Reaction mass of ethyl (1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate and ethyl (1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

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 27 April 2010 and 31 August 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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:

2010

Report date:

2010

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:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Molecular formula:

C10H14O2

IUPAC Name:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Test material form:

other: Liquid

Details on test material:

Identity: TM 09-218
Chemical name: Bicyclo[2,2,1]hept-5-ene-2-carboxylic acid ethyl ester
Molecular weight: 166.10
Appearance: Clear liquid
Storage conditions: Room temperature (ca. 20°C) in the dark
Date received: 18 February 2010

Method

Species / strain

Species / strain / cell type:

lymphocytes: Human lymphocytes in whole blood culture

Details on mammalian cell type (if applicable):

Culture of lymphocytes
Human blood was collected aseptically from two healthy, non-smoking male donors, pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM glutamine. Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL phytohaemagglutinin) of the cell suspension were placed in sterile universal containers and incubated at 37°C in a 5% CO2 atmosphere for approximately 48 hours. The cultures were gently shaken daily to resuspend the cells.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

First test
In the absence of S9 mix - 3 hour treatment, 18 hour recovery: 129.20, 215.30 and 358.80 μg/mL.
In the presence of S9 mix (2% v/v) - 3 hour treatment, 18 hour recovery: 480, 495 and 525 μg/mL.

Second test
In the absence of S9 mix - 21 hour continuous treatment: 80, 160, and 240 μg/mL.
In the presence of S9 mix (5% v/v) - 3 hour treatment, 18 hour recovery: 500, 550 and 575 μg/mL.

Vehicle / solvent:

Selection of solvent
At the Sponsor’s request, the selected solvent for this study was ethanol. Therefore, the solubility of TM 09-218 in ethanol was assessed within this test system. TM 09-218 was found to be miscible in ethanol at 166.10 mg/mL (1M). On dosing a 166.10 mg/mL solution at 1% v/v into aqueous tissue culture medium, giving a final concentration of 1661.0 μg/mL (10 mM), no precipitate was observed.
Concentrations with high ionic strength and osmolality may cause chromosomal aberrations (Galloway et al. 1987). Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test system.
The osmolality and pH of the test substance in medium was tested at 1661.0 μg/mL; no fluctuation in osmolality of more than 50 mOsm/kg and no change in pH of more than 1.0 unit were observed compared with the solvent control.
In this case, the highest final concentration used for subsequent testing was 1661.0 μg/mL (10 mM). This is the standard limit concentration within this test system as recommended in the regulatory guidelines. In this study TM 09-218 was added to cultures at 1% v/v (50 μL per 5 mL culture).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Treatment of cells with test substance – first test
Solvent, positive and treatment cultures were established approximately 48 hours after commencement of incubation of lymphocyte cultures. Duplicate cultures were prepared throughout for each 3 hour treatment in the absence and presence of S9 mix. All cultures were centrifuged and resuspended in fresh medium just before treatment. In the absence of S9 mix, 50 μL aliquots of TM 09-218 were added to give final concentrations of 16.70, 27.90, 46.50, 77.50, 129.20, 215.30, 358.80, 598, 996.60 and 1661 μg/mL. Ethanol was used as the solvent control and Mitomycin C at a final concentration of 0.2 μg/mL was the positive control.

For treatments in the presence of S9 mix, 1 mL of medium was removed from each culture and discarded. This was replaced with 1 mL of S9 mix (2% v/v final concentration), followed by 50 μL of each test substance dilution (giving the same series of final concentrations as above). Ethanol was used as the solvent control and Cyclophosphamide at a final concentration of 5 μg/mL was the positive control.
In both the absence and presence of S9 mix, no notable culture medium changes were observed either at or after 3 hour treatment.
Three hours after dosing, the cultures were centrifuged at 500g for 5 minutes. The supernatant removed and the cell pellets resuspended in fresh medium. They were then incubated for a further 18 hours.
In the absence of S9 mix the appropriate toxicity was achieved to permit metaphase analysis. In the presence of S9 mix, two additional tests were conducted to achieve the required toxicity. The concentrations of TM 09-218 selected for each test were as follows:
In the presence of S9 mix: 60, 120, 180, 240, 300, 360, 420, 480, 540 and 600 μg/mL.
In the presence of S9 mix: 300, 400, 420, 435, 450, 465, 480, 495, 510, 525 and 540 μg/mL.
No notable changes were observed either at or after 3 hour treatment.

Harvesting and fixation
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 μg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37°C. After a 10 minute period of incubation at 37°C, the suspensions were centrifuged at
500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear.

