Toxicity and Carcinogenicity Studies of Phenylephrine ...

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DRUG AND CHEMICAL TOXICOLOGY, 11(4), 355-370 (1988)

T O X I C I T Y AND CARCINOGENICITY STUDIES OF PHENYLEPHRINE HYDROCHLORIDE I N F344lN RATS AND B6C3F1 MICE John R. Bucher, James H u f f and Joseph K. Haseman N a t i o n a l Toxicology Program, N a t i o n a l I n s t i t u t e o f Environmental H e a l t h Sciences Research T r i a n g l e Park, North C a r o l i n a 27709 ABSTRACT Phenylephrine HC1 was i n c o r p o r a t e d i n t o feed g i v e n t o male and female F344/N r a t s and B6C3F1 mice i n s t u d i e s o f 14 days, 12 weeks, and 2 years d u r a t i o n . I n 12-week s t u d i e s , body w e i g h t g a i n s decreased w i t h dose, and deaths o f male r a t s and mice occurred a t c o n c e n t r a t i o n s o f 5,000 ppm and above; however, no o r g a n - s p e c i f i c t o x i c i t y was evident. During 2year s t u d i e s , body weights o f r a t s r e c e i v i n g d i e t s a t 620 and 1,250 ppm and mice a t 1,250 and 2,500 ppm ranged up t o 16% l e s s than c o n t r o l . S u r v i v a l o f h i g h dose male r a t s was substant i a l l y g r e a t e r than c o n t r o l s . S u r v i v a l s o f o t h e r dose groups o f r a t s and mice were s i m i l a r t o c o n t r o l s . Chronic f o c a l i n f l a m n a t i o n o f t h e l i v e r , and i n f l a m n a t i o n o f t h e p r o s t a t e were increased i n dosed r a t s . No increases i n neoplasms were observed i n r a t s o r mice consuming d i e t s c o n t a i n i n g phenylephrine H C l f o r 2 years. The incidences o f mononuclear c e l l leukemia and pheochromocytomas o f t h e adrendl g l a n d were decreased i n dosed male r a t s . Approximate time weighted average doses ranged up t o 54 mglkglday f o r r a t s and 280 mg/kg/day f o r mice d u r i n g t h e 2-year studies. INTRODUCTION Phenylephrine h y d r o c h l o r i d e i s a sympathomimetic amine used p r i m a r i l y as a nasal decongestant, and as a m y d r i a t i c i n ophthalmic a p p l i c a t i o n s .

I t i s a v a i l a b l e under a v a r i e t y o f

t r a d e names as a s t e r i l e s o l u t i o n f o r p a r e n t a l use, as an e l i x i r o r i n syrups, as v a r i o u s nasal and ophthalmic s o l u t i o n s , 355 Copyright @ 1988 by Marcel Dekker, Inc.

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BUCHER, HUFF, AND HASEMAN

and i n over t h e counter c o l d remedies (1).

T o t a l US human con-

sumption was estimated a t 20,000 kg i n 1977 (2).

Toxicities


r e s u l t i n g from systemic a b s o rp ti o n o f phenylephrine from o c u l a r o r nasal prepara ti o n s have been reported, and appear r e l a t e d t o t h e pharmacologic a c t i o n s o f t h e drug, r a t h e r than t o any i n t r i n s i c cytotoxicity.

Side e f f e c t s o f o c u l a r a d m i n i s t r a t i o n

a r e most o f t e n a s s o c i a te d w i t h t h e use o f 10% s o l u t i o n s , and i n c l u d e t r a n s i e n t p a i n , re l e a s e o f pigment from t h e i r i s , e l e v a t i o n o f i n t r a o c u l a r pressure, and headache ( 3 ) .

The most

s e rious r e p o r t e d sequelae i s a marked r i s e i n b l o o d pressure, which has l e d t o subarachnoid hemorrhage, v e n t r i c u l a r a r r h y t h mias, and cardi a c i n f a r c t i n s u s c e p t i b l e p a t i e n t s (4).

Most o f

these e f f e c t s can be r e l a t e d t o severe v a s o c o n s t r i c t i o n p r o duced by a-adrenerg ic s t imul a t ion. A v a i l a b l e c h ro n i c t o x i c i t y s t u d i e s ' o f phenylephrine HC1 i n animals were deemed inadequate by t h e Food and Drug A d minist rat ion.

