Subchronic studies of doxylamine in Fischer 344 rats

FUNDAMENTALANDAPPLIEDTOXlCOLCKiYl0,243-253(1988)

Subchronic

Studies of Doxylamine

CARLTON

D. JACKSON*

in Fischer 344 Rats’

AND BOON-NAM

BLACKWELL?

*Division of Comparative Toxicology and 7Pathology Associates Incorporated, National Center for Toxicological Research, Jeferson. Arkansas 72079 Received March 25, 1987; accepted October 30, 1987 Subchronic Studies of Doxylamine in Fischer 344 Rats. JACKSON, C. D., AND BLACKWELL, (1988). Fundam. Appl. Toxicol. 10,243-253. Doxylamine succinate was administered as an admixture in the feed to male and female Fischer 344 rats for either 14 or 90 days. The 14 day study included dose levels of 0,100,250,500,1000, or 2000 ppm doxylamine. Except for a 7% decrease in final body weight in female rats in the 2000 ppm group, there were no significant clinical observations made in the 1Cday study. Microscopic lesions judged to be treatmentrelated were limited to cytoplasmic vacuolization in the livers. The lesions were more numerous in the higher dose groups of males and present only in the 2000 ppm group of females. Dose levels of 0, 162, 405, 1012, 2530, and 6325 ppm doxylamine were administered in the 90-day study. There were no deaths during the study. Final body weights were decreased 13.3% in males of the 6325 ppm group and 5.2, 10.1, and 14.4% in females in the 1012,2530, and 6325 ppm groups, respectively. Liver/brain weight ratios were increased in all treated male groups and in the two highest dose groups of females. Other organ weight changes were decreases and believed to result from general reduction in weight gain in those groups where the decreases occurred. Treatment-related histological changes were identified in the liver and parotid salivary gland. Cytoplasmic vacuolization or fatty change of the liver was found in all groups of males but was more severe in the higher dose groups. In females, these liver lesions were observed only in the two highest dose groups. A dose-related change in the parotid salivary gland, consisting of cytomegaly with basophilic and coarsely granular or vacuolated cytoplasm, was observed. B.-N.

0 1988 Society ofToxicology.

Doxylamine (see Fig. 1) is used as an antihistamine in the treatment of allergies, rhinitis, and skin conditions. It also has sedative and mild local anesthetic properties. However, the major use of doxylamine has been as a sleep-inducing agent (AMA, 1971). The use of doxylamine is extensive, with approximately 165 metric tons of doxylamine succinate being produced in the United States in 1980 (Chemical Economic Handbook, 1980). Doxylamine is representative of the ethanolamine group of antihistamines which accounts for 37% of the antihistamines used. Acute toxicity studies of doxylamine were reported almost 40 years ago (Brown and ’ The results of this study were presented in part at the 24th Annual Meeting of the Society of Toxicology, San Diego, CA, March 19,1985. 243

Werner, 1948). The IV LD50 of doxylamine was found to be 62 + 4 and 49 + 1.4 mg/kg in mice and rabbits, respectively. Subcutaneous LD50 values were mice, 460 f 52 mg/kg; male rats, 440 f 34 mg/kg; and female rats, 445 f 38 mg/kg. The oral LD50 was found to be 470 + 32 and 250 f 42 mg/kg in mice and rabbits, respectively. Oral doses up to 45 mg/kg of doxylamine twice daily to rats for 38 days had no effect on food consumption, weight gain, or hematological parameters and caused no pathological changes in the tissues (Thompson and Werner, 1948). Doxylamine induced a weak response in the hepatocyte/ DNA repair assay at the highest nontoxic doses tested (Budroe et al., 1984). Teratogenicity studies on doxylamine in rabbits and rats were negative (Gibson et al., 1968). Likewise, no evidence was found that 0272-0590/88 $3.00 Copyright Q 1988 by the Society of Toxicology. AU rights of reprodwtioa in any form reserved.

