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Chronic toxicity and oncogenicity of inhaled methyl acrylate and n-butyl acrylate in Sprague-Dawley rats
Fd Chem. Toxic. Vol. 29, No. 5, pp. 329-339, 1991 Printed in Great Britain.All rights reserved
0278-6915/91 $3.00+ 0.00 Copyright © 1991PergamonPresspie
CHRONIC TOXICITY A N D ONCOGENICITY OF INHALED METHYL ACRYLATE A N D n-BUTYL ACRYLATE IN S P R A G U E - D A W L E Y RATS* W. REININGHAUS,A. KOESTNER'~and H.-J. KLIMISCH~ Institut fiir Biologische Forschung, Kfln, Germany, t'Department of Pathology, Michigan State University, East Lansing, USA and :~Department of Toxicology, BASF AG, Ludwigshafen, Germany (Accepted 6 February 1991) Abstract--No exposure-related clinical signs or lesions of systemic toxicity and no oncogenic responses were observed in male and female Spragne-Dawley rats exposed by inhalation to methyl acrylate (MA) or n-butyl acrylate (BA) vapours, at concentrations of 0, 15, 45 and 135 ppm. The rats were wholebody-exposed 6 hr/day, 5 days/wk, for 24 consecutive months. There was a 6-month post-exposure observation period for subgroups of BA-exposed rats. Atrophy of the neurogenie epithelial cells and hyperplasia of reserve cells were observed in the nasal mucosa of all MA- and BA-exposed groups. These changes were dose related and mainly affected the anterior part of the olfactory epithelium. Opacity and neovasculadzation of the cornea were seen in all MA-exposedgroups and in the group exposed to 135 ppm BA. These toxic effects of the olfactory epithelium and cornea were attributed to the known irritancy of MA and BA. In the BA subgroups kept for a 6-month post-exposure observation, reconstructive effects, such as replacement of altered olfactory epithelium with respiratory epithelium, and partial regression of corneal neovascularization were observed.
INTRODUCTION The use of acrylic acid esters is widespread in industrial and consumer products. Methyl acrylate (MA; CH2~-------CHCOOCH3) and n-butyl acrylate (BA; CH2=CH--COOC4H9) are mainly used for the production of synthetic fibres and for polymer dispersions. The risk of human exposure to vapours of these substances occurs particularly during processing, because of the high vapour pressure. The occupational health threshold limits for MA and BA were set at 10 ppm (American Conference of Governmental Industrial Hygienists, 1986) or at 5 ppm MA and 10 ppm BA (Deutsche Forschungsgemeinschaft, 1985). MA and BA are irritating to the skin and mucous membranes (Clayton and Clayton, 1981). No data concerning the subchronic or chronic toxicity of these acrylates have been published to date. MA and BA as well as acrylic acid, a metabolite common to this class of compounds (Miller et al., 1981b), were not found to be mutagenic in the Salmonella/microsome assay (Florin et al., 1980; Ishidate et al., 1981; Lijinskij and Andrews, 1980; McMahon et al., 1979; Waegemaekers et al., 1983). MA increased chromosomal aberrations in vitro (Ishidate et al., 1981) and tested positive in the micronucleus assay in mice at ip doses of 37.5 mg/kg body weight (Przybojewska et al., 1984). BA produced no chromosomal aberrations in the bone *Presented in part at the Annual Meeting of the Society of Toxicology, Atlanta, GA, USA, March 1984. Abbreviations: BA =n-butyl acrylate; MA = methyl acrylate.
marrow cells of Chinese hamsters (Engelhardt and Klimisch, 1983) and tested negative in a dermal oncogenicity study in mice (DePass et aL, 1984). Since inhalation is a likely exposure route, the present study was conducted to assess the chronic toxicity and oncogenic potential of inhaled MA and BA vapours. Based on the 3-month inhalation studies with MA and BA in Sprague--Dawley rats (H. H. Klimisch, K. Deckardt, K. O. Freisberg and D. Mirea, unpublished data, 1978), a vapour concentration of 135 ppm was selected for both substances as the 'maximum tolerated doses', in accordance with NCI guidelines (Sontag et al., 1976). These authors found that at MA and BA concentrations of 211 ppm, the body and organ weights were significantly affected. Toxic effects in organs and tissues at this concentration were severe enough to expect a shortening of the lifespan and weakening of the defence mechanisms, thereby interfering with the objective of this study. At 108 ppm marginal toxic effects were still consistently observed. MATERIALS AND METHODS Experimental design. Two independent whole-body inhalation studies of identical design were run in parallel. In one study, four groups of 86 male and 86 female rats were exposed to 0, 15, 45 and 135 ppm MA vapour (equal to 0, 58, 173 and 519 mg/m3); in the other study, the same number of rats was exposed to equal volume concentrations of BA vapour (equal to 0, 86, 258 and 773 rag/m3). During the initial 13-wk period, both the MA and BA concentrations were 0, 5, 15 and 45 ppm. All groups, including the control -groups exposed to air, were whole-body exposed
329
330
W . REININGHAUS el al.
Table 1. Number of rats killed at scheduled dissections in control, MA and BA inhalation groups No. of rats killed MA Exposure (month)
Group
12
BA
Male
Female
Male
Female
All
10
10
10
10
18
All
15
15
15
15
24
Control Low Medium High
46 50 53 52
49 48 47 46
10 10 10 10
10 10 10 10
24 + 6 (post-exposure)
Control Low Medium High
-----
-----
24 26 28 29
34 21 28 21
for 6 hr/day, 5 days/wk, for 24 consecutive months. For MA- and BA-exposed groups, interim kills were carried out after 12 and 18 months of exposure (Table 1). The final killing was carried out after 24 months of exposure, except for those BA rats kept for a 6-month post-exposure observation and recovery period (Table 1). Test substances. MA (purity >99.8%, main impurities methyl propionate and ethyl acrylate) and BA (purity >99.5%, main impurities butyl propionate and isobutyl acrylate), stabilized with 50 ppm monomethyl hydrochinone, were supplied in 5-kg lots (BASF, Ludwigshafen, Germany). Each lot was analysed by gas chromatograph (PEG-400, 50°C, isothermal, flame ionization detection) before and after use in order to confirm the purity and stability. Exposure and analyses. The vapours of the test substance were generated from the liquid form, which was introduced at a constant flow into heated evaporators (approx. 40°C for MA and 120°C for BA) through which a constant flow of air (0.3-3 m3/hr) was also passed. The inhalation chambers were constructed of stainless-steel and glass, each having a volume of 4.9 m 3 and a capacity for 172 rat (1 dose group). The chambers were ventilated with a continuous vertical stream of conditioned (21°C, 55% relative humidity) fresh air (approx. 60 m3/hr). The air was mixed with the generated vapours of the test substance in such a manner that the desired chamber concentrations of the substances could be obtained (Table 2). The control rats were exposed in identical chambers to fresh air only. During each daily exposure period the concentrations of the test substances in the chamber atmosphere were repeatedly determined for 10 min every hr, using an infra-red gas photometer (path length = 2.25 m at a wavelength of Table 2. Nominal and measured concentrations of MA or BA inside inhalation chambers of rats during months 3-24 of exposure Concn of test substance (ppm) MA Group Control Low Medium High
BA
Nominal Measured SD (M) (M) (%) 0 15 45 135
0 15.6 46.3 140.3
-5.1 5.0 5.1
M = mean
Nominal (M) 0 15 45 135
Measured SD (M) (%) 0 14.8 44.3 133.7
-5.9 5.5 6.3
8.3 #m; Miran IA, Foxboro Analytical, S. Norwalk, CT, USA) or a total hydrocarbon flame ionization detector (RS5; Ratfisch Instrumente, Miinchen, Germany). The vapour in the chambers was analysed by gas chromatography (PEG-400, 50°C, isothermal, flame ionization detection) in order to confirm the purity of the exposure atmosphere. Animals. Male and female Sprague-Dawley rats (WlGA, Versuchstier-Zuchtanstalt, Sulzfeld, Germany), approx. 35 days old, were randomly allocated to each group. The control, MA- and BA-exposed rats were housed and treated in three separate airconditioned rooms. Animal observation. All rats were checked for their general condition and for signs of toxicity before and after each daily exposure, and once daily during the post-exposure observation period. Body weight, food consumption, reflexes and any abnormal tissue masses were recorded weekly. Rats found dead were autopsied on the same day. Moribund rats were killed and autopsied. Haematology and urinalysis. Following anaesthesia with diethyl ether, administered shortly before the rats were killed, blood was collected from the orbital sinus of all rats, and erythrocyte and leucocyte counts were determined. In addition, those rats from the control groups and those exposed to MA and BA at 135ppm, which were killed as scheduled, were examined with respect to reticulocyte, normoblast and differential leucocyte counts as well as to number of erythrocytes with Heinz bodies, packed cell volume, erythrocyte volume, haemoglobin content and haemoglobin concentration. Bone marrow smears were prepared from the rats of these groups and also from all moribund rats. Urine was collected from all rats scheduled for autopsy. The determinations included volume, colour, transparency, pH and the concentrations of protein, glucose, bilirubin, urobilinogen as well as ketone bodies, occult blood and sediments. Ophthalmology. Shortly before the rats were autopsied, the eyes were examined for external changes and the pupillary reflex. Changes in the anterior part of the bulbus (slit lamp; Zeiss, Oberkochem, Germany) and changes of the fundus (ophthalmoscope; Zeiss, Jena, Germany) were examined in mydriasis (1% atropin). Gross autopsy. The rats were killed by exsanguination under diethyl ether anaesthesia (no previous diet deprivation). A complete autopsy was performed on all rats. Body weights and the absolute and relative weights (to body weight and brain weight) of all major organs were determined for each rat. All tissues with gross lesions and representative sections of organs and tissues (according to the OECD requirements for "combined chronic toxicity/carcinogenicity studies", with the exception of the accessory genital organs (OECD, 1981)) were preserved in a 4% neutral formaldehyde solution. The testes were preserved in Bouin's fixative and the lumbar vertebrae in Schaffer's fixative. Histopathology. Tissue sections were embedded in paraffin, cut at 5 #m, and stained with haematoxylin and eosin. The nasal cavity, liver, and kidney sections were additionally stained with periodic acid-Schiff stain (Horobin, 1988). The nasal cavity and lumbar
