Bicyclo[3.1.1]hept-2-ene-2-ethanol, 6,6-dimethyl-, 2-acetate, (1R,5S)-

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

comet assay in rats

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 April 2021 to (report date to be confirmed)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 489 with protocol deviations: 1-Formulation Analysis : Achieved concentration values were 78.8, 81.8 and 82.9% nominal at 25, 50 and 100 mg/mL and therefore fell below protocol specification criteria of 85-115% nominal. Homogeneity of the low concentration (25 mg/mL) also fell outside protocol specification (%CV ≤5%) at 7.89%. 2-Scoring of Comets : The codes G9, G10, G11, G12, G13 and G15 were scored without image capture. These deviations had no impact on the study.

Data source

Reference

Reference Type:

other: Draft 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:

yes

Remarks:

In formulation analysis and in scoring of Comets. However, these study deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

(1R,5S)-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl acetate

EC Number:

800-940-9

Cas Number:

35836-72-7

Molecular formula:

C13H20O2

IUPAC Name:

(1R,5S)-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl acetate

impurity 1

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No.: 1000099828
Purity: 99.5%
Name of test material (as cited in study report): NOPYL ACETATE
Physical state: colourless - slightly amber liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 19 October 2022

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DRT / 1000099828
- Purity: 99.5%
- Appearance: Colourless – slightly amber liquid
- Date received as two consignments on 09 December 2020 (used for the Range-Finder Experiment and Experiment 1) and 21 December 2020 (used for the Experiment 2)
- Expiration date of the lot/batch: 19 October 2022

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 2-8 °C, protected from light and under nitrogen with desiccant.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited.
- Age at study initiation: for males and females, 7 to 9 weeks old (during the Range-Finder Experiment) and 7 to 8 weeks old (during the Main Experiment 1 and Main Experiment 2).
- Housing: in wire topped, solid bottomed cages, with three animals of the same sex per cage.
- Diet (e.g. ad libitum): 5LF2 EU Rodent Diet, ad libitum
- Water (e.g. ad libitum): Mains water, ad libitum
- Acclimation period: at least 5 days
-Animals were not fasted prior to administration

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

IN-LIFE DATES: From 21 April 2021 to 22 July 2021

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

1% Methylcellulose (MC)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared once per experimental occasion by formulating (-)-NOPYL ACETATE in 1% MC as follows:The test article was weighed into a formulation bottle. Approximately 50% of the required final volume of vehicle was added and stirred to homogenise. Formulations were made up to final volume and stirred to mix. The final pH was recorded and formulations were aliquoted.

Two sets of duplicate samples (2 x 1.0 mL) were taken from the top, middle and bottom strata of each test article formulation together with duplicate 1.0 mL samples (taken from the middle) of the vehicle control. Samples were stored at 2-8°C, protected from light prior to dispatch to the analytical Test Site (under cool conditions) for analysis. Both sets of samples were shipped to the analytical Test Site in separate packages.

In both Main Experiments, a single set of samples was analysed in the first instance and the second set of samples were retained as reserves. However, in Main Experiment 1, due to lower than expected analytical results, the reserve samples were also analysed. The low results were subsequently confirmed. In Main Experiment 2, all samples met protocol acceptance criteria for achieved concentration and homogeneity. Therefore, the reserve samples were not required for analysis.

Duration of treatment / exposure:

21 hours.

Frequency of treatment:

Two administrations at 0 (Day 1) and 21 hours (Day 2). Necropsy at 24 hours (Day 2).

Post exposure period:

Main Experiments 1 and 2:
Day 1 (excluding positive control group) : Prior to dose, immediate, 1, 2 and 4 hours post dose
/ Day 2 : Prior to dose, immediate and prior to necropsy

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

197 mg/kg bw/day (nominal)

Remarks:

Experiment 1 - Group 2

Doses / concentrations 2

Dose / conc.:

409 mg/kg bw/day (nominal)

Remarks:

Experiment 1 - Group 3

Doses / concentrations 3

Dose / conc.:

829 mg/kg bw/day (nominal)

Remarks:

Experiment 1 - Group 4

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Experiment 2 - Group 7

No. of animals per sex per dose:

6 (three for the positive control groups)

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethylmethanesulphonate (EMS)
- Dose Volume : 10 mL/kg
- Concentration of EMS Solution: 20 mg/mL
- Dose level of EMS : 200 mg/kg bw/d

Examinations

Tissues and cell types examined:

The liver, duodenum, stomach and gonad (left testis) were removed from each control (vehicle and positive) and test article treated animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a single dose toxicity study in rats (Project Number 500-72) a group of 10 male Wistar rats were dosed via oral gavage at 5000 mg/kg. The animals were fasted for 16-18 hours prior to dosing. Nine deaths occurred within 6-48 hours of dose administration and clinical signs noted included ataxia, loss of righting reflex, piloerection, lethargy, urinary incontinence and comatose.

