TOXICOLOGY AND APPLIED PHARMACOLOGY 16, 100-107 (1970)

Acute Toxicity of Five Insect Chemosterilants, Hemel,

Hempa, Tepa, Metepa, and Methotrexate, for Cockerelsl

MARTIN SHERMAN AND R. B. HERRICK

College of Tropical Agriculture, University of Hawaii, Honolulu. Hawaii 96822

Received March 12,1969

Acute Toxicity of Five Insect Chemosterilants, Hemel, Hempa, Tepa, Metepa, and Methotrexate, for Cockerels. SHERMAN, MARTIN, and HERRICK, R. B. (1970). Toxicol. Appl. Pharmacol. 16, 100-107. The acute oral LDSO values in 11-day-old cockerels of the insect chemosterilants, hemel (hexamethylmelamine), hempa (hexamethylphosphoric triamide), tepa (tris(l-aziridinyl)phosphine oxide), metepa (tris(2-methyl-l-aziri- dinyl)phosphine oxide), and methotrexate (N-(p-(((2,4-diamino-6-pteri- dinyl)methyl)methylamino)benzoyl)glutamic acid), were calculated to be 341 (314373), 835 (771-932), 151 (130-169), 329 (306353), and ~1000 mg/kg, respectively. The LD30, LD70, and LD90 values were also com- puted. Methotrexate at 1000 mg/kg caused no detrimental effects in the treated birds. Leukopenia was marked in birds treated with 300 mg/kg metepa and 125 mg/kg tepa. Hemel at 300 mg/kg caused a moderate leukopenia, but hempa at 600 mg/kg had little or no effect on the number of leukocytes in the blood. Testes growth and spleen size were significantly restricted by treatment. Microscopic examination indicated that hempa at 500 mg/kg was highly inhibitory to testicular development.

Hemel (hexamethylmelamine), hempa (hexamethylphosphoric triamide), tepa (tris(l-aziridinyl)phosphine oxide), metepa (tris(2-methyl-I-aziridinyl)phosphine ox- ide), and methotrexate (N-(p-(((2,4-diamino-6-pteridinyl)methyl)methylamino)ben- zoyl)glutamic acid) have been reported to be effective chemosterilants of the house fly, Musca domestica L., and other insects (Borkovec, 1966; Chang and Borkovec, 1964; Chang et al., 1964; Painter and Kilgore, 1964). The effect of these compounds on mammals has also been investigated (Gaines and Kimbrough, 1964; Jasper et al., 1965; Kimbrough and Gaines, 1966). To further investigate the biological effects of these compounds, the present study was designed to determine their acute toxicity for cockerels.

METHODS

Single-comb White Leghorn cockerels obtained from a commercial hatchery were used in this study. The birds were housed in electrically heated battery brooders with

1 Presented in part at the annual meeting of the Entomological Society of America, New York, New York, November 27-30, 1967. Published with the approval of the Director of the Hawaii Agricultural Experiment Station as Journal Series No. 1088.

100

TOXICITY OF CHEMOSTERILANTS TO COCKERELS 101

raised wire floors, and received the University of Hawaii standard chick starter ration (Sherman et al., 1964) from the first day. Feed, water, and artificial light were furnished continuously. The experiments were initiated when the birds were 11 days of age.

Standard stock solutions of technical grade methotrexate were prepared in methanol while the others were prepared in chloroform. The required dosage was administered by gelatin capsule after the solvent was allowed to evaporate (Sherman and Rosenberg, 1953). Ten to 30 chicks were used at each of 4-7 doses. Dosage was calculated in terms of milligrams of technical material per kilogram of body weight. All birds were observed for 4 weeks following treatment. Weekly weight-gain ratios were determined by dividing the final body weight at the end of each week by the initial weight that week. These ratios were analyzed for significance by means of the one-tailed t test (Cochran and Cox, 1950). Signs of intoxication were recorded and the LD30, LD50, LD70, LD90, and confidence limits were computed by the IBM 360 utilizing a pro- gram written by Daum, Givens, and Beardon of Biometric Services, ARS, USDA (January 1962)2 and based on probit analysis as described by Finney (1952).

