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To: NCIC OPPT/DC/USEPA/US@EPA cc: Terry OBryan/DC/USEPA/US@EPA
Subject: AR226 and FYI Submission from DuPont Haskell
Please include the attached report in both the AR226 and TSCA Section 8 FYI files. Although the cover page of the report is captioned "Trade Secret," the company has confirmed that no confidentiality provisions apply, and that the document itself contains no trade secret or TSCA Confidential Business Information.
The study title of the report is "K-24921: Dermal Sensitization Test Buehler Method"
If you have any questions, please contact me.
Mary F. Dominiak
Oontai n
U.S. Environmental Protection Agency
EPA East, Room 4410-M, Mail Code 7405M
1200 Pennsylvania Avenue, NW
Washington, DC 20460
Phone: 202-564-8104 Fax: 202-564-4775
---- Forwarded by Mary Dominiak/DC/USEPA/US on 02/21/2002 02:52 PM
Jill H Hogan <Jill.H .Hogan-2 @usa.d Dominiak/DC/USEPA/US0EPA upont.com>
Gerry Kennedy
01/15/2002 09:02 AM
To: Mary
cc: Subject:
Re: Report from
in
Hi Mary,
Gerry checked and it is okay to send this report to you electronically. It's a pdf file so if you have trouble opening it, please let me know.
Thanks,
Jill
(See attached file: DuPont-7977.pdf) (See attached file: DuPont-7 977.pdf)
DuPont-7977.pdf
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FYI-00-001378
FYI-0 0 -0 0 1 3 7 8
85020000003
85020000003
I
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TRADE SECRET
STUDY TITLE
H -24921: Dermal Sensitization Test - Buehler Method
LABORATORY PROJECT IDENTIFICATION
DuPont-7977 PSL Study Number 11321 Work Request Number 13890 Service Code Number 641
DATA REQUIREMENT
U.S. EPA Health Effects Test Guidelines, OPPTS 870.2600, August, 1998
AUTHOR
George E. Moore, B.S.
STUDY COMPLETED ON
December 17, 2001
PERFORMING LABORATORY
Product Safety Labs 2394 Route 130
Dayton, New Jersey, 08810
SUBMITTER
DuPont Haskell Laboratory for Health and Environmental Sciences
Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050
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H-24921: Dermal Sensitization Test - Buehler Method
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GOOD LABORATORY PRACTICE STATEMENT
This study meets the requirements o f U.S. EPA Good Laboratory Practice Standards: Toxic Substances Control Act: 40 CFR 792 with the following exceptions:
1. Treatment solutions were not analyzed for concentration, uniformity or stability o f the test and control substances. The procedures used by trained personnel to prepare the treatment solutions insured:
a) The accuracy o f concentration because the test substance diluent (vehicle) was accurately measured with a graduated device. The test substance was weighed on a balance accurate to at least two decimal places
b) Uniformity, because all solutions were thoroughly mixed prior to administration to the test system; and
c) Stability, because treatment solutions were prepared just prior to use.
2. The stability, uniformity of mixture and verification of concentration o f HCA in its carriers were not determined.
Applicant/Sponsor: Study Director:
E.I. du Pont de Nemours and Company Newark, Delaware U.S.A.
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QUALITY ASSURANCE STATEMENT
The Quality Assurance Unit randomly selects intervals for QA inspections prior to study initiation. Records o f the findings o f these inspections are kept on file. The summary below provides verification of statements made in the final report section that addresses Quality Assurance audits.
Inspections were made of:
DATE 10/26/01 11/20/01 11/20/01 l> Jl 7 / s j
PROCEDURE INSPECTED 48 hour scoring (Induction #3) Raw data Draft report Final report
Findings reported to:
Study Director 11/20/01 Management 11/20/01
>4-----
Annamarie LaPorte Quality Assurance Auditor
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CERTIFICATION
We the undersigned declare that the methods, results and data contained in this report faithfully reflect the procedures used and raw data collected during the study.
