Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

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

Referenceopen allclose all

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
GLP compliance:
yes (incl. QA statement)
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:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True 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
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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

A Comet assay was conducted on (octahydro-4,7-methano-1H-indenyl) methyl acrylate, which showed that the test item did not induce DNA strand breaks in the liver, stomach or duodenum of male Sprague Dawley rats administered up to 2000 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date: 04th February 2022
Experimental Completion Date: 29th March 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was selected as there is a large volume of background data in this species and has been specifically requested by ECHA for this comet assay (ECHA decision number TPE-D-2114557356-42-01/F).
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: -6 male young adult out-bred Sprague Dawley rats (Crl:CD(SD)) were obtained from Charles River (UK)
Ltd., Margate, UK for use in the Range-Finder
-27 male young adult out-bred Sprague Dawley rats (HSD:Sprague DawleySD) were obtained from
Envigo, Blackthorn, UK for use in the Main Experiment
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: ca. 250 g
- Assigned to test groups randomly: animals were randomly allocated to cages. Male rats only due to the potential analysis of gonadal cells and to ethically minimise animal testing.
- Fasting period before study: no
- Housing: Animals were housed in wire topped, solid bottomed cages, with three animals per cage. Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd.,
Manchester) and provided with wooden Aspen chew blocks and rodent retreats.
-Diet:: ad libitum access to 5LF2 EU Rodent Diet
- Water ad libitum via water bottles


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 °C
- Humidity (%): 40-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light:

IN-LIFE DATES:
In-life Start Date: 07th February 2022
In-life End Date: 08th March 2022
Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
Formulations were freshly prepared prior to each dosing occasion for the Range-Finder (dose group 1RF) and prepared once for each dose group in the Main Experiment. (octahydro-4,7-methano-1H-indenyl) methyl acrylate was formulated in corn oil as follows:
The test article was weighed out into a pre-labelled bottle and vehicle was added to achieve the final volume. Formulations were stirred on a magnetic stirrer to homogenise and aliquoted (as required).


Duration of treatment / exposure:
21 hours
Frequency of treatment:
The test article and vehicle control were given as two administrations, at 0 and 21 hours; the positive control was administered once only at 21 hours.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control - vehicle only
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
positive control
No. of animals per sex per dose:
3 male animals were dosed during the Range-Finder Experiment
5 male animals were dosed during the Main Experiment, plus 3 males for the positive control

Main Experiment animals were dosed in replicate cage order i.e. cage 1 of Groups 1-4 dosed in ascending group order then cage 2 of Groups 1-4 in ascending group order. Group 5 was dosed at a time that allowed necropsy of these animals after Group 4 necropsy. Animals were not fasted prior to administration and were sampled at 24 hours.
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control:
The vehicle control group consisted of animals dosed with corn oil. The vehicle was selected as it has been used in previous in vivo studies with this compound.
The positive control, Ethyl Methanesulfonate (EMS, Sigma-Aldrich Chemical Co, Poole, UK) was freshly prepared in purified water as follows:
-dose volume (ml/kg): 10
-concentration of EMS solution (mg/ml): 20
-dose of EMS administered (mg/kg): 200
Tissues and cell types examined:
The liver, stomach, duodenum and gonad were removed from each control (vehicle and positive) and test article treated animal.
For histopathology, a sample of liver, stomach, duodenum and gonad from vehicle control and test article treated animals only was removed. Liver, stomach and duodenum samples were immediately preserved in neutral buffered formalin and stored at room temperature. Gonad samples were immediately preserved in modified Davidson’s fluid and stored at room temperature. No histopathology samples were preserved for the positive control animals.


Preserved liver, stomach, duodenum and gonad samples were embedded in wax blocks and sectioned at 5 µm nominal. Liver, stomach and duodenum slides were stained with haematoxylin and eosin and examined by the Study Pathologist. Gonad samples were not examined as the somatic tissues did not show genotoxic potential
Details of tissue and slide preparation:
  Preparation of Cell Suspensions
The comet liver samples were washed thoroughly in Merchants solution and placed in fresh buffer. The samples were cut into small pieces in Merchants solution and the pieces of liver were then pushed through bolting cloth (pore size of 150 µm) with approximately 4 mL of Merchants solution to produce single cell suspensions.

The comet stomach samples were washed in ice cold Merchants solution and then incubated on ice for 15 minutes prior to processing. After incubation the stomach samples were removed from the Merchants solution and the inner surface gently scraped (released material discarded) using the back of a scalpel blade. Cells were gently scraped from the inside surface of the stomach using the back of a scalpel blade in 200 µL of fresh Merchants solution to produce single cell suspensions.

The comet duodenum samples were washed thoroughly in ice cold Merchants solution; each sample was vortexed in ice cold Merchants solution for approximately 15 seconds. The tissue was removed from the Merchants solution and the inner surface gently scraped twice (released material discarded) using the back of a scalpel blade. The tissue was vortexed in ice cold Merchants solution for a further 15 seconds prior to gently scraping the inside of the duodenum three times with the back of a scalpel blade in 150 µL of fresh Merchants solution to produce single cell suspensions.

The comet gonad samples were prepared by making an incision along the length of a single gonad, removing the contents from the membrane and discarding the membrane. The remaining tissue was cut into small pieces and gently pushed through bolting cloth (pore size of 150 µm) with approximately 10 mL of Merchants solution to produce single cell suspensions.

All cell suspensions were held on ice prior to slide preparation.

Slide Preparation
Three slides, labelled ‘A’, ‘B’ and ‘C’ were prepared per single cell suspension per tissue. Slides were labelled with the study number, appropriate animal tag number and tissue. Slides were dipped in molten normal melting point agarose (NMA) such that all of the clear area of the slide and at least part of the frosted area was coated. The underside of the slides was wiped clean and the slides allowed to dry. 40 µL of each single cell suspension was added to 400 µL of 0.7% low melting point agarose (LMA) at approximately 37°C. 100 µL of cell suspension/agarose mix was placed on to each slide. The slides were then coverslipped and allowed to gel on ice

Unwinding and Electrophoresis
Following lysis, slides were washed in purified water for 5 minutes, transferred to electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH>13) at 2-8°C and the DNA unwound for 20 minutes (stomach and duodenum) or 30 minutes (liver and gonad). At the end of the unwinding period the slides were electrophoresed in the same buffer at 0.7 V/cm for 20 minutes (stomach and duodenum) or 40 minutes (liver and gonad). As not all slides could be processed at the same time a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run; i.e. for all animals the same number of triplicate slides was processed at a time.

Neutralisation
At the end of the electrophoresis period, slides were neutralised in 0.4 M Tris, pH 7.0 (3 x 5 minute washes). After neutralisation the slides were dried and stored at room temperature prior to scoring.

Staining
Prior to scoring, the slides were stained with 100 µL of 2 µg/mL ethidium bromide and coverslipped.
Evaluation criteria:
For valid data, the test article was considered to induce DNA damage if:
1. A least one of the test doses exhibited a statistically significant increase in tail intensity, in any tissue, compared with the concurrent vehicle control
2. The increase was dose related in any tissue.
3. The increase exceeded the laboratory’s historical control data for that tissue.
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 neither of the above criteria were met and target tissue exposure was confirmed.
Results which only partially satisfied the criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example comparison of the response against the historical control data, consistency of response within and between dose levels and any confirmatory experiments
Statistics:
Treatment of Data

After completion of microscopic analysis and decoding of the data the percentage tail intensity (i.e. %DNA in the tail) was calculated.