MA and BA inhalation in rats vertebrae were decalcified in an ultrasonic bath with 5% nitric acid before embedding. Frozen sections of the liver and the kidney were stained with Sudan III. The nasal cavity (levels 1, 2 and 6, according to Stromberg and Hebel, 1976), larynx, trachea, lungs and liver of all rats (344 rats/sex/test substance) as well as all tissues having gross lesions were examined. The heart, kidneys, urinary bladder, ovaries, pituitary gland, thyroid, adrenals, brain, spleen, lumbar vertebrae and lymph nodes from all rats of the control groups and those exposed to M A and BA at 135ppm (172 rats/sex/test substance) were also examined. In addition, at each scheduled autopsy 11 tissue samples from each of at least 10 randomly selected male and female rats from the control and 135-ppm groups were histologically examined (between 40 and 65 rats/sex/test substance). Furthermore, an extensive histological examination was performed on all rats found dead or moribund (98 MA- and 197 BA-exposed rats). All changes were graded according to severity (Thomas, 1978). All neoplastic changes were classified according to tissue of origin, type and biological behaviour if possible. Statistical analysis. The rats were allocated to groups at the beginning of the study and selected for scheduled autopsies using computer-assisted randomization procedures. A comparison of the exposure groups with their respective control group (each sex separately) was made setting the twosided fiducial limit ~ = 0.05 as a critical level of significance for all tests, without corrections for multiple testing. Mortality was analysed using the life-table method (Armitage, 1971), after accounting for non-spontaneous deaths. Body weight, food consumption, organ weights and all haematological parameters with a quasi continuous range of values were compared using Student's t-test or its modification according to Welch (Clauss and Ebner, 1970). Moribund rats, rats found dead and rats with apparently outlying values were excluded from routine statistics; findings in those rats were interpreted separately. The total incidences of frequently observed histological changes were analysed using contingency tables (Sokal and Rohlf, 1969). After classifying the tumours as 'incidental' or 'fatal', the incidence of tumour types and the incidence of turnouts in different organs were analysed using a two-sided chisquare test corrected for 'animal at risk', as suggested by Peto et al. (1980). This analysis is sensitive to heterogeneous total tumour incidences, as well as to time differences in tumour appearance. Additional non-parametric tests (Mann and Whitney, 1947; Pfanzagl, 1978) were used in order to back up the interpretation of the results after data screening.
331
30-
::: 135controls4515 ppmPPmPPm
.':t"
20-
10-
I
I
I
52
26
78
I
104
I
130
2 v
3O
20
ij
10 ¸ / 0
0
I
26
ff~l
52
I
78
I
I
104 130 Week of study
Fig. 1. Life-table corrected cumulative mortality of Sprague-Dawley rats exposed to MA vapour, +SE. Data were censored for scheduled killings. No significant differences were observed between groups. Overt signs of toxicity or reactions related to exposure were not observed. After 24 months, the cumulative incidence of rats with palpable tissue masses was approx. 6% for male rats and approx. 12% for females. No dose-related trends were observed. The mean life-table-corrected cumulative mortality was approx. 20% (Figs 1 and 2). During the 6-month post-exposure period (BA only) the cumulative mortality increased to approx. 45%. On the whole, the mortality data, though heterogeneous for BA-exposed female rats, did not indicate that exposure to the test substances had an adverse effect on longevity.
Body weight and food consumption
The actual and calculated concentrations agreed within a few per cent. The standard deviation was less than 10% (see Table 2).
At the start of the exposure period, the mean body weights were 183 g for male rats and 157 g for female rats. For both sexes the relative standard deviations were approx. 10% (Figs 3 and 4). From wk 1 to wk 13, during which the rats were exposed to 1/3 of the final test substance concentration, the bodyweight gain of all groups was practically identical. From wk 15 to the end of the exposure period, the body weights of the male and female rats exposed to M A at 135 ppm were slightly but significantly lower than those of other groups (approx. 4%). The food consumption of the male and female rats exposed to BA was consistently lower (approx. 4%) than that of the control groups. The food consumption returned to normal after the end of the exposure period. These effects indicate that the 135-ppm concentration meets the criteria for the maximum tolerable dose according to NCI guidelines.
Animal observation and mortality
Haematology and urinalysis
In general, the appearance and behaviour of the rats were normal. During exposure, all rats were quiescent.
With regard to the haematological parameters, there was no indication that the test substances
RESULTS Exposure concentrations
W. REININGHAUSel al.
332
T
controls
60040-
30400-
~[xMale / 20-
200-
10-
:
If~ f
0
15 ppm ppm ppm
: 45 : 135 i
i
26
i
52
i
78
i
104
130
Week of study
/~
Fig. 4. Body weights of rats exposed to BAvapour.
(Alt et al., 1980; Couser and Stilmont, 1975; Galaske et al., 1980; Turnbull et al., 1985).
40-
Organ weights and gross autopsy
30Female
20-
10-
0 0
2'~
s'2
48
164
i]0
Weekof study Fig. 2. Life-table-corrected cumulative mortality of Sprague--Dawley rats exposed to BA vapour, +SE. In female rats, groups exposed to 15 and 135 ppm differed statistically significantly from the other BA exposed groups, but not from MA groups.
Autopsy revealed only incidental patho-anatomical changes or gerontic patho-anatomical changes, typical of aged Sprague-Dawley rats. Gross changes generally correlated with the respective histological lesions. The autopsies performed after 24 months mainly revealed 5-17% lower weight of kidneys, thyroid, heart and liver in some of the M A - or BA-exposed rats, compared with respective controls. Despite their statistical significance, these findings were not considered to be toxicologically significant, because they were neither consistently dose dependent nor did they correlate with histopathological changes. Ophthalmology
caused any responses. Occasional differences between the control and exposed groups were interpreted to be incidental findings. With regard to the urine parameters, there was no indication of exposurerelated effects. Elevated urinary erythrocyte counts and protein concentrations were observed in most of the male and in several female rats of all groups including the control groups. These findings suggest degenerative and inflammatory changes such as nephrosis, nephritis and pyelitis, which are common spontaneous changes in ageing rats of this strain
v
Table 3. Frequency of corneal neovascularization or parenchymal cloudiness in rats (male plus female) after inhalation of MA or BA vapour Relative no. of rats (%) MA cones (ppm) BA cones (ppm) Exposure (month) 0 15 45 135 0 15 45 135 12 0 4 8 33 0 0 4 16 18 0 10 17 63 0 3 7 13 24 [ 10 30 59 3 4 2 34 24 + 6 (post3 24 3 28 exposure)
600
m
400
200 ~
o
l
s
: 15 :
ppm 45 p p m ppm
: 135 0
26
i
i
52
78
i
i
104 130 Week of study
Fig. 3. Body weights of rats exposed to MA vapour.
Centrally localized or diffuse stippling of the corneal epithelium and cloudiness of the corneal parenchyma with various degrees of neovascularization were observed in M A - and BA-exposed rats. The incidence of the changes in the corneal parenchyma increased with the concentration of the test substances and length of exposure, and was similar in male and female rats (Table 3). After 24 months, a significant increase of parenchymal changes was evident in all MA-exposed groups and in the groups exposed to BA at 135 ppm. The neovascularization
Absolute no. of rats~f
I
14"
37 ~
87 ~
3
5
5
42 ~"
t 150-157 Rats wereexamined per group, excludingrats re-examined after 6 months post-exposure. Those values marked with asterisks differ significantly(chi-square test) from the corresponding control value (*P < 0.05).