George E. Kioore, B .SC. Study Director
'
Gary WnorotfSki, B.A. Laboratory Director
___ VjL/lfr<- iCflLtLo
Carol Finlay, B.A. (
fl
Study Monitor
________ C u e - n x o o ! Date
h^Lc- I "1 ,l e c I
Date
0- ' Date
- 'V b b 'L
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TABLE OF CONTENTS
GOOD LABORATORY PRACTICE STATEMENT............................................................................................. 2 QUALITY ASSURANCE STATEMENT...............................................................................................................3 CERTIFICATION................................................................................................................................................... 4 TABLE OF CONTENTS....................................................................................................................................... 5 STUDY INFORMATION....................................................................................................................................... 6 H-24921: DERMAL SENSITIZATION TEST - BUEHLER METHOD.................................................................7 1. PURPOSE..................................................................................................................................................... 7 2. SUMMARY.....................................................................................................................................................7 3. MATERIALS.................................................................................................................................................. 8 4. M ETHO DS.................................................................................................................................................... 9 5. PROCEDURE............................................................................................................................................. 10 6. EVALUATION............................................................................................................................................. 11 7. HISTORICAL POSITIVE CONTROL VALIDATION STU D Y................................................................... 11 8. STUDY CONDUCT.....................................................................................................................................12 9. REFERENCES............................................................................................................................................ 12 10. QUALITY ASSURANCE.............................................................................................................................12 11. DEVIATION FROM THE FINAL PROTOCOL.......................................................................................... 12 12. FINAL REPORT AND RECORDS RETENTION...................................................................................... 12 13. RESULTS.....................................................................................................................................................12 14. CONCLUSION............................................................................................................................................ 13 TABLE 1: PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC (TEST SUBSTANCE)..................................................................................................................................................... 14 TABLE 1 (cont.): PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC (POSITIVE CONTROL-HCA).............................................................................................................................15 TABLE 2: INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g)........................................................................ 16 TABLE 2 (cont.): INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g )............................................................17 TABLE 3: INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g)........................................................................ 18 TABLE 4: INDUCTION PHASE SKIN REACTION SCORES........................................................................... 19 TABLE 4 (cont.): INDUCTION PHASE SKIN REACTION SCORES.............................................................. 20 TABLE 5: INDUCTION PHASE SKIN REACTION SCORES........................................................................... 21 TABLE 6: CHALLENGE PHASE SKIN REACTION SCORES........................................................................ 22 TABLE 6 (cont.): CHALLENGE PHASE SKIN REACTION SCORES............................................................ 23 TABLE 7: CHALLENGE PHASE SKIN REACTION SCORES........................................................................ 24 APPENDIX A: FEED AND WATER ANALYSES.............................................................................................. 25
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9th Collective Nomenclature: Svnonvms/Codes:
Haskell Number: CAS Reeistrv Number: ComDosition:
Puritv: Physical Characteristics: Stability: Sponsor: Study Initiated/Comnleted: In-Life Initiated/Comnleted:
STUDY INFORMATION
Octanoic acid, pentadecafluoro-, ammonium salt
Ammonium perfluorooctanoate FC-143 FLUORAD Brand Fluorochemical Surfactant
(3M Chemicals) C-8 Perfluorooctanoate, ammonium salt PFOA H-24921 Lot 332 (3M Specialty Materials) (Lot No.)
24921
3825-26-1
96.5-100% 0-1.5% 0-1 % 0-1%
Ammonium perfluorooctanoate [CAS # 3825-26-1] Ammonium perfluorohexanoate [CAS #21615-47-4] Ammonium perfluoroheptanoate [CAS #6130-43-4] Heptadecafluorononanoic acid, Ammonium Salt [CAS #4149-60-4]
95.2%
White solid
The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.
September 27, 2001/ (see report cover page)
October 10,2001 /November 9,2001
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H-24921 : DERMAL SENSITIZATION TEST - BUEHLER METHOD
PRO TO CO L NO.: AGENCY: PSL STUDY NUMBER: SPONSOR:
SU B M IT T E R :
TEST SUBSTANCE IDENTIFICATION: TEST SUBSTANCE DESCRIPTION: DATE RECEIVED: PSL REFEREN CE NO.: DATE OF PROTOCOL APPROVAL: EXPERIM ENTAL INITIATION DATE: EXPERIM ENTAL COM PLETION DATE: STUDY COM PLETION DATE: NOTEBO O K NO.:
P328 DUP
EPA (TSCA)
11321
E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.
