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Some pathological effects of hexachlorophene in the rat
J. COMP.
PATH.
1967.
VOL.
SOME OF
137
77.
PATHOLOGICAL
EFFECTS
HEXACHLOROPHENE
INTHE
RAT
BY
E. Department
of
Veterinary
THORPE
Pathology,
University
of Liverpool
INTRODUCTION
Since the trematocidal action of hexachlorophene (2,2, methylene bis [3, 5, 6 on Fasciola hepatica in sheep was reported (Hirschler, 1957, trichlorophenol]) cited by Bosman, Thorold and Purchase, 1961) there have been reports of toxicity in ruminants given the compound. Dorsman (1959), Federman (1959) and Guilhon and Graber (1961) recorded that hexachlorophene at 40 mg./kg. or more, could cause fatalities. Pugh and Crowley (1966) described liver damage in sheep following the administration of hexachlorophene. They found fatty change at the periphery of the hepatic lobule and concluded that the plane of nutrition of the animal was important in its response to hexachlorophene. Published data on the acute toxicity of hexachlorophene in mammals indicates speciesvariation. The LDGOfor mice has been variously reported as 80 mg./kg. (Florestano, 1949) and 187 mg./kg. (Nickerson, 1955). I n rats an LDw of 126 mg./kg. was determined (Thorpe, 1965). The LD5o in ruminants was reported as approximately 50 to 60 mg./kg. (Bosman et at., 1961). H exachlorophene was shown by Gould, Frigerio and Lebowitz (1955) to exert an inhibitory action on cytochrome oxidase, lactate dehydrogenase and the succinoxidases of cells of liver, kidney and heart, but there is little published data on the pathological effects of hexachlorophene in mammals. The present communication records an investigation of the changes in some of the tissuesof albino rats given single and repeated dosesof hexachlorophene.
MATERIALS
AND
METHODS
W&tar strain albino rats weighing 180 to 200 g. and bred in this laboratory were used. Hexachlorophene was administered orally by catheter as a 1 per cent. suspension in an aqueous solution of Lissapol-Dispersal OG (0.15 per cent. Lissapol w/v; 0.15 per cent. Dispersol OG w/v)* or given in the diet. In experiments 1 and 2, blocks of liver, kidney, small and large intestine were fixed in form01 saline and testes in Bouin’s fluid; they were processed for paraffin wax sections. Sections 4 t.~ thick were stained routinely with haematoxylin and eosin and the periodic acid-Schiff method was used on selected sections. Lesions in the testes in the first experiment were assessed quantitatively by a slight modification of the method of Chalkley (1943). One hundred measurements were made on sections of testes from each rat in the top dose group and the controls, recording hits on normal seminiferous tubules, abnormal tubules and interstitial tissue. *I.C.I.
(Dyestuffs
Division)
Ltd.
138
HEXACHLOROPHENE
IN
THE
RAT:
PATHOLOGY
RESULTS
Ef’ect
of Single Oral Doses on Male Rats (Experiment 1)
Three groups each of 30 male rats were dosed orally on one occasion at levels of 125 mg./kg., 75 mg./kg. and 25 mg./kg. respectively. A minimum of 4 animals from each group was killed, 1, 2, 5, 12 and 21 days after dosing. A control group of 20 rats dosed only with the vehicle solution was also killed at the above intervals. During the 6 days after dosing, a total of 6 rats died in the group dosed with 125 mg./kg. The survivors in the group and rats dosed with 75 mg./kg. showed diarrhoea for 3 to 4 days after receiving the compound. Microscopically the liver, kidneys and intestines of rats in the two lower dose groups did not differ significantly from the controls at any interval. In the top dose group, hepatic parenchymal cells showed an increased mitotic rate one and two days after dosing, although there was no evidence of degeneration or necrosis. The kidneys and intestines from this group showed no abormalities. TABLE ASSESSMENT
OF ABNORMAL
Group
SEMINIFEROUS
TUBULES
Day killed after dosing
treated
DOSED
WITH
125
MG./KG.
