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					HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                                  HET-CAM Test Method Protocols                                                                                                          March 27, 2009




                                                                                                                      Bagley et al.                                                                                    Balls et al. (1995) (Non-
                                                          CEC (1991)                      Gettings et al. (1991)                               Gettings et al. (1994)          Vinardell and Macián (1994)
                                                                                                                        (1992)                                                                                        Transparent Substances)
                                                                                    Lohmann's Selected White
                       Hen Strain                  Lohmann Selected Leghorn                                          White Leghorn                White Leghorn                         Leghorn SA31                         Not Noted
                                                                                    Leghorn LSL

                                                                                                                   Weight range of
                                              Weight range of eggs between 50 g
                  Egg Criteria for Use                                                         Not Noted           eggs between 50 g                 Not Noted                            Not Noted                          Not Noted
                                              and 60 g
                                                                                                                   and 60 g



                Egg Storage (Prior to use)                 Not Noted                           Not Noted               Not Noted                     Not Noted                            Not Noted                          Not Noted



              Incubation Temperature (0C)                  37.5 + 0.5                          Not Noted               37.5 + 0.5                        38                               Not Noted                          Not Noted

                 Relative Humidity (%)                     62.5 + 7.5                          Not Noted               62.5 + 7.5                        60                               Not Noted                          Not Noted

                     Egg Rotation?                              Yes                            Not Noted                  Yes                           Yes                               Not Noted                          Not Noted

                                              Eggs candled on Day 5 and every day
                                                                                                                                        Candled on Day 9 and non-viable
                 Checking Egg Viability       thereafter; non-viable embryos                   Not Noted               Not Noted                                                          Not Noted                          Not Noted
                                                                                                                                        embryos removed
                                              removed




                   Incubation Period                        10 Days                             10 Days                 10 Days                       9 Days                               10 Days                             9 Days




                                              Egg shell was scratched around the                                                                                             Egg shell was scratched around
                                                                                                                   Egg shell around
                                              air chamber with a rotating dentist                                                       Eggshell was scratched around the    the air cell by a dentist's rotary saw
                                                                                                                   the air pocket was
               Procedure for Opening Egg      saw blade and then pared off. The                Not Noted                                air cells and a small aperture was   and then pared off. After removal               Not Noted
                                                                                                                   removed with a
                                              inner shell was removed and the                                                           opened.                              of the inner membrane, the CAM
                                                                                                                   dental rotary saw
                                              CAM was layed open.                                                                                                            was exposed.




                 Manipulation of CAM                       Not Noted                           Not Noted               Not Noted                     Not Noted                            Not Noted                          Not Noted




                                                                                                                        B-5
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                                       HET-CAM Test Method Protocols                                                                                                                 March 27, 2009




                                                                                                                             Bagley et al.                                                                                       Balls et al. (1995) (Non-
                                                            CEC (1991)                        Gettings et al. (1991)                                   Gettings et al. (1994)          Vinardell and Macián (1994)
                                                                                                                               (1992)                                                                                           Transparent Substances)
                                                                                                                          Chemicals from                                                                                  Chemicals selected from the
                                                                                                                          various chemical                                                                                ECETOC database (acids, acyl
           Investigator Defined Test Substance Chemicals from various chemical                                            classes, commercial Oil/water -based personal care                                              halides, alcohols, aldehye, alkalis,
                                                                                           Hydro-alcoholic formulations                                                                  Commercial disinfectants
                         Classes               classes                                                                    products, and       formulations                                                                esters, hetercyclics, hydrocarbons,
                                                                                                                          personal care                                                                                   inorganic chemicals, ketones,
                                                                                                                          products                                                                                        organophosphates, pesticides,
             Total Test Substances Evaluated                     21                                     10                        32                             18                                   6                                     59



           Test Substance Quantity or Volume              0.3 mL or 0.1 g                           Not Noted               0.3 mL or 0.1 g                    0.3 mL                              0.3 mL                               Not Noted




                                                                                                                          Tested solutions at
                                                                                                                                              3 concentrations tested: threshold
             Test Substance Concentrations                                                                                concentrations that
                                                           0.1% to 100%                             Not Noted                                 concentration, 10% solution,                  Diluted or undiluted                        Not Noted
                        Tested                                                                                            were 10% of those
                                                                                                                                              undiluted
                                                                                                                          tested in vivo