Data were treated as follows:
1. The median value per slide was calculated
2. The mean of the slide medians was calculated to give the mean animal value
3. The mean of the animal means and standard error of the mean was calculated for each group.

Tail intensity data for each slide were supplied for statistical analysis. The median of the log-transformed tail intensities from each slide was averaged to give an animal summary statistic. Where the median value on a slide was zero, a small constant (0.0001) was added before taking the logarithm and calculating the average for the animal. This animal average was used in the statistical analysis.

Data was analysed using one-way analysis of variance (ANOVA) with the fixed factor for treatment group. The positive control group was excluded from this analysis. Levene’s test was used to test for equality of variances among groups. This showed no evidence of heterogeneity (P>0.01). Comparisons between each treated group and control were made using Dunnett’s test. The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p value are reported. In addition, a linear contrast was used to test for an increasing dose response.

The positive control group was compared to the control group using a two-sample t test. Levene’s test was used to test for equality of variances between the groups. This showed no evidence of heterogeneity (P>0.01). The test was one-sided looking for an increase in response with increasing dose. The back-transformed difference and p-value are reported.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDER RESULTS
(octahydro-4,7-methano-1H-indenyl) methyl acrylate at 2000 mg/kg/day. No clinical observations of toxicity were observed for any animal on Day 1. Decreased activity was noted for 2 out of 3 animals on Day 2, 2 hours post dose. No further clinical signs were observed for the remainder of the Range-Finder Experiment. There were only small changes in animal body weight between Day 1 and 2, with the group mean body weight percentage change value of +0.4%.
From these results, the current maximum recommended dose of 2000 mg/kg/day was tolerated and selected as the maximum dose for the Main Experiment. Two lower doses of 500 and 1000 mg/kg/day (equivalent to 25% and 50% of the maximum dose) were also selected.
Post dose observations and body weights are presented in below tables Individual Post Dose Observations and Individual and Group Mean Body Weights, respectively.


MAIN EXPERIMENT RESULTS
Formulations Analysis

Analyses demonstrated that the test article formulations at 50, 100 and 200 mg/mL were homogeneous (0.39-4.12% relative standard deviation (RSD), which fell within target criteria of =5%) and met criteria (100±15% of the nominal test article concentrations) for acceptable achieved concentration (mean values of 102-106%). The formulations were therefore considered acceptable. No test article was detected in the vehicle sample.

Post Dose Observations
There were no clinical observations of toxicity for any animal dosed with the vehicle, (octahydro-4,7-methano-1H-indenyl) methyl acrylate (at 500, 1000 or 2000 mg/kg/day) or the positive control (EMS). Please see table: Individual Post Dose Observations.

Body Weights
There was no clear test article related impact on animal body weights between Day 1 and Day 2 with group mean body weight change values of +2.5%, +2.9% and +3.6% for 500, 1000 and 2000 mg/kg/day, respectively, compared to +3.6% for the concurrent vehicle control group. Please see below tables: Individual and Group Mean Body Weights and Summary of Body Weight Percentage Change.

Bioanalysis
Plasma was processed from whole blood samples as a contingency for systemic exposure confirmation. Analysis of these samples was not performed

Clinical Pathology
Clinical Chemistry
Increased cholesterol was recorded for animals from all groups administered (octahydro-4,7-methano-1H-indenyl) methyl acrylate.
Increased urea was noted for animals administered 1000 or 2000 mg/kg/day (octahydro-4,7-methano-1H-indenyl) methyl acrylate and increased creatinine for animals administered 500, 1000, or 2000 mg/kg/day (octahydro-4,7-methano-1H-indenyl) methyl acrylate.
While the clinical chemistry changes were generally very small, based on the acute nature of this study (24 hours) at the maximum-tolerated dose, and because the values in (octahydro-4,7-methano-1H-indenyl) methyl acrylate-treated animals were outside the control range and often noted with a dose relationship, findings described were considered related to (octahydro-4,7-methano-1H-indenyl) methyl acrylate.
Other differences in individual clinical pathology parameters were observed for animals administered the test article; however, they were considered not test article related due to the negligible magnitude of the change, individual animal variability, and overlap of values for test article treated animals with concurrent control values. Please find below results table: Individual and Group Mean Clinical Chemistry.

Histopathology
On macroscopic examination, no changes were considered related to (octahydro-4,7-methano-1H-indenyl) methyl acrylate. Tissues were considered macroscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.
On microscopic examination, changes related to (octahydro-4,7-methano-1H-indenyl) methyl acrylate were recorded for the liver. Decreased hepatocyte glycogen was recorded with a dose relationship for animals administered 1000 or 2000 mg/kg/day.
Glycogen is normally stored in the hepatocytes in large, often perinuclear vacuoles with a granular or feathery appearance. Decreased glycogen vacuolation was noted in animals administered (octahydro-4,7-methano-1H-indenyl) methyl acrylate. Animals were not fasted prior to necropsy and were also sacrificed in replicate order (within 1 hour of each other); as such, this decrease in glycogen was attributed directly or indirectly to the effects of (octahydro-4,7-methano-1H-indenyl) methyl acrylate. This decrease may indicate an increased utilization of glycogen, possibly due to the increased metabolism in the liver or decreased food consumption.

Comet Analysis
Comet data for liver are presented in tables: Liver: Animal Comet Data (animal values) and Liver: Individual Slide Data (slide values), and summarised as group means in Table (octahydro-4,7-methano-1H-indenyl) methyl acrylate: Summary of Group Mean Data – Liver. Comet data for stomach are presented in Table Stomach: Animal Comet Data(animal values), Table Stomach: Individual Slide Data (slide values), and summarised as group means in Table (octahydro-4,7-methano-1H-indenyl) methyl acrylate: Summary of Group Mean Data – Stomach. Comet data for duodenum are presented in Table Duodenum: Animal Comet Data (animal values), Table Duodenum: Individual Slide Data (slide values), and summarised as group means in Table (octahydro-4,7-methano-1H-indenyl) methyl acrylate: Summary of Group Mean Data – Duodenum.







Validity of Data


The data generated in this study confirm that:



  1. The vehicle control data were comparable to laboratory historical control data for each tissue

  2. The positive control induced responses that were compatible with the laboratory historical control data and produces are statistically significant compared to the concurrent vehicle control



    1. Adequate numbers of cells and doses were analysed

    2. The high dose was considered to be the maximum recommended dose.


    As dosing was via oral gavage, exposure to the stomach and duodenum was assured. With regards to the liver, decreased hepatocyte glycogen indicated liver perturbations following dosing, which was attributed directly or indirectly to the effects of the test article. Furthermore, while the clinical chemistry changes were small, they were determined to be test article-related providing indirect evidence of a systemic response. Neither the clinical chemistry nor histopathology findings were notable, but combined provide indirect evidence of systemic test article exposure and therefore liver exposure as a well perfused tissue.


    The assay data were therefore considered valid.


assay data were therefore considered valid.


Data Analysis


There was no dose-related increase in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with (octahydro-4,7-methano-1H-indenyl) methyl acrylate did not cause excessive DNA damage that could have interfered with comet analysis.