244

JACKSON

AND BLACKWELL

FIG. 1. Structure of doxylamine.

doxylamine (Shapiro et al., 1977) or Bendectin (containing doxylamine) (Smithells and Sheppard, 1978) was teratogenic in humans. A recent study has demonstrated the production of heart abnormalities by Bendectin in monkeys (Hendrickx et al., 1982) examined prenatally but no effect was observed when the animals were examined at term (Hendrickx et al., 1985). The authors suggested that Bendectin produced a delay in closure of the ventricular system in treated animals. A second study confirmed the finding that Bendectin produced no evidence of teratogenicity or embryo, or fetal, or maternal toxicity in cynomolgus monkeys (Hendrickx et al.. 1985). Analysis of urinary metabolites in the rhesus monkey utilizing [ 14C]doxylamine indicated at least six radiolabeled components (Slikker et al., 1986). Identification of the urinary metabolites by high-performance liquid chromatography and mass spectrometry (Holder et al., 1985) indicated at least four metabolic pathways in the monkey (Slikker et al., 1986). The metabolic profile of plasma was found to be the same as that of urine, except for the absence of doxylamine N-oxide which was present in urine. The discovery that methapyrilene, another antihistamine, was a potent liver carcinogen in the rat (Lijinsky et al.. 1980) led to the study of several antihistamines for potential chronic toxicity. This report presents the results of 14-day and 90-day subchronic studies of doxylamine succinate in Fischer 344 rats. METHODS Doxylamine succinate (CAS No. 526-10-7) was obtained in bulk quantity from Richardson-Merrell, Inc.,

and compared to a reference standard of doxylamine succinate obtained from J. T. Baker Chemical Co. and to the chemical/physical properties published in the literature. Purity and structural identity were confirmed by gasand high-pressure liquid chromatography, melting point, and vacuum volatiles determination and spectral analysis by infrared, ultraviolet, mass spectra, and nuclear magnetic resonance. The results indicated the bulk sample to be essentially 100% pure. Doxylamine succinate was administered as an admixture in autoclaved Purina No. 5010 Rodent Laboratory Chow. The chemical was dissolved in 95% ethanol and mixed with the feed in a V-blender, and the ethanol was removed under vacuum with heating. Stability studies simulating the mixing. storage, and administration ofthe chemical demonstrated that doxylamine succinate was stable under the conditions of the study for at least 16 weeks (Thompson ef al.. 1982). For comparison with other studies. all dose levels were expressed as parts per million of the free amine, rather than the succinate salt. Microbiological integrity and dose level confirmation were performed on each batch of dosed and control feed. Fischer 344 rats, male and female. were used. The original breeding stock was obtained from the National Institutes of Health colonies and experimental animals were produced and maintained under specific pathogen free (SPF) conditions at the National Center for Toxicological Research. Animals approximately 110 g at allocation, were maintained three per cage on hardwood chip bedding in polycarbonate, shoebox cages covered with filter bonnets. Food and water were provided ad libitum. In the 1Cday study. groups of six animals per sex were maintained on feed containing dose levels of0, 100.250, 500, 1000, or 2000 ppm doxylamine for 14 days. In the 90-day subchronic study. groups of 12 animals per sex were used. Because of the paucity of toxic effects observed in the 14-day study. dose levels of 0, 162. 405, 1012,2530, or 6325 ppm doxylamine wereadministered in the 90-day study. Death checks were made twice daily. Clinical observations, body weights, and food consumption were determined weekly at the time the old cages, feeders, and water bottles were replaced with new ones. Animals were removed to control feed the day before being terminated by CO2 anesthesia. Blood samples were drawn from the orbital sinuses. A complete necropsy was performed on all animals. In the 90-day study. organ weights were determined for brain, heart, liver, lung. kidney, testis, and thymus. Approximately 30 tissues were processed for histopathological examination in the 90day study. All animals in the 14-day study were examined histologically. In the 90-day study. all animals in the control and highest dose groups (6325 ppm) were examined. Treatment-related lesions were identified in the liver and parotid salivary gland. Livers of all animals were then examined. Since the incidence of parotid gland changes in the third highest dose group was equal to that in controls, the parotid glands of the two lowest treat-

DOXYLAMINE:

SUBCHRONIC

TABLE 1 FINAL BODY WEIGHTS OF FISHER 344 RATS ADMINISTERED DOXYLAMINE FOR 14 DAYS Doxylamine concentration (mm) 0 100 250 500 1000 2000

Group mean body weight(g) + SD” Males 176.8 + 178.0 + 176.6 f 189.3 f 180.3 f 175.3 f

3.5 (-)b 18.0 (+0.7) 19.4(-0.1) 12.2 (+7.1) 8.8 (+2.0) 12.9 (-1.0)

Females 132.8 f 135.9 f 134.6 f 132.4 + 133.7 + 123.4 f

7.2 (-) 4.1 (+2.3) 8.0(+1.4) 8.6 (-0.3) 6.5 (+0.7) 6.0 (-7.1)

u Each group contained six animals. b Percentage difference from controls in parentheses.

ment groups were not examined. Tissue sections used in the histopathological evaluations of the 90-day subchronic study were also reviewed by the National Toxicology Program Subchronic Pathology Working Group which concurred with the pathology interpretations.

