Exo-1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was negative in two Ames tests.

The test substance did not show clasterogenic activity towards cultured CHO cells either in the presence or in the absence of S9 mix

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Not the recommended combination of strains according to guideline tested, positive controls missing

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats

Test concentrations with justification for top dose:

50, 167, 500, 1667 and 5000 µg/plate

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine (2-Aminoanthracene); with metabolic activation: all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: The plates were incubated for 48 - 72 hours in the dark at 37 °C

NUMBER OF REPLICATIONS: 3

PRELIMINARY TOXICITY SCREEN:
The preliminary toxicity screen for the Ames Assay performed without metabolic activation used two of the histidine auxotrophs of S. typhimurium, TA 1538 and TA 100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the S. typhimurium tester strains. The test compounds was prepared to a concentration of 50 mg/mL and five different levels tested for toxicity. Top agar, used as an overlay, was reconstituted into molten state and supplemented with 0.5 mM histidine - 0.5 mM biotin at a volume of 0.1 mL/mL of agar, and maintained at 45 °C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45 °C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 mL aliquotes of top agar solution, 0.1 mL of tester strain and 0.1 mL of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37 °C incubator. The plates were uncubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria.

Evaluation criteria:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies with at least one dose point inducing a mutant frequency value that is two-fold the solvent control value. Significance at the 95% confidence limit is determined by the program developed by Moore and Felton (1983). This program applies a linear regression analysis to the data points and any P value greater than 0.05 is considered significant. In addition, the greater the P value, the higher the probability that the data points fit a linear response. This dose-response relationship accasionally necessites slight modification of the original doses in a repeat assay. Alternatively, if the solvent control is within the 95% confidence limits of the test chemical procedures the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is definied as the absence of a reproducible increase in the number of histidine-independent colonies.

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: Results of the Ames test with the test item

Test item	Mean spondaneous revertants/plate
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
Without metabolic activation (-S9)
Solvent control; H2O	20±6	11±2	12±3	26±6	134±13
50	24±8	12±3	7±12	34±5	146±17
167	17±3	11±4	8±4	34±6	139±34
500	21±6	13±4	11±2	28±3	148±14
1667	18±1	10±2	8±3	25±6	149±17
5000	22±4	12±4	12±4	27±3	118±16
Positive controls
Sodium azide	443±30*				516±50*
9-Aminoacridine		520±100*
2-Nitrofluorene			319±26*	273±14*
With metabolic activation (+S9)
Solvent control; H2O	17±2	11±2	17±1	35±1	112±31
50	18±1	14±1	15±1	40±3	122±110
167	23±7	19±6	18±6	29±7	133±4
500	13±5	16±3	17±5	49±114	117±12
1667	15±2	10±4	17±3	34±6	113±10
5000	18±6	16±3	18±5	39±4	120±23

Conclusions:

Under the conditions of this Ames test die test substance did not induce gene mutation in strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987-10-22 to 1987-11-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

test was only conducted up to 4000 µg/plate. The recommended maximum test concentration is 5000 µg/plate.

GLP compliance:

yes

Type of assay:

bacterial forward mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test compound was stored in the dark at ambient temperature.

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and Escherichia coli WP2 uvrA pKM101

Metabolic activation:

with and without

Metabolic activation system:

Arocolor 1254 pre-treated rat liver S9 fraction

Test concentrations with justification for top dose:

31.25, 62.5, 125, 250, 500, 1000, 2000 or 4000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: Potassium dichromate: WP2 uvrA pKM101; Neutral Red: TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
The cultures were incubated at 37 °C for 48-72 h before the revertant colonies were counted

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- The test compound was totally miscible in the aqueous test system at all amounts tested.
- The addition of the test compound caused the pH of the medium to change from 7.43 to 7.48
- Microscopical evaluation of the background lawn shown no evidence of cytotoxicity at amounts up to 4000 µg test item per plate either in the presence or in the absence of rat liver S9 fraction.

Conclusions:

Under the conditions of this Ames test, the test substance did not induce gene mutation in strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium and in Escherichia coli WP2 uvrA pkM101 with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1987-10-01 to 1987-12-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

other: Genetic toxicity in vitro assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

10, 50, 100, 250, 500, 1000, 2000, 3000, 4000 and 5000 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methyl methanesulphonate (-S9 mix) and cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

Cytotoxicity studies: Two separate cytotoxicity assays were performed in order to determine the range of test substance concentrations to be used in the chromosome assay.