Animals treated with (octahydro-4,7-methano-1H-indenyl) methyl acrylate at all doses exhibited group mean tail intensities that were similar to the concurrent vehicle control group and that fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response.


There were individual animal liver %tail intensity values in each test article dosed group that exceeded the upper limit of the 95% reference range (1.80%). However, there was only one animal at 500 mg/kg/day (R0105) and one animal at 2000 mg/kg/day (R0304) that exceeded the upper limit of the observed range of all vehicle control dosed animals in the laboratory’s historical vehicle control data (2.37%). Furthermore, these small increases were not reproduced in all animals in any dose group, did not contribute to statistically significant increases for any group compared to the vehicle control group, and did not occur in a dose-related manner. As such, there were considered of no biological relevance.


These data were considered clearly negative in the liver, stomach and duodenum.


(octahydro-4,7-methano-1H-indenyl) methyl acrylate: Summary of Group Mean Data – Liver

























































































Group/Dose Level


(mg/kg/day)



Tail Intensity



Mean % Hedgehogs



Mean



SEM



Back-Transformed Difference from Vehicle



Ranked



P-value



Significance



 



 



 



 



 



 



 



 



1/ Vehicle (0)



0.98



0.22



-



-



-



-



0.39



2/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (500)



1.56



0.52



1.55



U



0.3636



NS



0.86



3/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (1000)



1.33



0.28



1.43



U



0.4367



NS



0.68



4/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (2000)



1.34



0.31



1.58



U



0.3481



NS



0.68



5/ EMS (200)



21.71



1.46



32.22



U



0.0001



***



0.89



Dose response: (groups 1,2,3,4 )



 



 



 



U



0.2026



NS



N/A



(octahydro-4,7-methano-1H-indenyl) methyl acrylate: Summary of Group Mean Data – Stomach

























































































Group/Dose Level


(mg/kg/day)



Tail Intensity



Mean % Hedgehogs



Mean



SEM



Back-Transformed Difference from Vehicle



Ranked



P-value



Significance



 



 



 



 



 



 



 



 



1/ Vehicle (0)



1.09



0.26



-



-



-



-



9.08



2/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (500)



0.75



0.19



0.63



U



0.9793



NS



12.32



3/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (1000)



0.56



0.12



0.53



U



0.9932



NS



8.01



4/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (2000)



0.76



0.17



0.72



U



0.9530



NS



9.18



5/ EMS (200)



9.79



2.04



11.07



U



0.0004



***



11.24



Dose response: (groups 1,2,3,4 )



 



 



 



U



0.8329



NS



N/A



 






























EMS



Ethyl Methanesulfonate



SEM



Standard Error of Mean



N/A



Not applicable



NS



Not significant (P>0.05)



***



P≤0.001



U



Unranked



(octahydro-4,7-methano-1H-indenyl) methyl acrylate: Summary of Group Mean Data – Duodenum

























































































Group/Dose Level


(mg/kg/day)



Tail Intensity



Mean % Hedgehogs



Mean



SEM



Back-Transformed Difference from Vehicle



Ranked



P-value



Significance



 



 



 



 



 



 



 



 



1/ Vehicle (0)



0.45



0.11



-



-



-



-



10.35



2/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (500)



0.85



0.22



1.89



U



0.2150



NS



8.78



3/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (1000)



0.72



0.20



1.70



U



0.2884



NS



10.34



4/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (2000)



0.64



0.28



1.28



U



0.5338



NS



9.92



5/ EMS (200)



6.93



1.76



20.60



U



0.0001



***



7.81



Dose response: (groups 1,2,3,4 )



 



 



 



U



0.3369



NS



N/A































EMS



Ethyl Methanesulfonate



SEM



Standard Error of Mean



N/A



Not applicable



NS



Not significant (P>0.05)



***



P≤0.001



U



Unranked



RANGE-FINDER TABLES


Individual Post Dose Observations











































































Group /
Treatment (mg/kg/day)



Animal ID and Sex



Clinical Sign



Day 1 (hours after administration)



Day 2 (hours after administration)



Prior to dose



Imm



0.5



1



2



4-6



Prior to dose



Imm



0.5



1



2



4-6



1RF / (octahydro-4,7-methano-1H-indenyl) methyl acrylate (2000)



R5001M



ü



ü



ü



ü



ü



ü



ü



ü



ü



ü



DA



ü



R5002M



ü



ü



ü



ü



ü



ü



ü



ü



ü



ü



ü



ü



R5003M



ü



ü



ü



ü



ü



ü



ü



ü



ü



ü



DA



ü


















Key to Range-Finder Clinical Observations



ü



No remarkable observations



DA



Reduced activity



Individual and Group Mean Body Weights










































Group / Sex



Treatment
(mg/kg/day)



Animal Number



Day 1
Body Weight
(g)



Day 2
Body Weight
(g)



Group Mean Weight Change


(Day 1-2)


(%)



1RF / M



(octahydro-4,7-methano-1H-indenyl) methyl acrylate


(2000)



R5001



248



247



 



 



R5002



254



255



 



 



R5003



242



246



 



Group mean (g)



248



249



+0.4



MAIN EXPERIMENT TABLES


Individual Post Dose Observations


Individual Physical Examinations




















































































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



Group                      1           2          3           4        5



Dose level (mg/kg/day)     0          500       1000         2000     200



session



Group/Sex



Animal Number



 



Observation



Phase



Day (s)



3


 



4


 



6



8



10



12



1/M



R0001



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



1/M



R0002



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



1/M



R0003



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



1/M



R0004



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



1/M



R0005



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



1/M



R0006



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



  p = present




















































































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



Group                      1           2          3           4        5



Dose level (mg/kg/day)     0          500       1000         2000     200



session



Group/Sex



Animal Number



 



Observation



Phase



Day (s)



3


 



4


 



6



8



10



12



2/M



R0001



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



2/M



R0002



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



2/M



R0003



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



2/M



R0004



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



2/M



R0005



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



2/M



R0006



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



p = present




















































































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



Group                      1           2          3           4        5



Dose level (mg/kg/day)     0          500       1000         2000     200



session



Group/Sex



Animal Number



 



Observation



Phase



Day (s)



3


 



4


 



6



8



10



12



3/M



R0001



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



3/M



R0002



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



3/M



R0003



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



3/M



R0004



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



3/M



R0005



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



3/M



R0006



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



p = present




















































































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



Group                      1           2          3           4        5



Dose level (mg/kg/day)     0          500       1000         2000     200



session



Group/Sex



Animal Number



 



Observation



Phase



Day (s)



3


 



4


 



6



8



10



12



4/M



R0001



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



4/M



R0002



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



4/M



R0003



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



4/M



R0004



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



4/M



R0005



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



4/M



R0006



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



p = present




















































































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



Group                      1           2          3           4        5



Dose level (mg/kg/day)     0          500       1000         2000     200



session



Group/Sex



Animal Number



 



Observation



Phase



Day (s)



3


 



4


 



6



8



10



12



5/M



R0001



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



5/M



R0002



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



5/M



R0003



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



5/M



R0004



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



5/M



R0005



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



5/M



R0006



Normal



No remarkable observations



DSNG



1/2



p/p



p/p



p/-



p/-



p/-



-/p



p = present


































Session



 



