N-[(1R)-2-[(Methylsulfonyl)oxy]-1-phenylethyl]carbamic acid 1,1-dimethylethyl ester

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12 November - 15 November, 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

During the last decade, the analysis of micronuclei (MN) has gained increased popularity as an alternative to classical chromosomal aberration analysis for detecting clastogenic agents. Since MN are formed from both acentric chromosome fragments and from lagging, intact chromosomes, this technique is the most reliable method currently available for evaluating the potential of a chemical to induce structural and/or numerical chromosomal damage. Among the various in vitro and in vivo MN assays used to detect genotoxic chemicals, the rodent erythrocyte micronucleus test is most commonly used. The assay is easily conducted by evaluating the frequency of MN in immature erythrocytes (polychromatic erythrocytes [PCE]) scored in blood preparations. The product of recent cell divisions, these enucleated cells are abundant, easily recognizable, and have a lifetime of approximately 2 days before maturing into normochromatic erythrocytes (NCE). The detection of a significantly elevated level of micronucleated PCE (MN-PCE) is considered an indicator of genetic damage in the bone marrow erythrocyte cell population.

The test article is an Ames-positive compound that is being tested to help assess potential worker safety issues related to exposure during the manufacturing process. To generate animal data, the test material is being studied to investigate possible genotoxic effects on polychromatic erythrocytes (PCE) in the bone marrow of adult Sprague Dawley® (SD) rats.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Remarks:

All data underwent a quality control review, ILS' standard operating procedures (SOPs) were followed, and this study was conducted in accordance with the highest standards of research practice. All changes to the protocol were approved by the Sponsor.

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl N-[(1R)-2-(methanesulfonyloxy)-1-phenylethyl]carbamate

EC Number:

855-580-5

Cas Number:

102089-75-8

Molecular formula:

C14H21NO5S

IUPAC Name:

tert-butyl N-[(1R)-2-(methanesulfonyloxy)-1-phenylethyl]carbamate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Hsd

Sex:

male

Details on test animals or test system and environmental conditions:

25 male Sprague Dawley® rats were allocated into 1 of 5 designated dose groups and administered the test article or the vehicle control once, or the ethyl methanesulfonate (EMS) positive control chemical daily for two consecutive days, via oral gavage.

The rats were acclimatized for at least 7 days and were 7-8 weeks of age and 253.4-284.5 g in weight at administration. Each animal was uniquely identified by ear tag prior to the start of the study. The animals were housed 2-3 per cage and were provided food and water ad libitum. The room temperature was kept at between 21.3-22.6 ̊C, humidity at 34.3-55.2%, and 12/12-hour light/dark cycle lighting.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.5% HPMC with 5% Kolliphor RH40 (w/w)

Details on exposure:

To select doses for the definitive study, a range finding study was conducted using a single dose regimen with 10 male Sprague Dawley® rats allocated to one of five dose groups. The dose levels for the range finder test were 250, 500, 1,000, 1,500, and 2,000 mg/kg. No toxicity was noted via clinical observations or body weight changes during the range finder.

The test article was prepared at ILS at concentrations of 12.5, 25, 50, 75, and 100 mg/mL. The dose levels for the five groups in the definitive study were 0, 150, 500, 1,000, 2,000 mg/kg/day.

Doses were chosen based on OECD guideline Test No. 474 (OECD, 2016) that requires a dose level of 2000 mg/kg/day or maximum tolerated toxicity in the test system. Dose levels for the definitive study were determined based on toxicity noted in the range finder (i.e., clinical signs, body weight changes). The EMS dose level was selected based on previous usage as a positive control. Similar dosing regimens resulted in no adverse clinical signs with a significant increase in MN frequency.

Dose formulations were administered via oral gavage once at a dose volume of 20 mL/kg body weight. EMS was dosed twice (24 hours ± 30 minutes apart) at 10 mL/kg. Dose volumes were based on individual animal daily body weight. Dose formulations were placed on a stir plate at least 30 minutes prior to dosing and continuously stirred.