MA and BA inhalation in rats Table 4. Frequency of reserve-cell hyperplasia in level 2 of the nasal mucosa of rats (male plus female) after inhalation of MA or BA vapour Relative no. of rats (%) MA conca (ppm) Exposure (month) 12 18 24 24 + 6 (postexposure) Absolute no. of rats'~
0
BA concn (ppm)
15
45
135
0
15
45
135
0 0 1
0 0 6
65 88 96
91 100 99 --
0 0 0 0
0 0 24 1
0 2 56 40
64 97 92 41
1
9
154"
168"
0
10"
65* 115"
t167-171 Rats were examined/group, and those values marked with asterisks differ significantly (chi-square test) from the corresponding control value (*P < 0.05).
regressed to empty 'ghost vessels' 6 months after the end of exposure. Other changes in the eye, in particular cataracts, which were observed sporadically in all groups, were not related to test substance exposure.
Non-neoplastic lesions The histological examinations revealed concentration-dependent lesions in the nasal mucosa at the level of the dorsal lamella of the second endoturbinate (level 2, according to Stromberg and Hebel, 1976). In a few male rats exposed to M A or BA at 15 ppm, a slight atrophy of the neurogenic part of the olfactory epithelium was observed (Plates 1-3 and Table 4). At 45 and 135 ppm, almost all MA-exposed rats developed a partial loss of the columnar cell layer, with an accompanying stratified reserve-cell hyperplasia. The lesions were mainly confined to the most anterior region of the olfactory epithelium, which lines the dorsal part of the nasal cavity at this level. Rats exposed to BA had distinctly less severe changes, which were qualitatively similar to the changes following M A exposure. Male and female rats were affected to a similar degree. Most changes developed during the first 12 months of exposure and increased only moderately with ongoing exposure.
333
During the 6-month post-exposure period, the altered olfactory epithelium was replaced by respiratory epithelium. No treatment-related changes were detected in the posterior nasal cavity, which is mainly lined with olfactory epithelium (level 6, according to Stromberg and Hebel, 1976). No irritative changes were observed in the larynx, trachea or lungs of the exposed rats. Occasional lesions in other organs did not indicate a trend related to the test substance concentration. Such lesions were considered to be strain- and agerelated.
Neoplastic lesions Up to the 18th month the cumulative incidence of primary tumours was only approx. 2% for male rats and approx. 4% for females. Such a low incidence precludes the use of further analysis of the time of tumour appearance. There was also no doserelated trend in the cumulated incidence of primary tumours. In Tables 5 and 6 the total numbers of rats with tumours, classified according to tumour types, are listed. Among the MA-exposed rats the relative tumour rates for the malignant epithelial and leukaemic changes were significantly heterogeneous (Table 5). The number of male rats with malignant epithelial tumours was lower in the 15- and 45-ppm groups than in the control and 135-ppm exposed groups. These tumours were located in the thyroid, testes and urinary bladder. Among female rats exposed to MA, the incidences of epithelial turnouts were homogeneously distributed. Since there was no dose response, these heterogeneous incidences of malignant epithelial turnouts among MA-exposed male rats are considered to be incidental. The number of MA- and BA-exposed male rats with leukaemic changes was higher than in the control groups. As shown in Table 7, the highest incidence of leukaemic changes in males occurred in the groups exposed to medium concentrations of M A and BA. For the male rats exposed to the high
Table 5. Total number of rats with tumourst after inhalation of MA vapour MA concn (ppm) Male rats Tumour type
Female rats
0
15
45
135
0
15
45
135
Benign Epithelial Mesenchymal Fibroadenoma~/ Other benign
30 20 5 l 5
24 12 6 0 8
21 17 3 0 3
24 16 4 0 4
33 16 5 14 2
39 27 4 19 1
34 27 2 l0 3
32 17 2 17 3
Malignant Epithelial Mesenchymal Leukaemic Other malignant
14 8* 6 0* 0
11 4* 5 3* 1
I1 3* 1 7* 1
19 12" 8 0* l
9 4 2 1 2
8 5 2 2 0
7 2 3 1 1
7 2 2 2 1
Total§
38
33
27
36
39
43
38
38
8
6
8
8
7
15
9
8
Two or more tumour types
t86 Rats were autopsied and examined per sex and group. ~Non-assignable regarding tissue type. §The number of rats with a specific tumour type was counted independently from the appearance of types in the same rat. Therefore, the line 'total" does not represent the sum of all listings, but the total number of rats beating a tumour of any type. Those values marked with asterisks differ significantly (chi-square test, after 'at risk' correction) in the heterogeneous distribution of relative tumour rate between groups (*P < 0.05).
W . R~tqINGtIAUS et al.
334
Table 6. Total number of rats with tumours* after inhalation of BA vapour BA conch (ppm) Male rats Tumour type
Female rats
0
15
45
135
0
15
45
Benign Epithelial Mesenchymal Fibroadenoma~: Other benign
34 23 2 3 I1
24 16 3 2 8
26 13 7 0 11
23 19 3 0 7
48 30 1" 26 4
48 25 7* 29 3
50 34 2* 20 7
135 39 23 1* 20 4
Malignant Epithelial Mesenchymal Leukaemic Other malignant Total§
16 7 10 0 2 41
19 9 9 3 0 35
18 2 10 5 1 39
14 5 7 3 0 34
15 6 10" 1 0 54
8 7 1" 0 0 50
11 7 4* 0 0 52
l0 7 4* 0 0 41
Two or more tumour types
13
12
10
10
21
21
19
17
t86 Rats were autopsied and examined per sex and group. :[Non-assignable regarding tissue type. §The number of rats with a specific tumour type was counted independently from the appearance of additional tumour types in the same rat. Therefore, the fine 'total' does not represent the sum of all listings but the total number of rats bearing a tumour of any type. Those values marked with asterisks differ significantly (chi-square test, after 'at risk' correction) in the heterogeneous distribution of relative tumour rate between groups (*P < 0.05).
BA concentration, the incidence was only 3.5% compared with 5.8% for the medium-concentration group. F o r the male rats exposed to the high M A concentration, the incidence was 0. For the females exposed to BA, only one case was recorded, which occurred in the control group. The incidence for the female rats exposed to M A was ncarly equally distributed among all groups. The overall incidence of leukaemic changes in the MA- and BA-exposed groups was well within the expected incidence of spontaneous leukaemia in Spragne-Dawley rats (Anver et al., 1982; Sher, 1982). Even the highest incidences for individual groups were comparable to the published incidences of spontaneous leukaemia in Sprague-Dawley rats. Furthermore, there was no dose-effect relationship. Based on published data, the incidence of leukaemic changes observed in the female rats was considerably lower than expected. The higher incidence of leukaemic changes in MA-exposed male rats does not seem to be an exposure-related finding. A statistical comparison of the BA-exposed groups (Table 6) showed that the number of female rats with benign and malignant mesenchymal tumours was heterogeneously distributed. These benign turnouts were mainly lipomas in the abdominal cavity and fibromas of the soft tissue (i.e. mainly skin/subcutis, as defined by Turusov, 1979). The malignant tumours were mainly sarcomas of the soft tissue. Since their incidence decreased with increasing BA concentration, they are considered to be incidental.
When primary tumours were grouped according to orgin and tumour type (Tables 8 and 9), there were only a few organs with significantly heterogeneous tumour incidences. Among the MA-exposed rats, the number of males with pituitary adenomas was distinctly higher in the control groups than in the exposed groups (Table 8). In female rats, the incidence of this type of tumour was higher in the 15- and 45-ppm groups than in the control and the 135-ppm groups. Based on the absence of a dose-effect relationship and the known incidence of spontaneous pituitary adenomas in ageing Spragne-Dawley rats, this statistical heterogeneity is considered to reflect random variability. Sarcomas had a random distribution. For instance, anaplastic soft tissue sarcomas (skin/subcutis) of male rats were restricted to groups exposed to M A at 15 and 135 ppm (Table 8). In BA-exposed male rats, the incidence of sarcomas in the thoracic cavity (fibro- lipo- and neurofibrosarcoma) was higher at 15 ppm than in all other groups in female rats. The incidence of soft-tissue sarcomas in fibrous connective tissue was highest in the controls (Table 9). On the whole, for both test substances the incidence of sarcomas varied significantly in all tissues or sites. There was no detectable dose dependency. Similarly, the incidence of all malignant mesenchymal tumours (Tables 5 and 6), which were mainly sarcomas of the soft tissue, does not indicate an exposurerelated tumour increase. Therefore, the varying rates of soft-tissue sarcomas are considered to reflect a
Table 7. Incidence of leukaemia, lymphoma and lymphnsarcoma in rats after inhalation of MA or BA vapour No. of rats with tumours* Test substance cnncn (ppm) Test substance
Sex
0
15
45
135
Total
No. of rats with turnouts in historical controlt
MA MA
Male Female
0 (0%) 1 (1.2%)
3 (3.5%) 2 (2.3%)
7 (8.1%) 1 (1.2%)
0 (0%) 2 (2.3%)
10 (2.9%) 6 (1.7%)
(3.7%) (3.0%)
BA BA
Male Female
0 (0%) 1 (1.2%)
3 (3.5%) 0 (0%)
5 (5.8%) 0 (0%)
3 (3.5%) 0 (0%)
I 1 (3.2%) 1 (0.3%)
(3.7%) (3.0%)
*Relative frequencies, given in parentheses, are based on 86 rats/group. t F r o m Sher (1982).
