Effects of exposure to furfural vapour in hamsters simultaneously treated with benzo[α] pyrene or diethylnitrosamine

Toxicology, 11 (1978) 127--144 © Elsevier/North-Holland Scientific Publishers Ltd.

E F F E C T S OF E X P O S U R E T O F U R F U R A L V A P O U R IN H A M S T E R S S I M U L T A N E O U S L Y T R E A T E D WITH BENZO[a] PYRENE OR DIETHYLNITROSAMINE* V.J. FERON and A. KRUYSSE Central Institute for Nutrition and Food Research TNO, Zeist (The Netherlands)

(Received April 21st, 1978) (Revision received June 19th, 1978) (Accepted July 24th, 1978)

SUMMARY Syrian golden hamsters were exposed t o 0 or 2 5 0 / 4 0 0 ppm furfural vapour, 7 h / d a y , 5 days / w eek for a period of 52 weeks. Simultaneously, a p r o p o r t i o n of the animals were given either intratracheal instillations o f b e n z o [ a ] p y r e n e (BP) or s u b c u t a n e o u s injections of di et hyl ni t rosam i ne (DENA). All t r e a t m e n t s were s t oppe d after 52 weeks. T he st udy was terminated after 81 weeks. Furfural e x p o s u r e caused yellowish discolouration of the animals' coat, irritation o f the nasal mucosa, growth ret ardat i on, a t r o p h y and d o w n w a r d growth o f sensory cells o f the o l f a c t o r y epithelium, degenerative changes in B o wman ' s glands, and the o c c u r r e n c e o f cyst-like structures in the lamina propria b en eath the o l f a c t o r y epithelium. T h e r e was no evidence o f furfural possessing carcinogenic activity. In a previous instillation e x p e r i m e n t with furfural, slight indications had been o b tain ed o f furfural p o t e n t i a t i n g the carcinogenic effect of BP, but in the present study the carcinogenic e f f e c t o f BP or DENA on the respiratory tract did n o t appear to be influenced by furfural exposure. It was c o n c l u d e d t h a t little, if any, significance can be at t ached t o furfural as a co - f acto r in respiratory t r a c t carcinogenesis.

INTRODUCTION Studies with furfural, acrolein and a c e t a l d e h y d e have been included in o u r * This study was supported by a grant from the research fund of the Scientific Advisory Committee on Smoking and Health. This fund has been established by the Dutch Cigarette Industry Foundation. Abbreviations: BP, benzo[a ] pyrene ; DENA, diethylnitrosamine. Address all correspondence to: Dr. V.J. Feron, Central Institute for Nutrition and Food Research TNO, P.O. Box 360, 3700 AJ Zeist, The Netherlands.

127

research programme on the significance of aldehydes occurring in cigarette smoke for the genesis of pulmonary cancer. Furfural was selected for a number of reasons, which have been given in a previous report [ 2 ] . The 4-h LCs0 o f furfural in Syrian golden hamsters has been found to be 12 500 mg/m 3 air (approx. 3250 ppm) [6]. On the basis o f this value furfural vapour may be classified as "slightly t o x i c " according to Sutton's classification o f acute inhalation toxicity of vapours [10]. The results o f a 13-week repeated exposure study (6 h / d a y ; 5 days/week) with furfural in hamsters, allowed the conclusion t h a t 20 ppm is a no-toxic effect level. The effects observed at the highest dose level of 552 ppm were slight growth retardation, irritation of eyes and nose, and degenerative and hyperplastic changes of the olfactory epithelium in the nasal cavity. The only effect observed at the intermediate level of 115 ppm was a minimal degree of degenerative changes of the olfactory epithelium (Feron, V.J. et al., unpublished). The present paper deals with a long-term inhalation s t u d y on furfural in hamsters, a number of which were given intratracheal instillations of BP or subcutaneous injections of DENA. The experiment was carried o u t to find o u t the effects o f long-term repeated exposures to furfural vapour, and the possible significance of this aldehyde as a co-factor in respiratory tract carcinogenesis. EXPERIMENTAL

