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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test and an in vitro micronucleus assay were performed on Tricyclodecanemonomethylol acrylate to evaluate the genotoxic potential. The Ames test was found negative, however positive results were obstained in the in vitro micronucleus test.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 February 2018 - 15 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
n/a
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
n/a
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
The selected dose levels were as follows:
- 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1537 (first experiment) and the TA 1535, TA 98 and TA 100 strains (both experiments),
- 0.34, 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1537 (second experiment),
- 31.25, 62.5, 125, 250, 500 and 1000 µg/plate in the TA 102 strain (both experiments).

Experiments with S9 mix
The selected dose levels were as follows:
- 250, 500, 1000, 2000 and 5000 µg/plate in the TA 1535, TA 98, TA 100 and TA 102 strains (first experiment using the direct incorporation method),
- 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate in the TA 1537 (first experiment using the direct incorporation method),
- 2.3, 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate in the TA 1537 (second experiment using the pre-incubation method)
- 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1535, TA 98 and TA 100 strains (second experiment using the pre-incubation method),
- 2.06, 6.17, 18.5, 55.6, 167 and 500 µg/plate in the TA 102 strain (second experiment using the pre-incubation method).
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); The preliminary test, all experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: observation of a decrease in number of revertant colonies and/or a thinning of the bacterial lawn.
Rationale for test conditions:
Not applicable.
Evaluation criteria:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
. a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
. and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
. neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
. nor any evidence of a dose-response relationship is noted.
Statistics:
no
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
other: No genotoxicity was obtained with the TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Emulsion

RANGE-FINDING STUDY:
A moderate emulsion was noted at dose levels = 500 µg/plate without S9 mix and at dose levels = 2500 µg/plate with S9 mix in the TA 98, TA 100 and TA 102 strains.
A moderate to strong toxicity was noted at dose levels = 100 µg/plate without S9 mix and at 5000 µg/plate with S9 mix, in both the TA 98 and TA 100 strains. No noteworthy toxicity was noted in the TA 102 strain, either with or without S9 mix.

RESULTS OF CYTOTOXICITY and GENOTOXICITY:
A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels = 500 µg/plate in the TA 102 strain without S9 mix and at dose levels = 2000 µg/plate in the TA 1535, TA 98, TA 100 and TA 102 strains with S9 mix (direct incorporation method).

Experiments without S9 mix:
In the first experiment, a moderate to strong toxicity was noted at dose levels = 27.8 µg/plate in the TA 1535 and TA 1537 strains and at dose levels = 83.3 µg/plate in the TA 98 and TA 100 strains.
In the second experiment, a slight to strong toxicity was noted at dose levels = 27.8 µg/plate in the TA 1535, TA 98 and TA 100 strains and at 250 µg/plate in the TA 1537 strain.
No noteworthy toxicity was noted towards the TA 102 strain, in both experiments.

Experiments with S9 mix:
In the first experiment (using the direct plate incorporation method), a slight to moderate toxicity was noted at 5000 µg/plate in the TA 1535 and TA 98 strains, at dose levels = 555.6 µg/plate in the TA 1537 strain and = 2000 µg/plate in the TA 100 strain. No noteworthy toxicity was noted towards the TA 102 strain in the first experiment.
In the second experiment (using the pre-incubation method), a slight to strong toxicity was noted at 250 µg/plate in the TA 1535 and TA 100 strains, at dose levels = 185.2 µg/plate in the TA 1537 strain, = 83.3 µg/plate in the TA 98 strain and = 500 µg/plate in the TA 102 strain.


The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, either with or without S9 mix. These results met thus the criteria of a negative response.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached
Conclusions:
Under the experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains (i.e. TA 1535, TA 1537, TA 98, TA 100 and TA 102), either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium.

 

The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose levels of the test item, dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The treatments of the TA 1537 strain [first experiment with S9 mix and second experiment (with and without S9 mix)] were performed at the test site.

Results

The test item was freely soluble in the vehicle at 100 mg/mL.

Consequently, using a maximum dose-volume of 50 µL/plate, the dose-levels used for the preliminary toxicity test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

 

Preliminary test results:

A moderate emulsion was noted at dose levels = 500 µg/plate without S9 mix and at dose levels = 2500 µg/plate with S9 mix in TA 98, TA 100 and TA 102 strains.

 In both the TA 98 and TA 100 strains, a moderate to strong toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at dose levels >= 100 µg/plate, without S9 mix and at 5000 µg/plate (decrease in the number of revertants), with S9 mix. No noteworthy toxicity was noted in the TA 102 strain, either with or without S9 mix.

