FIELD OF INVENTION
The present invention provides a method for in vitro screening of chemical compounds for predicting acute toxicity.
BACKGROUND OF INVENTION
The identification of a lead molecule is a very long and expensive process as the potential of an\ new chemical entity is evaluated by in-vivo toxicity studies, which are very lengthy to execute and a large number of animals are also required. (Bonkowsi Z and Howard N(1984) Biomedical Research Involving Animals, The Council for International organization of Medical Science) This animal testing is carried out because of belief that the effect produced by any new chemical entity in animals can be simulated in humans as well but many a times this is not the actual case. Therefore, there has been an increased awareness to address these problems.
Modern drug development process progresses through a series of stages in which a vast librarY of chemical compounds is screened to gradually narrow down to identify a lead molecule. The use of animals in the initial stage of drug development is an expensive and inefficient method of producing toxicological data for new compounds, as not all of them are potential drug candidates. In the past decade, a number of laws that mandate replacement, reduction and retlnement alternatives have been implemented in most of the advanced countries. Toxicity Testing in Animals: Alternative Models Environmental Health Perspective, 101, 1993) Earlier, classical I.D50 test procedure was used to establish LD50 values to calculate the safety index and provide the guidance lor dose selection in repeat-dose tests in animals but now OECD guidelines for acute toxicity by classical method has been deleted, in favor of refined guidelines e.g. fixed dose, up and down procedure, acute toxic class method to ensure that regulatory requirements are fulfilled using fewer animals.
A number of approaches for toxicological screening prior to the animal testing are available a* an alternative screening methods. One of them is to incorporate in vitro toxicity testing of compounds of interest into drug discovery process at the time when new compounds are being identified against some therapeutic targets. Quality toxicity data produced at this stage enable the scientist to design-out the toxicity and yet retain efficiency/potency at the same time.
Practically, there are many challenges need to be addressed in developing robust in vitro toxicological screening methods, which can be used as predictors of acute toxicity. Therefore, there is a need to develop a battery of rapid, cost-effective and accurate methods of in vitro screening employing appropriate cell lines to predict acute human toxicity at early stages of drug development

process in order to save the time and excessive use of animals for determining the safety of new chemical entities.
US 6,998.249 describe an in vitro method to determine the level of acute toxicity of a chemical compounds. A Report of the International Workshop NIH Publication No: 01-4499, August 2001, discloses In Vitro Methods for Assessing Acute Systemic Toxicity.
The present invention deals with evaluation of in vitro method and their validation for predicting acute toxicity.
DESCRIPTION OF THE INVENTION
The present invention describes in vitro methods using different primary and secondary cultures to predict the acute toxicity of chemical compounds by employing a method, which include generally:
1. Culturing primary rat hepatocytes. HepG2, CHO and C2C12 cell lines in the presence of plurality of concentrations of the said chemical compound
2. Assessment of cytotoxicity by using MTT assay
3. Calculating IC50
4. Calculating or collecting of LD50 data
5. Comparison of IC50 values with the LD50 values of the chemical compounds and calculation for regression analysis for IC50 and LD50 values
6. Prediction of GHS acute oral toxicity category by IC50 using regression equation
In another embodiment, in the in vitro test system, the cells are primary rat hepatocytes.
In another embodiment calculation of LD50 was done for the compounds for which acute toxicity was enaluated during the study or LD^ndata was collected from literature.
In yet another embodiment, the regression equation is log millimole regression equation
In yet another embodiment, the regression equation is
y = mx+c wherein, y = Log LD-50 (mmol/kg) and x " L.og IC-50 (mmol/1)

MATERIALS AND METHODS Chemicals
Hank's balanced salt solution (HBSS). minimum essential medium (MEM), triton X-100, ethylene glycol tetra acetic acid (EGTA) were obtained from Sigma-Aldrich, USA. Fetal bovine serum (FBS) was procured from Gibco. Collagenase and collagen type I were obtained from 1CN biomedics, USA.
Cell Lines
Cell lines viz. CHO,C2C12 and HepG2 were received from American type culture collection [Atcc],Manassas,VA,USA.
Compounds/Drugs
Cytotoxicity of a number of drugs/compounds was evaluated and IC50 values were calculated. Most of the compounds selected were drugs. These drugs belonged to various therapeutic classes viz. Analgesic. Antipyretic, Anti-inflammatory. Antacid, Anti a-Adrenergic, Antihistamine. Anliserotonergic, Anti Muscaranic, Anti Gastroenteritis. Antiallergic, Antibacterial, Anticancer, Anticonvulsant, Antifungal, Antidepressant, Antihypertensive, Antimalarial, Antipsychotic, Cholesterol Lowering, Anxiolytic and Gastric Secretion Stimulant. There were few other chemicals e.g. detergents, organic solvents, mutagens and mycotoxin. Maximum numbers of compounds were procured from Ranbaxy Research Laboratories; few compounds were procured from Sigma. For Mitomycin C and Diazepam marketed and approved formulations were used and procured from the local chemist.

