The food contaminant acetamide is not an in vivo clastogen, aneugen, or mutagen in rodent hematopoietic tissue

Acetamide (CAS 60-35-5) is classified by IARC as a Group 2B, possible human carcinogen, based on the induction of hepatocellular carcinomas in rats following chronic exposure to high doses. Recently, acetamide was found to be present in a variety of human foods, warranting further investigation. The regulatory body JECFA has previously noted conflicting reports on acetamide's ability to induce micronuclei (MN) in mice in vivo. To better understand the potential in vivo genotoxicity of acetamide, we performed acute MN studies in rats and mice, and a subchronic study in rats, the target species for liver cancer. In the acute exposure, animals were gavaged with water vehicle control, 250, 1000, or 2000 mg/kg acetamide, or the positive control (1 mg/kg mitomycin C). In the subchronic assay, bone marrow of rats gavaged at 1000 mg/kg/day (limit dose) for 28 days was evaluated. Both acute and subchronic exposures showed no change in the ratio of polychromatic to total erythrocytes (P/E) at any dose, nor was there any increase in the incidence of micronucleated polychromatic erythrocytes (MN-PCE). Potential mutagenicity of acetamide was evaluated in male rats gavaged with vehicle control or 1500 mg/kg/day acetamide using the in vivoPig-a gene mutation assay. There was no increase in mutant red blood cells or reticulocytes in acetamide-treated animals. In both acute and sub-chronic studies, elevated blood plasma acetamide in treated animals provided evidence of systemic exposure. We conclude based on this study that acetamide is not clastogenic, aneugenic, or mutagenic in vivo in rodent hematopoietic tissue warranting a formal regulatory re-evaluation.


EXECUTIVE SUMMARY:
This study was performed to evaluate the micronucleus induction potential of acetamide in mice. Sixty CD1 mice were divided into 5 groups, each group comprising 6 animals/sex. The main study was conducted at the dose levels of 250, 1000 and 2000 mg acetamide/kg body weight (Groups II, III and IV, respectively). A concurrent vehicle (distilled water) control group (Group I) was maintained along with the acetamide treated animals. Acetamide was dissolved in distilled water and administered orally for two consecutive days. Animals were sacrificed approximately between 18-24 hours after the final treatment. Before sacrifice blood samples were collected from each treatment group and vehicle control group to demonstrate the target organ exposure. A concurrent positive control group (Group V) was treated with a single intraperitoneal injection of Mitomycin-C at the dose level of 1 mg/kg body weight.
No toxicity to bone marrow [decrease in polychromatic to total erythrocytes ratio (P/E)] was observed in all animals treated at the dose levels of 250, 1000 and 2000 mg/kg body weight, when compared with the concurrent vehicle control group. All animals exhibited normal behavior and there were no mortalities. The number and percentage of micronucleated polychromatic erythrocyte (MNPCE) were not increased in animals treated with acetamide up to the dose level of 2000 mg/kg body weight when compared with the vehicle control group. No statistically significant effects on body weight were observed in any of the animals from positive control or treatment groups, when compared with the concurrent vehicle control group. The positive control group yielded a statistically significant increase in the number of micronucleated polychromatic erythrocytes (MNPCE) in comparison to the vehicle control group.
The dose formulation analysis revealed that the doses complied for the presence of test item for its nominal concentration (± 10%) of active ingredient (% CV < 10%). Plasma concentration of acetamide in different groups revealed that the test item reached the target tissue, i.e. bone marrow. Negative and positive control data were consistent with historical control distributions.
From the results of the present study, it is concluded that acetamide does not have micronucleus induction potential.

COMPLIANCE:
Signed and dated GLP and Quality Assurance statements are provided. There was no deviation from regulatory requirements.

Study Objective
This study was performed to evaluate the micronucleus induction potential of acetamide in mice.
The study was conducted in compliance with Principles of GLP (OECD 1998).

Study Guidelines
The present study was conducted according to: OECD, 2016: The Organisation for Economic Co-operation and Development (OECD), Guidelines for Testing of Chemicals, Volume II, OECD 474, Mammalian Erythrocyte Micronucleus Test, adopted by the Council on July 29, 2016.

Justification for Selection of the Test System
The mouse was selected as the test system of choice because it is a readily available rodent species. It has been historically shown to be a suitable model for assessing the micronucleus induction potential and is recommended by the OECD and other regulatory authorities. The results of the study are believed to be of value in predicting the micronucleus induction potential of the test item in humans.

Archives
All original raw data including any storage medium for electronically recorded data, documentation, the signed study plan, the study plan amendments, the draft report, one original final report, slides and the representative sample of the test item will be retained in the GLP Archives at Jai Research Foundation for a period of ten years. At the end of this period, the Sponsor's instructions will be sought to either extend the archiving period or return the archived material to the Sponsor or dispose of the material.