In an acute oral dose toxicity study conducted in rats (Project Number 695-72) groups of 10 male Wistar rats were dosed at 2.56, 3.2, 4.0 or 5.0 mL/kg. The animals were fasted for a minimum of 16 hours prior to dosing. Observations for mortality were made at 1 and 6 hours for up to 14 days. The LD50 was considered to be 3.0 mL/kg. Clinical signs included ataxia, loss of righting reflex, piloerection, lethargy, urinary incontinence and comatose.

Therefore, an initial dose of 1000 mg/kg/day was administered in a Range-Finder Experiment. An additional dose of 1400 mg/kg/day was tested in males only until an estimate of the Maximum Tolerated Dose (MTD) was determined for each sex (OECD, 2016).

From the results of the Range-Finder Experiment, dose levels of 250, 500 and 1000 mg/kg/day (-)-NOPYL ACETATE (equivalent to 25% MTD, 50% MTD and the MTD respectively) were tested in Main Experiment 1. Following consistent low recovery from the formulation analysis data, actual dose levels administered were 197, 409 and 829 mg/kg/day. Therefore, it was decided that the Main Experiment would be repeated at the high dose of 1000 mg/kg/day only (designated Main Experiment 2) to ensure that the MTD was achieved.

The Maximum Tolerated Dose (MTD) was defined as the highest dose that was tolerated without evidence of study-limiting toxicity, relative to the duration of the study period (for example, clear clinical signs such as abnormal behaviour or reactions, body weight depression or target tissue cytotoxicity, but not death or evidence of pain, suffering or distress necessitating humane euthanasia).

Both male and female animals were used in the Range-Finder Experiment. However, as there were no substantial sex differences in toxicity (considered to be a difference in MTD of 2-fold or greater), and the Sponsor confirmed there were no data available to suggest differences in metabolism or bioavailability, or sex specific human exposure, Main Experiments 1 and 2 were conducted in male animals only.

DETAILS OF 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 a chiller plate with ice packs.

METHOD OF ANALYSIS:
Liver, duodenum and stomach slides were analysed in the first instance. Following receipt of the results, the gonad was also analysed (Groups 6 to 8 only).
Scoring was carried out using fluorescence microscopy at an appropriate magnification and with suitable filters.
A slide from a vehicle and positive control animal were checked for quality and/or response prior to analysis. All slides were allocated a random code and analysed by an individual not connected with scoring of the study.
All animals per group were analysed.
Measurements of tail intensity (%DNA in tail) were obtained from 150 cells/animal/tissue. In general, this was evenly split over two or three slides.
The number of ‘hedgehogs’ (a morphology indicative of highly damaged cells sometimes associated with severe cytotoxicity, necrosis or apoptosis) observed during Comet scoring was recorded for each slide. To avoid the risk of false positive results ‘hedgehogs’ were not used for comet analysis. Each slide was scanned starting to the left of the centre of the slide.
The following criteria were used for analysis of slides:
1. Only clearly defined non overlapping cells were scored
2. Hedgehogs were not scored
3. Cells with unusual staining artefacts were not scored.

Slide analysis was performed by an analyst trained in accordance with Labcorp Early Development Laboratories Ltd. Standard Operating Procedures. Images of each comet scored for each tissue were retained electronically and archived.
Slides prepared from the left gonad were prepared but were only to be scored and evaluated if required (if liver, stomach or duodenum elicited a positive result). These slides were subsequently requested for analysis (Groups 6 to 8 only). All comet slides were retained until report finalisation; at this time the slides were discarded with SD approval. Due to the nature of the slides, long term storage is not recommended as comet integrity cannot be assured.

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 none 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 evidence for cytotoxicity, comparison of the response against the historical control data, consistency of response within and between individual animals and between dose levels and any additional experiments.