Growth inhibition assay-visual assessment:
This assay was performed to determine the toxicity of cineole alcohol over a wide range of concentrations, with and without S9 mix. The following method was used:
(i) 100,000 CHO cells were plated into a series of 15 mL volume glass Leighton tubes to test each of the following concentrations of cineole alcohol in triplicate; 10, 50, 100, 250, 500, 1000, 2000, 3000, 4000 and 5000 µg/mL. Assays were performed either in the presence or in the absence of S9 mix and untreated and solvent treated controls were included.
(ii) The tubes were incubated overnight at 37 °C in a 5% CO2 atmosphere.
(iii) 24 hours after cell plating, two series of test compound dilutions were prepared, the first in growth medium containing 5% FCS and the second in growth medium containing 2% FCS and 10% S9 mix.
The growth medium was removed from the tubes and the compound dilutions stated in (i) were applied to the appropriate tubes.
(iv) All tubes were incubated for 3 hours at 37 °C. The compound was then removed and fresh medium (5% FCS) was added for a further 21 hours.
(v) After this time, the medium was removed, the cultures fixed with methanol for 30 minutes, stained with 10% aqueous Giemsa for 10 minutes, rinsed and left to dry.
(vi) The degree of cell confluency was visually assessed in each tube to determine the compound concentration reducing the extent of cell sheet staining by approximately 50%.

Growth inhibition assay - cell counts:
This assay was performed to determine the relative cell numbers after treatment with a narrower range of test substance concentrations with and without S9 mix. The following method was used:
(i) A range of eight separate concentrations of cineole alcohol was selected covering the 50% endpoint derived from the previous experiment. These concentrations were 2250, 3000, 3750, 4500, 5250, 6000, 6750 and 7500 mg/mL for cultures with and without S9 mix.
(ii) 5 x 10^5 CHO cells were plated into a sufficient number of 100 mL volume glass Leighton tubes to assay all the above dose levels in duplicate. Untreated and solvent-treated controls were included.
(iii) Stages (ii), (iii) and (iv) of the first cytotoxicity assay were repeated.
(iv) The medium was removed from each tube and the cells detached from the glass by trypsin treatment. The total number of cells from each tube was counted. The concentrations of cineole alcohol that reduced the number of cells by approximately 50% compared to the controls were used as the top dose in the subsequent chromosome assays.

Chromosome assay:
(i) Experimental design
The test substance was assayed in the presence and in the absence of S9 mix. The following test and control cultures were set up:
(a) Three concentrations of the test chemical: The concentration that induced 50% inhibition of growth and 1:2 and 1:10 dilutions of this (3 cultures with S9, 3 cultures without S9 for each dose).
(b) A medium only control (3 cultures).
(c) A solvent control: growth medium containing solvent at the concentration present in the top dose of the test compound (3 cultures).
(d) A 10% S9 mix in medium control (3 cultures).
(e) A 10% S9 mix plus solvent in medium control (3 cultures).
(f) Positive controls -with S9 mix, the indirect acting mutagen cyclophosphamide (3 cultures), -without S9 mix, the direct acting mutagen, methyl methanesulphonate (3 cultures).
(g) Since the compound exposure time (3 h) was less than one cell cycle time (12 h) in duration, multiple sample times were employed. Cells were sampled 8, 12 and 24 hours after the initiation of compound exposure.
In some of the repeated experiments, a single 24 h sample time was employed.
(ii) The tests
(a) In the first experiment, a total of 36 separate cultures were prepared in 200 mL volume glass medical flats to represent the dose groups listed above. Approximately 1 x 10^6 CHO cells were added to each bottle and incubated at 37 °C in a 5% CO2 atmosphere overnight.
(b) After 24 hours the test and control substances were diluted in growth medium with or without S9 mix and then applied to the appropriate cultures. Growth medium containing 2% FCS was used in the presence of S9 mix.
(c) After 3 hours exposure, the medium was removed from all the tubes and fresh medium (5% FCS) was added to each culture. After 8 hours from the initiation of exposure, the metaphase cells were collected by mitotic shake off and fresh medium was added. This process was repeated at 12 hours. 24 hours from the initial of exposure, all the cells were removed mechanically from each culture with a rubber policeman.
2 hours before each sample time, Colcemid was added to each culture to give a final concentration of 0.2 µg/mL.
(d) Each culture from the three separate sample times was processed to prepare cells for metaphase analysis. This involved swelling the cells in hypotonic solution (0.56% KC1), fixing in methanol:acetic acid 3:1 and dropping cell suspensions onto glass microscope slides.
(e) All slides were coded, with the analysis commencing with slides prepared at 24 hours. Where possible, 300 metaphases were scored for each dose group. Only those cells showing the modal chromosome number (20) ± 2 centromeres were analysed for chromosome damage.
The vernier co-ordinates were recorded for damaged cells and the number of centromeres in each cell analysed were recorded.
(f) The mitotic index was assessed at the 24 hour sample time by counting the number of metaphases in a total of 500 cells from each slide. The mitotic index figure is presented as the fraction of mitotic cells per 500 cells counted.
Subsequent assays were carried out following the same procedure, with the number of sample times and dose groups varying between experiments.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
(a) The degree of cell confluency at the top dose of 5000 µg/mL was approximately 60% that of the control groups for experiments with and without S9 mix.