RESULTS 14-Day Study

There was little toxicity demonstrated in the 14-day study at dose levels up to 2000 ppm doxylamine. All the animals survived until the scheduled termination on Day 15. The only clinical observations were limited to one male rat in the 100 ppm group having rough hair and hunched posture and one female in the 500 ppm group having rough hair, hunched posture, red eyelids, and skinny appearance. There were no significant differences between groups in food consumption over the 2-week period and only the highest dose group females exhibited a decrease (7%) in body weight gain. Final body weights are presented in Table 1. Microscopic lesions which were judged to be treatment-related were confined solely to the liver of both male and female rats. The liver lesions were characterized by cytoplasmic vacuolization in both sexes. The hepatic vacuoles were generally similar in size in all

245

STUDIES IN RATS

animals in which they were seen. The incidence of liver lesions is presented in Table 2. The lesions were similar histologically to those observed in the 90-day subchronic study. The liver lesions were more numerous in the males in the higher dose levels and in females were found in the highest dose group only. In addition, the hepatic vacuoles were sufficiently coalescent to be recognized as fatty change in one male rat in each of the 100, 250, 1000, and 2000 ppm groups. Two of the six control males also had the same liver vacuolization but to a much lesser degree and none showed fatty change. Other lesions observed in the 14-day study, such as pulmonary edema and lung hemorrhage, occurred with nearly equal frequency in dosed and control groups and were not considered to be treatment-related. 90-Day Study

All animals in the 90-day study survived until termination. Body weight gain is presented in Figs. 2 and 3. Doxylamine produced a dose response with respect to de-

TABLE 2 HISTOPATHOLOGY LESIONSOF THE LIVERS OF FISCHER 344 BATS ADMINISTERED DOXYLAMINE FOR 14 DAYS Doxylamine dose level (ppm) 0

100 250

Males Cytoplasmic vacuolization Fatty change Chronic inflammation

001000

Females Cytoplasmic vacuolization Fatty change Congestion

0 0 0 0 000

2” 0

2 1

500

1000 2OCMI

3 1

5 0

4 1

4 1

0 0

0 0 10

0 0

3 0 0

’ Number of animals with lesion per group of six animals.

246

JACKSON

AND BLACKWELL

400 -

350

%.

300

A

-

:. 0

.

0.

%

1

0 .

5 2 250 E .P

-

2” 200

-

6 $ zl 150

I .

n

. .

.

.

.

Doxylamine Concentration

l

.

n 6325

-

ppm ppm

~2530

I $! u loo-

l

1012

0

405

ppm ppm

A

162

ppm

’ Control

I

501 0

2

6

4

8

Weeks

10

I

I

12

14

on Test

FIG. 2. Body weight gain of male Fischer 344 rats administered doxylamine in the feed for 90 days. All animals survived to termination. N = 12 animals per group.

decrease in the female 10 12 ppm group. Final body weights are presented in Table 3. Mean termination weights presented in Table 3

creased gain in body weight. Both males and females demonstrated a reduced weight gain at the two highest dose levels and a possible

Doxylamine Concentration m 6325 A 2530 l 1012 0 405 A 162 0

0

2

ppm ppm ppm ppm ppm

Control

% ? % 8 8. B .. . : B . * . f . . . . . . . .

4

6 Weeks

8

10

12

14

on Test

FIG. 3. Body weight gain of female Fischer 344 rats administered doxylamine in the feed for 90 days. All animals survived to termination. N = 12 animals per group.