Test Item
Details of the test item provided by the Supplier:

Principle
The mammalian micronucleus test is used to detect cytogenetic damage (which results in a chromosomal break, fragment or lagging whole chromosome) caused by the test item. The damaged chromosomal fragments remain in the anucleated cytoplasm of the erythrocyte and are visible, when stained, as a small round or oblong structure called micronuclei. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.

Animal Welfare
The study was undertaken in compliance with the 'Guidelines for Laboratory Animals Facility' issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. These guidelines promote the humane care of animals used in research by providing specifications that will enhance animal well-being and experimental quality for the advancement of biological knowledge that is relevant to humans and animals.
Project proposal for the experimentation was approved by Institutional Animal Ethics Committee (IAEC), Jai Research Foundation.
JRF is also accredited with Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) that promotes the humane treatment of animals in science.

Test Animals
For the main study, Hsd: ICR (CD1) mice (Mus musculus) were received from the Animal Breeding Facility, Jai Research Foundation. The animals were 8-10 weeks old on day 1 of dosing. The females used were nulliparous and non-pregnant. The male mice weighed between 33 and 43 g and the female mice weighed between 27 and 33 g on day 1 of the experiment (main study).

Acclimatisation
The animals were received into the experimental room and acclimatised for a period of six days (maximum 3 animals/cage). The animals were randomised into 5 groups using validated in-house developed software. The method of randomisation used was censored randomisation method ).

Identification
Before randomisation, animals were marked with nontoxic marker pen. After randomisation, each mouse was assigned a number, which was tattooed on its tail using a tattoo machine and appropriate labels were attached to the cages indicating the study number, test item code, group number and sex, dose, type of study, cage number and animal number.

Feed and Water
The quality of feed and water is regularly monitored at Jai Research Foundation. There were no known contaminants in the feed or water at levels that would have interfered with the experimental results obtained.
Water : UV sterilized drinking water filtered through Hi-Tech reverse osmosis water filtration system was provided ad libitum (APPENDIX 9).

Selection of Vehicle
Acetamide was found soluble in distilled water (stock A, 200 mg/mL). Hence distilled water was selected as the vehicle for oral gavage for the animals in the main study.

Rationale for Selection of Route of Administration
A potential route of human exposure is via the oral route. Therefore, the oral route of administration was selected for this study.

Main Study
Based on sponsor's suggestions and the published data from earlier studies , Mirkova, 1996, the main study was conducted with dose levels of 250, 1000 and 2000 mg/kg body weight. Five groups (comprising 6 animals/sex) were used for this study. Group I served as the vehicle (distilled water) control, Group II, III and IV were low, mid and high dose groups, respectively. Group V was the positive control group and received Mitomycin-C (1.0 mg/kg body weight on day 2 of treatment) in distilled water by the intraperitoneal route on a single occasion.
A quantity of 625, 2500 and 5000 mg of acetamide were weighed and dissolved in distilled water on day 1 of dosing . The volume was made up to 25 mL to obtain a concentration of 25, 100 and 200 mg/mL for male and female animals for groups II, III, and IV, respectively. A quantity of 250, 1000 and 2000 mg of acetamide were weighed and dissolved in distilled water on day 2 of dosing . The volume was made up to 10 mL to obtain a concentration of 25, 100 and 200 mg/mL for male and female animals for groups II, III, and IV, respectively. The dose volume was 10 mL/kg body weight for all the treatment groups including vehicle and positive control groups. The acetamide was administered orally to mice using a metal cannula attached to a BD 1 mL disposable syringe. Mice from the vehicle control group (Group I) received only distilled water orally on both the days.
The mice from the positive control group (Group V) received a single injection of Mitomycin-C intraperitoneally at the dose level of 1.0 mg/kg body weight on day 2 of treatment. Each day the dose solutions were freshly prepared prior to dosing.
Body weight was recorded before dosing on day 1, day 2 and before sacrifice. The clinical signs of toxicity were recorded before dosing, post dosing (up to four hours) and before sacrifice. The body temperatures of all the animals were measured before dosing and then approximately 2 and 5 hours after each dosing and before sacrifice using microprobe thermometer (Asanami and Shimono, 1997;).

Dose Formulation Preparation, Sampling and Analysis
For active ingredient concentration analysis, samples were collected from each prepared dose formulations along with vehicle (distilled water) during the main study following the detailed procedures below.
Two sets of three replicates of 2 mL each concentration (25, 100 and 200 mg/mL for male and female animals) were taken from middle portion along with vehicle (distilled water). First set of replicates (three replicates of 2 mL each) were sent to Department of Chemistry (JRF) for analysis and second set of replicates were stored in deep freezer (-70 ± 10 ºC) as backup. The unused aliquots will be discarded after receiving approval for finalisation of the report from the sponsor.