                                                                                                                          All tested
                                                                                                                                                All tested substances appear to be   All tested substances appear to be
              Application of Solids to CAM            Placed directly on CAM                        Not Noted             substances appear                                                                                             Not Noted
                                                                                                                                                solubilized                          solubilized
                                                                                                                          to be solubilized




                    Preferred Solvent                        Not Noted                              Not Noted                 Not Noted                      Not Noted                         Distilled water                          Not Noted




                                               Yes, for solids. 20 seconds after                                          20 seconds after
               Rinse after Test Substance      application of test substance, rinsed                                      test substance
                                                                                                    Not Noted                                                Not Noted                           Not Noted                       After 3 minute exposure
                      Application?             with 5 mL warm water or saline                                             applied rinsed with
                                               solution                                                                   5 mL warm water
                                                                                                                          At 0.5, 2, and 5
                                               Up to 300 seconds after test substance                                                           Up to 300 seconds after test         Up to 300 seconds after test
                   Evaluation Period                                                                Not Noted             minutes after test                                                                                  Within 30 seconds of rinsing
                                               applied                                                                                          substance applied                    substance applied
                                                                                                                          substance applied

                                                                                                                                                                                     0.1 M NaOH, 1% SDS, 0.9%             5% Texapon AV (internal reference
              Controls and Test Standards                    Not Noted                              Not Noted                   Vehicle                      Not Noted
                                                                                                                                                                                     NaCl, distilled water                standard)

                Number of Control Eggs                       Not Noted                              Not Noted                   2 eggs                       Not Noted                      2 eggs per substance                        Not Noted

                     Replicate Eggs                       Minimum 6 eggs                            Not Noted                   4 eggs             3 eggs per concentration tested                 6 eggs                                 6 eggs

            Number of Replicate Experiments                  Not Noted                              Not Noted                 Not Noted                           2                              Not Noted                              Not Noted


                                                                                                                          Hyperemia,
                                                                                        Hemorrhage, vascular lysis,                                                                  Hemorrhage, vasoconstriction,
                   Endpoints Assessed          Hemorrhage, lysis, and coagulation                                         hemorrhage,              Hemorrhage, coagulation, lysis                                            Hemorrhage, lysis, coagulation
                                                                                        coagulation                                                                                  coagulation
                                                                                                                          coagulation




                                                                                                                               B-6
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                                    HET-CAM Test Method Protocols                                                                                                            March 27, 2009




                                                                                                                             Bagley et al.                                                                                Balls et al. (1995) (Non-
                                                          CEC (1991)                       Gettings et al. (1991)                                     Gettings et al. (1994)       Vinardell and Macián (1994)
                                                                                                                               (1992)                                                                                    Transparent Substances)


                                              The starting second that each of the   The starting second that each of the Numerical time-                                        The starting second that each of
                                                                                                                                               Seconds for the three endpoints                                      Endpoints scored from 0 (no reaction)
                  Endpoint Evaluation         three endpoints is observed is         three endpoints is observed is       dependent scores                                       the endpoints is observed is
                                                                                                                                               recorded (see Kalweit, 1990)                                         to 3 (strong reaction)
                                              recorded.                              recorded.                            for three endpoints.                                   recorded




                                                                                                                               B-7
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                                        HET-CAM Test Method Protocols                                                                                                                     March 27, 2009




                                                                                                                                  Bagley et al.                                                                                        Balls et al. (1995) (Non-
                                                           CEC (1991)                          Gettings et al. (1991)                                      Gettings et al. (1994)             Vinardell and Macián (1994)
                                                                                                                                    (1992)                                                                                            Transparent Substances)