Preparation of Media And Solutions
DMEM (Dulbecco's Modified Eagle's Medium)
DMEM Powder I Sachet
Dextrose 4.5 gm
NallCO3 1.5 gm
Genamycin 0.05 gm
Sterile Water to make final volume 1L
MEM ( Minimum Essential Medium)
MEM Powder 1 Sachet
Nal IC03 2.2 gm
IIHPKS 2.38 gm
l.-Glutamine 0.292 gm
(ienlamycin 0.05 gm
Sterile Water to make final volume 1L
Perfusion Media
1 Bottle HBSS 1 L
Nal ICO., 2.2 g
IIEPHS 2.38 g
Kil'A 0.16 g
Sterile Water to make final volume 1 L
Digestion and Cell Wash Media
I Bottle HBSS I L
NaHCO, 2.2 g
CaCI;.2M2O 0.3 gm
Collagenase 0.05%
Sterile Water to make final volume 1L
Note: Collagenase was not added in cell wash media.
Phosphate Buffered Saline ( PBS)
NU2HPO4 2.38 g
KII2PO4 0.190 g
NaCl 8 g
Water to make final volume 1L

COLLAGEN COATING OF TISSUE CULTURE PLATES
Collagen Type I was dissolve in 0.02 N Acetic Acid to prepare a solution of cone. 50 u.g/ml. Collagen solution was poured into the 96-well plate at 50 u,l/well and then the plates were kept for at least 2 hours. After 2 hours the collagen solution was removed and the plates were washed with phosphate buffer saline. Coated plates were sealed with parafilm and stored in refrigerator till use.
Isolation, Culture and Maintenance of Cell Lines
CULTURES AND MAINTENANCE OF PRIMARY RAT HEPATOCYTES
Animals
Specific pathogen free (SPF), healthy male Wistar rats of 200-250 g were procured from Ranbaxy's Animal Breeding and Housing facility (ABHF). Animal room temperature and relative humidity was maintained at 22±3 °C and 50-70% humidity respectively. Illumination was controlled to give 12-hours light and 12 hours dark cycle. Prior to study, animals were subjected to veterinary examination for general health.
Isolation of hepatocytes
I he hepatocytes were isolated by two-step collagenase perfusion technique (Nicholas, 1992. Gomez l.echon et al., 1996, Seglen, 1994). The abdomen of the rats was opened under thiopentone sodium anesthesia and the portal vein cannulated with a catheter. The cells were isolated by a two-step collagenase perfusion technique. Liver was perfused with Ca++ and Mg++ free HBSS containing EGTA and HEPES pH 7.3 at 37 °C. Thereafter, a second perfusion with HBSS containing HEPES, Ca1 and 0.05% Collagenase at 37 °C was performed. The excised liver was transferred to Petri dish containing second perfusion media without collagenase, cooled on ice and cells were gently dispersed with forceps. The crude cell suspension was filtered through gauze, centrifuged at 200 rpm for two minutes and washed 3 times with second perfusion media without collagenase. Trypan blue dye exclusion method was used for viability testing.
Primary monolayer culture of rat hepatocytes
I lepatocytes with more than 80% viability were seeded in 96 well collagen coated plates at seeding density of 0.5x105 cells /well/ 100µl of MEM supplemented with 5% FBS in triplicate and were incubated for 2-3 hours in CO2 incubator maintained at 5% CO2, 98% humidity and 37°C

