(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:11)(cid:3)(cid:6)(cid:12)(cid:3)(cid:13)(cid:6)(cid:14)(cid:15)(cid:16)(cid:6)(cid:11)(cid:6)(cid:17)(cid:18)(cid:19)(cid:3)(cid:20)(cid:7)(cid:21)(cid:10)(cid:9)(cid:22)(cid:6)(cid:7)(cid:23)(cid:3)(cid:10)(cid:9)(cid:24)(cid:3)(cid:25)(cid:16)(cid:7)(cid:11)(cid:10)(cid:8)(cid:3)(cid:13)(cid:6)(cid:14)(cid:15)(cid:16)(cid:6)(cid:11)(cid:6)(cid:17)(cid:18)
`
`(cid:26)(cid:27)(cid:27)(cid:28)(cid:19)(cid:3)(cid:29)(cid:30)(cid:31)(cid:32)(cid:4)(cid:31)(cid:33)(cid:34)(cid:35)(cid:3)(cid:36)(cid:37)(cid:8)(cid:15)(cid:9)(cid:21)(cid:38)(cid:3)(cid:36)(cid:25)(cid:9)(cid:11)(cid:15)(cid:9)(cid:22)(cid:38)(cid:3)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:11)(cid:3)(cid:39)(cid:6)(cid:40)(cid:22)(cid:41)(cid:10)(cid:17)(cid:22)(cid:19)(cid:3)(cid:39)(cid:21)(cid:21)(cid:41)(cid:23)(cid:19)(cid:42)(cid:42)(cid:43)(cid:43)(cid:43)(cid:44)(cid:21)(cid:10)(cid:9)(cid:24)(cid:12)(cid:6)(cid:9)(cid:11)(cid:15)(cid:9)(cid:22)(cid:44)(cid:16)(cid:6)(cid:40)(cid:42)(cid:11)(cid:6)(cid:15)(cid:42)(cid:15)(cid:16)(cid:6)(cid:21)(cid:32)(cid:35)
`
`(cid:13)(cid:39)(cid:22)(cid:3)(cid:45)(cid:23)(cid:22)(cid:3)(cid:6)(cid:12)(cid:3)(cid:20)(cid:22)(cid:11)(cid:11)(cid:3)(cid:46)(cid:18)(cid:23)(cid:15)(cid:23)(cid:3)(cid:10)(cid:23)(cid:3)(cid:10)(cid:9)(cid:3)(cid:26)(cid:9)(cid:24)(cid:22)(cid:14)(cid:3)(cid:6)(cid:12)(cid:3)(cid:25)(cid:16)(cid:7)(cid:11)(cid:10)(cid:8)
`(cid:26)(cid:8)(cid:8)(cid:15)(cid:21)(cid:10)(cid:21)(cid:15)(cid:6)(cid:9)(cid:3)(cid:37)(cid:6)(cid:21)(cid:22)(cid:9)(cid:21)(cid:15)(cid:10)(cid:11)
`
`(cid:20)(cid:39)(cid:8)(cid:15)(cid:23)(cid:21)(cid:15)(cid:9)(cid:22)(cid:3)(cid:47)(cid:44)(cid:3)(cid:48)(cid:7)(cid:21)(cid:10)(cid:49)(cid:50)(cid:3)(cid:51)(cid:15)(cid:16)(cid:39)(cid:10)(cid:8)(cid:24)(cid:3)(cid:52)(cid:44)(cid:3)(cid:5)(cid:10)(cid:16)(cid:10)(cid:8)(cid:7)(cid:23)(cid:6)(cid:50)(cid:3)(cid:27)(cid:21)(cid:22)(cid:53)(cid:22)(cid:9)(cid:3)(cid:20)(cid:10)(cid:8)(cid:23)(cid:6)(cid:9)(cid:3)(cid:54)(cid:3)(cid:47)(cid:15)(cid:16)(cid:39)(cid:10)(cid:22)(cid:11)(cid:3)(cid:55)(cid:44)(cid:3)(cid:52)(cid:10)(cid:8)(cid:11)(cid:22)(cid:21)(cid:21)(cid:10)
`
`(cid:13)(cid:6)(cid:3)(cid:16)(cid:15)(cid:21)(cid:22)(cid:3)(cid:21)(cid:39)(cid:15)(cid:23)(cid:3)(cid:10)(cid:8)(cid:21)(cid:15)(cid:16)(cid:11)(cid:22)(cid:19)(cid:3)(cid:5)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:8)(cid:11)(cid:12)(cid:3)(cid:13)(cid:14)(cid:3)(cid:15)(cid:16)(cid:10)(cid:17)(cid:18)(cid:19)(cid:3)(cid:20)(cid:8)(cid:21)(cid:6)(cid:17)(cid:7)(cid:22)(cid:3)(cid:23)(cid:14)(cid:3)(cid:24)(cid:17)(cid:21)(cid:17)(cid:7)(cid:16)(cid:9)(cid:25)(cid:19)(cid:3)(cid:26)(cid:10)(cid:12)(cid:27)(cid:12)(cid:11)(cid:3)(cid:5)(cid:17)(cid:7)(cid:9)(cid:25)(cid:11)(cid:3)(cid:28)(cid:3)(cid:13)(cid:8)(cid:21)(cid:6)(cid:17)(cid:12)(cid:29)(cid:3)(cid:30)(cid:14)(cid:3)(cid:23)(cid:17)(cid:7)(cid:29)(cid:12)(cid:10)(cid:10)(cid:17)
