Reaction mass of 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane and cineole

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames Test conducted according to OECD Guideline 471 and GLP, Reaction mass of 1,4-cineole and 1,8-cineole was non mutagenic up to cytotoxic concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2uvrA.

In a Micronucleus test in cultured human lymphocytes conducted according to OECD Guideline 487 and GLP, Reaction mass of 1,4-cineole and 1,8 -cineole was not clastogenic.

In a Gene Mutation (HPRT) Assay in CHO cells conducted according to OECD Guideline 476 and GLP, Reaction mass of 1,4-cineole and 1,8-cineole was not mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-15 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

15 February 2016

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan for Salmonella typhimurium and Escherichia coli, respectively

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

First Test (Plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix
Second Test (Pre-incubation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test item was miscible at 400 mg/mL in DMSO. DMSO was, therefore, used as the vehicle for this study.
- Preparation of test formulation: The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log10 intervals. All concentrations were expressed in terms of the Reaction mass of 1,4-cineole and 1,8-cineole sample as received and containers of the neat test material were used within 7 days of opening for the first time.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM
Strains of S. typhimurium and E. coli were obtained from Moltox Inc.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: Test tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay.
- Exposure duration: Plates were incubated at 37 °C for 48-72 h

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test item may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.

OTHERS:
- Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light, and the pKM101 plasmid, which confers resistance to ampicillin. The responses of the strains to a series of reference mutagens were also assessed.
- Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer.
- After incubation period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

Rationale for test conditions:

The highest concentration of Reaction mass of 1,4-cineole and 1,8-cineole tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines.

Evaluation criteria:

- If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
- If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
- If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
- Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.

Statistics:

The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None
- Other confounding effects: None

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data:
Without S9 mix: 301-2776 (928 ± 350), 209-1255 (765 ± 214), 82-802 (235 ± 115), 87-1784 (305 ± 210), 438-3730 (2001 ± 743) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively
Without S9 mix: 338-4210 (1935 ± 802), 139-716 (375 ± 130), 90-648 (251 ± 91), 52-609 (143 ± 59), 352-1923 (956 ± 347) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively
- Negative (solvent/vehicle) historical control data:
Without S9 mix: 97-219 (154 ± 26), 8-44 (25 ± 7), 22-78 (40 ± 11), 5-41 (20 ± 7), 55-229 (165 ± 35) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively
With S9 mix: 91-237 (167 ± 30), 8-47 (21 ± 6), 24-84 (51 ± 15), 6-50 (29 ± 10), 97-280 (195 ± 32) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively

CYTOTOXICITY AND MUTAGENICITY:

First Test:
- No evidence of toxicity was obtained following exposure to the test item. A maximum exposure concentration of 5000 μg/plate was therefore selected for use in the second test.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in the presence or absence of S9 mix.

Second Test
- In the absence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in all strains at 500 μg/plate and above following exposure to the test item.
- In the presence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen at 1500 μg/plate and above in strain TA98 and 500 μg/plate and above in strains TA100, TA1535, TA1537 and WP2 uvrA (pKM101) following exposure to the test item.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in the presence or absence of S9 mix.

OTHERS:
- The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test item formulation.
- The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.

None

Conclusions:

The test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA (pKM101) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2uvrA (pKM101), were exposed to Reaction mass of 1,4-cineole and 1,8-cineole diluted in DMSO at the concentrations below. 

 

First Test (Plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Second Test (Pre-incubation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

 

Metabolic activation system used in this test is S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

 

In the first test, no signs of toxicity towards the tester strains were observed in either the absence or presence of S9 mix following exposure to the test item.

 

In the second test in the absence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in all strains at 500 μg/plate and above following exposure to the test item. In the presence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen at 1500 μg/plate and above in strains TA98 and 500 μg/plate and above in strains TA100, TA1535, TA1537 and WP2uvrA (pKM101) following exposure to the test item.

 

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in the presence or absence of S9 mix.