3



Prior to dose



4



Immediate



6



1 hour post dose



8



2 hours post dose



10



4 hours post dose



12



Prior to necropsy



Individual and Group Mean Body Weights

































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



Group                      1           2          3           4        



Dose level (mg/kg/day)     0          500       1000         2000     



Data presented in g



Group/Sex



Animal Number



Phase


Day        PRED 1         DSNG1    DSNG2



 



1/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



227.4


207.6


200.5


200.6


205.4


211.6


 


208.9


10.03


6


 



229.1


211.4


204.2


205.8


208.3


214.1


 


212.2


9.06


6



233.8


221.2


216.3


215.1


210.1


222.3


 


219.8


8.16


6



2/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



204.8


204.3


212.1


206.1


206.2


204.0


 


206.3


3.01


6



207.2


205.8


216.2


209.2


209.4


210.2


 


209.7


3.59


6



212.7


210.3


224.3


212.4


216.0


213.4


 


214.9


4.98


6



3/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N 03



 



204.8


196.2


196.1


201.1


212.8


216.1


 


204.5


8.42


6



212.4


202.7


203.0


206.4


213.6


223.7


 


210.5


8.08


6



220.4


208.1


208.9


210.2


218.1


233.7


 


216.6


9.81


6



4/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



200.8


199.8


193.4


207.4


192.0


207.8


 


200.2


6.69


6



200.6


197.0


197.7


208.8


194.1


208.3


 


201.1


6.14


6



204.7


208.4


204.2


223.4


199.2


209.6


 


208.3


8.27


6



5/M



R0001


R0002


R0003


 


Mean


SD


N



 



209.2


206.1


208.4


 


207.9


1.61


3



212.1


206.8


210.6


 


209.8


2.73


3



221.1


213.7


222.6


 


219.1


4.76


3



 



Summary of Body Weight Percentage Change
































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



Group                      1           2          3           4        



Dose level (mg/kg/day)     0          500       1000         2000     



Data presented in "%"  Interval X through X



Group/Sex



Animal Number



Phase


Day        DSNG 1 - 2 



 



1/M



Mean


SD


N



 



3.6


1.86


6



 



 



2/M



Mean


SD


N



 



2.5


0.89


6



 



 



3/M



Mean


SD


N



 



2.9


0.94


6



 



 



4/M



Mean


SD


N



 



3.6


2.39


6



 



 



5/M



Mean


SD


N



 



4.4


1.19


3



 



 



Individual and Group Mean Clinical Chemistry






































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



 



Group                      1           2          3           4        



 



Dose level (mg/kg/day)     0          500       1000         2000     



 



                                      ASTP IU/L


                         phase        Dosing



ALTP IU/L                           Dosing



HALP IU/L                           Dosing



CHOL mmol/L                           Dosing



T.BI umol/L                           Dosing



TP g/L                           Dosing



 



 



Group/Sex



Animal Number



Day



2



2



2



2



2



2



 



 



 



1/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



111


125


110


119


119


103


 


115


7.9


6



67


80


59


70


68


62


 


68


7.3


6



240


361


325


270


262


367


 


304


54.2


6



2.4


2.2


2.4


2.3


2.4


2.3


 


2.3


0.10


6


 



<1.7*


<1.7*


<1.7*


<1.7*


<1.7*


<1.7*


 


<1.7


0.00


6



52


52


51


53


55


52


 


53


1.4


6



 



 



 



2/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



119


115


114


101


107


103


 


110


7.2


2



74


89


74


65


68


59


 


72


10.3


6



403


390


342


344


363


287


 


355


41.2


6



2.7


2.4


2.5


2.3


2.8


2.5


 


2.5


0.19


6



<1.7*


<1.7*


<1.7*


<1.7*


<1.7*


<1.7*


 


<1.7


0.00


6



56


54


54


55


52


54


 


54


1.3


6



 



 



 



*= Value shown used in descriptive statistics






































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



 



Group                      1           2          3           4        



 



Dose level (mg/kg/day)     0          500       1000         2000     



 



                                      ASTP IU/L


                         phase        Dosing



ALTP IU/L                           Dosing



HALP IU/L                           Dosing



CHOL mmol/L                           Dosing



T.BI umol/L                           Dosing



TP g/L                           Dosing



 



 



Group/Sex



Animal Number



Day



2



2



2



2



2



2



 



 



 



3/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



109


131


121


107


104


103


 


113


11.1


6



69


86


58


74


82


79


 


75


10.1


6



279


269


291


284


309


351


 


297


29.6


6



2.7


2.5


2.5


2.7


2.5


2.5


 


2.6


0.10


6


 



<1.7*


<1.7*


<1.7*


<1.7*


<1.7*


<1.7*


 


<1.7


0.00


6



53


58


55


55


57


53


 


55


2.0


6



 



 



 



4/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



88


112


108


89


89


96


 


97


10.5


2



79


88


106


81


59


57


 


78


18.4


6



240


293


317


299


250


207


 


268


42.0


6



2.9


2.7


2.5


2.7


2.9


2.6


 


2.7


0.16


6



<1.7*


<1.7*


<1.7*


<1.7*


<1.7*


<1.7*


 


<1.7


0.00


6



55


59


54


54


56


55


 


56


1.9


6



 



 



 



*= Value shown used in descriptive statistic


 






































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



 



Group                      1           2          3           4       



 



Dose level (mg/kg/day)     0          500       1000         2000    



 



                                      ALB g/L


                         phase        Dosing



GLOB g/L                           Dosing



A/G Ratio                           Dosing



NA mmol/L                           Dosing



K mmol/L                           Dosing



CL mmol/L                           Dosing



 



 



Group/Sex



Animal Number



Day



2



2



2



2



2



2



 



 



 



1/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 


 



40


41


37


40


41


40


 


40


1.5


6



12


11


14


13


14


12


 


13


1.2


6



3.3


3.7


2.6


3.1


2.9


3.3


 


3.2


0.38


6



140


141


136


141


140


140


 


140


1.9


6



5.3


5.0


5.0


4.9


4.8


5.0


 


5.0


0.17


6



100


100


99


101


100


99


 


100


0.8


6



 



 



 



2/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



41


40


41


43


40


41


 


41


1.1


6



15


14


13


12


12


13


 


13


1.2


6



2.7


2.9


3.2


3.6


3.3


3.2


 


3.2


0.31


6



139


141


138


141


140


141


 


140


1.3


6



5.3


4.8


5.0


4.9


5.2


4.9


 


5.0


0.19


6



99


100


99


101


100


101


 


100


0.9


6



 



 



 







































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



 



Group                      1           2          3           4       



 



Dose level (mg/kg/day)     0          500       1000         2000    



 



                                      ALB g/L


                         phase        Dosing



GLOB g/L                           Dosing



A/G Ratio                           Dosing



NA mmol/L                           Dosing



K mmol/L                           Dosing



CL mmol/L                           Dosing



 



 



Group/Sex



Animal Number



Day



2



2



2



2



2



2



 



 



 



3/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 


 



40


44


40


42


43


40


 


42


1.8


6



13


14


15


13


14


13


 


14


0.8


6



3.1


3.1


2.7


3.2


3.1


3.1


 


3.1


0.18


6



140


140


139


139


140


140


 


140


0.5


6



5.0


5.1


4.8


5.4


5.1


5.1


 