`(cid:31)(cid:32)(cid:33)(cid:34)(cid:35)(cid:36)(cid:3)(cid:37)(cid:6)(cid:12)(cid:3)(cid:38)(cid:9)(cid:12)(cid:3)(cid:25)(cid:39)(cid:3)(cid:5)(cid:12)(cid:29)(cid:29)(cid:3)(cid:40)(cid:41)(cid:9)(cid:8)(cid:9)(cid:3)(cid:17)(cid:9)(cid:3)(cid:17)(cid:11)(cid:3)(cid:42)(cid:11)(cid:22)(cid:12)(cid:43)(cid:3)(cid:25)(cid:39)(cid:3)(cid:44)(cid:21)(cid:16)(cid:29)(cid:17)(cid:7)(cid:3)(cid:42)(cid:7)(cid:7)(cid:8)(cid:10)(cid:17)(cid:10)(cid:8)(cid:25)(cid:11)(cid:3)(cid:45)(cid:25)(cid:10)(cid:12)(cid:11)(cid:10)(cid:8)(cid:17)(cid:29)(cid:19)(cid:3)(cid:24)(cid:25)(cid:16)(cid:7)(cid:11)(cid:17)(cid:29)(cid:3)(cid:25)(cid:39)(cid:3)(cid:37)(cid:25)(cid:43)(cid:8)(cid:21)(cid:25)(cid:29)(cid:25)(cid:46)(cid:41)(cid:47)
`(cid:5)(cid:16)(cid:10)(cid:17)(cid:11)(cid:12)(cid:25)(cid:16)(cid:9)(cid:3)(cid:17)(cid:11)(cid:22)(cid:3)(cid:44)(cid:21)(cid:16)(cid:29)(cid:17)(cid:7)(cid:3)(cid:37)(cid:25)(cid:43)(cid:8)(cid:21)(cid:25)(cid:29)(cid:25)(cid:46)(cid:41)(cid:19)(cid:3)(cid:48)(cid:47)(cid:49)(cid:19)(cid:3)(cid:32)(cid:50)(cid:51)(cid:4)(cid:32)(cid:35)(cid:32)(cid:19)(cid:3)(cid:52)(cid:44)(cid:42)(cid:47)(cid:3)(cid:32)(cid:53)(cid:14)(cid:51)(cid:32)(cid:53)(cid:33)(cid:54)(cid:32)(cid:48)(cid:48)(cid:35)(cid:33)(cid:48)(cid:49)(cid:34)(cid:35)(cid:53)(cid:33)(cid:53)(cid:51)(cid:35)(cid:49)(cid:33)(cid:34)
`
`(cid:13)(cid:6)(cid:3)(cid:11)(cid:15)(cid:9)(cid:49)(cid:3)(cid:21)(cid:6)(cid:3)(cid:21)(cid:39)(cid:15)(cid:23)(cid:3)(cid:10)(cid:8)(cid:21)(cid:15)(cid:16)(cid:11)(cid:22)(cid:19)(cid:3)(cid:3)(cid:5)(cid:6)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:10)(cid:11)(cid:12)(cid:13)(cid:14)(cid:12)(cid:15)(cid:16)(cid:10)(cid:17)(cid:18)(cid:14)(cid:19)(cid:17)(cid:18)(cid:20)(cid:10)(cid:17)(cid:21)(cid:21)(cid:22)(cid:20)(cid:21)(cid:23)(cid:24)(cid:22)(cid:18)(cid:20)(cid:18)(cid:19)(cid:22)(cid:23)(cid:20)(cid:24)
`
`(cid:25)(cid:26)(cid:27)(cid:28)(cid:13)(cid:8)(cid:5)(cid:29)(cid:11)(cid:3)(cid:12)(cid:30)(cid:28)(cid:13)(cid:30)(cid:29)(cid:9)(cid:3)(cid:23)(cid:31)(cid:3)(cid:32)(cid:29)(cid:7)(cid:3)(cid:23)(cid:18)(cid:18)(cid:24)(cid:14)
`
`(cid:32)(cid:26)(cid:27)(cid:33)(cid:13)(cid:6)(cid:3)(cid:34)(cid:12)(cid:26)(cid:15)(cid:3)(cid:35)(cid:15)(cid:6)(cid:13)(cid:36)(cid:28)(cid:29)(cid:3)(cid:6)(cid:12)(cid:3)(cid:6)(cid:5)(cid:13)(cid:8)(cid:3)(cid:37)(cid:12)(cid:26)(cid:15)(cid:30)(cid:35)(cid:28)(cid:3)
`
`(cid:38)(cid:15)(cid:6)(cid:13)(cid:36)(cid:28)(cid:29)(cid:3)(cid:39)(cid:13)(cid:29)(cid:40)(cid:8)(cid:9)(cid:3)(cid:22)
`
`(cid:41)(cid:13)(cid:29)(cid:40)(cid:3)(cid:15)(cid:29)(cid:28)(cid:35)(cid:6)(cid:29)(cid:11)(cid:3)(cid:35)(cid:15)(cid:6)(cid:13)(cid:36)(cid:28)(cid:29)(cid:8)(cid:3)
`
`(cid:42)(cid:13)(cid:6)(cid:13)(cid:30)(cid:16)(cid:3)(cid:35)(cid:15)(cid:6)(cid:13)(cid:36)(cid:28)(cid:29)(cid:8)(cid:9)(cid:3)(cid:24)(cid:3)(cid:41)(cid:13)(cid:29)(cid:40)(cid:3)(cid:36)(cid:13)(cid:6)(cid:13)(cid:30)(cid:16)(cid:3)(cid:35)(cid:15)(cid:6)(cid:13)(cid:36)(cid:28)(cid:29)(cid:8)(cid:3)