WEXACHLOROPHENE
O/o normal tubules
0h abnormal tubules
0/O interstitial tissue
70.25
0
29.75
1
71.25
0
28.75
2
41
32.75
26.25
5
42.5
33.0
24.5
12
57.5
19.0
23.5
21
70
6.5
23.5
Control
Hexachlorophene
1
OF RATS
The ductus epididymis and testesfrom all rats killed 1 day after dosing showed no lesions. However, by the second day in rats given 125 mg./kg., there was a marked reduction in the sperm content of the tubules of the epididymis and an increase in the amount of cellular debris in the tubular lumen, some of which was recognisable as degenerate spermatids (Fig. 1). The seminiferous tubules of the testes showed lesions involving only the spermatogenic cells which were apparently randomly distributed. The proportion of tubules with these cellular changes is given in Table 1. Multinucleate giant cells, apparently of spermatid origin, were present in affected tubules, interspersed with the normal spermatogenie cells and mature sperms (Fig. 2). In a few tubules, large cells showing abnormal nuclear structures were found. In some of these cells this nuclear material resembled abnormal disrupted mitoses (Fig. 3), while in others the appearances clearly indicated karyorrhexis of mononuclear and multinuclear cells. In tubules where these latter cells were present there were degenerate spermatocytes and almost no sperms. No abnormality of the Sertoli cells was seen. Focally there was some increase in fluid in the interstitial tissue, but the interstitial
E.
139
THORPE
cells showed no changes. Reduction in the number of sperms in the ductus epididymis was more obvious 5 days after dosing, most tubules containing cells and cellular debris but few sperms. The Chalkley counts on the testes at this interval revealed little increase in the proportion of damaged seminiferous tubules from the previous interval. Affected seminiferous tubules showed a greater disruption of their cellular content. The abnormal cell types seen at the previous interval were still present, but some tubules were almost depleted of spermatocytes and spermatogonia (Fig. 4). Unaffected tubules showed spermatogenesis to the spermatid stage and some mature sperms. No other abnormalities in the testes were seen. Considerable regeneration of the seminiferous epithelium was present 12 days after dosing, but the numbers of sperms in testes and epididymis were still low compared to the controls. Multinucleate giant cells were less frequent and many showed nuclear pyknosis (Fig. 5), while degenerate spermatocytes were infrequent. By 21 days after dosing the epithelium in most of the seminiferous tubules had been almost completely restored to normal, although mature sperms were not as frequent as in the controls. The degenerative changes in the seminiferous epithelium in rats dosed with 75 mg./kg. were qualitatively similar to those described for the previous group but were less extensive. The timing of the recovery of the epithelium was similar. Rats given a single dose of 25 mg./kg. showed no lesions in any of the tissues examined. TABLE EFFECT
OF
REPEATED
GrOUp
OF
Mean weight at outset, g.
2
HEXACHLOROPHENE
Mean
after 5
ON
weight doses, g.
WEIGHT
GAIN
Percentage weight gain
Control
6
204.7
215.3
5.2
Control
?
209.8
219.1
4.4
J
210.5
213.9
1.14
75 mg./kg.
Efect
DOSES
75 mg./kg.
9
214.4
219.9
2.5
25 mg./kg.
$
219.9
230.1
4.6
25 mg./kg.
9
193
202.0
4.7
of 5 Doses (Experiment
2)
Two groups, each comprised of 6 male and 6 female rats were dosed orally on 5 consecutive days at levels of 75 mg./kg. and 25 mg./kg. respectively. A group of controls was dosed concurrently with the vehicle solution. All the animals were killed on the 5th day of the experiment. The animals that received daily doses of 75 mg./kg. developed diarrhoea by the second day and from the third day onward several had weakness of the hindquarters. Body weight measurements (Table 2) showed that the above group gained relatively and absolutely less weight than the controls whereas the lower
140
HEXACHLOROPHENE
IN
THE
RAT
:
PATHOLOGY
dose group (25 mg./kg.) had a percentage weight increase comparable with the controls. Microscopically, in the livers from the top dose group there were occasional large parenchymal cells with hyperchromatic nuclei, quite numerous binucleate cells and a few mitotic figures. The livers from rats dosed at 25 mg./kg. showed frequent mitoses among the parenchymal cells, most of the mitotic figures being at the stage of metaphase and telophase. No other changes were present in the livers of the treated rats, nor in sections of kidneys and intestines. The epididymis was almost devoid of sperms but showed numerous degenerate cells of spermatid origin (Fig. 6). The lesions in the seminiferous tubules resembled in most respects those found previously in rats given single doses of 125 mg./kg. The collections of multinucleate spermatids (Fig. 7) were more prominent, and tubules with marked atrophy of the seminiferous epithelium were quite common. These latter TABLE 3 in the diet throughout
Hexachlorophene
pregnancy.
Total No. Level
No. of females
Reabsorpion
of
.hfO.
pregnant
Litter
site
Abnormal
foetuses
0‘1%
4
3
31
4
10-3
0
0.05%
4
4
40
10
10-O
0
Control
3
3
33
1
11.0
0
Single
large
dose of hexachlorophene
on day 8.