                                                                                         Two different analyses were used.                                                                  Irritancy Potential calculated using Calculation of "S Score". "S Score" is
                                                                                                                               Scores are totaled
                                              Irritation Index is calculated using the   Both calculated an irritation index                                                                the formula: (301-sec                calculated using the most sensitive
                                                                                                                               to give a single
                                              formula: (301-sec H)/300*5+(301-sec        using time (seconds) of appearance                       Calculation of an Irritation Score for    H)/300*5+(301-sec v)/300*7=(301- endpoint (endpoint can change from
                                                                                                                               value (maximum of
                    Analysis Method           L)/300*7=(301-sec C)*9; where              of hemorrhage vascular lysis, or                         each egg. Mean value of individual        sec C)*9; where H=Hemorrhage, chemical to chemical). The scores
                                                                                                                               21). Mean value of
                                              H=Hemorrhage, L=Lysis;                     coagulation. The differences                             Irritation Scores calculated.             V=vasoconstriction;                  recorded for the most sensitive
                                                                                                                               4 eggs calculated
                                              C=Coagulation; sec=starting second         between the two methods was in                                                                     C=Coagulation of protein or          endpoint are summarized for the 6
                                                                                                                               for final value.
                                                                                         the particular calculations used.                                                                  blood; sec=starting second           eggs. (Maximum for 6 eggs is 18)




                                                                                                                                                    Practically None: 0-0.9; Slight         Practically None: 0-0.9; Slight
                                                                                                                                                                                                                                Non Irritation: S < 6 ; Moderately
                                                                                                                                                    Irritation: 1-4.9; Moderate             Irritation: 1-4.9; Moderate
                  Classification Scheme                     Not Noted                                Not Noted                     Not Noted                                                                                    Irritating: 6<= S <15; Severely
                                                                                                                                                    Irritation: 5-9.9; Strong Irritation:   Irritation: 5-8.9; Strong
                                                                                                                                                                                                                                Irritating: S >15
                                                                                                                                                    10-21                                   Irritation: 9-21




                   GLP Compliance?                          Not Noted                                Not Noted                     Not Noted                     Not Noted                              Not Noted                                Yes




                                                                                                                               Cites Luepke and                                             Cites Ergatt/Frame Data Bank
                                                                                         Cites Luepke (1985) as basis for                           Cites Luepke (1985) as basis for
                                                                                                                               Kemper (1986) as                                             (1990) as basis for protocol and
                                                                                         protocol used                                              scoring scheme
                                                                                                                               basis for protocol                                           analysis method



                         Notes

                                                                                                                                                    To study the effects of slow acting
                                                                                                                                                    materials, CAM scored 15 and 30
                                                                                                                                                    minutes after application on range
                                                                                                                                                    from 0 (no reactions) to 3 (strong
                                                                                                                                                    reactions)




                                                                                                                                    B-8
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                                 HET-CAM Test Method Protocols                                                                                                           March 27, 2009




                                             Balls et al. (1995) (Transparent                                       Gettings et al. (1996)     Gettings et al. (1996)
                                                                                       Kojima et al. (1995)                                                                          Gilleron et al. (1996)               Spielmann et al. (1996)
                                                        Substances)                                                  (HET-CAM I, II)            (HET-CAM III)
                                                                                                                    Lohmann's Selected
                   Hen Strain                          Not Noted                            Not Noted                                             White Leghorn                          White Essex                            Not Noted
                                                                                                                    White Leghorn


                                                                                                                                                                        Eggs were 7 days old prior to start of
               Egg Criteria for Use                    Not Noted                            Not Noted                    Not Noted                   Not Noted                                                                  Not Noted
                                                                                                                                                                        incubation and weighed 60 + 5 g




            Egg Storage (Prior to use)                 Not Noted                            Not Noted                    Not Noted                   Not Noted                            Not Noted                             Not Noted



           Incubation Temperature (0C)                 Not Noted                              37.6                       Not Noted                    38 + 0.5                            37.0 + 0.5                            Not Noted

              Relative Humidity (%)                    Not Noted                             about 70                    Not Noted                     60 + 5                             62.5 + 1.5                            Not Noted

                  Egg Rotation?                        Not Noted                        Yes, once per hour               Not Noted                      Yes                 Yes, with large ends upward for 9 days              Not Noted

                                                                                                                                             Candled on Day 9 and
              Checking Egg Viability                   Not Noted                            Not Noted                    Not Noted           returned to incubator in                     Not Noted                             Not Noted
                                                                                                                                             vertical position




                Incubation Period                        9 Days                              10 Days                      10 Days                     10 Days                              10 Days                              Not Noted