`
`(cid:43)(cid:26)(cid:28)(cid:28)(cid:3)(cid:44)(cid:29)(cid:15)(cid:33)(cid:8)(cid:3)(cid:45)(cid:3)(cid:42)(cid:12)(cid:30)(cid:11)(cid:13)(cid:6)(cid:13)(cid:12)(cid:30)(cid:8)(cid:3)(cid:12)(cid:46)(cid:3)(cid:35)(cid:36)(cid:36)(cid:29)(cid:8)(cid:8)(cid:3)(cid:35)(cid:30)(cid:11)(cid:3)(cid:26)(cid:8)(cid:29)(cid:3)(cid:36)(cid:35)(cid:30)(cid:3)(cid:27)(cid:29)(cid:3)(cid:46)(cid:12)(cid:26)(cid:30)(cid:11)(cid:3)(cid:35)(cid:6)
`(cid:5)(cid:6)(cid:6)(cid:7)(cid:8)(cid:9)(cid:10)(cid:10)(cid:40)(cid:40)(cid:40)(cid:14)(cid:6)(cid:35)(cid:30)(cid:11)(cid:46)(cid:12)(cid:30)(cid:28)(cid:13)(cid:30)(cid:29)(cid:14)(cid:36)(cid:12)(cid:33)(cid:10)(cid:35)(cid:36)(cid:6)(cid:13)(cid:12)(cid:30)(cid:10)(cid:37)(cid:12)(cid:26)(cid:15)(cid:30)(cid:35)(cid:28)(cid:47)(cid:30)(cid:46)(cid:12)(cid:15)(cid:33)(cid:35)(cid:6)(cid:13)(cid:12)(cid:30)(cid:48)(cid:37)(cid:12)(cid:26)(cid:15)(cid:30)(cid:35)(cid:28)(cid:42)(cid:12)(cid:11)(cid:29)(cid:49)(cid:13)(cid:36)(cid:12)(cid:6)(cid:23)(cid:18)
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`J. Toxico1.-Cut. & Ocular Toxicol. 5(2), 143-161 (1986)
`
`THE USE OF CELL LYSIS AS AN INDEX
`OF OCULAR IRRITATION POTENTIAL
`
`CHRISTINE M. HUTAK, M. Sc.
`RICHARD B. JACARUSO, M.Sc.
`STEVEN CARSON, Ph. D. *
`MICHAEL A. BARLEnA, Ph.D.
`St. John’s University
`Jamaica, New York
`
`Abstract
`We report an in vitro method for assessing ocular irritation by measuring
`cell lysis of mouse connective tissue (strain L, Clone 929) and rabbit cor-
`neal cell lines (SIRC). Lysis of corneal epithelial cells in vivo leads to swell-
`ing and subsequent opacification of the underlying corneal stroma. In vitro
`lysis was determined by the measurement of changes in absorbance (360
`nm) of cell suspensions over a 10-min exposure to test materials. These
`data were expressed as numbers of cells/ml using linear regression. The
`regression line obtained was linear and similar for both cell types. Loss
`of viability and spontaneous cell lysis were minimal over the assay inter-
`val. In a separate study, rabbit corneal cells were used to assess alteration
`of cell membrane integrity following 30-min exposures to test materials
`by measuring trypan blue dye exclusion in these cells. Activities of test
`materials were ranked according to the lowest concentration capable of pro-
`ducing statistically significant cell lysis as follows: triethanolamine lauryl
`sulfate (TLS) < triethanolamine (TEA) < propylene glycol (PG) (0.003,
`0.1, and lo%, respectively, p < 0.01). The order was similar when dye
`
`Address reprint requests to: S. Carson, Ph.D., St. John’s University, College of Pharmacy, Grand
`Central and Utopia Parkways, Jamaica, New York 11439.
`143
`
`Copyright 0 1986 by Marcel Dekker, Inc.
`
`073 1-3829/86/0502-0143$3 .SO10
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`144
`
`HUTAK ET AL.