(b) Growth inhibition assay -cell counts
The total cell counts were reduced by approximately 50% at cineole alcohol concentrations of around 5000 µg/mL in the presence of S9 and 3000 µg/mL in the absence of S9 mix, (see table 1). Thus, these two zest compound concentrations were taken as representing the LC50 doses and were used as the highest concentrations in the subsequent chromosome assays. However, at 5000 µg/mL in the presence of S9, too few metaphases were available for analysis in the chromosome assay due to compound toxicity. Thus both assays (+/- S9) were subsequently performed with 3000 µg/mL cineole alcohol as the top dose.

Remarks on result:

other: please refer to section 'Any other information on results incl.tables'

Chromosome assays

(i) The first experiment was performed with slides having been prepared at 8, 12 and 24 hours following the initiation of compound exposure.

(a) Without S9 mix

At the 24 hour sample time, cultures exposed for 3 hours to 3000 µg/mL cineole alcohol showed an increase in the number of gaps, breaks, acentric fragments and exchange figures compared to the controls. No consistent increase in damage was observed at 300 or 1500 µg/mL.

At the 8 and 12 hour sample times, too few metaphases were available for analysis.

(b)With S9 mix

At the 24 hour sample time, culture exposed for 3 hours to 3000 µg/mL of cineole alcohol showed a small increase in chromosome damage compared to the controls. Again, no consistent increase in chromosome damage was observed at 300 or 1500 µg/mL.

At the 12 hour sample time, there was no evidence of an increase in chromosome damage at any dose level of cineole alcohol. At the 8 hour sample time, there was a slight elevation in the number of gaps at 3000 µg/mL cineole alcohol compared to the solvent controls, but this was a less than two fold increase and was not considered significant.

(ii) A second experiment was carried out in order to substantiate the induction of chromosome damage at the top dose of the test compound at the 24 hours sample time. CHO cells were exposed to a single dose of cineole alcohol (3000 µg/mL) for 3 hours, the metaphases being sampled 24 hours following the initiation of exposure. Experiments were carried out with and without S9 mix, with solvent controls and positive controls included. In these experiments, there was no indication of a compound-induced increase in chromosome damage, either in the absence of S9 mix.

(iii) In order to clarify these data, a third experiment was performed using 3 doses of cineole alcohol (300, 1500 and 3000 µg/mL), sampled at the 24 hour interval. In this experiment, there was no evidence for an induced clastogenic response at any of the test compound concentrations either in the absence of S9 mix.

(iv) A final experiment was performed, without S9 mix, in which 3 sample times (8, 12 and 24 h) were employed. The positive control, solvent control and cineole alcohol (3000 µg/mL) dose groups were analysed. This experiment complemented the first assay in which too few metaphases were available for analysis at the 8 and 12 hour sample time without S9 mix.

The data showed no evidence of an increase in chromosome damage in cultures exposed to cineole alcohol at 3000 µg/mL. Throughout all the experiments performed, clear cut increases in chromosome damage with the positive control chemicals, cyclophosphamide and methyl methanesulphonate were observed at the 12 and 24 hour sample times. Although, in the initial experiment, clear cut increases in chromosome damage were observed at the top dose of cineole alcohol (3000 µg/mL) in the absence of S9 mix, this observation was not repeated in the three additional experiments that were performed.