Since the test item was found toxic and poorly soluble in the final treatment medium in the preliminary test, the selection of the highest dose level to be used in the main experiments was based on the level of toxicity and/or presence of emulsion, according to the criteria specified in the international guidelines.

 

Main experiments:

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

  

Experiments without S9 mix

The selected dose levels were:

.  1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1535, TA 98 and TA 100 strains for both mutagenicity experiments and in the TA 1537 strain for the first experiment,

. 0.34, 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1537 strain for the second experiment,

. 31.25, 62.5, 125, 250, 500 and 1000 µg/plate in the TA 102 strain for both mutagenicity experiments.

 

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose levels = 500 µg/plate in the TA 102 strain in both experiments.

In the first experiment, a moderate to strong toxicity was noted at dose levels = 27.8 µg/plate in the TA 1535 and TA 1537 strains and at dose levels = 83.3 µg/plate in the TA 98 and TA 100 strains.

In the second experiment, a slight to strong toxicity was noted at dose levels = 27.8 µg/plate in the TA 1535, TA 98 and TA 100 strains and at 250 µg/plate in the TA 1537 strain.

No noteworthy toxicity was noted towards the TA 102 strain, in both experiments.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Experiments with S9 mix

The selected dose levels were:

. 250, 500, 1000, 2000 and 5000 µg/plate for the first mutagenicity experiment in the TA 1535, TA 98, TA 100 and TA 102 strains,

. 6.9, 20.6, 61.7, 185.2, 55.6, 1666.7 and 5000 µg/plate in the TA 1537 for the first experiment,

. 2.3, 6.9, 20.6, 61.7, 185.2, 555.6 and 1666.7 µg/plate in the TA 1537 for the second experiment,

. 2.06, 6.17, 18.5, 55.6, 167 and 500 µg/plate in the TA 102 strain for the second mutagenicity experiment,

. 1.03, 3.09, 9.26, 27.8, 83.3 and 250 µg/plate in the TA 1535, TA 98 and TA 100 strains for the second experiment.

 

In the first experiment, a moderate emulsion was observed in the Petri plates when scoring the revertants at dose levels = 2000 µg/plate in the TA 1535, TA 98, TA 100 and TA 102 strains.

In the first experiment (using the direct plate incorporation method), a slight to moderate toxicity was noted at 5000 µg/plate in the TA 1535 and TA98 strains, at dose levels = 555.6 µg/plate in the TA 1537 strain and = 2000 µg/plate in the TA 100 strain. No noteworthy toxicity was noted towards the TA 102 strain in the first experiment.

In the second experiment (using the pre-incubation method), a slight to strong toxicity was noted at 250 µg/plate in the TA 1535 and TA 100 strains, at dose levels = 185.2 µg/plate in the TA 1537 strain, = 83.3 µg/plate in the TA 98 strain and = 500 µg/plate in the TA 102 strain.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains, either with or without S9 mix. These results met thus the criteria of a negative response.

 

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January - March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
Blood from two healthy, non-smoking male volunteers from a panel of donors at
Covance was used for each experiment. No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers ofalcohol. Donors were not taking any form of medication. The measured cell cycle time of the donors used at Covance, Harrogate falls within the range 13±2 hours. For
each experiment, an appropriate volume of whole blood was drawn from the
peripheral circulation into heparinised tubes on the day of culture initiation. Blood
was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by
placing 0.4 mL of pooled heparinised blood into 8.5 mL pre-warmed (in an incubator
set to 37±1°C) HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated
foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume
following addition of S-9 mix/KCl and the test article in its chosen vehicle was
10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the
culture medium at a concentration of approximately 2% of culture to stimulate the
lymphocytes to divide. Blood cultures were incubated at 37±1°C for approximately
48 hours and rocked continuously.
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v).
Test concentrations with justification for top dose:
Preliminary solubility data indicated that Tricyclodecanemonomethylol Acrylate (CAS 93962-84-6) was soluble in anhydrous analytical grade dimethyl sulphoxide
(DMSO) at a concentration of approximately 202.0 mg/mL. The solubility limit in culture medium was in the range of 63.12 to 126.2 µg/mL, as indicated by
precipitation at the higher concentration which persisted for approximately 24 hours after test article addition. A maximum concentration of 400.0 µg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to a precipitating concentration (OECD, 2016). Concentrations selected for the Micronucleus Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Treatment 3hr (without S9) : 4 to 50 µg/ml
Treatment 3hr (with S9) : 20 to 150 µg/ml
Treatment 24hr (without S9) : 4 to 50 µg/ml
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide (DMSO)
- Justification for choice: Based on available solubility data, the test item was dissolved in DMSO at 304 mg/mL. Therefore, using this stock solution at 304 mg/mL and a treatment volume of 1% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 3040 µg/mL) was achievable.
- Formulation : Test article stock solutions were prepared by formulating Tricyclodecanemonomethylol Acrylate under subdued lighting in DMSO, with the aid of vortex mixing, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 3.5 hours of initial formulation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C (-S9, 3hr); cyclophosphamide (+S9 mix); vinblastine (-S9, 24hr)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in culture medium
S-9 mix or KCl (1 mL per culture) was added appropriately. Cultures were treated with the test article, vehicle or positive controls (0.1 mL per culture).
The final culture volume was 10 mL.
For removal of the test article, cells were pelleted (approximately 300 g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 37±1°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin / streptomycin. Cyto-B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.

