The acute toxicity of pesticides to rats

TOXICOLOGY

AND

APPLIED

The Acute

2. 88-99

PHARMACOLOGY

Toxicity TI~OMAS Received

(

1960)

of Pesticides B. October

to Rats’

GAINES 5, 1959

The basic test for determining the relative acute toxicity of chemicals to animals is the LDjo determination. The oral LDso values for pesticides are generally available. However, it is recognized that the value for a given compound may vary not only for different species, but also for different strains and even for different laboratories. There is a need for strictly comparable values determined under as nearly similar conditions as possible. Furthermore, information on the acute dermal toxicity of pesticides frequently is lacking. This paper reports the results of LD;,(, studies with 42 pesticides and 2 metabolites of DDT (listed in Table 1) administered in single doses by the oral or dermal route to adult Sherman strain rats. METHODS

The rats used in these studies were reared in a separate building of the laboratory where the tests were made. They were at least 90 days old and had minimum body weights of 175 and 200 g for the males and females, respectively. The animals were individually caged during the study and fed Purina Laboratory Chow.” None of them was fasted prior to dosage. The survivors were held for daily observation until they appeared to have recovered completely or for a minimum of 14 days. Unless otherwise indicated, the compounds tested were of technical grade and were formulated just prior to administration. In order to give different dosage levels of the pesticides to rats, the concentrations of the formulations were usually varied and the volume given held constant. Because of the very low toxicity or poor solubility of a few of the com1 From the Technical Development Laboratories, Communicable Disease Center, Public Health Service, Bureau of State Services, United States Department of Health, Education, and Welfare, Savannah, Georgia. a Ralston Purina Co., St. Louis, Missouri. Use of trade names is for identification purposes only and does not constitute endorsement by the Public Health Service. 88

ACUTE

TOXICITY

OF

PESTICIDES

T O RATS

89

pounds, it was necessary to increase the volume as indicated in order to give some of the dosage levels. The compounds were given orally by means of a stomach tube. Except where otherwise indicated, peanut oil was used as the solvent or suspending agent. Dosing was done with a syringe with O.l-cc graduations and a blunt-pointed 17-gauge spinal needle which served as the stomach tube. The needle was 61 mm long, and the blunt end was built up with silver solder into a bead 3 mm in diameter to prevent injury to the esophagus. The tube did not actually reach the stomach of the rats, but extended far enough into the esophagus to prevent regurgitation. The poison formulations usually were given at the rate of 0.005 ml per gram of body weight. In the dermal LDno studies the compounds usually were dissolved in xylene and applied at the rate of 0.0016 ml per kilogram of body weight. The fur of the rats was clipped over the top of the shoulder and forward part of the back with an Ang-Ra No. 2 animal clipper to provide a clean area of about 3.0 X 4.5 cm for application of the toxic material. The clipped area of unbroken skin wa,s bathed with a 1: 1 solution of acetone and 95% ethyl alcohol to remove dirt and excess oils. None of the rats, except those dosed with Paris green, was restrained following treatment. No attempt was made to remove any of the poison residue following dosage. The toxic formulations were applied slowly to prevent run-off, using a l-ml pipette with O.Ol-ml graduations. The poisoned rats were observed at least once each hour during the first day after dosage, and twice a day thereafter, for symptoms of poisoning and time of death. The LD,,, values were determined by the method of Litchfield and Wilcoson ( 1949). RESULTS

The LD,,, values, with 19/20 confidence limits, and the survival time of rats which died from poisoning are presented in Tables 2-6. Although only one oral LD:,,, value is shown for DDT in male rats, this particular compound has been tested against this sex annuall,y for the past four years to check the possibility that the rat colony may change in susceptibility to this pesticide. Each successive LD;,a value for this compound fell within the 19/20 confidence limits of the original value. Symptoms noted in rats poisoned with chlorinated hydrocarbon pesticides included tremor, hyperexcitability, irritability, and convulsions. Many of the rats poisoned with dieldrin, aldrin, endrin, and isodrin were

