1 were negative, treatments in the presence of S-9 in experiment 2 included a pre-incubation step (incubation for 1 hour at 37±1°C).
Due to the suspected volatility of the test item, test article plates were incubated in sealed boxes with each concentration of test article in a separate box and the vehicle and positive controls were incubated in a separate incubator.
In the Range-Finder experiment, the vehicle and positive control plates were not incubated in a separate incubator to the test article plates in error. These data were used for toxicity assessment only.
In Experiment 1, the plates were not incubated in sealed boxes in error and were placed in the same incubator as the control plates. The controls are consistent with the historical control data and the toxicity profile is the same as that in the Range-Finder where the treatment plates for each concentration were placed in a sealed box. The suspected volatility of the test article has not affected the other plates and is considered not to have impacted on the integrity of the study.

DURATION
For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate. Incubation was carried out with shaking in an anhydric incubator. All treatments were completed within 6 hours of the end of the incubation period.
After plating with test substance or control, the plates were inverted and incubated at 37±1°C in the dark for 3 days.

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:
Range-finding test: triplicate plates; negative (vehicle) and positive controls were included in quintuplicate and triplicate, respectively.
Main experiments: triplicate plates; negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants; thinning of background bacterial lawn.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
- Dunnett's test gave a significant response (p=< 0.01) which was concentration related
- The positive trend/effects described above were reproducible.
Negative: If all of the above criteria were not met

Statistics:

Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additionnal information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additionnal information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Range-finder experiment: slight thinning of the background bacterial lawn was observed at 40 µg/plate and above with and without S-9

- Experiment 1: diminution of the background bacterial lawn: At 40 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 and strains TA98, TA1537 and TA102 in the presence of S-9; at 200 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S 9.
Reduction in revertant numbers: in strain TA1537 in the absence of S-9 at 200 µg/plate and above and in strains TA1537 and TA102 in the presence of S-9 at 1000 µg/plate

- Experiment 2: slight thinning of the background bacterial lawn: at 31.25 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 and strains TA98, TA537 and TA102 in the presence of S-9 and at 250 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9

None

Conclusions:

Under the test conditions, Delta-3-carene is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and TA102.

Executive summary:

In a GLP study performed according to OECD guideline 471, delta-3 -carene was tested for mutagenicity using Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with the plate incorporation and preincubation methods in the presence and absence of metabolic activation system (S-9 mix).

Based on the results of range-finder test, using TA100 tested at concentrations between 1.6 and 5000 µg/plate with and without S-9, all strains in experiment 1 were tested with and without S-9 at the concentration range of 0.064 - 1000 µg/plate. Due to the cytotoxicity found in experiment 1, the following concentration ranges were tested in the second experiment, using the preincubation method (1h at 37°C) when S-9 was added: 7.813 - 500 µg/plate (strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9) or 0.9766 - 62.5 µg/plate (strains TA98, TA100, TA1535 and TA1537 in the absence of S‑9 and strains TA98, TA1537 and TA102 in the presence of S-9).

In range-finder experiment, evidence of toxicity was observed at 40 µg/plate and above in the absence and presence of S-9. In experiment 1, evidence of toxicity was observed at 40 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S‑9 and strains TA98, TA1537 and TA102 in the presence of S‑9 and at 200 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9. In experiment 2, toxicity was observed at 31.25 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 and strains TA98, TA1537 and TA102 in the presence of S-9 and at 250 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9. No precipitation of test substance was observed at any of the doses used.

The positive controls induced the appropriate responses in the corresponding strains. Delta-3-carene showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S-9.

Under the test conditions, Delta-3-carene  is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and TA102.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

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:

Method: Plate incorporation assay method (Ames et al., 1973 and McCann et al., 1975)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of Aroclor 1254-induced adult male Sprague Dawley rat liver

Test concentrations with justification for top dose:

Up to 25000 µg or nL/plate

Vehicle / solvent:

Some insoluble materials were tested in aqueous or DMSO extracts

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: Two days (37 °C)

NUMBER OF REPLICATIONS: No data

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

None

None

Conclusions:

Under the test conditions, α-pinene is not considered as mutagenic in bacteria.

Executive summary:

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 100 and TA 98) were exposed to α-pinene at concentrations up to 25000 µg or nL/plate in both the absence and presence of metabolic activation (S9 fraction of Aroclor 1254-induced adult male Sprague-Dawley rat liver) according to the direct plate incorporation method for a preliminary cytotoxicity test.

