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REPORT TO MONSANTO CHEMICAL COMPANY SUBACUTE DERMAL TOXICITY OF
AROCLOR 1268
I. Introduction and Outline of Procedure A (ample identified a* Aroclor 1268 wa received from Monunto
Chemical Company for evaluation of 20-day aubacute dermal toxicity in albioo rabbit*.
Skin application* of th te*t material, in the form of a corn oil. solution, were made daily to four te*t group* con*iting of two rab bit* aach {ore male and one female) for a period of 20 day*. Thor teat group* corresponded to daily dermal dole* of 500, 1000, 1500, and 2000 rog/kg/lay ra*pectivcly.
In addition to the four teat group*, a treated control group of two' rabbit* all also employed. Tha animal* in thi* group received d^ily dermal application* of corn oil comparable to the volume administered to tha highest t*t dote group.
Th* rabbit* in all group* wer* houicd individually over the period of inv*tlgation and obaervation* ware made with ratpect to body weight affjct*, incidence of mortality, reactions diaplayed and itatu* of the
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TABLE 1
20-DAy Subacute Dermal Teeclcity - Albino Babbit*
D<i|i Schedule
Number oi
Do***
Animal*
_ fasAi/*y)
Number oi Scheduled Cos*acutivc Applicatioc*
2 20
Z 500
20 -
Z 1000
20
Z 1500
20
Z 2000
20
Administered a* a 50 par cant *olutioc in cons oil. ** The treated coctroi rabbit* received do*** oi cons oil comparable in
volume to that applied to th* rabbit* of the high do*t group.
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The animal* employed in th* 20-day subacute dermal toxicity tudy were adult. New Zealand train albino rabbit*- The body Wrights ol the teat animal* were in the two-to-thr*a kilogram range- A total of 10 adult rabbit* wer. selected from a larger population altar examina tion of each animal for general physical well-being. Each animal se lected for the experiment was boused individually in a wire-bottomed stainless steel rabbit cage for the duration of the test. All rabbit* were fed * standard laboratory rabbit ration* plus water ad libitum.
B. Method of Application In preparation for the test, the back of aach rabbit was shaved
using electric clipper*. The exposure site thus prepared on each ani mal constituted approximately ten per cent of the total body surface area. The animal* were then returned to tbeir stock cages, and 24 hour* were allowed to elaper before the first percutaneous applications were made- This period permitted the shin to recover from the slight disturbance of the stratum corneum caused by the clipping procedure and also permitted the he. ling of any microscopit abrasion* possibly produced during the shaving procedure.
At the end of the 24-hour recovery period, the calculated doses of th* test material in the form of a 50 per cent (w/v) corn oil
* Rockland Rabbit Ration, Rockland farms, New City, New York.
solution *cr gently distributed (without rubbing) over the entire *ppl> cation sites on the test rabbits.
The animals in tbs (retted control group were treated in the samr manner as those in the test group with the exception that corn 01! alone was applied to the siun.
Following the dermal applications, the animals were returned to their individual cages to await testing on the following day.
The above acheduls was followed every day, including weekends, for a total of 20 consecutive days,
C. Body Weight Effects On the morning of the first ,est day, the body weight of each
rabbit in every group was determined and recorded. Thereafter, indi vidual weighings were made weekly and the data recorded as an index to body weight effects. Dose calculations were made weekly to coin cide with changes in body weight.
D. Mortality and Reactions Checks for mortality and abnormal behavioral reactions were
made dally during the 20-day test period at well at during the 14-dav observation period. Special emphasis was placed on observations for local akin reactions in order to determine the degree of akin irritation produced by the teat material.
C. Hematologic Studies and Urine Analyses Blood studies including dsterminations of hemoglobin concent
ration, erythrocyte count, Md both total and differential leukocyte
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couats, were made at the beglaaiag of the study and at the *d of the 20-day teet period. Urine analysts for reducing substances and micro scopic elements were also foot at these time latervai*. The blood stud ies aad urine annlyse* ware doe* on the individual rabbits in each group,
r. Groee tad Microscopic Pathologic Studies
Arrangement* were made to subject aay animal which might die during the test to a gross autopsy. Also, la those instances where post-mortem changes wers not advanced, sections of representative tis sues and organs were scheduled to be taken for histopathologic study.
At the conclusion of the investigational period, all surviving rabbits In each group were sacrificed and gross and microscopic studio were performed. The following tissues and organs were Included 'a th. microscopic studies: heart, aorta, lung, trachea, liver, gall bladder, pancreas, esophagus, stomach, small Intestine, caecum, colon, spleen, cervical and mesenteric lymph node, kidney, urinary biadder, testis, ovary, prostate, uterus, pituitary, adrenal gland, tubmnxUlary salivsry gland, thyroid, parathyroid, akslstal muscle, bone marrow, peripheral nerve, spinal cord, brain, and skin.
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tu. R * m It * A. Body Weight Del Weekly Individual body weight* for each lrt group and the con
trol group are aeaembled in Table n.
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TABLE H
Body Weight Data
Animal Number and Sc x
Individual Body Weight* (Kilogram*) Teat Day Number:
0 7 14 20
l-M 2-F
2.31 2.50
2. 35 2.63
2.49 2.68
2.57 2.90
l-M 2-F
2. Jll 2.2"
2.58 2.50
2.55 2.53
2.63 2.61
l-M 2-F
2. It 2. 36
2.32 2.24
2.27 2.30
2. 18 2.24
l-M 2-F
2.21 2.72
1.70 2.39
(1.70) (2-J2)
_
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l-M 2-F
2. It. 2. l.H
1.98 1.53
(1.90) (1.50)
"
Oboe rvat ion Day
Number 14
2.74 2.9o
2.61 2.73
2. 30 2.21
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"
Note: Figure* in parenthe *r t repreent autopty weight*.
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B. Mortality The mortality data are summarised In Table HI- The data in
dicate the number dead and the total number of doses received prior to death.
The mortality data wsre statistically analysed employing thi technique of Litchfield and Wilcoxon*, and the mean subacute percu taneous lethal dose (LD50) was calculated together with the LD0 qj and the LDqqq^. For the purpose of these calculations the test per iod wss assumed to be 34 days, i.e. a 20-day application period plus a two-week observation period. All animals thst died during the inter val, at each dose level, were used in establishing the per cent mor tality for the dose level. The data are presented in Table IV and are graphically illustrated in Fig ure 1.
* J. T. Litchfield, Jr. and F. Wilcoxon, "A Simplified Method o( Evaluating Dose-Effect Experlmsnts", J. Phsrm. b Exp. Ther. , Vot. 9b, No. Z; pp. 99-113 (1949).
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TABLE IV
Statiatical Ana ly ala of Mortality Data
Dote mg/kg/day
Numbe r Dc*d
Number Taated
Oboervad Per Cent
Dead
Expected Per Cent
Dead
500 0
2
0
0.0
1000
0
2
0 11.5
1500
2
2 100
84.0
2000
2
2 100
99. 5
Mean Subacute Dermal Lethal Dole (LD^g) * 1250 mg/kg/day* 95 Per Cent Confidence Limit! of LD50 * 874 to 1788 mg/kg/da\ Subacute Dermal LDqqj * 835 mg/kg/day Subacute Dermal 1-099,99 * 2450 mg/kg/day
* The LD^q ipecifically refera to the dole which, adininiat. r<d each day over a 20-day period, would bo expected tv kill `0 per cent of the animala ao doled within the 20-day doling per iod plua an additional 14 days during which the leal material ia not applied.
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1. behavioral React!was
No behavioral reactions were m ale rc civlag doses of KK) mg/hg/dsry.
eraoag the nai
Generalised Inactivity, weakness, lassitude, and !* ef ap
petit# ware nxhlhited try - abb Its receiving doeee of 1900, 1500,
2000
mg/kg/day respectively. The severity of the reaction* wu rather mild
la the 1000 mg/kg/day does group. These eyrnjHotns prograised la lutan-
ally among rabbit* la the 1JOO and 2000 mg/kg/d*y dose groups aad even
tually aad ad In profound prostration aad death la all rabbits io tbasa dose groups. The surviving animals In tbs 1000 mg/kg/day dees group appeared
normal by the end of tbs 14-day observation period.
2. Local Skin Reactions
Local skin reactions first bee am a evident in all test groups
alter the second or third application of the test material. The reaction!'
began with alight erythema tad progress td to aubderraal hemorrhages
with drying, wrinkling sad eventual Assuring of the akin. The severity of
these local skin reactions was correlated with the magnitude of the does
level. During the 14-day observation period desquamation of the epithel
ium occarred and by the end of this period the akin of the surviving ani
mals appeared normal.
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Ne algaiAcaat hletapatholafllc tltaratiaae were ot*4 la eay at tie tiaeoen m4 orgaae at aaimale recalling Aaaae at $00 *o4 1000 mi As/^^r raapactiTely. MoAareta hepatic fcac reals wm aeteO moh male la tie 1500 aa4 1000 ntgAc/^T Oe* grosspa. Tile hepatic chaoge was considered tc h a direct effect at pa restate* owe alegogytlca at tie test material. All ether tie ease a<S or fas.* mmiui frosa &ee aatmale vara comparable to those at treated coatrel anten ale.
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IV. Summary A. Body Weight* Examination of the body weight data revealed ellght adver*e
effect* mon| animal* receiving do*e* of 1000 mg/kg/day during the do*ing period. Moderate to *v._re body weight tffecta wara noted among animal* in the 1500 and 2000 mg/kg/day doae group*. The bodyweight data of animal* receiving 500 m.j/kg/day compared well with thoae of the treated control group.
B. Mortality and Reaction* The 20-day *ubacute dermal mean lethal doae (LDjq) of Aro-
clor 1268 for the albino rabbit wa* found to be 1250 mg/kg/day. Generalired inactivity, weakne**, la*itude, and io** of appe
tite were exhibited by rabbit* receiving do*e* of 1000, 1500, and 2000 mg/kg/day re*pectively. No untoward reaction* were noted among ani mal* in the 500 mg/kg/day do*e group.
Local kin reaction* began with alight rytheroa and progre**ed to *ubdermal hemorrhage* with drying, wrinkling, and fiuring of the kin among nimal* of all tet group*. De*quamation of the epithelium occurred during the M-day obervatioo period. By the end of this period, the *kln or urvtving animate appeared normal.
C. Hematologic Studle* and Urine Analye Hematologic data and re*ult of the urine analyaa* for teat group
animal* compared well with tho*e of the treated control group animal*.
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D. Pathol Oron Pathologic Flndtaga No Lgaifleant iron pathologic finding* wr noted among
animal* In the 500 and 1000 mg/kg/day doe* group*. Aaimal* receiving do*e* of 1 500 and 2000 mg/kg/day respectively revealed liver dUcolor*tioc {lighter la color). All other ti u s and organ* examined from the*c animal* were comparable to those of treated control animal*.
2. Microscopic Pathologic Finding* No significant histopathologic tisane alteration* were notrd
among animal* receiving do*** of 500 and 1000 mg/kg/dav respectively. Moderate hepatic necrosi* u noted among animal* in the 1500 and 2000 mt/ki/d*y do*e group*. This finding wa* considered to be a direct ef fect of percutaneous absorption of the test material. Ail other tie a ue< and organa examined from these animal* w*r* comparable to those seen in the treated control animals.
Respectfully submitted, INDUSTRIAL BIO-TEST LA-BORATORIES, INC.
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Report prepared by:
Richard!. Palaaiblo, B.S. Departmental Director Acute Toxicity Department
Rep crt approved byl John H. Kay, Ph. D. Associate Director
March 2, 1961
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