Slide preparation
The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at 4°C until slide analysis was completed.

Microscopic examination
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures, or cultures where there were no signs of cytotoxicity. From these results the concentration causing a decrease in mitotic index of at least 50% (where possible) of the solvent control value was the highest concentration selected for metaphase analysis. Intermediate and low concentrations were also selected.

The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective. One hundred metaphase figures were examined from each culture, however, this number was reduced in cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed. Chromosome aberrations were scored according to the classification of the ISCN (1985). Only cells with 44 - 48 chromosomes were analysed. Polyploid and endoreduplicated cells were noted when seen.

The vernier readings of all aberrant metaphase figures were recorded.

The number of aberrant metaphase cells in each test substance group was compared with the solvent control value using the one-tailed Fisher exact test (Fisher 1973). A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

D20s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002).

The following model was used:
P = C+ (1 – C) / (1 + exp(-intercept – slope 1n(conc)))
P is the proportion of cells with aberrations, conc is the concentration of test substance. C is a parameter estimating the control proportion of aberrations.

Second test
Cultures were initiated and maintained as previously described. In this second test a 21 hour continuous treatment was used in the absence of S9 mix. In the presence of S9 mix, a 3 hour treatment was used, as in the first test. However, to modify study parameters the final concentration of S9 mix was increased from 2% v/v to 5% v/v. All cultures were centrifuged and resuspended in fresh medium just before treatment.
Concentrations of TM 09-218 were as follows:
In the absence of S9 mix: 20, 40, 80, 120, 160, 200, 240, 280, 320, 360 and 400 μg/mL.
In the presence of S9 mix (5% v/v): 300, 400, 430, 445, 460, 475, 490, 505, 520, 535 and 550 μg/mL.

Duplicate cultures were used for each treatment and two cultures were treated with the solvent control. Positive control cultures were treated as in the first test. Mitomycin C, at a final concentration of 0.1 μg/mL, and Cyclophosphamide, at a final concentration of 5 μg/mL, were added to duplicate cultures.
In both the absence and presence of S9 mix, no notable culture medium changes were observed either at or after treatment.

In the absence of S9 mix the appropriate toxicity was achieved to permit metaphase analysis.
In the presence of S9 mix, two additional tests were conducted to achieve the required toxicity. Concentrations of TM 09-218 were as follows:
In the presence of S9 mix: 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 and 1661 μg/mL.
In the presence of S9 mix: 450, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725 and 750 μg/mL.
In both the absence and presence of S9 mix, no notable changes were observed either at or after 3 hour treatment.
Three hours after dosing, the cultures containing S9 mix were centrifuged. The cell pellets were rinsed and resuspended in fresh medium. They were then incubated for a further 18 hours. Cultures treated in the absence of S9 mix were incubated continuously for 21 hours.
All cultures were treated with Colcemid®, at a final concentration of 0.1 μg/mL, two hours before the end of the incubation period. They were then harvested, fixed and the slides prepared as previously described. The slides were examined microscopically as previously described.

Evaluation criteria:

Assessment of Results
An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance is considered to cause a positive response if the following conditions are met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration. The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.
The increases are reproducible between replicate cultures.
The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response is considered to support the conclusion.
A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.

Results and discussion

Test results

Species / strain:

lymphocytes: Human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First test
Toxicity data
In the absence of S9 mix following 3 hour treatment, TM 09-218 caused a reduction in the mitotic index to 55% of the solvent control value at 358.80 μg/mL. The concentrations selected for metaphase analysis were 129.20, 215.30 and 358.80 μg/mL.
In the presence of S9 mix (2% v/v final concentration) following 3 hour treatment, TM 09-218 caused a reduction in the mitotic index to 56% of the solvent control value at 525 μg/mL. The concentrations selected for metaphase analysis were 480, 495 and 525 μg/mL.

Metaphase analysis
In both the absence and the presence of S9 mix, TM 09-218 caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.
All mean values for the solvent control (ethanol), and all TM 09-218 treatment concentrations were within the laboratory historical control range, when taken at the 99% confidence limit.
Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Second test
Toxicity data
In the absence of S9 mix following 21 hour continuous treatment, TM 09-218 caused a reduction in the mitotic index to 52% of the solvent control value at 240 μg/mL. The concentrations selected for metaphase analysis were 80, 160 and 240 μg/mL.
In the presence of S9 mix (5% v/v final concentration) following 3 hour treatment, TM 09-218 caused a reduction in the mitotic index to 50% of the solvent control value at 575 μg/mL. The concentrations selected for metaphase analysis were 500, 550 and 575 μg/mL.