 

α-Pinene showed no substantial increases in revertant colony numbers obtained with any of the tester strains at the concentration tested in either presence or absence of S9 mix.

 

Under the test conditions, α-pinene is not considered as mutagenic in this bacterial system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study conducted equivalent or similar to OECD Guideline 471 with deviations: no data on test material purity, number of cells per culture and number of replicates used; only 3 strains tested; no individual plate counts

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no data on test material purity, number of cells per culture and number of replicates used; only 3 strains tested; no individual plate counts

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

10% S9 fraction of induced male Sprague Dawley rat liver

Test concentrations with justification for top dose:

- E. coli WP2 uvr A pKM 101: 0, 100, 500, 1000, 5000 and 10000 µg/plate
- TA 100: 0, 5, 10, 25, 50, 75, 100, 200, 250, 400, 500, 1000, 1500, 2500, 5000 and 10000 µg/plate
- TA 98: 0, 5, 10, 20, 25, 30, 40, 50, 75, 100, 200, 250, 400, 500, 1000, 1500, 2500, 5000 and 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (or occasionally, sterigmatocystin)

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA100); 2-nitrofluorene or 4-nitro-o-phenylenediamine (TA98); methyl methanesulfonate (E.coli WP2 uvrA pKM101)

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes (37 °C)
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: No data

Evaluation criteria:

- Spontaneous mutations (those that occur by chance, not by chemical treatment) will appear as colonies on the control petri dishes. If the test chemical was mutagenic to any particular strain of bacterium, the number of histidine-independent colonies arising on those plates will be significantly greater than the corresponding control plates for that strain of bacteria.
- The positive control plates are also counted, and the number of mutant colonies appearing on them must be significantly increased over the spontaneous control number for the test to be considered valid. Failure of the positive control chemical to induce mutation is reason to discard the experiment.
- If no increase in mutant colonies is seen after testing several strains under several different culture conditions, the test chemical is considered to be non-mutagenic in the Ames test.

Statistics:

No data

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slightly toxic at 50, 100 and 200 µg/plate; toxic at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at 40 µg/plate when S. typhimurium TA 98 was exposed to the test material without metabolic activation

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity (slightly toxic at 50, 100 and 200 µg/plate and toxic at 500 µg/plate) was observed when S. typhimurium TA 98 was exposed to the test material without metabolic activation

Table 1: Mutagenicity of α-pinene in Escherichia coli strain WP2 uvrA pKM101

Dose	NA		10% RLI		NA		NA		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	181	46.1	266	53.5	136	1.5	161	7.2	204	12.3
100	90	7.8	210	1.5	139	4.4	141	5.2	224	4.1
500	89	1.9	208	3.3	175	31.4	135	11.8	193	13.1
1000	84	18.7	287	46	156	8.7	137	3.2	207	17.2
5000	94	4.7	162	10.5	148	3.1	132	4	184	20.8
10000	78	5.1	174	23.6	165	9.6	126	2.3	188	12.5
Positive Control	1183	140	710	32.1	930	19.3	1529	121.3	1171	9.9

Table 2: Mutagenicity of α-pinene in Salmonella typhimurium TA 100

Dose	10% RLI		NA		10% RLI		NA		10% RLI		NA		10% RLI		NA
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Not a Valid Test)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	70	2.2	49	2	63	3.8	57	7.2	56	1.8	38	3.8	48	9.7	0	0
5							48	2.3
10			46	5.3			49	4.3			45	5.9			0	0
25							52	1.8
50	65	5.5	44	3.5	57	5.2	29	2.9			41	13.4	50	6.7	0	0
75							14	0.6
100	80	1.2	63	4.5	85	15					54	14.7	40	3.5	0	0
200			57	4.2											0	0
250											19	5.8
400			10	0.9											0	0
500	63	5.9			48	4.4			49	4.4	12	3.7	52	5.5
1000	58	10.8			39	2.6							38	2.3
1500									46	2
2500									50	2
5000	33	3.2			90	4.3			21	0.9			27s	2.1
10000									11	0.7
Positive Control	738	30.5	428	39.3	1161	65.4	387	19.3	665	29.1	574	1.2	510	26.7	653	31.7