DuPont Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, DE 19714-0050
H-24921
White solid
September 19,2001
010919-19R
September 27,2001
October 10,2001
November 9, 2001
December 17,2001
01-55: pages 133-146
1. PURPOSE To assess the sensitization potential o f H-24921 after repeated topical applications.2
2. SUMMARY A dermal sensitization test was conducted with guinea pigs to determine the potential for H-24921 to produce sensitization after repeated topical applications.
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The test substance (90%' w/w mixture in distilled water) was topically applied for six hours to 20 healthy test guinea pigs, once each week for a three week induction period. A test vehicle control group (ten animals) was maintained under the same environmental conditions and treated with the vehicle (distilled water, 100%) for the induction phase. Twenty-seven days after the first induction dose, a challenge dose of the test substance (HNIC, 50% w/w solution in distilled water) and vehicle (distilled water, 100%) were applied to a naive site on each o f the test and test substance irritation control guinea pig. Approximately 24 and 48 hours after each induction and challenge dose, the test and test irritation control animals were scored for erythema.
A table summarizing the incidence and severity o f the sensitization response noted after challenge is found below.
50% w/w in distilled water Distilled water
Incidence with Skin Reactions2
Test Animals
Test Irritation Control Animals
Hours
24 48 24 48
0/20 0/20 0/10 0/10
0/20 0/20 0/10 0/10
50% w/w in distilled water Distilled water
Test Animals
24 48 0.13 0.05 0.00 0.00
Severity123 Test Irritation Control Animals
Hours 24 48 0.10 0.05 0.00 0.00
Based on the results o f this study, the test substance is considered not to be a contact sensitizer. The positive response observed in the historical positive control validation study with ahexylcinnamaldehyde, technical grade, 85% validates the test system used in this study (See Section 7).
3. MATERIALS
A. Test Substance
The test substance identified as H-24921, was received on September 19, 2001 and was further identified with PSL Reference Number 010919-19R. The test substance was stored at room temperature. The sample was a white solid.
1 The test substance, as received, was a solid. To enhance skin contact, the test substance was moistened with distilled water prior to application.
2 Animals with scores greater than 0.5 3Sum o f the erythema scores divided by the number of animals evaluated.
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Characterization o f the test substance provided to Product Safety Labs by the Sponsor was: Composition: 96.5-100% Ammonium perfluorooctanoate
pH: 5 (0.5% aqueous)
Solubility: Not applicable
Stability: The test substance is expected to be stable for the duration o f testing
Expiration Date: December 15, 2001
B. Animals
3.B. 1 Number o f Animals: 34
3.B.2 Number o f Groups: 3
3.B.3
Number o f Animals per Group: Preliminary Irritation Testing: 4 Test Group: 20 Test Vehicle Control Group: 10
3.B.4 Sex: Male
3.B.5 Species/Strain: Guinea pigs/Hartley albino
3.B.6
Age/Body weight: Preliminary Irritation Group; Young adult Test and Test Vehicle Control Groups: Young adult/349-443 grams at experimental start
3. B.7 Source: Received from Elm Hill Breeding Labs, Chelmsford, M A on October 3, 2001
4. METHODS
A. Husbandry
4.A.1
Housing: The animals were group housed in suspended stainless steel with mesh floors or plastic perforated bottom caging which conform to the size recommendations in the most recent Guide fo r the Care and Use o f Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at least three times per week.
4.A.2 Animal Room Temperature and Relative Humidity Ranges: 18-23 C and 30-68%
4.A.3 Photoperiod: 12 hour light/dark cycle
4A .4 Acclimation Period: 7 days
4A .5 Food: Pelleted Purina Guinea Pig Chow #5025
4A .6 Water: Filtered tap water was supplied ad libitum by automatic water dispensing system.
4.A.7 Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results o f this study. Analyses o f the food and water are conducted at least once a year and the
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DuPont-7977
records are kept on file at Product Safety Labs. The dates o f the most recent analyses are presented in Appendix A.
B. Identification
4.B. 1 Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex o f the animal.
4.B.2 Animal: Each guinea pig was marked with a color code and given a sequential animal number assigned to study #11321, which constituted unique identification.
5. PROCEDURE
A. Preparation and Selection of Animals
Within 24 hours prior to each application, the fur o f each guinea pig assigned to test and test vehicle control groups was removed by clipping the dorsal area and flanks. Care was taken to avoid abrading the skin. Prior to study initiation, the animals were weighed and the skin was checked for any abnormalities. Only healthy animals without pre-existing skin irritation were selected for test.