Chemicals and apparatus Furfural (obtained from Merck-Schuchardt, H o h e n b r u n n , G.F.R.) was distilled and analysed by gas c h r o m a t o g r a p h y to check its purity. Furfural was evaporated by fine dispersion by means of a CIVO-designed nebulizer. The nebulizer consists of a Lechler stainless steel aerodynamic nozzle atomizer which is m o u n t e d on a glass container for the liquid. It is provided with a stainless steel impaction-baffle to remove the larger particles, t h a t are recycled to the liquid in the container. The atomizer was operated with compressed air at a pressure of 2 kg/cm 2, the flow rate being 48 1 of air/min. The furfural mist from the nebulizer was passed into the inlet piece (at the top) of a 2.5 m 3 stainless steel/glass exposure chamber described in detail by Kruysse et al. [8]. In the inlet piece the mist was thoroughly mixed with a flow of conditioned air of 25 m3/h. Complete evaporation of furfural was obtained before the substance reached the upper conical part of the inhalation chamber. An exposure chamber of the same t y p e was supplied with air only, and contained the control animals. To m o n i t o r the furfural concentration in the test atmosphere, samples were taken automatically at regular intervals by means of a stainless steel sampling loop using a timer-controlled gas-sampling valve and analysed by gas-liquid chromatography. A 3 m × 4 mm helical A1 column, packed with LAC I-R 296 (Cambridge Industries Co. Inc., Cambridge, Mass., U.S.A.) on Gaschrom S (60--80 mesh), and a flame ionization d e t e c t o r were used.

128

Nitrogen was the carrier gas. The temperature o f the column was approx. 140°C. BP was used as supplied b y Fluka A.G., Buchs, S.G. Switzerland; its purity was checked b y thin-layer chromatography and appeared to be higher than 99%. It was ground in an agat mortar for a b o u t 0.5 h and then suspended in 0.9% NaC1 solution b y means o f ultrasonic vibration. 2 different BP suspensions containing 0.175 or 0.35% BP (w/v) respectively were prepared and stored at - 2 0 ° C in quantities sufficient for 1 t r e a t m e n t o f all animals in a group. Just prior to use the suspensions were again vibrated ultrasonically for 2 to 3 min. DENA was obtained from EGA-Chemie K.G., Keppler and Reif, Steinheim/Albuch, G.F.R. Its purity was checked gaschromatographically. Solutions of 0.0625% DENA in 0.9% NaC1 {v/v) were stored at 4~C (at most for a period o f 2 months) in quantities sufficient for 1 t r e a t m e n t of all animals in a group. 0.9% NaC1 solution (sterile, non-pyrogenic) was used as supplied by Baxter Laboratories, A. Christiaens N.V., Brussels, Belgium.

Experimental design and conduct Male and female Syrian golden hamsters, 6 weeks old, were obtained from the randomly bred colony of the Central Institute for the Breeding of L a b o r a t o r y Animals TNO, Zeist, The Netherlands. T h e y were housed 6 to a cage in tinned wire-screen cages, which w e r e suspended in a heptagonal central frame, rotatable around the vertical axis o f the inhalation chambers. The temperature in the chambers was 24°C, the relative h u m i d i t y o f the air inside was a b o u t 60%. In order to prevent contamination of the diet with furfural the animals were fed a fresh portion o f pelleted stock diet (Muracon I, T r o u w & Co. N.V., A m s t e r d a m / P u t t e n , The Netherlands) every day after the exposure. They received tap water ad lib., also during the exposures. 504 male and 504 female hamsters were evenly distributed according to b o d y weight over 4 inhalation chambers, 1 control c h a m b e r in which the animals were exposed to filtered and conditioned air, the others being test chambers in 1 o f which furfural vapour was added to the air supplied. The 2 other chambers were used for exposure to acetaldehyde and acrolein respectively. The results obtained with acrolein have already been described elsewhere [3] and those obtained with acetaldehyde will be the subject of a subsequent report. Furfural vapour was dosed at an average concentration of 400 p p m (1550 mg/m 3 air), 7 h/day, 5 d a y / w e e k , during the first 9 weeks, 330 ppm (1280 mg/m 3 air) during weeks 10--20 and 250 p p m (970 mg/m 3 air) during weeks 21--52. The dosage level was reduced twice, because o f considerable growth retardation and to avoid early mortality of test animals. Both the hamsters in the c o n t r o l chamber and those in the test chamber were divided according to b o d y weight into 5 groups. 2 groups (group 1 and 2) consisted o f 18 males and 18 females each, and each of the other 3 groups