 

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

 

Therefore, the test item is not considered as mutagenic in these bacterial systems.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 August to 27 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 476 Guideline 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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

15 February 2016

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test

Target gene:

Hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO-K1) cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CHO-KI cells were obtained from the European Collection of Cell Cultures.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
H0 Ham’s Nutrient Mixture F12, supplemented with 2 mM L glutamine and 50 µg/mL gentamicin.
H10: H0 medium supplemented with 10% heat inactivated fetal calf serum
H10 medium was used for cell culture unless otherwise specified.
The selective medium, in which only HPRT deficient cells will grow, consists of H10 supplemented with 6-TG at a final concentration of 10 µg/mL.
All cell cultures were maintained at 34-39 °C in a humidified atmosphere of 5% CO2 in air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes; Spontaneous mutants were eliminated from the cultures by 3-day incubation in the presence of methotrexate (0.3 μg/mL), thymidine (4 μg/mL), hypoxanthine (15 μg/mL) two days prior to storage at -196 °C, in heat-inactivated foetal calf serum (HiFCS) containing 10% dimethyl sulphoxide (DMSO).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix contains: S9 fraction (10% v/v) prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL, 3 h exposure without and with metabolic activation

Mutation tests:
3 h exposure without metabolic activation: 100, 300, 350, 375, 400, 425, 450, 475 and 500 µg/mL
3 h exposure with metabolic activation: 100, 300, 500, 550, 600, 650, 700, 750 and 800 µg/mL

Additional Mutation tests:
3 h exposure without metabolic activation: 100, 150, 200, 225, 250, 275, 300, 400 and 500 µg/mL
3 h exposure with metabolic activation: 50, 300, 350, 375, 400, 425, 450, 475 and 500 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test item was found to be soluble at 400 mg/mL in (DMSO).
- Test item was dissolved and formulated in DMSO, shortly before dosing. The final volume of DMSO added to the cultures was 1% v/v. All concentrations cited in this report are expressed in terms of Reaction mass of 1,4-cineole and 1,8-cineole as received and containers of the neat test material were used within 7 days of opening for the first time.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 1% (v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 1% (v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; H10 medium - H0 medium supplemented with 10% heat inactivated fetal calf serum.
- Cell density at seeding (if applicable): 2 x 10^6 cells from each culture were seeded into 150 cm2 flasks

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
All cell cultures were maintained at 34-39 °C in a humidified atmosphere of 5% CO2 in air.

SELECTION AGENT (mutation assays): The selective medium, in which only HPRT deficient cells will grow, consists of H10 supplemented with 6-TG at a final concentration of 10 µg/mL.

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Single culture for test item and duplicate cultures for vehicle controls
Main test: Quadruplicate cultures for vehicle and duplicate cultures for test item and positive controls

NUMBER OF CELLS EVALUATED:
200 cells/flask were seeded for cloning efficiency and 5 x 10^5 cells were analyzed for mutant frequencies.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency and Relative survival

- OTHER:
For each culture, three flasks were seeded with 200 cells each, to determine cloning efficiency and five flasks with 5 x 10^5 cells each in selective medium to determine cloning efficiency. The flasks were returned to the incubator for approximately seven days at 34-39 °C in a humidified atmosphere of 5% CO2 in air. At the end of this incubation period, colonies growing in the flasks were fixed and stained in a methanol:Giemsa solution (4:1 v/v) and counted.

Rationale for test conditions:

The highest final concentration used in the preliminary toxicity test was 2000 μg/mL. This is the standard limit concentration within this test system as recommended in the regulatory guidelines for test items of well-known composition.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
b) the increase is concentration-related when evaluated with an appropriate trend test
c) any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
b) there is no concentration-related increase when evaluated with an appropriate trend test
c) all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test item, for non-linearity and for the comparison of positive control and treated groups to vehicle control. The data was analyzed using SAS (SAS Institute Inc., 2002).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium were observed at 800 μg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: The osmolality of the test item in medium was tested at 800 μg/mL (the highest concentration tested in a main mutation test); no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Precipitation: Precipitate was observed by eye at the end of treatment at 2000 μg/mL.

PRELIMINARY TOXICITY TEST:
- Precipitate was observed by eye at the end of treatment at 2000 μg/mL and this was, therefore, the highest concentration plated for determination of Day 1 Relative Survival (RS) in both the absence and presence of S9 mix.
- Exposure to the test item for 3 h at concentrations from 15.63 to 2000 μg/mL in both the absence and presence of S9 mix resulted in RS values from 117 to 0% and from 112 to 0% respectively.
Concentrations for the main test were based upon these data.