Because o f t h i s , we have s t u d i e d phenylephrine

HC1 i n 14-day, 12-weekY and 2-year s t u d i e s u s i n g an o r a l dosed feed exposure regimen i n F344 r a t s and B6C3F1 mice o f b o t h

sexes.

The r e s u l t s o f these s t u d i e s (5) form t h e b a s i s o f t h i s

report. Chemical USP-grade phenylephrine H C l was o b t a i n e d from Ganes Chemical Co.,

New York, New York.

The i d e n t i t y o f pheny-

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TOXICITY AND CARCINOGENICITY STUDIES

l e p h r i n e H C l was confirmed by i n f r a r e d , u v / v i s and n u c l e a r resonance spectroscopy.

The p u r i t y was determined t o be


approximately 99% by elemental a n a l y s i s , HPLC, t i t r a t i o n of t h e amine group, and b r o m i n a t i o n o f t h e aromatic r i n g p o s i t i o n s (5).

Phenylephrine HC1 was s t a b l e when mixed i n feed.

feed m i x t u r e s were homogeneous t o f 1.5%.

Dosed

M i x t u r e s were ana-

l y z e d on 13 separate occasions d u r i n g t h e 2-year studies.

All

were determined t o be w i t h i n f 10% o f t h e t a r g e t concentrations. Experimental Design Four t o s i x week o l d male and female F344 r a t s and B6C3F1 mice were obtained from Charles R i v e r Breeding l a b o r a t o r i e s f o r a l l studies.

Animals were housed 5 p e r polycarbonate cage w i t h

heat t r e a t e d hardwood c h i p bedding. Ralston Purina Co., Z e i g l e r Bros.,

Feed (Rodent Chow 5001,

S t . Louis, MO- 14 day studies; NIH-07,

Gardeners, PA- 12-week and 2-year s t u d i e s ) and

water were a v a i l a b l e -ad l i b i t u m .

A f t e r 2-3 week a c c l i m a t i o n

periods, animals were d i s t r i b u t e d t o weight classes and assigned t o cages according t o a t a b l e o f random numbers. Numbers o f animals p e r dose group, and phenylephrine H C l conc e n t r a t i o n s i n t h e feed a r e g i v e n i n Table 1.

Body weights and

c l i n i c a l s i g n s were recorded weekly throughout t h e f i r s t 13-weeks and monthly t h e r e a f t e r throughout t h e studies.

Food

consumption was measured weekly by cage. A t study t e r m i n a t i o n , animals were k i l l e d w i t h carbon

dioxide.

Necropsies were performed on a l l animals, and t i s s u e s

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Table 1 D i e t a r y Concentrations Used i n Phenylephrine H C l S t u d i e s


14-day st u d i e s , n = 5 animals p e r sex, p e r dose l e v e l ratsmice-

0, 125, 250, 500, 1,000, 2,000 ppm 0, 63, 125, 250, 500, 1,000 ppm

12-week s tu d i e s , n = 10 p e r sex, p e r dose l e v e l ratsmice-

0, 1,250, 2,500, 5,000, 10,000, 20,000 ppm 0, 1,250, 2,500, 5,000, 10,000, 20,000 ppm

2-year s tu d i e s , n = 50 p e r sex, p e r dose l e v e l ratsmice-

0, 620, 1,250 ppm 0, 1,250, 2,500 ppm

were examined f o r gross lesions.

Approximately 40 t i s s u e s p e r

animal were preserved i n 10% n e u t r a l b u f f e r e d f o r m a l i n , embedded i n p a r a f f i n , sectioned, s t a i n e d w i t h hematoxylin and eosin, and examined m i c r o s c o p i c a l l y f o r nonneoplastic and n e o p l a s t i c lesio n s .

D e t a i l s o f pathology procedures and review

processes have been re p o rte d (5-7).

The i n l i f e p o r t i o n o f

these s t u d i e s was performed a t P h y s i o l o g i c a l Research L a borat ories, Minneapolis, MN.

Good Laboratory P r a c t i c e s were

i n e f f e c t d u r i n g t h e second y e a r o f t h e s t u d i e s and d u r i n g a l l p o s t l i f e phases. S t a t i s t i c a l Analyses D i f f e r e n c e s i n s u r v i v a l i n t h e 2-year s t u d i e s were anal y z e d by l i f e t a b l e methods (8).

