Reaction mass of ethyl (1S,2R,4S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate and ethyl (1R,2R,4R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 05 June 2012 and 19 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 475 using a mammablian bone marrow chromosome aberration method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Molecular formula:

C10H14O2

IUPAC Name:

ethyl (1R,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1R,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2R)-bicyclo[2.2.1]hept-5-ene-2-carboxylate; ethyl (1S,2S)-bicyclo[2.2.1]hept-5-ene-2-carboxylate

Test material form:

other: Liquid

Details on test material:

Name: TM 09-218c
Synonyms: Ethyl bicyclo [2.2.1]hept-5-ene-2- carboxylate
CAS Number: 10138-32-6
Description: Clear colorless liquid (BioReliance)
Storage Conditions: Room temperature, protected from light
Receipt/Login: 14 November 2011 / 15 November 2011

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

ICR mice were obtained from Harlan, Frederick, MD and were received on 05 June 2012 for DRF assay and on 22 June 2012 for definitive assay. At the time of dose administration for each phase of the study, the mice were 6 or 7 weeks old.

Animal Receipt and Acclimation
Virus antibody-free (VAF) mice were obtained from a supplier that monitored mice for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were acclimatized for 5 or 7 days after receipt. At BioReliance, mice were observed each day for signs of illness and other conditions of poor health. All mice were judged to be healthy prior to utilization in the study.

Animal Welfare Provisions and Animal Care
This study is not duplicative or unnecessary. The number of mice and the procedures and experimental design used for this study have been reviewed and were approved by the BioReliance Institutional Animal Care and Use Committee #8 and #9. All procedures involving mice performed at BioReliance follow the specifications recommended in The Guide for the Care and Use of Laboratory Animals (National Research Council, 2011).
Animals were housed in an AAALAC-accredited facility with a controlled environment of 50 ± 20% relative humidity, temperature of 72 ± 3°F and a 12 hour light/dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.
Mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. The purpose of this system was to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the cage.
Heat-treated Sani-Chip hardwood chips were used for bedding to absorb liquids (P.J. Murphy Forest Products, Montville, NJ). Bedding was analyzed by the Manufacturer for any contaminants.
Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards [water source is Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy metals, alkalinity and halogens.
A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients.
The results of bedding, food and water analyses are on file at BioReliance. There are no contaminants in the bedding, feed or water that are expected to interfere with the study.

Randomization
In each study, mice were assigned to the appropriate number of treatment groups using a randomization procedure based on equalization of group mean body weights. At the time of randomization, the weight variation of animals did not exceed ±20% from their group mean. Following randomization, animals were identified by sequentially numbered ear tags assigned to each animal during randomization process. The cage card contained the following information: animal number, sex, study number, test substance identification, treatment group number, dose level and route of administration. Cage cards were color coded as a function of treatment group. Raw data records and specimens were also identified by the unique test animal number.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Corn oil was used to deliver TM 09-218c to the test system based on the solubility of the test substance, compatibility with the test system and route of administration. Corn oil (CAS number: 8001-30-7, lot number: MKBD6671, expiration date: 24 September 2013) was obtained from Sigma-Aldrich.

Details on exposure:

DOSE FORMULATIONS
Preparation of Test Substance Dose Formulations
The test substance dose formulations were prepared fresh for each phase of the study prior to dose administration. All formulations at concentrations of 3.125, 6.25, 12.5, 25, 35, 50, 62.5, 75, and 100 mg/ml in the DRF assay, and all formulations at concentrations of 15.6, 31.25 and 62.5 mg/mL in the definitive assay were prepared by combining an appropriate amount of test substance with an appropriate volume of the vehicle. Each formulation was vortexed for 1 minute and stirred for a total of 20 minutes using a stir bar/plate. All formulations appeared as yellow solutions and were stirred prior to and during dose administration.

Synopsis
In the absence of toxicity data, a dose range finding (DRF) study was conducted exposing male and female mice to TM 09-218c up to 2000 mg/kg, the maximum regulatory recommended dose level. Based on the mortality and clinical signs of toxicity observed in the DRF study, a dose of 1250 mg/kg was determined to be the maximum tolerated dose and was selected as the high dose in the definitive assay. Two lower doses at 312 and 625 mg/kg were also tested. Since no differences in clinical signs of toxicity were observed between the sexes, only male mice were used in definitive study. At 2-4 hours prior to bone marrow collection, animals were administered colchicine to arrest cell division. Mitotic cells from the femoral bone marrow were collected at 18 ± 0.5 hours and at 42 ± 0.5 hours post dose administration, slides prepared and microscopically examined for structural and numerical aberrations. In addition, animals in the satellite groups were administered either the vehicle control or test substance at 312, 625 or 1250 mg/kg, and bled at different time points. The collected blood was processed to plasma and the plasma samples were analyzed to determine the concentration of test substance.