100 mg./kg.
6*
4
49
0
12.25
0
50 mg./kg.
5
3
33
0
1 I.0
0
Control
5
5
50
3
10-O
0
*2 females
died within
1 week
of dosing.
tubules were lined by Sertoli cells and a narrow incomplete rim of spermatocytes (Fig. 8). The testes from rats dosed with 25 mg./kg. showed no significant lesions or reduced numbers of sperms. Eflect on Pregnancy and the Foetus (Experiment 3) This experiment was designed to detect any teratogenic effect of hexachlorophene in rats and was carried out in two parts. In the first section hexachlorophene was given at levels of 0.1 per cent. and O-05 per cent. in the diet throughout pregnancy to mature female rats from the day of successful mating. To determine the effect of a single large dose, two levels, 100 mg./kg. and 50 mg./kg., were given by catheter on the 8th day following mating. Dissections of the foetuses were made on the 20th day after mating to demonstrate the presence and normal structure of the major viscera. The skeletons’ were then prepared by Dawson’s method of alizarin staining (Gatenby and Beams, 1950).
E.
THORPE
141
In neither experiment (Table 3) was there evidence of any specific interference with pregnancy or development of the rat foetus, although in the second experiment the higher dose level was close to the maximum tolerated, as 2 female rats died within a week of dosing. DISCUSSION
Several deaths occurred among the male rats given single oral doses of 125 mg./kg. hexachlorophene, but none of the group killed serially showed evidence of selective hepatotoxicity. No liver damage was reported in 4 cases of accidental ingestion of hexachlorophene in man (Wear, Shanahan and Ratcliffe, 1962). These findings are in contrast with the report of hepatic cellular damage in sheep dosed with hexachlorophene by Pugh and Crowley (1966). The liver lesions they described may indicate a species sensitivity to the compound, or may indicate that existing liver lesions in the sheep due to parasitism or the stress of late pregnancy significantly modified the response of the liver to hexachlorophene. The presence of previous lesions complicates a toxicity study, as the concurrent or sequential development of two differing forms of hepatic injury is not necessarily cumulative. For example, chloroform and urethane were found to be synergistic in causing liver damage (Lushbaugh and Storer, 1948), while carbon terachloride and alpha-naphthyl isothiocyanate had no interaction (Sutton, 1960), and the prior administration of beryllium sulphate inhibited chloroform-induced liver necrosis (Paget, 1961). The lack of morphological evidence of severe hepatic injury in rats given 5 doses of 75 mg./kg. hexachlorophene confirmed the absence of specific hepatotoxicity in this species. The presence of a few large cells, binucleate cells and increased mitotic figures among the hepatic parenchymal cells was considered to be a non-specific response to the administration of a compound at approximately the maximum tolerated dose. Diarrhoea following the ingestion of hexachlorophene had been reported for sheep and man and was found in those rats given large single and repeated doses, but no significant microscopic lesions were detected in the rat intestines. The lack of any degeneration in intestines contrasted with the changes observed in the seminiferous epithelium in these rats. The testicular lesions closely resembled those described by Kaufman, Klavins and Kinney (1956) following ethionine administration in the rat. Ribelin (1963) reported that a wide range of substances of differing biological properties was associated with similar forms of injury to the seminiferous epithelium and that the testes appeared to be capable of only a limited range of response to various types of injury, including prolonged inanition. Inanition was not considered to be a relevant factor in the present study because of the rapidity of onset of the changes. The degenerative changes in the seminiferous epithelium of the rat after a single dose of hexachlorophene appeared to be reversible. In testes of rats given 5 doses of 7.5 mg./kg. hexachlorophene, the seminiferous tubules ranged from those containing few viable spermatogenic cells, to others showing most of the normal stages of spermatogenesis as described by Leblond and Clermont (1952). The dosage schedule used, which was the maximum tolerated, did not appear likely to cause permanent impairment of spermatogenesis. The failure to detect any teratogenic properties of hexachlorophene in the rat foetus and the absence of lesions in the intestinal epithelium of dosed adult rats
142
HEXACHLOROPHENE
IN
THE
RAT
:
PATHOLOGY
indicated that the compound, at the levels used, had no uniform destructive action on actively dividing mammalian cells, As hexachlorophene has been shown to inhibit various oxidative enzymes (Gould et UC.,1955), this might be the mechanism causing the testicular lesions. Ribelin (1963) has indicated that atrophy of the seminiferous epithelium in the rat is a quite general manifestation of chemical toxicity. The rapidity of onset of the changes in the testes in the present study emphasises,therefore, the toxic nature of hexachlorophene. SUMMARY
The oral administration of single and repeated dosesof hexachlorophene (2,2, methylene bis [3, 5, 6 trichlorophenol]) to male rats resulted in atrophy of the seminiferous epithelium when high levels (75 mg./kg.) of the compound were given. The lesions in the testes were first detected 2 days after dosing and, when single doses were used, they were found to be reversible. The findings support the view that the seminiferous epithelium of the rat is a sensitive indicator of chemical toxicity. No evidence of specific hepatotoxicity or teratogenic activity due to hexachlorophene was found in rats dosed at the maximum tolerated level. ACKNOWLEDGMENTS
I wish Harling, Weston, from the
to thank Professor D. L. Hughes for advice and encouragement, Mrs. M. W. A.I.M.L.T., and Miss L. Andrews for technical assistance, and Mr. G. F.I.M.L.T., for the photomicrographs. The work was supported by a grant Agricultural Research Council. REFERENCES