Statistics:

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:

yes

Remarks:

In the liver, decreased hepatocyte glycogen, increased extramedullary haematopoiesis, increased hepatocyte mitotic figures, and diffuse hepatocyte vacuolation were recorded for animals from all groups administered test item, with a dose relationship.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

For both Main Experiments 1 and 2, there were no increases in mean %hedgehogs in the liver, stomach, duodenum or gonad that was considered evidence of excessive DNA damage due to treatment with (-)-NOPYL ACETATE that could have interfered with comet analysis. However, an apparent trend of increasing %hedgehogs was observed in Experiment 1, and an increase in %hedgehogs at 1000 mg/kg/day was observed in Experiment 2. The positive control substance did not reproducibly increase the frequency of %hedgehogs.

Any other information on results incl. tables

Clinical Chemistry

A small increase in albumin and a decrease in globulin were noted in animals administered 829/1000 mg/kg/day, which resulted in an increase in the albumin:globulin ratio.

Dose-related, decreased cholesterol was noted in animals administered 409 or 829 mg/kg/day.

A small, dose-related increase in sodium and a non-dose-related decrease in potassium were recorded for animals administered 409 or 829/1000 mg/kg/day.

Small increases in urea and creatinine were recorded for animals administered 829/1000 mg/kg/day and for occasional animals administered 409 mg/kg/day.

Increased glucose, considered secondary to stress and not a direct effect of the test article, was noted in animals in all groups, except group 7, administered (-)-NOPYL ACETATE, with no dose relationship.

Other differences in individual clinical pathology parameters were observed in animals administered (-)-NOPYL ACETATE; however, they were considered not (-)-NOPYL ACETATE related due to the negligible magnitude of the change, individual animal variability, and overlap of values for (-)-NOPYL ACETATE treated animals with the concurrent vehicle control values.

 

Macroscopic Observations

No (-)-NOPYL ACETATE-related macroscopic findings were recorded. 

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. 

 

Microscopic Observations

Upon microscopic examination, changes related to (-)-NOPYL ACETATE were recorded for the liver. 

In the liver, decreased hepatocyte glycogen, increased extramedullary haematopoiesis, increased hepatocyte mitotic figures, and diffuse hepatocyte vacuolation were recorded for animals from all groups administered (-)-NOPYL ACETATE, with a dose relationship. However, increased extramedullary haematopoiesis was not recorded for animals in Group 7 administered 1000 mg/kg/day. 

Glycogen, hepatocyte was characterized by generally perinuclear, clear, variably sized, indistinctly defined vacuoles. Mitosis, hepatocyte, increased was characterized by an increase (above the normally low background incidence) of mitotic figures within the liver parenchyma. 

Minimal inflammation was recorded for the forestomach of two Group 7 animals administered 1000 mg/kg/day. As this finding was not present in Group 4 animals administered 829 mg/kg/day, a relationship to the test article is uncertain, as this finding may have been due to gavage trauma.

Other tissues were considered microscopically unremarkable or the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, (-)-NOPYL ACETATE did not induce biologically relevant increases in DNA strand breaks in the liver, stomach, duodenum or gonad when tested up to 1000 mg/kg/day (the maximum tolerated dose level for this study). Any increases in DNA strand breaks were considered small, were variable in individual animals within the group and in the liver were generally concomitant with clinical chemistry and/or histopathological findings suggestive of hepatotoxicity and not genotoxicity.

Executive summary:

(‑)‑NOPYL ACETATE was tested for its potential to induce DNA strand breaks in the liver, duodenum and stomach of treated male rats. The gonad was also assessed, according to the OECD guideline 489 and under GLP conditions.

The test article and vehicle control (1% Methylcellulose or MC) were given as two administrations, at 0 and 21 hours; the positive control (Ethyl methanesulfonate or EMS) was administered once only at 21 hours. All animals were sampled on Day 2, equivalent to 24 hours after first administration. All doses were administered at a dose volume of 10 mL/kg. Individual dose volumes were based on individual body weight. In the first experiment, the achieved concentration values fell below the acceptable specification range. In this case,  a second experiment using only the high dose and control groups was initiated to ensure that the maximum tolerated dose level was achieved.

In the Range-Finder Experiment, groups of 3 female and/or 3 male rats were dosed by oral gavage with, (‑)‑NOPYL ACETATE at either 1000 (males and females) or 1400 mg/kg bw/day (males only). As no substantial difference in toxicity was observed between male and female animals in the Range-Finder Experiment, male animals only were used in the Main Experiments. Following consistent low recovery from the formulation analysis data, actual dose levels administered were 197, 409 and 829 mg/kg bw/day (Main Experiment 1, 6 male animals/dose) and 1000 mg/kg bw/d (Main Experiment 2, 6 male animals).