There is no ready explanation for the increase in chromosome damage in the first experiment, however, there is mounting evidence that at very high compound concentrations, caution should be exercised in attributing in vitro chromosome damage to genotoxic activity per se.

In view of all the data generated it was concluded that cineole alcohol did not induce chromosome damage in cultured CHO cells.

Table 1: Toxicity of the test substance to CHO cells in the absence or presence of S9 mix. Cultures of CHO cells were exposed to the test chemical for 3 hours in the absence or in the presence of S9 mix. The cells in each culture were counted 24 hours after the initiation of exposure (Cell count cytotoxicity assay)

Compound	Absence of S9 mix			Presence of S9 mix
	Conc. µg/mL	Mean cell count per mL (x10^4)	Percentage of solvent control	Mean cell count per mL (x10^4)	Percentage of solvent control
Test item	0 (a)	48.3	85	19.0	97
	0 (b)	57.0	100	19.5	100
	2250	45.5	80	20.1	103
	3000	31.0	54	13.5	69
	3750	27.0	47	15.8	81
	4500	27.6	48	14.5	74
	5250	15.8	28	9.0	46
	6000	25.4	45	13.4	68
	6750	10.8	19	1.5	7
	7500	6.0	11	0.4	2

(a) = untreated control

(b) = solvent control (solvent concentration was the same as that applied to the highest dose level used)

Table 2: Meatphase chromosome analysis of CHO cells after a 3 hour exposure to the test item or methyl metanesulphonate (MMS) in the absence of S9 mix (24 hour sample time), Summary table

Compound	Conc. (µg/mL)	CYT 733	Mitotic index	Total number of cells examined	*total No. of cells analysed for structural Aberrations	Aberrations
						Numerical	Structural
							Excluding Gaps			Including Gaps
						Percentage of polyploidy cells	Sum Total of Aberrations	Mean No. of Aberrations per cell	Percentage of cells with Aberrations	Sum total of Aberrations	Mean No. of Aberrations per cell	Percentage of cells with Aberrations
Untreated coltrol	0	008 010 017	0.004 0.008 0.008 Mean MI 0.007	220	205	6.8	7	0.034	1.95	13	0.063	4.88
Solvent control	0	001 005 011	0.030 0.008 0.004 Mean MI 0.014	239	226	5.4	1	0.004	0.44	8	0.035	3.54
Test item	300	003 014 016	0.014 0.010 0.014 Mean MI 0.013	283	260	8.1	5	0.019	1.92	26	0.100	9.23
Test item	1500	007 009 018	0.014 0.018 0.016 Mean MI 0.016	300	273	9.0	8	0.029	2.20	18	0.066	5.86
Test item	3000	002 012 015	0.010 0.014 0.008 Mean MI 0.011	229	204	10.9	36	0.176	12.75	93	0.456	28.43
MMS	60	004 006 013	0.006 0.018 0.022 Mean MI 0.015	177	174	1.7	137	0.787	46.55	186	1.069	53.45

CYT = Cytogenic code mumber

* = Total number of cells examined excluding polyploid cells.

Table 3: Metaphase chromosome analysis of CHO cells after a 3 hour exposure to the test substance or cyclophosphamide (CP) in the presence of S9 mix (24 hour sample time); Summary table

Compound	Conc. (µg/mL)	CYT 733	Mitotic index	Total number of cells examined	*total No. of cells analysed for structural Aberrations	Aberrations
						Numerical	Structural
							Excluding Gaps			Including Gaps
						Percentage of polyploidy cells	Sum Total of Aberrations	Mean No. of Aberrations per cell	Percentage of cells with Aberrations	Sum total of Aberrations	Mean No. of Aberrations per cell	Percentage of cells with Aberrations
Untreated coltrol	0	010 011 015	0.02 0.02 0.04 Mean MI 0.03	267	250	6.37	2	0.008	0.80	14	0.056	5.20
Solvent control	0	003 004 018	0.01 0.03 0.04 Mean MI 0.03	265	258	2.64	2	0.008	0.78	4	0.016	1.55
Test item	300	008 009 017	0.02 0.03 0.04 Mean MI 0.03	300	279	7.0	13	0.047	2.87	28	0.1	6.45
Test item	1500	001 012 016	0.02 0.02 0.04 Mean MI 0.03	300	289	3.7	3	0.01	1.04	17	0.059	5.54
Test item	3000	005 006 014	0.02 0.004 0.03 Mean MI 0.02	227	218	4.0	16	0.073	4.6	24	0.11	8.26
MMS	100	002 007 013	0.01 0.02 0.02 Mean MI 0.02	184	182	1.1	78	0.429	28.6	86	0.47	3.02