Dose Administration Procedure
All dose formulations were administered as a single intraperitoneal injection (IP) at a dose volume of 20 mL/kg body weight. The intraperitoneal route of administration has been routinely used, and is widely-accepted for use in the mammalian bone marrow chromosome aberration assay.
In order to arrest the cells in metaphase, all main study animals received a dose of 2 mg/kg of colchicine at 2-4 hours prior to bone marrow collection. Neat colchicine was diluted in Hank’s Balance Salt solution (HBSS) at a concentration of 0.5 mg/mL and was intraperitoneally administered to the animals at a dose volume of 4 mL/kg body weight.
All mice in the experimental groups were weighed immediately prior to dose administration, and the administered volume was based on individual body weight.

Dose Range- Finding Study
In the dose range- finding study, 3 mice/sex/group were exposed to TM 09-218c at doses of 62.5, 125, 250, 500, 700, 1000, 1250, 1500, or 2000 mg/kg. Mice were observed at ~1 to 3 hours after dose administration and each day thereafter for 3 days for clinical signs of toxicity. Body weights were recorded following dose administration on Study Day 0 and on Study Days 1, 2 and 3. Following the last observation on Study Day 3, animals were euthanized by exposure to CO2 verified by toe pinch reflex and discarded without further examination.

Definitive Study
The chromosome aberration study was conducted according to established procedures (Cohen, M.M. and K. Hirschorn. 1971; Preston R.J. et al., 1981) with appropriate Standard Operating Procedures (SOPs) implemented to ensure that the study was conducted in accordance with Good Laboratory Practice (GLP) regulations.

Design
Based on the mortality and clinical signs observed in the DRF study, mice were exposed to TM 09-218c at doses of 312, 625 or 1250 mg/kg. Since no differences were observed in the clinical signs of toxicity between sexes, only male mice were used in this study.

In-Life Evaluations
Mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity, which were recorded.


Plasma Analysis
Design
A total of 22 satellite groups consisting of three male mice each were dosed either with the vehicle or with TM 09-218c at 312, 625, or 1250 mg/kg. Additional 6 male mice were dosed at 1250 mg/kg.

Sample Collection and Storage
Prior to each bleeding time-point, mice were anesthetized with 70%CO2/30%O2 and blood (~ 0.5-0.7 mL) was collected via retro-orbital sinus, into collection vials containing K3EDTA as an anticoagulant. Immediately following blood collection, mice were euthanized by CO2 asphyxiation verified by toe pinch reflex and discarded without further examination. Blood samples were placed on wet ice pending centrifugation. Within 30 minutes of collection, blood samples were centrifuged for 10 minutes in a refrigerated centrifuge (~ 4°C) at 3000 rpm. After centrifugation, plasma samples were transferred into individually labeled vials, and then stored at -20°C or below until shipment to the sponsor for analysis.

Sample Shipment
Samples were shipped on dry ice to the Sponsor by overnight courier express to the attention of:
Dr. Richard Hiserodt IFF Inc. 1515 State Highway #36 Union Beach NJ 07735
The recipient (Principal Investigator), and the Study Director were notified of the shipment.

Bioanalysis
At the test site, the plasma samples were analyzed to confirm systemic exposure.

Duration of treatment / exposure:

Single administration

Frequency of treatment:

Single administration

Post exposure period:

42 ± 0.5 hours post dose administration

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
3.125, 6.25, 12.5, 25, 35, 50, 62.5, 75, and 100 mg/ml
Basis:
other: administered intraperitoneally in the range-finding study

Doses / concentrations 2

Remarks:

Doses / Concentrations:
15.6, 31.25 and 62.5 mg/mL
Basis:
other: administered intraperitoneally in the definitive study

No. of animals per sex per dose:

Range-finding study: 3 mice/sex
Definitive study: 5 males

Control animals:

yes

Positive control(s):

Characterization of Positive Control Substance
Cyclophosphamide monohydrate (CP, CAS number: 6055-19-2, lot number: 120M1253V, expiration date: 31 December 2013), was obtained from Sigma – Aldrich.

Preparation of Positive Control Substance Formulation
An aqueous dosing formulation of CP at a concentration of 2.5 mg/mL was prepared fresh on the day of dose administration. An appropriate amount of CP was dissolved in an appropriate volume of sterile water for injection (B. Braun Medical, CAS number: 7732-18-5, lot number: J1L003, expiration date: September 2013). The accuracy of preparation and stability of the CP formulation was demonstrated by acceptable results that met the criteria for a valid test.

