Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of several reliable in vitro studies, Isobornyl methacrylate shows no potential to induce gene mutations in bacterial cells nor does it induce chromosomal aberrations in cultured human lymphocytes.

There were no in vivo genetic toxicity studies identified for Isobornyl methacrylate.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

The bacterial reverse mutation test is able to identify substances that cause point mutations, by substitution, addition or deletion of one or a few
DNA base-pairs. Mutagenic substances can induce reversion in histidine-(for Salmonella typhimurium) or tryptophan-(for Escherichia coli)
deficient strains which are then able to grow and form colonies in a histidine- or tryptophan limited medium, while non-reverted strains cannot.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 7-8 March 2007

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Supplier : Rhodia Inc. (Cranbury, NJ USA)
- Name of test material: Isobornyl Methacrylate (IBOMA)
- CAS Registry No. : 7534-94-3
- Molecular weight : 222 g/mol
- Substance type: organic
- Physical state at room temperature: liquid

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix fraction of Aroclor 1254-induced rats

Test concentrations with justification for top dose:

Main experiment:
+/-S9 mix; 0, 156.3 (2nd experiment only), 312.5, 625, 1250, 2500 and 5000 µg/plate
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen.
The maximum concentration was 5000.0 µg/plate. The concentration range include two logarithmic decades. Five to six adequately spaced
concentrations were tested. Two independent experiments were performed.


Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in
the preliminary toxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its relative nontoxicity for the bacteria.

Untreated negative controls:

yes

Remarks:

Concurrent untreated and solvent control: DMSO were performed

Negative solvent / vehicle controls:

yes

Remarks:

vehicle: DMSO

True negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

for TA 1535, TA 100

Positive control substance:

sodium azide

Remarks:

sodium acide concentration: 1 µg/plate Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

for TA 1537

Positive control substance:

9-aminoacridine

Remarks:

9-aminoacridine concentration: 50 µg/plate Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

for TA 98

Positive control substance:

2-nitrofluorene

Remarks:

2-nitrofluorene concentration: 0.5 µg/plate Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

for WP2 uvrA strain

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

4-nitroquinoline-N-oxide concentration: 2.0 µg/plate Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

for TA 1535, TA 1537 and TA 98

Positive control substance:

other: 2-Anthramine; with metabolic activation

Remarks:

2-Anthramine concentration: 2 µg/plate

Positive controls:

yes

Remarks:

for TA 100

Positive control substance:

benzo(a)pyrene

Remarks:

benzo(a)pyrene concentration: 5 µg/plate Migrated to IUCLID6: with metabolic activation

Positive controls:

yes

Remarks:

for WP2 uvrA strain

Positive control substance:

other: 2-Anthramine; with metabolic activation

Remarks:

2-Anthramine concentration: 10 µg/plate

Details on test system and experimental conditions:

EXPERIMENTAL DESIGN
Treatment
The test item was tested in a preliminary test and two mutagenicity experiments. The preliminary test and the first experiment were performed
according to the direct plate incorporation method. The second experiment with and without S9 mix was performed according to the preincubation
method.

Salmonella typhimurium and E. coli strains were treated with Isobornyl methacrylate using the direct plate incorporation method with at least five dose levels, plated in triplicate, for the preliminary test and the first mutagenicity experiment with and without S9. The second experiment tested six dose levels with and without S9 mix and was performed according to the preincubation method. The dose range was determined in a preliminary toxicity assay in which the highest dose tested was 5000 µg/plate. Moderate toxicity was observed in the TA 100 stran at 5000 µg/plate dose level with and without S9 mix. For the plate incorporation method, 0.1 mL bacterial suspension and 2 mL of overlay agar were mixed with 0.05 mL S9 mix (when required) or phosphate buffer (pH 7.4). Following homogenization, the mixture was on minimal medium. Once solidified, the plates were inverted and incubated for 48 to 72 hours at 37 ± 2 °C.
In the preincubation method, 0.05 mL of IBOMA solution, vehicle control or positive control solution and 0.05 mL S9 mix (when required) or phosphate buffer (pH 7.4) were mixed with 0.1 mL bacterial suspension and added to a glass culture tube. The mixture was incubated for 60 ± 3 minutes at 37 ± 2 °C under shaking. After incubation, 2 mL of overlay agar were added and the mixture was poured onto minimum agar plate. Once solidified, the plates were inverted and incubated for 48 to 72 hours at 37 ± 2°C.