90

THOMAS

B. GAINES

TABLE LIST Comman

Chlorinated

OF COMMON

AND CHEMICAL

1 OF COMPOUNDS

NAMES

TESTED

name

hydrocarbon

compounds

Aldrin

1,2,5,4,10,10-Hexachloro-1,4,4a,5,8,8a-hexahydro-1,4endo,exo-5,8-dimethanonaphthalene

Chlordane

1,2,4,5,6,7,8,8-0ctachloro-3a,4,7,7a-tetrahydro-4,7methanoindan

Chlorobenzilate

Ethyl

DDA

bis( Chlorophenyl)

4,4’-dichlorobenzilate

DDE

l,l-Dichloro-Z,Z-bis(p-chlorophenyl)

DDT

l,l,l-Trichloro-2,2-bis(p-chlorophenyl)ethane

Dieldrin

1,2,3,4,10,10-Hexachloro-6,7-epoxy-1,4,4a octahydro-1,4-endo,exo-5,8-dimethanonaphthalene

, 5 f6 , 7 I8 1Xa-

Dilan

l,l-bis(p-Chlorophenyl) mixture

and

Endrin

1,2,3,4,10,10-Hexachloro-6,7-epoxy-1,4-4a,S,6,7,8,8aoctahydro-1,4-endo,endo-5,8-dimethanonaphthalene

Heptachlor

3a ,4 ,5 I 6 , 7>8 , %Heptachloro-3a,4,7,7a-tetrahydro-4,7methanoindene

Isodrin

1,2,3,4,10,10-Hexachloro-1,4,4a,5,8-8a-hexahydrc-l,4~5,8endo,endo-dimethanonaphthalene

Kelthane

l,l-bis(p-Chlorophenyl)

Lindane

1,2,3,4,5,6-Hexachlorocyclohexane, isomer

acetic

acid ethylene

-2-nitropropane

-2,2,2-trichlorcethancl 99%

Perthane

I,l-Dichloro-2,2-bis(p-ethylphenyl)ethane

Toxaphene

Chlorinated

Organic

phosphorus

butane

camphene

containing

67-69s

or more

gamma

chlorine

compounds

Bayer

21/199

0-(3-Chloro-4-methylumbelliferone)O,O-dethyl thioate

Bayer

29493

O,O-Dimethyl-O+(methyl thiophosphate

mercapto)

Chlorthion

0-(3-Chloro-4-nitrophenyl)

DDVP

O,O-dimethyl

2,2-Dichlorovinyl

Delnav

2,3-p-Dioxanedithiol thioate

Demeton

O,O-Diethyl with

Diazinon

O,O-Diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate

dimethyl

phosphoro-3-methyl

phenyl

phosphorothioate

phosphate

SJ-bis(O,O-diethyl)

phosphorodi-

S-(2.ethylthio)ethyl phosphorothioate mixture O,O-diethyl O-(2-ethylthio)ethyl phosphorothioate

ACUTE

TOXICITY

OF

TABLE Common

PESTICIDES

1 (Continued)

name

Chemical

name

Dicapthon

0-(Z-Chloro-4-nitrophenyl)

Dipterex

Dimethyl

EPK

O-Ethyl

Guthion

S-(3,4-Dihydro-4-oxo-l,2,3-benzotriazin-3-yl-methyl) O,O-dimethyl phosphorodithioate