DURATION
First experiment: 3 h treatment + 24 h recovery (with and without S9mix)
Second experiment: 3 h treatment + 24 h recovery (with S9mix); 24 h treatment + 20 h recovery (without S9mix)
NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

OTHERS
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in chromosome aberrations (Scott et al., 1991; Brusick, 1986). Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment.

Harvesting: At the defined sampling time, cultures were centrifuged at approximately 300 g for 10 minutes, the supernatant removed and discarded and cells resuspended in 4 mL
(hypotonic) 0.075 M KCl at 37±1°C for 4 minutes to allow cell swelling to occur. Cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (7:1, v/v). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets were clean.

Slides preparation: Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged
(approximately 1250 g, 2-3 minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and either stained immediately after drying or stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

Selection of Concentrations for the Micronucleus Experiment : Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined. Cytotoxicity (%) is expressed as (100 – Relative RI).
A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

Selection of Concentrations for Micronucleus Analysis (Micronucleus Experiment Only): Slides were examined, uncoded, for RI to a minimum of 500 cells per culture to determine whether chemically induced cell cycle delay or toxicity had occurred. The highest concentration selected for micronucleus analysis under each treatment condition gave approximately 50-60% cytotoxicity (OECD, 2016). Analysis of slides from highly toxic concentrations was avoided. Slides from the highest selected concentration and two lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity from maximum to little was covered.
The positive control concentrations analysed did not exceed the cytotoxicity limits for the test article concentration selection.

Slide Analysis
Scoring was carried out using fluorescence microscopy. Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.
For each treatment regime, two vehicle control cultures were analysed for micronuclei.
Slides from the positive control treatments were checked to ensure that the system was operating satisfactorily. One concentration from each positive control, which give satisfactory responses in terms of quality and quantity of binucleated cells and numbers of micronuclei, was analysed. This pre-analysis slide check was conducted
under non-blinded conditions.
All slides for analysis were coded by an individual not connected with the scoring of the slides, such that analysis was conducted under blind conditions. Labels with only the study number, assay type, experiment number, the sex of the donor and the code were used to cover treatment details on the slides.
One thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. The number of cells containing micronuclei on each slide was recorded.
Nucleoplasmic bridges (NPBs) between nuclei in binucleate cells were recorded during micronucleus analysis to provide an indication of chromosome rearrangement.
Various mechanisms may lead to NPB formation following DNA misrepair of strand breaks in DNA (Thomas et al., 2003). In this assay, binucleate cells with NPBs were
recorded as part of the micronucleus analysis.
Micronucleus analysis was not conducted on slides generated from the Range-Finder treatments.
Slide analysis was performed by competent analysts trained in the applicable Covance Laboratories standard operating procedures. The analysts were physically located
remote from the Covance facility, but were subject to Covance management and GLP control systems (including QA inspection). All slides and raw data generated by the
remote analysts were returned to Covance Laboratories for archiving on completion of analysis.