129

(group 3, 4 and 5) comprised 30 males and 30 females. In addition to exposure to air or furfural vapour the animals of the different groups were treated as follows: Group 1: no treatments; Group 2: 52 weekly intratracheal instillations of 0.2 ml 0.9% NaC1 solution; Group 3: 52 weekly intratracheal instillations of 0.2 ml 0.175% BP in 0.9% NaC1 solution; total a m o u n t of BP instilled/hamster was 18.2 mg; Group 4: 52 weekly intratracheal instillations of 0.2 ml 0.35% BP in 0.9% NaC1 solution; total a m o u n t of BP instilled/hamster was 36.4 mg; Group 5: 17 subcutaneous injections of 0.2 ml 0.0625% DENA in 0.9% NaC1 solution given once every 3 weeks; total volume of DENA injected/hamster was 2.1 pl. Before each instillation the animals were lightly anaesthetized with freshly distilled ether. The instillation procedure used has been described previously

[5]. Body weights were recorded fortnightly during the first 6 weeks and m o n t h l y thereafter. At the end of the exposure period (week 52) 3 males and 3 females, taken randomly out of each of the 2 groups which were not treated with BP or DENA, viz., groups 1 and 2, were killed by exsanguination from the abdominal aorta after anaesthesia with a barbiturate given intraperitoneally. Haematological studies were carried out on blood from the abdominal aorta, and comprised determinations of haemoglobin concentration, packed cell volume, and the numbers o f erythrocytes and total and differential leucocytes. In addition, determinations were made of total serum protein and serum albumin, and of the activities of serum glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and alkaline phosphatase. The weights of the following organs were recorded: heart, kidneys, liver, spleen, brain, gonads, lungs (+ trachea and larynx) and adrenals. Tissue samples of these organs and also of the gastrointestinal tract, thyroid, aorta, uterus, pancreas, mesenteric lymph nodes, skin, skeletal muscle, urinary bladder, salivary glands and head (after removal of the skin, brain and lower jaw) were fixed in a 4% neutral formaldehyde solution. The lungs were fixed by intratracheal infusion with the formaldehyde solution under 10 cm water pressure. Following fixation the heads were decalcified in nitric acid. The tissues and organs mentioned above of all animals were embedded in paraffin wax, and sections of 5 pm (4 transverse sections across the nasal cavity, and 3 longitudinal sections through larynx, trachea with main bronchi and the pulmonary lobes) were stained with haematoxylin and eosin, and examined microscopically. The experiment was terminated after 81 weeks when all survivors were killed and autopsied. Hamsters t h a t died spontaneously or were killed when moribund were also subjected to a thorough autopsy. A u t o p s y , fixation and histological techniques were the same as those mentioned above. Of all

130

animals the entire respiratory tract, grossly visible t u m o u r s and gross lesions suspected of being t u m o u r s were examined histologically. RESULTS

Condition and general appearance While being exposed to furfural the hamsters kept their eyes closed and many had " w e t noses", indicating irritating properties of furfural. After a test period of 4 months the animals' coat gradually became more and more yellowish-brown. The discolouration remained visible also after cessation of furfural exposures.

Body weights and mortality Clearly decreased b o d y weights occurred in males and females exposed to furfural vapour (Table I). During the post-exposure period (week 53--81) the differences in b o d y weight b e t w e e n furfural- and air-exposed animals generally diminished, b u t remained visible in most of the groups. Furfural exposure did not affect death rate (Table II). Neither was death rate clearly influenced by BP or DENA, although mortality increased slightly more rapidly in females treated with BP and exposed to either air or furfural than in females exposed to air or furfural alone.