Examinations

Details of tissue and slide preparation:

Bone Marrow Collection and Slide Preparation
Animals were administered colchicine intraperitoneally at 2-4 hours prior to scheduled bone marrow collection. At 18 ±0.5 hr or 42±0.5 hr post dose administration, mice were sacrificed by carbon dioxide asphyxiation verified by toe pinch reflex in the same sequence used for dosing. The femurs were exposed, cut just above the knee and bone marrow was aspirated into a syringe containing a small volume of HBSS. The bone marrow cell suspensions were transferred into appropriately labeled tubes containing 3 to 5 mL of HBSS. The tubes with bone marrow were kept on wet ice until centrifugation. The cells were collected by centrifugation (100 x g; 5-10 minutes; refrigerated centrifuge set at 2-8°C), re-suspended in 5 mL of warm (37°±1°C) hypotonic solution of 0.065M KCl, and then incubated for 10±1 minutes in a water bath at 37°C±1°C to swell the cells. Following this incubation period, a few drops of fixative (methanol: acetic acid, 3:1 v/v) were added and the cells were collected by centrifugation (100 x g; 5-10 minutes; refrigerated centrifuge). Following centrifugation, the supernatant was discarded and the cell pellets were re-suspended. Fresh fixative (5 mL) was added to each tube, tubes were centrifuged, supernatant was discarded and the cell pellets were re-suspended. Following another change of fixative, centrifugation and resuspension of the cells, fresh fixative was added and the tubes were capped and stored overnight at 2°C to 8°C. To prepare slides, the cells were collected by centrifugation (100 x g; 5-10 minutes; refrigerated centrifuge) and re-suspended to opalescence in fresh fixative. Two to four drops of fixed cells were dropped onto a wet slide and air dried. Each slide was identified by the study number and animal number. At least, two slides were prepared from each animal, air dried, stained with Giemsa stain and permanently mounted.

Scoring for Chromosome Aberrations (Bone Marrow Evaluation)
Slides were coded using a random number table by an individual not involved with the scoring process. Metaphase spreads (cells) were examined using a light microscope and under oil immersion (1000X) without prior knowledge of treatment groups.
The Mitotic Index (MI) was determined for each animal, across all treatment groups at both collection time points. The MI was determined as the percentage of cells in mitosis based upon 1000 cells counted per animal. The mean MI was calculated for each treatment group (including positive and negative control groups) and served as a parameter of cytotoxicity and inhibition of cell division.
A minimum of 100 metaphase spreads containing 40 chromosomes, were examined from each animal and scored for chromatid-type and chromosome-type aberrations. Fewer cells (50 metaphase spreads) were scored per animal in the cyclophosamide group when a higher percentage of aberrations, i.e. >10%, is observed during the scoring.
The following structural aberrations were recorded:
Chromatid-type aberrations: chromatid and isochromatid breaks and exchange figures such as quadriradials (symmetrical and asymmetrical interchanges), triradials and complex rearrangements.
Chromosome-type aberrations: chromosome breaks and exchange figures such as dicentric chromosomes and rings.
Fragments observed with an exchange figure were not scored as an aberration but not considered part of the incomplete exchange.
Pulverized chromosome(s), pulverized cells and severely damaged cells (≥ 10 aberrations) were also recorded.
Chromatid and isochromatid gaps were recorded but not included in the analysis.
The XY coordinates for each cell with structural aberrations were recorded using a calibrated microscope stage.
The percent polyploid and endoreduplicated cells were was determined per 100 metaphase cells per animal.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose Range Finding Study
Mortality was observed in 2/3 male and 2/3 female mice at 1500 mg/kg and in 2/3 male and 3/3 female mice at 2000 mg/kg. Lethargy and piloerection were observed in both male and female mice at doses of 62.5, 125, 250, 500, 700, 1000 mg/kg following dose administration and at 1250, 1500, and 2000 mg/kg on study Day 1. Piloerection persisted in the 1000, 1250, 1500 and 2000 mg/kg treatment groups throughout the study period. Prostration and irregular breathing were noted following dose administration at 1250, 1500, and 2000 mg/kg. Hunched position and/or tremors were among the clinical signs observed in the surviving mice in the 1500 and 2000 mg/kg treatment groups. No appreciable changes in group mean body weights were observed in any of the treatment groups with the only exception of females at 1500 mg/kg, which exhibited a group mean body weight loss of 11.8%.