Treatment of Data: After completion of scoring and decoding of slides, the numbers of binucleate cells with micronuclei (MNBN cells) in each culture were obtained.
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989). The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A
Cochran-Armitage trend test was applied to each treatment condition. Probability values of p=0.05 were accepted as significant.
Rationale for test conditions:
Acceptance Criteria: The assay was considered valid if the following criteria were met:
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures, particularly where no positive responses were seen
2. The frequency of MNBN cells in vehicle controls fell within the historical vehicle control (normal) ranges
3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the current historical vehicle control ranges
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at
the time of harvest
5.The highest concentration selected for micronucleus analysis under each treatment condition gave approximately 50-60% cytotoxicity
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met. Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at very toxic concentrations (Thybaud et al., 2007).
Statistics:
yes
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Treatment of cells for 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p=0.05), compared to those observed in
the concurrent vehicle controls, at the highest two concentrations analysed (24 and 30 µg/mL, giving 40% and 52% cytotoxicity, respectively). The MNBN cell frequencies exceeded the normal range of 0 to 0.7% in one or both cultures at all three concentrations analysed (12, 24 and 30 µg/mL) and there was a statistically significant linear trend (p=0.01). The data were indicative of a weak positive result.

Treatment of cells for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly higher, compared to those observed in the concurrent vehicle controls, at any concentration analysed. The MNBN cell frequencies in treated cultures were within the normal range at all concentrations analysed and there was no statistically significant linear trend. The data were indicative of a negative result.

Treatment of cells for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p=0.001), compared to those observed in the concurrent vehicle control, at all three concentrations analysed (21, 27 and 30 µg/mL, giving 10%, 35% and 48% cytotoxicity, respectively). The MNBN cell frequencies exceeded the normal range of 0 to 0.8% in both cultures at all concentrations analysed and there was a statistically significant linear trend (p=0.001). The data were indicative of a clear positive result.

No test article related increases in cells with NPBs were observed

The main study was considered as valid :
1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures for the 3+21 hour and 24+24 hour treatments in the absence of S-9. For the 3+21 hour treatment in the presence of S-9, statistically significant heterogeneity (p=0.05) was observed (Table 8.5) as there were marked differences in MNBN cell frequencies between replicate cultures at all test article concentrations analysed. However, these MNBN cell frequency values were all within the normal range, therefore this did not affect the interpretation of the data
2. The frequency of MNBN cells in vehicle controls fell within the normal range with the exception of the replicate A vehicle control following the 3+21 hour treatment in the absence of S-9, where the MNBN cell frequency of 0.8% was marginally outside the normal range of 0 to 0.7%. However, the mean vehicle control MNBN cell frequency (0.5%) was within the normal range, therefore this did not affect the interpretation of the data or the conclusions drawn for this treatment condition
3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest
5. The highest concentration selected for micronucleus analysis under each treatment condition gave approximately 50-60% cytotoxicity
Conclusions:
It is concluded that Tricyclodecanemonomethylol Acrylate induced micronuclei in cultured human peripheral blood lymphocytes when tested for 3+21 hours and 24+24 hours in the absence of a rat liver metabolic activation system (S-9). In the same test system, Tricyclodecanemonomethylol Acrylate did not induce micronuclei when tested up to toxic concentrations for 3+21 hours in the presence of S-9 under the experimental conditions described.
Executive summary:

Tricyclodecanemonomethylol Acrylate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats.

The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). The highest concentrations analysed in the Micronucleus Experiment were limited by toxicity and were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Tricyclodecanemonomethylol Acrylate on the replication index

(RI). Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in these cultures fell within (or very close to) the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control

chemicals, respectively, in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with

micronuclei. All acceptance criteria were considered met and the study was accepted as valid.

Treatment of cells for 3+21 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p=0.05), compared to those observed in the concurrent vehicle controls, at the highest two concentrations analysed (24 and 30 µg/mL, giving 40% and 52% cytotoxicity, respectively). The MNBN cell frequencies exceeded the normal range (0 to 0.7%) in one or both cultures at all three

concentrations analysed (12, 24 and 30 µg/mL) and there was a statistically significant linear trend (p=0.01). The data were indicative of a weak positive result.

Treatment of cells for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly higher, compared to those observed in the concurrent vehicle controls, at any concentration analysed. The MNBN cell frequencies in treated cultures were within the normal range at all concentrations analysed and there was no statistically significant linear trend. The data were indicative of a negative result.

Treatment of cells for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p=0.001), compared to those observed in the concurrent vehicle controls, at all three concentrations analysed (21, 27 and 30 µg/mL, giving 10%, 35% and 48% cytotoxicity, respectively). The MNBN cell frequencies exceeded the normal range (0 to 0.8%) in both cultures at all

concentrations analysed and there was a statistically significant linear trend (p=0.001).

The data were indicative of a clear positive result.