B. Induction Phase
Once each week for three weeks, 0.4 ml of a 90%' w/w mixture o f the test substance in distilled water was applied to the left side o f each test animal for six hours using an occlusive 25 mm Hill Top Chamber. The chambers were secured in place and wrapped with non-allergenic DuraporeTM adhesive tape to avoid dislocation of the chambers and to minimize loss o f the test substance. After the six hour exposure period, the chambers were removed and the test sites were gently wiped with water and a clean towel to remove any residual test substance. Approximately 24 and 48 hours after each induction application, readings were made of local reactions (erythema) according to the scoring system described in Section 5.D. The vehicle (distilled water, 100%) was used for the induction of the test vehicle control animals and scored as above.
C. Challenge Phase
Twenty-seven days after the first induction dose, 0.4 ml o f a 50% w/w solution o f the test substance (HNIC) in distilled water was applied to a naive site on the right rear flank o f each animal using the procedures described above. The vehicle (distilled water, 100%) was applied to the right front flank o f each test animal for the challenge phase. The test substance irritation control group was also treated with the test substance and vehicle for the challenge phase. These sites were evaluated for a sensitization response (erythema) approximately 24 and 48 hours after the challenge application according to the system described in Section 5.D.
D. Scoring System
0 - no erythema 0.5 - very faint erythema, usually non-confluent 1 - faint erythema, usually confluent 2 - moderate erythema 3 - severe erythema with or without edema1
1 The test substance, as received, was a solid. To enhance skin contact, the test substance was moistened with distilled water prior to application.
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E. Body Weights
Individual body weights o f the animals were recorded prior to the first induction, weekly and again on the day after challenge. The mean and standard deviation were calculated for all body weights and body weight gains. The overall mean body weight gains for test and test vehicle control animals were evaluated statistically by an unpaired t test (INSTAT Biostatistics, created by GraphPad Software, San Diego, and CA.).
F. Clinical Observations
The animals were observed daily. If any unusual clinical signs were observed, they were recorded.
6. EVALUATION
In order to evaluate the sensitization response noted during the challenge phase, two indices were used; one for incidence and one for severity. The incidence index was calculated to evaluate the incidence o f erythema (sensitization response) approximately 24 and 48 hours after challenge according to the following:
Scores o f 1 or greater in the test group are required to be indicative o f sensitization. If scores of one (1) or greater are seen on the control animals, then the reactions of the test substance group animals that exceed the most severe control reactions are considered to be positive scores.
Incidence is reported as the number o f positive animals in each group divided by the total number o f animals tested in that group.
Severity is reported as the sum o f the test grades divided by the total number o f animals tested in a given group determined for both 24 and 48 hours. All average grades are to be rounded off to the nearest tenth o f a unit.
7. HISTORICAL POSITIVE CONTROL VALIDATION STUDY
The procedures used in this study were validated using a-hexylcinnamaldehyde, technical grade, 85% (purity) as a positive control substance. The most recent validation, PSL Study #10548, was performed by Product Safety Labs and completed on M ay 10, 2001. The raw data and report for this study are archived in Product Safety Labs Historical Data Notebook No. 01, pages 20-28. This test was conducted at the Dayton Facility with HCA using Hartley strain albino guinea pigs from Elm Hill Breeding Labs following induction and challenge procedures similar to those described in Section 5. The data from this positive control study is summarized below.
Positive Control Animals Positive Vehicle Control Animals
Sensitization Response Indices
Incidence of Positive Response'
Severity2
Hours
24 48 24 48
5/10 2/10 0.70 0.40 0/5 0/5 0.45 0.30
1Animals with scores greater than 0.5. 2Sum o f the erythema scores divided by the number of animals evaluated.
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8. STUDY CONDUCT This study was conducted at Product Safety Labs, 2394 Route 130, Dayton, New Jersey, 08810 in compliance with the following regulation:
U.S. EPA Good Laboratory Practice Standards: Toxic Substances Control Act (TSCA): 40 CFR 792
The procedures as described in the protocol are based on the following testing guideline: U.S. EPA Health Effects Test Guidelines, OPPTS 870.2600, August, 1998 The primary technician for this study was Rolland Colis, B.S.
