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TOApproved for public release, distributionunlimited

FROMDistribution authorized to U.S. Gov't.agencies only; Administrative/OperationalUse; APR 1969. Other requests shall bereferred to Commanding Officer, EdgewoodArsenal, Attn: SMUEA-TSTI-T, EdgewoodArsenal, MD 21010.

AUTHORITY

USAEA notice, 7 Apr 1970

THIS PAGE IS UNCLASSIFIED

EDGEWOOD ARSENALTECHNICAL REPORT

EATR 4301

TOXICITY OF O-CHLOROBENZYLIDENE MALONONITRILE (CS)

IN TRIOCTYLPHOSPHATE (TOF) SOLUTIONS

by

J. T. Weimer

E. J. OwensT. A. Ballard

J. F. Ferrell, MAJ, VC

"J. S. Everts, CPT, VC

0 H. P. Averill, Ph. D.B. P. McNamora, Ph. D.

April 1969

DDC

A APR 151969

UULUU-DEPARTMENT OF THE ARMY '

EDGEWOOD ARSENAL

Reeac Laboratoriesb~kA Wosodc LAP*

Edgewood Arsemna, Marylmnd 21010

m o m .. .... ...m ' . . . . . .; . . . . . . . . .

EDGEWOOD ARSENAL TECHNICAL REPORT

EATR 4301

TOXICITY OF O-CHLOROBENZYLIDENE MALONONITRILE (CS)IN TRIOCTYLPHOSPHATE (TOF) SOLUTIONS

by

J. T. WeimerE. J. Owens

T. A. BallardJ. F. Ferrefl, MAJ, VCJ. S. Everts, CPT, VC

H. P. Averill, Ph.D.B. P. McNamara, Ph.D.

April 1969

Each transmittal of this document outside the agencies of the US Governmentmust have prior approval of the Commanding Officer, Edgewood Arsenal,ATTN: SMUEA-TSTI-T, Edgewood Arsenal, Maryland 21010.

Project 1B56260ZA079Task lB56260ZA07908

DEPARTMENT OF THE ARMYEDGEWOOD ARSENALResearch Laboratories

Medical Research LaboratoryEdgewood Arsenal, Maryland 21010

"I"l l 3

'"WllGUAouAtLMn, CODES

IS. AVAIL Andtr Sp3lgIAL

Distribution Statement

Each transmittal of this document outside the agencies of theUS Government must have prior approval of the Commanding Officer,Edrwood Arsenal, ATTN: SMUEA-TSTI-T, Edgewood Arsenal, Maryland21 0O.

Dis claimer

The findings in this report are not to be construed as an officialDepartment of the Army position, unless so designated by other authorizeddocuments.

Disposition

Destroy this report when no longer needed. Do not return itto the originator.

FOREWORD

The work described in this report was authorized under ProjectIB56260ZA079, Task 1B562602A07908, Nondefense Medical Aspects ofChemical Agents (U), Incapacitating and Riot Control Agents (U). The workwas started in January 1968 and completed in December 1968.

The volunteers in these tests are enlisted US Army personnel.These tests are governed by the principles, policies, and rules for medicalvolunteers as established in AR 70-25.

In conducting the research described in this report, the investi-gators adhered to the "Guide for Laboratory Animal Facilities and Care,"as promulgated by the Committee on the Guide for Laboratory AnimalResources, National Academy of Sciences-National Research Council.

Reproduction of this document in whole or in part is prohibitedexcept with permission of the Commanding Officer, Edgewood Arsenal,ATTN: SMUEA-TSTI-T, Edgewood Arsenal, Maryland Z1010; however,DDC is authorized to reproduce the document for US Government purposes.

The information in this document has not been cleared forrelease to the general public.

Acknowledgments

The authors acknowledge the assistance of W. U. Thomas,R. L. Fortier, S. G. Ryan, R. P. Merkey, and J. S. Olson in conducting theinvestigations reported herein. Special thanks are due to Mrs. Martha A.Langan who assisted greatly in the preparation of the tables and in thearrangement of the report.

2

DIGEST

As solutions containing 1% o-chlorobenzylidene malononitrile(CS) in trioctylphosphate (TOF) have been proposed for use in riot controlsituations, a series of toxicological tests was conducted in animals.Results showed that the doses tested are not permanently injurious to theeyes. Effects on skin ranged from none to necrosis; and the affected areasusually healed within 2 weeks. When the mixtures were directly injectedinto the trachea, some evidence of congestion and pneumonia was seen. Butthe severiy oand duration of signs were not in a category that would warrantveterinary treatment under conditions of normal animal care.

The reported studies indicate that no irreversible damage is

expected with 1% .CS/TOF solutions as long as the dose ranges shown to besafe in this paper are not exceeded.

I

S. . . . . ....... ....

CONTENTS

I. INTRODUCTION ........... ................. 7

II. TOXICOLOGY OF CS ................................. 7

A. Physical Properties ......... 7B. Airborne CS ........... ................ 7C. Patch Tests .............. ......................... 8D. Solutions of CS on the Skin of Animals ...... ........... 8E. Comparison of Skin and Eye Effects of CS With Those

Caused by Other Riot Control Compounds and Mixtures 9F. CS in Methylene Dichloride. . ................ 9

In. TOXICOLOGY OF TOF ............ ...................... 9

A. Physical Properties ............. .................... 9B. Summary of LD50's by Single Exposure.. .............. 9C. Single Inhalation Exposure ......... ................. 10D. Single Application to the Eyes of Rabbits ............. .10E. Single Doses in the Crops of Chickens .............. .. 11F. Repeated Inhalation in Guinea Pigs ...... ............. 11G. Repeated Applications to the Skin of Rabbits .......... ... 11H. Percutaneous Effects in Man .... ...... ..... 11

IV. TOXICOLOGY OF CS/TOF SOLUTIONS .................. .. 12

A. Materials ............ .......................... .. 12B. Eye Effects ........... ......................... .. 12C. Cutaneous Effects ............ ...................... 15D. Effects on Upper and Lower Respiratory Tract ........ .. 19