                                                                                                                                                                        Eggs were candled and non-viable eggs were
                                                                                                                                                                        discarded. The airspace delimited by the
                                                                                                                                                                        inner membrane at the large end of the egg
                                                                                A portion of the egg shell was                                                          was marked. The eggshell was removed
                                                                                                                                             Shell and inner shell
                                                                                removed and a drop of water was                                                         using a dentist's rotating saw blade. The inner
                                                                                                                                             membrane were removed
            Procedure for Opening Egg                  Not Noted                placed onto the shell membrane to        Not Noted                                      membrane was moistened with 1.5-2.0 mL of               Not Noted
                                                                                                                                             around the area defined by
                                                                                avoid bleeding. Then the CAM was                                                        0.9% NaCl and the egg was returned to the
                                                                                                                                             the air cell
                                                                                exposed with forceps.                                                                   incubator (at 37) for a maximum of 20 mins.
                                                                                                                                                                        After incubation, the NaCl solution was
                                                                                                                                                                        removed, using a vacuum pump, and the inner
                                                                                                                                                                        membrane was removed with forceps.


                                                                                                                                                                        A test substance applicator (TSA), which is
                                                                                                                                                                        comprised of a perlon mesh (pore diameter =
              Manipulation of CAM                      Not Noted                            Not Noted                    Not Noted                   Not Noted                                                                  Not Noted
                                                                                                                                                                        63 micron) locked between two Teflon rings,
                                                                                                                                                                        was placed on the CAM




                                                                                                                           B-9
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                                          HET-CAM Test Method Protocols                                                                                                                       March 27, 2009




                                                 Balls et al. (1995) (Transparent                                            Gettings et al. (1996)     Gettings et al. (1996)
                                                                                              Kojima et al. (1995)                                                                             Gilleron et al. (1996)                        Spielmann et al. (1996)
                                                            Substances)                                                       (HET-CAM I, II)            (HET-CAM III)
                                            Chemicals selected from the
                                            ECETOC database (acids, acyl
                                                                                                                             Surfactant-based
        Investigator Defined Test Substance halides, alcohols, aldehye, alkalis,                                                                      Surfactant-based personal                                                         Chemicals from various chemical
                                                                                      Surfactants, solvents, formaldehyde    personal care                                           Chemicals from various chemical classes
                      Classes               esters, hetercyclics, hydrocarbons,                                                                       care formulations                                                                             classes
                                                                                                                             formulations
                                            inorganic chemicals, ketones,
                                            organophosphates, pesticides,
         Total Test Substances Evaluated                        59                                     24                              25                         25                                     46                                            200


                                                                                                                                                                                   0.3 mL or 0.3 g of test substance placed inside
        Test Substance Quantity or Volume                   Not Noted                                0.2 mL                          0.3 mL                    0.1 mL                                                                               Not Noted
                                                                                                                                                                                                      the TSA



                                                                                                                             3 concentrations
          Test Substance Concentrations                                                                                      tested: threshold
                                                            Not Noted                             10% solution                                              10% solution                             Undiluted                               1-100% solutions tested
                     Tested                                                                                                  concentration, 10%
                                                                                                                             solution, undiluted


                                                                                                                             All test substances
                                                                                      All tested substances appear to be                              All test substances appear                                                     All tested substances appear to be
           Application of Solids to CAM                     Not Noted                                                        appear to be in liquid                                              Placed inside TSA
                                                                                      solubilized                                                     to be in liquid form                                                           solubilized
                                                                                                                             form




                 Preferred Solvent                          Not Noted                              Not Noted                       Not Noted                  Not Noted                             0.9% NaCl                                 0.9% NaCl or olive oil




                                                                                                                                                                                                                                     Yes, after 5 minutes (for substances that
            Rinse after Test Substance                                                Yes, after 20 seconds test substance                            After 20 seconds, with a     TSA (which contains the test substance) is
                                                            Not Noted                                                              Not Noted                                                                                         were insoluble in eater or oil and were
                   Application?                                                       was rinsed with water                                           saline rinse                 removed after 20 seconds
                                                                                                                                                                                                                                     colored).