Malathion

O,O-Dimethyl cinate O,O-Dimethyl

Methyl

parathion

XPD

O,O-dimethyl

phosphonate

phenylphosphorothioate

dithiophosphate

of diethyl

0-p-nitrophenyl

mercaptosuc-

phosphorothioate

dithionopyrophosphate

Parathion

O,O-Diethyl

Phosdrin

2-Methoxycarbonyl-1-methylvinyldimethyl

Ronnel

O,O-Dimethyl

0-p-nitrophenyl

phosphorothioate

O-2,4,5-trichlorophenyl

Schradan

Octamethylpyrophosphoramide

Thimet

O,O-Diethyl

Trithion

S-(p-Chlorophenylthiomethyl) thioate

phosphate phosphorothioate

S-(ethylthiomethyl)phosphorodithioate O,O-diethyl

phosphorodi-

compounds

Isolan

1-Isopropyl-3-methyl-S-pyrazolyl

Sevin

1-Naphthyl-N-methylcarbamate

Arsenic

phosphorothioate

(2,2,2-trichloro-l-hydroxyethyl) O-p-nitrophenyl

Tetra-n-propyl

Carbamate

91

T O RATS

dimethylcarbamate

Compounds

Calcium

arsenate

Lead

arsenate

Paris

green

Nicotine Anticoagulant

Calcium Lead Copper

sulfate

arsenate

arsenate acetoarsenite

Nicotine

sulfate

rodenticides

Diphacinone

2.Diphenyl-acetyl-1,3-indandione

Pival

2 -Pivalyl-I,3

Warfarin

3-(a-Acetonylbenzyl)-4-hydroxycoumarin

-indandione

observed to have several convulsions even though they eventually recovered. Weight loss as a result of anorexia was quite marked among the rats poisoned with the dieldrin group of compounds. Self-mutilation also was observed in many of these rats and in some of those poisoned with the closely related chlordane. In general, female rats were more susceptible than the males to poisoning by organic phosphorus compounds. However, schradan and ronnel, by the oral route, and schradan, by the dermal route, appeared to be appre-

60 90 60 60 70 89 30 67

Endrin Dieldrin (WWP)’ Heptachlor Isodrin Kelthane” Lindane Perthaneb Toxaphene

46

6

69 6 3 28 1 3

97 7 5 12 8 3 5

113

1

4 4

34cal 90

88

15.5 1100

100

48 17.8

880

740

1040

11

5

11

39 335

L=‘,o (w/kg)

18

Max. (daYSI

2

60

10

70

70 69

909-133 76-101 67-122

50 90

50 70 60 69 50 58 70

Number

of

-

4

-2 5 2 23 2

6 8

11

3

10

6 48

Min. (hr)

PESTICIDES

12.7-19.1

74-135

14.7-21.5 36-63

607-903 72 7-1065 87-147 41-51

972-1113

34-44 299-375

1

HYDROCARBON

19120 Confidenr<. limit3 (mdkg)

TABLE OF CZ~LORINATED

6 9 4 8

‘Ime

Snrrivsl

Males

TOXICITY

7 48

Min. thr)

ORAL

B Metabolites of DDT. DD.4 was administered as sodium salt in water. h Given at dosage rates as high as 0.010 ml per gram body weight. ” Water wettable powder givrn in water suspension.

70 70 86 70 60 68 70

Aldrin Chlordane Chlorobenzilate DDAa DDEajb DDT Dieldrin

of

ACUTE

Number

THE

Survival time

1

>4000

7.0

80

91

1000

8 18 7 -

162

- 73

46

118

1240

60 430 1220 600

11

-4

1 10

11

14 9 3 3

MKX. (days) L%O (mg/kg)

RATS

F.%male3

TO ADULT

70-91

s3-loo -

820-1220

6.0-8.1

140-188

b&S..1 -

41-51

106-13

992-1550

5672 391-473 107&1391 472-762

19/20 Confidenre limits (mdkgl

1

70 100

Kelthane” Lindane

a b (’ d

13 7

60 3 4 Undiluted technical grade. Given at dosage rates as high as 0.016 ml/g. Given at dosage rates as high as 0.0096 ml/g. Given at dosaFe rates as high as 0.0033 ml/g.