V. CHEMICAL STABILITY OF VARIOUS SOLUTIONS OFCS IN TOF ................ ............................. 20

VI. SUMMARY AND CONCLUSIONS ....... ................. .. 21

A. Oscular Effects of OS/TOF and TOF Alone ............. 21B. Cutaneous Effects of CS/TOF and TOF Alone ......... ... 21C. Effects of CS/TOF and TOF Alone on the Upper and

Lower Respiratory Tract ....... .................. 22D. Systemic Effects of CS/TOF and TOF Alone ............ 22

5

i i

CONTENTS Cont'd

LITERATURE CITED ......... ....................... .. 23

APPENDIX, Tables A-I Through A-XXII ............... 25

DISTRIBUTION LIST ....................... 47

LIST OF TABLES

Table

I. Approximate LD50 Doses for Single Exposure of TOF inAnimals .. .......... .................... 10

II. Experimental Design for 1% CS/TOF and TOF Alone Appliedto Rabbits' Eyes ........ .......................... ... 13

III. Experimental Design for Spraying 1% CS/TOF and TOFAlone Into Rabbits' Eyes ........ ..................... ... 14

IV. Experimental Design for 1% CS/TOF and TOF Alone Instilledin Monkeys' Eyes .................................... 15

V. Experimental Design for 1% CS/TOF and TOF Alone Appliedin Drops to the Clipped Backs of Rabbits ....... ............ 16

VI. Experimental Design for 1% CS/TOF and TOF Alone Appliedto the Clipped Backs of Monkeys ...... ................. .. 17

VII. Effects of 1% CS/TOF Solution on Human Skin ............. ... 18

VIII. Experimental Design for Determining Effects of 1% CS/TOFand TOF Administered to Dogs by Tracheal Intubation orIntratracheal Injection .............. ...................... 19

6

TOXICITY OF O-CHLOROBENZYLIDENE MALONONITRILE (CS)IN TRIOCTYLPHOSPHATE (TOF) SOLUTIONS

I. INTRODUCTION.

Solutions of o-chlorobenzylidene malononitrile (CS) andtrioctylphosphate (TOF) have been proposed for use in riot control situa-tions. This report brings together previously reported and new informationon the toxicological properties of CS, TOF, and CS/TOF solutions inanimals and man.

II. TOXICOLOGY OF CS.

A. Physical Properties.

The structural formula of CS is as follows:

C CN

C

H ' CN

CS is a white, crystalline solid that melts at 94*C and boils at 3100 to315*C (760 mm Hg). The solubility of CS in water is not listed becausethe compound hydrolyzes in that medium. Approximate solubilities of CSin organic solvents at room temperature are as follows:

Solvent % Dissolved by weight

Acetone 42Dioxane 33Ethyl acetate 25Methylene chloride 39Pyridine 46Chloroform 32Methyl isopropyl ketone 21

B. Airborne CS.

Previous studies have shown that CS is highly potent as anirritant and as a riot control compound and has a very low toxicity. 1-3

7

The effective dose for not control for 50%6 of a population (ED5O) is aCt (concentration x time) of 0. 1 to 10 mg min/cu m. The LD5O is estimatedto be 61, 000 mg min/cu m.

CS as an aerosol or vapor acts directly on mucous membranesto produce irritation, burning, and pain in the eyes and respiratory tract.The action on the eyes also causes lacrimation, blepharospasm, and con-junctivitis. The compound affects the upper and lower respiratory tracts,causing sneezing, coughing, salivation, congestion of the nose and wall ofthe pharynx, and a feeling of suffocation. The effects are immediate andpersist for 5 to 20 minutes after one leaves the contaminated atmosphere.

On the skin, these aerosols produce a burning sensation whichis greatly accentuated by moisture such as perspiration, lacrimation,rhinorrhea, and salivation. The burning sensation may persist for severalhours and recurs upon washing of the exposed area. 4 Exposure of the skinto CS aerosols in high concentrations of 300 mg/cu m at a wind velocity of5 mph, a temperature of 95°F, and a relative humidity of 95% for 30minutes produced irritation and burning, but no blisters. Continuation ofthese severe exposures for 45 minutes did produce vesication. 5

First and second degree burns were produced accidentally in12 officers participating in field exercises. The officers were thoroughlysoaked from a heavy continuous rain. They were subjected to micro-pulverized CSI dispersed from a modified flamethrower. The CS seemedto soak through the wet fatigues and burn the skin. From 7 to 10 hoursafter exposure, erythema appeared on the V-area of the neck, the forearms,the wrists, and the calves. In 14 to 16 hours after exposure, blisteringhad begun in the most severe cases. All vesicles healed in about 1 week,and no sequelae were reported. 5

Daily exposure of people to heavy concentrations of CS inmanufacturing plants resulted in erythema and vesiculation. 6

C. Patch Tests.

In 24-hour patch tests on men, dry CS on the forearm producedvarying skin reactions in both Negroes and Caucasians. The effectsvaried from no reaction to erythema, vesication, and sloughing. 4

D. Solutions of CS on the Skin of Animals.

The minimal dose of CS in corn oil, applied to the freshlyclipped backs of rabbits, that produced any sign of irritation was

8

20 mg/animal. Doses of 40 to 60 mg/animal caused mild to moderateerythema or necrosis. The results of these tests are shown in table A-I,appendix. (Tables A-I through A-XXII are in the appendix.)

E. Comparison of Skin and Eye Effects of CS With Those Causedby Other Riot Control Compounds and Mixtures.

The minimal dose of CS in corn oil, applied to the eyes ofrabbits, that produced any sign of irritation was 1. 0 mg/eye. Doses of upto 10 mg in the eye caused moderate to severe conjunctivitis and blepharitis.No corneal damage, ulceration, or other permanent damage was produced,and recovery from the minor signs was completed 9 days after application.The results of these tests are shown in table A-II.

