Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 25 January 2017 and 02 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene

EC Number:

951-275-7

Molecular formula:

C12H18O

IUPAC Name:

Reaction mass of Rel-(3aR,4S,7R,7aS)-1-(methoxymethylene)octahydro-1H-4,7-methanoindene and Rel-(3aR,4S,7R,7aS)-2-(methoxymethylene)octahydro-1H-4,7-methanoindene

Specific details on test material used for the study:

Physical state/Appearance: Clear colourless liquid
Storage Conditions: Approximately 4 ºC in the dark
The purity of the test item was accounted for in test item formulation

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: Whole blood cultures from the following donors: Preliminary Toxicity Test: male, aged 25 years Main Experiment: female, aged 24 years

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) 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. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration

Test concentrations with justification for top dose:

The molecular weight of the test item was given as 163, therefore, the maximum dose level was 1630 µg/mL, 10mM, the maximum recommended dose level.
Preliminary toxicity test: 6.37 to 1630 μg/mL (with and without S-9)
Main study: All groups: 0, 6.25, 12.5, 25, 37.5, 50, 75 and 100 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
The test item was insoluble in MEM at 16.3 mg/mL and in Acetone at 163 mg/mL but was soluble in DMSO at 163.0 mg/mL in solubility checks performed in house.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.4 µg/mL for 4-hour exposure and 0.2 µg/mL for 24-hour exposure

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

PREPARATION OF CULTURES: 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).

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 4 or 24 hours, 37 ºC
- Fixation time (start of exposure up to harvest of cells): 24 hrs

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 µg/mL) 2.5 hours
STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED:
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, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

OTHER EXAMINATIONS:
- In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of 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:

Criteria to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range. The level of spontaneous background aberrations was slightly elevated above the normal range and the experiment still considered valid.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations were analyzed.

Providing that the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, under any of the experimental conditions:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

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.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Results and discussion

Test results

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:

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 6.37 to 1630 µg/mL. The maximum dose was the 10 mM concentration.
A cloudy precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 407.5 µg/mL in the absence of S9 and at and above 203.75 µg/mL in the presence of S9, in the 4(20)-hour exposure groups.

Greasy/oily precipitate was also observed in the parallel blood-free cultures at the end of the exposure, at and above 203.75 µg/mL in the absence of S9 and at and above 815 µg/mL in the presence of S9, in the 4(20)-hour exposure groups. In the continuous exposure group cloudy precipitate was observed at and above 101.88 µg/mL.
Haemolysis was observed following exposure to the test item at and above 25.47µg/mL in the 4(20)-hour exposure groups and 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 50.94 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24 hour continuous exposure was also 50.94 µg/mL. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 100 µg/mL for all three exposure groups.

Chromosome Aberration Test – Main Experiment

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 in all three exposure groups. In the presence of metabolic activation (S9) and the continuous exposure group, the maximum dose level of the test item with metaphases suitable for scoring was 75 µg/mL. In the absence of metabolic activation (S9) the maximum dose level of the test item with metaphases suitable for scoring was 50 µg/mL.
Precipitate observations were made at the end of exposure and no precipitate was observed. Haemolysis was observed in the presence of metabolic activation (S9) and the continuous exposure group at and above 75 µg/mL and in the absence of metabolic activation (S9) at and above 50 µg/mL.
They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 55% mitotic inhibition was achieved at 50 µg/mL. In the presence of S9, 41% mitotic inhibition was achieved at 75 µg/mL. An inhibition of mitotic index of 43% was noted at 75 µg/mL in the 24-hour continuous exposure group.
The maximum dose level selected for metaphase analysis was 50 µg/mL in the 4(20)-hour exposure group in the absence of S9 and 75 µg/mL in the 4(20)-hour exposure group in the presence of S9 and 24-hour continuous exposure group.

The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
The required number of cells and concentrations were analyzed.
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 a statistically significant increase in the numbers of polyploid cells at any dose level in all of the exposure groups.