`
`exclusion ability was the index of activity except that benzafkonium chloride
`(BAK) was more active than the other materials. BAK and materials with
`cationic properties were not easily tested in the proposed suspension assay
`because of increased absorbance due to protein denaturation and subsequent
`masking of dissolution, if present. Six commercial formulations coded A
`through F were tested in the suspension assay as 1:3,ooO, 1:1,OOO, and 1:300
`dilutions and compared to similarly diluted standards to assess cell dissolu-
`tion ability. A shampoo concentrate acted similarly to TLS and an
`amphoteric-surfactant-based shampoo acted similarly to TEA at dilutions
`of 1:3,OOO. Effects of other formulations were intermediate between these
`extremes @ < 0.05), but similar to one another. At 1: 1,OOO dilutions, the
`effects converged, with all formulations having the same activity, which
`was significantly less than that of TLS, but significantly greater than that
`of TEA (p < 0.05). Over the next interval, the effects paralleled that of
`TLS but were significantly less except for that of the concentrate (p < 0.05).
`Another formulation showed a plateau-like effect while the remaining one
`showed a decreased effect owing to its inherent cationic nature. Results
`were in general agreement with in vivo and in v i m observations as well
`as with known chemical activities of the materials. A method for estimating
`the relative ability of materials and formulations to cause cell lysis is pro-
`posed which may serve as a model for the assessment of a form of corneal
`opacity initiated by the loss of integrity of limiting corneal layers.
`
`Introduction
`As noted in previous reports,'> there is great interest in the development of in
`vitro alternatives to testing procedures requiring animals. We have recently reported
`two in vitro methods for the assessment of irritancy potential of drug and cosmetic
`materials. One of these methods assessed the change in light absorbance at 360 nm
`by rabbit corneal cell monolayers (SIRC cell lines), which was considered to be an
`index of corneal opacity produced by the precipitation of cellular components,' Since
`initial monolayer absorbances were low, the method was insensitive to the effects
`of materials (detergents, anionic or nonionic surfactants) causing dissolution of the
`monolayer (unpublished data). Either mechanism (precipitation or dissolution) can
`
`cause corneal opacity,3* 4 which is a significant factor in ocular irritan~y.~ In the lat-
`ter case, the loss of limiting corneal layers has been shown to create opacity as a
`result of swelling of the underlying stromal layer^.^
`A number of cytotoxicity tests have reported the use of cell suspensions.6-10 With
`their use, the initial absorbance could be increased as required. Mouse connective
`tissue cells (strain L, clone 929) are known to grow readily in suspension and were
`used as suspensions in a cytotoxicity assay.9 Other reports111 have demonstrated
`the linear nature of the relationship between cell concentration and absorbance, one
`which satisfied Beer's law in low cell concentrations, that is, OD < 0.05.
`
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`OCULAR IRRITATION POTENTIAL
`
`145
`
`Cell suspension techniques have been used previously to assess cytotoxicity. They
`have been used to assess antimicrobial and/or surfactant hemolytic potential.6.10~ 13
`Suspensions have been used to assess ocular irritancy potential by measuring release
`of polymorphonuclear leukocyte granules into solution8 and by determining surfac-
`tant concentrations causing a loss of viability of 50% of suspended strain L, clone
`929 ce11s.9
`This paper reports the development of a method for assessing ocular irritation poten-
`tial in vitru using strain L, clone 929 cells and its subsequent modification to allow
`use of SIRC rabbit corneal cells, The test materials used were chosen on the basis
`of their known chemical activity and irritancy potential both in vitro and in vivu.
`They included benzalkonium chloride (BAK), triethanolamine lauryl sulfate (TLS),
`triethanolamine (TEA), and propylene glycol (PG). The ability of this method to
`be used in the testing of commercial formulations was assessed by exposing cell suspen-
`sions of rabbit corneal cells to fixed dilutions of commercial shampoos (1:3,OOO,
`1 : 1 ,OOO, 1 :300). Similarly diluted standards of high and low irritancy potential were
`included for relative comparisons.
`Finally, we also report results of parallel investigations of cytotoxicity which assessed
`the ability of rabbit corneal cell suspensions b exclude trypan blue dye following
`exposure to the above standards. This particular method was used to assess cell mem-
`brane integrity, the disruption of which may be an early event in cytotoxicity.14
`Presumably, changes in dye exclusion ability would precede or at least parallel the
`response of cell lysis.
`The method reported herein may be useful in screening compounds for their abili-
`ty to cause ocular irritation by their solubilizing effect on corneal cells. The present
`method was relatively easy to perform, was able to differentiate among the chosen
`test materials, and allowed for the testing of commercial formulations. It complements
`our methodology to assess irritation produced by materials causing cellular precipita-
`tion. The ability of rabbit corneal cells to exclude trypan blue dye following exposure
`to irritants both confirmed our observations and underscored the importance of cell
`membrane integrity as an index of irritancy potential.
`
`Materials and Methods
`
`Cells
`
`Two certified cell lines were used: SIRC rabbit corneal cells (ATCC CCL60) and
`strain L, clone 929 mouse connective tissue cells, clonal L-cell line (ATCC CCL1).