9. REFERENCES Robinson, M., Nusair, T., Fletcher, E., and Ritz, H., A Review o f the Buehler Guinea Pig Skin Sensitization Test And Its Use in a Risk Assessment Process for Human Skin Sensitization in Toxicology, 61, 91-107, 1990.
Ritz, H., and Buehler, E., Planning, conduct, and interpretation o f guinea pig sensitization patch tests, in Current Concepts in Cutaneous Toxicity, V.A. Drill and P. Lazar (Eds.), Academic Press, New York, 25, 1980.
10. QUALITY ASSURANCE The final report was audited for agreement with the raw data records and for compliance with the protocol and Product Safety Labs Standard Operating Procedures. Dates of inspections and audits performed during the study, and the dates of reporting of the inspection and audit findings to the Study Director and Facility Management are presented in the Quality Assurance Statement.
11. DEVIATION FROM THE FINAL PROTOCOL None
12. FINAL REPORT AND RECORDS RETENTION A copy o f the signed report, copies of all raw data generated at Product Safety Labs and a copy of the original signed protocol, will be maintained in the Product Safety Labs archives.
Laboratory-specific or site-specific raw data, such as personnel files and equipment records will be retained by the facility where the work was done.
The original raw data, the original final report and a copy o f the protocol will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware.
13. RESULTS Preliminary irritation testing scores for the test substance and historical positive control animals are presented in Table 1. Individual body weights and body weight gain for test and historical positive control animals are presented in Tables 2 and 3, respectively. Induction and Challenge Phase skin reaction scores for test and historical positive control anim als are presented in Tables 4 through 7. Page 12 of 25
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All test and control animals survived and appeared normal throughout the study. There were no statistically significant differences between the overall body weight gain o f the test and test vehicle control animals.
Induction Phase Test Animals (test substance applied as a 90%' w/w mixture in distilled water): Very faint to faint erythema (0.5-1) was noted for all test sites during the induction phase. Test Vehicle Control Animals (distilled water, 100%): No dermal irritation was noted for any test vehicle control site during the induction phase. Historical Positive Control Animals (HCA as received): Very faint to faint erythema (0.5-1) was noted for all test sites during the induction phase.
Challenge Phase Test Animals (test substance applied as a 50% w/w solution in distilled water): Very faint erythema (0.5) was noted for five of 20 test sites 24 hours after challenge. Irritation persisted at two o f these sites through 48 hours.
Test Substance Irritation Control Animals (test substance applied as a 50% w/w solution in distilled water): Very faint erythema (0.5) was noted for two o f ten test sites 24 hours after challenge. Irritation persisted at one of these sites through 48 hours.
Test Animals (distilled water, 100%): No dermal irritation was noted for any test site following the challenge phase.
Test Substance Irritation Control Animals (distilled water, 100%): No dermal irritation was noted for any test substance irritation control site following the challenge phase.
Historical Positive Control Animals (applied as a 75% w/w solution of HCA (as received) in mineral oil): Five o f ten animals exhibited signs o f a sensitization response (feint erythema [1]) 24 hours after challenge. Similar indications persisted at two o f these sites with very faint erythema (0.5) noted for most other sites through 48 hours.
Historical Positive Vehicle Control Animals (applied as a 75% w/w solution of HCA (as received) in mineral oil): Very faint erythema was noted for four o f five test naive control sites 24 hours after challenge. Irritation persisted at three o f these sites through 48 hours.
14. CONCLUSION Based on these findings and on the evaluation system used, H-24921 is considered not to be a contact sensitizer.
The positive response observed in the historical positive control validation study with ahexylcinnamaldehyde, technical grade, 85% (as received) validates the test system used in this study (See Section 7).1
1 The test substance, as received, was a solid. To enhance skin contact, the test substance was moistened with distilled water prior to application.
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TABLE 1: PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC1(TEST SUBSTANCE)
Animal No. Sex
2998 2999 3000 3001
M M M M
903 24 48
CONCENTRATION (% f 75 50
Hours after Patch Removal 24 48 24 48
0.5 0.5 0.5 0.5 0.5 0
0.5 0.5 0.5 0.5 0
0
0.5 0.5 0.5 0 0 0
1 1 1 1 0.5 0.5
25
24 48 00 00 00 00
1HNIC - Highest Non-Irritating Concentration
2 Four-tenths o f a milliliter o f the test substance was applied as w/w mixtures, suspensions or solutions in distilled water using an occlusive 25 mm Hill Top Chamber. 3 The test substance, as received, was a solid. To enhance skin contact, the test substance was moistened with distilled water prior to application.