Table 3: Mutagenicity of α-pinene in Salmonella typhimurium TA 98

Dose	NA		NA		NA		10% RLI		10% RLI		NA		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Not a Valid Test)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	22	2.7	15	2.9	21	2.4	30	4.2	28	5.3	36	2	22	2.1
5			10	3.2	23	3.2
10	25	3	9	0.6	19	4.4					25	1.8
20			8	3.1
25					18	0.3
30			6	0.9
40			4p	1.2
50	8s	1.7			18	2.7			20	2.9	11	2.2	22	1.2
75					9	1.9
100	15s	2.2							31	3.2	1	1.3	21	2.6
200											0	0
250	4s	1.2
400											3	0.9
500	t						32	0.7	25	2.3			19	2.4
1000									28	4.8			35	2.5
1500							24	5.4
2500							21	1.2
5000							13	2.7	15s	1.2			7	0.7
10000							9	0.6
Positive Control	468	17.3	506	17.4	295	14.3	418	21.8	1008	31.5	417	30.7	372	38.1

Abbreviations: RLI = induced male Sprague Dawley rat liver S9; p = Precipitate; s = Slight Toxicity; T = Toxic

Conclusions:

Under the test conditions, α-pinene is not considered as mutagenic in this bacterial system with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD guideline 471, strains of S. typhimurium (TA 100 and TA 98) and one Escherichia coli strain (WP2 uvrA pKM101) were exposed to α-pinene at concentration range of 0-10000 µg/plate in both the absence and presence of S9 metabolic activation from Sprague-dawley rats according to the pre-incubation method.

 

The positive controls induced the appropriate responses in the corresponding strains. α-Pinene showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S9 mix. When S. typhimurium TA 98 exposed to α-pinene without metabolic activation cytotoxicity was observed at 50 µg/plate and precipitation occurred at 40 µg/plate.

 

Under the test conditions, α-pinene is not considered as mutagenic in this bacterial system with and without metabolic activation.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Documentation insufficient for assessment: no data on test material purity, source and concentration units; conditions (temperature and CO2 concentration) during maintenance of cell cultures; details of metabolic activation systems; procedures used to block entry of cells into S-phase; negative/positive controls; grain count in individual culture

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:

Method: Unscheduled DNA synthesis assay (UDS) (Williams, 1977; Williams, 1980 and Butterworth et al., 1987)

GLP compliance:

not specified

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

Not applicable

Species / strain / cell type:

hepatocytes: isolated from adult male Fischer or Sprague-Dawley rats

Details on mammalian cell type (if applicable):

- Type and identity of media: Serum-free WME medium containing 5-10 µCi/mL H-TdR (20 Ci/mM)
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Metabolic activation system:

no data

Test concentrations with justification for top dose:

Up to 10000 µg or nL/mL

Vehicle / solvent:

Some insoluble materials were tested in aqueous or DMSO extracts

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18-20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 10-12 minutes

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: 75 or 150 cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: Relative cell survival was determined by concurrent cell counting or by measurement of LDH release from treated cells.

Evaluation criteria:

A positive UDS response at a given concentration was indicated by an increase in net nuclear grain count (NNG) of at least 6 grains per nucleus above the concurrent solvent control value and/or an increase in the percent of nuclei having 6 or more net grains to at least 10% above the concurrent negative control value .

Statistics:

No data

Species / strain:

hepatocytes: isolated from adult male Fischer or Sprague-Dawley rats

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

None

None

Conclusions:

Under the test conditions, α-pinene is not considered as mutagenic in the mammalian cell system with and without metabolic activation.

Executive summary:

In an Unscheduled DNA Synthesis (UDS) assay in mammalian cells, hepatocytes isolated from adult male Fischer or Sprague-Dawley rats were exposed to α-pinene at concentrations up to 10000 µg or nL/mL for 18-20 hours in serum-free WME medium containing 5-10 µCi/mL H-TdR (20 Ci/mM). Stained triplicate cultures were examined microscopically at 1500x under oil immersion and UDS was measured by automatic electronic counter and expressed as net nuclear grain count (NNG).

 

α-pinene was found to be negative in UDS assay.

 

Under the test conditions, α-pinene is not considered as mutagenic in this mammalian cell system with and without metabolic activation.

Reference 5

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:

weight of evidence

Justification for type of information:

See RAAF document.