DOXYLAMINE:

SUBCHRONIC

TABLE 3 FINAL BODY WEIGHTS OF FISCHER 344 RATS ADMINISTERED D~XVLAMINE FOR 90 DAYS”

Group mean body weight (g) + standard error

Dose level (ppm) 0 162 405 1012 2530 6325

Male 345.9 374.9 365.4 372.3 345.9 299.3

r 5.4 (-)* + 5.6 (f&4)* z!z 5.5 (+5.6)* + 5.5 (+7.6)* -c 5.0 (0) + 10.1 (-13.3)*

Female 206.7 203.2 204.7 196.0 185.8 177.0

+ f 2 + f +

2.6 (-) 2.8 (- 1.7) 2.8 (- 1.0) 2.7 (-5.2)* 2.6 (-lO.l)* 4.2 (-14.4)*

n Mean terminal weights were adjusted for differences between initial weights by analysis of covariance. Values significantly different (p < 0.05) from controls are indicated with an asterisk (*). * Percentage difference from controls in parentheses.

were adjusted for differences between initial body weights by analysis of covariance. The differences between initial body weights were eliminated by adjusting the termination means by regression where the estimates are obtained as if the treatment groups all had the same initial weights. A reduction in final body weight was observed only in the highest dose group of males, while the females exhibited a reduction in final body weight in the three highest dose groups. Food consumption by male rats, as determined from feeder weights, is presented in Fig. 4. When expressed as grams food/kilogram body weight/day, there was little or no difference between the control group and any treated group, except for the highest dose group. The highest dose group (6325 ppm) apparently consumed less food during the first week but then exhibited a marked increase (50%) in consumption the second week which was maintained throughout the study. The apparent increased food consumption occurred in this dose group even though this group exhibited a 13% decrease in weight gain compared to controls. Similar results were observed with the female rats but are not presented.

247

STUDIES IN RATS

Except for a few miscellaneous lesions, gross observations at necropsy (Table 4) were limited to a yellow appearance of the liver in the two highest dose groups of males (1 l/12 in 2530 ppm group and 2112 in 6325 ppm group) and the highest dose group (12/12 in 6325 ppm group) of females. Organ weights were determined and expressed as a ratio to brain weight. Statistical comparisons with appropriate control groups were made and are summarized in Table 5. Liver weight to brain weight ratios were increased in all treated groups in males but were increased only in the two highest dose groups of females. The thymus/brain weight ratio was decreased in the three highest dose groups of females but was decreased only in the highest dose group of males. Doxylamine produced a reduction in heart/brain weight ratio in the two highest dose groups in females but was effective only in the highest dose group of males. Kidney/brain weight ratio was decreased in the highest dose group of males but no significant effect was observed in females. Females exhibited a decreased lung/brain weight ratio in the highest dose group but no effect on this organ was observed in males. There was no significant

140

120

. ooxylun Conc 0 COntrc . PPfn . 1 .

0

2

A 162 ppm D 405 ppm

.

4

6 Weeks

6 on Study

1012 A 2530 pm n 8225 PPm

10

12

FIG. 4. Food consumption by male Fischer 344 rats administered doxylamine in the feed for 90 days. N = 4 feeders per group of 12 animals.

248

JACKSON

AND TABLE

GROSS

LESIONS

OBSERVED

IN FISCHER

BLACKWELL 4

344 RAT’S ADMINISTERED DOXYLAMINE FOR 90 DAYS

Males, dose levels (ppm) Observation Reddish mass on surface of liver Yellow liver Enlarged liver Yellow mass: Peritoneum Grey subcutaneous mass on neck

Females, dose levels (ppm)

0

162

405

1012

2530

6325

0

162

405

1012

2530

6325

0” 0 0

0 0 0

1 0 0

0 0 0

0 I1 0

1 2 0

0 0 0

0 0 0

0 0 0

0 0 0

0 0 0

0 12 1

0

0

0

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

0

0

’ Number of animals with lesion per group of 12 animals.

effect of doxylamine administration brain weight ratios in this study.