Duration of treatment / exposure:

Two consecutive days

Frequency of treatment:

once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 5

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5 males per dose group (5 dose groups total)

Control animals:

yes, concurrent no treatment

Positive control(s):

Ethylmethanesulphonate

Examinations

Details of tissue and slide preparation:

Blood was collected for potential test article bio-analysis at approximately 3 hours post-dose and for micronucleus (MN) frequency assessment at 48 and 71 hours post-dose (post initial dose for EMS group).

Blood Collection Tube: At least one day prior to scheduled collection, 350 μL of MicroFlowPLUS Kit Anticoagulant/Diluent was aliquoted into an appropriately labeled sterile microcentrifuge tube for each animal on study. The tubes were maintained at 1 – 10°C until needed.

Fixative Tubes: At least 1 day prior to blood collection, 2 mL of fixative (≥99.8% pure methanol) were aliquoted to 2 appropriately labeled 15 mL polypropylene conical tubes for each animal on study. The tubes were pre-chilled overnight (or longer) at -80 ± 5°C.

Blood Collection: Blood was collected at 48 hours (retro orbital sinus) and 71 hours (cardiac puncture) (± 30 minutes) post initial (or only) dose. Each tube was shaken immediately before bleeding each animal to coat the inside of the tube and the collection used a heparinized syringe or glass tube. Immediately thereafter, 60 – 120 μL of whole blood were dispensed into the corresponding microcentrifuge tube containing Anticoagulant/Diluent and mixed by inverting several times. The blood-Anticoagulant/Diluent mixture was maintained at room temperature until fixation (≤ 6 hours).

Fixing Blood Samples: Immediately prior to fixing, a microcentrifuge tube containing a blood-Anticoagulant/Diluent sample was inverted to ensure a homogeneous suspension. The corresponding pre-chilled 15 mL tubes containing fixative were quickly removed from the freezer, and 180 μL of the sample were forcefully dispensed into each duplicate tube and mixed, as described in the MicroFlowPLUSKit instruction manual. The tubes of fixed cells were quickly transferred back to the freezer and the process repeated for the remaining samples. The fixed blood samples were stored at -80 ± 5°C for at least 3 days prior to analysis.

Analysis: Flow cytometry analysis of erythrocytes was performed using MicroFlowPLUS Kit reagents (Litron Laboratories, Rochester, NY). The analysis was performed according to the kit’s instructional manual with minimal modification, including the following: 1) 1.0 mL of fixed cells for each experimental sample was transferred to a tube containing 12 mL of Solution C and washed in preparation for labeling and 2) the volumes of labeling solution I, labeling solution II, and DNA stain were adjusted when appropriate to minimize use of excess reagents.Unless precluded by excessive cytotoxicity, 20,000 (± 2000) immature CD71-positive PCE were analyzed to determine the frequency of normal and micronucleated PCE and NCE. The percentage of PCE was evaluated as a measurement of cytotoxicity.

Evaluation criteria:

The study met the criteria for a valid test.

Criteria for a Valid Test:

Negative Controls: Currently, there are no established limits for the group mean frequency of MN-PCE of rats treated with the vehicle control. The vehicle control MN-PCE data were acceptable for inclusion in the laboratory’s historical control database. However, an elevated baseline MN-PCE frequency deemed to be linked to the vehicle + excipient selected for this study would not invalidate the study.

Control Article: EMS induced an increase in the frequency of MN-PCE at p < 0.05 as compared to the negative control. Results were acceptable for inclusion in the laboratory’s historical control database.

Test Article: For negative studies, the highest test article concentration evaluated for MN must have, unless precluded by the use of the limit dose of 2000 mg/kg/day, induced bone marrow or systemic toxicity. A statistically positive (p < 0.05) change (typically a reduction) in the PCE frequency would be considered evidence of bone marrow toxicity. Clinical signs related to the central nervous system (e.g., change of spontaneous activity, ataxia, sedation, staggering), which would usually only occur if the test substance is systemically bioavailable, would be considered indicative of bone marrow exposure. Alternatively, detection (above the quantification limit) of the test article or a metabolite in plasma would be considered sufficient evidence of systemic bioavailability, and therefore, bone marrow exposure.