Evaluation criteria:

A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 and TA 98 and WP2 uvrA the number of reversions is at least twice as high and in strains TA 1535 and TA 1537 it is at least three times higher as compared to the vehicle controls. Also, a dose-dependent and reproducible
increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the
highest dose induced the above described enhancement factors or not. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic concentration: >= 5000 µg/plate for TA 1535

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

Remarks:

Cytotoxic concentration: >= 5000 µg/plate for TA 1537

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

Remarks:

Cytotoxic concentration: >= 5000 µg/plate for TA 98

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

Remarks:

Cytotoxic concentration: >= 2500 µg/plate for TA 100

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

Remarks:

Cytotoxic concentration: >= 5000 µg/plate for WP2 uvrA

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, Isobornyl methacrylate did not induce gene mutations by base pair changes or frameshifts in the genome of the strains tested.
Therefore, the test substance has to be judged as nonmutagenic up to 5000 µg/plate in the presence and absence of mammalian metabolic activation according to the Ames test results.

Executive summary:

In a reverse gene mutation assay (according to OECD 471) in bacteria (Ames test), strains TA1535, TA1537, TA98, TA100, and TA102 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to Isobornyl methacrylate (99.07 % stabilised with 143 ppm MEHQ) at concentrations of up to 5000 µg/plate in the presence and absence of mammalian metabolic activation S9 -mix. 

 

With the direct plate incorporation method, moderate to marked toxicity was noted in the TA 1535 strain at 5000 µg/plate and in the TA 100 strtain at dose levels of >= 2500 µg/plate with metabolic activation. Moderate toxicity was noted for TA 100 at 5000 µg/plate with metabolic activation. Exposure to graded doses of Isobornyl methacrylate in either the presence or the absence of metabolic activation. Exposure to graded doses of the test substance in either the presence or the absence of metabolic activation did not increase the reversion rates in any of the tester strains in either experiment.

Appropriate reference mutagens were used as positive controls.

The positive controls induced the appropriate responses in the corresponding strains.

There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable. This study satisfies the requirement for tests for in vitro mutagenicity (bacterial reverse gene mutation) data.

Conclusion:

Under the conditions of this study Isobornyl Methacrylate (IBOMA; CAS: 7534-94-3), did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli at the concentrations tested with and without metabolic activation.

Reference 2

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Commission Dirtective No. 2000/32/EC adopted 8 June 2000.

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Supplier : Rhodia Inc. (Cranbury, NJ USA)
- Name of test material: Isobornyl Methacrylate (IBOMA)
- CAS Registry No. : 7534-94-3
- Molecular weight : 222 g/mol
- Substance type: organic
- Physical state at room temperature: liquid

Species / strain / cell type:

lymphocytes: prepared from whole blood samples obtained from two healthy donors (one male and one female for each experiment)

Details on mammalian cell type (if applicable):

- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

Mammalian rat liver microsomal fraction induced with Acroclor 1254 (S9 mix)

Test concentrations with justification for top dose:

Experiment I: +/- S9 mix: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM (corresponding to 2220 μg/mL)
Experiment II: +/- S9 mix: 0.313, 0.625, 1.25, 2.5, 5 and 7.5 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (supplier: Merck, Clévenot, Chelles, France)
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

concurrent solvent control

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in: distilled water

Positive control substance:

mitomycin C

Remarks:

Final concentration: 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment) Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

dissolved in: distilled water

Positive control substance:

cyclophosphamide

Remarks:

Final concentration: 12.5 or 25 µg/mL (the concentration which gave a satisfactory response in terms of quality and quantity of metaphases and extent of chromosomal damage was selected for analysis) Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 20 h (with S9 mix); 3 and 20 hours (without S9 mix)
- Expression time (cells in growth medium): Cell cultures were established for 48 hours at 37 °C with the appropriate medium and supplements
- Fixation time (harvest of cells): 20 hours after beginning of treatment