`They were purchased from the American Type Culture Collection (Rockville, MD).
`These cultures were grown as adherent monolayers in a forced-air incubator main-
`tained at 37 "C. Growth medium was that specified by the supplier with the addition
`of 0.05 mg/ml gentamicin or 10,OO U/ml penicillin and 10,OOO pg/ml streptomycin
`
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`146
`
`HUTAK ET AL.
`to SIRC and strain L, clone 929 media, respectively. All manipulations were per-
`formed using sterile technique. Stock cultures were maintained in 80 cm2 sterile
`polystyrene T-flasks (Nunclon) and were fed once per week with growth medium.
`At this time flasks of SIRC and strain L, clone 929 cells were divided into two or
`three equal parts, respectively, and recultured. A mixture of 8% C02 in air was in-
`troduced into each flask containing mouse connective tissue cells before the flasks
`were sealed. All sera used were inactivated at 56°C for 30 min.
`Cell Lysis Determinations
`
`Susjxnsion Medium and Test Material Preparation
`
`The suspension medium used throughout this assay consisted of Dulbecco’s
`phosphate-buffered saline solution without calcium (calcium-free PBS), adjusted to
`pH 7.3, supplemented with 5% fetal bovine serum (K. C. Biologicals), and filtered
`through a 0.22-pm filter (Millipore Corp.). Concentrations of test materials in the
`calcium-free PBS medium were five times that desired in the assay tubes to allow
`for subsequent procedural dilution during the initiation of the assay. Materials tested
`included TLS, TEA, PG, and BAK in concentrations ranging from 0.001 to 10%.
`Shampoo formulations were those commercially available, diluted in calcium-free
`PBS-Ca so that cells were exposed to 1:300, 1:1,OOO, and 1:3.OOO dilutions. All other
`materials were of standard reagent or manufacturing grade.
`
`Preparation of Mouse Cell (Strain L, Clone 929) Suspnsions
`
`Cells were used after 6 days of growth as above. As a rule, one flask of cells was
`used for each concentration level tested. Flasks were treated with 0.125 % trypsin-
`EDTA and the cells obtained were suspended to 20 ml with calcium-free PBS medium,
`pooled, and maintained at 37°C until use.
`
`Preparation of Rabbit Corneal Cell (SZRC) Suspensions
`
`Cells were used after 5 days of growth. Suspensions were prepared as above ex-
`cept that two flasks of cells were used for each concentration level tested, each flask
`was washed three times with 0.1 % EDTA in phosphate-buffered saline, pH 7.4, before
`the addition of 0.125 % trypsin-EDTA, and the cells obtained were resuspended to
`10 ml with calcium-free PBS medium.
`Preliminary Experiments
`
`Viability was determined by trypan blue dye exclusion by cells before and after
`exposure to assay conditions. Results were analyzed by a one-way analysis of variance.
`
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`OCULAR IRRITATION POTENTIAL
`
`147
`
`The extent of spontaneous cell lysis was determined by comparing cell counts before
`and after exposure to assay conditions. Four separate trials were performed for both
`SIRC and strain L, clone 929 cells and data were analyzed statistically using Stu-
`dent's t-test. Data from the latter trials were used as global controls for all subse-
`quent assays, whereas simultaneous controls were included in assays using SIRC cells.
`Demonstration of a linear relationship between cell absorbance and concentration
`involved taking aliquots of a single suspension, recording the absorbance at 360 nm,
`and performing cell counts using a hemocytometer. Cells were maintained at 37 "C
`during all manipulations and a minimum of 300 cells/sample were used for counts.
`The regression line was determined mathematically for absorbance at 360 nm vs.
`cell concentration data and the correlation coefficient determined. Absorbance data
`could then be converted to numbers of cells/sample and correction for procedural
`dilution of samples by the addition of test material was possible.
`
`Exposures to Test Materials
`All tubes were maintained at 37°C during the procedure. Assay tubes were in-
`oculated with 4 ml of the cell suspension. The absorbance at 360 nm was determined
`on a spectrophotometer (Bausch & Lomb, model 332995) before addition of 1 ml
`concentrated test material and after 10 min of incubation at 37 "C. To account for
`spontaneous cell lysis due to procedural manipulation, simultaneously exposed suspen-
`sions of untreated SIRC cells were used. In assays using strain L, clone 929 cells,
`global controls were used as explained above.
`
`Dye Exclusion by SXRC Cells
`
`Suspension Medium and Test Material Preparation
`
`The test materials PG, TEA, TLS, and BAK were added to concentrated Tris-
`HCl, pH 7.3, and diluted to give a material concentration twice that required in the
`assay and 0 , l M Tris-HC1.
`
`Preparation of Cell Suspensions
`
`Corneal cells were washed three times with 0.1 % EDTA in phosphate-buffered
`saline, pH 7.4, suspended with 0.125% trypsin-EDTA (GIBCO), collected at 250
`g (5 min, 4"C), washed with O.1M Tris-HC1, pH 7.3 (Tris), and resuspended in
`Tris. The suspension was diluted to yield counts of about 300 cells/hemocytometer
`counting field and then divided into equal 0.5-ml aliquots and maintained at 37 "C
`until use.