Haematology, blood biochemistry and organ weights There were no significant differences in haematologolical or biochemical findings b e t w e e n air- and furfural-exposed hamsters. Relevant changes in organ-to-body weight ratios did not occur,

Pathology Effects of furfural exposure. The olfactory epitheliunl of the nasal cavity (Fig. 1 ) was severely damaged in animals exposed to tile furfural vapour (Tahle III). Regressive changes were seen as focal thinning of the layer of olfactory epithelium which was mainly clue to a nearly c o m p l e t e atrophy of the sensory cells in the affected areas. These degenerative alterations were paralleled b y accumulations o f sensory cells in the lamina propria (Fig. 2). The irregular groups o f sensory cells with their highly typical "owl e y e " nuclei were invariably seen to rest on a basal lamina, and often seemed to be part o f the lining of cyst-like or glandular structures which most likely represent endophytieally m o v e d surface epithelium. In several hamsters infolding and d o w n w a r d growth of the surface epithelium indeed was d e a r l y visible. Another, though less probable explanation, m a y be that these glandular structures represent altered B o w m a n ' s glands partly lined b y proliferated sensory cells. In a few animals exposed to furfural hyperplasia of olfactory epithelium was visible as small papillary formations or intra-epithelial cystlike structures (Fig. 3). Focal alterations of the glands o f B o w m a n occurred in many o f the hamsters exposed to furfural. The changes consisted of enlarged epithelial

131

c~ b~

-0.9% NaCI BP (18.2 mg) BP (36.4 rag) --

Furfural Furfural FurfuraI Furfural Furfural

-0.9% NaCl BP ( 1 8 . 2 m g ) BP (36.4 rag) --

Furfural Furfural Furfural Furfurat Furfural

----DENA

--DENA

--

----DENA

----DENA

86 86 86 86 87

86 86 87 87 87

85 84 84 85 84

85 84 85 85 85

88 b 85 d 85 d 84 d 84 c

96 95 95 96 98

94 c 99 95 c 94 96 c

108 108 105 101 104

4

104 103 d 103 c 98 d 103 d

115 119 115 116 115

90 d 91 d 85 d 85 d 86 d

102 106 102 101 107

14

110 b 106 c 106 b 102 d 109 d

119 116 117 116 123

955 93 d 86 d 91 d 86 d

106 112 103 105 108

26

104 107 d 102 b 1015 108 d

113 120 112 110 118

925 88 d 85 d 89 d 84 d

I01 106 100 105 103

42

108 b 111 105 103 108 b

119 119 106 106 117

98 91 d 94 d 94 d 89 d

102 110 103 107 104

52 e

AND TREATED

111 1095 104 107 112

115 120 108 108 116

102 99 c 101 c 101 95 c

98 113 112 106 108

66

108 104 99 108 115

123 118 108 110 121

109 100 c I04 b 108 109 c

102 116 116 113 112

80

INTRATRACHE-

a The various groups of furfural-exposed animals were compared with the corresponding groups of air-exposed animals. The symbols b, c a n d d i n d i c a t e s t a t i s t i c a l s i g n i f i c a n c e at t h e 5, 1 a n d 0 . 1 % l e v e l s r e s p e c t i v e l y ( S t u d e n t ' s t - t e s t ) . e A t w e e k 5 2 all t r e a t m e n t s w e r e s t o p p e d .