Plate I. Dorsomedial aspect of the nasal cavity of a control rat showing septum, intact olfactory epithelium, and the transitional region with the beginning of the respiratory epithelium (~7). Original magnification x 185.
~ ~ii~~!i!i~:~i:~i~!~ii~i~i~ii~i!!!i~i~i!i!~ii~ i~ i!i~
:
Plate 2. The same location after 24 months of exposure to 135 ppm BA vapour, showing a loss of columnar epithelium in the olfactory region with focal stratified reserve-cell hyperplasia and an intact respiratory epithelium (~7). Original magnification x 185.
Plate 3. The same location in a rat exposed to 135 ppm BA vapour for 24 months after a 6-month post-exposure period, showing respiratory epithelium (insert) in the olfactory region. Original mat,nification x 185.
MA and BA inhalation in rats
337
Table 8. Total incidence? of primary turnouts in rats after inhalation of MA vapour MA concn (ppm) Male rats 15 45
Female rats Tumour origin and type 0 135 0 15 45 135 Liver Adenoma, haemangioendothvlioma 2 1 0 0 2 5 3 5 Urinary bladder Papilloma 2 0 0 1 0 0 0 0 Carcinoma, sarcoma 1 2 0 3 0 0 I 0 Testis Leydig-cell adenoma, mesothelioma 3 6 7 6 . . . . Leydig-cell carcinoma 2 0 0 4 . . . . Ovary Thecoma, fibroma . . . . 0 1 0 3 Uterus Polyp . . . . 5 4 2 3 Carcinoma . . . . 0 0 0 1 Pituitary gland Adenoma 13" 2* 6* 6* 10" 21" 23* 9* Carcinoma 0 0 l 0 0 0 0 1 Thyroid Adenoma 0 2 3 1 0 2 2 I Carcinoma 3 1 0 2 l 0 0 0 Adrenal Adenoma, pheochromocytoma, unspecific benign tumour 5 6 4 3 1 0 0 0 Malignant pheochromocytoma 0 0 1 1 0 0 0 0 Soft tissue, unclassified Sarcoma 0* 4* 0* 6* l 1 1 0 Systemic, lymphoid tissue Lymphosarcoma, leukaemia 0 2 3 0 1 l 1 1 Non-lymphoid organ, parenchymal tissue Leukaemia, malignant lymphoma 0 0 3 0 0 0 0 0 Thymus, mesenchymal or epithelial tissue Thymoma 0 1 0 l 1 1 3 0 Mammary gland Adenoma, fibroadenoma l 0 0 0 14 19 II 17 Carcinoma 0 0 1 0 2 4 2 0 Abdominal cavity Lipoma I 3 0 2 5 1 2 2 Sarcoma, unspecific malignant tumour l 1 0 0 0 0 0 0 Other organs~ Benign tumour 9 6 4 4 2 2 1 1 Malignant tumour 5 4 3 6 2 3 1 3 ?86 Rats were autopsied and examined per sex and group. ~Cumulative incidence of tumours in organs with not more than two benign or two malignant tumours in any group. Those values marked with asterisks differ significantly(chi-square test, after 'at risk' correction) in the heterogeneous distribution of relative tumour rate between groups (*P < 0.05). heterogeneous s p o n t a n e o u s t u m o u r distribution a n d n o t a treatment-related incidence. DISCUSSION This 2 4 - m o n t h i n h a l a t i o n study in S p r a g u e Dawley rats revealed t h a t exposure to 15, 45 or 135 p p m M A or B A does n o t influence longevity or cause systemic toxicity detectable by haematological parameters, gross a n d histological examination, or urinalysis. This c o r r e s p o n d s to the results o f the subchronic ( 3 - m o n t h ) i n h a l a t i o n studies with the same c o m p o u n d s (H. H. Klimisch, K. Deckardt, K. O. Freisberg a n d D. Mirea, u n p u b l i s h e d data, 1978). In the nasal m u c o s a a n d c o r n e a b o t h test substances caused non-neoplastic changes, which were a t t r i b u t e d to the irritating effect o f acrylic esters. The changes were mainly restricted to a n a r r o w area o f the m o s t a n t e r i o r p a r t o f the olfactory epithelium. The sequence o f the observed changes p r o b a b l y begins with a t r o p h y of the neurogenic segment o f the epithelium, followed by progressive hyperplasia of the reserve cells a n d ultimately by a loss o f the u p p e r epithelial cell layer. A d v a n c e d changes are considered to be reversible pre-stages o f metaplasia.
Cellular atypia a n d infiltrative g r o w t h were n o t observed. D u r i n g the post-exposure period, the altered olfactory epithelium was replaced by respiratory epithelium. T h e changes in the nasal m u c o s a were qualitatively similar for b o t h substances, b u t less p r o n o u n c e d in the BA-exposed groups. It was s h o w n t h a t only m i n i m a l effects are caused by exposure to 15 p p m M A , a n d t h a t 15 p p m is the no-effect level for exposure to BA. Similar nasal changes have been observed in rats a n d mice in i n h a l a t i o n studies with ethyl acrylate a n d acrylic acid (Miller et al., 1981a a n d 1985). H u m a n exposure to M A or B A does n o t seem to pose a considerable health risk provided t h a t regulatory limit c o n c e n t r a t i o n s (Deutsche Forschungsgemeinschaft, 1985) are n o t surpassed for p r o l o n g e d periods o f time; even c o n c e n t r a t i o n s t h a t cause only reversible changes are so irritative t h a t a p r o l o n g e d exposure is precluded. C h a n g e s o f the eye were restricted to the corneal p a r e n c h y m a , where cloudiness with varying degrees o f neovascularization was observed. T h e incidence was time- a n d c o n c e n t r a t i o n - d e p e n d e n t in b o t h male a n d female rats. T h e no-effect level for M A was below 15 ppm, the level at which only m i n o r changes
W. REININOHAUSet al.
338
Table 9. Total incidencet of primary tumours in rats after inhalation of BA vapour MA concn (ppm) Male rats Female rats Tumour origin and type Liver Adenoma, angioma, haemangioendothelioma Sarcoma Urinary bladder Papilloma Carcinoma Testis Leydig-cell adenoma Leydig-cell carcinoma Ovary Fibroma, thecoma Uterus Polyp Sarcoma, carcinoma Pituitary gland Adenoma Carcinoma Thyroid Adenoma Carcinoma Adrenal Adenoma, gangiioneuroma, pheochromocytoma Malignant pheochromocytoma Pancreas (endocrine) Adenoma Unspecific malignant tumour Soft tissue, fibrous connective Fibroma Sarcoma Soft tissue Neurofibrosareoma Systemic, lymphoid tissue Leukaemia Thymus, mesenchymal or epithelial tissue Thymoma Mammary gland Adenoma, fibroma, fibroadenoma Carcinoma Thoracic cavity Sarcoma Abdominal cavity Lipoma, mesothelioma Sarcoma Other organs~/ Benign tumour Malignant tumour
0
15
45
135
0
15
45
135
l 2
0 0
0 0
I 0
4 0
2 0
4 0
0 0
2 0
1 1
1 0
3 0
0 0
0 0
1 0
0 0
7 1
2 3
4 l
5 1
. .
. .
. .
. .
.
.
.
.
l
0
4
l
. .
. .
. .
. .
7 2
5 1
7 2
6 1
8 1
8 l
3 0
4 l
12 0
17 1
22 l
14 0
2 2
3 0
2 1
7 1
4 4
2 l
6 0
1 3
Il 1
8 0
13 0
6 0
1 0
3 0
2 0
2 0
2 1
1 0
0 0
3 0
0 0
0 0
I 0
0 0
0 1
l 1
1 3
0 0
0 4*
2 0*
0 0*
0 1*
3 3 0
0 0 3
0 0 4
2 2 2
2 2 1
0 0 0
0 0 0
2 2 0
0
0
0
0
2
0
3
l
3 1
2 1
1 0
0 0
32 2
31 4
20 4
22 4
0*
3*
0*
0*
0
0
l
0
0 I
0 I
2 I
0 0
0 0
3 0
0 I
2 0
4 5
5 5
7 9
10 6
5 4
2 l
0 3
2 2
t86 Rats were autopsied and examined per group. :~Cumulative incidence of tumours in organs with not more than two benign or two malignant tumours in any group. Those values marked with asterisks differ significantly (chi-square test, after 'at risk' correction) in the heterogeneous distribution of relative tumour rate between groups (*P < 0.05).
