CD-ROM 1120 - 1 Revision 0 December 1996 METHOD 1120 DERMAL CORROSION

1.0 SCOPE AND APPLICATION 1.1 The dermal corrosion assay system is an in vitrotest method which determines th e corrosive potentia l of a substance toward human skin. The method is simple, rapid, accurate, and may be applied to both solids, liquids an d emulsions. The liquids may be aqueous or non-aqueous. Solids can be water-soluble or non-soluble. The samples may be pure chemicals, dilutions , formulations, or waste. No prior treatment of the sample is required. This method may be used to meet certain regulatory appl ications, e.g., DOT corrosivity measurement for Packing Groups, but is not required for determining if awaste passes or fails the characteristic of corrosivity per the RCRA definition

. 1.2 This method is applicabl e to a limited number of materials, specifically: Acids, inorganic and organic; acid derivatives (anhydride, haloacids, salts, etc.), inorganic and organic; acyl halides; alkylamines and polyalkylamines; bases, inorganic and organic; c hlorosilanes; and metal halides and oxyhalides. 2.0 SUMMARY OF METHOD 2.1 The assay system is an in vitrotest method which is composed of two components, a syn theticmacromolecular biobarrier and a Chemical Detection System (CDS). Test samples ar e applied o n top of the macromolecular biobarrier. Corrosive samples are able to disrupt th e macromolecularstructure of the biobarrier. A color change in the CDS, located beneath th e biob arrier, is detected visually and indicates that the test sample has altered the biobarrie r suffi cientlyto allow its passage through the full thickness of the biobarrier. The time it takes a sample to disrupt the b iobarrier is inversely proportional to the degree of corrosivity of the sample - the longer it takes to observe a color change, the less corrosive the substance is. Noncorrosiv e samples do not disrupt the biobarrier, or disrupt the biobarrier after a predetermined time period (see Section 2.4). Corrosive samples may be placed into three d ifferent classes of corrosivity, established by the time re quired for the sample to break through the biobarrier. These classes are called Packin g Groups by the U.S. Department of Transportation (DOT). Packing Groups are assigned according to the degree of danger presented by the corrosive material; Pac king Group I indicates great danger; Packin g Group II, medium danger; Packing Group III, minor danger. For consistency, these same definitions are used for this test method and are referred to as Group I, Group II, and Group III. 2.2 Priorto performing the assay, samples are pre-qualified to establish their compatibility with the assay system. The sample is placed in a small amount of CDS fluid. If any detectabl e change o ccurs in the CDS, the sample is qualified and can be analyzed by the test. If a sample is non-qualified, it is incompatible with the CDS and must be tested by another method. 2.3 Test samples are classified into categories by the screening test which is supplied with the assay kit. The category that a sample is assigned to will determine how the Groups will b e assigned. Test samples are classified by pH changes produced in two well-defined buffers - one designed to buffer acids and another that buffers bases. These buffers are supplied as part of the screening test. Four different categories are defined as follows:CD-ROM 1120 - 2 Revision 0 December 1996 2.3.1 CategoryA substances produce a large change in pH when they are added to 1 the acid buffer. This change in pH is indicated by a strong color change of the acid buffe r solution. 2.3.2 CategoryB substances produce a large change in pH when they are added to 1 the base buffer. This change in pH is indicated by a strong color change of the base buffer solution. 2.3.3 Category A substances produ ce little or no pH changes when added to the acid 2 buffer, and therefore, little or no color change in the buffer solution is observed. 2.3.4 Category B substances produce little or nopH changes when added to the base 2 buffer, and therefore, little or no color change in the buffer solution is observed. 2.4 Groups are assigned in the assay system by taking into account the category that i s assigned to a sample by the screening test, and the time it takes to det ect a color change in the CDS in the assay . Category A and B samples are assigned to Group I if a color change is observe d 1 1 between zero and three minutes, to Group II if a color change is observed after three minutes and up to one hour, and to Group III if a color change is observed after one hour and up to four hours. If no color change occurs in four hours, the chemical is classified as Noncorrosive. Category A and B samples are assigned to Group I if a color change is observed between

