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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

300-723-4

EC Name:

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

Cas Number:

127823-21-6

Molecular formula:

C14H20O2

IUPAC Name:

{tricyclo[5.2.1.0²,⁶]decan-8-yl}methyl prop-2-enoate

Test material form:

liquid

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control - vehicle only

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Doses / concentrations 5

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

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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


3. 
   
   
   
   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

 

 

Applicant's summary and conclusion

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).