Toxaphene

10 S

119 17

-

-

43 3

100 80

9 8

72

48

-

-

80

-

-

9 9

TOXICITY

40

DDT” Dieldrin

33 72

DERMAL

Dilan’ Endrin Heptachlor Isodrin

70 -

60

ACUTE

Aldrin Chlordan Chlordanea

C”mpound

THE

90

1075

1230 1000

195 3s

-

6900

-

--

98 840

3

717-1613

1060-1427 833-1200

119-320 30-41

-

5897-8073

60

so 70

60 so

50

50

30 90

69-117

60

-

PESTICIDES

60 70

HYDROCARBON

82-118 750-941

TABLE OF CIIL~RINATED

72

4

18

140

96 24 48 2 96 2

96 120

9

8

5

10

14

13 6 13 8

184

2s IS

T O ADULT

RATS

98

780

1000 900

2510 60 SWQ 1s 250 23

690 530

82-118

7

600-1014

776-1040

800-1250

52-70 5221-6666 12-19 200-313 20-2

1931-3263

580-82 431-652

1

CA

0” X 5

ii!

CT E

Y

M” ~

$

I<

E ;; z

a Died within an hour b Given in 10% water

Bayer 29493 Chlorthion DDVP Delnav Demeton Diazinon Dicapthon Dipterexh EPN Guthion Malathion Methyl parathion Parathion Phosdrin Ronnel Schradan Thimet Trithion

2

1

70

120

after dosed. solution at dosage

6

1

4

36 13

400 630

43 6.2 108

80

215 880

8.1-10.2 1.2-2.8 27-34

lb17 5.2-7.1 906-1725

as 0.01 ml/g.

2.3 30

9.1

13 6.1 1250

12-17

33-40 12-14 1206-1568

568-699

96-122 357-456

37-50 5.4-7.1

178-260 X16-915 62-104

limits (w/kg)

OF ORGANOPHOSPHORUS

1375 14

rates as high

6

4

5

0.5

12 s

1 1

50 70 50

10 1

2 1

5 2

1 1 5 2

5

4

5 0.5

2 7 II

Max. (days)

TOXICITY

11 1 0

Min. 0x1

ORAL

3 a

uf

ACUTE

1 n

50

70 60 68

70 80

49

80 70 150

79 59

58

Number

THE

TABLE

50 80 80

70 50 50

67 60

80 80

60 70

60 60

80 50

60 90

rats tested

PESTICIDES

2 2 2

a 8

1 1 2

18 2 2

5

3 a

3

2

1

4s

4 9 5

0.25

5 11

3 4 n

(days)

RATS

21 0.5

1

" 3 1

8 5

Min. (br)

T O ADULT

10.0

42 1.1

2630

3.6 3.7

1000 24

7.7 11

330 560

76

56 2.3 2.5

245 980

L% (m&z)

32-55 1.0-I .J 6.0-lS.O

3.2-4.0 3.0-4.5 2287-3025

22-28

IO-13 883-1130

284~-383 455-689 6.9-8.6

66-57

48-65 19-27 ?.2-2.S

213-282 899-1068

limits (dkg)

z

z

i

i

P

oi

ACUTE

ml/g. ml/g.

8

9 5 22 3 c 9 7 3

0.5

11

48

72 22 48 24 c 1 2 1

0

14 7 4 10 -

G

13

22 20

MaYi. (days)

Males

TABLE

5

L&o (w/kg)

202-262 183-264 63-72 1615-2730 14-34 4.3-5.1 13-17 5.3-7.3 44-66

d A 63% solution in 95% ethyl e 57% emulsifiable concentrate. f Given as an 8570 emulsifiable

220 > 4444 67 2100 21 4.7 15 6.2 54

230

concentrate

alcohol.

50 60 60 70 90 20 80 50 10 50 50 80 60 70 70 60

70

84-13 7 173-320 11-18 740-l 107 608-102 7

-

60

Number of rats tested

PESTICIDES

275-396 768-963

19120 Contidenw limits (w/kg)

OF ORGANOPHOSPHORUS

330 860 1500 4500 107 235 14 900 790 > 2000

TOXICITY

4 24 28 96

96

43 2

(hr)

Min.

SUNiV.31 time

DERMAL

a Given at dosage rates as high as 0.0096 JJ Given at dosage rates as high as 0.0073 0 Died within an hour after dosing.