were o b s e r v e d . F o r BA, t h e no-effect level was l o w e r t h a n 135 p p m . N e o v a s c u l a r i z a t i o n is a k n o w n n o n specific r e a c t i o n to c h r o n i c i r r i t a t i o n o f the c o r n e a ( H e n k i n d , 1978). I n a p r e v i o u s study, after t o p i c a l a p p l i c a t i o n o f m e t h y l m e t h a c r y l a t e similar c h a n g e s were i n d u c e d ( H o l y k a n d Eifrig, 1979). H o w e v e r , they were n o t o b s e r v e d in 2 7 - m o n t h i n h a l a t i o n studies w i t h ethyl acrylate o n F i s c h e r - 3 4 4 rats (Miller et al., 1985). A p e e r review s u m m a r y ( H e n s c h l e r , 1985) c o n c l u d e d t h a t such c o r n e a l c h a n g e s in rats c a n n o t be directly e x t r a p o l a t e d to h u m a n s . T h e fact t h a t such findings h a v e never b e e n o b s e r v e d in employees of an MA manufacturing company (G. Dietrich, p e r s o n a l c o m m u n i c a t i o n , 1985) f u r t h e r s u b s t a n t i a t e s this assessment. T h e h i g h e r irritancy o f M A as c o m p a r e d w i t h B A , i n d i c a t e d b y t h e different degree a n d f r e q u e n c y o f c h a n g e s in t h e o l f a c t o r y e p i t h e l i u m a n d in the c o r n e a l p a r e n c h y m a , m a y p a r t l y be c a u s e d by differences in
diffusibility a n d w a t e r / l i p i d solubility o f the t w o test s u b s t a n c e s (Tanii a n d H a s h i m o t o , 1982). I n general, the toxicity o f h o m o l o g o u s acrylates is r e p o r t e d to decrease w i t h i n c r e a s i n g m o l e c u l a r w e i g h t ( A u t i a n , 1975). O t h e r i n v e s t i g a t i o n s p o i n t to t h e i m p o r t a n t influence o f epithelial m e t a b o l i s m o n t h e u p t a k e a n d effect o f irritants (Stott a n d M c K e n n a , 1984). U n d e r the test c o n d i t i o n s o f t h e p r e s e n t study, e x p o s u r e to M A o r B A v a p o u r d i d n o t lead to a n i n c r e a s e d f r e q u e n c y o f any t u m o u r t y p e in a n y o r g a n t h a t c o u l d be a t t r i b u t e d to a n o n c o g e n i c p o t e n t i a l o f the test s u b s t a n c e . This is c o n s i s t e n t w i t h t h e results published on the absence of a dermal tumorigenic p o t e n t i a l o f B A ( D e P a s s et al., 1984).
Acknowledgements--This
study was sponsored by the BASF Aktiengesellschaft. The authors gratefully acknowledge the contributions of Professor H.-K. Koch, for ophthalmological evaluation (Universit~its-Augenklinik, Bonn,
MA and BA inhalation in rats Germany), and Professor C. Thomas, for histopathological evaluation (Pathologisches Institut, Universit~t Marburg, Germany). REFERENCES
Alt J. M., Hackbarth H., Dcerberg F. and Stolte H. (1980) Proteinuria in rats in relation to age-dependent renal changes. Laboratory Animals 14, 95-101. American Conference of Governmental Industrial Hygienists (1986) Documentation of Threshold Limit Values and Biological Exposure Indices. 5th Ed. ACGIH. Cincinnati, OH. Anver M. R., Cohen B. J., Lattuada C. P. and Foster S. J. (1982) Age-associated lesions in barrier-reared male Sprague--Dawley rats: a comparison between Hap: (SD) and Crl: COBS(R) CD(R) (SD) stocks. Experimental Aging Research 8, 3-24. Armitage P. (1971) Statistical Methods in Medical Research. pp. 408-414. Blackwell, Oxford. Autian J. (1975) Structure-toxicity relationships of acrylic monomers. Environmental Health Perspectives 11, 141-152. Clauss G. and Ebner H. (1970). Grundlagen der Statistik fiir Psychologen, P/idagogen and Soziologen. Harri Deutsch, Frankfurt a. M. Clayton G. D. and Clayton F. E. (Editors) (1981) Patty's Industrial Hygiene and Toxicology. pp. 2259. John Wiley & Sons, New York. Couser W. G. and Stilmant M. M. (1975) Mesangial lesions and focal glomerular sclerosis in the aging rat. Laboratory Investigations 33, 491-501. DePass L. W., Fowler E. H., Meckley D. R. and Well C. S. (1984) Dermal oncogenicity bioassays of acrylic acid, ethyl acrylate, and butyl acrylate. Journal of Toxicology and Environmental Health 14, 115-120. Deutsche Forschungsgemeinschaft (1985) Maximale Arbeitsplatz-Konzentration und biologische ArbeitsstoffToleranzwerte. Verlag Chemic, Weinheim. Engelhardt G. and Klimisch H. J. (1983) n-Butyl acrylate: cytogenetic investigations in the bone marrow of Chinese hamsters and rats after 4-day inhalation. Fundamental and Applied Toxicology 3, 640-641. Florin I., Rutberg L., Curvall M. and Enzell C. R. (1980) Screening of tobacco smoke constituents for mutagenicity using the Ames test. Toxicology 15, 219-232. Galaske R. G., Van Liew J. B. and Feld J. G. (1980) Urinary protein excretion in the rat: strain and age dependence. Contributions to Nephrology 19, 71-78. Henkind P. (1978) Ocular neovascularization. American Journal of Ophthalmology 85, 287-301. Henschler D. (Editor) (1985) Gesundheitsschiidliche Arbeitsstoffe. Toxikologisch-arbeitsmedizinische Begriindungen yon MAK-Werten: Methylacrylat. pp. 22. Verlag Chemie, Weinheim. Holyk P. R. and Eifrig D. E. (1979) Effects of monomeric methylmethacrylate on ocular tissues. American Journal of Ophthalmology 88, 385-395. Horobin R. W. (1988) In Understanding Histochemistry. pp. 119-121. Ellis Horwood, Chichester. Ishidate M., Jr, Sofuni T. and Yoshikawa K. (1981) Chromosomal aberration test in vitro as a primary screening tool for environmental mutagens and/or carcinogens. Gann Monographs on Cancer Research 27, 95-108. Lijinskij W. and Andrews A. W. (1990) Mutagenicity of vinyl compounds in Salmonella typhimurium. Teratogenesis, Carcinogenesis, Mutagenesis 1, 259-267. McMahon R. E., Cline J. C. and Thompson C. Z. (1979) Assay of 855 test chemicals in ten tester strains using a
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new modification of the Ames test for bacterial mutagens. Cancer Research 39, 582-693. Mann H. B. and Whitney D. R. (1947) On a test of whether one of two random variables is stochastically larger than the other. Annals of Mathematics and Statistics 18, 50-60. Miller, R. R., Ayres J. A., Jersey G. C. and McKenna M. J. (1981a) Inhalation toxicity of acrylic acid. Fundamental and Applied Toxicology 1, 271-277. Miller R. R., Ayres J. A., Rampy L. W. and McKenny M. J. (1981b) Metabolism of acrylate esters in rat tissue homogenates. Fundamental and Applied Toxicology 1, 410-414. Miller R. R., Young J. T., Kociba R. J., Keyes D. G., Bodner K. M., Calhoun L. L. and Ayres J. A. (1985) Chronic toxicity and oncogenicity bioassay of inhaled ethyl acrylate in Fischer-344 rats and B6C3FI mice. Drug and Chemical Toxicology 8, 1-42. OECD (I 981) OECD Guidelines for Testing of Chemicals, Section 4: Health Effects. Organization for Economic Co-operation and Development, Paris. Peto R., Pike M. C., Day N. E., Gray R. G., Lee P. N., Parish S., Peto J., Richards S. & Wahrendorf J. (1980) Guidelines for simple, sensitive significance tests for carcinogenic effects in long-term animal experiments. In Long-term and Short-term Screening Assays for Carcinogens, a Critical Appraisal pp. 311-426. International Agency for Research on Cancer, Lyon. Pfanzagl J. (1978) Allgemeine Methodenlehre der Statistik II. pp. 193-194. De Gruyter, Berlin. Przybojewska B., Dziubaltowska E. and Kowalski Z. (1984) Genotoxic effects of ethyl acrylate and methyl acrylate in the mouse evaluated by the micronucleus test. Mutation Research 135, 189-191. Sher S. P. (1982) Tumors in control hamsters, rats and mice: literature tabulation. In CRC Critical Reviews in Toxicology. pp. 49-79. CRC Press, Boca Raton, FL. Sokal R. R. and Rohlf F. J. (1969) Biometry: The Principles and Practice of Statistics in Biological Research. pp. 585-607. W. H. Freeman, San Francisco. Sontag J. M., Page N. P. and Saffiotti U. (1976) Guidelines for carcinogen bioassay in small rodents. National Cancer Institute, Bethesda, MD. Stott W. T. and McKenna M. J. (1984) The comparative absorption and excretion of chemical vapors by the upper, lower, and intact respiratory tract of rats. Fundamental and Applied Toxicology 4, 594-602. Stromberg M. W. and Hebel R. (1976) Anatomy of the Laboratory Rat. Williams & Wilkins Co., Baltimore, MD. Tanii H. and Hashimoto K. (1982) Structure-toxicity relationship of acrylates and methacrylates. Toxicology Letters 11, 125-129. Thomas C. (1978) Die histopathologische Diagnostik im Tierversuch. Arzneimittel-Forschung 28, 2002-2006. Turnbull G. J., Lee P. N. and Roe F. J. C. (1985) Relationship of body-weight gain to longevity and to risk of development of nephropathy and neoplasia in Sprague--Dawley rats. Food and Chemical Toxicology 23, 355-361. Turusov V. S. (1979) Tumours of the soft tissues. In Pathology of Tumours in Laboratory Animals. VoL 1I. Tumours of the Mouse. pp. 487-492. IARC Scientific publications no. 23. International Agency for Research on Cancer, Lyon. Waegemaekers T. H. J. M., Malten K. E. and Bensink T. (1983) Non-mutagenicity of a series of acrylate esters in the Ames Salmonella/microsome test. Mutation Research 113, 317-318.
