Hexahydro-4,7-methano-1H-indenyl acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Oct - 5 Dec 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline study conducted in compliance with GLP regulations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexahydro-4,7-methano-1H-indenyl acrylate

EC Number:

235-697-2

EC Name:

Hexahydro-4,7-methano-1H-indenyl acrylate

Cas Number:

12542-30-2

Molecular formula:

C13 H16 O2

IUPAC Name:

2-Propenoic acid hexahydro-4,7-methano-1H-indenyl ester

Details on test material:

- Name of the test substance used in the study report: Dihydrodicyclopentadienylacrylat
- Analytical Purity: 96.8% (sum of 4 isomers; determined by GC-FID)
- Impurities (identity and concentrations):
- 2-ethylhexanol 0.7 area-%,
- acrylic acid 0.06 g/100 g (0.11 g/100 g based on an acid value of 0,86 mg KOH/ g; determined by potentiographic titration),
- water 0.02 g/100 g
- hydroquinone-monomethylether 230 mg/kg.
- Purity test date: 20 Oct 1994; 94L00565
- Lot/batch No.: From vessel No. 27, August 30, 1994 G 403
- Test substance No.: 94/244

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 fraction

Test concentrations with justification for top dose:

0; 0.0001; 0.0005; 0.001; 0.005; 0.01 mg/mL in experiment 1 without metabolic activation and 0; 0.005; 0.01; 0.05; 0.1; 0.5 mg/mL in experiment 1 with metabolic activation.
0; 0.0005; 0.001; 0.005; 0.0075; 0.01 mg/mL in experiment 2 without metabolic activation and 0; 0.0005; 0.005; 0.05; 0.1; 0.5 mg/mL in experiment 2 with metabolic activation.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: without S-9 mix: Ethylmethanesulfonate; with S-9 mix: 3-Methylcholanthrene

Details on test system and experimental conditions:

DURATION
- Attachment period: 20-24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 1 week
- Selection time (if incubation with a selection agent): 1 week
- Fixation time (start of exposure up to fixation or harvest of cells): 2 weeks

SELECTION AGENT (mutation assays): 6-Thioguanine

DETERMINATION OF CYTOTOXICITY
- Method:
cloning efficiency (Cytotoxicity 1): 17 - 24 hours after termination of the exposure period approx. 200 cells of each replicate (pooled from 2 flasks) were taken in duplicates, seeded into Ham's F12 medium (25 cm2 flasks) and allowed for colony formation (incubator; 1 week). After the end of the incubation period the colonies were fixed, stained and counted.
cloning efficiency (Cytotoxicity 2):
-Same procedure as "Cytotoxicity 1".
-Timepoint: parallel to selection (7 days after exposure)
Other: examination of precipitation

Evaluation criteria:

The criteria for a positive response are:
- Increases of the corrected mutation frequencies above the concurrent negative control values and above 15 mutants per 10E +06 clonable cells and/or the evidence of a dose-response relationship in the increase in mutant frequencies.
- Evidence of reproducibility of any increase in mutant frequencies.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Reduced cell densities/cloning efficiencies were found at concentrations > 0.005 mg/mL in the experiments without metabolic activation. In the presence of S-9 mix cytotoxicity was observed at 0.5 mg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of test substance after addition to test medium was observed at concentrations >=0.1 mg/mL

RANGE-FINDING/SCREENING STUDIES:
-Treatment with Dihydrodicyclopentadienylacrylat at concentrations ranging from 0.005 to 5 mg/mL (with and without S-9 mix). Cytotoxicity was observed at concentrations >= 0.005 mg/mL in the absence of S9-mix, and at concentrations >=0.5 mg/mL in the presence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mutant frequencies-experiment 1 without metabolic activation.

Concentration (mg/ml)		No. of colonies^a						Mean mutant frequency (per 10^6cells)
								Not corrected	Corrected^b
0	A	0	0	0	0	0	0	2.22	2.73
	B	0	1	1	2	2	2
1% DMSO	A	0	0	0	0	1	1	1.95	2.15
	B	0	0	1	1	1	2
0.0001	A	0	0	0	0	0	0	0.56	0.68
	B	0	0	0	0	1	1
0.0005	A	0	0	0	0	0	0	0.56	0.78
	B	0	0	0	0	1	1
0.001	A	0	1	2	3	3	3	5.84	7.13
	B	0	1	1	2	2	3
0.005	A	0	0	0	0	0	0	3.06	4.01
	B	1	1	1	2	2	4
0.01	A	0	0	0	0	0	0	0.00	0.00
	B	0	0	0	0	0	0
EMS 0.3	A	22	27	27	29	30	40	98.06	116.56
	B	20	25	29	30	32	42
a: No of colonies 7 days after seeding, about 300000 cells/flask into selection medium (6 flasks/replicate)
b: corrected on the basis of the absolute cloning efficiency at the end of the expression period (cytotoxicity 2).