MAIN TEST:
3 h treatment in the absence of S9 mix: No precipitate was seen by eye at the end of treatment. The cell counts obtained immediately following treatment on Day 1 indicated that an appropriate toxicity profile would not be achieved in this experiment. Therefore, no survival plates were produced, the test was abandoned and an additional test was performed using modified dose concentrations.
Additional main test (3 h treatment in the absence of S9 mix): No precipitate was seen by eye at the end of treatment. Exposure to the test item resulted in RS values from 95 to 0%. Cultures treated at 100, 150, 200 and 225 μg/mL were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 250 μg/mL and above were not analysed for mutant frequency as RS was <10% at these concentrations. No significant increases in mutant frequency were observed after exposure to the test item at any concentration analysed.
3 h treatment in the absence of S9 mix: No precipitate was seen by eye at the end of treatment. RS values ranged from 87 to 0% relative to the vehicle control. As there were no concentrations showing a RS between 10 and 20%, the test was abandoned and an additional test was performed using modified dose concentrations.
Additional main test (3 h treatment in the presence of S9 mix): No precipitate was seen by eye at the end of treatment. Exposure to the test item resulted in RS values from 96 to 1%. Cultures treated at 50, 300, 350, 375 and 400 were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 425 μg/mL and above were not analysed for mutant frequency as RS was <10% at these concentrations. No significant increases in mutant frequency were observed after exposure to the test item at any concentration analysed.

HISTORICAL CONTROL DATA (mean and standard deviation)
- Positive historical control data:
Mean Mutant Frequency (x 10^-6): 284.7 ± 141 (Ethyl methanesulphonate, -S9); 360.4 ± 183 (3-Methylcholanthrene, +S9)
- Negative (solvent/vehicle) historical control data:
Mean Mutant Frequency (x 10^-6): 6.6 ± 5.9 (-S9); 7.2 ± 6.1 (+S9);
Mean Day 1 CE (%): 72 ± 0.1 (-S9); 73 ± 0.1 (+S9)

None

Conclusions:

The test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to Reaction mass of 1,4-cineole and 1,8-cineole for 3 h, with and without metabolic activation (10 % v/v S9); S9 fraction prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone.

 

Preliminary toxicity test

3 h exposure without and with metabolic activation: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL

 

Mutation tests

3 h exposure without metabolic activation: 100, 300, 350, 375, 400, 425, 450, 475 and 500 µg/mL

3 h exposure with metabolic activation: 100, 300, 500, 550, 600, 650, 700, 750 and 800 µg/mL

 

Additional mutation tests

3 h exposure without metabolic activation: 100, 150, 200, 225, 250, 275, 300, 400 and 500 µg/mL

3 h exposure with metabolic activation: 50, 300, 350, 375, 400, 425, 450, 475 and 500 µg/mL

 

In a preliminary toxicity test, precipitate was observed by eye at the end of treatment at 2000 μg/mL. Cytotoxicity was measured as Day 1 Relative Survival (RS). After exposure to the test item at concentrations from 15.63 to 2000 µg/mL RS values ranged from 117 to 0% and from 112 to 0%, in the absence and presence of S9 mix, respectively.

 

In the main mutation test in the absence of S9 mix, cells were exposed to the test item at concentrations of 100, 300, 350, 375, 400, 425, 450, 475 and 500 μg/mL. No precipitate was observed by eye at the end of treatment. The cell counts obtained immediately following treatment on Day 1 indicated that an appropriate toxicity profile would not be achieved in this experiment. Therefore, no survival plates were produced, the test was abandoned and an additional test was performed using modified dose concentrations. In the additional main mutation test in the absence of S9 mix, cells were exposed to the test item at concentrations of 100, 150, 200, 225, 250, 275, 300, 400 and 500 μg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 95 to 0% when compared with the vehicle control. Test item did not induce any statistically significant increases in mutant frequency at concentrations up to 225 μg/mL where RS was reduced to 20%.

 

In the main mutation test in the presence of S9 mix, cells were exposed to the test item at concentrations of 100, 300, 500, 550, 600, 650, 700, 750 and 800 μg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 87 to 0% when compared with the vehicle control. As there were no concentrations showing a RS between 10 and 20%, the test was abandoned and an additional test was performed using modified dose concentrations. In the additional main mutation test in the presence of S9 mix, cells were exposed to the test item at concentrations of 50, 300, 350, 375, 400, 425, 450, 475 and 500 μg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 96 to 1% when compared with the vehicle control. Test item did not induce any statistically significant increases in mutant frequency at concentrations up to 400 μg/mL where RS was reduced to 13%.