`
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`148
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`HUTAK ET AL.
`
`Preliminary Studies
`A study was performed to assess the change in dye uptake of untreated SIRC cells
`after 2 hr of incubation in Tris at 37"C, to determine cell stability for the duration
`of the assay period.
`
`Exposures to Test Materials
`
`The double-strength test materials were added to the aliquots of cell suspension
`in a 1 : 1 ratio to give final concentrations ranging from 0.00001 to 50%. Following
`30 min of incubation at 37"C, 25 pl cell suspension was removed and added to an
`equal volume of trypan blue. The resulting suspension was examined microscopical-
`ly to quantify the proportion of cells excluding dye. Dye exclusion by untreated con-
`trols was determined before and immediately after each trial. The proportion of cells
`excluding dye was expressed relative to that for similarly treated controls.
`
`Analysis of Data
`No statistical analysis was performed on data from the dye exclusion assay. Cell
`concentration data for suspensions exposed to test materials were statistically analyzed
`using the method of Williams.1s This method allowed for the determination of the
`lowest concentration producing a statistically significant effect compared to a zero-
`dose control. This method assumed that effects increased monotonically with con-
`centration. Analyses of variance were performed on all data analyzed in this way
`to obtain a mean square error term used in calculations. Both one- and two-way
`analyses of variance were used as indicated. Following the latter, Newman-Keuls
`post hoc comparisons were made among formulations at each concentration level.
`In all analyses, the criterion for statistical signficance was p < 0.05.
`
`Results
`
`Preliminary Studies
`The viability of both strain L, clone 929 mouse connective tissue cells and SIRC
`rabbit corneal cells in suspension medium was determined over a 20- or 30-min period
`at 37 "C. At least 300 cells were counted for each determination. At 0, 10, and 20
`min, theaverageviability was 86.8 f 0.9%, 87.2 f l.l%, and 87.0 f 1.4%, respec-
`tively for strain L, clone 929 cells. For SIRC cells at 0, 10,20, and 30 min the viability
`was 77.6 f 2.2%, 78.9 f 1.7%, 79.8 f 1.9%, and 78.0 f 1.8%, respectively.
`No statistically significant alterations in cell viability were noted for either line over
`the stated incubation intervals.
`
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`

`OCULAR IRRITATION POTENTIAL
`
`149
`
`I- a
`2 2 L b
`m E
`a
`c k
`P
`
`0.6 -
`0.5 -
`0.4 -
`0.3 -
`0.2 -
`0.1 -
`I
`
`1
`0.5
`
`I
`
`1 .o
`CELLS PER ML (x lo6)
`
`1
`1.5
`
`I
`
`Figure 1. Absorbance vs. concentration for strain L, clone 929 mouse connective tissue cells and
`SIRC rabbit corneal cells in suspension, measured at 360 nm. Cell suspensions were prepared as described
`in Methods section. Cell suspension absorbances were measured spectrophotometrically at 360 nm.
`Cell counts were determined by trypan blue dye exclusion using a hemocytometer grid with at least
`300 cells counted at each absorbance level.
`
`Analysis of the average cell numbers prior to and following the 10-min incubation
`period revealed no statistically significant spontaneous cell lysis in either cell line.
`Standard curves were constructed for the absorbance of cell suspensions at 360
`nm vs. cell concentration of both cell lines (Figure 1). The equations of the regres-
`sion lines, determined mathematically were: Y = 0.3834X + 0.0093, (r[7] = 0.9938,
`p < 0.01) and Y = 0.4242X - 0.0083, (r[30] = 0.9902, p < 0.01) for the strain
`L, clone 929 and SIRC cells, respectively.
`Eight aliquots of pooled SIRC cell suspensions were prepared for the assay and
`trypan blue dye exclusion was determined immediately upon suspension of cells and
`after 2 hr of incubation at 37°C in 0.lM Tris-HC1, pH 7.3. Overall, a net decrease
`in exclusion of 3.5 % was observed and considered acceptable.
`Cell Lysis by Test Materials
`
`Results of this study indicated that test materials produced characteristic, differen-
`tiable, and concentration-related effects on suspended cells of two types. The actual
`
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`HUTAK ET AL.
