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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three key in vitro studies are available to assess the genetic toxicity of dicyclopentadiene.

- OECD 471 (Bacterial Reverse Mutation Assay)

- OECD 476 (Mammalian Cell Gene Mutation Test)

- Chromosome aberration (according to Japan: Guidelines for Screening Mutagenicity Testing of Chemicals)

The above studies showed that dicyclopentadiene is not mutagenic in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
n/a
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Dicyclopentadiene resin grade
- Physical state: clear light yellow liquid
- Analytical purity: 75%
- Lot/batch No.: TNZ001
- Expiration date of the lot/batch: 1 April 2000
- Storage condition of test material: room temperature in dark
Target gene:
n/a
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Cytokinesis block (if used):
n/a
Metabolic activation:
with and without
Metabolic activation system:
S9 from Arochlor 1254 induced rat liver
Test concentrations with justification for top dose:
Dose range 1-666 µg/plate
Vehicle / solvent:
Vehicle(s)/solvent(s) used: ethanol
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, daunomycine, methylmethanesulfonate, 4-nitroquinoline N-oxide, 2 aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: observation of reduction of bacterial background lawn, reduction in revertant colonies
Evaluation criteria:
Negative (ie non-mutagenic) if:
a) total number of revertants in tester strain at any concentration is not > 2 x solvent control value for TA100 and 3 x solvent control value for TA1535, TA1537, TA98 and WP2uvrA +/- activation
b) Negative response should be repeatable in at least one independently repeated expt.
Positive (ie mutagenic) if:
a) it produces at least a 3-fold (TA1535, TA1537, TA98 and WP2uvrA) or 2-fold (TA100) dose-related increase in the number of revertants with respect to the number induced ny solvent control in TA100 +/- activation. However any mean plate count < 20 is considered to be not significant
b) Positive response should be repeatable in at least one independently repeated expt.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
n/a

n/a

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Dicyclopentadiene resin grade is not mutagenic in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay.
Executive summary:

In this OECD 471 guideline study, dicyclopentadiene resin grade did not induce a dose-related or a two-fold, increase in the number of revertant (His+) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) nor in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Therefore, it was concluded that dicyclopentadiene resin grade is not mutagenic in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study, guideline study, available as published report and summary information sheet (provided by Japan Chemical Industry Ecology-Toxicology and Information Center (JETOC)), minor restrictions in design and/or reporting but otherwise adequate for assessment.
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Dicyclopentadiene (DCPD)
- Analytical purity: 95%
Target gene:
n/a
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
no data
- Type and identity of media: Culture is foetal calf serum (FCS) supplemented with 10% Eagle MEM using the medium
Cytokinesis block (if used):
n/a
Metabolic activation:
with and without
Metabolic activation system:
Rat liver (strain not specified). Phenobarbital and 5,6-benzoflavone induced (Treatment not specified)
Test concentrations with justification for top dose:
Continuous treatment:
First experiment: 24 and 48 hour continuous treatment (-S9): 0.0, 0.014, 0.029, 0.057 mg/mL
Second experiment: 24 hour continuous treatment (-S9): 0.0, 0.029, 0.043, 0.057 mg/mL
Short-term treatment:
(-S9): 0.0, 0.014, 0.029, 0.057 mg/mL
(+S9): 0.0, 0.03, 0.05, 0.10 mg/mL
Vehicle / solvent:
Acetone
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: (-S9): 0.00005 mg/mL Mitomycin C, (+S9): 0.005 mg/mL cyclophosphamide
Remarks:
doses not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- The test material was incubated with CHL/IU cells in growth phase (2x10^4 cells/mL growth medium) for 24 hrs and 48 hrs continuous treatment without metabolic activation and for a shorter duration (6 hrs) with and without metabolic activation from rat liver S9, at 37°C in a 5% CO2 in air incubator.
- In accordance to Japanese guidelines, the dose range was selected to produce 50% or greater inhibition of cell growth or mitosis at the maximum dose level.
- Following short-term exposure, cultures containing S9 mix were washed and fresh medium added.
- All cultures were treated with Colcemid® approximately 2 hrs prior to harvest to arrest dividing cells in metaphase.
- Cells were fixed and slides stained with 3% Giemsa solution, a standard stain for metaphase chromosome spreads).
- All slides, including positive and negative controls were coded before microscopic analysis and read "blind".