A comparison of CS with other irritants in corn oil showed thatCS was less damaging to the eyes and skin than are CN (chloroaceto-phenone), DM (diphenylaminochloroarsine), or CN-DM mixtures. These

data are shown in table A-Ill.

F. CS in Methylene Dichloride.

Doses of 0. 05 and 0. 1 ml of 10% CS in methylene dichlorideplaced in rabbits' eyes caused immediate hyperemia, which disappearedin a few hours. There was no noticeable corneal damage. A dose of 0. 1 mlof a 50% mixture of CS in methylene dichloride was placed in the eyes of 10rabbits, but no eye damage was noted during a 14-day observation period.The eyes were treated daily with sodium sulfamide to prevent secondaryinfection. Meth lene dichloride alone produced no persistent effects oneyes or eyelids.

HI. TOXICOLOGY OF TOF.

A. Physical Properties.

Trioctylphosphate, tri-(Z -ethylhexyl)-phosphate, is a water-clear liquid with a specific gravity of 0. 9225 at Z00C; a boiling point ofZ20°C at 5 mm Hg; a vapor pressure of 1. 9 mm Hg at 200°C; and a pourpoint of -90°C.

B. Summary of LD50's by Single Exposure.

Approximate LD50 doses of TOF for a single exposure areshown in table I.

9

Table I. Approximate LD50 Doses for Single Exposureof TOF in Animals

Species Route Approximate LD50 Reference

Rabbits Intravenous >358 mg/kg 7Rabbits Intratracheal >1, 811 mg/kg 7Rabbits Intragastric 46 gm/kg 7Rabbits Cutaneous <20 ml/kg (19 gm/kg) 9

Rats Intragastric >36.8 gm/kg 7Rats Oral 37. 1 gm/kg 8Rats Oral 39.8 gmn/kg 9Rats Inhalation >93, 000 mg min/cu m 7Chickens Intracrop >2, 500 mg/kg 7Guinea pigs Inhalation ca. 30, 000 mg min/cu m 7Monkeys Cutaneous >75 mi/kg (70 gm/kg) This rpt

C. Single Inhalation Exposures.

One of two dogs died after a 4 -minute exposure to a concentrationof 250 mg/cu m of TOF (Ct = 1, 000 mg min/cu m). However, the two dogsexposed to a Ct of 500 and the two exposed to a Ct of L, 000 survived.

No deaths occurred when six rats were exposed for 8 hours tonearly saturated vapor concentrations of TOF. A mist of TOF generated at170*C killed no rats in 30 minutes, but killed two of six rats after 1 hour. 9

Ne..ropsy of rats that had inhaled TOF labeled with p3Z showeda high percentage of the compound in their stomachs with lesser amounts intheir lungs, livers, brains, spleens, kidneys, bones, muscles, and fat.The compound was excreted in both urine and feces. 7

D. Single Application to the Eyes of Rabbits.

A letter from Union Carbide Corporation9 states: "Eye injuryto rabbits: Definite Hazard." This caution has not been supported by

further study. 7

10

E. Single Doses in the Crops of Chickens.

One of eight chickens died after 2, 500 mg/kg of TOF was intro-

duced into their crops. All of eight chickens given a dose of 500 mg/kg

survived. Although demyelinization is produced by certain phosphates,

there was no gross or histological evidence of neurological damage. 7

F. Repeated Inhalation in Guinea Pigs.

Three groups of 20 guinea pigs each were exposed to TOF in

concentrations of 10.8, 26.4, and 85.0 mg/cu m, 6 hours/day, 5 days/weekfor 12 weeks. A control group of 20 unexposed animals was included. Death

rates were 30%, 46%, 25%, and 59%, respectively, for the controls, the low,

intermediate, and high doses. 7 The value of this experiment is doubtfulbecause of the presence of a respiratory infection. In the second experiment,

the death rates were 0/20, 1/20, and 1/20 for control animals and animals

exposed to concentrations of 1. 6 and 9. 6 mg/cu m, respectively. 7 Both

deaths occurred in the ninth week of exposure.

Two dogs and two monkeys at each of three concentrations, -

10.8, 26.4, and 85.0 mg/cu m, survived the 12 weeks of exposure. 7

G. Repeated Application to the Skin of Rabbits.

Repeated doses of 0. 1 rrl of TOF were applied to the clipped

backs of rabbits 5 days/week for Z to 4 weeks. Moderate erythema wasseen after the first application. Following the fourth and fifth applications,desquamation, cracking, and bleeding were noted in the skin. Microscopic

lesions in the skin included hyperkeratosis, parakeratosis, epidermal

thickening, dermatitis, focal atrophy of the hair follicles, some destruction

of the hair shafts, and slight degeneration of the sebaceous glands.

H. Percutaneous Effects in Man.

Union Carbide Corporation 9 reported that TOF applied to cloth

patches and worn on the skin for 5 days caused erythema in 1. 5% of Z00

volunteers. Three weeks later the material was retested on these 200 men

and removed after 48 hours. Five percent of the volunteers developed

e rythema.

11

IV. TOXICOLOGY OF CS/TOF SOLUTIONS.

A. Materials.

The CS used in these studies was obtained from EdgewoodArsenal stock, designated Lot No. 2011-73-1003. The purity was 95.4%as determined by chemical analysis performed by the Chemical ResearchLaboratory. The TOF used in this work was manufactured by Union CarbideCorporation. All solutions were prepared fresh each day.

B. Eye Effects.

Prior to dosing, both eyes of each animal (New Zealand whiterabbits and rhesus monkeys) were examined, and any animal with eyedefects or irritation was eliminated. Before and throughout the observationperiod, the animals were caged individually in raised pens, away frombedding and animal droppings. One day after exposure, the eyes wereflushed with isotonic saline. Clinical observation of the exposed andunexposed eyes were then recorded. Following this, one drop of fluoresceinsodium ophthalmic solution was instilled in each eye; then the eyes wereflushed with isotonic saline and treated with one drop of 15%6 sodiumsulfacetamide ophthalmic solution. On subsequent observations days, eyesthat needed treatment were flushed and treated with ophthalmic solution.

