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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (Ames): negative (OECD 471, GLP)

Gene mutation in mammalian cells (HPRT): negative (OECD 476, GLP)

Cytogenicity in mammalian cells (MNT in vitro): negative (OECD 487, GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-07-10 - 2012-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

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

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): Isobornyl acrylate
- 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

Additional strain / cell type characteristics:

other:

Remarks:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix fraction of Aroclor 1254-induced, male Sprague-Dawley rats and male Syrian hamster livers

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II; Salmonella typhimurium
without S9 mix: 0.3; 1; 3, 10, 33; 100; 333; and 1000 µg/plate
with S9 mix 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Escherichia coli
with and without S9 mix: 33: 100; 333; 1000; 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. 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, Merck, Darmstadt, purity > 99 %)
- 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 with 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 azide (purity: >= 99.0%, supplier: Serva, D-69042 Heidelberg, Germany) dissolved in aqua dest.; concentration: 10 µg/plate Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

for TA 1537, TA 98

Positive control substance:

other: 4-nitro-o-phenylenediamine, without metabolic activation

Remarks:

4-nitro-o-phenylenediamine (purity: > 99.9%, supplier: Fluka (Sigma Aldrich), 82024 Taufkirchen/Germany, dissolved in DMSO; concentration: 10 µg/plate

Positive controls:

yes

Remarks:

for E. coli WP2 uvr A

Positive control substance:

methylmethanesulfonate

Remarks:

methylmethanesulfonate, MMS (purity: > 99.0%, Supplier: Sigma Aldrich, 82024 Taufkirchen/Germany Migrated to IUCLID6: without metabolic activation

Positive controls:

yes

Remarks:

for TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvr A

Positive control substance:

other: 2-aminoanthracene, with metabolic activation

Remarks:

2-aminoanthracene (purity: 97.5%, supplier: Sigma-Aldrich, D-82024 Taufkirchen, Germany) dissolved in DMSO; concentration: 2.5 µg/plate (10 µg/plate in TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation assay as described by Haworth et al. 1983, with some differences.

- Salmonella typhimurium strains were obtained from Dr. Bruce Ames (University of California, Berkeley, U.S.A.) and were stored as recommended (Maron and Ames, 1983).
- Cultures were grown overnight with shaking at 37 °C in Oxoid No. 2 broth, and their phenotypes were analyszed prior to their use for mutagenicity assays.

Test conditions:
System of testing:
-Metabolic activation system: S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone.
Administration:
-Number of replicates: 2
-Plate per test: 3
-Application: pre-incubation

Evaluation criteria:

Validity of the data is confirmed by the laboratory´s historical control data from January 2011 until December 2011 representing approx.550 experiments (WP2 uvrA the historical data are based on approx. 200 experiments).

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was not observed below 100 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

In conclusion, it can be stated that during the decribed mutagenicity test with Salmonella typhimurium and Escherichia coli and under the experimental conditions reported, Isobornyl acrylate did not induce gene mutations by base pair changes or frame shifts 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.

Executive summary:

The study was performed to investigate the potential of Isobornyl acrylate to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment /Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II; Salmonella typhimurium

without S9 mix: 0.3; 1; 3, 10, 33; 100; 333; and 1000 µg/plate

with S9 mix 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Escherichia coli

with and without S9 mix: 33: 100; 333; 1000; 2500; and 5000 µg/plate

Reduced background growth was observed at higher concentrations with and without

metabolic activation in strains TA 1535, TA 1537, TA 98, and TA 100 in both independent

experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication

factor of 0.5), were observed at higher concentrations in strains TA 1535, TA 1537, TA 98,

and TA 100 with and without metabolic activation in both independent experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Isobornyl acrylate at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Isobornyl acrylate is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia colireverse mutation assay.

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-20 - 2013-01-04

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)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Supplier: Evonik Industries AG, D-64293 Darmstadt, Germany
- Name of test material: Isobornyl acrylate
- 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

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: 2, 4, 8, 16, 24 and 32 µg/mL
+S9 mix: 8, 16, 32, 64, 96 and 128 µg/mL

Experiment II:
-S9 mix (24 hours): 1, 2, 4, 8, 16, and 24 µg/mL
+S9 mix (4 hours): 4, 8, 16, 32, 64, and 96 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

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 CELLS EVALUATED: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation
microscope.

Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 (3).
Cell Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni¬cal University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before free¬zing, the level of spontaneous mutants was depressed by treatment with HAT-medium as described (4). Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks. About 5105 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1 %). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37°C in a 1.5 % carbon dioxide atmosphere (98.5 % air).
Mammalian Microsomal Fraction S9 Mix
Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays with cell cultures as many chemicals only develop a mutagenic potential when they are me¬tabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with mammalian liver micro¬some preparations (S9 mix).

S9 (Preparation by Harlan CCR)
Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 were prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU, weight approx. 220 – 320 g, Harlan Laboratories B.V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin, 22335 Hamburg, Germany) and by peroral administrations of 80 mg/kg b.w. -naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules were kept at –20 °C for up to one week. Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.
The protein concentration of the S9 preparation was 29.8 mg/mL (Lot. No.: 280912) in the pre-experiment and the main experiments


S9 MIX

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solu-tion to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were ad¬ded to the S9 supernatant to reach following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath.

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 2010 - 2011.
- 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:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. 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.

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

Conclusions:

Interpretation of results: negative

In conclusion it can be stated that under the experimental conditions reported, Isobornyl acrylate 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:

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

The study was performed in three independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The experimental part of the first experiment with metabolic activation was prematurely terminated due to microbial contamination and repeated as experiment IA. The data of experiment IA are reported as experiment I with metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The cell cultures were evaluated at the following concentrations:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	2.0		4.0		8.0		16.0
4 hours	+	8.0	16.0		32.0		64.0		96.0
		Experiment II
24 hours	-	2.0	4.0		8.0		16.0		24.0
4 hours	+	8.0	16.0		32.0		64.0		96.0

 

No precipitation or phase separation of the test item was observed up to the maximum concentration of the main experiments.

Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 16.0 µg/mL without metabolic activation and at 96.0 µg/mL with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 24.0 µg/mL without metabolic activation and at 96.0 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of three times the mutation frequency of the corresponding solvent control at any of the test points.

In the second experiment the mutation frequency exceeded the historical range of solvent controls at several concentrations. However, since the induction factor was not exceeded and statistical analysis showed no dose dependent increase, the increased values were judged as biologically irrelevant fluctuation.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.4 up to 51.7 mutants per 106 cells; the range of the groups treated with the test item was from 7.9 up to 52.7 mutants per 106 cells. The highest level of the solvent controls (51.7 mutants per 106 cells) exceeded the historical range of solvent controls with metabolic activation (up to 36.6 mutants per 106 cells). This deviation is judged as irrelevant as the mutation frequency in the solvent control of the parallel culture and the mean value of both parallel cultures (51.7 and 22.7 equal to a mean of 37.2) was fully acceptable.

EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-23 - 2012-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD 487, GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted July 22, 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Supplier: Evonik Industries AG, D-64293 Darmstadt, Germany
- Name of test material: Isobornyl acrylate
- 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

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a female donor* (33 or 34 years old, respectively) for Experiment IA and IIB, from a 33 year-old female donor for Experiment IB, from a 34 year-old female donor for Experiment IIA. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.

*The blood samples for Exp. IA and IIB were taken from the same person at different time points.

Cytokinesis block (if used):

Cytochalasin B was used (4 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver microsomal fraction S9 mix Type and composition of metabolic activation system:
- source of S9: male rat liver (phenobarbital/beta-naphthoflavone treated)
- method of preparation of S9 mix . The S9 was prepared from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, weight approx. 220 - 320 g, Harlan Laboratories B. V.) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital and by oral administrations of beta-naphthoflavone each, on three consecutive days. The livers were excised and processed 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week without deterioration of the enzymatic activities. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
- concentration or volume of S9 mix and S9 in the final culture medium : final protein concentration of 0.75 mg/mL in the cultures (5 %).