Metaphase analysis
In the absence of S9 mix following 21 hour continuous treatment, TM 09-218 caused statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at concentrations of 80 μg/mL (p<0.001 including gaps and p<0.01 excluding gaps) and 160 μg/mL and 240 μg/mL (p<0.01 both including and excluding gaps), when compared with the solvent control.
All mean values for the solvent control (ethanol) were within the laboratory historical control range, when taken at the 99% confidence limit. All mean values for TM 09-218 treatment concentrations were outside the laboratory historical control range, when taken at the 99% confidence limit.

In the presence of S9 mix, TM 09-218 caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any concentration, when compared with the solvent control.
All mean values for the solvent control (ethanol), and all TM 09-218 treatment concentrations were within the laboratory historical control range, when taken at the 99% confidence limit. Both positive control compounds, Mitomycin C and Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

Polyploid analysis
No statistically significant increases in polyploid metaphases were observed during metaphase analysis in either test.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results

First Test

 

Exposure period	S9 mix	Nominal concentration of TM 09-218	Cells with aberrations excluding gaps			Cells with aberrations including gaps			Relative mitotic index (%)	Polyploidy mean incidence (%)
(hours)	(v/v)	(µg/mL)	Individual values (%)		Mean (%)	Individual values (%)		Mean (%)
3	-	0 (Ethanol)	0.0	0.0	0.0	2.0	1.0	1.5	100	0.5
		129.2	0.0	1.0	0.5	1.0	2.0	1.5	99	0.5
		215.3	0.0	0.0	0.0	2.0	1.0	1.5	70	0.0
		358.8	1.0	4.0	2.5	1.0	8.0	4.5	55	0.5
		0.2 (Mitomycin C)	21.3	27.8	24.1***	29.8	27.8	28.9***	-	0.0
3	+	0 (Ethanol)	2.0	1.0	1.5	2.0	1.0	1.5	100	0.5
	(2%)	480	0.0	0.0	0.0	0.0	1.0	0.5	108	2.5
		495	1.0	0.0	0.6	2.0	1.3	1.7	69	2.2
		525	4.1	2.0	2.7	4.1	2.0	2.7	56	2.0
		5 (Cyclophosphamide)	22.7	31.3	26.3***	25.0	31.3	27.6***	-	0.0

 

One-tailed Fisher's exact test

***  p<0.001

Otherwise  p>0.01

 

Second Test 

Exposure period	S9 mix	Nominal concentration of TM 09-218	Cells with aberrations excluding gaps						Cells with aberrations including gaps			Relative mitotic index (%)	Polyploidy mean incidence (%)
(hours)	(v/v)	(µg/mL)	Individual values (%)				Mean (%)		Individual values (%)		Mean (%)
21	-	0 (Ethanol)	1.0		0.0		0.5		2.0	1.0	1.5	100	1.5
		80	7.0		3.0		5.0**		11.0	8.0	9.5***	100	3.0
		160	6.0		3.0		4.5**		8.0	6.0	7.0**	78	2.0
		240	6.0		4.0		5.0**		6.0	7.0	6.5**	52	4.5
		0.1 (Mitomycin C)	47.6		40.0		43.5***		47.6	40.0	43.5***	-	0.0
3	+	0 (Ethanol)		0.0		0.0		0.0	0.0	0.0	0.0	100	1.5
	(5%)	500		1.0		0.0		0.5	1.0	0.0	0.5	99	0.5
		550		1.0		0.0		0.5	1.0	1.0	1.0	78	0.5
		575		0.0		2.0		1.0	0.0	3.0	1.5	50	1.0
		5 (Cyclophosphamide)		7.0		6.0		6.5***	11.0	7.0	9.0***	-	0.5

 

One-tailed Fisher's exact test

*** p<0.001

**  p<0.01

Otherwise p>0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

It is concluded that, under the experimental conditions described, TM 09-218 has shown evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix following 21 hour continuous exposure only, in this in vitro cytogenetic test system. Under all other experimental conditions in this in vitro cytogenetic test system, TM 09-218 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations.

Executive summary:

The genotoxic potential of the test substance, TM 09-218, was assessed according to OECD Test Guideline 473 using an In vitro Mammalian Chromosome Aberration method. The test substance showed evidence of causing an increase in the frequency of structural chromosome aberrations in the absence of S9 mix following 21 hour continuous exposure only, in this in vitro cytogenetic test system. Under all other experimental conditions, the test substance showed no evidence of causing an increase in the frequency of structural chromosome aberrations.