                                                                                                                             Up to 300 seconds
                                              Up to 300 seconds after test substance At 0.5, 2, and 5 minutes after test                              At 0.5, 2, and 5 minutes                                                   Up to 300 seconds after test substance
                 Evaluation Period                                                                                           after test substance                                 Up to 300 seconds after test substance applied
                                              applied                                substance rinsed                                                 after test substance rinsed                                                applied
                                                                                                                             applied

                                              5% Texapon AV (internal reference                                                                                                    Positive Controls: benzalkonium chloride,
           Controls and Test Standards                                                             Not Noted                       Not Noted                  Not Noted                                                                             Not Noted
                                              standard)                                                                                                                            dimethylformamide, imidazole

              Number of Control Eggs                        Not Noted                              Not Noted                       Not Noted                  Not Noted                              Not Noted                                      Not Noted
                                                                                                                             3 eggs per
                   Replicate Eggs                             6 eggs                                 4 eggs                                                   Not Noted                                  3                                          Not Noted
                                                                                                                             concentration tested
         Number of Replicate Experiments                    Not Noted                              Not Noted                       Not Noted                  Not Noted                                  3                                          Not Noted


                                                                                                                             Hemorrhage, lysis,       Dilation, hemorrhage,
                Endpoints Assessed                Hemorrhage, lysis, coagulation      Hyperemia, hemorrhage, coagulation                                                                  Hemorrhage, lysis, coagulation                 Hemorrhage, coagulation, lysis
                                                                                                                             coagulation              coagulation




                                                                                                                                    B-10
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                            HET-CAM Test Method Protocols                                                                                                         March 27, 2009




                                             Balls et al. (1995) (Transparent                            Gettings et al. (1996)   Gettings et al. (1996)
                                                                                  Kojima et al. (1995)                                                                 Gilleron et al. (1996)                     Spielmann et al. (1996)
                                                        Substances)                                       (HET-CAM I, II)          (HET-CAM III)

                                                                                                         The starting second
                                                                                                         that each of the      Numerical time-
                                           The starting second that each of the                                                                           The starting second that each of the endpoints The starting second that each of the
               Endpoint Evaluation                                                    Not Noted          endpoints is observed dependent scores for three
                                           endpoints is observed is recorded                                                                              is observed is recorded                        endpoints is observed is recorded
                                                                                                         is recorded (see      endpoints.
                                                                                                         Kalweit 1990)




                                                                                                               B-11
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                                      HET-CAM Test Method Protocols                                                                                                                March 27, 2009




                                             Balls et al. (1995) (Transparent                                           Gettings et al. (1996)     Gettings et al. (1996)
                                                                                          Kojima et al. (1995)                                                                          Gilleron et al. (1996)                     Spielmann et al. (1996)
                                                        Substances)                                                      (HET-CAM I, II)            (HET-CAM III)

                                                                                                                        Mean Irritation Score
                                           Computer program calculates an
                                                                                                                        (IS; determined at
                                           Irritation Index. Irritation Index Is                                                                                                                                           Irritation Score calculated ( (301-sec
                                                                                                                        10% concentration) of
                                           used to calculate a "Q Score". "Q                                                                                                                                               H)/300*5+(301-sec L)/300*7=(301-sec
                                                                                                                        3 eggs is calculated.                             Irritation Index calculated using the formula:
                                           Score" is a comparison of the                                                                                                                                                   C)*9; where H=Hemorrhage, L=Lysis;
                                                                                                                        The Irritation           Time-dependent scores    (301-sec H)/300*5+(301-sec L)/300*7=(301-
                                           Irritation Index of a test chemical with Score was calculated based on the                                                                                                      C=Coagulation; sec=starting second) for
                                                                                                                        Threshold                were used to calculate a sec C)*9; where H=Hemorrhage, L=Vessel
                 Analysis Method           that of the reference chemical. If the time of onset for each endpoint.                                                                                                         10% solution calculated (IS10).
                                                                                                                        Concentration (ITC;      single value (maximum of Lysis; C=Coagulation; sec=starting second.
                                           effect of test chemical is identical to Mean value of 4 eggs calculated.                                                                                                        Irritation Threshold (ITC; lowest
                                                                                                                        lowest concentration     21).                     Mean of 3 assays were calculated.
                                           reference, Q is 1.0. If effect of test                                                                                                                                          concentration of a test substance to
                                                                                                                        producing a slight                                Reproducibility also was assessed.
                                           chemical is less irritating, Q is lower.                                                                                                                                        induce an irritant reaction on the CAM)
                                                                                                                        reaction during
                                           If effect of test chemical is more                                                                                                                                              calculated.
                                                                                                                        observation period) is
                                           irritating, Q is higher.
                                                                                                                        calculated.