2 2 zero and three minutes, to Group II if a color change is observed after three minutes and up to 30 minutes, and to Group III if a color chang e is observed after 30 minutes and up to 60 minutes. If no color change occurs in 60 minutes, the chemical is classified as Noncorrosive. 3.0 INTERFERENCES 3.1 The test is not subject to interference from color, turbidity, co lloidal matter or high salinity. 3.2 The Pre-qualification Test, the Screening Test and the Assay must be performed at room temperature. The samples must also be at room temperature (17- 25 E C). 4.0 APPARATUS AND MATERIALS 4.1 Corrosi texAssay Kit (InVitro International, 16632 Millikan Avenue, Irvine, CA 92714)

. The following three items are supplied in the Corrositex Assay Kit:

4.1.1 Four racks holding seven vials with black caps. 4.1.2 One tray of 24 membrane discs. 4.1.3 Four data sheets (color charts). 4.2 Combina tion hot plate/stir plate or equivalent - able to heat to 75 E C. Stirring spee d should be adjustable. 4.3 Digital thermometer - able to read to 75 E C. 4.4 Timers (6) - able to measure hours, minutes and seconds.CD-ROM 1120 - 3 Revision 0 December 1996 4.5 Repeat pipettor - this pi pet is different than the pipet specified in Section 4.12. Delivers 200 μL repeatedly, without refilling between individual deliveries. 4.6 2.5 mL combitip for repeat pipettor. 4.7 La bIndustries or equivalent sample pipettor - a positive displacement pipettor usefu l when pipetting viscous samples. 4.8 Pipet tips for Lab Industries, or equivalent, pipettor. 4.9 Test tubes 4.10 Balance - capable of weighing 100 mg accurately. 4.11 Spatula - capable of transferring 0.1 - 0.5 g. 4.12 Pipets - microliter, with disposable tips. Should be able to measure 100 μL accurately. 4.13 Tweezers. 4.14 Permanent marker pens. 4.15 Plastic wrap. 5.0 REAGENTS 5.1 All reagents listed below are provided in the Corrositex Assay Kit except for the positive and negative controls mentioned in Section 5.7. The Corrositex Assay Kit is available from InVitro International, 16632 Millikan Avenue, Irvine, CA 92714. 5.2 Chemical Detection System (CDS). 5.3 Screening test buffer solutions. 5.4 Confirmation Test Solution. 5.5 One gram of the biobarrier matrix and a microstir bar. 5.6 10 mL of biobarrier diluent. 5.7 Positive and negative controls, if desired, for GLP purposes. 6.0 SAMPLE COLLECTION, PRESERVATION, AND HANDLING 6.1 Appropriate precautions should be taken for handling potentially corrosive substance s such as wearing gloves and having proper eye protection. 6.2 Samples should be analyzed as soon as possible after collection.CD-ROM 1120 - 4 Revision 0 December 1996 7.0 PROCEDURE 7.1 Follow the established laboratory procedures for working with hazardous test samples. Wear lab coat, gloves and safety glasses when working with any potentially corrosive material. 7.2 Pre-Qualification Test 7.2.1 Add 100 mg or 150 μL of sample to 1.0 mL of CDS in duplicate test tubes. 7.2.1.1 Sample qualifies if there is a color reaction within 5 minutes: proceed with assay. 7.2.1.2 If no reaction is observed, the sample is non-qualified. Seek othe r methods to determine corrosivity. 7.3 Screening Test 7.3.1 Liquid samples 7.3.1.1 Add150 μL of sample to Test Tubes A and B. Cap the test tubes and shake vigo rously for 10 seconds. Read the color change of the mixture within on e minute. If the sample is immiscible in the solution, wait one minute and then read the color change at the interface. 7.3.1.2 Assign the category. If an intense color chang e (similar to the Category 1 color chart) is observed in Tube A or Tube B, assign the sample to Category 1. If a less int ense color change (similar to the Category 2 color chart) is observed in eithe r Tube A or Tub e B, assign the sample to Category 2 . If no color change is observed in either Tube A or Tube B, proceed to the next step. 7.3.1.3 Confirm test. Add two drops of the Confirm reagent to Tube B. Cap the testtube and shake vigorously for 5 seconds. The color of the solution will match one of the colo rs shown in the accompanying color chart, confirming that the sample i s Category 2 material. 7.3.2 Solid samples 7.3.2.1 Add 100 mg of sample to TestTubes A and B. Cap the test tubes and shake vigorously for one minute. Wait another minute and read the color change of the mixture. If the sample is insoluble in the solution, allow the mixture to settle and read the color change at the interface of the solution and the solid. 7.3.2.2 Assign the category. If an intense color chang e (similar to the Category 1 color chart) is observed in either Tube A or Tube B, assign the sample to Category 1. If a less intense color change (similar to the Category 2 color chart) is observed in either Tube A or Tube B, assign the sample to Category 2. If no color change is observed in either Tube A or Tube B, proceed to the next step. 7.3.2.3 Confirm test. Add two drops of the Confirm reagent to Tube B. Cap the testtube and shake vigorously for 5 seconds. The color of the solution will match one of the colo rs shown in the accompanying color chart, confirming that the sample i s Category 2 material.CD-ROM 1120 - 5 Revision 0 December 1996 7.4 Assay 7.4.1 Biobarrier preparation 7.4.1.1 Biobarrier matrix preparation - must be completed at least two hours prior to running assay. 7.4.1.2 Place scintillation vial containing biobarrier matrix powder on the ho t plate pad. Begin spinning the stir bar before adding the diluent. 7.4.1.3 Add the entire contents of the biobarrier diluent vial slowly an d constantly to the vial of biobarrier matrix powder. Make sure that the stir bar is turning while adding the diluent. The stir bar should be turning rapidly, but not so fast that the solution foams. 7.4.1.4 Turn the he at on low; monitor the temperature of the solution as it i s warming. Gradually increase the heat as necessary towarm the solution to 68 E C (±1 E C) to solub ilize the matrix. This may take approximately 20 minutes. DO NOTallow th e temperature to exceed 70 E C. 7.4.1.5 W h ile the solution is warming, remove the tray of 24 membrane discs from the refrigerator. Remove the tray lid. 7.4.1.6 Af ter the biobarrier matrix solution reaches 68 E C and has bee n completelysolubilized, turn off the heat and move the vial toward the edge of the heat pad to keep it warm while aliquotting into the membrane discs. 7.4.1.7 Dispense solubilized solution into membrane discs. Using the repeat pip ettor, set to dispense 200 μL. Slowly fill the pipette tip with biobarrier solution , avoiding air bubbles. Dispense one a liquot back into the biobarrier vial to ensure proper subsequent volume delivery. Wipe the tip before dispensi ng each aliquot. Dispense 200 μL into each disc, ensuring that the entire membraneis covered and no air bubbles have formed. Any air bubbles in the gel will alter the results of the test, therefore the dis c cannot be used. 7.4.1.8 Label the lid with the date, time, lot number, and initials of the technician p reparing the biobarrier. Wrap the filled tray evenly with plastic wrap; do not bunch up the plastic wrap underneath the plate. 7.4.1.9 Store the tray at 2 - 8 E C for at least two hours before beginning an y testing. The biobarrier is stable for seven days if wrapped and stored at 2 - 8 E C. 7.4.2 Running the assay. 7.4.2.1 On the da ta sheet, complete the lot number, date, time, name o f technician, name of chemical, whether it is solid or liquid, and pH of a 10% solutio n diluted in water or appropriate solvent. 7.4.2.2 When ready to test, remove one tray of seven pre-filled black-capped scintillation vials from the kit box.CD-ROM 1120 - 6 Revision 0 December 1996 7.4.2.3 Remove the tray of 24 membrane d iscs from the refrigerator. Place on a tray of ice. 7.4.2.4 Place disc into first scintillation vial. Do not allow the discs to be i n contact with the CDS for longer than two minutes before applying the test sample