Any other information on results incl. tables

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

Group	Final concentration ofFRET 14-0383(µg/mL)
4(20)-hour without S9	0*, 6.25, 12.5*, 25*, 37.5*, 50*, 75, 100, MMC0.4*
4(20)-hour with S9 (2%)	0*, 6.25, 12.5, 25*, 37.5*, 50*, 75*, 100, CP2*
24-hour without S9	0*, 6.25, 12.5, 25*, 37.5*, 50*, 75*, 100, MMC0.2*

MMC = Mitomycin C

*  = Dose levels selected for metaphase analysis

CP = Cyclophosphamide

 

Mitotic Index - Preliminary Toxicity Test

Dose 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	6.50	100	8.45	100	8.45	100
6.37	-	-	-	-	-	-
12.73	9.75	150	7.05	83	7.25	86
25.47	7.90 H	122	5.15 H	61	6.45 H	76
50.94	2.25 H	35	5.60 H	66	3.90 H	46
101.88	NM H	NM	NM H	NM	NM H G/O	NM
203.75	NM H G/O	NM	NM H C	NM	NM H G/O	NM
407.5	NM H G/O C	NM	NM H C	NM	NM H G/O	NM
815	NM H G/O C	NM	NM H G/O C	NM	NM H G/O	NM
1630	NM H G/O C	NM	NM H G/O C	NM	NM H G/O	NM

-      = Not assessed for mitotic index

NM   = No metaphases or insufficient metaphases suitable for scoring

H     = Hemolysis observed at the end of exposure in blood cultures

G/O= Greasy/oily precipitate observed at end of exposure period in blood-free cultures

C     = Cloudy precipitate observed at end of exposure period in blood-free cultures

Mitotic Index – Main Experiment (4(20)-hour Exposure Groups)

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	9.45	6.90	8.18	100	7.80	9.20	8.50	100
6.25	-	-	-	-	-	-	-	-
12.5	8.20	9.45	8.83	108	-	-	-	-
25	7.50 H	9.90 H	8.70	106	6.85	8.40	7.63	90
37.5	8.00 H	8.90 H	8.45	103	9.55 H	9.85 H	9.70	114
50	2.50 H	4.90 H	3.70	45	9.20 H	7.00 H	8.10	95
75	NM H	NM H	NM	NM	5.20 H	4.80 H	5.00	59
100	NM H	NM H	NM	NM	NM H	NM H	NM	NM
MMC 0.4	2.00	2.25	2.13	26	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	3.90	3.45	3.68	43

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

H = Haemolysis

 

 

Mitotic Index – Main Experiment (24-hour Exposure Group)

Dose Level (µg/mL)	24-Hour Without S9
	A	B	Mean	% of Control
0	4.50	6.05	5.28	100
6.25	-	-	-	-
12.5	-	-	-	-
25	6.70	4.50	5.60	106
37.5	3.80 H	5.80 H	4.80	91
50	4.00 H	3.50 H	3.75	71
75	2.20 H	3.80 H	3.00	57
100	NM H	NM H	NM	NM
MMC 0.2	1.45	2.85	2.15	41

MMC = Mitomycin C

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

H = Haemolysis

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the test conditions, FRET 14-0383 is not considered as clastogenic in human lymphocytes according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to FRET 14-0383 in DMSO at concentration range of 6.25 - 100 μg/mL, for 4 + 24 h (treatment + recovery) with metabolic activation (2% S-9), and for 4 + 20 h or 24 + 0 h (treatment + recovery) without metabolic activation for a preliminary cytotoxicity test.

 

In the main test, all experiments were performed at concentrations up to 100 µg/mL without S-9 and with S-9 and the following concentrations were selected for analysis:

4(20)-hour without S9: 0, 12.5, 25, 37.5, and 50 μg/mL

4(20)-hour with S9 (2%): 0, 25, 37.5, 50 and 75 µg/ml

24-hour without S9: 0, 25, 37.5, 50 and 75 µg/ml

 

Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control ranges. Positive controls (Mitomycin C at 0.4 and 0.2 µg/mL (4 and 24 hr treatment groups respectively) without S-9 and cyclophosphamide at 2 µg/mL with S-9) induced the appropriate response. Treatment of cells with FRET 14-0383 in the presence or absence of S-9 in both experiments resulted in frequencies of cells with structural or numerical aberrations that were generally similar to those observed in concurrent vehicle controls for all concentrations analysed.

 

Under the test conditions, FRET 14-0383 is not considered as clastogenic in human lymphocytes in vitro.