-0.9% NaCl BP (18.2 mg) BP (36.4 mg) --

Air Air Air Air Air

Females

-0.9% NaCl BP (18.2 rag) BP ( 3 6 . 4 r a g ) --

Air Air Air Air Air

Males

Subcutaneous injection of

0

Intratracheal instillation of

Inhalation of

VAPOUR

A v e r a g e b o d y w e i g h t s (g) a t e n d o f w e e k

TO AIR OR FURFURAL

Treatments e

AVERAGE BODY WEIGHTS OF HAMSTERS EXPOSED ALLY WITH BP OR SUBCUTANEOUSLY WITH DENA a

TABLE I

TABLE H

-0.9% NaCl

BP (18.2 rag) BP (36.4 rag) --

Furfural Furfural

Furfural Furfural Furfural

BP (18.2 rag) BP (36.4 rag) --

-0.9% NaCI

BP (18.2 m g ) BP (36.4 rag) --

Air Air Air

Furfural Furfural

Furfural Furfural Furfural

--DENA

-- 1 -

--DENA

-- I •

--DENA

~}

--DENA

-- }

30 30 30

30 b

30 30 30

30 b

30 30 30

30 b

30 30 30

30 b

0 0 1

0

0 0 0

0

0 0 0

1

0 0 0

0 1 1

0

1 1 1

0

0 0 0

3

0 0 0

1

1

1 2 1

0

5 2 1

0

1 0 0

3

0 0 0

1 2 1

1

9 7 3

2

1 0 1

3

3 3 0

2

3 7 4

2

13 10 3

4

1 0 1

3

6 4 0

4

52 a

8 16 7

6

16 12 6

5

2 2 5

4

6 6 0

5

66

13 22 21

12

21 18 11

16

4 7 8

4

8 11 1

7

80

a A t w e e k 52 all t r e a t m e n t s w e r e s t o p p e d . b Initially b o t h g r o u p s t o g e t h e r c o m p r i s e d 36 males and 36 females. A t w e e k 52 6 males and 6 f e m a l e s w e r e killed. T h e s e animals are n o t i n c l u d e d in t h e table.

-0.9% NaCi

Air Air

Females

BP (18.2 rag) BP (36.4 rag) --

0

0.9% NaCl

42

Air Air Air

26

Males Air Air

14

4

Number of animals/ group

Intratracheal instillation of

Inhalation of

Subcutaneous injection of

N u m b e r o f d e a t h s at end o f w e e k

Treatments a

C U M U L A T I V E M O R T A L I T Y O F H A M S T E R S E X P O S E D TO A I R O R F U R F U R A L V A P O U R A N D T R E A T E D I N T R A T R A C H E A L L Y WITH BP O R S U B C U T A N E O U S L Y WITH D E N A

o~

T A B L E III

Small h a e m o r r h a g e s Small p n e u m o n i c loci F o c a l s u b p l e u r a l fibrosis Accumulations of macrophages T h i c k e n e d alveolar s e p t a Heart-failure lung

Lungs

H y p e r p l a s i a : slight moderate

73"achea

Slight h y p e r p l a s i a

Larynx

Marked atrophy of olfactory epithelium along with downward growth of sensory cells M a r k e d d e g e n e r a t i o n o f B o w m a n ' s glands Cyst-like s t r u c t u r e s in l a m i n a p r o p r i a Rhinitis Thickened submueosa

Nasal cavity

No. o f a n i m a l s e x a m i n e d c

Site and t y p e o f lesions

0 1 1 6 0 0

2 0

1

0 0 0 2 0

36

18

0 2 0 8 3 0

0 0

0

9 12 26 8 1

33

15

17

2 2 0 0 2 3

0 0

0

0 0 0 0 0

34

17

17

2 0 0 1 0 0

1 1

0

14 9 20 8 1

34

17

__a 0.9% NaCI b

___a 0.9% NaC1 b

a 18

Air

Furfural

Air

18

_ a 0.9% NaCI b

Inhalation of

Inhalation of

0.9% NaCI b

Furfural

Females

Males

I n c i d e n c e o f lesions

SITE, T Y P E A N D I N C I D E N C E O F N O N - N E O P L A S T I C C H A N G E S IN H A M S T E R S E X P O S E D T O A I R O R F U R F U R A L

C~ o1

0

0

0

1

0

1 2 0 0 0

3 1

0 3 1

0 0 0 2

0

2

0

0

3 2 1 2 2

0 0

1 3 2

1 0 0 0

a No further treatment. b Given intratracheally (0.2 ml), weekly during 52 weeks. c A few animals were lost through cannibalism or autolysis. d These organs were examined in only 6 animals/sex/group killed at the end of the exposure period (week 52).

Endometritis

Uterus d

Prostatitis

Prostate d

1

2

A tr ophy

Testes d

0

0

Myodegeneration

S k e l e t a l muscle d

Infiltrates of l y m p h o c y t e s

Sublingual salivary glands d

Dilated glands

1

0 0 0 0 0

Bile duct proliferation Kupffer cells laden with pigment Eosinophilic droplets in hepatocytes Amyloidosis Foci of clear cells

Stomach d

2 3

0 3 0

0 1 1 0

Cysts Accumulations of RES-cells

Liver d

Slight amyloidosis Tubular nephrosis: slight moderate

Kidneys d

Focal e m p hys em a Oedema Thickened wall o f arteries Minimal degree of peribronchiolar adenomatoid proliferation

1

0

0

0

1 1 0 1 0

1 0

2 2 2

0 1 0 1

Fig. 1. Normal olfactory epithelium in the nasal cavity. Male hamster, air exposure, H & E, x 400.