Analytical Acceptance Criteria
The following criteria for acceptable specification for the concentration of the test item in the vehicle were used to determine a valid assay: 90 to 110% of nominal concentration with <10% coefficient of variance (%CV) of each concentration (Whitmire et al., 2010).

Evidence of Tissue Exposure
Blood samples were withdrawn from each animal in each treatment group and vehicle control group at the time of sacrifice before bone marrow collection. Blood samples were collected in heparinised (20 IU/mL) micro-centrifuge tubes. Blood samples were collected from orbital plexus under very light isoflurane anesthesia. To separate out the plasma, blood samples were centrifuged at 3000 rpm for 15 minutes at 4 °C. The plasma samples were stored at -70 ± 10 °C until analysis. The plasma samples were analysed for determination of test item concentration at Department of Chemistry, JRF.

Slide Preparation
Within 18 -24 h following the last treatment, mice from the vehicle control and the treatment groups (group I -group IV) were sacrificed by CO 2 asphyxiation (MacGregor et al. 1987) and the positive control group (group V) was sacrificed 24 hour after the last treatment by CO 2 asphyxiation . Femur bones from the sacrificed animals were excised and the epicondyle tips were removed. The bone marrow content was expelled by flushing and aspirating approximately 3 mL of foetal bovine serum using a 1 mL syringe and 24 gauge needle into centrifuge tubes. The aspirated bone marrow content was mixed using the syringe to dissociate the cells in order to avoid cell clump formation.
The tubes were centrifuged at around 1500 rpm for 10 minutes and the supernatant was discarded leaving about 0.2 -0.3 mL of medium with the cell pellet. The cell pellet was dissociated thoroughly using a Pasteur pipette and a drop of suspension was placed on a clean slide. A smear was prepared and allowed to air dry.
The slides were marked with study number, animal number and slide number. Two slides were prepared per animal and the cells were fixed with methanol and allowed to air dry for 20 minutes. Slides were stained using 5% Giemsa in phosphate buffer for 25 minutes. Subsequently the slides were rinsed in distilled water, air-dried and mounted. In order to prevent bias in the scoring, the slide numbers were masked with code numbers provided by the Department of Bio-statistics and Systems Information, Jai Research Foundation.

Scoring of Bone Marrow Micronucleus
One out of two slides from each animal was used for screening of micronucleated erythrocytes whereas the other slide was kept as back up, to be used for scoring when required. The slides were examined for the presence of micronuclei in polychromatic and normochromatic erythrocytes under microscope [Nikon Eclipse E600, Nikon Eclipse 80i, Nikon Eclipse Ni-U (Fluorescence) and Nikon Eclipse Ci]. A minimum of 4000 polychromatic erythrocytes were screened per animal to evaluate the incidence of micronuclei. A minimum of 500 normochromatic erythrocytes to its corresponding polychromatic erythrocytes were recorded to determine the P/E ratio. The masked labels were removed and all the slides were decoded after scoring.

Calculation
The P/E ratios were calculated from polychromatic to total (polychromatic + normochromatic) erythrocytes. The percentage of micronucleated polychromatic erythrocytes was also calculated.

Statistical Evaluation of Results
The data of percent micronucleated polychromatic erythrocytes (% MNPCE), P/E ratio and body weight of both the sexes were statistically analysed for normality using Shapiro-Wilk's test.
Where results of normality test were significant, non-parametric test (Kruskal-Walis test) was performed. Where results of normality test were non-significant then Bartlett test was performed to meet the homogeneity of variance before conducting ANOVA test followed by Dunnett's ttest. T-test was also performed to determine the level of significant difference between the vehicle control and the treated groups and positive control group.

Historical control data
Jai Research Foundation (JRF) has conducted more than 500 GLP studies for regulatory submission as per OECD TG 474 and established a strong historical control data base. JRF used quality control methods, such as control charts to identify data variability and to show that the methodology was 'under control'. Quality control charts (QC charts) have been added in APPENDIX 8 demonstrating the JRFs established historical positive control ranges and distribution, and a historical negative control ranges and distribution. Results of negative and positive control were within historical distribution limits. Overall results of treatment group was also within historical control limits.

Assay Acceptance and Evaluation Criteria
Before assay data were evaluated, criteria for a valid assay had to be met. The following criteria were used to determine a valid assay:

Acceptance Criteria
i. The vehicle (or negative) controls values were in the range of historical control data. ii.
The positive controls has produced responses that were compatible with that of the historical data and has produce statistically significant responses compared with the concurrent negative control.
iii. Mortality was not observed in control or treatment group and six animals per sex per group (group I to V) were evaluated for micronucleus induction potential of the test item in all the groups.
iv. The highest dose was a limit dose, maximum tolerable dose (MTD) which did not cause distress or death to the animal or produce toxicity to bone marrow.
v. PCE to erythrocyte ratio was more than the 20% of the vehicle control.

