Document NeMEgxrZE7Zw9ROn3w8kx238w
H-24921: Dermal Sensitizatfon Test - Buehler Method
AR226-2804
DuPont-7977
TRADE SECRET
STUDY TITLE
H-24921: Dermal Sensitization Test - Buehler Method
LABORATORY PROJECT IDENTIFICATION
DuPont-7977 PSL Study Number 11321
Work Request Numbet----^J
Service Code Numberfatf)
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
cora^ S-n,.,^. o,., ,,,, ^ ^ ^
H-24921: Dennal Sensitization Teat - Buehler Method
DuPont-7977
GOOD LABORATORY PRACTICE STATEMENT
This study meets the requirements of 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 of the test and control substances. The procedures used by trained personnel to prepare the treatment solutions insured:
a) The accuracy of 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 preparedjust prior to use.
2.
The stability, uniformity of mixture and verification of concentration of HCA in its carriers were
not determined.
Applicant/Sponsor: Study Director:
E.L du Font de Nemours and Company Newark, Delaware U.S.A.
George EAIoore, B.S.
ZZ/L^L
Date
^ed.Do651
con^88"'
Page 2 of 25
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
QUALITY ASSURANCE STATEMENT
The Quality Assurance Unit randomly selects intervals for QA inspections prior to study initiation. Records of the findings of 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
/y/7/fl/
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
(jz^-m^xj/ z^yj^agr
Annamarie LaPorte Quality Assurance Auditor
s^^- ,0^ no^c A OO^
Coi^
Page 3 of 25
H-24921: Dennal Sensitization Test - Buchler Method
DuPont-7977
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.
^^^^^w^^^ l^ George E.Woore,B.S.
Study Director
Gary Wnonnreki, BA.
Laboratory Director
^ L ^ ______^L jt.f-/filf.e0^c- /^L/rH-ft-b
Carol Furiay, B.A. (
(
Study Monitor
d^ Date n ^f------
1>.c.
Date
n . toc^
/!. 44_H -^<S6^
Date
TSC Ooe500A con1Aa\"
^
San^-0
COlOP8^
Page 4 of 25
H-24921: Dcnnal Senaitfzation Test - Buehler Method
DuPont-7977
TABLE OF CONTENTS
GOODUWORATORYPI^CTICESTATEMENT.......................................................................................^ QUALITY ASSURANCE STATEMENT ....................................................................................................................3
TABLE OF CONTENTS.................................................,,..........,,,...,,,,..........,,...,.,,.,,,,,.,...,,,,.,,..,,,,.,.....,,...,,,,.....,,...5 STUDY INFORMATION ..............................................................................................................................................6 H-24921: DERMAL SENSmZATION TEST - BUEHLER METHOD.....................................................................? 1. PURPOSE...............................................................................................................................................7 2. SUMMARY........................................................................................................................,.....................? 3. MATERIALS............................................................................................................................................8 4. METHODS.................................................................................................................,.....,,.....................9 5. PROCEDURE.......................................................................................................................................10 6. EVALUATION.......................................................................................................................................11 7. HISTORICAL POSITIVE CONTROL VALIDATION STUDY................................................................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: PREUMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC (TEST
TABLE 1 (cent): PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC (POSITIVE CONTROL-HCA)........................................................................................................................... 15 TABLE 2: INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (fl)............................................................................ 16 TABLE 2 (cent): 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
Page 5 of 25 Company Seized. Does not contain TSCACBI
H-24921: Dermal Sensirization Test - Buehler Method
9th Collective Nomenclature: Synonyms/Codes:
STUDY INFORMATION
| |
Haskell Number. CAS Registry Number: Composition:
DuPont-7977
Purity: Physical Characteristics: Stability:
Sponsor
Study Initiated/Completed: m-Life Initiated/Completed:
White solid
The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.
E. I. du Font de Nemours and Company Wihnington, Delaware 19898
U.SA.