Irritation of the eye was evaluated according to the modifiedDraize technique as described in the "Illustrated Guide for Grading EyeIrritation by Hazardous Substances, " published by the Food and DrugAdministration. The grades of ocular lesions are shown in table A-IV.

In most studies, animals were sacrificed and necropsied 3 and30 days after exposure. To date, only the gross observations of theapplication sites on the 3-day animals have been completed by the patholo-

gists, and these are reported here. (This is also true of the skin studies,which folow.)

1. Instillation in Rabbits' Eyes.

In pilot studies, 0. 2 ml of 0. 05%0 and 0. 1% (w/v) solutions ofCS/TOF produced no more than mild chemosis and hyperemia wheninstilled in the eyes of rabbits (table A-V).

12

__ __

A 1% CS/TOF .olution* and TOF alone were tested as shkwmj .table II. Some animals were killed and necropsied 3 days after dosing.The rest were observed for 14 days.

Table IL. Experimental Design for 1%16 CS/TOF and TOF AloneApplied to Rabbits' Eyes

No. of rabbits Dose Necropsied 3 daysexposed CS/TOF TOF Saline after dosing

ml10 0.2 4

8 0.2 22 0.2 26 0.12 0.16 0.052 0.1

10 0.025 44 0.025 22 0.025 26 0.01 42 0.01

10 0.005 44 0. 005 2

2 0.005 2

None of the CS/TOF mixtures produced more than mildchemosis and hyperemia. At all but the highest dose, these mild signsof irritation were gone within a week. At the highest dose, mild chemosis

persisted in one rabbit for 14 days. Individual gross observations duringthe 14-day period are presented in table A-VI.

The highest dose of TOF alone, 0. 2 ml, caused mild tomoderate chemosis and hyperemia which disappeared in 13 days. The two

* The volume of each dose here and elsewhere may be converted to milli-grams as follows: specific gravity of CS = ca. 1, of TOF = 0. 9225; a 1%solution of CS/TOF contains 10 mg of CS per milliliter. Therefore,0. 2 ml of 1% CS/TOF contains 2 mg of CS and 186 mg of TOF.

13mp_ _

rabbits that received 0. 1 ml displayed mild chemosis for 1 week. Noirritation was seen in rabbits that received lower doses. Individual obser-vations are presented in table A-VII.

Gross pathological examination of the eyes of the rabbits thatwere killed 3 days after dosing showed mild hyperemia of the conjunctivaein two of the four animals that received the lowest dose of 1% CS/TOF.Conjunctivitis, usually mild, was seen in all four rabbits that received the

intermediate dose of CS/TOF and in both TOF controls. At the high doseof the mixture, all four test animals had moderate to severe conjunctivitisand chemosis; the TOF controls had moderate conjunctivitis. With theexception of very mild conjunctivitis in one rabbit, no significant changeswere seen grossly in the eyes of the saline control animals. The findingsare summarized in table A-VIII.

2. Spray in Rabbits' Eyes.

To simulate an operational spray device, a hypodermic syringe

with a 21-gauge needle was used to spray 1 or 10 ml of CS/TOF and TOFalone into one eye of restrained rabbits. (Accuracy at 6 feet was limited.)The experimental design is shown in table M.

Table IMI. Experimental Design for Spraying 1% CS/TOF andTOF Alone Into Rabbits' Eyes

No. of Distance from Dose Necropsied 3 days

rabbits which sprayed CS/TOF TOF after dosingft ml

4 1 108 3 10 44 3 1 4

4 6 102 1 104 3 10 22 3 1 22 6 10

The mixture of CS/TOF and TOF alone (I and 10 ml) producedmoderate to severe chemosis and hyperemia, which persisted for 7 days in

14

4

the animals that were not sacrificed (tables A-IX and A-X). There wereerythematous reactions on the head and ears of all rabbits.

Necropsy showed that three of the four rabbits sprayed withI ml of the mixture from a distance of 3 feet had mild conjunctival hyper-

emia, as did one of the two TOF controls. Three of the four sprayed with10 ml of the mixture from the same distance had mild conjunctivitis, andthe fourth had only mild hyperemia of the conjunctivae. The eyes of bothTOF controls sprayed with 10 ml were normal (table A-X).

3. Instillation in Monkeys' Eyes.

The ocular effects of a 10% CS/TOF solution were studied inrhesus monkeys according.to the design shown in table IV.

Table IV. Experimental Design for 1% CS/TOF and TOFAlone Instilled in Monkeys' Eyes

No. of monkeys Dose Necropsied 3 daysexposed CS/TOF TOF after dosing

ml

8 0.1 4

8 0.025 48 0.005 44 0.1 24 0.025 24. 0.005 2

Lacrimation and conjunctival hyperemia on the day of exposurewere the only signs of irritation shown during the 14-day observationperiod, and all monkeys were affected. Necropsy of animals killed 3 daysafter dosing showed all eyes to be normal.

C. Cutaneous Effects.

New Zealand white rabbits and rhesus monkeys were preparedfor exposure by clipping the hair from their backs. Any animal with skinabnormalities (abrasions, discoloration, etc.) was excluded from the study.

The grading system used to assess skin irritation is given intable A-XI.

15

1. Drops.

Discrete drops of 1% CS/TOF or TOF alone were placed on thebacks of the animals with a syringe.

a. Rabbits.

In preliminary studies, the skin of groups of six rabbits eachwas exposed to drops (1. 0 mnl) of 0. 05%0 and 0. 1% CS/TOF solution. Bothdoses produced severe erythema and edema, followed by dehydration andnecrosis. These signs disappeared in the low-dose group within 12 days.Erythemra in two of the high-dose groups was still evident at the end of the14-day observation period (table A-XII).