Fig. 2. Thinned layer of irregular olfactory epithelium almost completely devoid of sensory cells. Note the groups of sensory cells in the lamina propria. Male hamster, furfural exposure, week 22, H & E, × 375.

136

Fig. 3. Papillary furfural exposure,

and intraepithelial cyst-like week 81, H & E, X 200.

formations.

Nasal

septum,

male

hamster,

cells with huge round nuclei occasionally containing many nucleoli (Fig. 4). In a few animals the lumen of some of the glands seemed to be slightly distended. A much more striking finding in furfural-exposed hamsters was the occurrence of large cyst-like, glandular structures in the lamina propria beneath the olfactory epithelium. Their lumen was often filled with homogeneous, eosinophilic, periodic acid-Schiff-positive material often containing cellular debris (Fig. 5). These structures were either lined by flat or cuboidal epithelium, or most often by tall epithelial cells with cilia or many long cilialike projections. In several instances the lining epithelium, in which goblet cells were never encountered, was found to be continuous with the surface epithelium (Fig. 6). This finding suggests that infolding and downward growth of surface epithelium are main factors in the formation of these cystlike structures. On the other hand, genesis from Bowman’s glands cannot be excluded. The cyst-like formations were, along with normal or fairly normal glands of Bowman, almost exclusively found beneath surface epithelium which resembled more an irregular type of respiratory epithelium devoid of goblet cells than olfactory epithelium. However, since Bowman’s glands occur only in areas covered with olfactory epithelium, the respiratory-like epithelium very probably represents olfactory epithelium having lost its sensory cells and, therefore, mainly consisting of basal and sustentacular cells, the latter of which may easily be mistaken for ciliated cells. On the other hand, the

137

Fig. 4. Greatly enlarged epithelial cells of the glands of Bowman. Note the huge nucleus in one of the enlarged cells and the irregular and damaged olfactory epithelium. Male hamster, furfural exposure, week 81, H & E, X 375.

Fig. 5. Cyst-like structures in the lamina propria of the nasal septum. Note the thin layer of olfactory epithelium and the large cell with big nucleus of Bowman’s gland (arrow). Male hamster, furfural exposure, week 81, H & E, X 160.

138

Fig. 6. Surface epithelium continuous with epithelium lining a cyst-like structure. Note the presence of both Bowman's glands and tall epithelia] cells with cilia or cilia-like projections. Male hamster, furfural exposure, week 81, H & E, X375. possibility t h a t the o l f a c t o r y epithelium in these regions was replaced by a respiratory t y p e o f epithelium c a n n o t be excluded. It may even be considered an obvious alternative because the respiratory epithelium present in the nasal cavity and o t h e r segments o f the respiratory tract was n o t affected by furfural, very p r o b a b l y because this t y p e o f epithelium is less sensitive or even insensitive to the irritating action o f furfural. Comparison o f the alterations seen in the nasal cavity o f animals killed at the end of the exposure period (week 52) with those f o u n d in hamsters killed terminally (in week 81 after a n o n - e x p o s u r e period o f 6 m o n t h s ) did n o t p r o d u c e evidence of recovery of the furfural-induced lesions. On the o t h e r hand, there was also no evidence o f progression of the lesions, and neoplasms in the nasal cavity were n o t observed. There were no changes in o t h e r parts o f the respiratory tract or outside the airway system t h a t could be ascribed to furfural (Table III). Respiratory tract tumours. There were no significant differences in incidence, site and t y p e of BP- and DENA-induced respiratory tract t u m o u r s b e t w e e n hamsters exposed to air and those exposed to furfural vapour (Tables IV and V). F r o m calculations (according to the m e t h o d described by Saffiotti et al. [9] ) o f the probabilities for observation o f a respiratory tract t u m o u r at death of the animals, it appeared t h a t there were no appreciable differences in latent period o f BP- or DENA-induced t u m o u r s b e t w e e n air and furfural exposed animals.