 

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and the metabolizing system.

 

Therefore, the test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August to 12 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 487 Guideline 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 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

15 February 2016

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Following treatment (3 h exposure) or during treatment (20 h exposure), cytokinesis was blocked using the inhibitor Cytochalasin B at 6 μg/mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL; 3 h exposure with and without S9-mix; 20 h continuous exposure without S9-mix

Main Test
3 h exposure to the test item formulations without S9-mix: 100, 200, 300, 400, 450, 500 and 550 µg/mL
3 h exposure to the test item formulations with S9-mix: 400, 600, 800, 1000 and 1200 µg/mL
20 h continuous exposure to the test item without S9-mix: 50, 100, 200, 250, 275, 300, 325, 350, 400, 450 and 500 µg/mL
Additional main test: 3 h exposure to the test item formulations without S9-mix: 125, 250, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Test formulation preparation: The solubility of the test item in a vehicle compatible with this test system was assessed. Reaction mass of 1,4-cineole and 1,8-cineole was found to be soluble at 400 mg/mL in DMSO which resulted in a final concentration of 2000 μg/mL when dosed at 0.5% v/v.
Test item was dissolved and diluted in DMSO, shortly before dosing. The final volume of DMSO added to the cultures was 0.5% v/v. All concentrations cited in this report are expressed in terms of Reaction mass of 1,4-cineole and 1,8-cineole as received and containers of the neat test item were used within 7 days of opening for the first time.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (0.5% v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Colchicine

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (0.5% v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

CULTURE OF LYMPHOCYTES: Human blood was collected aseptically from two healthy, non-smoking, adult (between 18-35 years of age) donors, pooled (in equal volumes from each donor) and diluted with HML media. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each culture contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 34-39 °C, and the cells were resuspended (twice daily) by gentle inversion.

METHOD OF APPLICATION: in medium
HML Media RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine.

DURATION
- Exposure duration: 3 h (± S9) and 20 h continuous exposure (-S9) in preliminary toxicity test; 3 h (± S9) and 20 h continuous exposure (-S9) in main tests
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Following treatment (3 h exposure) or during treatment (20 h exposure), cytokinesis was blocked using the inhibitor Cytochalasin B at a final concentration of 6 μg/mL.

STAIN (for cytogenetic assays): Slides were stained with Acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures were prepared for each treatment level and duplicate cultures were prepared for vehicle controls.
- Main test: Duplicate cultures were prepared for each treatment level and positive control cultures; quadruplicate cultures were prepared for vehicle controls; two slides were prepared from each culture.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Harvesting and Fixation: The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 34-39 °C, cultures were then incubated for 3 minutes at 34-39 °C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix. The cultures were centrifuged at 500 g for five minutes. The supernatant was removed, and the cell pellet re-suspended. A further 4 mL of fresh fixative was then added and the cells stored at 4 °C until slide preparation.
Slide Preparation: The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared. Pre-cleaned microscope slides were prepared for each culture by aliquoting the re-suspended cells onto the slides, and allowing the slides to air-dry. One slide was prepared from each culture. The remaining cell cultures were stored at approximately 4 °C until slide analysis was complete.
Slide Staining: Slides were stained with Acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes
Microscopic Examination: The prepared slides were examined by fluorescence microscopy. The incidences of mononucleate, binucleate and polynucleate cells were assessed per culture. The presence of an unusual number of, for example, cells undergoing mitosis, polyploid cells, necrotic cells and debris, if any, was also noted.

NUMBER OF CELLS EVALUATED:
- Scoring of Micronuclei: Interphase cells were examined by fluorescence microscopy and the incidence of micronucleated cells per 1000 binucleate cells per culture were scored where possible.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The analysis for micronucleated cells was based on the following criteria (Fenech and Morley 1985 and Fenech, 1993):
Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
% Cytostasis = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test item treatment culture
C = solvent control culture
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

OTHER:
At least three concentrations were selected for micronucleus analysis. The highest concentration was intended to be that which caused a depression in the cytokinesis-block proliferative index (CBPI) equivalent to 55 ± 5% cytotoxicity (approximately) when compared with the concurrent vehicle control or, where no cytotoxicity was observed, the maximum concentration as recommended in the test guidelines or the limit of solubility.