5.1


0.19


6



99


99


100


99


101


100


 


100


0.8


6



 



 



 



4/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



42


45


42


40


42


42


 


42


1.6


6



13


14


12


14


14


13


 


13


0.8


6



3.2


3.2


3.5


2.9


3.0


3.2


 


3.2


0.21


6



141


142


141


141


139


140


 


141


1.0


6



5.2


5.1


4.8


4.8


5.3


4.9


 


5.0


0.21


6



102


101


101


101


100


100


 


101


0.8


6



 



 



 







































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



 



Group                      1           2          3           4       



 



Dose level (mg/kg/day)     0          500       1000         2000    



 



                                     CAL mmol/L


                         phase        Dosing



P mmol/L                           Dosing



HCRE umol/L                      Dosing



UREA mmol/L                           Dosing



GLUC mmol/L                           Dosing



 



 



 



Group/Sex



Animal Number



Day



2



2



2



2



2



 



 



 



 



1/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 


 



2.59


2.64


2.57


2.65


2.61


2.62


 


2.61


0.030


6



2.8


3.0


2.7


2.7


2.5


2.6


 


2.7


0.17


6



12


16


17


15


14


14


 


15


1.8


6



3.4


3.6


4.0


3.7


3.3


4.1


 


3.7


0.32


6



11.5


9.2


11.0


11.9


11.7


13.5


 


11.5


1.40


6



 



 



 



 



2/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



2.67


2.64


2.67


2.57


2.67


2.61


 


2.64


0.041


6



3.0


2.8


3.1


2.8


3.0


3.1


 


3.0


0.14


6



21


19


18


18


18


20


 


19


1.3


6



4.1


4.6


3.9


3.0


3.6


3.3


 


3.8


0.58


6



8.6


9.2


10.7


9.3


10.3


10.1


 


9.7


0.79


6



 



 



 



 







































































Test Article        vehicle (octahydro-4,7-methano-1H-indenyl) methyl acrylate  EMS



 



Group                      1           2          3           4       



 



Dose level (mg/kg/day)     0          500       1000         2000    



 



                                     CAL mmol/L


                         phase        Dosing



P mmol/L                           Dosing



HCRE umol/L                      Dosing



UREA mmol/L                           Dosing



GLUC mmol/L                           Dosing



 



 



 



Group/Sex



Animal Number



Day



2



2



2



2



2



 



 



 



 



3/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 


 



2.60


2.71


2.60


2.63


2.50


2.64


 


2.61


0.069


6



2.8


2.9


2.7


2.9


2.8


3.3


 


2.9


0.21


6



19


19


18


18


19


18


 


19


0.5


6



4.0


4.4


4.8


4.2


3.9


3.5


 


4.1


0.45


6



10.6


11.2


12.2


11.5


11.6


12.0


 


11.5


0.57


6



 



 



 



 



4/M



R0001


R0002


R0003


R0004


R0005


R0006


 


Mean


SD


N



 



2.60


2.64


2.46


2.68


2.63


2.53


 


2.59


0.081


6



2.8


3.0


3.2


2.7


3.0


2.8


 


2.9


0.18


6



19


22


15


17


18


17


 


18


2.4


6



4.4


6.3


2.7


4.5


3.2


4.0


 


4.2


1.25


6



10.4


9.2


10.9


10.5


11.7


10.8


 


10.6


0.82


6



 



 



 



 



 





















































Clinical Chemistry



Code



Parameter



ASTP



Aspartate aminotransferase



NA/K/CL



Sodium /potassium /chloride



ALB



Albumin



GLUC



Glucose




ALTP



Alanine aminotransferase



CAL



Calcium



GLOB



Globulin



UREA



Urea



HALP



Alkaline phosphatase



P



Inorganic phosphorous



A/G RATIO



Albumin/globulin ratio



HCRE



Creatinine



T.BI



Bilirubin



TP



Total protein



CHOL



Total cholesterol



 



 



Liver: Animal Comet Data




























































































































































































































































































































Group/ Dose Level



Animal



Slide



Total



Median Tail



Hedgehogs



(mg/kg/day)



Number



Number



Comets



Intensity



(%)



 



 



 



 



 



 



1/ Vehicle (0)



R0001



L-19A



50



0.64



0.00



 



R0001



L-19B



50



0.60



1.79



 



R0001



L-19C



50



1.29



1.85



 



R0002



L-74A



50



0.36



1.67



 



R0002



L-74B



50



0.20



0.00



 



R0002



L-74C



50



2.55



0.00



 



R0003



L-102A



50



0.15



0.00



 



R0003



L-102B



50



0.14



0.00



 



R0003



L-102C



50



0.14



0.00



 



R0004



L-16A



50



0.37



0.00



 



R0004



L-16B



50



1.41



0.00



 



R0004



L-16C



50



0.43



0.00



 



R0005



L-29A



50



0.89



0.00



 



R0005



L-29B



50



1.72



0.00



 



R0005



L-29C



50



1.53



0.00



 



R0006



L-27A



50



2.06



1.64



 



R0006



L-27B



50



1.04



0.00



 



R0006



L-27C



50



2.06



0.00



2/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (500)



R0101



L-59A



50



0.28



0.00



R0101



L-59B



50



0.21



1.72



R0101



L-59C



50



0.80



0.00



 



R0102



L-10A



50



0.56



0.00



 



R0102



L-10B



50



0.19



1.89



 



R0102



L-10C



50



0.18



1.89



 



R0103



L-8A



95



0.94



1.92



 



R0103



L-8B



 NE-A, NE-PS



 NE-A, NE-PS



 NE-A, NE-PS



 



R0103



L-8C



55



0.76



3.28



 



R0104



L-82A



50



2.46



0.00



 



R0104



L-82B



50



1.29



0.00



 



R0104



L-82C



50



1.81



0.00



 



R0105



L-62A



50



3.29



0.00



 



R0105



L-62B



50



4.02



0.00



 



R0105



L-62C



50



3.59



0.00



 



R0106



L-65A



50



2.16



0.00



 



R0106



L-65B



50



2.28



1.75



 



R0106



L-65C



50



2.42



1.47



































































































































































































































































































































































































Group/ Dose Level



Animal



Slide



Total



Median Tail



Hedgehogs



(mg/kg/day)



Number



Number



Comets



Intensity



(%)



3/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (1000)



R0201



L-93A



50



1.48



1.79



R0201



L-93B



50



1.04



1.89



R0201



L-93C



50



1.49



1.64



 



R0202



L-56A



50



0.66



0.00



 



R0202



L-56B



50



0.29



1.64



 



R0202



L-56C



50



0.22



0.00



 



R0203



L-17A



50



2.03



3.39



 



R0203



L-17B



50



0.73



0.00



 



R0203



L-17C



50



3.27



0.00



 



R0204



L-43A



50



2.23



0.00



 



R0204



L-43B



50



1.55



0.00



 



R0204



L-43C



50



1.75



0.00



 



R0205



L-68A



50



0.11



0.00



 



R0205



L-68B



50



2.40



1.89



 



R0205



L-68C



50



2.85



0.00



 



R0206



L-57A



50



0.36



0.00



 



R0206



L-57B



50



0.58



0.00



 



R0206



L-57C



50



0.85



0.00



4/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (2000)