Bosm;;‘2:jJ.,
Thorold,
P. W., and Purchase,
H. S. (1961). J. S. Afr.
vet. med. Ass.,
Chalkle’y, H: W. (1943-44). J, Nat. Cancer Inst., 4, 47. Dorsman, W. (1959). Proc. 16th Int. vet. Congress, Madrid, 2, 609. Federman, M. (1959). Dtsch. tieriirztl. Wschr., 66, 526. Florestano, H. J. (1949). J. Pharm. expt. Ther., 96, 238. Gatenby, J. B., and Beams, H. (1950). The Microtomists vade Mecum, p. 451. Churchill; London. Gould, B., Frigerio, N. A., and Lebowitz, W. B. (1955). Arch. Biochem.
11th
Ed.,
Biophys.,
56, 476. Guilhon, J., and Graber, M. (1961). Bull. Acad. vkt. Fr., 34, 119. Kaufman, N., Klavins, J. V., and Kinney, T. D. (1956). Amer. J. Path., 32, 105. Leblond, C. P., and Cleremont, Y. (1952). Amer. J. Anat., 90, 167. Lushbaugh, C. C., and Storer, J. B. (1948). A.M.A. Arch. Pathol., 45, 494. Nickerson, M. (1955). Sindar Corp. Tech. Bull., 113. Paget, G. E. (1961). Toxicol. upp. Pharmacol., 3, 595. Pugh, D. M., and Crowley, J. (1966). lret Rec., 78, 86. Ribelin, W. E. (1963). A.M.A. Arch. Puthol., 75, 229. Sutton, P. M. (1960). J. Path. Back, 79, 157. Thorpe, E. (1965). J. camp. Path., 75,45. Wear, J. B., Shanahan, R., and Ratcliffe, R. K. (1962). J. Amer. med. Ass., 181, 587. [Received
for pubZication,
Jnne
fOth,
19661
E.
Fig.
1.
Fig.
2.
Fig.
3.
Fig.
1.
THORPE
Epididymis of rat 2 days after a dose of 125 mg./kg. There is a reduced content of sperms, and degenerate spermatids and cellular debris are present. H. & E. x 100. Testis of rat 2 days after a dose of 125 mg./kg. Multinucleate cells of spermatid origin are present. H. & E. x 100. Testis from same rat illustrated in Fig. 2, showing large cells with condensed nuclear material resembling abnormal mitosis. Some increase in interstitial fluid is present. H. & E. x 100. Testis of rat 5 days after a dew of 125 mg.kg. The 3 tubules show changes that rang<- from normal sprrmatogenesis, (lj to spermatld giant cell formation (2) and to almost rompletc atrophy of seminiferous epithelium (3). H. & E. x 100. l-0 ./uce prr,ve 142
HEXACHLOROPHENE
Fig.
5.
Fig.
6.
Fig.
7.
Fig.
8.
Testis of matogenic x 200. Epididymis degenerate Testis of H. & E. Testis of H. & 12.
rat
12 days epithelium
IN
THE
RAT
:
PATHOLOGY
after a single dose of 125 mg./kg., showing partial restoration and giant cells with pyknotic nuclei in lumen of the tub&.
of sperH. & E.
of a rat given 5 doses of 75 mg./kg. The lumen of the tubules contains numerous spermatids and cellular debris but no sperms. H. & E. x 100. rat given 5 doses of 75 mg.;‘kg. showing abnormal multinucleate spermatid cells. x 200. rat in samr group as in Fig. 7, showing massive atrophy of seminiferous epithclium. x 200.