For analyses o f tumor i n c i -

dence data, two procedures were used t o assess dose-response

359


t r e n d s and t o t e s t f o r p a i r w i s e d i f f e r e n c e s between dosed groups and c o n t r o l s : [l]L i f e t a b l e analyses ( a p p r o p r i a t e f o r


f a t a l tumors) and [ E l t h e i n c i d e n t a l tumor t e s t ( a p p r o p r i a t e f o r tumors observed i n animals d y i n g from an u n r e l a t e d cause). For f u r t h e r d i s c u s s i o n o f these s t a t i s t i c a l methods see Haseman

(9) RESULTS 14-Day Studies The d i e t a r y c o n c e n t r a t i o n s o f phenylephrine H C l used i n 14-day s t u d i e s ranged up t o 2000 ppm f o r r a t s and 1000 ppm f o r mice (Table 1).

None o f t h e r a t s o r mice d i e d b e f o r e t h e end

of t h e studies, and t h e r e were no b i o l o g i c a l l y s i g n i f i c a n t e f f e c t s on body weights, c l i n i c a l signs, o r feed consumption. No compound-related e f f e c t s were observed a t necropsy o r upon microscopic examination o f t i s s u e s from a randomly s e l e c t e d 10% o f t h e study animals. 12-Week Studies Since no t o x i c e f f e c t s were seen i n t h e 14-day studies, d i e t a r y c o n c e n t r a t i o n s were increased f o r t h e 12-week s t u d i e s (Table 1).

Four of 10 male r a t s t h a t received 20,000 ppm, 2/10

males t h a t received 10,000 ppm and 1/10 males t h a t received

5,000 ppm phenylephrine H C l i n t h e d i e t d i e d b e f o r e t h e end o f t h e studies.

No female r a t s d i e d early.

F i n a l mean body

weights o f a l l dosed males and a l l b u t t h e lowest group of female r a t s were more than 10% lower than those o f t h e

360


c ont rols. 5,000,

The f i n a l mean body weights o f r a t s t h a t r e c e i v e d

10,000 o r 20,000 ppm were 57%, 45%, o r 35% t h a t o f


c o n t r o l s f o r males and 70%, 58% o r 51% t h a t o f c o n t r o l s f o r females.

Feed consumption by dosed r a t s was c o n s i s t e n t l y lower

than t h a t by t h e c o n t r o l s and decreased as dose increased suggesting p o s s i b l e p a l a t a b i l i t y problems. 10,000 o r 20,000 ppm were hyperexcitable.

Rats t h a t r e c e i v e d Chronic k e r a t i t i s o f

t h e eye was observed i n 4 /8 males and 8/10 females a t 20,000 ppm and i n 4/10 males and 6/10 females a t 10,000 ppm.

MinSmal

t o m i l d t e s t i c u l a r a tro p h y was observed i n 4 / 8 males, and seminal v e s i c l e a tro p h y was observed i n 5 / 6 males t h a t r e c e i v e d 20,000 ppm; m i l d t o moderate o v a ri a n atrophy was observed i n 5/10 females t h a t re c e i v e d 20,000 ppm.

No o t h e r o b s e r v a t i o n s

appeared t o be compound-related. Three of 10 male mice r e c e i v i n g 20,000 ppm and 2/10 r e c e i v i n g 10,000 ppm d i e d b e fo re t h e end o f t h e study. female mice s u r v i v e d t o t h e end o f t h e study.

All

The f i n a l mean

body weights o f males t h a t received 10,000 o r 20,000 ppm were 18 and 25% lower than t h a t o f c o n t r o l s r e s p e c t i v e l y .

Final

mean body weights o f dosed female mice were 10-32% lower than t h a t o f t h e con tro l s . r e l a t e d t o dose.

Feed consumption d i d n o t appear t o be

Rough h a i r coats and l e t h a r g y were considered

compound-related e f f e c t s .

Inflammatory eye l e s i o n s (acute

k e r a t i t i s , pano p th a l mi ti s , o r p h t h i s i s b u l b i ) were observed i n 3/10 males and 2/10 females t h a t re c e i v e d 20,000 ppm.

No


361

h i s t o p a t h o l o g i c changes were considered compound-related i n o t h e r tissues.