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Test substance was tested for 3 h with S9 mix only, scored 300 metaphases

GLP compliance:

yes

Type of assay:

other: genotoxic in vitro assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Bush Boake Allen Ltd,
- Code Number: SD93853
- ST Number ST87/161
- Purity: 99.4% (+/- 0.5%), racemic mixture

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in the dark under ambient temperature
- Stability under test conditions: given

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Source: Flow Laboratories Ltd., Irvine, Scotland
Passage number: 36-40
Representative sample was free from mycoplasma contamination

Cytokinesis block (if used):

Colcemid at final concentration of 0.2 ug/mL

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from Arocolor 1254 induced rat liver

Test concentrations with justification for top dose:

10, 100, 250, 500, 1000, 2000, 3000, 4000 and 5000 ug/ml
Growth inhibition assays were performed to determine top dose for main experiments (set at LC50): up to 2500 ug/mL without S9 mix and 4500 ug/mL with S9 mix

Vehicle / solvent:

DMSO
concentrations of cineole alcohol of up to 500 mg/ml could be attained using this carrier.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

For further information on results and tables please refer to attached background information.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The mutagenic activity of the test substance was investigated a study equivalent to OECD guideline 471. The bacterial strains TA 1535, TA 1537, TA 1538, TA98 and TA 100 of S. Salmonella typhimurium and strain WP2 uvrA pKM101 of Escherichia coli were exposed to the test item concentrations of 31.25, 62.5, 125, 250, 500, 1000, 2000 or 4000 µg/plate with and without metabolic activation.

The test compound was totally miscible in aqueous test system at all amounts. Microscopical evaluation of the background lawn showed no evidence of cytotoxicity at amounts up to 4000 µg test item/ plate either in the presence or in the absence of rat liver S9 fraction. Under the conditions of this test, the test item did not increase the reverse mutation frequency in any of the strains either in the presence or in the absence of rat liver S9 fraction.

 

Another reverse mutation assay with the test item equivalent to OECD guideline 471 is available. In a Preliminary Toxicity Screening test two bacterial strains of Salmonella typhimurium, TA 1538 and TA 100, were tested at dose levels of 50, 167, 500, 1667 and 5000 µg/plate. The strains exhibited normal growth of the bacterial lawn and of the revertant colonies at all dose levels tested. Based on these results, the same dose levels were used for the Plate Incorporation Mutation Assay (main test). In the main test, the test item was evaluated in strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium with and without metabolic activation. Positive control substances were included for samples without metabolic activation, but were missing for conditions with metabolic activation.

Under the conditions of this Ames test the test substance did not induce gene mutation in strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium with and without metabolic activation. All solvent and positive controls used in the evaluation of the test item induced mean mutation frequency values, which were within the acceptable range of historical data.

The clasterogenic potential of the test substance was assessed from assays designed to monitor chromosome damage in Chinese Hamster Ovary (CHO) cells. Cultures were grown in glass bottles and incubated in medium containing the test substance for 3 hours, either in the presence or in the absence of S9 mix. Metaphase cells were prepared on glass microscope slides for the analysis of chromosome aberrations at 8, 12 or 24 hours following the initiation of compound exposure. The conclusion drawn from a number of individual experiments was that the test substance did not show clasterogenic activity towards cultured CHO cells either in the presence or in the absence of S9 mix, under the experimental conditions described.

In a second clastogenicity study, CHO cells were incubated with test substance either in the absence of S9 mix for 24 hours and sampled at 24 hours, or the cells were cultured in the presence of S9 mix for 3 hours and sampled at 8, 12, and 24 hours following the start of exposure. No consistent increase of chromosomal aberrations was observed in this experiment.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.