PATH.
1967.
VOL.
SOME OF
137
77.
PATHOLOGICAL
EFFECTS
HEXACHLOROPHENE
INTHE
RAT
BY
E. Department
of
Veterinary
THORPE
Pathology,
University
of Liverpool
INTRODUCTION
Since the trematocidal action of hexachlorophene (2,2, methylene bis [3, 5, 6 on Fasciola hepatica in sheep was reported (Hirschler, 1957, trichlorophenol]) cited by Bosman, Thorold and Purchase, 1961) there have been reports of toxicity in ruminants given the compound. Dorsman (1959), Federman (1959) and Guilhon and Graber (1961) recorded that hexachlorophene at 40 mg./kg. or more, could cause fatalities. Pugh and Crowley (1966) described liver damage in sheep following the administration of hexachlorophene. They found fatty change at the periphery of the hepatic lobule and concluded that the plane of nutrition of the animal was important in its response to hexachlorophene. Published data on the acute toxicity of hexachlorophene in mammals indicates speciesvariation. The LDGOfor mice has been variously reported as 80 mg./kg. (Florestano, 1949) and 187 mg./kg. (Nickerson, 1955). I n rats an LDw of 126 mg./kg. was determined (Thorpe, 1965). The LD5o in ruminants was reported as approximately 50 to 60 mg./kg. (Bosman et at., 1961). H exachlorophene was shown by Gould, Frigerio and Lebowitz (1955) to exert an inhibitory action on cytochrome oxidase, lactate dehydrogenase and the succinoxidases of cells of liver, kidney and heart, but there is little published data on the pathological effects of hexachlorophene in mammals. The present communication records an investigation of the changes in some of the tissuesof albino rats given single and repeated dosesof hexachlorophene.
MATERIALS
AND
METHODS
W&tar strain albino rats weighing 180 to 200 g. and bred in this laboratory were used. Hexachlorophene was administered orally by catheter as a 1 per cent. suspension in an aqueous solution of Lissapol-Dispersal OG (0.15 per cent. Lissapol w/v; 0.15 per cent. Dispersol OG w/v)* or given in the diet. In experiments 1 and 2, blocks of liver, kidney, small and large intestine were fixed in form01 saline and testes in Bouin’s fluid; they were processed for paraffin wax sections. Sections 4 t.~ thick were stained routinely with haematoxylin and eosin and the periodic acid-Schiff method was used on selected sections. Lesions in the testes in the first experiment were assessed quantitatively by a slight modification of the method of Chalkley (1943). One hundred measurements were made on sections of testes from each rat in the top dose group and the controls, recording hits on normal seminiferous tubules, abnormal tubules and interstitial tissue. *I.C.I.
(Dyestuffs
Division)
Ltd.
138
HEXACHLOROPHENE
IN
THE
RAT:
PATHOLOGY
RESULTS
Ef’ect
of Single Oral Doses on Male Rats (Experiment 1)
Three groups each of 30 male rats were dosed orally on one occasion at levels of 125 mg./kg., 75 mg./kg. and 25 mg./kg. respectively. A minimum of 4 animals from each group was killed, 1, 2, 5, 12 and 21 days after dosing. A control group of 20 rats dosed only with the vehicle solution was also killed at the above intervals. During the 6 days after dosing, a total of 6 rats died in the group dosed with 125 mg./kg. The survivors in the group and rats dosed with 75 mg./kg. showed diarrhoea for 3 to 4 days after receiving the compound. Microscopically the liver, kidneys and intestines of rats in the two lower dose groups did not differ significantly from the controls at any interval. In the top dose group, hepatic parenchymal cells showed an increased mitotic rate one and two days after dosing, although there was no evidence of degeneration or necrosis. The kidneys and intestines from this group showed no abormalities. TABLE ASSESSMENT
OF ABNORMAL
Group
SEMINIFEROUS
TUBULES
Day killed after dosing
treated
DOSED
WITH
125
MG./KG.