September 27,2001/ (see report cover page)
October 10,2001 /November 9.2001
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Company Sanitized. Does not contain TSCA CBI
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
H-24921: DERMAL SENSmZA-nON TEST BUEHLER METHOD
PROTOCOL NO.: AGENCY; PSL STUDY NUMBER: SPONSOR:
SUBM1TTER;
TEST SUBSTANCE IDENTIFICATION: TEST SUBSTANCE DESCRIPTION; DATE RECEIVED: PSL REFERENCE NO.: DATE OF PROTOCOL APPROVAL: EXPERIMENTAL INITIATION DATE: EXPERIMENTAL COMPLETION DATE: STUDY COMPLETION DATE: NOTEBOOK NO.:
P328DUP
EPA(TSCA)
11321
EJ. du Font de Nemours and Company Wilmington, Delaware 19898 U.SA.
DuPont HaskeU Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50
Newark, DE 19714-0050
H-24921
White solid
September 19,2001
1.
PURPOSE
To assess the sensitization potential of H-24921 after repeated topical applications.
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.
Page 7 of 25 Company Sanitized. Does not contain TSCA CBI
t)
H-24921: Dermal Sensitizarion Test - Buehler Method
DuPont-7977
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 of 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 of the sensitization response noted after challenge is found below.
-
50% w/w in distilled water Distilled water
Incidence with Skin Reactions2
TestAnimals"
-Tesrlrritation'ConfforAnimals
Hours
24
48
24
48
0/20 0/20
0/20 0/20
0/10 0/10
0/10 0/10
50% w/w in distilled water Distilled water
Test Animals
24
0.13 0.00
48 0.05 0.00
Severity3
Test Irritation Control Animals
H ours
24
48
0.10 0.00
0.05 0.00
Based on the results of 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-249
identified with PSL Reference Numb temperature. The sample was a white sd
on September 19, 2001 and was further test substance was stored at room
' The test substance, as received, was a solid. distilled water prior to application.
To enhance skin contact, the test substance was moistened with
2- Animals with scores greater than 0.5
ISpmpany SamSseS. Does not contain TSCA CBI
3 Sum of the erythema scores divided by the number of animals evaluated.
Page 8 of 25
H-24921: Dermal Sensitizarion Test - Buehler Method
DuPont-7977
Characterization of the test substance provided to Product Safety Labs by the Sponsor was:
pH: 5 (0.5% aqueous)
Solubility: Not applicable
Stability: The test substance is expected to be stable for the duration of testing
Expiration Date: December 15,2001
B.
Animals
3B. 1 3B.2
3JB.3
Number of Animals: 34
Number of Groups: 3
Number of Animals per Group: Preliminary Irritation Testing: 4
Test Group: 20 Test Vehicle Control Group: 10
3B.4 3B.5
3JB.6
3.B.7
Sex: Male
Species/Strain:Guinea pigs/Hartleyalbino
Age/Body weight: Preliminary Irritation Group; Young adult Test and Test Vehicle Control Groups: Young adult/349-443 grams at experimental start
Source: Received from Elm Hill Breeding Labs, Chelmsford, MA on October 3,
2001
4.
METHODS
A.
Husbandry
4A.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/or the Care and Use of Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage and was changed at
least three times pCT week.
4A^ 4AJ
Animal Room Temperature and Relative Humidity Ranges: 18-23 C and 30-68% Photoperiod; 12 hour light/darkcycle
4A.4 Acclimation Period: 7 days
4A.5 4A.6
Food: Pelleted Purina Guinea Pig Chow #5025
Water Filtered tap water was supplied ad libitum by automatic water dispensing
system.
4A.7
Contaminants: There were no known contaminants reasonably expectedto be found
in the food or water at levels which would have interfered with the results of this study. Analyses of die food and water are conducted at least once a year and the
Page 9 of 25 Company SanrKzed. Does net contenT TSCA CSI
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
records are kept on file at Product Safety Labs. The dates of the most recent analyses
are presentedin Appendix A.
B.
Identification
4.B.1
Cage: Each cage was identified with a cage card indicatingat least the studynumber and identification and sex of the animal.
42
Animal: Each guinea pig was marked with a color code and given a sequential animal number assignedto study #11321, which constituted unique identification.
5.
PROCEDURE
A.