Evaluation and Interpretation Criteria
Once criteria for a valid assay had been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were, i. At least one of the treatment groups exhibits statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared to concurrent negative control. ii.
A positive result was defined as a dose-dependent, significant increase in the incidence of micronuclei when evaluated with an appropriate trend test e.g. Chi-square trend analysis.
iii. Statistical and biological relevance was considered in data interpretation.
iv. Any of the results falling outside the distribution of the historical negative control data i.e. Poisson based 95% control limits.
The test item was considered clearly negative, if, in all experimental conditions examined: i. None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control. ii.
There was no dose-related increase at any sampling time when evaluated by an appropriate trend test.
iii. All results were inside the distribution of the historical negative control data (e.g. Poissonbased 95% control limits), and iv. Bone marrow exposure to the test item(s) occurred v.
There is no requirement for verification of a clear positive or clear negative response.

Clinical Observations, Body Temperature and Body Weight
All animals were normal in the vehicle control group (Group I) and treatment groups II, III and IV (250, 1000, 2000 mg/kg body weight, respectively) and positive control group (Group V), both post-treatment and pre-sacrifice.
Significant decrease or increase in body temperature was not observed after day 1 and day 2 of dosing in both male and female animals from treatment groups, when compared with the concurrent vehicle control group.
No statistically significant effect on mean body weight was observed in positive control or treatment groups, when compared with the concurrent vehicle control group. No mortalities were observed.
Individual clinical observations are provided in APPENDIX 1. The summary of mean body temperature and individual body temperature are provided in TABLE 1 and APPENDIX 2, respectively. The summary of mean body weight and individual body weight are provided in TABLE 2 and APPENDIX 3, respectively.

Micronucleated Polychromatic Erythrocytes
Values of % MNPCE and P/E ratio for vehicle and positive controls were within the range of historical control data limits (APPENDIX 8).
No toxicity to bone marrow [decrease in polychromatic to total erythrocytes ratio (P/ Group-wise total polychromatic erythrocytes (PCE), micronucleated polychromatic erythrocytes (MNPCE), percent MNPCE and mean P/E ratio in bone marrow cells are given in TABLE 3 with individual data presented in APPENDIX 4 and APPENDIX 5.

Dose Formulation Analysis
The dose formulations complied with the presence of test item for its nominal concentration of (±10) active ingredient (% CV < 10%). Mean recoveries were 99.69, 100.57 and 105.41% at the prepared concentrations of 25, 100 and 200 mg/mL, respectively for both male and female animals (APPENDIX 7).

Evidence of Tissue Exposure
The plasma samples were analysed to demonstrate the target organ exposure, i.e., for test item concentration in blood. Dose dependent increase in concentration was observed in plasma samples (APPENDIX 7

CONCLUSION
From the results of the present study, it is concluded that acetamide does not have micronucleus induction potential in male and female mice up to the dose level of 2000 mg/kg body weight, following oral administration for two consecutive days.

5.
Mirkova, E.T., 1996: Activities of the rodent carcinogens thioacetamide and acetamide in the mouse bone marrow micronucleus assay,  Note : Temperature of positive control animals was not recorded on day one since positive control animals were not treated on day one. Note : Temperature of positive control animals was not recorded on day one since positive control animals were not treated on day one.

Micronucleus Test of Acetamide in Mice
Keys: F = Female, 1 = Normal, -= Not applicable (Animals of positive control group were not treated on day one). Note: Body weight of positive control animals was not recorded on day one since positive control animals were not treated on day one.

Test Facility Management
Dr. Manish V. Patel

Study Schedule
Study

Study Plan and Amendment (if any) Distribution
a. Original copy in Archive and study Sponsor; b. Photocopy to Study Director, QAU, and Residue Chemistry.

Objective
The objective of this study is to evaluate the micronucleus induction potential of acetamide in mice.

Regulatory Guidelines
This study is intended for regulatory submission and will be conducted in accordance with the known requirement of international guidelines: OECD

Principle of the Test Method
The mammalian micronucleus test is used to detect cytogenetic damage (which results in a chromosomal break, fragment or lagging whole chromosome) caused by the test item. The damaged chromosomal fragments remain in the anucleated cytoplasm of the erythrocyte and are visible, when stained, as a small round or oblong structure called a micronucleus during the maturation of erythrocytes. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.

Test Item
The

GLP Compliance
This study will be conducted in compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17, N° 1, Environment Directorate, the Organisation for Economic Co-operation and Development, Paris (1998) and all subsequent OECD consensus documents.

Standard Operating Procedures (SOP)
Unless otherwise specified all procedures mentioned in the study plan are subject to detailed Standard Operating Procedures of Jai Research Foundation.