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TABLE 1 (cont): PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC1 (POSITIVE CONTROL-HCA)
Historical Positive Control Validation Study
A nim al No. Sex
0210 0211 0212 0213
M M M M
Concentration (%)2
100 75 50 25
0.5 0.5
0
0
0.5 0
0
0
0.5 0
0
0
0.5 0.5
0
0
1HNIC - Highest Non-Initating Concentration 2 Four-tenths o f a milliliter o f HCA was applied as received and as w/w solutions in mineral oil using an occlusive 25 mm Hill Top Chamber.
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TABLE 2: INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g) Test Substance Group
Animal No.
Sex Initial
Week 2
Gain (0 to wk 2)
Week 3
Gain (wk 2 to 3)
Week 4
Gain (wk 3 to 4)
Day After Challenge
3148 M 419 459 40 536 77 594 58
652
Total Gain W eight (wk 4 to S) Gain
(wk 0 to 5)
58 233
3149 M 371 403 32 471 68 516 45
567
51 196
3150 M 385 421 36 485 64 525 40
567
42 182
3151 M 381 413 32 464 51 525 61
562
37 181
3152 M 443 484 41 552 68 628 76
664
36 221
3153 M 427 482 55 548 66 615 67
681
66 254
3154 M 389 428 39 493 65 553 60
603
50 214
3155 M 375 422 47 481 59 536 55
586
50 211
3156 M 352 405 53 463 58 516 53
566
50 214
3157 M 396 458 62 527 69 589 62
647
58 251
3158 M 372 395 23 444 49 482 38
532
50 160
3159 M 373 413 40 463 50 514 51
552
38 179
3160 M 404 471 67 527 56 592 65
628
36 224
3161 M 381 443 62 505 62 554 49
596
42 215
3162 M 396 434 38 473 39 537 64
566
29 170
3163 M 421 472 51 539 67 601 62
668
67 247
3164 M 388 429 41 491 62 531 40
584
53 196
3165 M 404 444 40 511 67 544 33
595
51 191
3166 M 374 444 70 516 72 582 66
653
71 279
3167 M 394 458 64 490 32 526 36
581
55 187
M ean 396.2 449.4 S ta n d a r d 17.61 16.27 D eviation
53.2 13.48
509.4 20.23
60.0 16.05
556.8 33.04
47.4 15.42
616.2 41.12
59.4 220 8.99 40.98
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TABLE 2 (coni): INDIVIDUAL BODY WEK3HTS/WEIGHT GAIN (g) Test Vehicle Control Group
Animal No.
Sex
Initial
W eek 2
Gain (0 to wk 2)
Week 3
Gain (wk 2 to 3)
Week Gain 4 (wk3to4)
Day After Challenge
3168 M 392 434 42 502 68 547 45
603
Gain (wk 4 to 5)
Total W eight
Gain (wk 0 to 5)
56 211
3169 M 398 439 41 497 58 553 56
616
53 218
3170 M 385 419 34 482 63 540 58
590
50 205
3171 M 401 452 51 522 70 581 59
585
4 184
3172 M 381 409 28 459 50 503 44
542
39 161
3173 M 381 439 58 497 58 566 69
606
40 225
3174 M 368 408 40 456 48 502 46
533
31
165
3175 M 382 431 49 484 53 540 56
577
37
195
3176 M 349 409 60 479 70 526 47
583
57 234
3177 M 398 461 63 531 70 592 61
633
41 235
M ea n 375.6 429.6 54.0 489.4 59.8 545.2 55.8
Standard
Deviation
18.28
22.18
9.41
27.57
9.96
34.95
9.68
586.4 37.11
41.2 210.8 9.65 30.30
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TABLE 3: INDIVIDUAL BODY WEK3HTS/WEIGHT GAIN (g) Historical Positive Control Validation Study1
Animal Sex Initial
No.