110 50 40 60 SO 10 loo 50 10 69 40 50 49 150 100 70

DDVP Delnav Demeton Diazinon Dicapthon Dipterexd EPN Guthion Malathionc Methyl parathion NPDf Parathion Phosdrin Schradan Thimet Trithion

60 80

loo

29493 21/1998

Chlorthion”

Bayer Bayer

tested

Number rats

THE

6

c 8 3 20 17 7 7 9 5 22 4 ‘ 7 4 3

18

-

Max. (days)

4100

330 -

LDx, (wdkg)

levels

22-29 176-275 63-72 1224-2646 4.9-9.5 3.5-5.0 37-52 2.3-2.6 22-32

-

59-96 53-76 7.8-8.7 379-546 217-6625

36284633

-

275-396

19120 Contidvnr< limits (w/kg)

as high as 0.064 ml/g.

75 63 8.2 455 1250 > 2000 25 220 > 4444 67 1800 6.8 4.2 44 2.5 27

Fenlal~~

RATS

at dosage

0 6 24 13 4.5 1 5 48 42 17 1 C 2 1 2

72

43 -

Min. (hr)

Survival time

T O ADULT

route

10 -

120 70 ---. 59 70 60

-

-

-

9 7 13

2

1

48 9 60

-

,L

n

” Died within one hour. b Given at dosage rates as high as 0.014 ml/g. L’ Suspension in 955% ethyl alcohol. Cl Given at dosage rates as high as 0.02 ml/g. (’ Suspension in water-lead arsenate and calcium

Isolan SevinfL Calcium arsenate” Lead arsenate” Paris green” Nicotine sulfate”

route

Isolan Sevinb Calcium arsenateC Lead arsenateC Paris green’ Nicotine sulfate” Diphacinone Pival Warfarin

Dermal

Oral

COlllpOt,“d

Survival

arsenate

--

given

48 7

-

-

10 20 20 10 70

-

80 -

1

i

7 -

4

ml,‘g.

-

-

-

-

2 9 15 2

1 28 26 6

60 69 70

a

O1

RATS

80

ADULT

70

TO

rates as high as 0.0096

PESTICIDES

at dosage

-

-

-

1.4-2.5 233-336 2.3-3.8

1.9 280 3.0

-

-

-

21-25 733-986

-

23 850

TABLE 6 OF MISCELLANEOUS

>4000

THF, ACUTE TOXICITY

> 4000 > 2400 >2400 > 2400 285

-

83

100

298 1050

SO0

13

LD,,, (w/kg)

22%.i.ii,

5.6-6.5

-

X30-1334 x0-125 75-91

11-16 307-815 261-340

2 i % m 0 2z z

ACUTE

TOXICITY

OF

PESTICIDES

T O RATS

97

ciably more toxic to male rats than to females. There was some indication that methyl parathion, by the oral route, was more toxic to males than to female rats. The organophosphorus compounds Thimet, demeton, Phosdrin, and parathion were more tosic than any of the chlorinated hydrocarbons to rats. On the other hand, Chlorthion. ronnel, and malathion were among the least toxic of the pesticides tested. Rats poisoned with the organophosphorus insecticides and those poisoned with the carbamates Sevin and Isolan exhibited symptoms typical of cholinergic poisoning. These symptoms varied in intensity with the dosage given and included muscle fasciculation, excessive salivation and lacrimation, tremor, diarrhea, and involuntary urination. The Diazinon sample used for determining the dermal LD,n values shown for this compound in Table 5 was obtained in the summer of 1959. Similar studies conducted with a sample of this compound when it was received fresh in 1953 indicated that the dermal LD;,#, was 180 mg/kg with 19/20 confidence limits of 150-203 ma/kg for female rats and approximately 200 mg/kg for males. Soon after the 1953 sample of Diazinon was received, the amber-colored liquid began to crystallize. At the same time considerable variation was observed in the mortality among different groups of poisoned rats, with a tendency for the compound to become more toxic with increased crystallization. A small amount of this sample was transferred to an unsealed bottle and became filled with crystals after standing for several weeks. The dermal LDr,() of this crystallized Diazinon to male rats was about 34 mg/k,v. A small portion of the original sample was placed in an open petri dish and allowed to stand exposed to the air for a period of 19 days. The Diazinon crystallized into a solid cake with about a lo:/,. increase in sample weight. Thus, the crystallization was not due to loss of solvent with resultant settling out of crystals from a. supersaturated solution. No chemical tests were undertaken. When similar studies concerning the effect of air exposure were made on the Diazinon received in 1959, there was no crystallization of the exposed material and no increase in its dermal toxicity to rats. This is an example of the fact that chemicals, especially those sold as mixtures of a principal compound and related compounds, may vary either as the result of intentional or unintentional changes in manufacture. Tt was recognized that, when rats were dosed with very large quantities of a I)oison by the dermal route, some of the material might be ingeste(\. However: there was no indication that ingestion of the material was an