                                                                                                                                                                                                                           BGA Classification Model:
                                                                                                                                                                                                                           Non/Slight: ITC > 10 and IS10 <16;
                                                                                                                                                                                                                           Moderate: (1) ITC > 16 and IS10 > 16
                                                                                                                                                                                                                           or (2) ITC < 10 and IS10 < 16; Irritant
                                           Non Irritating: Q < 1.5; Moderately                                          Irritant (According
                                                                                                                                              Irritant (According to                                                       (R36): (1) ITC < 10 and IS10 > 16 or
              Classification Scheme        Irritating: 1.5 < Q <2.0; Severely                   Not Noted               to FHSA): IS ≥ 5.1 or                               Non Irritant: 0-4.9; Irritant: 5.0-21
                                                                                                                                              FHSA): Score ≥ 4.83                                                          (2) ITC < 2.5 and IS10 < 16; Severe
                                           Irritating: Q < 2.0                                                          IS/ITC ≥ 3.0
                                                                                                                                                                                                                           (R41): (1) ITC < 1% or (2) ITC between
                                                                                                                                                                                                                           1 and 2.5% and IS10 > 16. Proposed
                                                                                                                                                                                                                           model using mtc10 (see notes below):
                                                                                                                                                                                                                           R41: mtc10 < 50 seconds.



                GLP Compliance?                             Yes                                 Not Noted                     Not Noted                 Not Noted                            Not Noted                                       Yes


                                                                                                                                                                                                                           Cites Luepke (1985) as basis for
                                                                                                                                                                                                                           protocol. Cites Kalweit (1990) and
                                                                                  Cites Luepke (1985) and Luepke and
                                                                                                                                                                                                                           Spielmann (1991) as publishing the
                                                                                  Wallat (1985) as basis for scoring
                                                                                                                                                                                                                           protocol used. Cites the standard
                                                                                  scheme and analysis method.
                                                                                                                                                                                                                           protocol for the test method as InVitox
                                                                                                                                                                                                                           Protocol.

                      Notes
                                                                                                                                                                                                                           Nine additional endpoints were
                                                                                                                                                                                                                           conducted using the raw data and the IS
                                                                                                                                                                                                                           and IT scores in this analysis. Of these
                                                                                                                                                                                                                           the endpoints evaluated, that best
                                                                                                                                                                                                                           correlated with in vivo classification
                                                                                                                                                                                                                           was mtc10 (mean detection time for
                                                                                                                                                                                                                           appearance of coagulation when using a
                                                                                                                                                                                                                           10% solution).

                                                                                                                                                                            Abbreviations: CAM = Chorioallantoic membrane, ECETOC = European Centre for Ecotoxicology and Toxicology of
                                                                                                                                                                            Hazardous Substances Act (1964), IS = Irritation score, IT = Irritation threshold concentration, NaCl = Sodium chloride
                                                                                                                                                                            SDS = Sodium dodecyl sulfate, Sec = seconds.




                                                                                                                              B-12
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                HET-CAM Test Method Protocols                                                    March 27, 2009




                                                                                                 Gilleron et al. (1997)           Hagino et al. (1999)


                                                                        Hen Strain                   White Essex                    White Leghorn



                                                                    Egg Criteria for Use         Eggs were 7 days old                  Not Noted




                                                                 Egg Storage (Prior to use)           Not Noted                        Not Noted



                                                                Incubation Temperature (0C)            37 + 0.5                           37.6

                                                                   Relative Humidity (%)              52.5 + 2.5                        about 70

                                                                      Egg Rotation?                   Not Noted                    Yes, once an hour



                                                                  Checking Egg Viability              Not Noted                        Not Noted




                                                                     Incubation Period                 10 Days                          10 Days




                                                                                                                          Portion of the egg shell above the
                                                                Procedure for Opening Egg             Not Noted
                                                                                                                          air space was removed.




                                                                                                                          Drop of water is placed on the
                                                                                              A test substance applicator
                                                                                                                          membrane (to avoid capillary
                                                                   Manipulation of CAM        (TSA) was placed on the
                                                                                                                          bleeding). A silicone rubber ring is
                                                                                              CAM
                                                                                                                          placed on the CAM.