. Within two minutes, add 5 00 μL (using the Lab Industries pipettor, or equivalent) or 500 mg (using spatula or tweezers) of test sampl e to disc. Start timer the instant the sample is added. Note : Do not cap the vials while test is in progress due to potential pressur e build-up. 7.4.2.5 Watch the vial for threeminutes, ensuring that the color reaction is not missed if it is a Group I sample. Changes in the CDS m ay include various color changes, flaking or precipitation. 7.4.2.6 Add three more discs and samples to vials, staggering each start time so that the most accurate reaction times are recorded. 7.4.2.7 Allow assay to run until color or physical reaction occurs. Category 1 samples should be chec ked for reactions at 3 minutes, 1 hour and 4 hours. Category 2 samples should be checked for reactions at 3, 30 and 60 minutes. 7.4.2.8 At the first indication of the presence of a ch emical reaction in the CDS, there will be a color change produced beneath the bottom-center of each biobarrier disc. As soon as a reaction is observed, immediately record net time of each vial on the data sheet. 7.4.2.9 Run positive and negat ive controls in the other two vials, if desired, for GLP purposes. 7.4.3 Assignment of Groups. 7.4.3.1 Category 1 samples are assigned to Group I if a color change i s observed between zero and three minutes, to Group II if a color change is observed after three minutes and up to one hour, and to Group III if a color cha nge is observed after one hourand u p to four hours. If no color change occurs in four hours, the chemical i s classified as Noncorrosive. 7.4.3.2 Category 2 samples are assigned to Group I if a color change i s observed between zero and three minutes, to Group II if a color change is observed after three minutes and up to 30 minutes, and to Group III if a color change is observed after 30 minutes and up to 60 minutes. If no colo r change occurs in 60 minutes, the chemical is classified as Noncorrosive. 8.0 QUALITY CONTROL 8.1 Samples should b e analyzed in quadruplicate. The test may be analyzed in duplicate if a simple screening of corrosives and noncorrosives is all that is required. However, it i s recommended that for greater accuracy, sample s be analyzed in quadruplicate. It is suggested that positive and negative controls be analyzed to conform with GLP.CD-ROM 1120 - 7 Revision 0 December 1996 9.0 METHOD PERFORMANCE 9.1 Interlaboratory and intralaboratory studies were conducted with five different laboratories. Ten different chemicals were tested with six replicates. The data are presented in Table 1. 9.2 More than 200 data points have been collected at InVitro International for six reference samples. Statistical analysis of this data shows the standard deviation for ethylenediamine and ferric chl oride is about 5% of their respective assay times, and about 10% for maleic anhydride, sodium h y droxide, and dicyclohexylamine. The standard deviation for sulfuric acid approaches 18% of its assay time, but when taken into account that the mean assay time is less than 1 minute, a standard deviation of 0.13 minutes is actually a reflection of the diff iculty of measuring such brief time periods. 9.3 The Corrositex assay has been used by more than 300 la boratories to test approximately 4, 000 test materials in its first phase of utilization in industry. Diverse chemicals and formulations which include liquids, solids, insolubles and immiscibles have been studied from many majo r industries includ ing petrochemical, agrochemical, surfactant, textile, paper and pulp, electroplating and water treatment. Examples of dermal corrosion values comparedto pH for selected compounds are shown in Table 2. 9.4 Data results from 1,050 samples that have been tested using the assay system wer e complied and compared with in vivo data. Ninety-two percent of the samples (965 samples) passed the Pre-qual ification Test and were then analyzed in the screening test and the assay. Assay was found to be highly concordant with corrosive/noncorrosive in vivoresults. Of 406 corrosive samples with in vivodata, 377 (93%) were correctly identified as corrosive by assay. Of 296 noncorrosive samples with in vivo