Test concentrations with justification for top dose:

DOSE SELECTION
At least three test item groups were evaluated for genotoxic effects. Evaluation of cytogenetic damage was performed in two independent cultures per concentration. For freely soluble non-cytotoxic test items the maximum concentration will be 5 µL/mL, 5 mg/mL or 10 mM, whichever is the lowest. For cytotoxic test items the highest concentration should show cytotoxicity indicated as cytostasis of 55 ± 5 %.
With respect to the molecular weight of the test item, 2083.0 µg/mL of Isobornyl acrylate (approx. 10 mM) was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 2.0 and 2083.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, no precipitation of the test item was observed. Independent of the usage of S9 mix, phase separation was observed at concentrations above 125 µg/mL. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment IA. The experimental part without S9 mix was repeated with a top dose of 500.0 µg/mL and narrow concentration spacing to obtain evaluable concentrations in a cytotoxic range (Exp. IB).
Using a reduced CBPI as an indicator for toxicity in Experiment IA and IB, clear toxic effects were observed after 4 hrs treatment in the absence of S9 mix with 130.2 µg/mL and above (Exp. IA) and 60.0 µg/mL and above (Exp. IB). In the presence of S9 mix clear toxic effects were observed after 260.4 µg/mL and above.
Therefore, 100.0 µg/mL (without S9 mix) and 400.0 µg/mL (with S9 mix) were chosen as top concentrations in Experiment IIA.
Due to a slight positive effect in Experiment IIA in the presence of S9 mix a confirmatory experiment (IIB) was performed with a top dose of 400.0 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; it was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

concurrent solvent control (0.5% DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 4:


Without S9 mix With S9 mix
Exposure period Exp. IA & IB Exp. IIA Exp. IA, IIA & IIB
Recovery 4 hrs 20 hrs 4 hrs
Cytochalasin B exposure 16 hrs - 16 hrs
Preparation interval 20 hrs 20 hrs 20 hrs
Total culture period 40 hrs 40 hrs 40 hrs
88 hrs 88 hrs 88 hrs

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in suspension

TREATMENT AND HARVEST SCHEDULE:

Exposure time 4 hours
About 48 hours after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test group. The culture medium was replaced with serum-free medium (in the absence and presence of S9 mix) containing the test item. In the case of metabolic activation, 50 µL S9 mix per mL medium was used. Concurrent solvent and positive controls were performed. After 4 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period.

Exposure time 20 hours
About 48 hours after seeding for each test group two blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks (Nunc GmbH & Co. K, 65203 Wiesbaden, Germany). The culture medium was replaced with complete medium containing 10 % FBS (v/v) and the test item. Concurrent solvent and positive controls were performed. After 20 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in and washed with "saline G". The washing procedure was repeated once as described. After washing the cells were re-suspended in complete culture medium containing 10 % FBS (v/v).

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation (true for both exposure times)
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cultures were harvested by centrifugation 40 hrs after beginning of treatment. The cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation for 5 minutes. Then the cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 1000 binucleate cells per culture were evaluated for cytogenetic damage, except for the positive control in Experiment IIB with S9 mix, where only 500 binucleate cells per culture were evaluated.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mean Proliferation Index CBPI: 1.86

Rationale for test conditions:

This in vitro test is an assay for the detection of micronuclei. The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of chromosomal damage and aneugenicity. Micronuclei arise from chromosomal fragments or whole chromosomes. Micronuclei rarely occur spontaneously but are inducible by clastogens or agents affecting the spindle apparatus. (see also "Test concentrations")

Evaluation criteria:

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data (see ANNEX II) and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Key result

Species / strain:

lymphocytes: human healthy volunteer

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH/ Data on osmolality: The osmolarity and pH-value were determined in the solvent control and the maximum concentration without metabolic activation in experiments IA, IB and IIA: pH values were always closely comparable; the osmolarity of IBOA treatments differs to the solvent controls in a range of +/- 11%; see table in "Any other information"
- Possibility of evaporation from medium: not relevant due to low VP
- Water solubility: limited. The solvent DMSO was chosen due to its solubility properties (and its relative non-toxicity to the cell cultures.)
- Precipitation and time of the determination: No precipitation of the test item in the culture medium was observed. Phase separation was observed in Exp. IA at 130 µg/mL (with and without S9 mix), in Exp. 1B at 125 µg/mL (without S9 mix), in Exp. IIA at 100 µg/mL (with and without S9 mix) and in Exp. IIB at 140 µg/mL (with S9 mix).