Weekly observations were made of body weight and feed consumption, blood was collected prior to treatment and at varying intervals thereafter to determine the effect of treatment on the total leukocyte count. The method of Natt and Herrick (1952) was used with slight modification. Blood was obtained from the brachial vein and was diluted 1: 200.

At the end of 4 weeks, all surviving birds were sacrificed and the testes and spleen were removed and weighed. Samples of the testes were fixed in 10% formalin and examined histologically on slides stained with hematoxylin and triosin. Statistical analysis was made on the above data when applicable, using analysis of variance and Duncan’s multiple range test (Steel and Torrie, 1960).

RESULTS

Table 1 summarizes the acute toxicity of single oral doses of the 5 insect chemo- sterilants for 11-day-old cockerels over a 4-week period. Methotrexate caused no chick mortality at 1000 mg/kg, the highest dose administered.

No symptoms of intoxication were noted in any of the methotrexate-treated chicks. The general symptoms of poisoning exhibited by the birds treated with the other chemosterilants were ataxia and paralysis followed by death. Symptoms of intoxication were prolonged in the chicks given doses approximating the LD50 as follows: hemel, 5 days; hempa, 4 days; metepa, 7 days; and tepa, 9 days.

The ranges of time required for all mortality to occur at those doses causing death were: hemel, ~18 hours-26 days; hempa, (18 hours-3 days; metepa, 2 hours-7 days; and tepa, 1 hour-8 days.

During the first week after treatment, all the treated chicks except those receiving the lowest doses (100 mg/kg) of hemel and hempa gained significantly less weight than the control birds. However, during the second week, all chicks except those receiv- ing the higher doses of hemel (300-400 mg/kg) attained weight-gain ratios equal to or greater than untreated chicks. These hemel-treated cockerels were gaining normally by the third week.

2 Form C201603-8, Catalogue of programs for IBM 1620 and 1710. June 1968 edition.

102 SHERMAN AND HERRICK

Twenty-eight days after treatment, the survivors were weighed, sacrificed, and their testes and spleen were removed and weighed (Table 2). Despite the fact that the weight- gain ratios of all surviving birds were normal 3 weeks after treatment, the majority of treated chicks were significantly smaller than the control birds. Testes growth was significantly restricted by all treatments except the 2 highest levels of methotrexate. The inhibition of testes weight was not necessarily due to body weight depression since birds receiving 100 mg/kg metepa and 100, 500, 600 mg/kg hempa showed testicular arrest despite normal body weights.

Microscopic examination of testis tissue revealed that in untreated cockerels mean tubule diameter was 0.099 mm; lumina were conspicuous; and spermatogonia and spermatocytes were present. This configuration was similar to that found in 8- to

TABLE 1

ACUTE ORAL TOXICITY OF CHEMOSTERILANTS FOR 11-DAY-OLD WHITE LEGHORN COCKERELS, 4 WEEKS AFTER TREATMENT

Chemosterilant LD30 LD.50

tmdkd (w/kg) LD70

~~g/kd LD90

@x/kg)

Hemel

Hempa

Metepa

Tepa

Methotrexatc

308 (275-333)

771 (691-836)

305 (278-326)

127 (101-144)

>looO

341 (314-373)

835 (771-932)

329 (306-353)

151 (130-169)

11000

378 (349-430)

905 (835-1070)

355 (332-388)

179 (160-209)

>lOOO

439 (396-541)

1016 (916-1335)

396 (367453)

230 (200-307)

>lOOO

4 95 % confidence limits.

12-week-old chicks by Parker et al. (1942). Chicks treated with 100 mg/kg tepa had a mean tubule diameter of 0.081 mm and had testicular tissue similar to that found in the control birds but with fewer spermatocytes present. The mean testis tubule dia- meters in chicks treated with 250 mg/kg metepa and hemel were 0.067 and 0.052 mm, respectively. There were less conspicuous lumina and far fewer spermatocytes than were found in the tissues of the control birds. Hempa at 500 mg/kg was highly inhibi- tory to testicular development. The testis tubules were small, with a mean diameter of 0.046 mm, and were not well defined. There were large intertubular spaces filled with connective tissue. The tubule lumina were inconspicuous; spermatogonia were observed, but no spermatocytes were present.