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): colcemid solution (10 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Analysis of 200 metaphases/concentration (with 44 to 46 chromosomes) was made, with 100 metaphases/culture, whenever possible. Only 50 metaphases/culture were analyzed when at least 10% of the cells with structural
chromosome aberrations were observed. All metaphase anayses were performed blind.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether the test item induces mitotic inhibition.
Mitotic index was determined without blind scoring.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of historical laboratory control of CIT-data
b) The positive control substances should produce significant increases of the number of cells with structural chromosomal aberrations compared to the vehicle controls and should be consistent with CIT historical data.

EVALUATION OF RESULTS
A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural
chromosomal aberrations or a significant and reproducible positive response for at least one of the test concentrations and one of the two harvest
times.

A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor
a significant and reproducibly positive response at any one of the test points is considered nonmutagenic in this system.
Reference to historical data or other biological relevance, and statistical significance should be taken into account in the evaluation of the findings, if necessary.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was
compared to that of the vehicle control cultures. If necessary, the results were compared using the 2 test, in which p = 0.05 was used as the lowest
level of significance.

Key result

Species / strain:

lymphocytes: prepared from whole blood samples obtained from two healthy donors (one male and one female for each experiment)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

-S9mix: moderate to marked cytotoxicity ≥5 mM; +S9mix: slight to marked cytotoxicity ≥ 2.5 mM

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Isobornyl methacrylate was freely soluble in the vehicle (DMSO) at 444 mg/mL.
In the culture medium, the concentration of 10 mM (corresponding to 2220 μg/mL) showed a slight to moderate emulsion. At this concentration, the pH was approximately 7.4 (7.4 for the vehicle control) and the osmolality equal to 322 mOsm/kg H2O (306 for the vehicle control).

Conclusions:

Interpretation of results: negative

In the Chromosomal aberration test with cultured human lymphocytes according to OECD 473 in the presence and absence of mammalian activation
structural chromosomal aberrations were not observed with Isobornyl methacrylate. Therefore, the test substance is considered to be
non-mutagenic in vitro under the experimental conditions reported.

Executive summary:

In a mammalian cell cytogenetics assay primary lymphocyte cultures were exposed to Isobornyl methacrylate (purity: 99.66%, solvent:Dimethylsulfoxid (DMSO)) at concentrations of 0, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5.0 and 10 mM with and/or without metabolic activation (S9 -mix). Isobornyl methacrylat was tested in two independent experiments, both with and without a liver

metabolizing system (S9 mix). The following results were received:

Experiments without S9 mix:

Moderate to marked cytotoxicity was noted at concentrations ≥5 mM as shown by a 42-95% decrease in mitotic index (MI). 

The metaphase analysis showed a 42% decrease in mitotic index at ≥5 mM and 10 mM being too toxic (3 -hour treatment). For the 20-hour treatment 7.5 mM induced a 47% decrease in mitotic index.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3 or 20-hour treatments.

Experiments with S9 mix:

A slight to marked cytotoxicity was noted at concentrations ≥2.5 mM as shown by a 35-92% decrease in mitotic index.

The metaphase analysis showed also a 42% decrease in mitotic index at ≥5 mM and 10 mM being too toxic (20-hour treatment, first experiment) and 60% decrease in mitotic index at 2.5 mM and higher concentrations being too toxic (20 -hour treatment, second experiment).

The concentrations selected for metaphase analysis were 1.25, 2.5 and 5 mM without and with S-9.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments.

The frequency of cells with structural chromosome aberrations for the vehicle and positive control tests was classified as acceptable and therefore considered valid.

This study satisfies the requirement for Test Guideline (In vitro mammalian cytogenetics according to OECD 473, EU Method B.10, and OPPTS 870.5375) for in vitro cytogenetic mutagenicity data.

Conclusion:

Under the conditions of this study, Isobornyl Methacrylate did not induce chromosome aberrations in cultured human lymphocytes in vitro.