Rationale for test conditions:

The maximum final concentration tested in the preliminary toxicity test was 2000 μg/mL as this is the standard limit concentration within this test system as recommended in the regulatory guidelines.

Evaluation criteria:

Criteria for Assessing Genotoxic Potential
Providing that all of the acceptance criteria have been met, the test item was considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control.
- The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical negative control data.
If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.
Providing that all of the acceptance criteria have been met, a negative response will be claimed if, in all of the experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical negative control data.
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.

Statistics:

The analysis assumed that the replicate was the experimental unit. An arcsine square-root transformation was used to transform the data. Test item was compared to control using Williams’ tests (Williams 1971, 1972). Positive controls were compared to control using t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only three groups.
Statistical significance was declared at the 5% level for all tests.
The data were received in an Excel spread sheet and analyzed using SAS 9.1.3 (SAS Institute 2002) and Quasar 1.4.1 (Quasar 1.4.1 2016).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium were observed at 2000 μg/mL of more than 1.0 unit compared with the vehicle control.
- Effects of osmolality: The osmolality of the test item in medium was tested at 2000 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.
- Precipitation: Precipitate was observed by eye at the end of treatment at 1000 μg/mL and above.

PRELIMINARY TOXICITY TEST:
- In all exposure conditions the highest concentration tested was 2000 μg/mL and precipitate was observed by eye at the end of treatment at 1000 μg/mL and above.
- After 3 h treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 94.2% cytotoxicity, was obtained at 1000 μg/mL.
- After 3 h treatment in the presence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 37.2% cytotoxicity, was obtained at 1000 μg/mL.
- After 20 h treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 36.2% cytotoxicity, was obtained at 250 μg/mL. At higher tested concentrations overt toxicity was observed.
These results were used to select concentrations for the main test.

MAIN TEST
3 h treatment in the absence of S9 mix:
Cytotoxicity: The required toxicity profile was not achieved in the main test; therefore an additional main test was performed with modified dose levels.
Additional 3 h treatment in the absence of S9 mix
Cytotoxicity: Precipitate was observed by eye at the end of treatment at 1000 μg/mL. A reduction in CBPI compared to vehicle control values, equivalent to 57% cytotoxicity, was obtained at 900 μg/mL. Concentrations selected for micronucleus analysis were 550, 800 and 900 μg/mL.
3 h treatment in the presence of S9 mix:
Cytotoxicity: Precipitate was observed by eye at the end of treatment at 1000 μg/mL and above. No reduction in CBPI compared to vehicle control values was obtained at concentrations up to 1000 μg/mL. Concentrations selected for micronucleus analysis were 600, 800 and 1000 μg/mL.
20 h treatment in the absence of S9 mix:
Cytotoxicity: No precipitate was observed by eye at the end of treatment at any concentration tested. A reduction in CBPI compared to vehicle control values, equivalent to 50% cytotoxicity, was obtained at 500 μg/mL. Concentrations selected for micronucleus analysis were 50, 250 and 500 μg/mL.

MICRONUCLEUS ANALYSIS
Test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
Mean micronucleus induction in the vehicle control were within the historical control range.
- Positive historical control data:
Binucleate Individual MN/1000 cells (3 h treatment in the absence of S9 mix): 16-67 (31.9 ± 10.6) - Mitomycin C; 15-43 (24.1 ± 6.5) - Colchicine
Binucleate Group MN (3 h treatment in the absence of S9 mix): 18-64.5 (31.9 ± 10.3) - Mitomycin C; 17.5-38 (24.1 ± 5.7) – Colchicine
Binucleate Individual MN/1000 cells (20 h treatment in the absence of S9 mix): 15-41 (23 ± 6.6) - Mitomycin C; 13-23 (17.7 ± 2.1) - Colchicine
Binucleate Group MN (20 h treatment in the absence of S9 mix): 16-37.5 (23 ± 6.2) - Mitomycin C; 14-21.5 (17.7 ± 1.9) - Colchicine
Binucleate Individual MN/1000 cells (3 h treatment in the presence of S9 mix): 12-29 (18.6 ± 4.1) - Cyclophosphamide
Binucleate Group MN (3 h treatment in the presence of S9 mix): 13-27 (18.6 ± 3.8) - Cyclophosphamide