0278-6915/91 $3.00+ 0.00 Copyright © 1991PergamonPresspie
CHRONIC TOXICITY A N D ONCOGENICITY OF INHALED METHYL ACRYLATE A N D n-BUTYL ACRYLATE IN S P R A G U E - D A W L E Y RATS* W. REININGHAUS,A. KOESTNER'~and H.-J. KLIMISCH~ Institut fiir Biologische Forschung, Kfln, Germany, t'Department of Pathology, Michigan State University, East Lansing, USA and :~Department of Toxicology, BASF AG, Ludwigshafen, Germany (Accepted 6 February 1991) Abstract--No exposure-related clinical signs or lesions of systemic toxicity and no oncogenic responses were observed in male and female Spragne-Dawley rats exposed by inhalation to methyl acrylate (MA) or n-butyl acrylate (BA) vapours, at concentrations of 0, 15, 45 and 135 ppm. The rats were wholebody-exposed 6 hr/day, 5 days/wk, for 24 consecutive months. There was a 6-month post-exposure observation period for subgroups of BA-exposed rats. Atrophy of the neurogenie epithelial cells and hyperplasia of reserve cells were observed in the nasal mucosa of all MA- and BA-exposed groups. These changes were dose related and mainly affected the anterior part of the olfactory epithelium. Opacity and neovasculadzation of the cornea were seen in all MA-exposedgroups and in the group exposed to 135 ppm BA. These toxic effects of the olfactory epithelium and cornea were attributed to the known irritancy of MA and BA. In the BA subgroups kept for a 6-month post-exposure observation, reconstructive effects, such as replacement of altered olfactory epithelium with respiratory epithelium, and partial regression of corneal neovascularization were observed.
INTRODUCTION The use of acrylic acid esters is widespread in industrial and consumer products. Methyl acrylate (MA; CH2~-------CHCOOCH3) and n-butyl acrylate (BA; CH2=CH--COOC4H9) are mainly used for the production of synthetic fibres and for polymer dispersions. The risk of human exposure to vapours of these substances occurs particularly during processing, because of the high vapour pressure. The occupational health threshold limits for MA and BA were set at 10 ppm (American Conference of Governmental Industrial Hygienists, 1986) or at 5 ppm MA and 10 ppm BA (Deutsche Forschungsgemeinschaft, 1985). MA and BA are irritating to the skin and mucous membranes (Clayton and Clayton, 1981). No data concerning the subchronic or chronic toxicity of these acrylates have been published to date. MA and BA as well as acrylic acid, a metabolite common to this class of compounds (Miller et al., 1981b), were not found to be mutagenic in the Salmonella/microsome assay (Florin et al., 1980; Ishidate et al., 1981; Lijinskij and Andrews, 1980; McMahon et al., 1979; Waegemaekers et al., 1983). MA increased chromosomal aberrations in vitro (Ishidate et al., 1981) and tested positive in the micronucleus assay in mice at ip doses of 37.5 mg/kg body weight (Przybojewska et al., 1984). BA produced no chromosomal aberrations in the bone *Presented in part at the Annual Meeting of the Society of Toxicology, Atlanta, GA, USA, March 1984. Abbreviations: BA =n-butyl acrylate; MA = methyl acrylate.
marrow cells of Chinese hamsters (Engelhardt and Klimisch, 1983) and tested negative in a dermal oncogenicity study in mice (DePass et aL, 1984). Since inhalation is a likely exposure route, the present study was conducted to assess the chronic toxicity and oncogenic potential of inhaled MA and BA vapours. Based on the 3-month inhalation studies with MA and BA in Sprague--Dawley rats (H. H. Klimisch, K. Deckardt, K. O. Freisberg and D. Mirea, unpublished data, 1978), a vapour concentration of 135 ppm was selected for both substances as the 'maximum tolerated doses', in accordance with NCI guidelines (Sontag et al., 1976). These authors found that at MA and BA concentrations of 211 ppm, the body and organ weights were significantly affected. Toxic effects in organs and tissues at this concentration were severe enough to expect a shortening of the lifespan and weakening of the defence mechanisms, thereby interfering with the objective of this study. At 108 ppm marginal toxic effects were still consistently observed. MATERIALS AND METHODS Experimental design. Two independent whole-body inhalation studies of identical design were run in parallel. In one study, four groups of 86 male and 86 female rats were exposed to 0, 15, 45 and 135 ppm MA vapour (equal to 0, 58, 173 and 519 mg/m3); in the other study, the same number of rats was exposed to equal volume concentrations of BA vapour (equal to 0, 86, 258 and 773 rag/m3). During the initial 13-wk period, both the MA and BA concentrations were 0, 5, 15 and 45 ppm. All groups, including the control -groups exposed to air, were whole-body exposed
329
330
W . REININGHAUS el al.
Table 1. Number of rats killed at scheduled dissections in control, MA and BA inhalation groups No. of rats killed MA Exposure (month)
Group
12
BA
Male
Female
Male
Female
All
10
10
10
10
18
All
15
15
15
15
24
Control Low Medium High
46 50 53 52
49 48 47 46
10 10 10 10
10 10 10 10
24 + 6 (post-exposure)
Control Low Medium High
-----
-----
24 26 28 29
34 21 28 21
for 6 hr/day, 5 days/wk, for 24 consecutive months. For MA- and BA-exposed groups, interim kills were carried out after 12 and 18 months of exposure (Table 1). The final killing was carried out after 24 months of exposure, except for those BA rats kept for a 6-month post-exposure observation and recovery period (Table 1). Test substances. MA (purity >99.8%, main impurities methyl propionate and ethyl acrylate) and BA (purity >99.5%, main impurities butyl propionate and isobutyl acrylate), stabilized with 50 ppm monomethyl hydrochinone, were supplied in 5-kg lots (BASF, Ludwigshafen, Germany). Each lot was analysed by gas chromatograph (PEG-400, 50°C, isothermal, flame ionization detection) before and after use in order to confirm the purity and stability. Exposure and analyses. The vapours of the test substance were generated from the liquid form, which was introduced at a constant flow into heated evaporators (approx. 40°C for MA and 120°C for BA) through which a constant flow of air (0.3-3 m3/hr) was also passed. The inhalation chambers were constructed of stainless-steel and glass, each having a volume of 4.9 m 3 and a capacity for 172 rat (1 dose group). The chambers were ventilated with a continuous vertical stream of conditioned (21°C, 55% relative humidity) fresh air (approx. 60 m3/hr). The air was mixed with the generated vapours of the test substance in such a manner that the desired chamber concentrations of the substances could be obtained (Table 2). The control rats were exposed in identical chambers to fresh air only. During each daily exposure period the concentrations of the test substances in the chamber atmosphere were repeatedly determined for 10 min every hr, using an infra-red gas photometer (path length = 2.25 m at a wavelength of Table 2. Nominal and measured concentrations of MA or BA inside inhalation chambers of rats during months 3-24 of exposure Concn of test substance (ppm) MA Group Control Low Medium High
BA
Nominal Measured SD (M) (M) (%) 0 15 45 135
0 15.6 46.3 140.3
-5.1 5.0 5.1
M = mean
Nominal (M) 0 15 45 135
Measured SD (M) (%) 0 14.8 44.3 133.7
-5.9 5.5 6.3
8.3 #m; Miran IA, Foxboro Analytical, S. Norwalk, CT, USA) or a total hydrocarbon flame ionization detector (RS5; Ratfisch Instrumente, Miinchen, Germany). The vapour in the chambers was analysed by gas chromatography (PEG-400, 50°C, isothermal, flame ionization detection) in order to confirm the purity of the exposure atmosphere. Animals. Male and female Sprague-Dawley rats (WlGA, Versuchstier-Zuchtanstalt, Sulzfeld, Germany), approx. 35 days old, were randomly allocated to each group. The control, MA- and BA-exposed rats were housed and treated in three separate airconditioned rooms. Animal observation. All rats were checked for their general condition and for signs of toxicity before and after each daily exposure, and once daily during the post-exposure observation period. Body weight, food consumption, reflexes and any abnormal tissue masses were recorded weekly. Rats found dead were autopsied on the same day. Moribund rats were killed and autopsied. Haematology and urinalysis. Following anaesthesia with diethyl ether, administered shortly before the rats were killed, blood was collected from the orbital sinus of all rats, and erythrocyte and leucocyte counts were determined. In addition, those rats from the control groups and those exposed to MA and BA at 135ppm, which were killed as scheduled, were examined with respect to reticulocyte, normoblast and differential leucocyte counts as well as to number of erythrocytes with Heinz bodies, packed cell volume, erythrocyte volume, haemoglobin content and haemoglobin concentration. Bone marrow smears were prepared from the rats of these groups and also from all moribund rats. Urine was collected from all rats scheduled for autopsy. The determinations included volume, colour, transparency, pH and the concentrations of protein, glucose, bilirubin, urobilinogen as well as ketone bodies, occult blood and sediments. Ophthalmology. Shortly before the rats were autopsied, the eyes were examined for external changes and the pupillary reflex. Changes in the anterior part of the bulbus (slit lamp; Zeiss, Oberkochem, Germany) and changes of the fundus (ophthalmoscope; Zeiss, Jena, Germany) were examined in mydriasis (1% atropin). Gross autopsy. The rats were killed by exsanguination under diethyl ether anaesthesia (no previous diet deprivation). A complete autopsy was performed on all rats. Body weights and the absolute and relative weights (to body weight and brain weight) of all major organs were determined for each rat. All tissues with gross lesions and representative sections of organs and tissues (according to the OECD requirements for "combined chronic toxicity/carcinogenicity studies", with the exception of the accessory genital organs (OECD, 1981)) were preserved in a 4% neutral formaldehyde solution. The testes were preserved in Bouin's fixative and the lumbar vertebrae in Schaffer's fixative. Histopathology. Tissue sections were embedded in paraffin, cut at 5 #m, and stained with haematoxylin and eosin. The nasal cavity, liver, and kidney sections were additionally stained with periodic acid-Schiff stain (Horobin, 1988). The nasal cavity and lumbar
