REPORT ON INTACUTANEOUS IRRITATION TEST OF INDELIBLE INK
IN ALBINO RABBITS
VIMTA LAB LTD 142,IDA, PHASE II
CHERLAPALLY, HYDERABAD -500 051
INDIA.
Study Number: 1965/2003-04
Date: June 20, 2003
SPONSOR
RAYUDU LABORATORIES PVT LIMITED
2-3-703/1/B, TIRUMALA NAGAR
POST BOX NO. 1502,AMBERPET
HYDERABA - 500 013,INDIA.
TESTING FACILITY
TOXICOLOGY DIVISION
VIMTA LABS LTD,142, IDA, PHASE-II
CHERLAPALLY,HYDERABAD-500 051
INDIA.
CERTIFICATE
The present study entitled-"Indelible Ink: Intracutaneous Test in Albino Rabbits" was sponsored by Rayudu Laboratories Pvt. Limited, Hyderabad vide letter No. Nil dated June 13, 2003 and letter Ref No. RL/209/03-04 dated June 14, 2003.
The study was conducted as per IS 12418, Part 4, 1991 [reaffirmed 1996]
The registration number of the above sample at Vimta Labs Ltd is 1965 dated June 13, 2003.
Institutional Animal Ethics Committee approved the protocol of the study.
The study was conducted between June 14 and 17, 2003.
The study complies with the regulations of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Dr. D. Mohana Ranga Rao, M.Sc.,Ph.D.
Study Director
June 20, 2003.
ABSTRACT
The present work was carried out to evaluate the safety of the extracts (Intracutaneous irritation test) of the test substance, Indelible Ink by conducting intracutaneous irritation test in albino rabbits as per IS 12418 (Part 4) 1991 [reaffirmed1996].
Extracts of the test substance were prepared in sodium chloride solution (0.9% w/v) and 1 in 20 solution of alcohol in sodium chloride solution.
The test extracts were injected intracutaneously (0.2 ml/site), on each side of the spinal column, using one side for test extract while the other side was used for blank (control) extract. The test extract was injected at 10 sites of each of the two rabbits. On the other side, blank extract was injected at 5 sites in a similar fashion. The injected sites were observed at 4, 24, 48 and 72 hours after the injection for gross evidence of tissue reactions such as erythema and oedema.
The results indicate administration of sodium chloride extract or 1 in 20 solution of alcohol in sodium chloride solution extract of the test substance did not produce any evidence of irritation, viz, erythema or oedema at the injection sites. The results of the test sites were comparable to that of the control (Blank) sites.
Thus the test substance, Indelible Ink did not cause any irritation when injected intracutaneously in albino rabbits. It meets the requirements of intracutaneous test in albino rabbits as per IS 12418 (Part 4): 1991 [reaffirmed 1996].
IDENTIFICATION AND DETAILS OF THE TEST SUBSTANCE
[AS PROVIDED BY THE SPONSOR]
Identity: Indelible Ink
Intended Use: Marker
Batch Number :005/03-04
Date of Manufacture: June 2003
Date of Expiry: not provided
Color Tinted : voilet Liquid
Acidity (pH) : between 1 to 3 (in gerneral it will be less than 2)
Photosensitve chemicals % (total solids): not less tha 10%
Drying time : with in 40 seconds
Performance : Test Rectified spirit, trichloroethylene
Storage conditions : store in a cool and dark place away from light and fire
Quantity Supplied : 2 bottles
Type of Packing : In a plastic bottle
Date of Sample Receipt : June 13, 2003.
Other details : if any Nil
Animal Management
Healthy albino rabbits of both sexes (New Zealand strain), in the weight range of 1.8 to 2.0kg, procured from the Mahaveera Enterprises, Hyderabad [CPCSEA Registration Number 146/1999/CPCSEA] were used in the present study. The rabbits were housed individually in stainless steel cages in the animal house. The animals were provided with commercially available laboratory pellet diet (Amrut Laboratory Animal Feed) and drinking water ad libitum. In addition, the rabbits were provided with Lucerne grass daily. Animal room temperature was maintained at 22 ± 3° C and relative humidity at 30-70% The Photoperiodicity was maintained at 12 hours of artificial light (0700-1900 Hours) in each 24 hours period.
PROCEDURE
Sample Preparation
The test substance (~ 4ml) was applied to a glass plate and allowed to dry. The dried film was extracted with 20 ml of the extracting medium using an autoclave at 121.0 ± 2° C for one hour. The extracting ratio was ~0.2ml of dried film per ml of extracting liquid. The extracted liquid was allowed to cool, shaken vigorously and decanted and stored into dry sterile vessel until injection.
Grouping of animals
For each extraction vehicle 2 animals were used. Each group comprised of both male and female animals.
Identification of Animals
Each animal was identified by color codes. Through out the study each cage was identified by a label having the recording of the study number, animal numbers, sex, body weight and details of treatment.
Method
About 24 hours before testing, fur from back of rabbit on both sides of the spinal column was removed close to the skin and the clipped area was cleaned with surgical spirit and allowed to dry. The dosage and other details are given as below
Two animals were used for each extract. Extracts were injected intracutaneously, on each side of the spinal column, using one side for the test extract and the other side for control (blank) extract. The test extract was injected, at a dose of 0.2 ml per site, at 10 sites on one side of each of the two rabbits. Similarly at 5 sites on the other side of each rabbit the corresponding blank was injected in a similar fashion.
Observation Period
The injected sites were examined at 4, 24, 48 and 72 hours after the injection for gross evidence of tissue reaction, such as, necrosis, erythema and oedema. The observations were rated on a numerical scale for the test extract of the sample and for the corresponding blank using the following guidelines.
Interpretation
The requirements of the test are met if the difference between average test and blank (control) mean score is 1.0 or less.
RESULTS
Intracutaneous administration of the extract of the test substance to albino rabbits failed to cause any erythema or oedema in animals. The results were comparable to that of the blank extract (Table 1)
Table 1: Summary of Intracutaneous Test of Extracts of Indelible Ink to Albino Rabbits.
Interpretation
A comparison of the scoring of the intracutaneous test of test extract and blank extract either in sodium chloride or 1 in 20 solution of alcohol in sodium chloride solution did not indicate any difference in albino rabbits (Table 2).
Table 2: Comparison of the Scoring of the Test and Blank Extracts in Albino Rabbits
CONCLUSION
The sites treated with different test extracts showed no signs of erythema and oedema following the intracutaneous injection to albino rabbits. The difference between test and blank mean scores, for both the sodium chloride extract and 1 in 20 solution of alcohol in sodium chloride solution extract, was found to be zero. Thus, the test substance Indelible Ink did not cause any evidence of irritation. The test substance meets the requirements of Intracutaneous test in albino rabbits
REFERENCES
Extractables Test Annex A: Systemic Injection Test and Intracutaneous Test, IS 12418 (Part 4). 1991.Biological Reactivity Tests IN VIVO, Appendix 88, USP 24, 2000.