W eek 2
Gain (0 to wk 2)
Week 3
Gain (wk 2 to 3)
277 M 396 448 52 508
60
Week Gain Day After 4 (wk 3 to 4) Challenge
550 42
588
Gain (wk 4 to 5)
Total W eight
Gain (wk 0 to 5)
38 192
278 M 407 470 63 535 65 610 75
619
9 212
279 M 397 456 59 512 56 565 53
598
33 201
280 M 393 452 59 496 44 558 62
624
66 231
281 M 407 466 59 512 46 585 73
639
54 232
282 M 396 444 48 490 46 549 59
613
64 217
283 M 392 473 81 524 51 590 66
630
40 238
284 M 357 426 69 465 39 540 75
597
57 240
285 M 399 472 73 511 39 561 50
625
64 226
286 M 364 430 66 473 43 519 46
588
69 224
M ean 390.8 453.7 62.9 502.6 48.9 562.7 60.1
S ta n d a rd 16.84 16.96 D ev iatio n
9.79
21.80
8.88
26.42 12.15
Vehicle
C o n tro l
287 M 367 424 57 484 60 540 56
612.1 18.22
588
49.4 19.10
221.3 15.78
48 221
288 M 374 431 57 480 49 547 67
598
51 224
289 M 359 421 62 499 78 559 60
619
60 260
290 M 386 453 67 506 53 554 48 291 M 411 479 68 543 64 598 55
624 664
70 238 66 253
M ean 379.4 441.6 62.2 502.4 60.8 559.6 57.2
S tandard 20.26 24.37 D e v ia tio n
5.26
25.07
11.26
22.63
6.98
618.6 29.37
59 239.2 9.43 17.22
1PSL Study #10548, performed by PSL and completed on May 10, 2001. Page 18 of 25
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 4: INDUCTION PHASE SKIN REACTION SCORES
Test Substance Group1
Induction Number Hours*2
Animal No. 3148 3149 3150 3151 3152 3153 3154 3155 3156 3157 3158 3159 3160 3161 3162 3163 3164 3165 3166 3167
1 24 48
0.5 0.5 0.5 0.5 0.5 0
1 0.5 00 0.5 0.5 00 0.5 0 0.5 0 0.5 0.5 0.5 0.5 0.5 0 00 00 11 0.5 0.5 1 0.5 0.5 0.5 0.5 0.5 0.5 0
2 24 48
0.5 0 0.5 0.5 0.5 0 0.5 0 0.5 0.5 0.5 0 0.5 0 0.5 0.5
1 0.5 0.5 0 0.5 0
1 0.5 0.5 0.5 0.5 0 0.5 0 0.5 0.5
1 0.5 0.5 0 0.5 0 0.5 0.5
3 24 48
0.5 0 0.5 0.5 0.5 0 0.5 0 0.5 0
1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0 0.5 0 0.5 0.5 0.5 0 0.5 0 0.5 0.5 0.5 0 1 0.5 1 0.5 0.5 0 0.5 0.5
'Four-tenths of a milliliter of the test substance was applied as a 90% w/w mixture in distilled water, using an occlusive 25 mm Hill Top Chamber. 2Hours after induction dose.
Page 19 of 25
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 4 (cont): INDUCTION PHASE SKIN REACTION SCORES
Test Vehicle Control Group1
Induction Number Hours12
Animal No. 3168 3169 3170 3171 3172 3173 3174 3175 3176 3177
1 24 48
00 00 00 00 00 00 00 00 00 00
2 24 48
00 00 00 00 00 00 00 00 00 00
3 24 48
00 00 00 00 00 00 00 00 00 00
1Four-tenths of a milliliter of the vehicle (distilled water, 100%), was applied using an occlusive 25 mm Hill Top Chamber. 2Hours after induction dose.
Page 20 of 25
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 5: INDUCTION PHASE SKIN REACTION SCORES Historical Positive Control Validation Study1 Positive Control Group123
Induction Number Hours1
Animal No. 277 278 279 280 281 282 283 284 285 286
1 24 | 48
0.5 0 0.5 0.5 0.5 0.5 00 0.5 0.5 0.5 0 00 0.5 0 0.5 0.5 00
2 24 I 48
0.5 0 1 0.5
0.5 0 0.5 0 0.5 0.5 0.5 0 0.5 0 0.5 0 0.5 0.5 0.5 0
3 24 48
0.5 0 1 0.5 1 0.5
0.5 0.5 1 0.5 1 0.5
0.5 0.5 1 0.5 1 0.5
0.5 0
1PSL Study #10548, performed by PSL and completed on May 10,2001. 2 Four-tenths o f a milliliter o f HCA was applied as received using an occlusive 25 mm Hill Top Chamber. 3Hours after induction dose.