Table 2: Mutant frequencies-experiment 1 with metabolic activation.

Concentration (mg/ml)		No. of colonies^a						Mean mutant frequency (per 10^6cells)
								Not corrected	Corrected^b
0	A	0	0	1	1	2	2	6.39	8.23
	B	1	3	3	3	3	4
1% DMSO	A	0	0	0	0	1	2	0.84	1.12
	B	0	0	0	0	0	0
0.005	A	0	0	0	0	0	0	0.00	0.00
	B	0	0	0	0	0	0
0.01	A	0	1	1	2	2	2	3.33	3.95
	B	0	0	1	1	1	1
0.05	A	0	0	0	0	0	1	0.56	0.69
	B	0	0	0	0	0	1
0.1	A	0	0	1	1	2	3	3.61	4.69
	B	0	0	1	1	2	2
0.5	A	0	0	0	0	0	0	3.34	4.51
	B	1	1	2	2	2	4
MCA 0.01	A	37	40	45	49	50	51	129.17	188.84
	B	27	29	30	33	34	40
a: No of colonies 7 days after seeding, about 300000 cells/flask into selection medium (6 flasks/replicate)
b: corrected on the basis of the absolute cloning efficiency at the end of the expression period (cytotoxicity 2).

 

Table 3: Mutant frequencies-experiment 2 without metabolic activation.

Concentration (mg/ml)		No. of colonies^a						Mean mutant frequency (per 10^6cells)
								Not corrected	Corrected^b
0	A	0	0	0	1	1	1	2.50	3.64
	B	0	0	0	1	2	3
1% DMSO	A	0	0	0	0	0	0	1.67	2.30
	B	0	0	1	1	1	3
0.0005	A	0	0	0	0	0	0	0.00	0.00
	B	0	0	0	0	0	0
0.001	A	0	0	0	0	0	1	3.06	4.72
	B	1	1	2	2	2	2
0.005	A	0	0	0	0	1	1	1.67	2.5
	B	0	0	0	1	1	2
0.0075	A	0	0	0	0	0	0	2.78	4.26
	B	0	1	1	2	2	4
0.01	A	0	0	0	c	c	c	0.00	0.00
	B	0	0	c	c	c	C
EMS 0.3	A	34	37	41	43	50	50	149.45	262.33
	B	26	41	44	50	58	64
a: No of colonies 7 days after seeding, about 300000 cells/flask into selection medium (6 flasks/replicate)
b: corrected on the basis of the absolute cloning efficiency at the end of the expression period (cytotoxicity 2).
c: not seeded because not enough cells (due to toxicity)

 

Table 4: Mutant frequencies-experiment 2 with metabolic activation.

Concentration (mg/ml)		No. of colonies^a						Mean mutant frequency (per 10^6cells)
								Not corrected	Corrected^b
0	A	0	0	0	0	0	1	2.78	4.22
	B	0	1	1	1	2	4
1% DMSO	A	0	0	0	0	0	0	0.00	0.00
	B	0	0	0	0	0	0
0.0005	A	1	2	2	3	3	5	5.56	9.25
	B	0	0	0	1	1	2
0.005	A	0	0	0	0	0	1	1.12	1.74
	B	0	0	0	1	1	1
0.05	A	0	0	0	0	0	1	1.12	1.70
	B	0	0	0	1	1	1
0.1	A	1	1	1	3	3	5	5.28	8.01
	B	0	0	1	1	1	2
0.5	A	0	c	c	c	c	c	0.00	0.00
	B	0	c	c	c	c	c
MCA 0.01	A	2	5	6	7	9	13	27.78	43.53
	B	7	9	9	9	11	13
a: No of colonies 7 days after seeding, about 300000 cells/flask into selection medium (6 flasks/replicate)
b: corrected on the basis of the absolute cloning efficiency at the end of the expression period (cytotoxicity 2).
c: not seeded because not enough cells (due to toxicity)

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative