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H-24921: Dermal Sensitization Test - Buehler Method
AR226-2804
DuPont-7977
TRADE SECRET
STUDY TITLE
H -24921: Dermal Sensitization Test - Buehler M ethod
LABORATORY PROJECT IDENTIFICATION
DuPont-7977 PSL Study Num ber 11321 Work Request N u m b e^jiB M Q Service Code N u m b e r ^ p
DATA REQUIREMENT
U.S. EPA H ealth Effects Test Guidelines, OPPTS 870.2600, A ugust, 1998
AUTHOR
George E. M oore, B.S.
STUDY COMPLETED ON
December 17,2001
PERFORMING LABORATORY
Product Safety Labs 2394 Route 130
Dayton, New Jersey, 08810
S U B M IT T E R
DuPont Haskell Laboratory for H ealth 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 m eets the requirem ents o f U.S. EPA Good Laboratory Practice Standards: Toxic Substances C ontrol Act: 40 CFR 792 with the following exceptions:
1. Treatm ent solutions were not analyzed for concentration, uniform ity o r stability o f the test and control substances. The procedures used by trained personnel to prepare the treatm ent solutions insured-
a) The accuracy o f concentration because the test substance diluent (vehicle) was accurately m easured w ith a graduated device. The test substance was weighed on a balance accurate to at least two decim al places
b) U niform ity, because all solutions were thoroughly m ixed prior to adm inistration to th e test system; and
c) Stability, because treatm ent solutions w ere prepared ju st prior to use. .
2. The stability, uniform ity o f m ixture and verification o f concentration o f HCA in its carriers were not determ ined.
A pplicant/Sponson Study D irector
E.L du Pont de Nemours and Company Newark, Delaware U .S.A .
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QUAUTY ASSURANCE STATEMENT
The Q uality A ssurance U nit random ly 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 o f statem ents m ade in th e final report section that addresses Quality Assurance audits.
Inspections were m ade of:
DATE
PROCEDURE INSPECTED
10/26/01
48 hour scoring (Induction #3)
11/20/01
Raw data
11/20/01
Draft report
!> /l 7/0J Final report
Findings reported to:
Study D irector 11/20/01 M anagement 11/20/01
A nnam arie LaPorte Q uality Assurance A uditor
.ed poe 1 S ari'"1' C o,'tnPa ^
,n TSC a c b i ,rrialtv
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H-24921: Dcnnal Sensitization Test - Buehler Method
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CERTIFICATION
W e the undersigned declare that the m ethods, results and data contained in this report faithfully reflect the procedures used and raw data collected during the study.
^ f c --------------- -
G eorge E. M oore, B .S. '
Study D irector
G ary W norot^Ski, B A . Laboratory D irector
______C o - A M - I l c i a U u
C arol Finlay, B.A. (
f
Study M onitor
D ate
tX c . 11 , lo f t | D ate
h - < U > b'L
COt^P
in TSC l^ B ' n ta ' BO01
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H-24921: Dermal Sensitization Test - Buehler Method
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TABLE OF CONTENTS
GOOD LABORATORY PRACTICE STATEMENT.............................
QUALITY ASSURANCE STATEMENT.............................................. CERTIFICATION.................................................................................
TABLE OF CONTENTS...................................................................... STUDY INFORMATION......................................................................
H-24921: DERMAL SENSITIZATION TEST - BUEHLER METHOD... 1. PURPOSE....................................................................................
2. SUMMARY....................................................................................
3. MATERIALS.................................................................................. 4. METHODS....................................................................................
5. PROCEDURE............................................................................... 6. EVALUATION......................................................................
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: PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC {TEST SUBSTANCE)............................................................................................................................... ' ____
TABLE 1 (coni): 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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Company
H-24921: Dermal Sensitization Test - Buehler Method
9th Collective Nomenclature: S vnonvm s/C odes:
STUDY INFORMATION
H askell N um ber CAS Registry Number: C om position:
DuPont-7977
Purity: Physical Characteristics: Stability: Sponsor
Study Initiated/Com nleted: In-Life Initiated/Coronleted:
W hite solid
The test substance appeared to be stable under the conditions o f the study; no evidence o f instability was observed.