- Negative (solvent/vehicle) historical control data:
Binucleate Individual MN/1000 cells (3 h treatment in the absence of S9 mix): 1-12 (6.2 ± 2.4)
Binucleate Group MN (3 h treatment in the absence of S9 mix): 2.5-8.8 (6.2 ± 1.5)
Binucleate Individual MN/1000 cells (20 h treatment in the absence of S9 mix): 0-12 (6.9 ± 2.7)
Binucleate Group MN (20 h treatment in the absence of S9 mix): 2-10.5 (6.9 ± 2.3)
Binucleate Individual MN/1000 cells (3 h treatment in the presence of S9 mix): 0-13 (6.2 ± 2.7)
Binucleate Group MN (3 h treatment in the presence of S9 mix): 2.5-10.5 (6.2 ± 2.0)

Table 7.6.1/1: Main test results

Treatment/Concentration (μg/mL)	CBPI	Mean CBPI	Mean Cytotoxicity (%)	Binucleated cells containing micronuclei
				per 1000 cells	Mean	p-value^b	Trend test p-value^c
3 h treatment in the absence of S9 mix
Vehicle^a	1.92	1.90	0	4	3.5	-	-
	1.86			3
	1.90			4
	1.92			3
125	1.87	1.82	8.3	NA	-	-	-
	1.78			NA
250	1.65	1.71	21.2	NA	-	-	-
	1.76			NA
500	1.43	1.39	57.1	NA	-	-	-
	1.34			NA
550	1.88	1.88	2.2	4	4.0	0.495
	1.88			4
600	1.85	1.86	4.0	NA	-	-	-
	1.88			NA
650	1.86	1.86	3.8	NA	-	-	-
	1.87			NA
700	1.82	1.81	9.6	NA	-	-	-
	1.80			NA
750	1.76	1.77	14.3	NA	-	-	-
	1.78			NA
800	1.56	1.59	34.4	5	5.5	0.105	0.034*
	1.62			6
850	1.69	1.59	34.4	NA	-	-	-
	1.49			NA
900	1.36	1.39	57.0	4	4.5	0.105	0.105
	1.41			5
950	1.50	1.44	51.5	NA	-	-	-
	1.37			NA
1000	1.23	1.22	75.5	NA	-	-	-
	1.21			NA
Mitomycin C 0.3	1.57	1.56	37.6	46	48.5	<0.001***	-
	1.55			51
Colchicine 0.07	1.70	1.62	31.0	31	34.5	<0.001***	-
	1.55			38
3 h treatment in the presence of S9 mix
Vehicle^a	1.88	1.88	0	7	6.3	-	-
	1.96			6
	1.84			6
	1.85			6
400	1.83	1.86	2.9	NA	-	-	-
	1.89			NA
600	1.85	1.83	6.0	6	5.5	0.804	-
	1.81			5
800	1.77	1.81	8.6	7	6.0	0.804	0.759
	1.84			5
1000	1.86	1.84	5.3	5	6.0	0.804	0.901
	1.81			7
1200	-	-	-	-	-	-	-
Cyclophosphamide 5	1.52	1.54	38.8	26	24.0	<0.001***	-
	1.56			22
20 h treatment in the absence of S9 mix
Vehicle^a	1.99	1.99	0.0	8	7.0	-	-
	1.97			4
	2.02			6
	1.96			10
50	1.90	1.92	7.0	5	4.5	1.000	-
	1.93			4
100	1.91	1.90	8.4	NA	-	-	-
	1.89			NA
200	1.77	1.75	23.6	NA	-	-	-
	1.74			NA
250	1.73	1.71	27.8	9	9.5	1.000	0.174
	1.69			10
275	1.69	1.70	29.4	NA	-	-	-
	1.70			NA
300	1.62	1.44	54.9	NA	-	-	-
	1.27			NA
325	1.63	1.65	34.3	NA	-	-	-
	1.67			NA
350	1.65	1.62	36.9	NA	-	-	-
	1.59			NA
400	1.58	1.57	42.4	NA	-	-	-
	1.55			NA
450	1.53	1.52	47.2	NA	-	-	-
	1.52			NA
500	1.52	1.49	50.0	4	3.5	0.054	0.247
	1.46			3
Mitomycin C 0.1	1.82	1.79	20.3	15	16.0	0.002**	-
	1.75			17
Colchicine 0.02	1.52	148	51.7	18	15.5	0.003**	-
	1.43			13

a. Vehicle control = DMSO (0.5% v/v)

b. p-values are for comparisons to control using Williams' test for test item and the t-test otherwise

c. Trend test p-values are for the linear contrast including the control group and lower concentrations of the same compound

*p<0.05, ** p <0.01; ***p<0.001

CBPI: Cytokinesis block proliferative index

NA: Not analysed for micronucleus frequency

Conclusions:

The test substance did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to Reaction mass of 1,4-cineole and 1,8-cineole in the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 10% (v/v) S9 fraction; S9 fraction was obtained from the liver homogenates of male Sprague-Dawley derived rats induced with phenobarbital and 5,6-benzoflavone.

