Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 17 June 2014 and 13 November 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 473 using an In Vitro Mammalian Chromosome Aberration method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

EC Number:

943-623-1

Molecular formula:

C13 H24 O

IUPAC Name:

Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

Test material form:

other: Liquid

Details on test material:

Identification: IFF 215 (Floriane)
Physical state / Appearance: Clear colorless liquid
Storage conditions: Refrigerated at approximately 4 °C in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.


Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

4 (20)-hour without S9: 0, 7.5, 15, 30, 60, 80, 100, 120 µg/mL.
4 (20)-hour with S9: 0, 7.5, 15, 30, 60, 80, 100, 120 µg/mL.
24-hour without S9: 0, 3.75, 7.5, 15, 30, 60, 80, 100, 120 µg/mL.
4 (20)-hour with S9 (1 %): 0, 3.75, 7.5, 15, 30, 45, 60, 80 µg/mL.

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Without S9: Mitomycin C at 0.4 and 0.2 µg/mL in Experiment 1 and 2 respectively dissolved in Minimal Essential Medium. With S9: cyclophosphamide at 5 µg/mL dissolved in dimethyl sulphoxide.

Details on test system and experimental conditions:

Test Item
The molecular weight of the test item was given as 196.2, therefore, the maximum dose level was 1962 µg/mL, which was calculated to be equivalent to the 10mM dose level. The purity of the test item was 87.4 % and was accounted for in the test item formulations.

The test item was insoluble in aqueous media at 19.62 mg/mL and in dimethyl sulphoxide at 196.2 and 98.1 mg/mL, however, the test item was miscible in acetone at 196.2 and 392.4 mg/mL in solubility checks performed in house. Acetone is toxic to human lymphocytes at 100 µL; therefore all of the formulations were prepared at concentrations two times greater than required in the culture flasks. To compensate, each formulation was dosed using 50 µL (0.05 mL) aliquots. The highest achievable concentration was 392.4 mg/mL which when dosed into culture flasks, gave a concentration of 1962 µg/mL. Prior to each experiment, the test item was accurately weighed and formulated in acetone and serial dilutions prepared.

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Microsomal Enzyme Fraction
Lot No’s. PB/βNF S9 01/06/14 and PB/βNF S9 27/07/14 were used in this study. The S9 Microsomal fraction was prepared in house from male rats induced with Phenobarbitone/β Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (10 to 20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2% in the Preliminary Toxicity Test and Experiment 1 and 1% in Experiment 2.

This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.

Test Procedure
Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood


With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.

In Experiment 2, 1 mL of 10% S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.

The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4 hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

The dose range of test item used was 0, 7.66, 15.33, 30.66, 61.31, 122.63, 245.25, 490.5, 981, 1962 µg/mL.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1
Two exposure groups were used for Experiment 1:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 7.5, 15, 30, 60, 80, 100 and 120 µg/mL.

ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 7.5, 15, 30, 60, 80, 100 and 120 µg/mL.


Experiment 2
Two exposure groups were used for Experiment 2:
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0, 3.75, 7.5, 15, 30, 60, 80 and 120 µg/mL.

ii) 4-hour exposure to the test item with S9-mix (1%) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0, 3.75, 7.5, 15, 30, 45, 60 and 80 µg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) and cells with endoreduplicated chromosomes was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Evaluation criteria:

The following criteria were used to determine a valid assay:

Negative Control
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.

Positive Control
All the positive control chemicals must induce a clear positive response (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

Results and discussion

Test results

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 7.66 to 1962 µg/mL. The maximum dose was the 10 mM concentration.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 245.25 µg/mL in the 4(20)-hour exposure group in the absence of S9-mix, 490.5 µg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at and above 122.63 µg/mL in the continuous exposure group.

Haemolysis was also observed following exposure to the test item at and above 30.66 µg/mL in the 4(20)-hour exposure group in the absence of S9-mix, at and above 15.33 µg/mL in the 4(20)-hour exposure group in the presence of S9-mix and at and above 61.31 µg/mL in the 24-hour continuous exposure group. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 61.31 µg/mL in all three exposure groups. The mitotic index data confirms that the test item induced excessive toxicity with a sudden onset in all of the exposure groups. Based on these observations it was considered that it would be difficult to achieve optimum toxicity.

The selection of the maximum dose level was based on toxicity for both Experiments 1 and 2.


Chromosome Aberration Test - Experiment 1
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 30 µg/mL in both exposure groups.
No precipitate observations were made at the end of exposure in either exposure group. Haemolysis was observed following exposure to the test item at and above 7.5 µg/mL in both exposure groups.

The mitotic index data confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed prior to the excessive toxicity observed at and above 60 µg/mL. Therefore, the maximum dose level selected for metaphase analysis was 30 µg/mL in both the absence and presence of S9-mix.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The qualitative assessment of the slides determined that the toxicity was not as severe as the previous experiment and there were metaphases suitable for scoring present up to 120 µg/mL and 80 µg/mL in the absence and presence of S9-mix, respectively.