WEXACHLOROPHENE
O/o normal tubules
0h abnormal tubules
0/O interstitial tissue
70.25
0
29.75
1
71.25
0
28.75
2
41
32.75
26.25
5
42.5
33.0
24.5
12
57.5
19.0
23.5
21
70
6.5
23.5
Control
Hexachlorophene
1
OF RATS
The ductus epididymis and testesfrom all rats killed 1 day after dosing showed no lesions. However, by the second day in rats given 125 mg./kg., there was a marked reduction in the sperm content of the tubules of the epididymis and an increase in the amount of cellular debris in the tubular lumen, some of which was recognisable as degenerate spermatids (Fig. 1). The seminiferous tubules of the testes showed lesions involving only the spermatogenic cells which were apparently randomly distributed. The proportion of tubules with these cellular changes is given in Table 1. Multinucleate giant cells, apparently of spermatid origin, were present in affected tubules, interspersed with the normal spermatogenie cells and mature sperms (Fig. 2). In a few tubules, large cells showing abnormal nuclear structures were found. In some of these cells this nuclear material resembled abnormal disrupted mitoses (Fig. 3), while in others the appearances clearly indicated karyorrhexis of mononuclear and multinuclear cells. In tubules where these latter cells were present there were degenerate spermatocytes and almost no sperms. No abnormality of the Sertoli cells was seen. Focally there was some increase in fluid in the interstitial tissue, but the interstitial
E.
139
THORPE
cells showed no changes. Reduction in the number of sperms in the ductus epididymis was more obvious 5 days after dosing, most tubules containing cells and cellular debris but few sperms. The Chalkley counts on the testes at this interval revealed little increase in the proportion of damaged seminiferous tubules from the previous interval. Affected seminiferous tubules showed a greater disruption of their cellular content. The abnormal cell types seen at the previous interval were still present, but some tubules were almost depleted of spermatocytes and spermatogonia (Fig. 4). Unaffected tubules showed spermatogenesis to the spermatid stage and some mature sperms. No other abnormalities in the testes were seen. Considerable regeneration of the seminiferous epithelium was present 12 days after dosing, but the numbers of sperms in testes and epididymis were still low compared to the controls. Multinucleate giant cells were less frequent and many showed nuclear pyknosis (Fig. 5), while degenerate spermatocytes were infrequent. By 21 days after dosing the epithelium in most of the seminiferous tubules had been almost completely restored to normal, although mature sperms were not as frequent as in the controls. The degenerative changes in the seminiferous epithelium in rats dosed with 75 mg./kg. were qualitatively similar to those described for the previous group but were less extensive. The timing of the recovery of the epithelium was similar. Rats given a single dose of 25 mg./kg. showed no lesions in any of the tissues examined. TABLE EFFECT
OF
REPEATED
GrOUp
OF
Mean weight at outset, g.
2
HEXACHLOROPHENE
Mean
after 5
ON
weight doses, g.
WEIGHT
GAIN
Percentage weight gain
Control
6
204.7
215.3
5.2
Control
?
209.8
219.1
4.4
J
210.5
213.9
1.14
75 mg./kg.
Efect
DOSES
75 mg./kg.
9
214.4
219.9
2.5
25 mg./kg.
$
219.9
230.1
4.6
25 mg./kg.
9
193
202.0
4.7
of 5 Doses (Experiment
2)
Two groups, each comprised of 6 male and 6 female rats were dosed orally on 5 consecutive days at levels of 75 mg./kg. and 25 mg./kg. respectively. A group of controls was dosed concurrently with the vehicle solution. All the animals were killed on the 5th day of the experiment. The animals that received daily doses of 75 mg./kg. developed diarrhoea by the second day and from the third day onward several had weakness of the hindquarters. Body weight measurements (Table 2) showed that the above group gained relatively and absolutely less weight than the controls whereas the lower
140
HEXACHLOROPHENE
IN
THE
RAT
:
PATHOLOGY
dose group (25 mg./kg.) had a percentage weight increase comparable with the controls. Microscopically, in the livers from the top dose group there were occasional large parenchymal cells with hyperchromatic nuclei, quite numerous binucleate cells and a few mitotic figures. The livers from rats dosed at 25 mg./kg. showed frequent mitoses among the parenchymal cells, most of the mitotic figures being at the stage of metaphase and telophase. No other changes were present in the livers of the treated rats, nor in sections of kidneys and intestines. The epididymis was almost devoid of sperms but showed numerous degenerate cells of spermatid origin (Fig. 6). The lesions in the seminiferous tubules resembled in most respects those found previously in rats given single doses of 125 mg./kg. The collections of multinucleate spermatids (Fig. 7) were more prominent, and tubules with marked atrophy of the seminiferous epithelium were quite common. These latter TABLE 3 in the diet throughout
Hexachlorophene
pregnancy.
Total No. Level
No. of females
Reabsorpion
of
.hfO.
pregnant
Litter
site
Abnormal
foetuses
0‘1%
4
3
31
4
10-3
0
0.05%
4
4
40
10
10-O
0
Control
3
3
33
1
11.0
0
Single
large
dose of hexachlorophene
on day 8.