It is concluded that Tricyclodecanemonomethylol Acrylate induced micronuclei in cultured human peripheral blood lymphocytes when tested for 3+21 hours and 24+24 hours in the absence of a rat liver metabolic activation system (S-9). In the same test system, Tricyclodecanemonomethylol Acrylate did not induce micronuclei when tested up to toxic concentrations for 3+21 hours in the presence of S-9 under the experimental conditions described.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo Comet assay was proposed in order to evaluate the relevance of the results obtained in the in vitro studies.

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

In vitro gene mutation study in bacteria (Valin 2018):

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium (OECD 471).

The test item was freely soluble in the vehicle at 100 mg/mL. Consequently, using a maximum dose-volume of 50 µL/plate, the dose-levels used for the preliminary toxicity test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Emulsions were observed in the Petri plates when scoring revertants at dose-levels = 500 µg/plate for the TA 98, TA 100 and TA 102 without S9 mix or at dose-levels = 2500 µg/plate for the TA 98, TA 100 and TA 102 strains with S9 mix (direct incorporation method).

A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacteria lawn) was noted at dose-levels = 100 µg/plate in the TA 98 and TA 100 strains without S9 mix.

No noteworthy toxicity was noted in TA 102 without S9 mix or in TA 98, TA 100 and TA 102 with S9 mix (direct incorporation method).

Since the test item was freely soluble but toxic in the preliminary test, the selection of the highest dose level for the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

  

Experiments without S9 mix: Emulsions were observed in the Petri plates when scoring the revertants at dose-levels =500 µg/plate in the TA 102.

A moderate to strong toxicity (thinning of the bacteria lawn) was noted at dose-levels =27.8 µg/plate in the TA 98, TA 1535, TA 1537 and TA 100 strains without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, in any of the TA 1535, TA 98, TA 100 and TA 102 strains, in any experiments.

 

Experiments with S9 mix: Emulsions were observed in the Petri plates when scoring the revertants at dose-levels = 2000 µg/plate for the TA 1535, TA 98, TA 100 and TA 102 strains with S9 mix (direct incorporation method). A moderate toxicity (decrease in the number of revertants and/or thinning of the bacteria lawn) was noted at dose-levels = 2000 µg/plate in the TA 100 strain according to the direct incorporation method.A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacteria lawn) was noted at dose-levels = 83.3 µg/plate in the TA 98, TA 1535, TA 100 and TA 102 strains according to the pre-incubation method.  

The test item did not induce any noteworthy increase in the number of revertants, in any of the TA 1535, TA 98, TA 100 and TA 102 strains, in any experiments.

The first experiment with S9 mix and the second experiment with and without S9 mix are still pending for the TA 1537 strain. These intermediate results met the criteria for a negative response.

In conclusion : Under the experimental conditions, and pending results of the TA 1537 strain (first experiment with S9 mix and second experiment with and without S9 mix), the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains (i.e. TA 1535, TA 98, TA 100 and TA 102), either in the presence or in the absence of a rat liver metabolizing system.

In vitro micronucleus test (Lloyd, 2019)

Tricyclodecanemonomethylol Acrylate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two male donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats.

The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO). The highest concentrations analysed in the Micronucleus Experiment were limited by toxicity and were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatment of cells for 3+21 hours in the presence of S-9 resulted in frequencies of MNBN cells that were similar to and not significantly higher, compared to those observed in the concurrent vehicle controls, at any concentration analysed. The MNBN cell frequencies in treated cultures were within the normal range at all concentrations analysed and there was no statistically significant linear trend. The data were indicative of a negative result.

Treatment of cells for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were significantly higher (p=0.001), compared to those observed in the concurrent vehicle controls, at all three concentrations analysed (21, 27 and 30 µg/mL, giving 10%, 35% and 48% cytotoxicity, respectively). The MNBN cell frequencies exceeded the normal range (0 to 0.8%) in both cultures at all

concentrations analysed and there was a statistically significant linear trend (p=0.001).

The data were indicative of a clear positive result.

It is concluded that Tricyclodecanemonomethylol Acrylate induced micronuclei in cultured human peripheral blood lymphocytes when tested for 3+21 hours and 24+24 hours in the absence of a rat liver metabolic activation system (S-9). In the same test system, Tricyclodecanemonomethylol Acrylate did not induce micronuclei when tested up to toxic concentrations for 3+21 hours in the presence of S-9 under the experimental conditions described.

Justification for classification or non-classification

Based on the available data, it is not possible to conclude on the genotoxicity classification of the registered substance according to the Regulation EC N°1272/2008.