E. I. du Pont de Nemours and Company W ilmington, Delaware 19898 U .SA .
September 27,2001/ (see report cover page)
October 10,2001 /Novem ber 9,2001
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H-24921: Dennal Sensitization Test - Buehler Method
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H-24921 : DERMAL SENSiTIZAHON TEST - BUEHLER METHOD
PR O T O C O L NO .: AGENCY: PSL STUDY NUMBER: SPONSOR:
SUBM ITTER:
TEST SUBSTANCE IDENTIFICATIO N: TEST SUBSTANCE DESCRIPTION: DATE RECEIV ED : P SL R EFE R EN C E N O .: DATE O F PROTOCOL APPROVAL: EX PERIM EN TA L IN ITIA TIO N DATE: EX PERIM EN TA L CO M PLETIO N DATE: STUDY CO M PLETIO N DATE: N O TEB O O K N O .:1
P328 DUP EPA (TSCA)
11321 ET. du Pont de Nemours and Company W ilmington, D elaw are 19898 U .S A DuPont Haskell Laboratory for H ealth and Environm ental Sciences Elkton Road, P.O . B ox SO Newark, DE 19714-0050 H-24921 W hite solid Septem ber 19,2001
Septem ber 27,2001 October 10,2001 November 9,2001 Decem ber 17,2001 01-55: pages 133-146
1. PURPOSE To assess the sensitization potential o f H-24921 after repeated topical applications.
2. SUMMARY A dermal sensitization test was conducted with guinea pigs to determ ine the potential for H -24921 to produce sensitization after repeated topical applications.
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The test substance (90% ' w/w m ixture 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 anim als) was maintained under the same environm ental conditions and treated w ith the vehicle (distilled water, 100%) for the induction phase. Twenty-seven days after th e first induction dose, a challenge dose o f 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. Approxim ately 24 and 48 hours after each induction and challenge dose, the test and test irritation control anim als were scored for erythem a.
is found below,
--
50% w/w in distilled w ater D istilled w ater
Incidence w ith Skin Reactions2
TestA nim als
T e^Im tationC ontrorA nim flls
E burs
24 48
24
0/20 0/20
0/10
0/20 0/20
0/10
48 0/10 0/10
50% w/w in distilled w ater D istilled w ater
Test Animals
24 48 0.13 0.05 0.00 0.00
Severity3 Test Irritation Control Anim als
E ours 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 w ith ahexylctnnam aldehyde, 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 -2492X ^gygrerrecL on Septem ber 19, 2001 and was further
identified w ith PSL R eference N u i n b e |j ^ [ H |^ ^ p |r h e test substance was stored at room
tem perature. T he sample was a white
' *
The test substance, as received, was a solid. To enhance skin contact, the test substance was moistened with distilled water prior to application.
Animals with scores greater than 0.5
Com pany Sanffizetf, D oe? rrof co n tain TSCA CBI
3Sum of the erythema scores divided by the number of animal evaluated.
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C haracterization o f the test substance provided to Product Safety Labs by the Sponsor was:
Cni*>ositioi 8 H B H H H ^ H I H H ! ^ pH: 5 (0.5% aqueous) Solubility: N ot applicable Stability: T he test substance is expected to be stable for the duration o f testing Expiration Date: December 15,2001 B. Animals
3 3 .1 Number o f Animals: 34
3 3 .2 Number o f Groups: 3
3 3 .3
Number o f Animals per Group: Prelim inary Irritation Testing: 4 Test Group: 20 Test Vehicle Control Group: 10
3 3 .4 Sex: Male
3 3 .5 Species/Strain: Guinea pigs/Hartley albino
3 3 .6
Age/Body weight: Preliminary Irritation Group; Young adult
Test and Test V ehicle Control Groups: Young adult/349-443 grains at experim ental start
3 3 .7 Source: Received from Elm Hill Breeding Labs, Chelmsford, M A 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 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 p tr week.
4A_2 Animal Room Temperature and Relative Humidity Ranges: 18-23 C and 30-68%
4A 3 Photoperiod: 12 hour lighl/dark cycle
4A .4 Acclimation Period: 7 days
4A .5 Food: Pelleted Purina Guinea Pig Chow #5025
4A .6 W ater Filtered tap water was supplied ad libitum by automatic water dispensing system.4
4A .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 die food and water are conducted at least once a year and the
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records are kept on file at Product Safety Labs. The dates o f the most recent analyses are presetted in Appendix A.
B. Identification
4.B.1 Cage: Each cage was identified with a cage card indicating pt least tin study rmmlwr and identification and sex o f the animal
4 3 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
W ithin 24 hours prior to each application, the fur o f each guinea pig assigned to test nd test vehicle control groups was removed by clipping the dorsal area and flanks. C are was tk<>n to avoid abrading th e skin. Prior to study initiation, the anim als w ere weighed and the Inn was checked for any abnorm alities. Only healthy animals w ithout pre-existing skin irritation w ere selected for test.
B. Induction Phase
Once each w eek for three weeks, 0.4 ml o f a 90%' w/w m ixture o f the test substance in distilled w ater w as applied to the left side o f each test animal for six hours using an occlusive 25 mm H ill Top Chamber. The chambers were secured in place and wrapped w ith non-allergenic DuraporeTM adhesive tape to avoid dislocation o f the chambers and to m inim ize loss o f the test substance. A fter the six horn exposure period, the chambers w ere removed and the test sites w ere gently w iped w ith water and a clean tow el to remove any.residual test substance. Approxim ately 24 and 48 hours after each induction application, readings w ere m ade o f local reactions (erythem a) according to the scoring system described in Section 5.D. The vehicle (distilled water, 100%) was used for the induction o f the test vehicle control anim als and scored as above.
C. Challenge Phase
Twenty-seven days after the first induction dose, 0.4 m l o f a 50% w/w solution o f the test substance (HNIC) in distilled w ater was applied to a naive site on the right rear flank o f each anim al 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 w ith the test substance and vehicle for the challenge phase. These sites w ere evaluated for a sensitization response (erythem a) approxim ately 24 and 48 hours after the challenge application according to the system described in Section 5.D.
D. Scoring System
0 - no erythem a 0.5 - very faint erythema, usually non-confluent 1 - faint erythem a, usually confluent 2 - m oderate erythema 3 - severe erythem a w ith or without edema
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 anim als w ere recorded prior to the first induction, weekly and again on the day after challenge. The mean and standard deviation w ere calculated for all body weights and body weight gains. The overall m ean body weight fflins for test and test vehicle control anim als were evaluated statistically by an unpaired t test (INSTAT B iostatistics, created by GraphPad Software, San D iego, and CA.).
F. Clinical Observations
The anim als w ere observed daily. If any unusual clinical signs w ere observed, they w ere recorded.
6. EVALUATION
In order to evaluate the sensitization response noted during the challenge phase, tw o indices w ere used; one for incidence and one for severity. The incidence index was calculated to evaluate the incidence o f erythem a (sensitization response) approxim ately 24 and 48 hours after challenge according to the following:
Scores o f 1 o r greater in the test group are required to be indicative o f sensitization. If scores o f one (1) or greater are seen on the control anim als, then the reactions o f the test substance group anim als that exceed the most severe control reactions are considered to be positive scores.
Incidence is reported as the num ber o f positive anim als in each group divided by the total num ber o f animals tested in that group.
Severity is reported as the sum o f the test grades divided by the total num ber o f animals tested in a given group determ ined for both 24 and 48 hours. A ll average grades are to be rounded o ff to the nearest tenth o f a unit.
7. HISTORICAL POSITIVE CONTROL VALIDATION STUDY
The procedures used in this study were validated using a-hexylcinnam aldehyde, technical grade, 85% (purity) as a positive control substance. The most recent validation, PSL Study #10548, was perform ed 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 H istorical D ata N otebook N o. 01, pages 20-28. This test was conducted at the Dayton Facility w ith HCA using H artley strain albino guinea pigs from Elm H ill Breeding Labs following induction and challenge procedures sim ilar to those described in Section 5. The data from this positive control study is sum m arized 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 animal evaluated.
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8. STUDY CONDUCT This study w as conducted at Product Safety Labs, 2394 Route 130, Dayton, New Jersey, 08810 in com pliance w ith the follow ing regulation:
U.S. EPA Good Laboratory Practice Standards: Toxic Substances Control A ct (TSCA): 40 CFR 792
The procedures as described in the protocol are based on the following testing guideline: U.S. EPA H ealth Effects Test Guidelines, OPPTS 870.2600, August, 1998 The prim ary technician for this study was Rolland Colis, B.S.
9. REFERENCES Robinson, M ., Nusair, T., Fletcher, E., and R itz, H., A Review o f the B uehler Guinea Pig Skin Sensitization Test And Its U se 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 . D rill 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 com pliance w ith the protocol and Product Safety Labs Standard Operating Procedures. D ates o f inspections and audits perform ed during the study, and the dates o f reporting o f the inspection and audit findings to the Study D irector and Facility M anagement are presented in the Q uality A ssurance Statement.
11. DEVIATION FROM THE FINAL PROTOCOL None
12. FINAL REPORT AND RECORDS RETENTION
A copy o f the signed report, copies o f all raw data generated at Product Safety Labs and a copy o f the original signed protocol, w ill be m aintained in the Product Safety Labs archives.
Laboratory-specific or site-specific raw data, such as personnel files and equipm ent records w ill be retained by the facility w here the work was done.
The original raw data, the original final report and a copy o f the protocol w ill be retained at
Haskell Laboratory, Newark, Delaware, or at Iron M ountain Records M anagement, W ilmington, D elaw are.
13. RESULTS
Prelim inary irritation testing scores for the test substance and historical positive control anim als
are presented in Table 1. Individual body weights and body w eight gain for test and historical
positive control anim als 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.
ftTSCACBi
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A ll test and control anim als survived and appeared norm al throughout the study. There w ere no statistically significant differences between the overall body weight gain o f the test and test vehicle control anim als.
Induction Phase
Test Animals (test substance applied as a 90%' w/w mixture in distilled water): Very feint to feint erythem a (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 feint to feint 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 feint erythema (0.5) was noted for five o f 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 feint erythem a (0.5) was noted for tw o o f ten test sites 24 hours after challenge. Irritation persisted at one o f these sites through 48 hours.
Test Animals (distilled water, 100%): No dermal irritation was noted for any test site follow ing the challenge phase.
Test Substance Irritation Control Animals (distilled water, 100%): No dermal irritation was noted for any test substance irritation control site follow ing the challenge phase.
Historical Positive Control Animals (applied as a 75% w/w solution o f HCA (as received) in mineral oil): Five o f ten anim als exhibited signs o f a sensitization response (feint erythem a [1]) 24 hours after challenge. Sim ilar indications persisted at tw o o f these sites with very feint erythem a (0.5) noted for most other sites through 48 hours.
Historical Positive Vehicle Control Animals (applied as a 75% w/w solution o f HCA (as received) in mineral oil): Very feint 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 w ith ahexylcinnamaldehyde, technical grade, 85% (as received) validates the test system used in this study (See Section 7).
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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H-24921: Dermal Sensitization Test - Buehler Method
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TABLE 1: PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC1(TEST SUBSTANCE)
Anim al No. Sex
2998 2999 3000 3001
M M M M
90r 24 48
CONCENTRATION (%Y 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 of a milliliter of the test substance was applied as w/w mixtures, suspensions or solutions in HiatillwH water using an occlusive 25 mm Hill Top Chamber.