temperature. After 2-3 hours of plating, primary hepatocytes got adhered and attained their epithelial morphology.
Procedures for maintenance of cell lines
All the cell lines used in the study were routinely grown as a monolayer in tissue culture grade flasks (e.g., 75 or 25 cm2) at 37 °C ±1 °C\ 90% ±10% humidity, and 5.0% ±1.0% C02/air.Cell were examined routinely (i.e., on workdays) basis under a phase contrast microscope. All cell culture studies followed good cell culture practices (llartung et al. 2002).
Receipt of Cell Lines
Upon receipt, vials of cryopreserved cells were stored in a liquid nitrogen cylinder until needed.
Thawing of Cells
frozen cells were removed from liquid nitrogen cylinder and thawed by putting ampoules immediately into a water bath at 37 °C ±1 °C. As soon as the cells thawed, they were resuspended into pre-warmed Culture Medium and transferred to tissue-culture flask.
Masks were incubated at 37 °C ±1 °C. 90% ±10% humidity, and 5.0% ±1.0% C02/air. After 24 hours of incubation, when the cells had attached to the bottom of the flask supernatant was removed and replaced with fresh pre-warmed (37°C) culture medium.
Routine Culturing of Cells Lines
I rypsinization of cells was done whenever they exceeded 50% confluency (but were less than 80% confluent. For trypsinization culture medium was removed from the culture flask and cell monolayer was briefly rinsed with PBS gently, to remove any remaining serum that might inhibit the action of the trypsin. Trypsin-EDTA solution was added and cultures were incubated at room temperature for few seconds and then observed under the phase contrast inverted microscope for rounding of cells. After 2-3 minutes, the flasks were lightly tapped to detach the cells into a single cell suspension. After the cells were detached, monolayer was dispersed by gentle trituration to. obtain a single cell suspension for exaci counting. The cell suspension was counted using a hemocytometer.

Subculture of Cells
Cell lines were routinely sub-cultured into other flasks or seeded into 96-well microtiter plates and passaged at suggested cell densities as mentioned in the subsequent sections.
Cryopreservation of Cells
Cell suspension of lxl06 cells/ml was made in freezing media (culture media with 10% DMSO). Cell were kept in the 1° cryo cooler and brought to the freezing temperature.
Containers were kept in freezer (-70 to -80 °C) for 24 hours (-freezing rate of 1 °C/minute) and then the cryovials were transferred to liquid nitrogen for storage.
Cultures and Maintenance of HepG2 Cells
IIepG2 is human hepatocellular carcinoma cell line. HepG2 cells were isolated from a liver biopsy of a 15-year-old male Caucasian with a well-differentiated hepatocellular carcinoma. These cells have an epithelial-like morphology. Routinely HepG2 cells were maintained as monolayer culture in complete medium at 37°C in a humidified 5% CO3 incubator with medium changed every 3-4 days. Cultures were sub cultivated after every 6-8 days in the ratio of 1:4 to 1:6. For calculating IC50, cells were suspended in MEM supplemented 10% FBS and 50 mg /L gentamycin at lx104 cells /ml final density and seeded in 96 well plates at seeding density of 0.1-0.2x104 cells /well/ 100µl of MEM in triplicate and incubated for 24 hrs in CO2 incubator maintained at 5% CO2, 98% humidity and 37 °C temperature.
Cultures and Maintenance of CHO Cells
I he Chinese hamster ovary (often abbreviated CHO) tissue is commonly cultured as individual cells in a monolayer and studied worldwide. Chinese hamster ovary (CHO) cells were introduced in the early 1960s as a viable epithelial cell line containing twin female X chromosomes. Doubling time for CHO cell is 14-17 hours. Cultures were sub cultivated at semi confluent stage in the ratio of 1:6 to 1:10 after every 3-4 days. CHO cells were suspended in MEM supplemented 10% FBS and 50 mg /L gentamycin at lx104 cells /ml final density. Cells were seeded in 96 well plates at seeding density of 0.1-0.2 x104 cells /well/ 100µl of MEM in triplicate and incubated for 24 hrs in CO2 incubator maintained at 5% CO2, 98% humidity and 37°C temperature.
Cultures and Maintenance C2C12 cell line
C2C12 is Mouse myoblast cell line. C2Cl2 are fibroblastic which subclone from myoblast line established from normal adult C3H mouse leg muscle. These cells differentiates rapidly; produces