Statistics:

Individual animal data, group means, and standard deviations were calculated and reported. Final body weight and body weight gain measurements, and MN-PCE and PCE frequency data were analyzed using Statistical Analysis System version 9.2 (SAS Institute, Cary, NC). Homogeneity of variance was analyzed using Levene’s test, and normality of the vehicle control data was assessed using a Shapiro-Wilk test. Homogeneous data were analyzed using a one-way analysis of variance (ANOVA), and test-article administered groups were compared to the appropriate control group using Dunnett’s test. Dose-dependent changes were evaluated using a linear regression model.

Data that were not homogeneous and normally distributed were transformed and re-assessed. In the event that data could not be transformed to be homogeneous and normally distributed, the data were analyzed using the appropriate non-parametric Dunn’s test. Dose-dependent changes were evaluated using an appropriate non-parametric trend test.

Positive control data were analyzed by an appropriate t-test to determine whether there was a significant difference from the vehicle control group.

The confidence level for the statistical analyses was p < 0.05.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Remarks:

There was no evidence of induction of micronuclei in the animals administered the test material at either time point.

Toxicity:

yes

Remarks:

Under the conditions of the MN assay, there was a dose dependent reduction in % polychromatic erythrocytes (PCE) at both the 48 and 71 hour time points, indicative of bone marrow cytotoxicity.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There were no significant clinical signs of toxicity exhibited during the study. However, there was statistically less body weight gain in the 2000 mg/kg dose group (i.e., no weight gain). Exposure to EMS led to reduced body weight as compared to the vehicle control group.

Under the conditions of the MN assay, there was a dose dependent reduction in % polychromatic erythrocytes (PCE) at both the 48 and 71 hour time points, indicative of bone marrow cytotoxicity. There was no evidence of induction of micronuclei in the animals administered the test material at either time point. Administration of EMS, the positive control, resulted in a statistical decrease in PCE and increase in micronucleated PCE (MN-PCE).

Interpretation of Results

Negative: No dose level exhibits a statistically significant increase compared to the concurrent control group, there is no dose-related trend response, and bone marrow exposure to the test article occurred as observed as a change in PCE frequency in bone marrow or other appropriate measurement (unless precluded by the selection of the limit dose). In addition, all results should be within the historical negative control range; however, this final criterion will not be compulsory should an elevated baseline MN-PCE frequency be observed that is deemed to be linked to the vehicle + excipient selected for this study.

Positive:At least one dose level exhibits a statistically significant increase compared to the concurrent vehicle control group, there is a dose-related trend response, and any significant results are outside the range of historical negative control data.

Any other information on results incl. tables

In-Life Animal Observations:

Mortality/Moribundity: All animals survived to the scheduled termination with no animals showing signs of moribundity.

Clinical and Cage-Side Observations: Clinical observations remained normal for each animal during the course of the study.

Body Weights: As compared to the vehicle control group, there was statistically less body weight gain in the top dose group (2000 mg/kg) and a corresponding positive downward trend test. The top dose group animals did not gain weight over the course of the study. The EMS group animals also lost weight compared to the vehicle control animals.

Confirmation of a Valid Test:

Negative Controls: The vehicle control MN-PCE frequency data were acceptable for inclusion in the laboratory’s historical control database.

Control Article: Under the conditions of this assay, EMS induced a statistically significant (p < 0.05) increase in MN-PCE frequency.

Test Article: The maximum dose of 2000 mg/kg was tested, meeting the criteria for exposure to the test article.

Applicant's summary and conclusion

Conclusions:

The test material was considered to be negative for genotoxicity, as there were no significant increases in MN-PCE in the animals administered the test material at either time point. There was a dose dependent reduction in %PCE at both the 48 and 71 hour time points, with statistical decreases in %PCE measured in the middle (1000 mg/kg) and top (2000 mg/kg) dose groups, indicative of bone marrow cytotoxicity.