`150
`Table 1. Total Number of Strain L, Clone 929 Cells in Suspension Following 10-Min Exposures to
`Test Materials
`Total Number of Cells in Suspension ( X lo6)
`10 Min
`Initial
`Average Change
`3.468 (0.069)
`3.733 (0.081)
`0.266
`
`Test Material
`Global Control
`
`n
`19
`
`Propylene glycol (46)
`0.01
`0.03
`0.1
`0.3
`1 .o
`
`5
`5
`5
`5
`4
`
`3.309 (0.019)
`3.274 (0.037)
`2.892 (0.272)
`3.119 (0.015)
`3.281 (0.018)
`
`2.996 (0.014)
`3.036 (0.024)
`2.697 (0.284)
`2.924 (0.018)
`3.146 (0,011)
`
`0.313
`0.238
`0.195
`0.195
`0.138
`
`Triethanolamine
`lauryl sulfate (5%)
`0.786a
`3.113 (0.016)
`3.899 (0.013)
`5
`0.01
`0.786a
`2.977 (0.059)
`3.763 (0.052)
`0.03
`5
`0.743 (0.059)
`3.753 (0.019)
`3 .008a
`5
`0.1
`3 .4018
`0.356 (0.021)
`3.763 (0.029)
`0.3
`5
`0.188 (0.008)
`3.028a
`3.218 (0.017)
`4
`1 .o
`Bp < 0.01.
`Exposures wen performed at 37°C for 10 min following initial determination of total cells in suspension obtained
`by linear regression from absorbance at 360 nm. All test mnterials were prepared in the exposure medium: Dulbec-
`CO'S phosphate-buffered calcium-free saline, pH 7.3, supplemented with 5% fetal bovine serum. Values represent
`average numbers of cells in suspension f (standard error of the mean). The significance of the average change
`is denoted.
`
`~
`
`~
`
`results can be seen in Tables 1 and 2 for strain L, clone 929 and SIRC cells, respec-
`tively. Data here represent average changes in cell number over the 10-min incuba-
`tion period. Figures 2 and 3 show values that are corrected for the contribution of
`spontaneous lysis using controls. In all cases, cell numbers were derived from ab-
`sorbance measurements at 360 nm using the previously determined regression lines.
`Propylene glycol and TLS were common test materials to both systems. The PG
`produced no statistically significant cell lysis in concentrations ranging from 0.01
`to 1 .O% in strain L, clone 929 cell suspensions and 0.1 to 1.0% in SIRC cell suspen-
`sions, Statistically significant cell lysis first occurred at concentrations of 10% PG
`(p < 0.01) in the latter system (see Tables 1 and 2).
`Triethanolamine lauryl sulfate produced statisticaliy significant cell lysis at con-
`centrations of 0.01-1.0% @ < 0.01) in strain L, clone cell suspensions and 0.003%
`(p < 0.05) and 0.01-1 .OR (p < 0.01) in SIRC cell suspensions. No significant ef-
`fect was noted in concentrations of 0.0012 in the latter cell type.
`
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`

`OCULAR IRRITATION POTENTIAL
`151
`Table 2. Total Number of SIRC Rabbit Corneal Cells in Suspension Following 10-Min Exposures
`to Test Materials
`
`Test Material
`Propylene glycol (%)
`Control
`0.1
`0.3
`1 .o
`10
`
`Triethanolamine ( % )
`Control
`0.01
`0.03
`0.1
`0.3
`1 .o
`3 .O
`10
`
`Triethanolamine
`lauryl sulfate (%)
`Control
`0.001
`0.003
`0.01
`0.03
`0.1
`0.3
`1 .o
`
`n
`
`15
`15
`15
`14
`10
`
`20
`10
`10
`10
`10
`10
`10
`10
`
`20
`10
`10
`1s
`10
`10
`10
`10
`
`Total Number of Cells in Suspension ( X lo6)
`Initial
`10 Min
`Average Change
`
`3.075 (0.031)
`3.172 (0.046)
`3.086 (0.022)
`3.090 (0.034)
`2.049 (0.019)
`
`3.398 (0.065)
`3.633 (0.090)
`3.538 (0.062)
`3.396 (0.024)
`3.265 (0.033)
`3.220 (0.045)
`3.293 (0.032)
`2.305 (0.198)
`
`3.195 (0.062)
`3.083 (0.014)
`3.1 19 (0.013)
`3.213 (0.083)
`3.344 (0.136)
`3.266 (0.121)
`3.297 (0.133)
`3.217 (0.148)
`
`2.950 (0.023)
`2.905 (0.054)
`2.826 (0.081)
`2.840 (0.075)
`2.542 (0.034)
`
`3.243 (0.062)
`3.448 (0.074)
`3.294 (0.055)
`3.021 (0.071)
`2.261 (0.052)
`1.950 (0.050)
`1.658 (0.053)
`1.354 (0.172)
`
`3.054 (0.082)
`2.920 (0.020)
`2.834 (0.022)
`2.252 (0.079)
`1.690 (0.072)
`0.845 (0.020)
`0.558 (0.016)
`0.488 (0.038)
`
`0.124
`0.158
`0.260
`0.250
`O.62la
`
`0.155
`0.185
`0.244
`0.375a
`1.004a
`1.271a
`1 .634a
`1.95 la
`
`0.141
`0.163
`0.286b
`0.965a
`1 .653a
`2.421a
`2.738a
`2.729a
`
`Benzalkoniurn
`chloride (%)
`Control
`0.002
`0.006
`0.01
`0.02
`0.2
`a p < 0.01.
`bp < 0.05.
`Exposures and test material preparation were performed as described in Table 1. Values represent average numbers
`of cells in suspension f (standard error of the mean). The significance of the average change is denoted.