Spleen size (Table 2) was also significantly affected in all treated birds except those receiving the 2 highest levels of methotrexate, the lowest level of hemel and metepa and 600 mg/kg hempa.

Figure 1 summarizes the effect of treatment with selected doses of chemosterilant on the circulating leukocytes of the blood. There was a rapid decline in total leukocyte level in treated cockerels, especially those treated with 300 mg/kg metepa and 125 mg/kg tepa. Limited differential counts taken 1 and 2 days after treatment indicated

TOXICITY OF CHEMOSTERILANTS TO COCKERELS 103

that the leukopenia was due to a reduction in the number of lymphocytes. This leuko- penia in the metepa-treated birds was apparent 2 days after treatment, and recovery began to take place 5 days later. Recovery was slow, taking at least 28 days. Tepa caused as rapid a decline as metepa, but recovery of leukocyte level began earlier and was more rapid. Hemel at 300 mg/kg caused a moderate leukopenia, particularly 2 to 4 days following treatment. Hempa at 600 mg/kg caused a slight, probably insignifi- cant, decline in white cells 1 day after treatment, followed by normal levels of leuko- cytes. Methotrexate at 1000 mg/kg also caused a slight leukopenia which lasted for at least 3 days.

DAYS

,’ ,j’ - CONTROL

,,A’ o- HEMP* 600mg/kg /’

/’

l - - - HEMEL 300mgh.g

,,’

- TWA 125mg1kg

I - - - METEPA 300rrqlkg

- METHOTREXATE lOOOmpltg

i_ 1 I I I / I I 8 IO 12 14

OF EXPERIMENT

FIG. 1. Effect of selected oral dosages of chemosterilants on circulating leukocytes in blood of cockerels.

DISCUSSION

In comparing the acute oral LD.50 values of the alkylating agents and their analogs reported in this study, there appears to be an excellent correlation in the order of their relative toxicities to cockerels and rats. The reported LD50 values in the rat of tepa, metepa, hemel, and hempa were 37, 136, 350, and >2500 mg/kg (Gaines and Kim- brough, 1964; Jasper et al., 1965; Kimbrough and Gaines, 1966). The chick, however, was more susceptible than the rat to hempa, of equal susceptibility to hemel, and less susceptible than the rat to tepa and metepa. Sherman and Herrick (1966) found ll- day-old cockerels and lCday-old Japanese quail of both sexes to be less susceptible than the rat to another alkylating chemosterilant, apholate.

As Sherman and Herrick (1966) and others (Hayes, 1964; Gaines and Kimbrough, 1964) have stated previously, the effects of single oral doses of the alkylating agents are often delayed and long lasting. Mortality among the chicks treated with the alkyl- ating agents tepa and metepa was delayed in some instances until at least 7 days after treatment. A similar delay in mortality occurred with the chicks treated with the

TABL

E 2

TEST

ES A

ND

SPL

EEN

WEI

GHT

S O

F CO

CKER

ELS

GIV

EN

SING

LE O

RALD

OSE

S O

F CH

EMO

STER

ILAN

TS'

Inse

ct

Dos

e N

umbe

r of

ch

emos

teril

ant

(mg/

kg)

cock

erel

s -

Hem

el

400

3 28

3’

f 6

27’

zt

7 35

0 4

314’

*

31

58”

i 9

300

9 36

3’

z!c

8 61

’ +

6 25

0 18

40

3f

It 21

65

’ f

6 10

0 10

40

3f

+ 9

96’

f 10

Hem

pa

1000

1

317

20

800

7 31

8’zk

5

38’i

6 70

0 8

318’

f

11

59’

f 5

600

10

446

f12

73’

f 5

500

10

409

i 8

67’

f 4

250

10

396f

f

16

95’

f 16

10

0 10

42

4 !I

10

loo’

& 5

Met

hotre

xate

10

00

500

250

100

390’

i

10

120

i 6

0.03

1 43

1 xt

17

120

f 10

0.

027

390’

f

9 83

’ -I

8 0.

021

396s

z!z

12

75’f

3 0.