Preliminary Toxicity Test

3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL;

3 h exposure with and without S9-mix; 20 h continuous exposure without S9-mix.

Main Test

100, 200, 300, 400, 450, 500 and 550 µg/mL;

3 h exposure to the test item formulations without S9-mix.

Additional main test:

3 h exposure to the test item formulations without S9-mix: 125, 250, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 µg/mL

3 h exposure to the test item formulations with S9-mix: 400, 600, 800, 1000 and 1200 µg/mL

20 h continuous exposure to the test item without S9-mix: 50, 100, 200, 250, 275, 300, 325, 350, 400, 450 and 500 µg/mL

Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.

 

In all exposure conditions the highest concentration tested was 2000 μg/mL and precipitate was observed by eye at the end of treatment at 1000 μg/mL and above. In the preliminary toxicity test, after 3 h of treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 94.2% cytotoxicity, was obtained at 1000 μg/mL. After 3 h of treatment in the presence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 37.2% cytotoxicity, was obtained at 1000 μg/mL. After 20 h treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 36.2% cytotoxicity, was obtained at 250 μg/mL. At higher tested concentrations overt toxicity was observed.

 

Three concentrations were assessed for the determination of the induction of micronuclei. For the 3 h and 20 h treatments in the absence of S9-mix, the highest concentration selected (900 and 500 μg/mL, respectively) was that which caused a reduction in cytokinesis-block proliferative index (CBPI) equivalent to 55 ± 5% cytotoxicity. For the 3 h treatment in the presence of S9-mix, the highest concentration selected (1000 μg/mL) was that showing visible precipitate (observed by eye) at the end of the treatment period.

 

Following 3 h of treatment in the absence of S9 mix, a reduction in CBPI equivalent to 57% cytotoxicity was obtained at 900 μg/mL. Concentrations selected for micronucleus analysis were 550, 800 and 900 μg/mL. Following 3 h of treatment in the presence of S9 mix, no reductions in CBPI were obtained at any concentrations up to 1000 μg/mL. Concentrations selected for micronucleus analysis were 600, 800 and 1000 μg/mL. In the absence of S9 mix following 20 h of treatment, a reduction in CBPI equivalent to 50% cytotoxicity was obtained at 500 μg/mL. Concentrations selected for micronucleus analysis were 50, 250 and 500 μg/mL.

 

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

 

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

 

Therefore, the test item did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2uvrA (pKM101), were exposed to Reaction mass of 1,4-cineole and 1,8-cineole diluted in DMSO. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in the presence or absence of S9 mix. The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory. Therefore, test item is not considered as mutagenic in these bacterial systems.

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to Reaction mass of 1,4-cineole and1,8-cineole in the presence and absence of a metabolic activation system. In both the absence and presence of S9 mix, following 3 h of treatment, and in the absence of S9 mix, following 20 h of treatment, the test item did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls. The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system. Therefore, the test item did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to Reaction mass of 1,4-cineole and 1,8-cineole for 3 h, with and without metabolic activation. The test item did not induce any statistically significant increases in mutant frequency at concentrations up to 225 μg/mL in absence of metabolic activation system, where RS was reduced to 20%. The test item did not induce any statistically significant increases in mutant frequency at concentrations up to 400 μg/mL in presence of S9, where RS was reduced to 13%. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and the metabolizing system. Therefore, the test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.

Justification for classification or non-classification

As negative results were obtained with the registered substance in an Ames test, in an in vitro gene mutation test (HPRT) in CHO cells and in an in vitro micronucleus test in cultured peripheral human lymphocytes, Reaction mass of 1,4-cineole and 1,8-cineole has no genotoxic potential and is therefore not classified according to Regulation (EC) No. 1272/2008 (CLP).