Amendment to Study Plan
This study plan may be subjected to amendment. Amendment to study plan, whether initiated by the Sponsor or the Study Director will be generated, authorized by the Study Director and will be sent to the Sponsor for approval.
In the event that circumstances dictate immediate action, the nature of these circumstances will be communicated to the Sponsor as soon as practicable (by telephone, facsimile transmission or e-mail) and will be confirmed as soon as possible by way of formal study plan amendment.

Deviation(s)
Any deviation(s) will be documented in the study file and reported in the study report.

Quality Assurance
This study plan has been verified by JRF Quality Assurance Unit (QAU) and documented (Number 94462).
The QAU JRF will inspect the critical phase(s) of the study by study based inspection and/or process based inspection. The raw data, draft and final reports will be audited to ensure that the final report accurately reflects the raw data. The audit/inspection reports will be provided to the Study Director and the Test Facility Management. The date of audits/inspections and reporting of findings to the Study Director and the Test Facility Management will be incorporated in the study report.

ANIMAL WELFARE
The study will be undertaken in compliance with the 'Guidelines for Laboratory Animals Facility' issued by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. These guidelines promote the humane care of animals used in research by providing specifications that will enhance animal well-being and experimental quality for the advancement of biological knowledge that is relevant to humans and animals.
Jai Research Foundation is committed to enhancing animal welfare and ensures that studies are designed and conducted to cause the minimum suffering or distress to animals, consistent with the scientific objectives and in accordance with Jai Research Foundation's policy on animal welfare.
Project proposal for the experimentation is subject to the approval by the Institutional Animal Ethics Committee (IAEC), Jai Research Foundation.
JRF is accredited with Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) that promotes the human treatment of animals in science.

Humane Endpoint
Moribund animals or animals obviously in pain or showing signs of severe and enduring distress shall be humanely killed. Depending on the time, since dose administration, and the circumstances of death, the bone marrow may be removed and used as part of the interpretation of the results, (at the discretion of the study director).

Initial Considerations
Test item, at doses, that causes marked pain and distress due to corrosive or severely irritant actions, will not be administered. If required, study will be terminated.

Reason for Selection of the Test System
The mouse is selected as a test system because it is readily available laboratory rodent species. It has been shown to be a suitable model for mutagenicity studies and is also recommended by the OECD and other regulatory authorities. The results of this study are believed to be of value in predicting the potential of the test item to cause cytogenetic damage in humans.

Animals
Healthy and young Swiss albino mice or Hsd:ICR(CD1) (Mus musculus) will be obtained from the Animal Breeding Facility, JRF or any other CPCSEA approved source. The animals, 6 -10 weeks old on the first day of dosing will be used in the study. The female mice used will be nulliparous and non-pregnant. Body weight variation among the animals should not exceed ±20% of the mean body weight for each sex at the time of initiation of dosing. The animals will be identified with unique numbers by tattooing.

Acclimatisation
The animals, after veterinary examination for good health, will be acclimatised to the laboratory conditions for a minimum period of 5 days prior to commencement of treatment and they will be observed for clinical symptoms daily. After acclimatisation, the animals will be randomized using Censored Randomization Method  using validated in-house developed software.

Housing and Animal Identification
The mice will be housed (no more than four per cage) in polypropylene mice cages provided with rice husk as the bedding material. Each day cages will be supplied with a polypropylene water bottle fitted with a stainless steel nozzle.
Individual mouse will be identified with a unique number tattooed on the tail using a tattoo machine. The cages will be labeled with details of the study number, test item code, group number, sex, dose, type of study, cage number, and animal numbers. The labels used will be of different colours for different dose groups.

Animal Room Sanitation
Each day, the floor of the experimental procedure room will be swept and all worktops and the floor will be mopped with disinfectant solution.

Feed and Water
The mice will be provided with laboratory mice pellet feed (mice standard feed) and reverse osmosis water, filtered through reverse osmosis water purification system, ad libitum.

Environmental Conditions
The temperature of the experimental procedure room will be maintained at 22 ± 3 °C and the relative humidity between 40 and 70%. The photoperiod will be 12 h light and 12 h darkness, light hours being 06:00 -18:00 h approximately, and air exchanges will be a minimum of 15 volumes /hour.

Selection of Vehicle
Acetamide is highly water soluble, so solubility will be first tested with distilled water first. In case of insolubility, test item will be suspended in vegetable oil or 0.5% carboxymethyl cellulose (CMC), unless otherwise recommended by sponsor. Fresh dose formulations will be prepared daily and administered within 2 hours of preparation. The concentration of the test item will be adjusted so as to permit constant dosing volume. All animals will receive a single standard volume of 10 mL/kg body weight by oral gavage administration. Vehicle control animals will receive the vehicle alone.