                                                                                                      B-13
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                    HET-CAM Test Method Protocols                                                        March 27, 2009




                                                                                                       Gilleron et al. (1997)            Hagino et al. (1999)


                                                                                                                                 Surfactant, polyols, color additives,
                                                                                                                                 organic salts, PABA derivative,
                                                                Investigator Defined Test Substance Chemicals from various
                                                                                                                                 esters, inorganic chemicals,
                                                                              Classes               chemical classes
                                                                                                                                 alcohols, amines, alkanolamines,
                                                                                                                                 carboxylic acids

                                                                 Total Test Substances Evaluated                 60                               39
                                                                                                                              0.2 mL (placed inside the rubber
                                                                                                  0.3 mL or 0.3 g of test     ring on the CAM). Solids (0.2 g)
                                                                Test Substance Quantity or Volume substance placed inside the are reduced to a fine powder with a
                                                                                                  TSA                         No. 200 sieve and placed inside a
                                                                                                                              rubber ring on the CAM.


                                                                  Test Substance Concentrations
                                                                                                             Not Noted                   0.1, 1, 10, and 100%
                                                                             Tested




                                                                   Application of Solids to CAM          Placed inside TSA             Placed inside rubber ring




                                                                         Preferred Solvent                   Not Noted                        Not Noted




                                                                                                    TSA (which contains the
                                                                    Rinse after Test Substance                                   Yes, after 20 seconds with distilled
                                                                                                    test substance) is removed
                                                                           Application?                                          water
                                                                                                    after 20 seconds


                                                                                                    Up to 300 seconds after test At 0.5, 2, and 5 minutes after test
                                                                        Evaluation Period
                                                                                                    substance applied            substance applied


                                                                   Controls and Test Standards               Not Noted                        Not Noted


                                                                     Number of Control Eggs                  Not Noted                        Not Noted

                                                                          Replicate Eggs                     Not Noted                          4 eggs

                                                                 Number of Replicate Experiments             Not Noted                        Not Noted


                                                                                                    Hemorrhage, lysis,           Hyperemia, hemorrhage,
                                                                        Endpoints Assessed
                                                                                                    coagulation                  coagulation




                                                                                                            B-14
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                            HET-CAM Test Method Protocols                                           March 27, 2009




                                                                                         Gilleron et al. (1997)         Hagino et al. (1999)



                                                                                      The starting second that
                                                                                                                  Numerical time-dependent scores
                                                                Endpoint Evaluation   each of the endpoints is
                                                                                                                  for three endpoints.
                                                                                      observed is recorded




                                                                                              B-15
HET-CAM Draft Mild/Moderate Ocular Irritants BRD: Appendix B1
DO NOT CITE, QUOTE, OR DISTRIBUTE
                                                                                                     HET-CAM Test Method Protocols                                                  March 27, 2009




                                                                                                                         Gilleron et al. (1997)          Hagino et al. (1999)



                                                                                                                      Irritation Index calculated
                                                                                                                      using the formula: (301-sec
                                                                                                                      H)/300*5+(301-sec
                                                                                                                      L)/300*7=(301-sec C)*9;
                                                                                                                      where H=Hemorrhage,
                                                                                         Analysis Method                                                      Not Noted
                                                                                                                      L=Vessel Lysis;
                                                                                                                      C=Coagulation;
                                                                                                                      sec=starting second. Mean
                                                                                                                      value and SEM of are
                                                                                                                      calculated.




                                                                                                                      Non Irritant: 0-4.9;
                                                                                       Classification Scheme                                                  Not Noted
                                                                                                                      Irritant: 5.0-21




                                                                                        GLP Compliance?                       Not Noted                       Not Noted




                                                                                                                      Cites Gilleron 1996 as basis
                                                                                                                                                   Cites Luepke 1985 as basis for
                                                                                                                      for protocol and analysis
                                                                                                                                                   scoring method
                                                                                                                      method



                                                                                               Notes




                                Abbreviations: CAM = Chorioallantoic membrane, ECETOC = European Centre for Ecotoxicology and Toxicology of Chemicals, FHSA = Federal
                                Hazardous Substances Act (1964), IS = Irritation score, IT = Irritation threshold concentration, NaCl = Sodium chloride, NaOH = Sodium hydroxide,
                                SDS = Sodium dodecyl sulfate, Sec = seconds.




                                                                                                                              B-16

				
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