Because o f weight g a i n depression and t h e few


deaths observed a t t h e h i g h e r d i e t a r y l e v e l s , t h e phenylephrine H C l c o n c e n t r a t i o n s s e l e c t e d f o r r a t s f o r t h e 2-year s t u d i e s were 620 and 1,250 ppm, and f o r mice, 1,250 and 2,500 ppm i n feed. Two Year Studies I n Rats For r a t s , t h e p r e s t u d y mean body weight o f h i g h dose males was 5% lower than t h a t o f c o n t r o l s , and mean weights were 4-16% lower throughout t h e study.

The mean body weight o f low dose

male r a t s ranged from 3 t o 8% lower than t h a t o f t h e c o n t r o l s . The body weight o f h i g h dose female r a t s was 4 t o 10% lower, and t h a t o f low dose female r a t s was s i m i l a r t o c o n t r o l s throughout t h e study.

No s p e c i f i c c l i n i c a l s i g n s o f t o x i c i t y

o r ophthalmologic f i n d i n g s noted i n any group o f dosed r a t s r e r e a t t r i b u t e d t o phenylephrine HCl.

The average d a i l y f e e d

consumption was s i m i l a r f o r dosed and c o n t r o l male and female rats.

The average amount o f phenylephrine H C l consumed p e r day

was approximately 22 o r 47 mg/kg f o r low o r h i g h dose male r a t s , and 26 o r 54 mg/kg f o r low o r h i g h dose female r a t s . Estimates o f t h e p r o b a b i l i t i e s o f s u r v i v a l f o r male and female r a t s i n t h e v a r i o u s groups a r e shown i n F i g u r e 1.

The

s u r v i v a l o f h i g h dose male r a t s was s i g n i f i c a n t l y g r e a t e r than t h a t o f t h e c o n t r o l s a f t e r week 98.

S u r v i v a l s o f low dose male

WEEKS ON STUDY



363

r a t s and female r a t s were n o t s t a t i s t i c a l l y d i f f e r e n t from c o n t r o l s [ f i n a l s u r v i v a l r a t s : males- c o n t r o l 30/50, low dose


33/50, h i g h dose 42/50; females, c o n t r o l 42/50,

low dose 34/50,

h i g h dose 36/50]. H i s t o p a t h o l o g i c e v a l u a t i o n of r a t s revealed an increase i n c h ronic f o c a l i n f l a m n a t i o n o f t h e l i v e r and i n f amnation o f t h e p r o s t a t e gland i n dosed r a t s (Table 2).

These

esions d i d n o t

d i f f e r i n chara c te r from t h e l e s i o n s t h a t occur i n u n t r e a t e d c o n t r o l F344 r a t s .

The l i v e r l e s i o n was c h a r a c t e r i z e d by t h e

presence o f mononuclear c e l l s s c a t t e r e d around b i l e d u c t s i n the portal triad.

Small granulomas were f r e q u e n t l y adjacent t o

o r replaced b i l e d u c ts i n t h e p o r t a l regions. appeared t o c o n s i s t e n t i r e l y o f macrophages.

The granulomas No o t h e r b i o l o g i -

c a l l y s i g n i f t c a n t increased incidences o f nonneoplastic o r n e o p l a s t i c l e s i o n s were seen i n dosed male o r female r a t s . Several n e o p l a s t i c and nonneoplastic l e s i o n s were decreased i n dosed r a t s versus c o n t r o l s .

Most n o ta b l e was a decreased i n c i -

dence o f mononuclear c e l l leukemia i n males, and a l e s s e r decrease i n females (Table 2).

Adrenal g l a n d m e d u l l a r y

h yperplasia and pheochromocytomas were decreased i n dosed male r a t s , and b i l e d u c t h y p e rp l a s i a was decreased i n t h e l i v e r s o f dosed male and female r a t s . FIGURE 1 Kaplan-Meier S u r v i v a l Curves f o r Male and Female Rats.

364


Table 2


Number o f Rats With Selected Lesions i n t h e 2-Year Phenylephrine HC1 S t u d i e s

Ma1es

Females

0

620

1,250

0

620

1,250

Number Examined

50

50

50

50

50

50

Liver chronic inflamnation b i l e duct hyperplasia

2 46

13a 16a

17a 1l a

17 20

28b 5a

35" 2a

Hemat o p o i e t ic Sys tem leukemia

24

9a

5a

15

14

10

Adrenal Gland medullary hyperplasia pheochromocytoma

10 14

4 11

6 2a

2 6

3 6

1 2

Prostate in f 1amnati on

10

24a

24a

S i te/Lesion

a. p b. p

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