Dose Formulation Preparation, Sampling and Analysis
Since test item will be prepared freshly and will be used within 2 hours of preparation, stability of the test item in the selected vehicle will not be tested separately.
Dose formulation will be prepared as per JRF/TOX/SOP-260 and JRF/TOX/SOP-266. For active ingredient concentration and homogeneity (in case of suspension) analysis, required samples will be collected from the prepared dose formulations (high, mid, and low dose) along with vehicle during the main study following the detailed procedures below. The size of samples will be determined by the study director/study person for feasibility considerations and to allow sufficient amount for analysis.
If dose formulations are solutions, required aliquots of the vehicle and all dose formulations will be collected from the middle portion. If dosing formulations are suspensions, aliquots from the top (T), middle (M), and bottom (B) of required dose formulation will be collected for homogeneity and concentration verification immediately following the preparation of the dose formulation during main study. The vehicle control will be sampled from the middle portion only.
In all cases, 2 sets of samples per dose formulation will be collected: the 1 st set of aliquots of selected dose formulations will be analyzed for homogeneity (in case of suspension) and active ingredient concentration.
The 2 nd set of aliquots of selected dose formulation will be stored in the deep freezer (-70 ± 10 °C) at JRF as backup and will be analyzed only if needed.

APPENDIX 6 (Continued) JRF Study Number: 485-1-06-17727 Page 10 of 18
Unless otherwise requested by the Sponsor, required samples will be collected from any partial retest of main study. These samples will be held at JRF as backup and only analyzed as would be required in the amendment. Any repetition of the affected portion of the study will be specified by study plan amendment.
In all cases, any unused aliquots will be discarded after receiving approval for finalization of the report from the sponsor.
All analytical work will be conducted by the Department of Chemistry, JRF, under GLP compliance. The detailed method together with the sample preparation procedure will be fully documented in the study records and described in the final report. Analytical parameters used for analysis of prepared dose formulations for the active ingredient will be added through study plan amendment. All unused samples will be handled as per the relevant Standard Operating Procedures.

Analytical Acceptance Criteria
The acceptable specification for the concentration of the test item in the dose formulation will be as described/mentioned below: Solutions: 90 to 110% of nominal with <10% coefficient of variance (% CV) of each concentration.
In the event of a sample being outside the acceptable specification range, the study director will: a) Justify the acceptability of the results, b) Suggest re-analysis of the backup samples, or c) Retest the affected portion of the study.

Main Study
Five groups (comprising 6 animals/sex/group) will be used for this study. Group I will serve as the vehicle control, Group II (250 mg/kg), III (1000 mg/kg) and IV (2000 mg/kg) will be low, mid and high dose groups, respectively. Group V will be the positive control and will receive mitomycin-C (1.0 mg/kg body weight on Day 2 of treatment) in distilled water by the intraperitoneal route on a single occasion. The rectal temperature of the treated animals will be monitored during the main study using digital laboratory thermometer. The temperatures will generally be measured before dosing (Day 1), approximately 2, 5 and 24 hours after each dosing. Dose levels are selected based on sponsor's suggestions and the published data from earlier studies , Mirkova, 1996.

APPENDIX 6 (Continued) JRF Study Number: 485-1-06-17727 Page 11 of 18
Mortality, severity of clinical symptoms, change in body temperature for up to 48 h after the initial dose and reduction in the immature to total (immature + mature) erythrocyte ratio will be considered for the evaluation of toxicity to bone marrow. For changes in thermal regulation, the body temperature rise by, at least, 1 °C or fall by, at least, 3 °C for five or more hours will be declared as having exceeded MTD. Body temperature changes, outside this range, have been previously reported to cause an increase in micronucleus formation in absence of chemical treatment (Asanami and Shimono, 1997;.

Evidence of Tissue Exposure
To demonstrate target organ exposure, plasma analysis will be performed along with main study to demonstrate the absorption of test item after oral dosing as well as to demonstrate the target organ exposure i.e. test item concentration in the blood samples.
It may be inferred from the published literature (Zhao et al., 2007;Putcha, Griffith and Feldman (1984)), that (i) acetamide fed orally to rats and mice is likely to rapidly reach the bloodstream and be transported throughout the body, (ii) at the proposed regimen of two daily back-to-back doses followed by bone marrow harvest around 24 hours after last dose, significant exposure of acetamide is expected to occur, and (iii) determination of acetamide levels in blood plasma should be a suitable method to provide evidence of exposure for the micronucleus assays.
Therefore in this study, blood samples will be withdrawn from each animal in each treatment group and vehicle control group at the time of sacrifice before bone marrow collection. Blood samples will be collected in heparinised (20 IU/mL) micro-centrifuge tubes. Blood samples will be collected from orbital plexus under very light isoflurane anesthesia. To separate out the plasma, blood samples will be centrifuged at 3000 rpm for 15 minutes at 4 °C. The plasma samples will be stored at -70 ± 10 °C until analysis. The plasma samples will be analysed for determination of test item concentration at Department of Chemistry, JRF. The details of bioanalytical method and results will be presented in the report. Bioanalytical parameters used for analysis of plasma samples will be added through study plan amendment.