RANGE-FINDING/SCREENING STUDIES:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-experiment was performed with 11 concentrations of the test item and the solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix) and the cells were prepared 40 hrs after start of the exposure.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : see table in "Any other information"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
- In the case of the cytokinesis-block method: Mean Proliferation Index CBPI: 1.86

HISTORICAL CONTROL DATA

Historical control data without S9
-----------------------------------------------------------------------------------------------------------------------
Micronucleated cells [%]
-----------------------------------------------------------------------------------------------------------------------
Test group concentration No. of experiments Range Mean Standard deviation
-----------------------------------------------------------------------------------------------------------------------
Solvent control (pulse treatment)
-----------------------------------------------------------------------------------------------------------------------
Total 26 0.15-1.40 0.72 ± 0.38
-----------------------------------------------------------------------------------------------------------------------
Solvent control (continuous treatment)
-----------------------------------------------------------------------------------------------------------------------
Total 23 0.05-1.45 0.75 ± 0.23
-----------------------------------------------------------------------------------------------------------------------
Positive control (pulse treatment)
-----------------------------------------------------------------------------------------------------------------------
Mitomycin C
1.0-3.0 µg/ml 11 6.75-25.10 13.91 ± 4.81
-----------------------------------------------------------------------------------------------------------------------
Positive control (continuous treatment)
-----------------------------------------------------------------------------------------------------------------------
Demecolcin
0.05-0.1 µg/ml 12 1.80-5.55 3.18 ± 1.05
-----------------------------------------------------------------------------------------------------------------------
Demecolcin#
0.05-1.0 µg/ml 5 2.25-5.45 3.47 ± 1.00
-----------------------------------------------------------------------------------------------------------------------
# Micronucleated mononucleate cells


Historical control data with S9
------------------------------------------------------------------------------------------------------------------------
Micronucleated cells [%]
------------------------------------------------------------------------------------------------------------------------
Test group concentration No. of experiments Range Mean Standard deviation
------------------------------------------------------------------------------------------------------------------------
Solvent control (pulse treatment)
------------------------------------------------------------------------------------------------------------------------
Total 22 0.20-1.70 0.78 ± 0.31
------------------------------------------------------------------------------------------------------------------------
Positive control (pulse treatment)
------------------------------------------------------------------------------------------------------------------------
CPA
7.5-12.5 µg/ml 21 2.85-11.05 5.98 ± 1.85
------------------------------------------------------------------------------------------------------------------------

The micronucleus rates of the cells after treatment with the test item (0.10 – 1.45 % micronucleated cells) were slightly above the range of the solvent control values (0.40 – 0.90 % micronucleated cells), but within the range of the laboratory historical control data (0.05-1.45 %; mean 0.75+-0.23% micronucleated cells). In Experiment IIA, in the presence of S9 mix one single statistically significant increase in the number of micronucleated cells (2.75 %) clearly exceeding the laboratory historical solvent control data range (0.20 – 1.70 %; mean 0.78+-0.31% micronucleated cells) was observed at the highest evaluated concentration (175.0 µg/mL). In the confirmatory experiment IIB statistically significant increases (0.55, 0.85 and 0.65 % micronucleated cells, respectively) were observed after treatment with 120.0, 140.0 and 160.0 µg/mL. These values were clearly within the historical solvent control data range of 0.20 – 1.70 % micronucleated cells and therefore biologically not relevant. Thus, the positive finding of Experiment IIA could not be confirmed.

The number of micronucleated cells after pulse treatment with the test item vary from 0.1-0.65%. After continuous treatment without S9 mix, the number of micronucleated cells range between 0.6 and 1.45%. Independent of the treatment, the number of micronucleated cells is always within the range of the historical control data (0.15-1.4% for pulse treatment and 0.05-1.45% for continuous treatment, respectively).
In Exp. IA with S9 mix, the number of micronucleated cells increased, but even at the highest dose reported, which is already in the range of observable phase separation, the number of micronucleated cells is comparable to that observed in the solvent control and clearly within the historical control range of the solvent control (0.2 - 1.70 % micronucleated cells).
In Experiment IIA, in the presence of S9 mix one single statistically significant increase in the number of micronucleated cells (2.75 %) clearly exceeding the laboratory historical solvent control data range (0.20 – 1.70 % micronucleated cells) was observed at the highest evaluated concentration (175.0 µg/mL). Since the number of micronucleated cells in the solvent control (1.68% micronucleated cells) is at the upper limit of the historical control data range (0.2 - 1.70 % micronucleated cells) but still inside, this experiment meets all criteria for validity.
A confirmatory experiment (Exp. IIB) was performed to investigate the equivocal results of Exp. IA and IIA (with S9 mix).
In the confirmatory Experiment IIB statistically significant increases (0.55, 0.85 and 0.65 % micronucleated cells, respectively) were observed after treatment with 120.0, 140.0 and 160.0 µg/mL caused by a low response of the solvent control. Since all three values were clearly within the historical solvent control data range of 0.20 – 1.70 % micronucleated cells, these findings can be considered as biologically irrelevant. The value of the solvent control (0.1% micronucleated cells), however, is smaller than the lower limit of the historical control data range (0.2 - 1.70 % micronucleated cells). Since the investigation of a potential increase in micronucleated cells is the relevant endpoint of this test, the undercutting of the historical control data range has no relevance for the assessment of this experiment and this experiment meet all criteria for validity.
Thus, the positive finding of Experiment IIA was not confirmed.

Footnote regarding the historical control data (HCD) by the test laboratory:
Regarding the OECD guideline 487, the HCD are used as cut-off criteria. The generation of HCD includes the data for a single test system in a single laboratory for a defined period. The volume includes normally manageable sample sizes (regarding the OECD guideline 487: minimum 20 data sets). In case of the current study 48 single experiments (with S9 mix) from 2009 and 2010 were included. Therefore, the HCD cannot describe the whole range of variation for test system but is rather an estimation. Furthermore, the use of human primary lymphocytes includes additional variation factors caused by sex, age and constitution on the day of donation.
With regard to the endpoint (induction of micronucleated cells) the upper limit of the HCD is the important threshold to estimate the validity of experiments related to the laboratory standard. A result below the HCD is not a sign of an insufficient or invalid experiment. Rather it is a parameter for the suitability of the cell system used in relation to a low background level.


Historical control data without S9
-----------------------------------------------------------------------------------------------------------------------
Micronucleated cells [%]
-----------------------------------------------------------------------------------------------------------------------
Test group concentration No. of experiments Range Mean Standard deviation
-----------------------------------------------------------------------------------------------------------------------
Solvent control (pulse treatment)
-----------------------------------------------------------------------------------------------------------------------
Total 26 0.15-1.40 0.72 ± 0.38
-----------------------------------------------------------------------------------------------------------------------
Solvent control (continuous treatment)
-----------------------------------------------------------------------------------------------------------------------
Total 23 0.05-1.45 0.75 ± 0.23
-----------------------------------------------------------------------------------------------------------------------
Positive control (pulse treatment)
-----------------------------------------------------------------------------------------------------------------------
Mitomycin C
1.0-3.0 µg/ml 11 6.75-25.10 13.91 ± 4.81
-----------------------------------------------------------------------------------------------------------------------
Positive control (continuous treatment)
-----------------------------------------------------------------------------------------------------------------------
Demecolcin
0.05-0.1 µg/ml 12 1.80-5.55 3.18 ± 1.05
-----------------------------------------------------------------------------------------------------------------------
Demecolcin#
0.05-1.0 µg/ml 5 2.25-5.45 3.47 ± 1.00
-----------------------------------------------------------------------------------------------------------------------
# Micronucleated mononucleate cells


Historical control data with S9
------------------------------------------------------------------------------------------------------------------------
Micronucleated cells [%]
------------------------------------------------------------------------------------------------------------------------
Test group concentration No. of experiments Range Mean Standard deviation
------------------------------------------------------------------------------------------------------------------------
Solvent control (pulse treatment)
------------------------------------------------------------------------------------------------------------------------
Total 22 0.20-1.70 0.78 ± 0.31
------------------------------------------------------------------------------------------------------------------------
Positive control (pulse treatment)
------------------------------------------------------------------------------------------------------------------------
CPA
7.5-12.5 µg/ml 21 2.85-11.05 5.98 ± 1.85
------------------------------------------------------------------------------------------------------------------------

Osmolarity and pH

Table 1

		Concentration [µg/mL]	Solvent control	Isobornyl acrylate
Exp.	Osmolarity[mOsm]	2083.0	393	361
	pH-value		7.9	7.9
Exp. IB	Osmolarity[mOsm]	500.0	383	392
	pH-value		7.6	7.7
Exp. IIA	Osmolarity[mOsm]	100.0	344	389
	pH-value		7.6	7.6

Summary of Results

Summary of results of the in vitro micronucleus test in human lymphocytes with Isobornyl acrylate

Table 2:  Exposure period 4 hrs without S9 mix

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
IA	40 hrs	Solvent control1	1.76		0.45
		Positive control2	1.92	n.c.	3.85S
		16.3	1.71	7.0	0.65
		32.5	1.69	10.0	0.55
		65.1	1.49	36.2	0.45
IB	40 hrs	Solvent control1	1.97		0.40
		Positive control2	1.75	22.6	5.25S
		12.5	1.87	10.2	0.15
		25.0	1.80	17.6	0.10
		50.0	1.63	35.5	0.10
IIA	40 hrs	Solvent control1	1.70		0.90
		Positive control3	1.43	38.4	4.50S
		18.7	1.59	16.3	0.60
		32.7	1.55	21.0	0.95
		57.1	1.25	63.8	1.45

*         For the positive control groups, the relative values are related to the negative controls;
for the test item treatment groups the values are related to the solvent controls

**       The number of micronucleated cells was determined in a sample of 2000 binucleated cells

S         The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.     Not calculated as the CBPI is equal or higher than the solvent control value

1         DMSO           0.5 % (v/v)
2         MMC 2.0 µg/mL

3         Demecolcin     150.0 ng/mL

Table 2 (cont.):  Exposure period 4 hrs with S9 mix

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
IA	40 hrs	Solvent control1	2.00		0.80
		Positive control2	1.68	32.1	3.60S
		65.1	1.80	20.2	0.35
		130.2PS	1.57	43.2	0.70
		260.4PS	1.20	80.3	0.85
IIA	40 hrs	Solvent control1	2.01		1.68***
		Positive control3	1.57	43.4	8.50S
		50.0	2.02	n.c.	1.35***
		125.0PS	1.84	16.9	1.48***
		175.0PS	1.45	55.8	2.75S***
IIB	40 hrs	Solvent control1	1.86		0.10
		Positive control4	1.21	76.0	4.30S****
		50.0	1.76	11.4	0.30
		100.0	1.56	34.7	0.20
		120.0	1.36	57.7	0.55S
		140.0PS	1.25	70.7	0.85S
		160.0PS	1.26	69.8	0.65S

*         For the positive control groups and the test item treatment groups the values are related to the solvent controls

**       The number of micronucleated cells was determined in a sample of 2000 binucleated cells

***     The number of micronucleated cells was determined in a sample of 4000 binucleated cells

****   The number of micronucleated cells was determined in a sample of 1000 binucleated cells
due to strong toxic effects

PS      Phase separation occurred at the end of treatment

S         The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.     Not calculated as the CBPI is equal or higher than the solvent control value

1         DMSO           0.5 % (v/v)

2         CPA   20.0 µg/mL

3         CPA   17.5 µg/mL

4         CPA   15.0 µg/mL

Conclusions:

It can be stated that under the experimental conditions reported, that Isobornyl acrylate did not induce micronuclei in human lymphocytes in vitro when tested up to cytotoxic or the highest evaluable concentrations. Therefore, the substance is considered to be non-mutagenic in this in vitro micronucleus test.

Executive summary:

Isobornyl acrylate, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in four independent experiments. The following study design was performed:

	Without S9-Mix		With S9-Mix
	Exp.IA & IB	Exp. IIA	Exp. IA, IIA & IIB
Exposure period	4 hrs	20 hrs	4 hrs
Recovery	16 hrs	-	16 hrs
Cytochalasin B exposure	20 hrs	20 hrs	20 hrs
Preparation interval	40 hrs	40 hrs	40 hrs
Total culture period	88 hrs	88 hrs	88 hrs

In each experimental group two parallel cultures were analysed. At least 1000 binucleate cells per culture were evaluated for cytogenetic damage, except for the positive control in Experiment IIB with S9 mix, where only 500 binucleate cells per culture were evaluated.

The highest applied concentration in the pre-test on toxicity (2083.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 (adopted 2010).

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In IA and IB in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IIA in the absence and presence of S9 mix and in IIB in the presence of S9 mix, cytotoxicity was observed at least at the highest evaluated concentrations.

In Exp. IA, IB and IIA (without S9 mix), no relevant increase of micronucleated cells was observed after treatment with the test item.

Exp. IA (with S9 mix):

In Exp. IA with S9 mix, the number of micronucleated cells increased, but even at the highest dose reported due to the mentioned phase separation at higher doses, the number of micronucleated cells is comparable to the solvent control and clearly within the historical control range of the solvent control (0.2 - 1.70 % micronucleated cells).

Exp. IIA (with S9 mix):

In Experiment IIA, in the presence of S9 mix one single statistically significant increase in the number of micronucleated cells (2.75 %) clearly exceeding the laboratory historical solvent control data range (0.20 – 1.70 % micronucleated cells) was observed at the highest evaluated concentration (175.0 µg/mL). Since the number of micronucleated cells in the solvent control (1.68% micronucleated cells) is at the upper limit of the historical control data range (0.2 - 1.70 % micronucleated cells) but still inside, this experiment meets all criteria for validity.

A confirmatory experiment (Exp. IIB) was performed to investigate the equivocal results of Exp. IA and IIA (with S9 mix).

Exp. IIB (with S9 mix):

In the confirmatory Experiment IIB statistically significant increases (0.55, 0.85 and 0.65 %

micronucleated cells, respectively) were observed after treatment with 120.0, 140.0 and 160.0 µg/mL caused by a low response of the solvent control. Since all three values were clearly within the historical solvent control data range of 0.20 – 1.70 % micronucleated cells, these findings can be considered as biologically irrelevant. The value of the solvent control (0.1% micronucleated cells), however, is smaller than the lower limit of the historical control data range (0.2 - 1.70 % micronucleated cells). Since the investigation of a potential increase in micronucleated cells is the relevant endpoint of this test, the undercutting of the historical control data range has no relevance for the assessment of this experiment and this experiment meet all criteria for validity.

Thus, the positive finding of Experiment IIA was not confirmed.

It can be stated out that under the experimental conditions reported, Isobornyl acrylate did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Therefore, the substance is considered to be non-mutagenic in this in vitro micronucleus assay, when tested up to cytotoxic and/ or the highest evaluable concentration.

Footnote historical control data (HCD):

Regarding the OECD guideline 487, the HCD are used as cut-off criteria. The generation of HCD includes the data for a single test system in a single laboratory for a defined period. The volume includes normally manageable sample sizes (regarding the OECD guideline 487: minimum 20 data sets). In case of the current study 48 single experiments (with S9 mix) from 2009 and 2010 were included. Therefore, the HCD cannot describe the whole range of variation for test system but is rather an estimation. Furthermore, the use of human primary lymphocytes includes additional variation factors caused by sex, age and constitution on the day of donation.
With regard to the endpoint (induction of micronucleated cells) the upper limit of the HCD is the important threshold to estimate the validity of experiments related to the laboratory standard. A result below the HCD is not a sign of an insufficient or invalid experiment. Rather it is a parameter for the suitability of the cell system used in relation to a low background level.

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)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

Based on the results of several reliable in vitro studies, Isobornyl acrylate shows no potential to induce gene mutations in bacteria nor does it induce gene mutations or chromosomal damage in mammalian cells.

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

Gene mutation in bacteria

The study was performed to investigate the potential of Isobornyl acrylate to induce genemutations in the plate incorporation test (experiment I) and the pre-incubation test(experiment II) using theSalmonella typhimurium strains TA 1535, TA 1537, TA 98, andTA 100, and the Escherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments both with and without livermicrosomal activation. Each concentration, including the controls, was tested in triplicate.

Reduced background growth was observed at higher concentrations with and without metabolic activation in strains TA 1535, TA 1537, TA 98, and TA 100 in both independent experiments.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations in strains TA 1535, TA 1537, TA 98,

and TA 100 with and without metabolic activation in both independent experiments. No substantial increase in revertant colony numbers of any of the five tester strains wasobserved following treatment with Isobornyl acrylate at any dose level, neither in thepresence nor absence of metabolic activation (S9 mix). There was also no tendency ofhigher mutation rates with increasing concentrations in the range below the generallyacknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under theexperimental conditions reported, the test item did not induce gene mutations by base pairchanges or frameshifts in the genome of the strains used.Therefore, Isobornyl acrylate is considered to be non-mutagenic in this Salmonellatyphimurium and Escherichia coli reverse mutation assay.

Gene mutation in mammalian cells

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

The study was performed in three independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The experimental part of the first experiment with metabolic activation was prematurely terminated due to microbial contamination and repeated as experiment IA. The data of experiment IA are reported as experiment I with metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

No precipitation or phase separation of the test item was observed up to the maximum concentration of the main experiments. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 16.0 µg/mL without metabolic activation and at 96.0 µg/mL with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 24.0 µg/mL without metabolic activation and at 96.0 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of three times the mutation frequency of the corresponding solvent control at any of the test points. In the second experiment the mutation frequency exceeded the historical range of solvent controls at several concentrations. However, since the induction factor was not exceeded and statistical analysis showed no dose dependent increase, the increased values were judged as biologically irrelevant fluctuation. E

MS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion it can be stated that under the experimental conditions reported, Isobornyl acrylate 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.

Isobornyl acrylate, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytesin vitroin four independent experiments. The cells were exposed to the test item for 4 or 20 hrs without S9 -mix as metabolic activation or for 4 hrs with S9 -mix. In each experimental group two parallel cultures were analysed. At least 1000 binucleate cells per culture were evaluated for cytogenetic damage, except for the positive control in Experiment IIB with S9 mix, where only 500 binucleate cells per culture were evaluated. The highest applied concentration in the pre-test on toxicity (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 (adopted 2010). Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In IA and IB in the absence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage. In Experiment IIA in the absence and presence of S9 mix and inand IIB in the presence of S9 mix,cytotoxicity was observed at least at the highest evaluated concentrations.

In IA, IB and IIA in the absence of S9 mix and inin the presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. In Experiment IIA, in the presence of S9 mix one statistically significant increase in the number of micronucleated cells (2.75 %) clearly exceeding the laboratory historical solvent control data range (0.20 – 1.70 % micronucleated cells) was observed at the highest evaluated concentration (175.0 µg/mL). In the confirmatory experiment IIB statistically significant increases (0.55, 0.85 and 0.65 % micronucleated cells, respectively) clearly within the historical solvent control data range were observed after treatment with 120.0, 140.0 and 160.0 µg/mL. Thus, the positive finding of Experiment IIA could not be confirmed.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

It can be stated that under the experimental conditions reported, Isobornyl acrylate did not induce micronuclei as determined by thein vitromicronucleus test in human lymphocytes.

Therefore, the substance is considered to be non-mutagenic in thisin vitromicronucleus test, when tested up to cytotoxic and/or the highest evaluable concentration.

Justification for classification or non-classification

GHS classification according to Annex I 1272/2008 CLP (EU GHS) and UN GHS:

- No classification required.