139

o

b-a

-0.9% NaCl BP (18.2 rag) BP (36.4 rag) --

Furfural Furfural Furfural Furfural Furfural

0.9% NaCl BP (18.2 rag) BP (36.4 m g ) --

--

--

---DENA

----DENA

----DENA

---DENA

--

~44t

14 ) 14

15) 12

15 I

15 ) 0

(0%)

(0%)

0

5 (19%) 7 (23%) 7 (27%)

27 30 26

(0%)

3 (11%) 7 (29%) 11 ( 4 1 % )

o

28

27 24 27

28

0 (0%) 2 (7%) 14 (47%) 11 (41%)

29 30 27

4 (14%) 19 (63%) 12 (41%)

27

29 30 29

30

0 0 0

0

0 0 0

o

0 0 0

0

0 0 2

0

nasal cavity

0 1 2

0

1 0 3

o

0 0 3

0

0 1 7

0

larynx a

a T u r n o u t s f o u n d in t h e i n t e r l a r y n g e o t r a c h e a l z o n e are classified as laryngeal n e o p l a s m s .

Furfural Furfural Furfural Furfural Furfural

Air

Air Air Air Air

-0.9% NaCl BP (18.2 rag) BP (36.4 m g )

0.9% NaC1 BP (18.2 m g ) BP (36.4 m g ) --

Air Air Air Air

Females

--

Air

Males

Effective number of animals

1 4 5

0

0 3 8

o

2 8 5

0

2 8 3

0

trachea

respiratory tract (total)

Subcutaneous injection of

Inhalation of

Intratracheal instillation of

N u m b e r o f animals w i t h t u m o u r s o f t h e

Treatments

0 1 0

0

1 1 2

o

0 2 3

0

1 3 3

0

bronchi

4 5 0

0

1 5 0

o

0 6 0

0 "

2 13 0

0

lungs

5 12 7

0

3 9 13

o

2 16 11

0

5 27 15

0

Total number of respiratory tract turnouts

I N C I D E N C E A N D SITE O F R E S P I R A T O R Y T R A C T T U M O U R S IN H A M S T E R S E X P O S E D TO A I R O R F U R F U R A L V A P O U R A N D T R E A T E D I N T R A T R A C H E A L L Y WITH BP O R S U B C U T A N E O U S L Y WITH D E N A

T A B L E IV

Papillomas and carcinomas of the trachea and pulmonary adenomas were the most common types of tumours induced by BP (Table V). In the low BP-dose group only 1 malignant t u m o u r was found in the respiratory tract viz., a pulmonary adenosquamous carcinoma of a female exposed to furfural and killed in week 78. Polyps and papillomas of the larynx and trachea were most frequently encountered in animals treated with DENA (Table V). Only 1 malignant turnout was found, viz., an adenocarcinoma in the nasal cavity of a male exposed to air. T u m o u r s outside the respiratory tract. Benign tumours found in organs other than the respiratory tract were: 1 light cell tumour and 1 papillary adenoma of the thyroid and 1 haemangioma of the spleen. Malignant tumours observed were: 2 lymphoreticular malignancies of the thymus, 1 osteosarcoma of a rib, 1 hepatocellular carcinoma and 1 melanosarcoma of the skin. 3 of these malignant tumours occurred in the low BP-dose group, the 2 others in the high BP
141

b~

b.a

DENA e

0 0

0 0

Bronchi Polyp/Papilloma Adenocarcinoma

0

0

Trachea Polyp/Papilloma S q u a m o u s cell carcinoma Anaplastic carcinoma Sarcoma

0

Larynx Polyp/Papilloma

30

0 0

15

15

1 1

0 0

2 1

1

0

1 0

5

1

0 0

30

2

0

0 0

29

3 0

0 0

0

3

7

1 1

29

15

0 0

0 0

0

0

0

0 0

27

12

0 0

0 0

0

2

0

0 0

29

BP c (18.2 mg)

___a 0.9% NaC1 b

Bpd (36.4 rag)

_ a 0.9% NaCI b

BP c (18.2 mg)

Inhalation of furfural

I n h a l a t i o n o f air

Incidence of tumours

Nasal cavity Polyp]Papilloma Adenocarcinoma

No. o f a n i m a l s examinedf

Males

Site a n d t y p e of tumours

2 0

0 0

4

4

0

0 0

30

BP d (36.4 m g )