Study Performance
The test item will be dissolved or suspended in a selected vehicle . Fresh dose formulation will be prepared, on the day of dose administration. Animals will be fasted prior to dosing (feed, but not water, will be withheld for approximately 2-3 hours). Animals will be dosed (10 mL/kg body weight) by oral intubation for 2 consecutive days, approximately 24 hours (± 1 hour) apart. The body weight will be recorded prior to the dosing on each day and also before sacrifice. Clinical signs will be recorded after dosing, each day, and before sacrifice. The animals will be sacrificed by CO 2 asphyxiation between 18 and 24 h following the last treatment of the test item . Animals in the positive control group will be sacrificed by CO 2 asphyxiation approximately 24 hours after the last treatment . Femur bones from the sacrificed animals will be excised and the epicondyle tips will be removed. The bone marrow content will be expelled by flushing with foetal bovine serum. The aspirated bone marrow content will be mixed using a syringe to dissociate the cells. Cell clump formation will be avoided, and the content will be centrifuged. The supernatant will be discarded. A minimum number of two slides will be prepared, per animal, with the cell pellet, fixed with methanol.
Slides will be stained using 5% Giemsa . In order to prevent bias in the scoring procedure, the slide numbers will be masked with code numbers provided by the Department of Biostatistics and Systems Information, Jai Research Foundation.

Historical control data
Jai Research Foundation (JRF) has conducted more than 500 GLP studies for regulatory submission as per OECD TG 474 and established a strong historical control data base. JRF uses quality control methods, such as control charts to identify data variability and to show that the methodology is 'under control'. Quality control charts (QC charts) will be added in the report demonstrating the JRFs established historical positive control ranges and distribution, and a historical negative control ranges and distribution.

Microscopic Observation
Slides will be observed under a light microscope. The proportion of immature erythrocytes among the total (immature + mature), i.e., P/E ratio will be determined for each animal by counting a minimum of 500 erythrocytes. A minimum of 4500 polychromatic (immature) erythrocytes, per animal, will be scored for the incidence of micronuclei. Additional information may be obtained by scoring mature erythrocytes for micronuclei.

Statistical Analysis
The data of percent micronucleated polychromatic erythrocytes (% MNPCE), P/E ratio and body weight for both the sexes will be subjected to normality test using Shapiro-Wilk's test and Bartlett's test to assess homogeneity of variance. The data will be analyzed by Chi-square and Fisher's exact test or Student's t-test depending on the nature of the data (Richardson, C. et al, 1989). If the data do show suitable homogeneity of variance, the data will be subjected to Analysis of Variance (ANOVA) followed by Dunnett's t-test . Depending upon the nature of data non-parametric tests will be performed if applicable. If increase in % micronucleated cells are statistically significant, then dose response will be evaluated with an appropriate trend test i.e. Chi-square trend analysis.

Acceptance Criteria
The study will be considered valid as the following criteria are met: i. The vehicle (or negative) controls should be in the range of historical control data. ii.
The positive controls should produce responses that are compatible with that of the historical data and should produce statistically significant responses compared with the concurrent negative control. iii.
Appropriate number of animals, doses and cells has been analysed.

iv.
A minimum of three treatment groups including controls are analysed if the test item produces toxicity. v.
The highest dose should be a limit dose, maximum tolerable dose (MTD) without causing distress or death to the animal or produce toxicity to bone marrow. vi.
The PCE to total erythrocyte ratio should not be less than 20 % of the negative control.

Evaluation and Interpretation Criteria
After fulfilling the acceptability criteria, the test item will be considered clearly positive if: i. At least one of the treatment groups exhibits statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared to concurrent negative control.

ii.
A positive result is defined as a dose-dependent, significant increase in the incidence of micronuclei when evaluated with an appropriate trend test e.g. Chi-square trend analysis.
iii. Statistical and biological relevance will be considered in data interpretation.
iv. Any of the results falling outside the distribution of the historical negative control data i.e. Poisson based 95% control limits.

APPENDIX 6 (Continued) JRF Study Number: 485-1-06-17727 Page 14 of 18
The test item will be considered clearly negative, if, in all experimental conditions examined: i. None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control. ii.
There is no dose-related increase at any sampling time when evaluated by an appropriate trend test. iii.
All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and iv.
Bone marrow exposure to the test item(s) occurred v.
There is no requirement for verification of a clear positive or clear negative response.