data, 83% were identified as noncorrosives, demonstrating the ability of this in vitro method to correctly identify corrosives and noncor rosives. Assay was also shown to accurately predict Packing Groups. Six hundred out of 7 02 samples (85.5%) were placed in the same Packing Group as that indicated by in vivo testing. Only 38 test samples out of 702 samples that had in vivo data were found to underestimate (5.4%). Of these 38 samples, 28 were distinct samples and the remainderwe re samples that had been tested in more than one laboratory. When taking thi s information into account, the percent of underestimation decreased to about 4%. 10.0 REFERENCES 1. Code of Federal Regulations, Transportation Title 49, Hazardous Materials Table, Sectio n 172.101 (1991). 2. Code of Federal Regulations, Transportation Title 49, Method of Testing Corrosion to the Skin, Part 173, Appendix A (1991). 3. Schless elman, J.J. (1973) Planning a Longitudinal Study: I. Sample Size Determination. J

. Chron. Dis. 26 , 553-560. 4. ASTM Stan dards on Precision and Bias for Various Applications, “Standard Practice fo r Conducting an Interlaboratory Study to Determine the Precision of a Test Method”; ASTM : Philadelphia, PA, 1992; E 691-92. 5. Gordon, V.C., Harvell, J., and Maibach, H. (1994). Dermal Corrosion, The Corrositex System, A DOT Accepted Method to Predict Corrosivity of Test Materi als. In VitroToxicology . Ed. Mary Ann Liebert, 1994.CD-ROM 1120 - 8 Revision 0 December 1996 TABLE 1 LABORATORY DATA Dichloroacetyl chloride Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 3.30 0.59 17.88% 2 2.99 0.30 10.03% 3 3.88 0.47 12.11% 4 2.50 0.26 10.40% 5 3.30 0.28 8.48%