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additionnal information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see additionnal information on results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Range-finder experiment: slight thinning of the background bacterial lawn was observed at 40 µg/plate and above with and without S-9

- Experiment 1: diminution of the background bacterial lawn: At 40 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 and strains TA98, TA1537 and TA102 in the presence of S-9; at 200 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S 9.
Reduction in revertant numbers: in strain TA1537 in the absence of S-9 at 200 µg/plate and above and in strains TA1537 and TA102 in the presence of S-9 at 1000 µg/plate

- Experiment 2: slight thinning of the background bacterial lawn: at 31.25 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 and strains TA98, TA537 and TA102 in the presence of S-9 and at 250 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9

None

Conclusions:

Based on the read-across approach, the target substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and TA102.

Executive summary:

In a GLP study performed according to OECD guideline 471, delta-3 -carene was tested for mutagenicity using Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with the plate incorporation and preincubation methods in the presence and absence of metabolic activation system (S-9 mix).

Based on the results of range-finder test, using TA100 tested at concentrations between 1.6 and 5000 µg/plate with and without S-9, all strains in experiment 1 were tested with and without S-9 at the concentration range of 0.064 - 1000 µg/plate. Due to the cytotoxicity found in experiment 1, the following concentration ranges were tested in the second experiment, using the preincubation method (1h at 37°C) when S-9 was added: 7.813 - 500 µg/plate (strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9) or 0.9766 - 62.5 µg/plate (strains TA98, TA100, TA1535 and TA1537 in the absence of S‑9 and strains TA98, TA1537 and TA102 in the presence of S-9).

In range-finder experiment, evidence of toxicity was observed at 40 µg/plate and above in the absence and presence of S-9. In experiment 1, evidence of toxicity was observed at 40 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S‑9 and strains TA98, TA1537 and TA102 in the presence of S‑9 and at 200 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9. In experiment 2, toxicity was observed at 31.25 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 and strains TA98, TA1537 and TA102 in the presence of S-9 and at 250 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9. No precipitation of test substance was observed at any of the doses used.

The positive controls induced the appropriate responses in the corresponding strains. Delta-3-carene showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S-9.

Based on the read-across approach, the target substance is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100 and TA102.

Reference 6

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:

weight of evidence

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:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

None

None

Conclusions:

Based on the read-across approach, the target substance is not considered as mutagenic in bacteria with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, strains of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 100 and TA 98) were exposed to α-pinene at concentrations up to 25000 µg or nL/plate in both the absence and presence of metabolic activation (S9 fraction of Aroclor 1254-induced adult male Sprague-Dawley rat liver) according to the direct plate incorporation method for a preliminary cytotoxicity test.

 

α-Pinene showed no substantial increases in revertant colony numbers obtained with any of the tester strains at the concentration tested in either presence or absence of S9 mix.

 

Based on the read-across approach, the target substance is not considered as mutagenic in bacteria with and without metabolic activation.

Reference 7

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:

weight of evidence

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:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slightly toxic at 50, 100 and 200 µg/plate; toxic at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at 40 µg/plate when S. typhimurium TA 98 was exposed to the test material without metabolic activation

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity (slightly toxic at 50, 100 and 200 µg/plate and toxic at 500 µg/plate) was observed when S. typhimurium TA 98 was exposed to the test material without metabolic activation

Table 1: Mutagenicity of α-pinene in Escherichia coli strain WP2 uvrA pKM101

Dose	NA		10% RLI		NA		NA		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	181	46.1	266	53.5	136	1.5	161	7.2	204	12.3
100	90	7.8	210	1.5	139	4.4	141	5.2	224	4.1
500	89	1.9	208	3.3	175	31.4	135	11.8	193	13.1
1000	84	18.7	287	46	156	8.7	137	3.2	207	17.2
5000	94	4.7	162	10.5	148	3.1	132	4	184	20.8
10000	78	5.1	174	23.6	165	9.6	126	2.3	188	12.5
Positive Control	1183	140	710	32.1	930	19.3	1529	121.3	1171	9.9

Table 2: Mutagenicity of α-pinene in Salmonella typhimurium TA 100

Dose	10% RLI		NA		10% RLI		NA		10% RLI		NA		10% RLI		NA
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Not a Valid Test)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	70	2.2	49	2	63	3.8	57	7.2	56	1.8	38	3.8	48	9.7	0	0
5							48	2.3
10			46	5.3			49	4.3			45	5.9			0	0
25							52	1.8
50	65	5.5	44	3.5	57	5.2	29	2.9			41	13.4	50	6.7	0	0
75							14	0.6
100	80	1.2	63	4.5	85	15					54	14.7	40	3.5	0	0
200			57	4.2											0	0
250											19	5.8
400			10	0.9											0	0
500	63	5.9			48	4.4			49	4.4	12	3.7	52	5.5
1000	58	10.8			39	2.6							38	2.3
1500									46	2
2500									50	2
5000	33	3.2			90	4.3			21	0.9			27s	2.1
10000									11	0.7
Positive Control	738	30.5	428	39.3	1161	65.4	387	19.3	665	29.1	574	1.2	510	26.7	653	31.7