100 mg./kg.
6*
4
49
0
12.25
0
50 mg./kg.
5
3
33
0
1 I.0
0
Control
5
5
50
3
10-O
0
*2 females
died within
1 week
of dosing.
tubules were lined by Sertoli cells and a narrow incomplete rim of spermatocytes (Fig. 8). The testes from rats dosed with 25 mg./kg. showed no significant lesions or reduced numbers of sperms. Eflect on Pregnancy and the Foetus (Experiment 3) This experiment was designed to detect any teratogenic effect of hexachlorophene in rats and was carried out in two parts. In the first section hexachlorophene was given at levels of 0.1 per cent. and O-05 per cent. in the diet throughout pregnancy to mature female rats from the day of successful mating. To determine the effect of a single large dose, two levels, 100 mg./kg. and 50 mg./kg., were given by catheter on the 8th day following mating. Dissections of the foetuses were made on the 20th day after mating to demonstrate the presence and normal structure of the major viscera. The skeletons’ were then prepared by Dawson’s method of alizarin staining (Gatenby and Beams, 1950).
E.
THORPE
141
In neither experiment (Table 3) was there evidence of any specific interference with pregnancy or development of the rat foetus, although in the second experiment the higher dose level was close to the maximum tolerated, as 2 female rats died within a week of dosing. DISCUSSION
Several deaths occurred among the male rats given single oral doses of 125 mg./kg. hexachlorophene, but none of the group killed serially showed evidence of selective hepatotoxicity. No liver damage was reported in 4 cases of accidental ingestion of hexachlorophene in man (Wear, Shanahan and Ratcliffe, 1962). These findings are in contrast with the report of hepatic cellular damage in sheep dosed with hexachlorophene by Pugh and Crowley (1966). The liver lesions they described may indicate a species sensitivity to the compound, or may indicate that existing liver lesions in the sheep due to parasitism or the stress of late pregnancy significantly modified the response of the liver to hexachlorophene. The presence of previous lesions complicates a toxicity study, as the concurrent or sequential development of two differing forms of hepatic injury is not necessarily cumulative. For example, chloroform and urethane were found to be synergistic in causing liver damage (Lushbaugh and Storer, 1948), while carbon terachloride and alpha-naphthyl isothiocyanate had no interaction (Sutton, 1960), and the prior administration of beryllium sulphate inhibited chloroform-induced liver necrosis (Paget, 1961). The lack of morphological evidence of severe hepatic injury in rats given 5 doses of 75 mg./kg. hexachlorophene confirmed the absence of specific hepatotoxicity in this species. The presence of a few large cells, binucleate cells and increased mitotic figures among the hepatic parenchymal cells was considered to be a non-specific response to the administration of a compound at approximately the maximum tolerated dose. Diarrhoea following the ingestion of hexachlorophene had been reported for sheep and man and was found in those rats given large single and repeated doses, but no significant microscopic lesions were detected in the rat intestines. The lack of any degeneration in intestines contrasted with the changes observed in the seminiferous epithelium in these rats. The testicular lesions closely resembled those described by Kaufman, Klavins and Kinney (1956) following ethionine administration in the rat. Ribelin (1963) reported that a wide range of substances of differing biological properties was associated with similar forms of injury to the seminiferous epithelium and that the testes appeared to be capable of only a limited range of response to various types of injury, including prolonged inanition. Inanition was not considered to be a relevant factor in the present study because of the rapidity of onset of the changes. The degenerative changes in the seminiferous epithelium of the rat after a single dose of hexachlorophene appeared to be reversible. In testes of rats given 5 doses of 7.5 mg./kg. hexachlorophene, the seminiferous tubules ranged from those containing few viable spermatogenic cells, to others showing most of the normal stages of spermatogenesis as described by Leblond and Clermont (1952). The dosage schedule used, which was the maximum tolerated, did not appear likely to cause permanent impairment of spermatogenesis. The failure to detect any teratogenic properties of hexachlorophene in the rat foetus and the absence of lesions in the intestinal epithelium of dosed adult rats
142
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indicated that the compound, at the levels used, had no uniform destructive action on actively dividing mammalian cells, As hexachlorophene has been shown to inhibit various oxidative enzymes (Gould et UC.,1955), this might be the mechanism causing the testicular lesions. Ribelin (1963) has indicated that atrophy of the seminiferous epithelium in the rat is a quite general manifestation of chemical toxicity. The rapidity of onset of the changes in the testes in the present study emphasises,therefore, the toxic nature of hexachlorophene. SUMMARY
The oral administration of single and repeated dosesof hexachlorophene (2,2, methylene bis [3, 5, 6 trichlorophenol]) to male rats resulted in atrophy of the seminiferous epithelium when high levels (75 mg./kg.) of the compound were given. The lesions in the testes were first detected 2 days after dosing and, when single doses were used, they were found to be reversible. The findings support the view that the seminiferous epithelium of the rat is a sensitive indicator of chemical toxicity. No evidence of specific hepatotoxicity or teratogenic activity due to hexachlorophene was found in rats dosed at the maximum tolerated level. ACKNOWLEDGMENTS
I wish Harling, Weston, from the
to thank Professor D. L. Hughes for advice and encouragement, Mrs. M. W. A.I.M.L.T., and Miss L. Andrews for technical assistance, and Mr. G. F.I.M.L.T., for the photomicrographs. The work was supported by a grant Agricultural Research Council. REFERENCES