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 (co n t): PRELIMINARY IRRITATION TESTING SCORES FOR DETERMINATION OF HNIC1(POSTTIVE CONTROL-HCA)
H istorical Positive Control V alidation Study
A nim al No. Sex
0210 0211 0212 0213
M M M M
C oncentration (% ?
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 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 WEK3HTS/WEIGHT GAIN (g) Test Substance Group
Animal Ses
No.
Initial
W eek 2
Gain (0tow k 2)
W eek
3
Gain (wk 2 to 3)
W eek 4
G ain (wk 3 to 4]
Day After C h a llen g e
3148 M 419 459 40 536 77 594 58
652
G ain (w k 4toS )
T o tal
W eight G ain
(w k0to5)
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 tandard D eviation
17.61
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
59.4 220 8.99 40.98
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H-24921: Dermal Sensitization Test - Buehler Method
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TABLE 2 (cont): INDIVIDUAL BODYW EIGHTS/W EIGHT GAIN (g) T est V ehicle Control Group
A nim al No. Sex Initial
W eek Gain
2 (0towk2)
W eek
3
Gain (w k 2to3)
W eek Gain 4 (wk 3 to 4)
Day After Chidlenge
3168 M 392 434 42 502 68 547 45
603
G ain (w k 4to5)
Total W eight
G ain (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 ean 375.6 429.6 54.0 489.4 59.8 545.2 55.8
S tandard D eviation
18.28
22.18
9.41
27.57
9.96
34.95
9.68
586.4 37.11
41.2 210.8 5 30.30
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TABLE 3: INDIVIDUAL BODY WEK3HTS/WEIGHT GAIN (g) H istorical Positive Control Validation Study1
Animal
Sex Initial No.
W eek
2
Gain (0 to wk 2)
W eek
3
Gain (wk 2 to 3)
W eek Gain 4 (wk 3 to 4)
Day After C h a llen g e
G ain (wk 4 to 5)
Total W eight
G ain (w k0to5)
277 M 396 448 52 508 60 550 42
588
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 e a n 390.8 453.7 62.9 502.6 48.9 562.7 60.1
S ta n d a r d 16.84 16.96 9.79 21.80 8.88 26.42 12.15
D eviation V ehicle C ontrol
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
624
70 238
291 M 411 479 68 543 64 598 55
664
66 253
M e a n 379.4 441.6 62.2 502.4 60.8 559.6 57.2 S ta n d a r d 20.26 24.37 5.26 25.07 11.26 22.63 6.98
D eviation
618.6 29.37
59 239.2 9.43 17.22
1PSL Study #10548, performed by PSL and completed on May 10,2001. Com paq
Page 18 of 25
in TSCA CBI
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 4: INDUCTION PHASE SKIN REACTION SCORES
Test Substance Group1
Induction N um ber
1
H ours2
24 48
A nim al No.
3148
0.5 0.5
3149 3150 3151 3152
0.5 0.5 0.5 0
1 0.5 00
3153
0.5 0.5
3154
00
3155 3156
0.5 0 0.5 0
3157 3158
0.5 0.5 0.5 0.5
3159
0.5 0
3160
00
3161 3162
00 11
3163 3164 3165 3166
0.5 0.5 1 0.5
0.5 0.5 0.5 0.5
3167
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 o f 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 of25
Sanitized. Does n o t contain TSCA CBl
C om pany
H-24921: Dermal Sensitization Test Buehler Method
DuPont-7977
TABLE 4 (cont): INDUCTION PHASE SKIN REACTION SCORES
T est Vehicle Control Group1
Induction Number Hours2
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 o f the vehicle (distilled water, 100%), was applied using an occlusive 25 mm Hill Top Chamber*.
2Hours after induction dose.