Table 3: Mutagenicity of α-pinene in Salmonella typhimurium TA 98

Dose	NA		NA		NA		10% RLI		10% RLI		NA		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Not a Valid Test)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	22	2.7	15	2.9	21	2.4	30	4.2	28	5.3	36	2	22	2.1
5			10	3.2	23	3.2
10	25	3	9	0.6	19	4.4					25	1.8
20			8	3.1
25					18	0.3
30			6	0.9
40			4p	1.2
50	8s	1.7			18	2.7			20	2.9	11	2.2	22	1.2
75					9	1.9
100	15s	2.2							31	3.2	1	1.3	21	2.6
200											0	0
250	4s	1.2
400											3	0.9
500	t						32	0.7	25	2.3			19	2.4
1000									28	4.8			35	2.5
1500							24	5.4
2500							21	1.2
5000							13	2.7	15s	1.2			7	0.7
10000							9	0.6
Positive Control	468	17.3	506	17.4	295	14.3	418	21.8	1008	31.5	417	30.7	372	38.1

Abbreviations: RLI = induced male Sprague Dawley rat liver S9; p = Precipitate; s = Slight Toxicity; T = Toxic

Conclusions:

Based on the read-across approach, the target substance is not considered as mutagenic in bacteria with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed similarly to the OECD guideline 471, strains of S. typhimurium (TA 100 and TA 98) and one Escherichia coli strain (WP2 uvrA pKM101) were exposed to α-pinene at concentration range of 0-10000 µg/plate in both the absence and presence of S9 metabolic activation from Sprague-dawley rats according to the pre-incubation method.

 

The positive controls induced the appropriate responses in the corresponding strains. α-Pinene showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S9 mix. When S. typhimurium TA 98 exposed to α-pinene without metabolic activation cytotoxicity was observed at 50 µg/plate and precipitation occurred at 40 µg/plate.

 

Based on the read-across approach, the target substance is not considered as mutagenic in bacteria with and without metabolic activation.

Reference 8

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

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

Adequacy of study:

weight of evidence

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:

hepatocytes: isolated from adult male Fischer or Sprague-Dawley rats

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

None

None

Conclusions:

Based on the read-across approach, the target substance is not considered as mutagenic in the mammalian cell system with and without metabolic activation.

Executive summary:

In an Unscheduled DNA Synthesis (UDS) assay in mammalian cells, hepatocytes isolated from adult male Fischer or Sprague-Dawley rats were exposed to α-pinene at concentrations up to 10000 µg or nL/mL for 18-20 hours in serum-free WME medium containing 5-10 µCi/mL H-TdR (20 Ci/mM). Stained triplicate cultures were examined microscopically at 1500x under oil immersion and UDS was measured by automatic electronic counter and expressed as net nuclear grain count (NNG).

 

α-pinene was found to be negative in UDS assay.

 

Based on the read-across approach, the target substance is not considered as mutagenic in the mammalian cell system with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no study available

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Several in vitro genetic toxicity data were available on the main components of cypress oil: Carene-3-delta and Pinene alpha. Both components are bicyclomonoterpen molecules, are very similar in structure and have the same toxicity profile regarding the genetic toxicity potential.

A weight of evidence of all the available data was therefore followed to complete the 7.6.1 endpoint.

Three Ames tests were available on delta-3 -carene and alpha pinene and were all negative. All the tests were performed with and without metabolic activation and were negative in both metabolic conditions. The bacterial strains tested in the three tests included: S. tiphymurium TA1535, TA1537, TA1538, TA98, TA100, TA102 and E. coli WP2. Therefore all the strains mandatory by the REACh regulation were tested.

An UDS test was also available on alpha pinene. The assay was performed in vitro on rat hepatocyte and gave negative results.

It can therefore be considered that Cypress oil is not mutagenic in bacteria and in mammalian cells based on data on its main components.

Justification for classification or non-classification

Harmonized classification:

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

Self-classification:

Based on the data available, Cypress oil is not classified as genotoxic according to the Regulation (EC) No. 1272/2008 and UN GHS criteria.