Bosm;;‘2:jJ.,
Thorold,
P. W., and Purchase,
H. S. (1961). J. S. Afr.
vet. med. Ass.,
Chalkle’y, H: W. (1943-44). J, Nat. Cancer Inst., 4, 47. Dorsman, W. (1959). Proc. 16th Int. vet. Congress, Madrid, 2, 609. Federman, M. (1959). Dtsch. tieriirztl. Wschr., 66, 526. Florestano, H. J. (1949). J. Pharm. expt. Ther., 96, 238. Gatenby, J. B., and Beams, H. (1950). The Microtomists vade Mecum, p. 451. Churchill; London. Gould, B., Frigerio, N. A., and Lebowitz, W. B. (1955). Arch. Biochem.
11th
Ed.,
Biophys.,
56, 476. Guilhon, J., and Graber, M. (1961). Bull. Acad. vkt. Fr., 34, 119. Kaufman, N., Klavins, J. V., and Kinney, T. D. (1956). Amer. J. Path., 32, 105. Leblond, C. P., and Cleremont, Y. (1952). Amer. J. Anat., 90, 167. Lushbaugh, C. C., and Storer, J. B. (1948). A.M.A. Arch. Pathol., 45, 494. Nickerson, M. (1955). Sindar Corp. Tech. Bull., 113. Paget, G. E. (1961). Toxicol. upp. Pharmacol., 3, 595. Pugh, D. M., and Crowley, J. (1966). lret Rec., 78, 86. Ribelin, W. E. (1963). A.M.A. Arch. Puthol., 75, 229. Sutton, P. M. (1960). J. Path. Back, 79, 157. Thorpe, E. (1965). J. camp. Path., 75,45. Wear, J. B., Shanahan, R., and Ratcliffe, R. K. (1962). J. Amer. med. Ass., 181, 587. [Received
for pubZication,
Jnne
fOth,
19661
E.
Fig.
1.
Fig.
2.
Fig.
3.
Fig.
1.
THORPE
Epididymis of rat 2 days after a dose of 125 mg./kg. There is a reduced content of sperms, and degenerate spermatids and cellular debris are present. H. & E. x 100. Testis of rat 2 days after a dose of 125 mg./kg. Multinucleate cells of spermatid origin are present. H. & E. x 100. Testis from same rat illustrated in Fig. 2, showing large cells with condensed nuclear material resembling abnormal mitosis. Some increase in interstitial fluid is present. H. & E. x 100. Testis of rat 5 days after a dew of 125 mg.kg. The 3 tubules show changes that rang<- from normal sprrmatogenesis, (lj to spermatld giant cell formation (2) and to almost rompletc atrophy of seminiferous epithelium (3). H. & E. x 100. l-0 ./uce prr,ve 142
HEXACHLOROPHENE
Fig.
5.
Fig.
6.
Fig.
7.
Fig.
8.
Testis of matogenic x 200. Epididymis degenerate Testis of H. & E. Testis of H. & 12.
rat
12 days epithelium
IN
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:
PATHOLOGY
after a single dose of 125 mg./kg., showing partial restoration and giant cells with pyknotic nuclei in lumen of the tub&.
of sperH. & E.
of a rat given 5 doses of 75 mg./kg. The lumen of the tubules contains numerous spermatids and cellular debris but no sperms. H. & E. x 100. rat given 5 doses of 75 mg.;‘kg. showing abnormal multinucleate spermatid cells. x 200. rat in samr group as in Fig. 7, showing massive atrophy of seminiferous epithclium. x 200.