Preparation and Selection of Animals
Within 24 hours prior to each application, the fur of 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 of the test substance in distilled water was applied to the left side of 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 of 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 of a 50% w/w solution of the test substance (HNIC) in distilled water was applied to a naive site on the right rear flank of each animal using the procedures described above. The vehicle (distilled water, 100%) was applied to the right front flank of 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 edema
The test substance, as received, was a solid. To enhance skin contact, the test substance was moistened with distilled water prior to application.
Page 10 of 25
company sanfzed D^ not contain TSCA CBI
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
E.
Body Weights
Individual body weights of 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. recorded.
If any unusual clinical signs were observed, they were
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 of erythema (sensitization response) approximately 24 and 48 hours after challenge according to the following:
Scores of 1 or greater in the test group are required to be indicative of 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 of positive animals in each group divided by the total number of annuals tested in that group.
Severity is reported as the sum of the test grades divided by the total number of 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 of 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 May 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 0/5
2/10 0/5
0.70 0.45
0.40 0.30
' Animals with scores greater than 0.5.
2 Sum of the erythema scores divided by the number of animals evaluated.
Page 11 of 25
^ompan*?ST7feaf. Vifffsnorconrarn TSCA GOT
H-24921: Dermal Sensitizatfon Test - Buehler Method
DuPont-7977
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 of 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 of guinea pig sensitizadon patch tests, in Current Concepts in Cutaneous Tenacity, VA. 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 RNAL PROTOCOL
None
12. FINAL REPORT AND RECORDS RETENTION
A copy of 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 of 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 animals are presented in Tables
4tbrough7-
Page 12 of 25
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H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
All test and control animals survived and appeared normal throughout the study. There were no statistically significant differences between the overall body weight gain of the test and test
vehicle control ^r"Trials
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 Anhnali; (test substance applied as a 50% w/w solution in distilled water): Very &int erythema (0.5) was noted for five of 20 test sites 24 hours after challenge. Irritation persisted at two of these sites through 48 hours.
Test Substance Irritation Control Animals (test substance applied as a 50% w/w solution in distffled water): Very &int erythema (0.5) was noted for two of 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 often animals exhibited signs of a sensitization response (&int erythema [1]) 24 hours after challenge. Similar indications persisted at two of these sites with very &int
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 &int erythema was noted for four of five test naive control sites 24 hours after challenge. Irritation persisted at three of 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 The test substance, as received, was a solid. distilled water prior to application.
To enhance skin contact, the test substance was moistened with
Page 13 of 25
Company SanHfzed. Ehws not contain TSCA CB(
H-24921: Dennal Sensitizadon Test - Buehler Method
DuPont-7977
TABLE 1: PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC1 (TEST SUBSTANCE)
Animal No. Sex
2998
M
2999
M
3000
M
3001
M
903
24 48
0.5 0.5
0.5 0.5
0.5 0.5
1
1
CONCENTRATION (%)2
75
50
Hours after Patch Removal 24 48 24 48
0.5 0.5 0.5
0
0.5 0.5
0
0
0.5
0
0
0
1
1 0.5 0.5
25
24 48
0
0
0
0
0
0
0
0
' HNIC - Highest Non-Iiritating Concentration
2 Four-tenths of a milliliter of the test substance was applied as w/w mixtures, suspensions or solutions in distilled water using an occlusive 25 nun Hill Top Chamber.
3 The test substance, as received, was a solid. distilled water prior to application.
To enhance skin contact, the test substance was moistened with
Page 14 of 25
tCompaaiySariSfeaA &3esno?con(a;n TSCA CBI
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 1 (cont): PREUMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC1 (POSTTTVCEONTROL-tiCA)
Historical Positive Control Validation Study
Animal No. Sex
0210
M
0211
M
0212
M
0213
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
' HNIC - Highest Non-Irritating Concentration
2 Four-tenths of a milliliter ofHCA was applied as received and as w/w solutions in mineral oil using an occhisive 25 mm Hill Top Chamber.
Page 15 of 25
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H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 2: INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g)
Test Substance Group
Anima Sei Initial Week Gain Week Gain Week Gain Day After
No.