`
`5
`4
`5
`5
`5
`5
`
`2.860 (0.015)
`2.900 (0.030)
`2.935 (0.012)
`2.945 (0.023)
`2.879 (0.024)
`2.84 1 (0.024)
`
`2.726 (0.024)
`2.647 (0.015)
`3.622 (0.012)
`3.939 (0.019)
`5.426 (0.100)
`6.592 (0.142)
`
`0.134
`0.249
`-0.686
`-0.984
`-2.547
`-3.751
`
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`

`152
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`HUTAK ET AL.
`
`-
`
`10
`
`0.3
`
`0.03
`0.1
`0.01
`MATERIAL CONCENTRATION (%)
`Figure 2. Dissolution (lysis) of strain L, clone 929 mouse connective tissue cells in suspension expos-
`ed to triethanolamine lauryl sulfate and propylene glycol. Exposures and test material preparation were
`as described in Methods section. Mean cell lysis values were corrected for spontaneous cell lysis and
`expressed as the percent decrease of the initial number of cells in suspension prior to exposure to the
`test materials. Error bars indicate standard errors of mean cell lysis values. TLS, triethanolamine lauryl
`sulfate; PG, propylene glycol.
`
`1 .o
`
`Triethanolamine and BAK were used as test materials in the SIRC cell system only
`(see Table 2). Triethanolamine first produced statistically significant effects at a con-
`centration of 0.1 % @ < 0.01). Benzalkonium chloride produced an atypical response
`in which the number of cells artificially appeared to increase with exposure due to
`a change in the optical characteristics of the cells (whitening) that was grossly visible.
`
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`

`OCULAR IRRITATION POTENTIAL IN RABBIT CORNEAS
`
`153
`
`PG -
`
`TLS D---c)
`TEA M
`
`MATERIAL CONCENTRATION (%)
`Figure 3. Lysis of SIRC rabbit corneal cells in suspension, exposed to triethanolamine lauryl sulfate,
`triethanolamine, and propylene glycol. Exposures and test material preparation were as described in
`Methods section. Mean cell lysis values were corrected for spontaneous cell lysis and expressed as
`the percent decrease of the initial number of cells in suspension prior to exposure to the test materials.
`Error bars indicate standard errors of mean cell lysis values. TLS, triethanolamine lauryl sulfate; TEA,
`triethanolamine; PG, propylene glycol.
`
`It is apparent from Figures 2 and 3 that the effects of these materials are differen-
`tiable: characteristically different and discrete curves are seen. Although the rank-
`ing of these materials is obvious, using the objective criterion of the lowest concen-
`tration producing a statistically significant effectls we see that TLS < TEA < PG
`(0.003, 0.1, and 10% respectively, Table 2).
`The system reported was able to accommodate commercial formulations. Six com-
`mercial shampoos were obtained and used in fixed dilutions of 1 :300, 1 : 1 ,OOO, and
`1:3,OOO with suspension medium. Included in each trial were similarly diluted samples
`of TLS and PG (1 : 1,OOO) for reference purposes. Results obtained are listed in Table
`3. Figure 4 shows these results relative to effects of the standard test materials. A
`two-way analysis of variance was performed on the data and indicated a significant
`effect of concentration, F(2,170) = 169, p < 0.001, and of test material type, F(8,170)
`= 189, p < 0.001. A significant interaction between material concentration and type
`
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`

`

`154
`
`HUTAK ET AL.
`Table 3. Total Number of SIRC Rabbit Corneal Cells in Suspension Following 10-Min Exposure
`to Shampoo Formulations
`Total Number of Cells in Suspension (X 106)
`Mean Change
`10 Min
`(% Control)
`3.151 (0.013)
`0.113 3.45
`3.127 (0.030)
`0.095 2.93
`3.054 (0.014)
`0.056 1.82
`3.313 (0.012)
`0.137 3.96
`3.009 (0.010)
`0.164 5.16
`3.979 (0.041)
`0.062 1.75
`
`DilutionlFormulation
`Control A
`B
`C
`D
`E
`F
`
`n
`5
`5
`5
`5
`5
`5
`
`Initial
`3.264 (0.021)
`3.222 (0.022)
`3.11 1 (0.009)
`3.450 (0.022)
`3.173 (0.009)
`3.535 (0.020)
`
`1:3,000 A
`B
`C
`D
`E
`F
`
`1:1,OOO A
`B
`C
`D
`E
`F
`
`5
`5
`5
`5
`5
`5
`
`5
`5
`5
`5
`5
`5
`
`3.265 (0.028)
`3.290 (0.024)
`3.098 (0,004)
`3.475 (0.002)
`3.165 (0.012)
`3.637 (0.018)
`
`3.156 (0.019)
`3.233 (0.021)
`3.098 (0.004)
`3.403 (0.030)
`3.156 (0.010)
`3.680 (0.020)
`
`2.837 (0.023)
`2.453 (0.065)
`2.052 (0.024)
`2.686 (0.030)
`2.752 (0.069)
`2.920 (0.017)
`
`1.934 (0.055)
`1