Table V gives the design of the experiment with 1% CS/TOFsolutions.

Table V. Experimental Design for 106 CS/TOF and TOF AloneApplied in Drops to the Clipped Backs of Rabbits

No. of rabbits Dose Necropsied 3 days

exposed CS/TOF TOF after dosing

ml

10 1.0 44 1.0 26 0.56 0.25

10 0.10 44 0.10 26 0.05

10 0.025 44 0.025 2

All doses of CS/TOF and TOF alone produced severe erythemaand edema, followed by dehydration and necrosis. At the end of the 14-dayobservation period, a few rabbits still had areas of cutaneous necrosis anderythema (tables A-XM and A-XIV). The rabbits necropsied 3 days afterexposure had only mild to severe erythema (table A-XV).

16

L

b. Monkeys.

The local and systemic effects of cutaneously applied 1%CS/TOF solutions were studied in monkeys according to the design shownin table VI.

Table VI. Experimental Design for 1% CS/TOF and TOF AloneApplied to the Clipped Backs of Monkeys

No. of monkeys Dose Necropsied 3 daysexposed CS/TOF TOF after dosing

Local effects8 1.Oml 44 1.Oml 28 0.1lml 44 0.1 rl 28 0. OZ5 ml 44 0.OZ5 ml 2

Systemic effects2 1 Z. 5 ml/kg*2 25.0 ml/kg2 50.0 nml/kg*z 75. Oml/kg*

*The monkeys weighed about 2 kg.

The monkeys exposed to 1. 0, 0. 1, and 0.025 ml of CS/TOFand TOF alone showed no signs of skin irritation during the 14-day obser-vation period. The skin of those monkeys necropsied 3 days after dosingwas normal.

All the monkeys that received the large cutaneous doses of TOFdeveloped mild erythema within 24 hours. In those that received doses of12. 5, 25, or 50 ml/kg, the erythema progressed to drying and wrinklingof the skin by the end of the first week, and the skin was normal by 3 weeks.The monkeys that received the highest dose, 75 mnl/kg, developed scatteredareas of necrosis, but these had healed by the end of 3 weeks. No physicalor behavioral signs of neurologic damage or systemic signs were noted.The cutaneous signs are listed in table A-XVI.

17

2. Patch Tests.

a. Rabbits.

Patches of sateen cloth were contaminated with 0. 10 and 1. 0 mlof 1% CS/TOF and taped to the backs of eight rabbits per dose for 24 hours.The same procedure was followed for TOF alone except that four rabbitsper dose were used. Half of the rabbits at each dose were killed andnecropsied 3 days after dosing.

The CS/TOF mixture produced mild to severe erythema in allrabbits within 48 hours. Necrosis appeared as early as the third day, buthad healed by the ninth day. Mild erythema was still present 10 days afterdosing (table A-XVII).

Results with the patches contaminated with TOF alone were thesame as those with the mixture except that necrosis appeared a day later.

Rabbits dosed with 0. 1 ml of CS/TOF had mild to moderatelysevere erythema; the skin of one was slightly thickened. The TOF controlsdosed with 0. 1 ml also had mild erythema. Those rabbits that received1. 0 ml of the mixture had mild to moderate erythema, and one had a slightthickening of the skin. Both of the TOF controls at the 1. 0-ml dose hadmoderate erythema, and the skin of one was slightly thickened.

b. Men.

Table VII shows the design and the results of a study in whichI% CS/TOF was placed on the volar surface of the forearm of humansubjects. The environmental temperature during these exposures was 59°F(56" to 61") and the relative humidity was 95% (83% to 99%0).

Table VII. Effects of 1% CS/TOF Solution on Human Skin

No. of Dose Material applied Resultssubjects on

ml2 0. 1 Bare skin No effect2 0. 025 Bare skin No effect2 0. 1 Sateen patch* Mild stinging

for 8 hr (one subject)2 0. 025 Sateen patch* No effect

*Patch taped to skin and left in place for 24 hours.

18

D. Effects on Upper and Lower Respiratory Tract.

Beagle hounds that were less than 2 years old were used inthese studies. Preexposure physical examinations included counts of redand white blood cells and measurement of packed cell volume, blood ureanitrogen, and rectal temperature. These measurements were repeated 72or 96 hours after exposure. Each animal was found to be in good healthbefore it was used.

The protocol for this series of tests is shown in table VIII.

Table VIII. Experimental Design for Determining Effects of1% CS/TOF and TOF Administered to Dogs by Tracheal

Intubation or Intratracheal Injection

No. of Dose Necropsied 3 days Necropsied 30 daysdogs CS/TOF Saline TOF after dosing after dosing

mlIITracheal intubation

8 0.50 4 48 0.25 4 4j 8 0.10 4 44 0.50 2i4 0.25 14 0.10 28 0. 50o 4

Intratracheal injection

8 05001 I I4 0. 50 ?

I. Tracheal Intubation.

After the. dogs had been given a short-acting barbiturate, alaryngoscope was inserted and a polyethylene tube fitted to a 21-gaugeneedle was passed 2 inches posterior to the larynx, where the dose wasdeposited. The only sign was coughing when the trachea was pressed andcongestion of the lungs, revealed by auscultation (table A-XVm). Red andwhite blood cell count, packed cell volume, blood urea nitrogen, and rectaltemperature were not affected (tables A-XIX and XX).

19

Mild tracheal hyperemia and minimal pulmonary congestionwere seen in two of the four dogs necropsied 3 days after receiving 0. 1 mlof the CS/TOF mixture (table A-XXI). A small pneumonic area was presentin the lung of one dog that received 0. 25 ml, and discolored areas, probablyrepresenting congestion, were seen in two other dogs. At the highest dose,three of four dogs had some hyperemia of the tracheal mucosa and differentdegrees of pneumonia. One of these dogs had a single consolidated nodule2 cm in diameter while the other two had larger areas of consolidationscattered throughout the lungs. The fourth dog had patchy discolorationthat was probably due to congestion but could represent early pneumonia.Two of the six saline control animals had mild hyperemia in the trachealmucosa, while two others had only pulmonary congestion. There was nosign of tracheal damage in the TOF controls. The lungs of all four, however,showed varying degrees of congestion and pneumonia. A small pneumonicarea was detected grossly in only one of the 12 dogs necropsied 30 daysafter receiving CS/TOF. Some evidence of pneumonia was present in thosethat had received TOF alone. All of these dogs were clinically normal.

2. Intratracheal Injection.

The investigators prepared this group of dogs by clipping thehair on the ventral neck and scrubbing the skin with surgical soap. Theanimals were tranquilized with chlorpromazine hydrochloride. Dosing wasaccomplished by inserting a 21-gauge needle into the trachea 1 to 2 inchescaudal to the larynx and injecting the test solution.

Lesions in this group were the same as those in the group thatwas given the materials by intubation (table A-XXI). Blood values were notaffected (table A-XIX). Moderate tracheitis was seen in one dog killed 3days after dosing, but mild, focal pneumonia was seen in all four.Ecchymotic hemorrhages were present in all lobes of the lungs of one salinecontrol animal. Three of the four dogs necropsied 30 days after receivingCS/TOF had at least microscopic evidence of pneumonitis, as did bothsaline controls.

V. CHEMICAL STABILITY OF VARIOUS SOLUTIONS OF CS IN TF.

The stability of CS/TOF solutions stored under various con-ditions for 15 days was determined by ultraviolet analysis. The solventsystems were checked daily, and the changes in ultraviolet absorbancepeaks at 300 nrp (peak for CS) and 228 np (secondary activity) wererecorded. The concentrations of unreacted CS found in the solutions werecompared with the control peak and the percentage of this value recorded.

20

4.4

The details of this experiment and the results are shown intable A-XXII. The CS/TOF solutions containing 1% CS were relativelystable when stored for various time periods at either Z5* or IZC withfluorescent light, but were degraded when the temperature was raised to90°C. Concentrations of 0. 10/ or 0. 06% were degraded at 25° and 12°C inroom light in direct proportion to solution strength. Samples containing1. 0% CS stored in darkness were less stable than those stored at the sametemperature (25°C) under fluorescent light.

VI. SUMMARY AND CONCLUSIONS.

A. Oscular Effects of CS/TOF and TOF Alone.

The highest dose (0. 1 ml) of 1% CS/TOF and TOF alone instilledin the eyes of monkeys had no effect. The highest dose (0. 2 ml) put in theeyes of rabbits produced very mild effects, which regressed within 2 weeks.

Severe chemosis and redness, which persisted for 1 week, wasproduced when 10 ml of a 1%/6 CS/TOF solution or TOF alone was sprayedinto the eyes of rabbits from a distance of 1, 3, and 6 feet. No corneallesions were seen.

B. Cutaneous Effects of CS/TOF and TOF Alone.

The lowest dose (0. 025 ml) of 1%6 CS/TOF and TOF aloneapplied in drops to the skin of rabbits caused immediate erythema thatprogressed to necrosis in 8 to 10 days. Higher doses caused the samereaction with the area affected being more extensive due to the greaterspread of the liquid over the skin. In most instances the skin had healed bythe end of 2 weeks. The highest dose (I ml) of the CS/TOF mixture orTOF alone had no effect on the skin of monkeys.

Patch tests with 0. 1 and 1. 0 ml of 1% CS/TOF caused necrosisof rabbit skin that healed in 9 days. The same doses of TOF produced noreaction.

When 0.025 ml of 1%6 CS/TOF was applied to the bare skin ofthe volar forearm of men or was placed on a sateen patch that was taped tothe skin of the forearm, no reactions were seen. When the dose wasincreased to 0. 1 ml, one of two subjects experienced a mild stinging forabout 8 hours.

21

C. Effects of CS/TOF and TOF Alone on the Upper and LowerRespiratory Tract.

When 1%6 CS/TOF was introduced into the trachea of dogs byeither intubation or injection, only mild clinical signs were noted. Thesesigns were not dose-related; in fact, they were most frequent at the lowestdose.

The results indicate that injection of 0. 5 ml of CS/TOF or TOFalone into the trachea of the dog does not cause enough damage to bediscernible by clinical observation. This dose did not result in secondaryinfection severe enough to cause fever, pronounced dehydration, spontaneouscoughing, generalized auscultatory sounds, or elevation in white blood ceUcount. In general, the severity and duration of signs were not in a categorythat would warrant veterinary treatment under the conditions of normalanimal care. This is especially significant in view of the method ofexposure, i.e., direct injection into the trachea.

D. Systemic Effects of CS/TOF and TOF Alone.

The only effects seen in the dogs, rabbits, and monkeys used inthe eye, skin, and lung studies were mild respiratory signs in the dogsexposed by instilling the materials directly into the trachea.

The reported studies indicate that no irreversible damage isexpected with 1%16 CS/TOF solutions as long as the dose ranges shown to besafe in this paper are not exceeded.

22

ILITERATURE CITED

1. Gongwer, L. E., Ballard, T. A., Gutentag, P. J.,Punte, C. L., Owens, E. J., Wilding, J. L., and Hart, J. W. CWLTechnical Memorandum 24-18. The Comparative Effectiveness of FourRiot Control Agents. November 1958. UNCLASSIFIED Report.

2. USA CWL Human Estimates Committee: Minutes ofMeeting Held 26 August 1959. UNCLASSIFIED Report.

3. Farrand, R. L. CRDLR 3043. The Cause of Death and thePhysiological Responses in Anesthetized Dogs During Exposure to an

Aerosol of CS. January 1961. UNCLASSIFIED Report.

4. Gutentag, P. J., Owens, E. J., and Punte, C. L.CWLR 2365. The Evaluation of CS Aerosols as a Riot Control Agent inMan. April 1960. UNCLASSIFIED Report.