2 (Otowk2) 3 (wk2to3) 4 (wk3to4) Challenge
3148 M 419 459
40
536
77
594
58
652
Gain (wk4to5)
58
Total Weight
Gain (wk0to5)
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
Mean
Standard Deviation
396.2 17.61
449.4 16.27
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 8.99
220 40.98
Page 16 of 25
^"Pany sanded. Does,
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 2 (cont): INDIVIDUAL BODY WEIGHTS/WEIGHT GAIN (g)
Test Vehicle Control Group
Animal Sex Initial Week Gain Week Gain Week Gain Day After
No.
2 (Otowk2) 3 (wk2to3) 4 (wk3to4) Challenge
3168 M 392 434
42
502
68
547
45
603
Gain (wk4to5)
56
Total Weight
Gate (wk0to5)
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
Mean 375.6 429.6 Standard 18.28 22.18 Deviation
54.0 9.41
489.4 27.57
59.8 9.96
545.2 34.95
55.8 9.68
586.4 37.11
41.2 9.65
210.8 30.30
Page 17 of 25
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H-24921: Dennal Sensirization Test - Bushier Method
DuPont-7977
TABLE 3: INDIVIDUAL BODYWEtGHTS/WEIGKT GAIN (g)
Historical Positive Control Validation Study'
Animal Sex Initial Week Gain Week Gain Week Gain Day After
NO.
2 (Otowk2) 3 (wk2to3) 4 (wk3to4) Challenge
277 M 396 448
52
508
60
550
42
588
Gain (wk4to5)
38
Total |
Weight Gain
(wkOtoS)
192
278 M 407 470
63
535
65
610
75
619
279 M 397 456
59
512
56
565
53
598
9
212
33
201
280 M 393 452
59
496
44
558
62
624
281 M 407 466
59
512
46
585
73
639
282 M 396 444
48
490
46
549
59
613
66
231
54
232
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
286 M 364 430
66
473
43
519
46
588
64
226
69
224
Mean 390.8 453.7
Standard Deviation
Vehicle Control
16.84
16.96
287 M 367 424
288 M 374 431
289 M 359 421
290 M 386 453
291 M 411 479
Mean 379.4 441.6 Standard 20.26 24.37 Deviation
62.9 9.79
57 57 62 67 68 62.2 5.26
502.6 21.80
484 480 499 506 543 502.4 25.07
48.9 8.88
60 49 78 53 64 60.8 11.26
562.7 26.42
540 547 559 554 598 559.6 22.63
60.1 12.15
56 67 60 48 55 57.2 6.98
612.1 18.22
588 598 619 624 664 618.6 29.37
49.4 19.10
221.3 15.78
48 51 60 70 66 59
9.43
221 224 260 238 253 239.2 17.22
' PSL Study #10548, performed by PSL and completed on May 10,2001. Page 18 of 25
SaniltZ11'Do5'
mpany
H-24921: Dermal Sensitizarion Test - Buehler Method
DuPont-7977
TABLE 4: INDUCTION PHASE SKIN REACTION SCORES
Test Substance Group'
Induction Number Hours2
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
0
0
0.5
0.5
0
0
0.5
0
0.5
0
0.5
0.5
0.5
0.5
0.5
0
0
0
0
0
1
1
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 occhisive 25 nun Hill Top Chamber*'.
Hours after induction dose.
pwl90"5
c.mp.ws""1'"-1'00"'
H-24921: Dermal Sensitizatkm Test Buehler Method
DuPont-7977
TABLE 4 (cont): INDUCTION PHASE SKIN REACTION SCORES
Test Vehicle Control Group'
Induction Number Hours2
1
24
48
2
24
48
3
24
48
Animal No.
3168
0
0
0
0
0
0
3169 3170
0
0
0
0
0
0
0
0
0
0
0
0
3171
0
0
0
0
0
0
3172
0
0
0
0
0
0
3173
0
0
0
0
0
0
3174
0
0
0
0
0
0
3175
0
0
0
0
0
0
3176
0
0
0
0
0
0
3177
0
0
0
0
0
0
' Four-tenths of a milliliter of the vehicle (distilled water, 100%), was applied using an occlusive 25 nun Hill Top
Chamber*. 2 Hours after induction dose.