MA and BA inhalation in rats vertebrae were decalcified in an ultrasonic bath with 5% nitric acid before embedding. Frozen sections of the liver and the kidney were stained with Sudan III. The nasal cavity (levels 1, 2 and 6, according to Stromberg and Hebel, 1976), larynx, trachea, lungs and liver of all rats (344 rats/sex/test substance) as well as all tissues having gross lesions were examined. The heart, kidneys, urinary bladder, ovaries, pituitary gland, thyroid, adrenals, brain, spleen, lumbar vertebrae and lymph nodes from all rats of the control groups and those exposed to M A and BA at 135ppm (172 rats/sex/test substance) were also examined. In addition, at each scheduled autopsy 11 tissue samples from each of at least 10 randomly selected male and female rats from the control and 135-ppm groups were histologically examined (between 40 and 65 rats/sex/test substance). Furthermore, an extensive histological examination was performed on all rats found dead or moribund (98 MA- and 197 BA-exposed rats). All changes were graded according to severity (Thomas, 1978). All neoplastic changes were classified according to tissue of origin, type and biological behaviour if possible. Statistical analysis. The rats were allocated to groups at the beginning of the study and selected for scheduled autopsies using computer-assisted randomization procedures. A comparison of the exposure groups with their respective control group (each sex separately) was made setting the twosided fiducial limit ~ = 0.05 as a critical level of significance for all tests, without corrections for multiple testing. Mortality was analysed using the life-table method (Armitage, 1971), after accounting for non-spontaneous deaths. Body weight, food consumption, organ weights and all haematological parameters with a quasi continuous range of values were compared using Student's t-test or its modification according to Welch (Clauss and Ebner, 1970). Moribund rats, rats found dead and rats with apparently outlying values were excluded from routine statistics; findings in those rats were interpreted separately. The total incidences of frequently observed histological changes were analysed using contingency tables (Sokal and Rohlf, 1969). After classifying the tumours as 'incidental' or 'fatal', the incidence of tumour types and the incidence of turnouts in different organs were analysed using a two-sided chisquare test corrected for 'animal at risk', as suggested by Peto et al. (1980). This analysis is sensitive to heterogeneous total tumour incidences, as well as to time differences in tumour appearance. Additional non-parametric tests (Mann and Whitney, 1947; Pfanzagl, 1978) were used in order to back up the interpretation of the results after data screening.
331
30-
::: 135controls4515 ppmPPmPPm
.':t"
20-
10-
I
I
I
52
26
78
I
104
I
130
2 v
3O
20
ij
10 ¸ / 0
0
I
26
ff~l
52
I
78
I
I
104 130 Week of study
Fig. 1. Life-table corrected cumulative mortality of Sprague-Dawley rats exposed to MA vapour, +SE. Data were censored for scheduled killings. No significant differences were observed between groups. Overt signs of toxicity or reactions related to exposure were not observed. After 24 months, the cumulative incidence of rats with palpable tissue masses was approx. 6% for male rats and approx. 12% for females. No dose-related trends were observed. The mean life-table-corrected cumulative mortality was approx. 20% (Figs 1 and 2). During the 6-month post-exposure period (BA only) the cumulative mortality increased to approx. 45%. On the whole, the mortality data, though heterogeneous for BA-exposed female rats, did not indicate that exposure to the test substances had an adverse effect on longevity.
Body weight and food consumption
The actual and calculated concentrations agreed within a few per cent. The standard deviation was less than 10% (see Table 2).
At the start of the exposure period, the mean body weights were 183 g for male rats and 157 g for female rats. For both sexes the relative standard deviations were approx. 10% (Figs 3 and 4). From wk 1 to wk 13, during which the rats were exposed to 1/3 of the final test substance concentration, the bodyweight gain of all groups was practically identical. From wk 15 to the end of the exposure period, the body weights of the male and female rats exposed to M A at 135 ppm were slightly but significantly lower than those of other groups (approx. 4%). The food consumption of the male and female rats exposed to BA was consistently lower (approx. 4%) than that of the control groups. The food consumption returned to normal after the end of the exposure period. These effects indicate that the 135-ppm concentration meets the criteria for the maximum tolerable dose according to NCI guidelines.
Animal observation and mortality
Haematology and urinalysis
In general, the appearance and behaviour of the rats were normal. During exposure, all rats were quiescent.
With regard to the haematological parameters, there was no indication that the test substances
RESULTS Exposure concentrations
W. REININGHAUSel al.
332
T
controls
60040-
30400-
~[xMale / 20-
200-
10-
:
If~ f
0
15 ppm ppm ppm
: 45 : 135 i
i
26
i
52
i
78
i
104
130
Week of study
/~
Fig. 4. Body weights of rats exposed to BAvapour.
(Alt et al., 1980; Couser and Stilmont, 1975; Galaske et al., 1980; Turnbull et al., 1985).
40-
Organ weights and gross autopsy
30Female
20-
10-
0 0
2'~
s'2
48
164
i]0
Weekof study Fig. 2. Life-table-corrected cumulative mortality of Sprague--Dawley rats exposed to BA vapour, +SE. In female rats, groups exposed to 15 and 135 ppm differed statistically significantly from the other BA exposed groups, but not from MA groups.
Autopsy revealed only incidental patho-anatomical changes or gerontic patho-anatomical changes, typical of aged Sprague-Dawley rats. Gross changes generally correlated with the respective histological lesions. The autopsies performed after 24 months mainly revealed 5-17% lower weight of kidneys, thyroid, heart and liver in some of the M A - or BA-exposed rats, compared with respective controls. Despite their statistical significance, these findings were not considered to be toxicologically significant, because they were neither consistently dose dependent nor did they correlate with histopathological changes. Ophthalmology
caused any responses. Occasional differences between the control and exposed groups were interpreted to be incidental findings. With regard to the urine parameters, there was no indication of exposurerelated effects. Elevated urinary erythrocyte counts and protein concentrations were observed in most of the male and in several female rats of all groups including the control groups. These findings suggest degenerative and inflammatory changes such as nephrosis, nephritis and pyelitis, which are common spontaneous changes in ageing rats of this strain
v
Table 3. Frequency of corneal neovascularization or parenchymal cloudiness in rats (male plus female) after inhalation of MA or BA vapour Relative no. of rats (%) MA cones (ppm) BA cones (ppm) Exposure (month) 0 15 45 135 0 15 45 135 12 0 4 8 33 0 0 4 16 18 0 10 17 63 0 3 7 13 24 [ 10 30 59 3 4 2 34 24 + 6 (post3 24 3 28 exposure)
600
m
400
200 ~
o
l
s
: 15 :
ppm 45 p p m ppm
: 135 0
26
i
i
52
78
i
i
104 130 Week of study
Fig. 3. Body weights of rats exposed to MA vapour.
Centrally localized or diffuse stippling of the corneal epithelium and cloudiness of the corneal parenchyma with various degrees of neovascularization were observed in M A - and BA-exposed rats. The incidence of the changes in the corneal parenchyma increased with the concentration of the test substances and length of exposure, and was similar in male and female rats (Table 3). After 24 months, a significant increase of parenchymal changes was evident in all MA-exposed groups and in the groups exposed to BA at 135 ppm. The neovascularization
Absolute no. of rats~f
I
14"
37 ~
87 ~
3
5
5
42 ~"
t 150-157 Rats wereexamined per group, excludingrats re-examined after 6 months post-exposure. Those values marked with asterisks differ significantly(chi-square test) from the corresponding control value (*P < 0.05).