3 0

0 0

0

5

3

0 0

27

DENA e

SITE, T Y P E A N D I N C I D E N C E O F R E S P I R A T O R Y T R A C T T U M O U R S IN H A M S T E R S E X P O S E D T O A I R O R F U R F U R A L V A P O U R A N D T R E A T E D I N T R A T R A C H E A L L Y W I T H BP O R S U B C U T A N E O U S L Y W I T H D E N A

TABLE V

¢,o

14

1 0 0

0 0 0

0 0

5

0 1

2

1

0

24

1

1

11 2

a No f u r t h e r t r e a t m e n t . b G i v e n i n t r a t r a c h e a l l y (0.2 ml), w e e k l y d u r i n g 52 weeks. c G i v e n i n t r a t r a c h e a l l y in 52 w e e k l y doses o f 0 . 3 5 m g / h a m s t e r . d G i v e n i n t r a t r a c h e a l l y in 52 w e e k l y doses o f 0 . 7 0 m g / h a m s t e r . e G i v e n s u b c u t a n e o u s l y in 17 3 - w e e k l y doses o f 0 . 1 2 5 u l / h a m s t e r . f A few h a m s t e r s w e r e lost t h r o u g h c a n n i b a l i s m o r autolysis.

Adenoma Adeno-squamous carcinoma Adenocarcinoma

Lungs

Polyp/Papilloma Adenocareinoma

Bronchi 1 0

0

0

0 0

0

0

Trachea

Polyp/Papilloma S q u a m o u s cell carcinoma

1

27

0

0

28

0

0

0

14

2 0

0 0

Polyp/Papilloma

Larynx

No. o f a n i m a l s examined f

Females

Adenoma Adenocarcinoma S q u a m o u s cell carcinoma Anaplastic carcinoma

Lungs

0 0

0

0

8

27

0

0

0 0

14

0 0

0

0

0

28

0

0

0 0

14

1 0

4

0

1

27

0

0

0 0

0 1

5

2

2

30

0

0

6 0

0 0

0

0 0

0

5

26

0

0

0 0

weekly by intratracheal instillation during a period of 36 weeks, and in which no furfural-related tumours were observed [2]. In this instillation experiment slight indications had been obtained of furfural potentiating the carcinogenic effect of BP on the respiratory tract. However, in the present study there was no evidence of an enhancing effect of furfural on either the BP- or the DENA-carcinogenesis. It seems, therefore, justified to attach little, if any, significance to furfural as a co-factor in respiratory tract carcinogenesis. ACKNOWLEDGEMENTS

The authors thank Mrs. A. Smit-Spaapen, Messrs. P. van den Heuvel, F. Hendriksma, Th. Dirksen, J. Catsburg and H. van Trierum for excellent technical assistance, Miss F. de Vrijer for help with the preparation of the manuscript, and Drs. A.P. de Groot and R. Kroes for critically reviewing the manuscript. REFERENCES 1 R.F. Brenkman, Regeneratie en metaplasie van respiratoir epitheel, Thesis, University of Leiden, 1970. 2 V.J. Feron, Cancer Res., 32 (1972) 28. 3 V.J. Feron and A. Kruysse, J. Toxicol. Environ. Health 3 (1977) 379. 4 V.J. Feron, A. Kruysse, H~P. Til and H.R. Immel, Toxicology, 9 (1978) 47. 5 D. de Jong and V.J. Feron, Biotechniek, 10 (1971) 66. 6 A. Kruysse, Acute inhalation toxicity of furfural in hamsters, Central Institute for Nutrition and Food Research TNO, Report no. R 3853, 1972. 7 A. Kruysse, V.J. Feron and H.P. Til, Arch. Environ. Health, 30 (1975) 449. 8 A. Kruysse, V.J. Feron and A.M. Verbeek, TNO-Nieuws, 25 (1970) 177. 9 U. Saffiotti, R. Montesano, A.R. Sellakumar, F. Ceils and D.G. Kaufman, Cancer Res., 32 (1972) 1073. 10 W.L. Sutton, J. Occup. Med., 5 (1963) 212.

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