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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Dicyclopentadienyl acrylte was negative in a bacterial and mammalian cell gene mutation assay. Furthermore, no clastogenic effects of dicyclopentadienyl acrylate was observed in an in-vitro chromosome aberration assay. Based on these results dicyclopentadienyl acrylate has no genotoxic potential.

Link to relevant study records

Reference

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

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

Target gene:

HPRT locus

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

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.

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'.

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)

 

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In-vitro studies: Bacterial systems

One bacterial gene mutation assay according to OECD guideline 471 is available with dihydrodicyclopentadienyl acrylate (BASF SE, 1994). The assay was negative at doses up to 5000 μg/plate (standard plate test) and 120 µg/plate (preincubation test), respectively with and without S-9 mix in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537. The selected Salmonella strains are not capable to detect oxidizing or cross-linking damage, thus leading to a limited conclusion. Since the experiment was negative in the strains tested, with and without metabolic activation, there is no indication for a mutagenic potential of the test substance in bacterial cells, not taking in account oxidizing or cross-linking damage as the underlying mechanism.

In-vitro studies: Mammalian cell gene mutation test

One mammalian cell gene mutation test (HPRT test, according to OECD TG No. 476) is available for dihydrodicyclopentadienyl acrylate (BASF AG, 1994).

Doses of up to 0.01 mg/mL (without S-9 mix) or 0.5 mg/mL (with S-9 mix from Aroclor-induced rat livers) were tested in a 4-hour treatment. 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. No significant increase of mutant frequencies nor a dose-response relationship were observed, indicating that the substance has no mutagenic potential in mammalian CHO cells.

In-vitro studies: chromosomal aberration

For analysis of chromosomal aberrations, V79 hamster lung fibroblasts were treated with 1.25 -15 µg/mL (without S9-mix) and 25 – 200 µg/mL (with S9-Mix), respectively. Cells were treated for 18 and 28 h (without S9-Mix), and were sampled afterwards. Treatment with S9-mix was 4 h with subsequent sampling 18 and 28 h after beginning of treatment. 1000 cells were examined for mitotic index and 100 mitoses per dose were analyzed for chromosomal damage. No increase in aberration frequency was observed in treated cultures, thus there is no evidence for clastogenicity of dihydrodicyclopentadienyl acrylate.

 

 

Justification for selection of genetic toxicity endpoint
The key study was selected

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

No classification required for genetic toxicity.

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008 (last amended by EC/286/2008 (2011-03-10)):

No classification required for genetic toxicity.