5. Hellreich, A., Goldman, R. H., Bottiglieri, N. G., andWeimer, J. T. EATR 4075. The Effects of Thermally-Generated CSAerosols on Human Skin. January 1967. UNCLASSIFIED Report.

6. Bowers, M. J., Owens, E. J., and Punte, C. L. CWLTechnical Memorandum 34-50. Interim Report of CS Exposures in PlantWorkers. June 1960. UNCLASSIFIED Report.

7. MacFarland, H. N., and Punte, C. L., Jr. ToxicologicalStudies on Tri-(Z-Ethylhexyl)-Phosphate. Arch. Environ. Health. 13,13-20 (1966).

8. Smyth, H. F., Jr., and Carpenter, C. P. FurtherExperience With the Range Finding Test in Industrial ToxicologyLaboratory. J. Indus. Hyg. Toxicol. 39, 63-68 (1948).

9. Union Carbide Corp. Toxicology Studies, FlexolPlasticizer TOF: Industrial Medicine, Toxicology Dept, 30 Oct 1967.

23

I----- -- -- ___ I �-.

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APPENDIX

TABLES

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Appendix 27

Table A-IV. Guide for Grading Eye Irritation

Part, of Condition found Ratingeye scale

Cornea No ulceration or opacity 0

Scattered or diffuse areas of opacity (other than [l]slight dulling of normal luster), details of irisclearly visible

Easily discernible translucent areas, details of iris 2slightly obscured

Nacreous areas, no details of iris visible, size of 3pupil barely discernible

Complete corneal opacity, iris not discernible 4

Iris Normal 0

Markedly deepened folds, congestion, swelling, [I *moderate circumcorneal injection (any of these or

combination of any thereof), iris still reacting tolight (sluggish reaction is positive)

No reaction to light, hemorrhage, gross destruction 2(any or all of these)

Conjunctivae Redness (refers to palpebral and bulbar conjunctivaeexcluding cornea and iris)

Vessels normal 0

Some vessels definitely injected 1

Diffuse, crimson red, individual vessels not [21*easily discernible

Diffuse beefy red 3

Chemosis

No swelling 0

Any swelling above normal (includes nictitating 1membrane)

Obvious swelling with partial eversion of lids [21*

Swelling with lids about half-closed 3

Swelling with lids more than half-closed 4

* Bracketed figures indicate lowest grade considered positive under Section

191. 12 of the Federal Hazardous Substances Labeling Act Regulations.

Appendix 28

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Apeni 31

j Table A-VIII. Effects of 1% CS/TOF and TOF Alone Instilled in Eyes of Rabbits

(These rabbits were sacrificed and necropsied 3 days after dosing)

Ocular effects

RabbitDose Ro.Iit 2* 3* Necropsy findings

CH R C CH R C G H R C

ml

CS/TOF

0.2 119 2 0 0 0 0 0 0 <1 0 4/4 Moderate to severe

0.2 120 1 0 0 0 0 0 <1 0 0 conjunctivitis and

0.2 121 1 0 0 0 0 0 <1 0 0 chemosis

0.2 122 1 0 0 <1 0 0 1 0 0

0.025 123 0 1 0 0 0 0 0 <1 0 4/4 Mild conjunctivitis

0.025 124 <1 0 0 0 0 0 1 <1 0

0.025 125 1 0 0 0 1 0 <1 0 0

0.025 126 1 0 0 0 1 0 0 1 0

0.005 I 127 0 1 0 0 0 0 0 1 0 2/4 Conjunctival

0.005, 128 0 0 0 0 0 0 0 0 0 hyperemia

0.005 129 0 0 0 0 0 0 0 0 0

0.005 1 130 <1 0 0 0 0 0 0 <1 0

TOF

0.2 137 1 0 0 0 0 0 0 <1 0 2/2 Moderate

0.2 138 1 0 0 0 0 0 < 1 0 0 conjunctivitis

0.025 139 0 1 0 0 < 1 0 0 < 1 0 2/2 Mild conjunctivitis

0.025 140 0 <1 0 0 0 0 0 <1 0

0.005 141 0 0 0 0 0 0 0 0 0 1/2 Mild conjunctivitis

0.005 142 0 0 0 0 0 0 0 < 1 0

Saline

0.2 131 0 0 0 0 o 0 0 0 0 None

S0.2 132 0 0 0 0 "0 0 0 0 0

0.025 133 0 0 0 0 0 0 0 0 None

0.025 134 0 0 0 0 0 0 0 0

0.005 135 0 0 0 0 0 0 0 0 None

0.005 136 0 0 0 0 0 0 0 0

NOTES: CH = ChemosisR a Redness

C = Cornea

The grading system used in this table is shown in table A-IV.

* Number of days after dosing

Appendix 32

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Appendix 34

A

Table A-XI. Grading System for Assessing Skin Irritation

Effect Degree of effect Ratingscale

Erythema No erythema 0

Mild erythema I

Moderate erythema 2

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Erythema with edema 4

Necrosis No necrotic tissue 0

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100% necrotic tissue with well defined 3eschar formation

Dehydration and/or No dehydration or desquamation 0desquamation Mild dehydration or desquamation 1

Moderate dehydration or desquamation 2

Severe dehydration or desquamation 3

Appendix 35

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Table A-XV. Cutaneous Effects of 1% CS in TOF and TOF Alone in Rabbits

(These rabbits were sacrificed and necropsied 3 days after dosing)

Cutaneous effects

Dose 1* 2* 3* Necropsy findings

E N D E N D E N Di ml

1.0 3 0 0 2 0 0 2 0 0 Moderate to severe3 0 0 3 0 0 3 0 0 erythema3 0 0 4 0 0 3 0 02 0 0 2 0 0 3 0 0

1.0 1 0 0 2 0 0 3 0 0 Moderate erythema1 0 0 2 0 0 3 0 02 0 0 2 0 0 2 0 03 0 0 3 0 0 3 0 0

0.025 1 0 0 1 0 0 3 0 0 Moderate erythema.1 0 0 1 0 0 3 0 01 0 0 3 0 0 3 0 01 0 0 2 0 0 3 0 0

TOF

1.0 1 0 0 1 0 0 2 0 0 Moderate erythema1 0 0 3 0 0 2 0 0

S0.1 0 0 0 1 0 0 3 2 0 Moderate erythema

1 0 0 3 0 0 3 2 0

0. 025 0 0 0 1 0 0 3 0 0 Mild to moderate0 0 0 3 0 0 3 0 0 erythema.