on testis/

Histopathologv Treatment-related lesions were identified in the liver and parotid salivary gland. The liver lesions consisted of cytoplasmic vacuolization which progressed to severe hepatocelMar fatty change in the two highest dose groups. A typical example is compared to control liver in Fig. 5. In the more severely affected animals the fatty change was characterized by many medium-sized cytoplasmic vacuoles which surrounded the nucleus and severely distended the cytoplasm of the affected hepatocytes. Fatty change was more prominent in the midzonal area. In the lower dose groups, hepatic cytoplasmic vacuolization was considered to be a less severe form of fatty change and was characterized by numerous small vacuoles dispersed throughout the amphophilic or eosinophilic cytoplasm of slightly enlarged hepatocytes. The incidence of hepatic lesions is presented in Tables 6 and 7. A dose-related change in the parotid salivary glands was observed. The change consisted of cytomegaly with basophilic and coarsely granular or vacuolated cytoplasm. A

typical example is shown and compared to a normal gland in Fig. 6. This change was observed throughout the entire parotid gland in some animals, while in others, the distribution was more patchy and multifocal. In some animals, only one or two small foci were present. The incidence of cytomegaly is presented in Table 8. Although this change was observed in an occasional control animal, the lesion was much more prominent in the treated animals and the incidence was dose-related. DISCUSSION In the 14-day study, doxylamine at dose levels up to 2000 ppm produced essentially no signs of clinical toxicity except for a small decrease in final body weight in the 2000 ppm female group. Histologically, the only treatment-related changes observed were the occurrence of cytoplasmic vacuolization which, on coalescing, was diagnosed as fatty change. This minor toxic change suggested that higher dose levels should be used in the 90day subchronic study and dose levels of 162 up to 6325 ppm were used. Even at the high dose all animals survived and, except for decreased weight gain in the higher dose groups, there was little evidence of clinical toxicity.

DOXYLAMINE:

SUBCHRONIC

249

STUDIES IN RATS

TABLE 5 ORGANWEIGHTSANDORGAN/BRAINWEIGHTRATIOSINRSCHER D~XYLAMINEFOR 90 DAYS

344 RAT~ADMINISTERED

Dose level (ppm) 0

162

405

1012

2530

6325

Males Mean brain weight’ Mean liver weight Liver/brain ratio Mean thymus weight Thymusfbrain ratio Mean heart weight Heart/brain ratio Mean kidney weight Kidney/brain ratio Mean lung weight Lung/brain ratio Mean testis weight Testis/brain ratio

1.995 12.086 6.058 0.3347 0.1678 1.095 0.5489 1.289 0.646 1 1.516 0.7600 1.475 0.7393

2.003 13.483 6.731tb 0.3765 0.1880 1.150 0.5741 1.392 0.6950 1.627 0.8123 1.498 0.7479

1.990 15.301 7.689t 0.3575 0.1796 1.146 0.5759 1.408 0.7075 1.577 0.7924 1.493 0.7503

I .947 13.420 6.893t 0.3363 0.1727 1.192 0.6122 1.328 0.6820 1.511 0.776 1 1.479 0.7596

1.943 19.829 10.205t 0.2945 0.1516 1.000 0.5147 1.434 0.7380 1.465 0.7540 1.453 0.7478

1.815 18.063 9.952t 0.2047 0.11284 0.789 0.4347+ 1.015 0.55924 1.241 0.6837 1.298 0.7152

Females Mean brain weight Mean liver weight Liver/brain ratio Mean thymus weight Thymus/brain ratio Mean heart weight Heart/brain ratio Mean kidney weight Kidney/brain ratio Mean lung weight Lung/brain ratio

1.772 6.270 3.538 0.2770 0.1563 0.726 0.4097 0.726 0.4100 1.090 0.6151

1.800 6.258 3.477 0.2479 0.1377 0.714 0.3967 0.746 0.4144 1.110 0.6167

1.810 7.237 3.998 0.2643 0.1460 0.729 0.4028 0.828 0.4575 I .046 0.5779

1.789 6.079 3.398 0.2442 0.13654 0.686 0.3825 0.734 0.4103 1.050 0.5869

1.738 8.339 4.798t 0.2093 0.12044 0.624 0.35904 0.77 I 0.4436 0.966 0.5558

1.704 9.654 5.665t 0.1865 0.10944 0.542 0.31814 0.674 0.3955 0.871 0.51124

’ Weight in grams. b Significant differences from control value (two-tailed t test) of decrease (4) or increase (t) withp < 0.05.