98

THOMAS

B. GAINliS

important factor, except in rats dosed with Paris green. In order to prevent ingestion of this poison the rats were placed in restraining cages for 24 hours following dosage,and at the end of that period they were bathed to remove any residue. The “cage” for each rat was simply a roll of hardware cloth about 1.5 inches in diameter into which the animal was introduced and held by wire laced through the ends. DISCUSSION

A comparison of laboratory results and use experience indicates that there is a much closer relationship between dermal LD,,, values and the occurrence of occupational poisoning than between oral LDa,, values and occupational poisoning. This is emphasizedby a study of groups of compounds in which the oral toxicities are of similar order of magnitude but the dermal toxicities are significantly different. Two such comparisons are made in Table 7. RELATIONSHIP

TABLE 7 TOSICITY AKD SAFETY

BETWEEN

DDT Lindane Dieldrin

-.-..Methyl parathion Guthion Parathion Thimet Phosdrin

18

65

13 13

220 21

2

6

6

5

OF PESTICIDES

Ingestion Ingestion Ingestion and occupational Occupational Occupational Ingestion and occupational Occupational Occupational

Severe Mild Mild Severe Severe Severe

Over 100 casesof occupational poisoning by dieldrin have been reported (Hayes, 1959), and others are known to have occurred. By contrast, DDT and lindane, which are much less toxic by the dermal route (but have an oral toxicity of the sameorder of magnitude as dieldrin), are not known to have produced systemic occupational poisoning, although crude benzene hexachloride may be a source of dermatitis or mild systemic effects, and both compounds have produced poisoning when they were

swallowed. In a similar

way.

severe

occupational

poisoning

has occurred

with

ACUTE

TOXICITY

OF PESTICIDES

99

TO RATS

parathion, but not with methyl parathion or Guthion. A few cases of mild poisoning have followed occupational exposure to methyl parathion and Guthion, but their limited use is not the only reason for their present record. Thimet and Phosdrin have had even more limited use, but both already have been the cause of severe occupational poisoning. The results indicate that Phosdrin is slightly more poisonous by the dermal route than by the oral route to male rats. The difference is small and may not be significant. However, Isolan was considerably more toxic by the dermal route to both males and females. The basis of this unexpected result has not been explored. SUMMARY LD,, values for 42 pesticides and 2 metabolites of DDT administered in a single dose by the oral or dermal route to Sherman strain adult rats have been determined. 2. A comparison of laboratory results and use experience indicates that there is a much closer relationship between dermal LD,,, values and the occurrence of occupational poisoning than between oral LD,, values and occupational poisoning. 3. The carbamate pesticide Isolan was observed to be different from other compounds tested in that it was considerably more toxic by the dermal route than by the oral route to rats. 1.

ACKNOWLEDGMENT The author wishes to express care and technical assistance.

his

appreciation

to Richard

L.

Mocre

fcr

animal

REFERESCES W. J., JR. (1959). The tcxicity of dieldrin to man: report Bull. World Health Organization 20, 891-912. LITCHFIELD, J. J., and WILCOXOX, F. (1949). -4 simplified method dose-effect experiments. J. Pharnzacol. &ptZ. Therap. 96, 99-133. HAYES,

cn

a survey,

of evaluating