Definitive Study
Clinical Signs
No mortality was observed in any of the treatment groups. All mice in the control substance groups appeared normal during the study period. Piloerection and lethargy were among the clinical signs observed in the test substance treatment groups. In addition, prostration and irregular breathing were noted in the 1250 mg/kg treatment group, following dose administration.

Bone Marrow Evaluation
Microscopic evaluation of bone marrow cells indicated the following:
No appreciable reductions in the mean Mitotic Index (MI) compared to the vehicle control were observed in any of the treatment groups.
No statistically significant increase in the number of cells with structural aberrations compared to the vehicle control were noted in any of the test substance treatment groups (Fisher’s exact test; p> 0.05). In contrast, CP induced a statistically significant increase in the number of structurally aberrant cells compared to the vehicle control (Fisher’s exact test; p< 0.05). No numerical aberrations were observed in the test substance groups or the control groups.

Systemic Exposure
Plasma Analysis
Plasma samples collected from the vehicle control group at 18 hours post dose, and from the test substance treatment group animals at 15 minutes, 30 minutes, 1, 2, 4, 8, and 18 hours post-dose, were analyzed by the Sponsor using LC-MS/MS method (A163.DR). The analysis results indicated the following:
A plasma sample from one of the vehicle control treated animals (animal number# 41) was free of interferences.


Toxicokinetic Evaluation
Mean plasma concentrations (bio-analytical data) of TM 09-218c were used to determine toxicokinetic parameters using Microsoft Office Professional, Excel 2010. The mean toxicokinetic parameters such as Cmax (maximum plasma concentration), Tmax (time at which Cmax was observed). AUC0-thr (the area under the concentration vs time curve), estimated total systemic clearance (CLsys) were calculated.

The results indicate an apparent increase in Cmax values trended toward linearity with increases in the relative ratio of dose of 1:2:4 and values of 1:2.2:5.9. The systemic exposure was greater than predicted with relative ratio increases in AUC (0-18hr) of 1:6:20, which also followed the CLsys observed and the suggestion of significantly greater clearance at the low dose relative to the mid and high dose groups.

Any other information on results incl. tables

Dose Formulation Analysis

Aliquots of vehicle control and test article formulations used in the dose administration were analyzed by BioReliance analytical chemistry department. The analyses were performed by gas chromatography (GC) using a method validated under BioReliance Study Number AD39TT.GCGTCHEM.BTL. The results of this analysis are indicated as follows: No test substance was detected in the vehicle control sample. All test substance formulations met the protocol specified acceptance criteria of 85.0% -115.0% of target concentrations and ≤5.0% relative standard deviation (%RSD). Test article formulations from the low and high concentrations were held at room temperature for 4.5 hours and re-analyzed to assess the formulation stability. The results indicated that formulations were stable at room temperature for at least 4.5 hours. Based on these results, all formulations were accurately prepared and were stable for the purpose of this study.

Systemic Exposure

Plasma Analysis

Plasma concentrations of the test substance treated animals at different time intervals are summarized in the following table:

Group / Treatment	Average test substance concentrations (ng/mL) Post dose
	15 minutes	30 minutes	1 hours	2 hours	4 hours	8 hours	18 hours
1 / vehicle control	NS	NS	NS	NS	NS	NS	*
2 / 312 mg/kg	700	361	102	28.3	0	0	0
3 / 655 mg/kg	1256	1545	667	92.7	362	3.3	0
4 / 1250 mg/kg	4074	3534	2262	1159	255	292	0

NS = Not sampled;

* data from one animal (animal #41) free of test substance.

Toxicokinetic Evaluation

The mean toxicokinetic parameters are presented in the following table:

Group	Dose (mg/kg)	Cmax (ng/mL)	Tmax (hr)	AUC(0-t) (ng/mL·hr)	CL(sys) (mL/kg/min)
2	312	700.3	0.25	429.7	12250
3	625	1583	0.4	2643	4753
4	1250	4127	0.3	8585	2639

Cmax (maximum plasma concentration); Tmax (time at which Cmax was observed) AUC0-t (the area under the concentration vs time curve) CL (sys) estimated total systemic clearance

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this study, a single intraperitoneal administration of TM 09-218c at doses up to and including a dose of 1250 mg/kg, did not induce a significant increase in the frequency of cells with structural or numerical aberrations in the bone marrow of male ICR mice. Therefore, TM 09-218c was concluded to be negative in the mouse chromosome aberration test.

Executive summary:

The genotoxic potential of the test substance, TM 09-218, was assessed according to OECD Test Guideline 475 using a mammalian bone marrow chromosome aberration method. There was no significant increase in the frequency of cells with structural or numerical aberrations in the bone marrow of male ICR mice.