extensive contracting myotubes expressing characteristic muscle proteins. Doubling time for C2C12 cell is 20 hours. Cultures were sub cultivated at semi confluent stage in the ratio of 1:6 to 1:10 after every 2-4 days. Cells were suspended in MEM supplemented 10% FBS and 50 mg/L gentamycin at a final density of 7.5 x 103 cells /ml. Cells were seeded in 96 well plates at seeding density of 0.75x10J cells /well/ 100jal of MEM in triplicate and incubated for 24 hrs in COT incubator maintained at 5% COi, 98% humidity and 37°C temperature.
4. Drug Concentration Preparation
Appropriate solvent were used for different compounds and different drug concentrations were chosen on the basis of solubility and precipitation test in the MEM and also based on the preliminary experiments. The final concentration tested for different drugs are mentioned in results.
5. Drug treatment
After appropriate incubation period culture media was aspirated and replaced with an equal volume of the media supplemented with different drug concentrations followed by incubation in COT incubator for 24 hours. Different drug concentrations were added to 96-well plate as mentioned below in Figure I.

(Figure Removed)

figure I: Treatment of Cells with Different Drug Concentrations W here.
VC- Vehicle Control
VCB- Blank (Vehicle containing no cells) l)NC\B- Blank (Drug concentrations containing no cells) D1.4- lour Different drugs

(1-5- Different concentrations of drug
Assessment of Cytotoxicity by MTT Assay
Viability of cells was assessed after approximately 24 hours incubation with test drug by MTT reduction assay. The MTT assay is based on metabolic reduction of 3'(4.5 diethyl thiazolyl-2-yl)-2.5 diphenyl tetrazolium bromide. 50u.l of MTT solution (Img/ml) in MEM without serum was added directly to the microtitre plate wells containing cells complete medium and test drugs. The culture was then incubated for approximately 3-4 hours to allow for MTT metabolism to formazan, following incubation the supernatant was aspirated and 150 µl of DMSO added to dissolve the formazan. Plates were agitated on a plate shaker in order to ensure that a homogenous solution is formed. Thereafter, the optical density was measured on Elisa reader (Medispec) at a single wavelength of 540nm (Monks et al., 1991).
lQo Calculation
I o calculate ICso value (concentration that produced 50% cell death) the results were transformed to % of controls and dose - response curve were made and the IC50 values were interpolated mathematically by linear regression method as well as non linear regression method. For all the compounds linear method was used for ICso calculations. However, only for the compounds which showed more than 50% cytotoxicity at the highest concentration non linear method was used.

EXPERIMENTAL PROCEDURE
Study Design
An outline of the study design is presented in the following table:
Table 1 - Study Design

(Table Removed)

Justification for Dose Selection
For Dose Range Finding Study does were selected based on the literature or efficacy data. For Confirmatory Study doses were selected based on the results of dose range finding study.
Dose Volume
The dose volume was 10 or 20 ml/kg body weight for all animals.
Route and Method of Administration
The volume lo be administered was calculated based on the treatment day body weight of the animals. The test item and vehicle were administered to the animals by oral route using intubation needle. Control group received vehicle only whereas other groups received the test item in vehicle.

Formulation Preparation
The required formulations was prepared by dissolving/suspending the drug in appropriate vehicle recommended for oral administration. The concentration verification of the all the dose formulations was performed
OBSERVATIONS
I he following observations were made during the course of study for calculation of LD-50.
Clinical Signs and Mortality
Animals were examined for clinical signs and mortality at 10-15 min after each dose administration, thereafter at 30-40 min, 1 hr ± 10 min. 4 hr ± 10 min and 24 hr ± 10 min post dosing followed by once daily throughout the observation period. Any animal showing signs of severe debility or intoxication, particularly if death appeared imminent were euthanized.
Calculations
Percent mortality was calculated in different treatment groups as compared to control and LD-50 values were calculated depending on the dose response data using dose response curve by non linear regression method.
8. Calculation for regression analysis for ICso and LD50 values
linear regression analysis was performed for the calculated IC50 values from each of the in-vitro test system used viz. primary rat hepatocytes, CHO Cell line, C2C12 cell line and HepG2 cell line and oral LD-50 values for mice and rat. IC-50 values in (u,g/ml) were converted into the mmol/l and both the values were transformed into log values. LD-50 values in mg/kg were also transformed to mmol/kg and both mg/kg as well as mmol/kg values were further transformed to log values.
To evaluate and compare the predictivity and correlation between the IC-50 values calculated by different models and LD-50 values in rat and mice, separately, regression analysis was performed for the following models:

r IC -50 values (µg/ml) by linear method versus the corresponding oral LD- 50 (mg/kg)
values (Weight Regression -Linear) r IC -50 values (mmol/l) by linear method versus the corresponding oral LD- 50 (mmol/kg)
values (Millimole Regression - Linear) r Log IC -50 values (µg/ml) by linear method versus the corresponding oral Log LD- 50
(mg/kg) values (Log Weight Regression -Linear) r IC -50 values (mmol/l) by linear method versus the corresponding oral Log LD- 50
(mmol/kg) values (Log Millimole Regression - Linear) r- IC -50 values (µg/ml) by non linear method versus the corresponding oral LD- 50 (mg/kg)
values (Weight Regression - Non Linear) r IC -50 values (mmol/l) by non linear method versus the corresponding oral LD- 50
(mmol/kg) values (Millimole Regression - Non Linear) r Log IC -50 values (µg/ml) by non linear method versus the corresponding oral Log LD-
50 (mg/kg) values (Log Weight Regression - Non Linear) r IC -50 values (mmol/l) by non linear method versus the corresponding oral Log LD- 50
(mmol/kg) values (Log Millimole Regression -Non Linear)
I hese regression analyses were performed for LD-50 values in rat and Mice separately.
In addition, the mean IC-50 values were calculated for each reference substance considering the individual IC-50 values for each in-vitro test system (Primary rat hepatocytes, CHO Cell line, C2Cl2 cell line and HepG2 cell line) and a combined regression equation was generated using the mean IC- 50 values and corresponding LD-50 values.

Table 2 - Regression Equation Components For Log Millimole Regression -Linear
Using Rat LD-50 Values

(Table Removed)

Table 3 - Regression Equation Components For Log Millimole Regression -Linear
Using Mice LD-50 Values

(Table Removed)

IC -50 values (linear) in mmol/1 and LD50 values of mice and rat in mmol/kg were transformed into log values and then regression analysis was performed. Results of regression Analysis showed that in this model also best correlation was observed for primary rat hepatocytes. Correlation for CHO. C2C12 and HepG2 cell lines was not much different. The

order of correlation for all cell lines was Primary rat hepatocytes > CHO> C2C12> HepG2 as observed for millimole regression.
In case of mice also the results were similar to rat, for order of correlation as well as goodness of fitting of data.
Table 4 - Regression Equation Components For Log Millimole Regression - Non Linear
Using Rat LD-50 Values

(Table Removed)

Table 5 - Regression Equation Components For Log Millimole Regression - Non Linear
Using Mice LD-50 Values

(Table Removed)

10. Prediction of GHS acute oral toxicity category by IC30 using regression equation.
further to the regression analysis, using the IC-50 values generated during the study by MTT method for different test systems, LD-50 values were predicted by using respective regression equations. These predicted LD-50 values for different compounds/drugs were then used to categorize the compounds into different categories as per the Global Harmonization System (GHS) of classification and Labeling of Chemicals. Then the predicted GHS categories were compared with the actual category of the compound as per their actual LD-50 values generated by the in-vivo test, to find what percentage of the compound was categorized correctly, as well as what percentage of compound was categorized in ± I category. This prediction was done for each of the category using each of he regression model and each in-vitro test system, to find out beal model and best in-vitro test system
Log Millimole - Linear vs Rat
(Table Removed)

Log Millimole - Linear vs Mice
(Table Removed)

Log Millimole - Non Linear vs Rat

(Table Removed)
Log Millimole - Non Linear vs Mice

(Table Removed)

WE CLAIM:

1. An in-vitro method using different primary and secondary cultures to predict the acute
toxicity of chemical compounds by employing a method, which include generically:
a) Culturing primary rat hepatocytes. HepG2, CHO and C2C12 cell lines in the presence of plurality of concentrations of the said chemical compound
b) Assessment of cytotoxicity by using MTT assay
c) Calculating IC50
d) Calculating or collecting of LD50data
e) Comparison of IC50 values with the LD50 values of the chemical compounds and
calculation for regression analysis for IC50 and LD50 values
0 Prediction of GHS acute oral toxicity category by IC50 using regression equation
2. The cells of in-vitro test system according to claim I are primary rat hepatocytes.
3. The regression equation according to claim 1 is log millimole regression equation for all cell lines and combined equation
4. The regression equation according to claim 1 is
y = mx+c wherein, y = Log LD-50 (mmol/kg) and x = Log IC-50 (mmol/I)