No precipitate of the test item was observed at the end of exposure, however, haemolysis was observed at and above 30 µg/mL and 15 µg/mL in the absence and presence of S9-mix, respectively.

The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed in both exposure groups. In the absence of S9-mix 38% and 64% mitotic inhibition was achieved at 60 and 80 µg/mL, respectively. In the presence of S9-mix, 40% mitotic inhibition was achieved at 60 µg/mL only.

The maximum dose level selected for metaphase analysis was, therefore, based on the concentrations approaching the optimum toxicity limit of 50% and was 60 µg/mL in both exposure groups.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Chromosome Aberration Test - Experiment 1

The dose levels of the controls and the test item are given in the table below:

 

Group	Final concentration ofIFF215 (Floriane)(µg/mL)
4(20)-hour without S9	0*, 7.5*, 15*, 30*, 60, 80, 100, 120, MMC 0.4*
4(20)-hour with S9 (2%)	0*, 7.5*, 15*, 30*, 60, 80, 100, 120, CP 5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Chromosome Aberration Test - Experiment 2

The dose levels of the controls and the test item are given in the table below:

 

Group	Final concentration ofIFF215 (Floriane)(µg/mL)
24-hour without S9	0*, 3.75, 7.5, 15*, 30*, 60*, 80, 120, MMC0.2*
4(20)-hour with S9	0*, 3.75, 7.5, 15, 30*, 45*, 60*, 80, CP5*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Mitotic Index - Preliminary Toxicity Test

4-Hour Treatment, 20-Hour Recovery -S9

4-Hour Treatment, 20-Hour Recovery +S9

24-Hour Treatment -S9

 

se Level (µg/mL)	4(20)-Hour Without S9		4(20)-Hour With S9		24-Hour Without S9
	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	2.10	100	3.20	100	5.20	100
7.66	-	-	-	-	-	-
15.33	2.30	110	2.15 H	67	3.60	69
30.66	2.90 H	138	2.40 H	75	2.85	55
61.31	1.75 H	83	3.80 H	119	2.60 H	50
122.63	- NM H	-	- NM H	-	- NM H P	-
245.25	- NM H P	-	- NM H	-	- NM H P	-
490.5	- NM H P	-	- NM H P	-	- NM H P	-
981	- NM H P	-	- NM H P	-	- NM H P	-
1962	- NM H P	-	- NM H P	-	- NM H P	-

Mitotic Index - Experiment 1

Dose Level (mg/mL)	4(20)-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	2.15	3.35	2.75	100	5.10	5.15	5.13	100
7.5	2.30 H	2.25 H	2.28	83	6.20 H	7.05 H	6.63	129
15	1.20 H	3.20 H	2.20	80	9.10 H	8.15 H	8.63	168
30	1.45 H	3.50 H	2.48	90	3.80 H	5.35 H	4.58	89
60	- NM H	- NM H	-	-	- NM H	- NM H	-	-
80	- NM H	- NM H	-	-	- NM H	- NM H	-	-
100	- NM H	- NM H	-	-	- NM H	- NM H	-	-
120	- NM H	- NM H	-	-	- NM H	- NM H	-	-
MMC 0.4	1.55	2.25	1.90	69	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	0.65	0.70	0.68	13

Mitotic Index - Experiment 2

Dose Level (µg/mL)	24-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	9.15	9.55	9.35	100	5.70	6.55	6.13	100
3.75	-	-	-	-	-	-	-	-
7.5	-	-	-	-	-	-	-	-
15	9.95	12.45	11.20	120	- H	- H	-	-
30	9.55 H	8.50 H	9.03	97	4.70 H	6.05 H	5.38	88
45	NA	NA	NA	NA	5.85 H	5.60 H	5.73	93
60	5.75 H	5.80 H	5.78	62	4.55 H	2.85 H	3.70	60
80	3.45 H	3.20 H	3.33	36	- NM H	- NM H	-	-
120	- NM H	- NM H	-	-	NA	NA	NA	NA
MMC 0.2	2.90	3.70	3.30	35	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	1.50	1.85	1.68	27

Mean Frequency of Polyploid Cells (%)

Experiment 1 

Dose Level (µg/mL)	Exposure Group
	4(20)-Hour Without S9	4(20)-Hour With S9
0	0	0
7.5	0	0.5
15	0	0
30	0	0
MMC 0.4	0	NA
CP 5	NA	0

 

Experiment 2 

Dose Level (µg/mL)	Exposure Group
	24-Hour Without S9	4(20)-Hour With S9
0	0	0
15	0	NA
30	0	0
45	NA	0
60	0	0
MMC 0.2	0	NA
CP 5	NA	0

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item, IFF 215 (Floriane), did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The genotoxic potential of the test substance, IFF 215 (Floriane) was assessed according to OECD Test Guideline 473 using an In Vitro Mammalian Chromosome Aberration method. The test item, IFF 215 (Floriane), did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.