Reference 3

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:

2009-10-01 - 2010-02-04

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:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 15 August and 27 - 29 October 2008

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Supplier: Evonik Röhm GmbH, D-64293 Darmstadt, Germany
- Name of test material: Isobornyl Methacrylate (IBOMA)
- CAS Registry No. : 7534-94-3
- Molecular weight : 222 g/mol
- Substance type: organic
- Physical state at room temperature: liquid
- Stability under test conditions: stable
- Storage condition of test material: At room temperature (+15 to +25°C), light protected

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (each batch is screened)
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes/no

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

Experiment I (4 hours):
-S9 mix: 0.6, 1.3, 2.5, 5.0 and 10.0 µg/ml
+S9 mix: 17.5, 35.0, 70.0, 140 and 280 µg/ml

Experiment II:
-S9 mix (24 hours): 1.1, 2.2, 4.4, 8.8 and 17.5 µg/ml
+S9 mix (4 hours): 17.5, 35.0, 70.0, 140 and 280 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent DMSO was chosen due to its solubility properties and its non-toxicity to the cells. The final
concentration of DMSO in the culture medium was 0.5 % v/v.

Negative solvent / vehicle controls:

yes

Remarks:

concurrent solvent control (DMSO)

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

EMS dissolved in: Nutrient medium, Final concentration: 0.15 mg/mL = 1.2 mM Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

DMBA dissolved in: DMSO (final concentration in nutrient medium 0.5 %), Final concentration DMBA: 1.1 µg/mL = 4.3 µM Migrated to IUCLID6: with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
Experiment II: 4 hours with and 24 hours without metabolic activation. The experimental parts of the second experiment with
and without metabolic activation were performed in two separate experiments (experiment II and IIA) for technical reasons. The
results are combined and reported as experiment II.
NUMBER OF REPLICATIONS:


NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation
microscope (Nikon, 40407 Düsseldorf, Germany).

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10exp+6 cells found in the solvent controls fall within the laboratory historical control data
range of 2006 – 2009.
- the positive control substances must produce a significant increase in mutant colony frequencies (Historical data).
- the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.

Evaluation of Results
A test item is regarded as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive
response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test
points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is regarded as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency
at least at one of the concentrations in the experiment.
The test item is regarded as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be
considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low
spontaneous mutation rate in the range normally found ( 0.6 - 33.2 mutants per 10exp+6 cells) a concentration-related increase of the mutations
within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

Statistical Analysis
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT®11 (SYSTAT
Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the
groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability
value) is below 0.05. However, both, biological and statistical significance were considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in forward gene mutations in mammalian cells

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Results and Disscussions

Isobornyl methacrylate was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The assay was performed in two independent experiments with identical experimental procedures, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The maximum dose of the pre-test was 2240 µg/mL corresponding to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxic effects and phase separation. In the pre-experiment no relevant toxic effect was observed in the experimental part with metabolic activation up to a concentration of 1120 µg/mL (4 h treatment). At the maximum concentration of 2240 µg/mL the cell growth was completely inhibited. Following 4 h treatment without metabolic activation the cell growth was completely already inhibited at 70 µg/mL and above. After 24 hours of treatment the cell growth was also completely blocked at 140 µg/mL and above.

In the first experiment the cultures at 15.0 and 20.0 µg/mL withought metabolic activation and 560 and 1120 µg/mL with metabolic activation were not continued due to exceedingly strong effects. In the second main experiment the maximum concentration of 35 µg/mL without metabolic activation and 560 and 1120 µg/mL with metabolic activation were not continued for the same reason. At 0.5 µg/mL in experiment without metabolic activation were not continued since a minimum of only four analysable concentrations are required by the guideline.

Phase separation of the test item was observed at 70 µg/mL and above in experiment I with S9 and at 140 µg/mL and above in experiment II in the presense of S9.