R0301



L-50A



50



0.56



0.00



R0301



L-50B



50



1.69



0.00



R0301



L-50C



50



1.65



1.69



 



R0302



L-86A



50



1.42



1.72



 



R0302



L-86B



50



0.78



1.69



 



R0302



L-86C



50



0.79



0.00



 



R0303



L-72A



50



0.50



0.00



 



R0303



L-72B



50



0.11



0.00



 



R0303



L-72C



50



0.47



1.64



 



R0304



L-41A



50



2.54



0.00



 



R0304



L-41B



50



1.96



0.00



 



R0304



L-41C



50



3.43



0.00



 



R0305



L-9A



50



1.39



0.00



 



R0305



L-9B



50



0.82



1.96



 



R0305



L-9C



50



1.41



0.00



 



R0306



L-53A



50



1.09



1.72



 



R0306



L-53B



50



0.80



1.79



 



R0306



L-53C



50



2.74



0.00



5/ EMS (200)



R0401



L-97A



50



23.54



1.75



 



R0401



L-97B



50



23.27



0.00



 



R0401



L-97C



50



23.44



0.00



 



R0402



L-90A



50



22.28



0.00



 



R0402



L-90B



50



20.26



1.59



 



R0402



L-90C



50



26.22



3.23



 



R0403



L-32A



50



17.29



0.00



 



R0403



L-32B



50



14.57



0.00



 



R0403



L-32C



50



24.53



1.56



 


 

 



 



 



 



















NE-A



Not evaluated due to agarose detachment from slide



NE-PS



Not evaluated due to partial gel detachment



EMS



Ethyl Methanesulfonate



Stomach: Animal Comet Data























































































































































































































































Group/ Dose Level



Animal



Total



Tail Intensity (%)



Hedgehogs



(mg/kg/day)



Number



Comets



Mean



SD



(%)



 



 



 



 



 



 



1/ Vehicle (0)



R0001



150



0.68



0.44



8.33



 



R0002



150



2.09



0.63



9.73



 



R0003



150



1.43



0.73



10.83



 



R0004



150



1.28



0.56



7.87



 



R0005



150



0.35



0.20



6.83



 



R0006



150



0.69



0.37



10.54



2/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (500)



R0101



150



0.44



0.12



8.44



R0102



150



1.01



0.50



11.55



R0103



150



0.68



0.50



7.61



 



R0104



150



0.19



0.19



11.31



 



R0105



150



0.66



0.28



18.15



 



R0106



150



1.51



0.80



15.53



3/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (1000)



R0201



150



0.55



0.47



3.69



R0202



150



0.60



0.35



5.34



R0203



150



0.99



0.31



9.51



 



R0204



150



0.41



0.27



10.46



 



R0205



150



0.27



0.12



10.49



 



R0206



 NE-NC



 NE-NC



 NE-NC



 NE-NC



4/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (2000)



R0301



150



0.71



0.14



10.73



R0302



150



1.41



0.66



10.10



R0303



150



1.07



0.73



10.07



 



R0304



150



0.58



0.24



7.61



 



R0305



150



0.34



0.12



10.20



 



R0306



150



0.43



0.19



6.90



5/ EMS (200)



R0401



150



7.92



0.40



8.98



 



R0402



150



13.87



2.96



13.40



 



R0403



150



7.58



0.96



12.19



 



 



 



 



 



 



















SD



Standard Deviation



EMS



Ethyl Methanesulfonate



NE-NC



Not evaluated due to no cells



 


Upon slide analysis, it was noted that there were no cells on any slide prepared for animal R0206. As there were at least 5 animals analysed for the intermediate dose group, the requirements of OECD test guideline have been met (OECD, 2016). In addition, the data are clearly negative, therefore there is no impact on data interpretation or study conclusion


Stomach: Individual Slide Data




























































































































































































































































































































Group/ Dose Level



Animal



Slide



Total



Median Tail



Hedgehogs



(mg/kg/day)



Number



Number



Comets



Intensity



(%)



 



 



 



 



 



 



1/ Vehicle (0)



R0001



S-76A



50



0.51



9.90



 



R0001



S-76B



50



0.35



5.81



 



R0001



S-76C



50



1.18



8.99



 



R0002



S-96A



50



1.75



9.65



 



R0002



S-96B



50



1.70



8.33



 



R0002



S-96C



50



2.81



11.21



 



R0003



S-28A



50



2.21



7.22



 



R0003



S-28B



50



0.77



10.28



 



R0003



S-28C



50



1.31



14.55



 



R0004



S-25A



50



0.97



6.52



 



R0004



S-25B



50



0.95



9.09



 



R0004



S-25C



50



1.93



8.05



 



R0005



S-12A



50



0.55



9.00



 



R0005



S-12B



50



0.34



5.32



 



R0005



S-12C



50



0.15



6.06



 



R0006



S-66A



50



1.09



8.33



 



R0006



S-66B



50



0.35



12.50



 



R0006



S-66C



50



0.64



10.00



2/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (500)



R0101



S-107A



50



0.41



9.18



R0101



S-107B



50



0.34



8.65



R0101



S-107C



50



0.57



7.55



 



R0102



S-45A



50



0.56



10.53



 



R0102



S-45B



50



0.92



12.84



 



R0102



S-45C



50



1.55



11.11



 



R0103



S-46A



50



0.55



9.00



 



R0103



S-46B



50



0.25



5.56



 



R0103



S-46C



50



1.23



8.14



 



R0104



S-58A



50



0.41



10.29



 



R0104



S-58B



50



0.04



12.66



 



R0104



S-58C



50



0.13



10.81



 



R0105



S-63A



50



0.59



19.51



 



R0105



S-63B



50



0.42



17.27



 



R0105



S-63C



50



0.97



17.48



 



R0106



S-38A



50



2.28



16.00



 



R0106



S-38B



50



0.69



11.65



 



R0106



S-38C



50



1.56



18.87



































































































































































































































































































































































































Group/ Dose Level



Animal



Slide



Total



Median Tail



Hedgehogs



(mg/kg/day)



Number



Number



Comets



Intensity



(%)



3/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (1000)



R0201



S-20A



50



1.09



3.23



R0201



S-20B



50



0.23



2.82



R0201



S-20C



50



0.32



4.76



 



R0202



S-21A



50



0.82



6.25



 



R0202



S-21B



50



0.20



4.49



 



R0202



S-21C



50



0.78



5.21



 



R0203



S-60A



50



0.66



7.87



 



R0203



S-60B



50



1.26



10.84



 



R0203



S-60C



50



1.06



9.89



 



R0204



S-3A



50



0.45



11.11



 



R0204



S-3B



50



0.13



12.20



 



R0204



S-3C



50



0.65



7.89



 



R0205



S-83A



50



0.39



11.34



 



R0205



S-83B



50



0.27



8.89



 



R0205



S-83C



50



0.14



11.11



 



R0206



S-36A



 NE-NC



 NE-NC



 NE-NC



 



R0206



S-36B



 NE-NC



 NE-NC



 NE-NC



 



R0206



S-36C



 NE-NC



 NE-NC



 NE-NC



4/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (2000)



R0301



S-85A



50



0.80



10.98



R0301



S-85B



50



0.77



11.24



R0301



S-85C



50



0.54



10.00



 



R0302



S-39A



50



1.64



11.54



 



R0302



S-39B



50



0.66



6.10



 



