Summary: The Agar Overlay test was designed to determine the cytotoxicity of diffusible components
from materials or solutions. A layer of agar was added over a cell monolayer to act as a cushion to
protect the cells from mechanical damage while allowing the diffusion of leachable materials. The test
articles were then placed on top of the agar layer and incubated. The cell monolayers were examined
and scored based on the degree of cellular destruction. All test method acceptance criteria were met.
The test procedure(s) listed above were followed without deviation.
Results:
Test Article:
Results Scores
Amount Tested
Pass/Fail #1 #2 #3 Average
Pass 0 1 1 1 = 100 mm? per well
Controls:
aR] Scores
Identification Amount Tested
#1 #2 #3 Average
Negative Control — 2
Polypropylene Pellets 0 Y 4 4 = 100 mm” per well
Positive Control = 4 4 4 4 2 100 mm? per well
Latex Natural Rubber
Acceptance Criteria: The United States Pharmacopeia & National Formulary (USP <87>) states that the
test article meets the requirements if the reactivity grade is not greater than grade 2 or a mild reactivity.
The ANSI/AAMIZISO 10993-5 standard states that the achievement of a numerical grade greater than 2 is
considered a cytotoxic effect. Nelson Laboratories acceptance criteria was based upon the negative
control receiving “0″ reactivity grades and positive control receiving 3-4 reactivity grades (moderate to
severe).
(aatass
WE /3
Study Director Chad Summers, A.S., ASQ CQA Study Completion Date
20. Box 571830 | Murray, UT 84157-1830 USA – 6280 South Redwood Road | Salt Lake City, UT 84123-8600 USA
www.nelsonlabs.com – Telephone 801 200 7500 – Fax 801 290 7998 . sales@nelsoniats.com
aly FRT0031-0001 Rev 3
Page 1 of 3
Ties? results late andy to (he tast artice ksted 0 this report. Reports may not be (eproduced excep! m ihe antitety. Subject to NL terms and corations al www nelsentabs cons
Laboratory Number 709563
Agar Overlay GLP Report
LABORATORIES
Procedure: Six well cell culture plates were seeded with a verified quantity of industry standard L-929
cells (ATCC CCL-1) and incubated at 37 + 1°C with & + 1% CO, until approximately 80% confluent. The
agar overlay consisted of an equal mixture of INAGAR (1.0%) and 2X MEM + 10% bovine calf serum.
Liquid or gel test articles were applied to sterile filter discs testing no less than 0.1 mL per well. Positive
and negative reference controls were included with each assay.
All tests were performed using three test wells per test article. After the addition of the test articles, the
cell culture plates were incubated as described above for 24-26 hours. Following incubation, cells were
evaluated microscopically using the evaluation criteria outline below:
Grade Description Of Zone
0 No detectable zone around or under the test article.
1 Some malformed or degenerate cells under the test article.
2 Zone limited to area under the test article and less than 0.45 cm beyond the test article.
3 Zone extends 0.45 to 1.0 cm beyond the test article.
4 Zone extends greater than 1 cm beyond the test article.
The results from the three wells were averaged to give an average cytotoxicity score.
PO. Box 571830 | Murray, UT 84157-1830 USA, – 6280 South Redwood Road | Salt Lake Criy, UT 84123-6600 USA.
aly FRT0031-0001 Rev 3
www.nelsonlabs.com + Telephone 801 2980 7600 – Fax B01 230 7998 . sales@nelsonlabs com
Page 2 of 3
Laboratory Number 709563
NE ON Agar Overlay GLP Report
Compliance Statement: The test was conducted in accordance with the USFDA (21 CFR Part 58)
Regulations. This final report reflects the raw data.
Activity Date
Study Initiation 04 Sep 2013
Phase Inspected by ality Assurance: 05 Sep 2013
Interpretation
Audit Resuits Reported to Study Director 06 Sep 2013
Audit Results Reported to Management 06 Sep 2013
Scientists Title
Michelle Lee Supervisor
Chad Summers Study Director
Data Disposition: The study plan, raw data and final report from this study are archived at NLI or an
approved off-site location.
: ier = is 56° 271
2 Bang
valify Assuré&nce Date
P.O. Box 571830 | Murray, UT 84157-1830 USA. – 6280 South Redwood Road | Salt Lake City, UT 84123-6600 USA. aly FRT0031-0001 Rev 3
www.nelsontabs.com – Telephone 801 290 7500 – Fax 801 280 7998 – sales@nelsonlabs.com
Page 3 of 3