Cytotoxic effects as indicated by a relative cloning efficiency I of less than 50 % in both parallel cultures occurred at 5.0 µg/mL and above in experiment I without metabolic activation (4 hours treatment). In the second experiment cytotoxic effects as described above were determined at 17.5 µg/mL and above without metabolic activation. Although the relative cloning efficiency I (survival) was below 10% or even 0% at 10.0 µg/mL in the first experiment and at 17.5 µg/mL in the second experiment without metabolic activation the data are judged acceptable. The mean values of the corresponding cell counts at the first subcultivation following treatment remained well above 10%. In the first experiment without metabolic activation the cell counts of 32.1 % and 41.8% were equal to a mean of 37%. In the second experiment without metabolic activation the cell counts of 68.8% and 92.3% were equal to a mean of 81%.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration. All mutant frequencies remained well within the historical range of solvent controls (with a single exception in the second culture of experiment II at 1.1 µg/mL). The induction factor exceeded the threshold of three times the corresponding solvent control in the second culture of experiment I without metabolic activation at 2.5 and 5.0 µg/mL. This effect however, was judged to be based upon the rather low solvent control of 5.4 mutant colonies/10⁶cells.

Compared to the corresponding solvent control of culture I

(18.9 mutant colonies/10E+6 cells) no increase occurred. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT ® statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of < 0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.4 up to 28.7 mutants per 10⁶ cells; the range of the groups treated with the test item was from 3.8 up to 34.2 mutants per 10⁶cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. This showed the sensitivity of the test system and the activity of the S9 mix.

Summary of results

			relative	relative	mutant		relative	relative	mutant
	Conc.	S9 mix	cloning	cloning	Colonies/	induction	cloning	cloning	Colonies/	induction
	µg/mL		Efficientcy I	Efficientcy II	10^6cells	factor	efficiency I	efficiency II	10^6cells	factor
			%	%			%	%
Column	1	2	3	4	5	6	7	8	9	10
Exp.I  4 hours			Culture I				Culture II
Solvent contr. DMSO		-	100.0	100.0	18.9	1.0	100.0	100.0	5.4	1.0
Pos. contr. EMS	150.0	-	96.1	81.9	103.3	5.5	108.4	87.9	92.5	17.2
Isobornyl methacrylate	0.6	-	104.3	93.0	15.1	0.8	119.6	88.7	10.8	2.0
Isobornyl methacrylate	1.3	-	80.9	112.6	4.3	0.2	98.6	80.6	15.9	2.9
Isobornyl methacrylate	2.5	-	3.5	87.5	13.5	0.7	80.5	80.8	21.0	3.9
Isobornyl methacrylate	5.0	-	0.0	77.2	24.8	1.3	45.3	99.8	21.5	4.0
Isobornyl methacrylate	10.0	-	0.0	83.2	12.7	0.7	0.0	100.9	6.0	1.1
Isobornyl methacrylate	15.0	-	0.0	Culture was not continued#			0.0	Culture was not continued#
Isobornyl methacrylate	20.0	-	0.0	Culture was not continued#			0.0	Culture was not continued#