Saline

1.0 0 0 0 0 0 0 0 0 0 None0 0 0 0 0 0 0 0 0

S0.1 0 0 0 0 0 0 0 0 0 None

0 0 0 0 0 0 0 0 0

0. 025 0 0 0 0 0 0 0 0 0 None

0 0 0 0 0 0 0 0 0

NOTES: E = ErythemaN = NecrosisD = Dehydration and/or desquamation

The grading system used in this table is shown in table A-XI.

* Number of days after dosing

Appendix 39

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Appendix 413

Table A-XX. Rectal Temperatures of Dogs AfterAdministration of 1% CS/TOF andTOF Alone by Tracheal Intubation

Rectal temperature (average)Test No. ofDose solution animals Preexposure 72 Hours after

exposure

ml OF

S0.5 CS/TOF 8 102.3 102.5

0.25 CS/TOF 8 102.0 102.6

0.1 CS/TOF N 102.2 101.7

0.5 TOF Not taken 101.6

Appendix 44

Table A-XXI. Gross Pathological Observations in Dogs FollowingAdministration of 1% CS/TOF and TOF Alone byTracheal Intubation and Intratracheal Inection

(3 Days after dosing)

Gross pathological observations

Solution DoseTrachea No. Lungs No.

r es ponding responding

mlrr1Tracheal Intubation

CS/TOF 0. 1 Mild hyperemia 2/4 Slight con- 2/4gestion

0.25 No significant Focal pneu- 1/4lesions monia

Congestion 2/4

0.5 Mild to moderate 3/4 Pneumonia 3/4hyperemia Congestion 1/4

TOF 0.5 No significant Pneumonia 4/4lesions

Saline All Mild hyperemia 2/6 Congestion 2/6dose

Intratracheal Injection

CS/TOF 0. 5 Moderate 1/4 Mild, focal 4/4tracheitis pneumonia

Saline 0.5 None Ecchyrnotic 1/2hemorrhage

Appendix 45

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UNCLASSIFIEDSecuritty Clsassification

DOCUMENT CONTROL DATA.- R & D(S-cwiti, ci...Jfic.ia, of 1i00. body of absftrac w ind oxhnd aR~ otatio, SRst * be sw .e h"e .1. w ~ owe,*fl **.t oeseAl

3,ORIGINATING ACTIVITY (CotpowieshAtor) 8RPRT SECUR1ITY CLASSUPIC&flei

CO. Edgewood Arsenal UN CLASSIFIEDATTN: SMUEA-RMT ab, GROUPEdgewood Arsenal, Maryland 21010 FNA3- REPORT TITLE

TOXICITY OF O-CHLOROIBENZYLIDENE MALONONITRILE (CS) INTRIO CT YLPI-T0SPHATE (TOF) SOLUTIONS

4. DESCRIPTIVE NOTES (r~peof .1rpoll edrinetIve. doete)

This work was started in January 1968 and completed in December 1968.S. AUTHOR($) (First. rMoRiddle Initill lost liesr)

Weimer, J. T., Owens, E. J., Ballard, T. A., Ferrell, J. F., MAJ, VC,Everts, J. S., CPT, VC, Averill, H. P., Ph.D., and McNamara, B. P., Ph. D.

S. REPORT DATE 746 TOTAL NO. OF PAGES bNOOPRP

April 1969 529S.CON TRACT ORt GRAN T No. 9.1 ORIOINATORS REPORT NU~MUERIS,

b PROJECT NO. 1B56260ZA079 EATR 4301

c~Task lB562602A07908 "bOHRRPORTN040161(AnF.Owdnb0se Nes.^**lewdep

10. DISTRtIBUTION STATEMENT

Each transmittal of this document outside the agencies of the US Government musthave prior approval of the Commanding Officer, Edgewood Arsenal, ATTN:SMUEA-TSTI-T, Edgewood Arsenal, Maryland 21010.It- SUPPLEMENTARY NOTES 12n. SIPONSRING MILITARY ACTIVITY

Nondefense medical aspects of chemicalagentsI

Inýcapacitating and riot control agents ______________________3) As$YRCT

(U) IAs solutions containing 1% o-chlorobenzylidene malononitrile (CS) intrioctylphosphate (TOF) have been proposed for use in riot control situations, aseries of toxicological tests was conducted in animals. Results showed that thedoses tested are not permanently injurious to the eyes. Effects on skin ranged fromnone to necrosis, and the affected areas usually healed within 2 weeks. When themixtures were directly injected into the trachea, some evidence of congestion and

* . pneumonia was seen. But the severity and duration of signs were not in a categorythat would warrant veterinary treatment under conditions of normal animal care.The reported studies indicate that no irreversible damage is expected with 1%CS/TOF solutions as long as the dose ranges shown to be safe in this paper are not

* exceeded.;

DDIMLA 4'... 17 JAN: 44am. USRN IsoDD "e"V61473UNCLASSIFIEDseuwfity mssdlffealý

UNCLASSIFIED

KEY onc LINK Ak LINK 6 LINK C

KEY WORDSROLE WY ROLE. WT ROLE NY

CSTOFo-chlorobenzalmalononit rileo- chlorobenzylidene malononit rile

TrioctylphosphateRiot controlCutaneous effectsOcular effectsUpper and lower respiratory tract effectsTracheal intubation

Tracheal injectionToxicityDogsRabbitsMonkeysMan

II

UNCLASSIFIEDUseewtt Clasa"Lesta