Page 21 of 25
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABUE 6: CHALLENGE PHASE SKIN REACTION SCORES Test Substance Group1
Animal No.
3148 3149 3150 3151 3152 3153 3154 3155 3156 3157 3158 3159 3160 3161 3162 3163 3164 3165 3166 3167
Honrs after Dosing
24
50%
Distilled water (100%)
00
48
50%
Distilled water (100%)
00
0 0 00
0 0 00
0 0 00
0 0 00
0.5 0 0 0
0.5 0 0 0
0 0 00
0 0 00
0 0 00
0.5 0 0.5 0
0 0 00
0 0 00
0.5 0 0 0
0.5 0 0.5 0
0 0 00
0 0 00
0 0 00
0 0 00
0 0 00
1Four-tenths of a milliliter of the test substance was applied as a 50% w/w solution in distilled water, using an occlusive 25 mm Hill Top Chamber to the right rear flank and the vehicle (distilled water, 100%) was applied to the right front flank.
Page 22 of 25
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 6 (cont): CHALLENGE PHASE SKIN REACTION SCORES Test Substance Irritation Control Group1
Animal No.
3168 3169 3170 3171 3172 3173 3174 3175 3176 3177
Hours after Dosing
24
50%
Distilled water (100%)
0.5 0
48
50%
Distilled water (100%)
00
0 0 00
0 0 00
0 0 00
0.5 0 0.5 0
0 0 00
0 0 00
0 0 00
0 0 00
0 0 00
1Four-tenths of a milliliter of the test substance was applied as a 50% w/w solution in distilled water, using an occlusive 25 mm Hill Top Chamber to the right rear flank and the vehicle (distilled water, 100%) was applied to the right front flank.
Page 23 of 25
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 7: CHALLENGE PHASE SKIN REACTION SCORES1 Historical Positive Control Validation Study12
Anim al No. Positive Control
277 278 279 280 281 282 283 284 285 286 Vehicle Control 287 288 289 290 291
H ours3 24 48 1 0.5 0.5 0.5 0.5 0.5 11 1 0.5 11 0.5 0 0.5 0 1 0.5 00
0.5 0.5 00 0.5 0.5 0.5 0.5 0.5 0
1Four-tenths of a milliliter of a 75% w/w solution of HCA (as received) in mineral oil using an occlusive 25 mm Hill Top Chamber. 2PSL Study #10548, performed by PSL and completed on May 10,2001. 3Hours after challenge dose.
Page 24 of 25
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
APPENDIX A: FEED AND WATER ANALYSES
On June 21, 2001 animal feed was analyzed for the presence of the following contaminants-
Aldrin BHC-A (Alpha-Hexachlorocyclohexane) BHC-B (Beta-Hexachlorocyclohexane) BHC-D (Delta-Hexachlorocyclohexane) BHC-G (Lindane) Captan Chlordane Chlorpyrifos-Methyl Chlorpyrifos (Duraban) 4,4 DDD 4,4 DDE 4,4 DDT Dieldrin
Dylox (Trichlorfon) Endosulfan I & II Endsolfan Sulfate Endrin Endrin Aldehyde Esfenvalerate Fenvalerate Heptachlor Heptachlor Epoxide Mavrik (Tau-Fluvalinate) Methoxychlor Mirex Quintozene
None o f the above compounds were present above the limit o f detection (0.005 ppm)
LABORATORY:
FOOD PRODUCTS LABORATORY, INC. 12003 N.E. Ainsworth Circle Suite 105 Portland, OR 97220
On June 21,2001, water was analyzed for NJDEPE Safe Drinking Water Act parameters.
LABORATORIES:
NEW JERSEY LABORATORIES NJDEPE LAB I.D. #15001 A.A. Labs Division 222 Easton Avenue New Brunswick, NJ 08901
SELLDOER LABORATORIES OF NEW JERSEY, INC. 400 South Avenue Garwood, NJ 07027
Results of water analysis for possible contaminants were acceptable within regulatory standards.
Page 25 of 25