When final body weights were corrected for small differences in initial body weights, only the highest dose (6325 ppm) had an effect on weight gain in males (Table 3). In females, there was a more linear dose effect on weight gain, with the top three dose levels producing reductions of 5 to 14% in final body weights. Except for the highest dose group, there was no effect of doxylamine on food consumption. The highest dose group apparently consumed less food during the first week and then exhibited a marked increase in food consumption over other groups. This increased consumption was then sustained for the du-

ration of the study. Although no determinations of food spillage were made, the results are consistent with spillage as an explanation for the apparent increased consumption by the highest dose group. The marked reduction in food consumption during the first week suggests that the 6325 ppm dose level could have been unpalatable and was rejected by the animals in that group. Subsequently, the animals may have wasted feed due to its objectionable taste which led to an apparent increased consumption. In a subsequent feeding study in this laboratory (unpublished) with the antihistamine pyrilamine,

250

JACKSON

FIG. 5. Histology doxylamine-dosed

AND

BLACKWELL

of the liver of a male Fischer 344 rat administered (A) control feed or (B) 6325 ppm feed for 90 days. Note the extensive fatty change in the treated animal. (H & E, X85).

food spillage was determined and found to account for 50 to 75% of the food consumption at the higher dose levels. If this were true in the present study, the decreased weight gain observed in the highest dose group could have been due, at least in part, to an actual reduced food intake rather than a direct effect of the chemical. That these animals were

TABLE

affected by the chemical was demonstrated, however. by the histological changes observed in this group, and in lower dose groups as well. The lack of gross lesions observed at necropsy was consistent with the survival rate and scarcity of clinical observations. The livers of the two highest dose groups of males

6

LIVERLESIONSINMALEFISCHER~~~RATSADMINISTEREDDOXYLAM~NEFOR

90

DAYS

Dose level (ppm) 0 Cytoplasmic vacuolization” Fatty change Chronic inflammation Granulomatous inflammation Necrosis

1 b (2.0) 0 0 0 0

162

405

1012

2530

6325

1 (2.O)C 0 3 0 0

2 (2.0) 0 1 0 1

3 (2.3) 9 (2.0) 3 0 1

0 12 (4.0) 3 0 2

0 12 (4.0) 0 0 1

’ Sections diagnosed as “fatty change” were not scored as “cytoplasmic vacuolization” although ’ Number of animals with lesion per group of I2 animals. ’ Average degree of severity scored as 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe.

it might

be present.

DGXYLAMINE

SUBCHRONIC

251

STUDIES IN RATS

TABLE I LIVER LESIONS IN FEMALE

FISCHER

344 RATS ADMINISTERED

D~XYLAMINE

FOR 90 DAYS

Dose level (ppm)

Congestion Cytoplasmic vacuolization * Fatty change Chronic intlammation Granulomatous inflammation Necrosis

0

162

405

1012

2530

6325

1” 0 0 0 0 0

0 0 0 0 0 0

0 0 0 2 1 0

0 0 0 0 2 0

0 5 (3.O)C 1 (2.0) 2 0 0

0 0 12 (2.8) 0 0 0

’ Number of animals with lesion per group of 12 animals. * Sections diagnosed as “fatty change” were not scored as “cytoplasmic vacuolization” although it might be present. ’ Average degree of severity scored as 1 = minimal, 2 = mild, 3 = moderate, and 4 = severe.

and the highest dose group of females were yellow, otherwise, there were few gross observations. The yellow appearance of livers in the higher dose groups was consistent with the histological diagnosis of fatty livers in these dose groups. There were no treatment-

related gross lesions observed in animals administered dose levels below 2530 ppm doxylamine. Several organ weights were affected by doxylamine administration; however, except for liver, all the other changes observed appeared

FIG. 6. Histology of the parotid salivary gland of male Fischer 344 rats administered (A) control or (B) 6325 ppm doxylamine-dosed feed for 90 days. Note the marked cytomegaly and basophilic and coarsely granular or vacuolated cytoplasm in the treated animal. (H & E, X85).