Formic acid Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 6.32 0.51 8.07% 2 5.08 0.46 9.06% 3 5.18 0.77 14.86% 4 4.82 0.29 6.02% 5 4.02 0.29 7.21%

Dichloroacetic acid Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 6.92 0.32 4.62% 2 5.21 0.25 4.80% 3 6.32 0.98 15.51% 4 5.78 0.26 4.50% 5 5.65 0.46 8.14%

Chloroacetic acid Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 5.46 0.36 6.59% 2 7.83 0.00 0.00% 3 4.95 0.35 7.07% 4 6.91 0.94 13.60% 5 4.95 0.34 6.87%TABLE 1 (continued)

CD-ROM 1120 - 9 Revision 0 December 1996 Dodecyltrichlorosilane Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 10.78 0.05 0.46% 2 11.59 0.36 3.11% 3 11.22 0.82 7.31% 4 11.96 0.56 4.68% 5 10.98 0.29 2.64% Ammonium hydrogen sulfate Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 10.47 0.56 5.35% 2 9.02 0.33 3.66% 3 13.82 1.0 7.24% 4 11.17 0.93 8.33% 5 7.88 0.26 3.30% Ethylenediamine Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 17.24 0.38 2.20% 2 21.33 0.53 2.48% 3 26.12 1.30 4.98% 4 20.76 0.19 0.92% 5 22.48 1.40 6.23%

Aluminum chloride Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 11.91 0.41 3.44% 2 21.33 0.53 2.48% 3 26.12 1.30 4.98% 4 20.76 0.19 0.92% 5 22.48 1.40 6.23%TABLE 1 (continued)

CD-ROM 1120 - 10 Revision 0 December 1996 Acetic acid Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 28.52 0.87 3.05% 2 27.00 0.00 0.00% 3 34.98 0.22 0.63% 4 36.30 0.41 1.13% 5 29.67 0.62 2.09% Dicyclohexylamine Laboratory Results (minutes) Deviation

Mean of 6 Test Standard Deviation Relative Standard

1 181.73 0.47 0.26% 2 168.83 9.11 5.40% 3 210.70 7.68 3.64% 4 159.04 7.58 4.77% 5 126.75 0.62 0.49%CD-ROM 1120 - 11 Revision 0 December 1996 TABLE 2. EXAMPLES OF DERMAL CORROSION VALUES FOR SELECTED COMPOUNDS

Compound Name Concentration pH Time (weight %) (minutes) 1

Acetic acid 99+ 0.00 29.31 Aluminum chloride pure 2.92 16.50 Ammonium hydroxide 10.00 12.37 5.41 Bromoacetic acid 55.60 0.93 9.17 Butylamine 40.00 12.96 >240 Citric acid 20.00 1.28 47.65 1,2-Diaminopropane 99+ 12.06 21.67 Dichloroacetic acid 3.10 0.98 37.63 Dicyclohexylamine 99.00 9.57 210.00 Diethylamine 98.00 13.86 5.89 Diethylene triamine 99.00 12.01 34.00 Ethanolamine 99+ 11.82 21.68 Ferric chloride 98.00 3.00 21.30 Formic acid 33.90 0.62 >240 Hydrochloric acid 35.00 0.00 5.80 Hexanoic acid 99.00 3.00 149.00 Maleic acid 99.00 1.30 15.55 Mercaptoacetic acid 15.10 1.60 42.09 Nitric acid 90.00 0.00 0.57 Phosphoric acid 85.00 0.00 15.00 Potassium hydroxide pellets 14.00 6.82 Propionic acid 99+ 0.35 34.59 Sodium hydroxide pellets 13.81 14.67 Sodium metasilicate 20.00 13.28 17.17 Sulfuric acid 15.00 0.00 11.48 Thiophosphoryl chloride 98.00 5.81 10.13 Tributylamine 99+ 10.70 >240 Trichlorotoluene 99.00 3.32 >240 Triethanolamine 60.00 11.02 41.03 Triphosphoryl chloride 98.00 5.80 10.25 pH of a 10% solution of the compound in water. 1CD-ROM 1120 - 12 Revision 0 December 1996 METHOD 1120 DERMAL CORROSION