 

Main Experiment 1

In the liver, animals treated with (‑)‑NOPYL ACETATE at 197 and 409 mg/kg/day exhibited group mean and individual animal %TI values that were similar to the concurrent vehicle control group values and fell within the laboratory’s historical vehicle control data ranges. At 829 mg/kg/day, there were two animals with slightly elevated %TI values which contributed to statistical significance for the group (P≤0.01) and a statistically significant linear trend (P≤0.05). These animals were R0302 and R0303 (%TI values of 2.18 and 2.39, respectively. However, with the exception of these, all other (‑)‑NOPYL ACETATE treated animals at 829 mg/kg/day and the group mean value fell within the historical control 95% reference range of 0.04 to 1.80 %TI (it should be noted that vehicle animal R0006 elicited a %TI value of 3.73). The small increases in DNA strand breaks were also concomitant with histopathological findings suggestive of adaptive changes and/or mild hepatotoxicity.

In the duodenum and stomach, animals treated with (‑)‑NOPYL ACETATE at all doses exhibited group mean and individual animal %TI values that were generally similar to the concurrent vehicle control group and which fell within the laboratory’s historical vehicle control ranges. There were no statistically significant increases in %TI for any of the groups receiving the test article, compared to the concurrent vehicle control group and no evidence of a dose-response.

 

Main Experiment 2

In the liver, animals treated with (‑)‑NOPYL ACETATE at 1000 mg/kg/day exhibited group mean and individual animal (three individuals) %TI values which exceeded the concurrent vehicle control group, which contributed to statistical significance for the group (p≤0.001). Individual vehicle control animals demonstrated %TI values of between 0.16 to 0.76 %TI and the animals treated at 1000 mg/kg/day demonstrated %TI values of between 1.19 to 5.33 %TI. It should be noted that the animal with the highest %TI value (animal R0602) also demonstrated a marked elevation in %hedgehogs of 11.44% (historical vehicle control maximum %hedgehog cells was 7.89%) suggestive of toxicity. As seen in Experiment 1, these findings were also concomitant with histopathological findings suggestive of adaptive changes/mild hepatotoxicity. In addition, clinical chemistry changes of increases in ALTP and ALP enzyme activities and decreased cholesterol and globulin indicated early onset test article-related liver changes suggestive of heptatotoxicity and therefore any increases in DNA strand breaks are not likely due to genotoxicity.

In the stomach, animals treated with (‑)‑NOPYL ACETATE at 1000 mg/kg/day exhibited a small, but statistically significant (p≤0.05) increase in group mean %TI of

0.68 %TI. However, this was primarily due to a single animal with a slightly elevated %TI value (animal R0602 with a %TI value of 1.33) when compared to the vehicle control group animals. However, all (‑)‑NOPYL ACETATE treated animals at 1000 mg/kg/day and the group mean value fell well within the historical control 95% reference range of 0.16 to 8.29 %TI for the stomach. Therefore, the increases in %TI were considered to be within the normal variation for the assay and the statistical significance was considered not biologically relevant.

In the duodenum, animals treated with (‑)‑NOPYL ACETATE at 1000 mg/kg/day exhibited group mean and individual animal %TI values that were similar to the concurrent vehicle control group and which fell within the laboratory’s historical vehicle control 95% reference range. There was no statistical significance at 1000 mg/kg/day.

In the gonad, animals treated with (‑)‑NOPYL ACETATE at 1000 mg/kg/day exhibited a small increase in group mean %TI value which gave rise to statistical significance (P≤0.01) for the group. Individual vehicle control animals demonstrated %TI values of between 0.16 to 0.32 %TI and the animals treated at 1000 mg/kg/day demonstrated %TI values of between 0.24 to 0.39 %TI. However, as all animals at 1000 mg/kg/day fell within the historical vehicle control range of 0.04 to 0.45 %TI it is considered that the %TI values were within the normal variation for the assay and the statistical significance not biologically relevant.

 

For both Main Experiments 1 and 2, there were no increases in mean %hedgehogs in the liver, stomach, duodenum or gonad that were considered evidence of excessive DNA damage due to treatment with (‑)‑NOPYL ACETATE that could interfered with comet analysis.

 

It was concluded that under the conditions of this study, (‑)‑NOPYL ACETATE did not induce biologically relevant increases in DNA strand breaks in the liver, stomach, duodenum or gonad when tested up to 1000 mg/kg/day (the maximum tolerated dose level for this study). Any increases in DNA strand breaks were considered small, were variable in individual animals within the group and in the liver were generally concomitant with clinical chemistry and/or histopathological findings suggestive of hepatotoxicity and not genotoxicity.