R0302



S-39C



50



1.91



11.88



 



R0303



S-14A



50



1.44



11.11



 



R0303



S-14B



50



1.54



10.59



 



R0303



S-14C



50



0.23



8.60



 



R0304



S-52A



50



0.30



8.14



 



R0304



S-52B



50



0.76



7.61



 



R0304



S-52C



50



0.69



7.14



 



R0305



S-92A



50



0.25



10.87



 



R0305



S-92B



50



0.48



3.17



 



R0305



S-92C



50



0.30



14.00



 



R0306



S-99A



50



0.23



6.06



 



R0306



S-99B



50



0.60



6.36



 



R0306



S-99C



50



0.47



8.18



5/ EMS (200)



R0401



S-103A



50



7.84



9.70



 



R0401



S-103B



50



8.36



8.49



 



R0401



S-103C



50



7.57



8.51



 



R0402



S-101A



50



16.53



15.49



 



R0402



S-101B



50



10.69



11.59



 



R0402



S-101C



50



14.41



13.04



 



R0403



S-26A



50



8.64



12.50



 



R0403



S-26B



50



7.35



8.57



 



R0403



S-26C



50



6.76



15.53



 



 



 



 



 



 



 














NE-NC



Not evaluated due to no cells



EMS



Ethyl Methanesulfonate



Duodenum: Animal Comet Data























































































































































































































































Group/ Dose Level



Animal



Total



Tail Intensity (%)



Hedgehogs



(mg/kg/day)



Number



Comets



Mean



SD



(%)



 



 



 



 



 



 



1/ Vehicle (0)



R0001



150



0.21



0.06



2.94



 



R0002



150



0.83



0.17



8.54



 



R0003



150



0.19



0.19



8.94



 



R0004



150



0.45



0.34



6.45



 



R0005



150



0.31



0.30



5.38



 



R0006



150



0.70



0.47



30.23



2/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (500)



R0101



150



0.36



0.42



3.55



R0102



150



0.39



0.32



11.96



R0103



150



0.87



0.53



17.11



 



R0104



150



1.42



0.63



2.67



 



R0105



150



0.46



0.11



5.29



 



R0106



150



1.58



0.79



11.35



3/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (1000)



R0201



150



1.15



0.59



15.61



R0202



150



0.30



0.06



9.39



R0203



150



1.15



0.61



7.02



 



R0204



150



0.32



0.15



9.44



 



R0205



150



1.18



0.69



5.53



 



R0206



150



0.23



0.18



14.29



4/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (2000)



R0301



150



0.57



0.26



13.66



R0302



150



2.01



0.54



7.43



R0303



150



0.27



0.11



6.84



 



R0304



150



0.35



0.41



18.69



 



R0305



150



0.12



0.06



7.34



 



R0306



150



0.52



0.03



5.03



5/ EMS (200)



R0401



150



4.61



1.31



13.33



 



R0402



150



10.37



1.85



4.17



 



R0403



150



5.80



3.30



5.14



 



 



 



 



 



 















SD



Standard Deviation



EMS



Ethyl Methanesulfonate



Duodenum: Individual Slide Data




























































































































































































































































































































Group/ Dose Level



Animal



Slide



Total



Median Tail



Hedgehogs



(mg/kg/day)



Number



Number



Comets



Intensity



(%)



 



 



 



 



 



 



1/ Vehicle (0)



R0001



D-87A



50



0.16



3.06



 



R0001



D-87B



50



0.28



3.33



 



R0001



D-87C



50



0.20



2.38



 



R0002



D-54A



50



0.97



6.45



 



R0002



D-54B



50



0.64



10.61



 



R0002



D-54C



50



0.88



8.45



 



R0003



D-61A



 NE-NC



 NE-NC



 NE-NC



 



R0003



D-61B



75



0.05



10.87



 



R0003



D-61C



75



0.32



6.90



 



R0004



D-18A



50



0.24



6.45



 



R0004



D-18B



50



0.26



5.17



 



R0004



D-18C



50



0.83



7.58



 



R0005



D-7A



50



0.64



3.17



 



R0005



D-7B



50



0.27



7.81



 



R0005



D-7C



50



0.03



5.08



 



R0006



D-81A



50



0.89



23.08



 



R0006



D-81B



50



0.16



32.43



 



R0006



D-81C



50



1.05



34.21



2/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (500)



R0101



D-64A



50



0.07



5.36



R0101



D-64B



50



0.17



1.79



R0101



D-64C



50



0.84



3.51



 



R0102



D-77A



50



0.70



13.11



 



R0102



D-77B



50



0.06



6.56



 



R0102



D-77C



50



0.42



16.13



 



R0103



D-24A



50



1.33



20.63



 



R0103



D-24B



50



0.29



14.52



 



R0103



D-24C



50



0.99



16.13



 



R0104



D-98A



50



1.01



4.84



 



R0104



D-98B



50



1.11



0.00



 



R0104



D-98C



50



2.15



3.13



 



R0105



D-89A



50



0.33



5.45



 



R0105



D-89B



50



0.54



5.00



 



R0105



D-89C



50



0.50



5.45



 



R0106



D-2A



50



2.07



12.70



 



R0106



D-2B



50



0.66



8.47



 



R0106



D-2C



50



2.00



12.70



 


































































































































































































































































































































































































Group/ Dose Level



Animal



Slide



Total



Median Tail



Hedgehogs



(mg/kg/day)



Number



Number



Comets



Intensity



(%)



3/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (1000)



R0201



D-44A



50



1.81



15.07



R0201



D-44B



50



0.98



14.71



R0201



D-44C



50



0.67



17.19



 



R0202



D-71A



50



0.34



8.62



 



R0202



D-71B



50



0.33



6.67



 



R0202



D-71C



50



0.24



12.70



 



R0203



D-48A



50



0.51



7.41



 



R0203



D-48B



50



1.22



3.51



 



R0203



D-48C



50



1.73



10.00



 



R0204



D-91A



50



0.44



10.34



 



R0204



D-91B



50



0.37



6.67



 



R0204



D-91C



50



0.15



11.29



 



R0205



D-1A



50



0.51



10.81



 



R0205



D-1B



50



1.89



4.76



 



R0205



D-1C



50



1.13



0.00



 



R0206



D-55A



50



0.43



18.03



 



R0206



D-55B



50



0.16



10.77



 



R0206



D-55C



50



0.10



14.29



4/ (octahydro-4,7-methano-1H-indenyl) methyl acrylate (2000)



R0301



D-47A



50



0.31



16.13



R0301



D-47B



50



0.83



11.86



R0301



D-47C



50



0.57



12.90



 



R0302



D-30A



50



2.63



4.41



 



R0302



D-30B



50



1.70



8.62



 



R0302



D-30C



50



1.71



9.21



 



R0303



D-69A



50



0.23



4.69



 



R0303



D-69B



50



0.19



7.94



 



R0303



D-69C



50



0.40



7.94



 



R0304



D-40A



50



0.82



20.63



 



R0304



D-40B



50



0.16



19.40



 



R0304



D-40C



50



0.08



16.18



 



R0305



D-5A



50



0.09



3.77



 



R0305



D-5B



50



0.19



7.94



 



R0305



D-5C



50



0.08



9.84



 



R0306



D-49A



50



0.49



5.00



 