REPORT
Unless otherwise instructed by the Sponsor, one copy of the final report will be issued along with one soft copy in PDF. The report will include the following information:

ARCHIVES
On completion of the study all original raw data including any storage medium for electronically recorded data, documentation, the signed study plan, the study plan amendment, slides, the draft report, one original final report, and the representative sample of the test item will be retained in the GLP Archives at Jai Research Foundation for a period of ten years. At the end of this period, the Sponsor's instructions will be sought to either extend the archiving period or return the archived material to the Sponsor or dispose of the material.  A calibration curve of acetamide considered as reference standard concentration ranging from 1.01 to 50.59 ppm was prepared for dose formulation analysis. The coefficient of determination (r 2 ) was 0.99968339 (acceptance criteria: r 2 ≥ 0.98).

APPENDIX 7 (Continued) C. Plasma sample analysis
Five groups (comprising 6 animals/sex/group) were used for this study. Group I was serve as the vehicle control, Group II (250 mg/kg), III (1000 mg/kg) and IV (2000 mg/kg) were low, mid and high dose groups, respectively. Group V was the positive control and received mitomycin-C (1.0 mg/kg body weight on Day 2 of treatment) in distilled water by the intraperitoneal route on a single occasion. Blood samples were withdrawn from each animal in each treatment group and vehicle control group at the time of sacrifice before bone marrow collection. Blood samples were collected in heparinised (20 IU/mL) micro-centrifuge tubes. Blood samples were collected from orbital plexus under very light isoflurane anesthesia. To separate out the plasma, blood samples were centrifuged at 3000 rpm for 15 minutes at 4 °C. The plasma samples were stored at -70 ± 10 °C until analysis.The experimental outline and samples details are as below:

APPENDIX 7 (Continued)
The dose formulation analysis was performed following the validated method (JRF Study N° 228-2-14-17729; "Validation of Analytical method for Determination of Acetamide Concentration, Homogeneity and Stability in Vehicle").
The plasma samples analysis was performed following the validated method (JRF Study N°228-2-14-18476; "Validation of Bioanalytical Method for Determination of Acetamide Concentration using Acetamide-D3 as Reference Standard in Mice and Rat Plasma"). Concentrations were obtained as per below Acetamide was quantified based on the response factor of xanthyl-acetamide (area of xanthyl-acetamide over the area of xanthyl-propionamide) against a calibration plot of response factor of xanthyl-3Dacetamide (area of xanthyl-3D-acetamide over the area of xanthyl-propionamide).

Preparation of working solutions for linearity
The stock dilutions were prepared with diluent as per the table given below from the stock solution 'A'. These solutions were stored at 2 -8 ºC in refrigerator.

Preparation of solutions of quality controls
Weight (

Preparation of spiked matrix CC standards
The above prepared CC spiking solutions were spiked in the interference free blank mice plasma in order to ranged the matrix standards concentrations as per below

Preparation of spiked matrix quality control samples
The above prepared QC spiking solutions were spiked in the interference free blank mice plasma in order to ranged the concentrations as per below

0.5 M Hydrochloric acid solution
50 mL of methanol was transferred in to 100 mL volumetric flask. 4.125 mL of 37 % HCl solution was added in the same volumetric flask. Volume was made equal to mark with methanol. Solution was mixed well.

0.7 M KOH solution
3.9 g of KOH was transferred to 100 mL glass bottle and 100 mL of Milli-Q water was added by using measuring cylinder. Solution was mixed well. Solution was stored in refrigerator until use.

5% Xanthydrol solution
5 g of Xanthydrol was transferred to 100 mL volumetric flask. 50 mL of methanol was added to it and solution was mixed well. Volume was made equal to mark with methanol. Solution was stored in refrigerator until use.

Diluent solution [Methanol: Milli-Q water (50:50), % v/v]
100 mL of Methanol and 100 mL of Milli-Q water were mixed in 200 mL of glass bottle using measuring cylinder and mixed well.

Saturated sodium chloride solution
71g of sodium chloride in 200 mL of Milli-Q water were mixed in 200 mL of glass bottle using measuring cylinder and mixed well.
2.0 mL of 0.7 M KOH was added to each sample tube to neutralize.

8.
3.0 mL of ethyl acetate was added to each tube. Vortexed, sonicated and centrifuged all samples at 10,000 rpm for 5 min.

9.
1.3 mL of supernatant was transferred from each sample to a new ria vial.

10.
Samples were placed on speedovap nitrogen evaporator until all of the ethyl acetate has been removed.
11. 130 μL of ethyl acetate were added to each RIA vial. Sonicated, vortexed and centrifuged at 10000 rpm for 10 min.
12. 100 μL of the supernatant was carefully removed and placed in a GC-MS vial and injected on to GC-MS.  Remarks: * GI-T1_M was identified for repeat analysis due to variation in internal standard response and GII-T14_M was also identified for repeat analysis due to concentration was found to be beyond linearity range.