Page 20 of 25
,W ean^rTSCJiCB;
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
% TABLE 5: INDUCTION PHASE SKIN REACTION SCORES H istorical Positive Control Validation Study12 Positive Control Group2
Induction N um ber
1
H onrs3
24 | 48
A nim al No. 277 0.5 0
278 0.5 0.5
279 0.5 0.5
280 0 0
281 0.5 0.5
282 0.5 0
283 0 0
284 0.5 0
285 0.5 0.5
286 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 1 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.
2Four-tenths of a milliliter o f HCA was applied as received using an occlusive 25 mm Hill Top Chamber. 3Hours after induction dose.
Sc pin any S a n ita r i, Cz&c noi cc-ntstn Page 21 of 25
Cy C&i
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 6: CHALLENGE PHASE SKIN REACTION SCORES Test Substance Group1*
A nim al No.
3148 3149 3150 3151 3152 3153 3154 3155 3156 3157 3158 3159 3160 3161 3162 3163 3164 3165 3166 3167
H onrs a fte r D osing
24 50% D istilled w ater
(100% ) 00
48 D istilled w ater
50% (100% ) 00
0 0 00
0 0 00
0 0 00
0 0 00
0.5 0
0 .0
0.5 0
00
0 0 00
0 000
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
0 000
1Four-tenths of a milliliter o f the test substance was applied as a 50% w/w solution in distilled water, using an
occlusive 25 nun Hill Top Chamber9 to the right rear flank and the vehicle (distilled water, 100%) was applied to the right front flank.
Page 22 of 25
Company Sanitized. Does not contain
TSCACBg
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
Honrs after Dosing
24
50%
Distilled water (100%)
0.5 0
00
00
00
48
50%
Distilled water (100%)
00
00
00
00
0.5 0 0.5 0 0 0 00 0 0 00
0 0 00
0 0 00 0 000
1Four-tenths of a milliliter o f die 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 o f 25
Company Sanitized. D oes not contain TSCACBI
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
TABLE 7: CHALLENGE PHASE SKIN REACTION SCORES1 H istorical Positive Control V alidation Study12
Animal No. Positive Control
277 278 279 280 281 282 283 284 285 286 Vehicle Control 287 288 289 290 291
Honrs3 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 o f 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.
Company Sanitized. Does not contain TSCA c b i
Page 24 o f 25
H-24921: Dermal Sensitization Test - Buehler Method
DuPont-7977
APPENDIX A: FEED AND WATER ANALYSES
On June 21,2001 anim al feed was analyzed for the presence o f the following contam inants:
A ldrin BHC-A (Alpha-Hexachlorocyclohexane) BHC-B (Beta-Hexachlorocyclohexane) BHC-D (Delta-Hexachlorocyclohexane) BHC-G (Lindane) C ap tan C h lo rd an e Chlorpyrifos-M ethyl Chlorpyrifos (Dursban) 4,4 DDD 4,4 DDE 4,4 DDT D ieldrin
Dylox (Trichlorfon) Endosulfan I & II Endsolfan Sulfate Endrin Endrin Aldehyde E sfe n v alera te F e n v alerate H eptachlor H eptachlor Epoxide M avrik (Tau-Fluvalinaie) M ethoxychlor M irex Q uintozene
N one o f the above compounds w ere present above the lim it o f detection (0.005 ppm)
LABORATORY:
FOOD PRODUCTS LABORATORY, INC. 12003 N.E. Ainsworth Circle Suite 105 Portland, OR 97220
O n June 21,2001, w ater was analyzed for NJDEPE Safe Drinking W ater A ct param eters.
LABORATORIES:
NEW JERSEY LABORATORIES NJDEPE LAB ID . #15001 A A . Labs D ivision 222 Easton Avenue New Brunswick, N J 08901
SILLIKER LABORATORIES OF NEW JERSEY, INC. 400 South Avenue Garwood, NJ 07027
Results o f w ater analysis for possible contam inants were acceptable w ithin regulatory standards.
Page 25 o f 25 C o m p an y S an itized . D oes not c o n ta in TSCA CBI