NUMBER OF REPLICATIONS: 2 cultures per dose level

NUMBER OF CELLS EVALUATED:
- Japanese guidelines specify that 100 metaphase spreads should be counted and analyzed for structural aberrations (gaps, breaks, exchanges) and polyploids, and the percentage of cells with aberrations (with and without gaps) calculated.
Rationale for test conditions:
Study performed according to JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals.
Evaluation criteria:
Chromosome analysis was done according to the Environmental Mutagen Society of Japan, mammalian test (MMS) Session 1 and was based on the taxonomy of the gap or chromatid-type chromosomal pattern, cut and the presence/absence of abnormal ploidy structure.
The following were recorded: number of cells observed, number and type of structural abnormality, total number of cells for ploidy.
Statistics:
Fischer's Exact test - frequency of cells with chromosomal abnormalities
Kastenbaum & Bowman method - micronucleus test
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dicylcopentadiene did not induce structural chromosomal aberrations or polyploidy in CHL/IU cells up to a concentration causing more than 50% cell growth inhibition with or without metabolic activation. Structural chromosomal aberrations were marginally induced at the highest dose –S9, 0.057mg/mL, after 24 hr continuous exposure.

n/a

Conclusions:
Interpretation of results: negative

Dicyclopentadiene did not induce significant cytogenetic damage to mammalian cells in vitro under conditions of this assay. Although some marginal chromosome damage occurred at the highest –S9 dose after 24 hrs continuous exposure, the test material was confirmed to be negative for clastogenicity in an in vitro micronucleus assay.
Executive summary:

Dicyclopentadiene (DCPD) was assessed for mutagenic properties by evaluating increases in the chromosome aberrations in Chinese hamster lung (CHL/IU) cells. DCPD did not induce significant cytogenetic damage to mammalian cells in vitro under conditions of this assay. Although some marginal chromosome damage occurred at the highest –S9 dose after 24 hrs continuous exposure, the test material was confirmed to be negative for clastogenicity in an in vitro micronucleus assay.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Version: July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Version: May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Deviations:
no
Principles of method if other than guideline:
n/a
GLP compliance:
yes (incl. QA statement)
Remarks:
Exception: no analysis was done on homegeneity, concentration, or stability of the test substance formulation. The test item was formulated within 2 hours of it being applied to the test system and it was assumed to be stable for this duration.
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Identification: Dicyclopentadiene
Physical state / appearance: clear colourless liquid
Batch: Ex tank 10621-04-2014; Size 0.1L W23B400081
Purity: Approximately 95%
Expiry date: 01 August 2014
Storage conditions: Approximately 4°C
Molecular weight: 132.2 g/mol
Target gene:
n/a
Species / strain / cell type:
mouse lymphoma L5178Y cells
Cytokinesis block (if used):
n/a
Metabolic activation:
with and without
Metabolic activation system:
PB/BNF S9 fraction prepared in-house from the livers of male Sprague-Dawley rats following three consecutive daily doses of phenobarbital/ß-naphthoflavone (80/100 mg/kg bw/day). The S9 was stored in a liquid nitrogen freezer at approximately -196°C.
Test concentrations with justification for top dose:
Initial toxicity test: 0, 5.16, 10.31, 20.63, 41.25, 82.5, 165, 330, 660, 1320 µg/mL
Experiment 1: (10, 15, 20, 25, 30, 35 µg/mL (4h -S9)
Experiment 1: 10, 20, 30, 40, 50, 60 µg/mL (4h +S9)
Experiment 2: 5, 10, 20, 30, 40, 50 µg/mL (24h -S9)
Experiment 2: 10, 20, 30, 40, 45, 50 µg/mL (4h +S9)
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
positive controls were formulated in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (24 hours in experiment 2 in the absence of S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: 2
Rationale for test conditions:
Study performed according to OECD 476 test guideline
Evaluation criteria:
Majority of plates for viability or TFT resistance are analysable
Viability of solvent controls: 65-120%
Total suspension growth of the solvent control over 4h should be in the range 8-32.
In-house vehicle control MF in the range 50-170x10-6
Positive control chemicals should induce at least 3-5 fold increase in MF
The upper limit of cytotoxicity in the positive control and test substances should be the same
Highest concentration of test substance should be 10mM/5000µg/mL unless limited by cytoxicity or solubility.
Statistics:
n/a
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the preliminary cytotoxicity test there was marked reduction in relative suspension growth of the cells at concentrations of ca. 80µg/mL and above, and cloudiness was observed at and above 330µg/mL. The maximum dose level in the subsequent mutagenicity experiments was therefore limited by test item-induced toxicity.

Two subsequent mutagenicity experiments were undertaken.

There was evidence of marked toxicity following exposure to the test item in the absence and presence of S9. Near optimum levels of toxicity were achieved in the absence of S9, but not in the presence of S9, despite a narrow concentration selection, due to the steep toxicity curve. A dose level that exceeded the upper limit for toxicity was plated for viability and TFT resistance as sufficient cells were available.
The vehicle controls had mutation frequencies (MFs) that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both positive controls induced marked increases in MF.
The test item did not induce any statistically significant or dose-related increases in the mutation frequency, either in the absence or presence of S9.

n/a

Conclusions:
Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Dicyclopentadiene did not induce a significant increase in mutant frequencies at the TK locus in the L5178Y mouse lymphoma cells. It was therefore concluded that DCPD is non-mutagenic under the conditions of the experiment.
Executive summary:

Dicyclopentadiene (DCPD) was assessed for mutagenic properties by evaluating increases in the mutant frequency at the TK locus of L5178Y mouse lymphoma cells. The application of the test substance was limited by a steep toxicity dose-response curve. The test substance did not cause a statistically significant or dose-related increase in mutant frequency either in the absence or presence of PB/BNF S9, following incubation for 4 hours (24 hours in one experiment in the absence of S9). The positive control substances (EMS and CP) gave the expected increases in mutation frequency.

