PREPARED FOR:
KEITH MOELLER
INTRODUCTION:
The MEM Elution test was designed to determine the cytotoxicity of extractable substances. An
extract of the sample was added to cell monolayers and incubated. The cell monolayers were
examined and scored based on the degree of cellular destruction.
ACCEPTANCE CRITERIA:
The United States Pharmacopeia & National Formulary states that the sample meets the
requirements if the reactivity grade is not greater than grade 2 or a mild reactivity. The
AAMI/ISO 10993-5 standard states that the overall assessment of the results shall be made by
capable persons based upon the data and results. Nelson Laboratories acceptance criteria was
based upon the negative and media controls receiving “0″ reactivity grades and positive results
receiving a 3-4 reactivity grades (moderate to severe).
PROCEDURE:
The amount of test material extracted was based on ANSI/AAMI/ISO and USP surface area
recommendations or weight (0.20 g/mL extract fluid for polymers and plastic). The sample was
extracted for 24-25 hours at 37 + 1°C in 1X Minimal Essential Media with 5% calf serum.
Positive (Latex Natural Rubber) and negative (Polypropylene Pellets) controls were extracted
and included in the assay. A blank of extraction media (media control) was also included in the
assay.
Page 2 of 5
NELOAON
LABORATORIES
American Biotech Labs MEM Elution
Lab Number 452240
Multiple well cell culture plates were seeded with a verified quantity of L-929 cells and incubated
until 80-90% confluent. The cell culture media was removed from the plates. The test extracts
were filtered and the appropriate amount of extract was added to each well on the cell culture
plates. Each extract was tested on three wells of cells. The cells were incubated at 37 + 1°C
with 5 £ 1% CO; for 72 £ 3 hours.
The cell monolayers were examined microscopically. The wells were scored as to the degree of
discernable morphological cytotoxicity on a relative scale of 0 to 4:
CONDITIONS OF ALL CULTURES REACTIVITY GRADE
No cell lysis, intracytoplasmic granules. NONE 0
Not more than 20% rounding, occasional lysed cells. SLIGHT 1
Not more than 50% rounding, no extensive cell lysis. MILD 2
Not more than 70% rounding and lysed cells. MODERATE 3
Nearly complete cell destruction. SEVERE 4
The results from the three wells were averaged to give a final cytotoxicity score.
RESULTS:
The resuits are summarized in Table 1. The test is acceptable if all three of the negative control
and medium control test wells have a score of 0 and all three of the positive control test wells
have a score of 3 or higher.
The sample meets USP requirements if none of the cell culture exposed to the sample shows
greater than a mild reactivity (grade 2).
STATEMENT OF UNCERTAINTY:
If applicable, the statement of uncertainty is available to sponsors upon request.
Technical Rexiewer Bobbi Rushton-Castro
Study Director
o\ Det 2006
Study Completion Date
rsh
Page 3 of 5
NEDON
LABORATORIES
American Biotech Labs MEM Elution
Lab Number 452240
TABLE 1. Results
IDENTIFICATION SCORE #1 | SCORE #2 | SCORE #3 | AVERAGE
Negative Control
Media Control
Positive Control
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Page 4 of 5
NEDON
LABORATORIES
American Biotech Labs MEM Elution
Lab Number 452240
All reports and letters issued by Nelson Laboratories, Inc. are for the exclusive use of the
sponsor to whom they are addressed. These results relate only to the samples tested. Reports
may not be reproduced except in their entirety. No quotations from reports or use of the
corporate name is permitted except as expressly authorized by Nelson Laboratories, Inc. in
writing. The significance of any data is subject to the adequacy and representative character of
the samples tendered for testing. Nelson Laboratories, Inc. warrants that all tests are performed
in accordance with established laboratory procedures and standards. Nelson Laboratories, Inc.
makes no other warranties of any kind, express or implied. Nelson Laboratories, Inc. expressly
states that it makes no representation or warranty regarding the adequacy of the samples
tendered for testing for any specific use of application, that determination being the sole
responsibility of the sponsor. Nelson Laboratories’ liability for any loss or damage resulting from
its actions or failure to act shall not exceed the cost of tests performed, and it shall not be liable
for any incidental or consequential damages.
AMERICAN BIOTECH LABS
80 W. CANYON CREST RD.
ALPINE UT 84004
SUBMITTED BY:
NELSON LABORATORIES, INC.
6280 S. REDWOOD RD.
SALT LAKE CITY UT 84123-6600
801-290-7500
Page 1 of 5
LABORATORIES
MEM ELUTION
LABORATORY NUMBER: 452240
PROCEDURE NUMBER: STP0032 REV 03
SAMPLE SOURCE: American Biotech Labs
TYPE OF TEST: Solid
SAMPLE IDENTIFICATION: Refer to Table 1
DEVIATIONS: None
CELL LINE: Mouse Heteroploid Connective Tissue (1-929)
INCUBATION PERIOD: 72+ 3 hours at 37 £ 1°C
METHOD OF SCORING: Cytopathic Effect (0-4)
AMOUNT TESTED/SAMPLE EXTRACT: 3g/15.0mL
SAMPLE RECEIVED DATE: 21 Nov 2008
LAB PHASE START DATE: 24 Nov 2008
LAB PHASE COMPLETION DATE: 29 Nov 2008
REPORT ISSUE DATE: 01 Dec 2008