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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.
REFERENCES
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toxicity for mammals. Science 144, 57-58. COCHRAN, W. G., and Cox, G. M. (1950). Experimental Designs, p. 18. Wiley, New York. FINNEY, D. J. (1952). Probit Analysis. A Statistical Treatment of the Sigmoid Response Curve,
2nd ed., pp. 236-245. Cambridge Univ. Press, London and New York. GAIND, T. B., and KIMBROUGH, R. D. (1964). Toxicity of metepa to rats. With notes on two
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NAP, M. P., and HERRICK, C. A. (1952). A new blood diluent for counting the erythrocytes and leucocytes of the chicken. Poultry Sci. 31, 735-738.
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PARKER, J. E., MCKENZIE, F. F., and KEMPSTER, H. L, (1942). Development of the testes and combs of White Leghorn and New Hampshire cockerels. Poultry Sci. 21,35-44.
SHERMAN, M., and HERRICK, R. B. (1966). Acute and subacute toxicity of apholate to the chick and Japanese quail. Toxicol. Appl. Pharmacol. 9, 279-292.
TOXICITY OF CHEMOSTERILANTS TO COCKERELS 107
SHERMAN, M., and ROSENBERG, M. M. (1953). Acute toxicity of four chlorinated dimethano- naphthalene insecticides to chicks. J. Econ. Entomol. 46, 1067-1070.
SHERMAN, M., Ross, E., and CHANG, M. T. Y. (1964). Acute and subacute toxicity of several organophosphorus insecticides to chicks. Toxicol. Appl. Pharmacol. 6, 147-153.
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