018

Met

epa

450

350

300

250

100

5 10

10

10 1 2 9 17

10

295

60

0.02

0 30

7-f

f 47

45

’ f

14

0.01

4 35

8”

f 11

67

’ f

6 0.

019

383’

x!

z 10

66’

It 5

0.01

8 0.

067

424

zt 1

5 88

’4

6 0.

021

Mea

n bo

dy

Mea

n te

stes

we

ight

b we

ight

b k)

(m

g)

Mea

n te

stes

we

ight

’ (%

>

Mea

n te

stes

tu

bule

di

amet

erd

(mm

)

0.01

0 0.

019

0.01

7 0.

016

0.02

3 0.

052

0.00

6 0.

012

0.01

9 0.

016

0.01

6 0.

046

0.02

3 0.

023

Mea

n sp

leen

M

ean

sple

en

weig

htb

weig

ht’

(mid

(%

I

327’

+

69

480’

z~

63

609”

A

66

672/

-+

74

752

11~7

75

440

374’

f

26

448’

zt

25

921

* 88

72

7f

i 30

63

2’

zt 4

6 66

4’

+ 14

752

i41

776

f 37

68

2’

zt 5

4 68

2’

i 62

250

405’

zt

1

486’

f

32

680f

zt

65

820

1113

0.12

0.

16

ii4

0.17

g

0.23

0.

19

2

0.14

fi

0.12

X

0.14

0.

21

E

0.18

E

0.16

0.

16

0.19

0.

18

0.17

0.

17

0.08

0.

13

0.14

0.

18

0.19

Tepa

Con

trol

225

2 28

1”

47 5

8 40

’ +

10

0.01

4 25

5”

i 10

0.

09

200

3 39

0”

f 30

77

’ i

15

0.02

0 52

7’

f 75

0.

13

175

2 39

3e

It 13

65

” f

14

0.01

6 54

0’

f 11

6 0.

14

150

5 38

2e

+ 25

80

’ i

10

0.02

1 53

8’

i 91

0.

14

125

7 34

9e

f 19

69

’ 3.

1 10

0.02

0 43

6”

z!z 38

0.

12

100

8 36

6’

f 16

66

’~~

8 0.

018

0.08

1 60

5’

f 67

0.

16

0 20

45

1 +

8 13

1 f

5 0.

029

0.09

9 93

5 I6

8 0.

21

n D

ata

take

n 28

day

s af

ter

treat

men

t. *

Mea

n &

SE.

c As

per

cent

of

bod

y we

ight

. d

Mea

n va

lues

bas

ed o

n 50

mea

sure

men

ts

per

bird

an

d 3

bird

s pe

r do

sage

. e

Sign

ifica

ntly

le

ss th

an

cont

rol

at P

x 0

.01.

’

Sign

ifica

ntly

le

ss th

an

cont

rol

at P

-C

0.05

.

106 SHERMAN AND HERRICK

analogs of the alkylating agents, particularly hempa. The depressant effect of treatment on the circulating leukocytes was particularly great and long lasting with tepa and metepa, and less with hemel. Hempa treatment had only a short depressing effect on the leukocyte count.

There is a paucity of information on the dose-mortality response of animals to the antimetabolite, methotrexate. Krakoff and Karnofsky (1965) found a reduction in leukocytes of humans treated with this carcinostat. In our study, no toxic effects were detected in chicks given as much as 1000 mg/kg of this material except a possible leukopenic response and weight depression. The apparent inverse effect of dosage on testes and spleen weights is difficult to explain.

The feeding and weight-gain patterns of the birds treated with the alkylating agents and their analogs were similar to those in birds treated with apholate (Sherman and Herrick, 1966). The chicks were generally off-feed only during the first week after treatment and subsequently recovered appetite so that growth paralleled that of the control birds. However, at the end of the experiment, body weights were still signifi- cantly lower than those of the untreated control birds.

ACKNOWLEDGMENTS

The authors wish to thank Miss Joan Takahama for her valuable technical assistance. This investigation was supported in part by Public Health Service Research Grant UI-00111 from the National Center for Urban and Industrial Health.

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TOXICITY OF CHEMOSTERILANTS TO COCKERELS 107

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