R0306



D-49B



50



0.55



5.00



 



R0306



D-49C



50



0.51



5.08



5/ EMS (200)



R0401



D-13A



50



5.86



14.29



 



R0401



D-13B



50



4.71



14.08



 



R0401



D-13C



50



3.24



11.48



 



R0402



D-84A



50



9.14



3.70



 



R0402



D-84B



50



12.50



5.36



 



R0402



D-84C



50



9.48



3.45



 



R0403



D-11A



50



4.57



3.70



 



R0403



D-11B



50



3.29



5.08



 



R0403



D-11C



50



9.54



6.45



 


 



 



 



 



 



 



 














NE-NC



Not evaluated due to no cells



EMS



Ethyl Methanesulfonate



 


 


Historical Control Ranges: Comet Assay Data


Data generated from studies performed within the GLP laboratory, by GLP trained staff, whether a claim of GLP compliance was made or not, were included in the compilation of the historical control ranges without bias.
























































































































RAT LIVER COMET HISTORICAL CONTROL RANGES



Vehicle Control Data


   
  

Tail Intensity (%)



Hedgehogs (%)



Number of Animals


 

161



161



Mean


 

0.41



1.52



Standard Deviation


 

0.45



1.35



Observed Range



Minimum



0.01



0.00


 

Maximum



2.37



7.89



95% Reference Range



Lower Limit



0.04



0.00


 

Upper Limit



1.80



5.36



Positive Control Data


   
  

Tail Intensity (%)



Hedgehogs (%)



Number of Animals


 

82



82



Mean


 

26.43



1.57



Standard Deviation


 

7.92



1.40



Observed Range



Minimum



9.52



0.00


 

Maximum



43.42



5.98



95% Reference Range



Lower Limit



10.53



0.00


 

Upper Limit



37.00



5.41



Range compiled Aug 2020; generated from 25 experiments (vehicle data) or 26 experiments (positive data) analysed between September 2018 and August 2020



 
























































































































RAT STOMACH COMET HISTORICAL CONTROL RANGES



Vehicle Control Data


   
  

Tail Intensity (%)



Hedgehogs (%)



Number of Animals


 

45



45



Mean


 

1.40



4.71



Standard Deviation


 

2.18



3.11



Observed Range



Minimum



0.15



0.60


 

Maximum



11.46



14.05



95% Reference Range



Lower Limit



0.16



1.04


 

Upper Limit



8.29



12.63



Positive Control Data


   
  

Tail Intensity (%)



Hedgehogs (%)



Number of Animals


 

21



21



Mean


 

12.68



5.50



Standard Deviation


 

4.36



2.84



Observed Range



Minimum



4.18



1.23


 

Maximum



20.54



10.93



95% Reference Range



Lower Limit



N/A



N/A


 

Upper Limit



N/A



N/A



Range compiled Aug 2020; generated from 7 experiments (vehicle data) or 6 experiments (positive data) analysed between September 2018 and August 2020



 


 
























































































































RAT DUODENUM COMET HISTORICAL CONTROL RANGES



Vehicle Control Data


   
  

Tail Intensity (%)



Hedgehogs (%)



Number of Animals


 

50



50



Mean


 

0.95



5.95



Standard Deviation


 

0.86



3.94



Observed Range



Minimum



0.14



0.49


 

Maximum



4.50



20.71



95% Reference Range



Lower Limit



0.20



0.54


 

Upper Limit



3.14



13.22



Positive Control Data


   
  

Tail Intensity (%)



Hedgehogs (%)



Number of Animals


 

27



27



Mean


 

11.29



6.32



Standard Deviation


 

3.09



3.62



Observed Range



Minimum



3.19



2.01


 

Maximum



17.49



16.67



95% Reference Range



Lower Limit



N/A



N/A


 

Upper Limit



N/A



N/A



Range compiled Aug 2020; generated from 9 experiments analysed between December 2018 and August 2020



 


 

Conclusions:
It is concluded that, under the conditions of this Comet assay, (octahydro-4,7-methano-1H-indenyl) methyl acrylate did not induce DNA strand breaks in the liver, stomach or duodenum of male Sprague Dawley rats administered up to 2000 mg/kg/day (the maximum recommended dose for in vivo Comet studies).
Executive summary:

The in vivo alkaline comet (single cell gel electrophoresis) assay is used for the detection of DNA strand breaks (apparent as an increase in DNA migration) in cells or nuclei isolated from tissues of animals that have been exposed to potentially genotoxic material(s). Under alkaline conditions (>pH 13), the comet assay can detect single and double stranded breaks, resulting, for example, from direct interactions with DNA, alkali labile sites or as a consequence of transient DNA strand breaks resulting from DNA excision repair. These strand breaks may be repaired, resulting in no persistent effect, may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change. They may also lead to chromosomal damage which is also associated with many human diseases including cancer.


(octahydro-4,7-methano-1H-indenyl) methyl acrylate was tested for its potential to induce DNA strand breaks in the liver, stomach and duodenum of treated rats. As there was no strand break induction observed in any of the somatic tissues, the gonad was not assessed.


There were no dose-related increases in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with (octahydro-4,7-methano-1H-indenyl) methyl acrylate did not cause excessive DNA damage that could have interfered with comet analysis.


Animals treated with (octahydro-4,7-methano-1H-indenyl) methyl acrylate at all doses exhibited group mean tail intensities that were similar to the concurrent vehicle control group and that fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response.


These data were considered clearly negative in the liver, stomach and duodenum.


 


It is concluded that, under the conditions of this comet assay, (octahydro-4,7-methano-1H-indenyl) methyl acrylate did not induce DNA strand breaks in the liver, stomach or duodenum of male Sprague Dawley rats administered up to 2000 mg/kg/day (the current maximum recommended dose for in vivo Comet studies).


 

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

Additional information

In vitro gene mutation study in bacteria (CitoxLab, 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 (Covance, 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.


 


Comet assay (Labcorp, 2022)
(octahydro-4,7-methano-1H-indenyl) methyl acrylate was tested for its potential to induce DNA strand breaks in the liver, stomach and duodenum of treated rats. As there was no strand break induction observed in any of the somatic tissues, the gonad was not assessed.
There were no dose-related increases in %hedgehogs in liver, stomach or duodenum, thus demonstrating that treatment with (octahydro-4,7-methano-1H-indenyl) methyl acrylate did not cause excessive DNA damage that could have interfered with comet analysis.
Animals treated with (octahydro-4,7-methano-1H-indenyl) methyl acrylate at all doses exhibited group mean tail intensities that were similar to the concurrent vehicle control group and that fell within the 95% reference range of the laboratory's historical vehicle control data. There were no statistically significant increases in tail intensity for any of the groups receiving the test article, compared to the concurrent vehicle control, with no evidence of a dose response.
These data were considered clearly negative in the liver, stomach and duodenum.
It is concluded that, under the conditions of this comet assay, (octahydro-4,7-methano-1H-indenyl) methyl acrylate did not induce DNA strand breaks in the liver, stomach or duodenum of male Sprague Dawley rats administered up to 2000 mg/kg/day (the current maximum recommended dose for in vivo Comet studies).


 

Justification for classification or non-classification

Based on the available in vitro and in vivo experimental data, the registered substance does not meet the criteria for classification according to Regulation (EC) No 1272/2008.