Solvent contr. DMSO		+	100.0	100.0	16.3	1.0	100.0	100.0	18.7	1.0
Pos.contr. DMBA	1.1	+	43.1	81.8	896.7	54.9	43.5	81.8	758.7	40.6
Isobornyl methacrylate	17.5	+	95.9	120.6	18.6	1.1	93.8	92.8	21.7	1.2
Isobornyl methacrylate	35.0	+	90.4	121.6	21.1	1.3	85.8	99.1	12.2	0.7
Isobornyl methacrylate	70.0 (p)	+	80.0	115.8	22.4	1.4	86.6	113.8	15.9	0.9
Isobornyl methacrylate	140.0 (p)	+	78.2	128.9	16.8	1.0	78.6	127.4	3.8	0.2
Isobornyl methacrylate	280.0 (p)	+	63.8	114.3	12.8	0.8	47.3	109.3	19.0	1.0
Isobornyl methacrylate Isobornyl methacrylate	560.0 (p) 1120.0 (p)	+ +	37.9 0.0	Culture was not continued#   Culture was not continued#			22.6 0.0	Culture was not continued#   Culture was not continued#
Exp.II  24 hours			Culture I				Culture II
Solvent contr. DMSO		-	100.0	100.0	23.0	1.0	100.0	100.0	28.7	1.0
Pos. contr. EMS	75.0	-	107.9	88.5	284.9	12.4	106.5	84.7	489.3	17.1
Isobornyl methacrylate	0.5	-	100.0	Culture was not continued##			97.9	Culture was not continued##
Isobornyl methacrylate	1.1	-	102.6	104.4	17.4	0.8	92.3	94.5	34.2	1.2
Isobornyl methacrylate	2.2	-	105.8	94.5	28.3	1.2	106.0	93.8	25.9	0.9
Isobornyl methacrylate	4.4	-	112.2	115.8	11.1	0.5	99.1	96.2	6.2	0.2
Isobornyl methacrylate	8.8	-	104.9	110.9	28.9	1.3	96.5	100.1	14.7	0.5
Isobornyl methacrylate	17.5	-	5.9	99.2	28.4	1.2	8.3	90.4	18.0	0.6
Isobornyl methacrylate	35.0	-	0.0	Culture was not continued##			0.0	Culture was not continued##

Exp.II 4 hours			Culture I				Culture II
Solvent contr. DMSO		+	100.0	100.0	10.8	1.0	100.0	100.0	12.3	1.0
Pos. contr. DMBA	1.1	+	64.1	60.0	679.1	62.6	87.1	89.0	620.8	50.4
Isobornyl methacrylate	17.5	+	93.2	110.0	11.8	1.1	95.4	116.0	27.5	2.2
Isobornyl methacrylate	35.0	+	94.5	87.7	17.6	1.6	96.2	114.0	25.5	2.1
Isobornyl methacrylate	70.0	+	98.9	95.0	11.0	1.0	107.9	121.5	16.4	1.3
Isobornyl methacrylate	140.0 (p)	+	70.8	111.6	8.9	0.8	95.1	103.4	13.7	1.1
Isobornyl methacrylate	280.0 (p)	+	73.9	62.8	29.1	2.7	50.5	122.8	20.0	1.6
Isobornyl methacrylate	560.0 (p)	+	44.6	Culture was not continued#			39.7	Culture was not continued#
Isobornyl methacrylate	1120.0 (p)	+	0.0	Culture was not continued#			0.0	Culture was not continued#

# culture not continued due to exceedingly strong toxic effects or phase separation

## culture was not continued since a minimum of only four analysable concentrations is required

p phase separation

Conclusions:

Interpretation of results: negative

In conclusion it can be stated that under the experimental conditions reported the test item Isobornyl methacrylate did not induce gene mutations at
the HPRT locus in V79 cells of the Chinese hamster.
Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a mammalian cell gene mutation assay HPRT locus using V79 cells of the Chinese hamster cultured in vitro were exposed to Isobornyl methacrylate (99.35 %) dissolved in DMSO at concentrations of

Experiment I:

-S9 mix: 0.6, 1.3, 2.5, 5.0 and 10.0 µg/ml

+S9 mix: 17.5, 35.0, 70.0, 140 and 280 µg/ml

Experiment II:

-S9 mix: 1.1, 2.2, 4.4, 8.8 and 17.5 µg/ml

+S9 mix: 17.5, 35.0, 70.0, 140 and 280 µg/ml

in the presence and absence of mammalian metabolic activation S9 mix. 

The assay was performed in two independent experiments. The cells were exposed to for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The maximum dose was 2240 μg/mL (pre-test) corresponding to a molar concentration of about 10 mM.

The positive controls did induce the appropriate response. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments up to the maximum concentration.

There was no evidence of induced mutant colonies over background. 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Based on the results of several reliable in vitro studies, Isobornyl methacrylate shows no potential to induce gene mutations in bacterial cells nor does it induce chromosomal aberrations in cultured human lymphocytes.

There were no in vivo genetic toxicity studies identified for Isobornyl methacrylate.


Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

GHS classification according toAnnex I 1272/2008 CLP (EU GHS):

- No classification required.