252

JACKSON

AND BLACKWELL

TABLE 8 PAROTID GLAND LESIONSIN FISCHER344 RATS ADMINISTERED DOXYLAMINE FOR 90 DAYS Dose level (ppm) Cytomegaly

0

162

405

1012

2530

6325

Males Females

2” 1

NEh NE

NE NE

2 I

9 7

12 12

’ Number of animals with lesion per group of I2 animals. b NE = not examined histologically.

to be a secondary effect of general weight loss at the high doses, rather than a direct effect of the chemical. When liver/brain weight ratios were compared to those of controls, all dose levels were effective in increasing the ratio in male animals. This observation is consistent with the histopathology results (compare Tables 5 and 6) in which all dose groups of males exhibited some degree of cytoplasmic vacuolization or fatty change. A similar correlation was observed in females where the two highest dose groups had increased liver/brain weight ratios (Table 5) and only the same two highest dose groups exhibited cytoplasmic vacuolization or fatty change. Significant treatment-related effects on organ/brain weight ratios observed in the thymus, heart, kidney, and lung were all decreases compared to controls and occurred only in treatment groups exhibiting decreased weight gain. The changes observed in the parotid salivary gland were considered unusual. Although occasional small patches of acinar cells of increased size, containing basophilic, cytoplasmic granularity, were observed in controls and low dose group animals, the incidence and severity of the change were doserelated. The interpretation of these changes was difficult, however, knowing the physiology of the parotid gland. First, the secretion of granules by the parotid glands during salivation is affected by a number of factors such as diurnal variation, texture and moistness of feed, and length of time since last eating. In

addition, the salivary gland is responsive to other physiological changes such as hormone levels, and in the rodent, the glands serve as regulators for serum levels of sodium and other ions. The salivary glands are also extremely sensitive to autolysis and other fixation artifacts. Thus, while there was a definite treatment-related effect, the toxicological significance of the changes was unclear. It would be of interest to determine if a long-term, chronic administration of doxylamine would produce any permanent, irreversible changes in the salivary gland. In summary, except for a slight decrease in final body weight in females and cytoplasmic vacuolization or fatty change in livers of males, there was little toxicity of doxylamine observed at dose levels below 2530 ppm. Even at the highest dose levels, doxylamine did not produce many of the changes observed with methapyrilene, such as increased mitosis in the hepatocytes (Reznik-Schiiller and Lijinsky, 198 1) or biliary hyperplasia (Jackson and Sheldon, 1984). However, the fatty changes in the liver and the morphological changes in the parotid salivary gland suggested that doxylamine should be tested in a long-term chronic study in the rat. ACKNOWLEDGMENTS The authors are indebted to Ms. Cindy Hewitt for the statistical analyses and Mrs. Ruth York for typing the manuscript.

REFERENCES AMA (197 1). Drug Evaluations, 1st ed., p. 367. American Medical Association, Chicago. IL. BROWN, B. B.. AND WERNER, H. W. (1948). The pharmacologic parameters of 2-[u-(2-dimethylaminoethoxy)-ol-methylbenzyll-pyridine succinate, a new antihistaminic agent. J. Lab. Clin. Med. 33,325-33 1. BUDROE, J. D.. SHADDOCK. J. G.. AND CASCIANO, D. A. (1984). A study ofthe potential genotoxicity of methapyrilene and related antihistamines using the hepatocyte/DNA repair assay.Mrctat. Rex 135, 13 I - 137. Chemical Economic Handbook (1980). 565. 3000 B. April, SRI International, Medicinal Chemicals.

DOXYLAMINE:

SUBCHRONIC

GIBSON, J. P., STAPLES, R. E., LARSON, E. J., KUHN, W. L., HOLTKAMP, D. E., AND NEWBERNE, J. W. (1968). Teratology and reproduction studies with an antinauseant. Toxicol. Appl. Pharmacol. 13,439-447. HENDRICKX, A. G., CUKIERSKI, M., PRAHALADA, S., JANOS, G., AND ROWLAND, J. (1985). Evaluation of Bendectin embryotoxicity in nonhuman primates. I. Ventricular septal defects in prenatal macaques and baboon. Teratology 32, 179- 189. HENDRICKX, A. G., CLJKIERSKI, M., PRAHALADA, S., JANOS, G., BOOHER, S., AND NYLAND, T. (1985). Evaluation of Bendectin embryotoxicity in nonhuman primates. II. Double-blind study in term cynomolgus monkeys. Teratology 32, 19 1- 194. HENDRICKX, A. G., PRAHALADA, S., AND ROWLAND, J. M. (1982). Embryotoxicity studies on Bendectin in cynomolgus monkeys (Macaca fascicularis). Teratology 25,47A.

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