Page 20 of 25
H-24921: Dermal Scnsitization Test - Buehler Method
DuPont-7977
TABLE 5: INDUCTION PHASE SKIN REACTION SCORES Historical Positive Control Validation Study" Positive Control Group2
Induction Number Hours3
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
0
0
0.5
0.5
0.5
0
0
0
0.5
0
0.5
0.5
0
0
2
24
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
' PSL Study #10548, perfonned by PSL and completed on May 10,2001.
2 Four-tenths of a milliliter ofHCA was appliedas received usingan occtusive 25 nun Hill Top Chamber.
3 Hours after induction dose.
''^^osftv Sani'.ESd, O-:^ nj; ^cr'isn 'i ^Cs ^fci;
Page 21 of 25
H-24921: Domal Sensitization Test - Buehler Method
t
DuPont-7977
TABLE 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
50% 0 0 0
0 0 0.5 0.5 0 0 0 0.5 0 0 0.5 0.5 0 0 0 0 0
Hours after Dosing
24
Distilled water (100%)
50%
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.5
0
0
0
0
0
0
0
0.5
0
0
0
0
0
0
0
0
0
0
48 Distilled water (100%) 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
' Four-tenths of a milliliter of the test substance was appliedas a 50% w/w solution in distilled water, using an occlusive 25 mm Hill Top Chamber9 to the right rear flank and the vehicle (distilled water, 100%) was applied to
the right front flank.
pw220f25
^-^--------"------c^^,
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 6 (cont): CHALLENGE PHASE SKIN REACTION SCORES
Test Substance Irritation Control Group'
Animal No.
3168 3169 3170 3171 3172 3173 3174 3175 3176 3177
50%
0.5 0 0 0 0.5 0 0 0 0 0
Hours after Dosing
24
Distilled water (100%)
0
50% 0
0
0
0
0
0
0
0
0.5
0
0
0
0
0
0
0
0
0
0
48 Distilled water (100%)
0 0 0 0 0 0 0 0 0 0
' Four-tenths of a milliliter of the test substance was appliedas a 50% w/w solution in distilled water, using an
occlusive 25 nun Hill Top Chamber* to the right rear Sank and the vehicle (distilled water, 100%) was applied to the right front flank.
Page 23 of 25
^mpany Sanitized. Does "otcontamTSCACBl
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 7: CHALLENGE PHASE SKIN REACTION SCORES1 Historical Positive Control Validation Study2
Animal No. Positive Control
277 278 279 280 281 282 283 284 285 286 Vehicle Control 287 288 289 290 291
Hours3
24
48
1
0.5
0.5
0.5
0.5
0.5
1
1
1
0.5
1
1
0.5
0
0.5
0
1
0.5
0
0
0.5
0.5
0
0
0.5
0.5
0.5
0.5
0.5
0
' Four-tenths of a milliliter of a 75% w/w solution of HCA (as received) in mineral oil using an occlusivc 25 mm Hill Top Chamber.
2 PSL Study#10548, performedby PSL and completedon May 10,2001.
3 Hours after challenge dose.
Page 24 of 25
company samt'28d. Does not contain TSCA CBI
H-24921: Dermal Sensitizarion 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
Dylox (Trichlorfon)
BHC-A (Alpha-Hexachlorocyclohexane)
Endosulfan I & n
BHC-B (Beta-Hexachlorocyclohexane)
Endsolfan Sulfate
BHC-D (Delta-Hexachlorocyclohexane) BHC-G (Lindane)
Endrin Endrin Aldehyde
Captan
Esfenvalerate
Chlordane
Fenvalerate
Chlorpyrifos-Methyl
Heptachlor
Chlorpyrifos (Dursban)
Heptachlor Epoxide
4,4 DDD 4,4 DDE 4,4 DDT Dieldrin
Mavrik (Tau-Fhivalinate) Methoxychlor Mirex Qumtozene
None of the above compounds were present above the limit of 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 ID. #15001 AA. Labs Division
222 Easton Avenue
New Bmnswick, NJ 08901
SILLKER 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
Company Sanitized. Does not contain TSCA CB1