In conclusion, DCPD does not cause gene mutation in mammalian cells in vitro, either without or with metabolic activation, under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A supporting study OECD 474 (Mammalian Erythrocyte Micronucleus Test) on dicyclopentadiene/Codimer Concentrate showed that the test item is not mutagenic in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Non-human information

In Vitro data

The key studies are considered to be a bacterial mutation assay (NOTOX, 2000), a mammalian cell cytogenetic assay (JETOC, 1998b), and a mammalian gene mutation assay (Harlan, 2014). These are three recognised core assay types for investigating mutation in vitro.

In Vitro Gene Mutation in Bacterial Cells

 

In a key OECD 471 guideline study (Notox, 2000), dicyclopentadiene resin grade did not induce a dose-related or a two-fold, increase in the number of revertant (His+) colonies in any of the four Salmonella typhimurium tester strains (TA1535, TA1537, TA98 and TA100) nor in the number of revertant (Trp+) colonies in Escherichia coli tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Therefore, it was concluded that dicyclopentadiene resin grade is not mutagenic in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay.

 

In Vitro Chromosome Aberration in Mammalian Cells

 

Dicyclopentadiene (DCPD) was assessed for mutagenic properties by evaluating increases in the chromosome aberrations in Chinese hamster lung (CHL/IU) cells (JETOC, 1998). DCPD did not induce significant cytogenetic damage to mammalian cells in vitro under conditions of this assay. Although some marginal chromosome damage occurred at the highest –S9 dose after 24 hrs continuous exposure, the test material was confirmed to be negative for clastogenicity in an in vitro micronucleus assay.

 

In Vitro Gene Mutation in Mammalian Cells

 

Dicyclopentadiene (DCPD) was assessed for mutagenic properties by evaluating increases in the mutant frequency at the TK locus of L5178Y mouse lymphoma cells in an OECD Guideline 476 study (Harlan Laboratories Ltd., 2014). The application of the test substance was limited by a steep toxicity dose-response curve. The test substance did not cause a statistically significant or dose-related increase in mutant frequency either in the absence or presence of PB/BNF S9, following incubation for 4 hours (24 hours in one experiment in the absence of S9). The positive control substances (EMS and CP) gave the expected increases in mutation frequency. In conclusion, DCPD does not cause gene mutation in mammalian cells in vitro, either without or with metabolic activation, under the conditions of this test.

Additional studies showing negative results for dicyclopentadiene in an in vitro mammalian cell micronucleus assay (JETOC, 1998b); in the Ames test (+/- S9) in Salmonella strains TA1535, TA1537, TA1538, TA98 and TA100 (Litton Bionetics 1980); in Salmonella strains TA1535, TA1537, TA98 and TA100 (Litton Bionetics 1980 ); and in S cerevisiae D4 (+/- S9) (Litton Bionetics, 1980) and S cerevisiae D3 (Litton Bionetics, 1980) are available.The data from these assays support the above conclusion that dicyclopentadiene is not mutagenic in vitro.

In Vivo data

There is no in vivo genetic toxicity data available for dicyclopentadiene. However, there is a supporting bone marrow micronucleus study in the mouse available for dicyclopentadiene / codimer concentrate, containing 29.175% dicyclopentadiene (DuPont, 2004). This is a recognised core assay type for investigating in vivo gene mutation.

DCPD/Codimer Concentrate was evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) in male and female mice (5-7 mice/sex) in an OECD Guideline 474 study (EI DuPont de Nemours and Company, 2004). Animals received 2 doses at 24 hour intervals and were assessed 24 hours after the second dose.

 

DCPD/Codimer Concentrate did not induce a statistically significant increase in micronucleated polychromatic erythrocytes in male or female mouse bone marrow. The highest dose administered on the study (1750 mg/kg body weight) gave clear evidence of clinical signs (both sexes) and exposure of the bone marrow to the substance (decreased PCE/NCE ratio) in females.

 

Based on these findings, DCPD/Codimer Concentrate was considered negative in this in vivo assay.

 

Human information

There is no information indicating any adverse effects of dicyclopentadiene. 

Justification for classification or non-classification

Based on the available in vitro and in vivo data, Dicyclopentadiene does not meet the criteria to warrant classification for mutagenicity under CLP.