MA and BA inhalation in rats Table 4. Frequency of reserve-cell hyperplasia in level 2 of the nasal mucosa of rats (male plus female) after inhalation of MA or BA vapour Relative no. of rats (%) MA conca (ppm) Exposure (month) 12 18 24 24 + 6 (postexposure) Absolute no. of rats'~
0
BA concn (ppm)
15
45
135
0
15
45
135
0 0 1
0 0 6
65 88 96
91 100 99 --
0 0 0 0
0 0 24 1
0 2 56 40
64 97 92 41
1
9
154"
168"
0
10"
65* 115"
t167-171 Rats were examined/group, and those values marked with asterisks differ significantly (chi-square test) from the corresponding control value (*P < 0.05).
regressed to empty 'ghost vessels' 6 months after the end of exposure. Other changes in the eye, in particular cataracts, which were observed sporadically in all groups, were not related to test substance exposure.
Non-neoplastic lesions The histological examinations revealed concentration-dependent lesions in the nasal mucosa at the level of the dorsal lamella of the second endoturbinate (level 2, according to Stromberg and Hebel, 1976). In a few male rats exposed to M A or BA at 15 ppm, a slight atrophy of the neurogenic part of the olfactory epithelium was observed (Plates 1-3 and Table 4). At 45 and 135 ppm, almost all MA-exposed rats developed a partial loss of the columnar cell layer, with an accompanying stratified reserve-cell hyperplasia. The lesions were mainly confined to the most anterior region of the olfactory epithelium, which lines the dorsal part of the nasal cavity at this level. Rats exposed to BA had distinctly less severe changes, which were qualitatively similar to the changes following M A exposure. Male and female rats were affected to a similar degree. Most changes developed during the first 12 months of exposure and increased only moderately with ongoing exposure.
333
During the 6-month post-exposure period, the altered olfactory epithelium was replaced by respiratory epithelium. No treatment-related changes were detected in the posterior nasal cavity, which is mainly lined with olfactory epithelium (level 6, according to Stromberg and Hebel, 1976). No irritative changes were observed in the larynx, trachea or lungs of the exposed rats. Occasional lesions in other organs did not indicate a trend related to the test substance concentration. Such lesions were considered to be strain- and agerelated.
Neoplastic lesions Up to the 18th month the cumulative incidence of primary tumours was only approx. 2% for male rats and approx. 4% for females. Such a low incidence precludes the use of further analysis of the time of tumour appearance. There was also no doserelated trend in the cumulated incidence of primary tumours. In Tables 5 and 6 the total numbers of rats with tumours, classified according to tumour types, are listed. Among the MA-exposed rats the relative tumour rates for the malignant epithelial and leukaemic changes were significantly heterogeneous (Table 5). The number of male rats with malignant epithelial tumours was lower in the 15- and 45-ppm groups than in the control and 135-ppm exposed groups. These tumours were located in the thyroid, testes and urinary bladder. Among female rats exposed to MA, the incidences of epithelial turnouts were homogeneously distributed. Since there was no dose response, these heterogeneous incidences of malignant epithelial turnouts among MA-exposed male rats are considered to be incidental. The number of MA- and BA-exposed male rats with leukaemic changes was higher than in the control groups. As shown in Table 7, the highest incidence of leukaemic changes in males occurred in the groups exposed to medium concentrations of M A and BA. For the male rats exposed to the high
Table 5. Total number of rats with tumourst after inhalation of MA vapour MA concn (ppm) Male rats Tumour type
Female rats
0
15
45
135
0
15
45
135
Benign Epithelial Mesenchymal Fibroadenoma~/ Other benign
30 20 5 l 5
24 12 6 0 8
21 17 3 0 3
24 16 4 0 4
33 16 5 14 2
39 27 4 19 1
34 27 2 l0 3
32 17 2 17 3
Malignant Epithelial Mesenchymal Leukaemic Other malignant
14 8* 6 0* 0
11 4* 5 3* 1
I1 3* 1 7* 1
19 12" 8 0* l
9 4 2 1 2
8 5 2 2 0
7 2 3 1 1
7 2 2 2 1
Total§
38
33
27
36
39
43
38
38
8
6
8
8
7
15
9
8
Two or more tumour types
t86 Rats were autopsied and examined per sex and group. ~Non-assignable regarding tissue type. §The number of rats with a specific tumour type was counted independently from the appearance of types in the same rat. Therefore, the line 'total" does not represent the sum of all listings, but the total number of rats beating a tumour of any type. Those values marked with asterisks differ significantly (chi-square test, after 'at risk' correction) in the heterogeneous distribution of relative tumour rate between groups (*P < 0.05).
W . R~tqINGtIAUS et al.
334
Table 6. Total number of rats with tumours* after inhalation of BA vapour BA conch (ppm) Male rats Tumour type
Female rats
0
15
45
135
0
15
45
Benign Epithelial Mesenchymal Fibroadenoma~: Other benign
34 23 2 3 I1
24 16 3 2 8
26 13 7 0 11
23 19 3 0 7
48 30 1" 26 4
48 25 7* 29 3
50 34 2* 20 7
135 39 23 1* 20 4
Malignant Epithelial Mesenchymal Leukaemic Other malignant Total§
16 7 10 0 2 41
19 9 9 3 0 35
18 2 10 5 1 39
14 5 7 3 0 34
15 6 10" 1 0 54
8 7 1" 0 0 50
11 7 4* 0 0 52
l0 7 4* 0 0 41
Two or more tumour types
13
12
10
10
21
21
19
17
t86 Rats were autopsied and examined per sex and group. :[Non-assignable regarding tissue type. §The number of rats with a specific tumour type was counted independently from the appearance of additional tumour types in the same rat. Therefore, the fine 'total' does not represent the sum of all listings but the total number of rats bearing a tumour of any type. Those values marked with asterisks differ significantly (chi-square test, after 'at risk' correction) in the heterogeneous distribution of relative tumour rate between groups (*P < 0.05).
BA concentration, the incidence was only 3.5% compared with 5.8% for the medium-concentration group. F o r the male rats exposed to the high M A concentration, the incidence was 0. For the females exposed to BA, only one case was recorded, which occurred in the control group. The incidence for the female rats exposed to M A was ncarly equally distributed among all groups. The overall incidence of leukaemic changes in the MA- and BA-exposed groups was well within the expected incidence of spontaneous leukaemia in Spragne-Dawley rats (Anver et al., 1982; Sher, 1982). Even the highest incidences for individual groups were comparable to the published incidences of spontaneous leukaemia in Sprague-Dawley rats. Furthermore, there was no dose-effect relationship. Based on published data, the incidence of leukaemic changes observed in the female rats was considerably lower than expected. The higher incidence of leukaemic changes in MA-exposed male rats does not seem to be an exposure-related finding. A statistical comparison of the BA-exposed groups (Table 6) showed that the number of female rats with benign and malignant mesenchymal tumours was heterogeneously distributed. These benign turnouts were mainly lipomas in the abdominal cavity and fibromas of the soft tissue (i.e. mainly skin/subcutis, as defined by Turusov, 1979). The malignant tumours were mainly sarcomas of the soft tissue. Since their incidence decreased with increasing BA concentration, they are considered to be incidental.
When primary tumours were grouped according to orgin and tumour type (Tables 8 and 9), there were only a few organs with significantly heterogeneous tumour incidences. Among the MA-exposed rats, the number of males with pituitary adenomas was distinctly higher in the control groups than in the exposed groups (Table 8). In female rats, the incidence of this type of tumour was higher in the 15- and 45-ppm groups than in the control and the 135-ppm groups. Based on the absence of a dose-effect relationship and the known incidence of spontaneous pituitary adenomas in ageing Spragne-Dawley rats, this statistical heterogeneity is considered to reflect random variability. Sarcomas had a random distribution. For instance, anaplastic soft tissue sarcomas (skin/subcutis) of male rats were restricted to groups exposed to M A at 15 and 135 ppm (Table 8). In BA-exposed male rats, the incidence of sarcomas in the thoracic cavity (fibro- lipo- and neurofibrosarcoma) was higher at 15 ppm than in all other groups in female rats. The incidence of soft-tissue sarcomas in fibrous connective tissue was highest in the controls (Table 9). On the whole, for both test substances the incidence of sarcomas varied significantly in all tissues or sites. There was no detectable dose dependency. Similarly, the incidence of all malignant mesenchymal tumours (Tables 5 and 6), which were mainly sarcomas of the soft tissue, does not indicate an exposurerelated tumour increase. Therefore, the varying rates of soft-tissue sarcomas are considered to reflect a
Table 7. Incidence of leukaemia, lymphoma and lymphnsarcoma in rats after inhalation of MA or BA vapour No. of rats with tumours* Test substance cnncn (ppm) Test substance
Sex
0
15
45
135
Total
No. of rats with turnouts in historical controlt
MA MA
Male Female
0 (0%) 1 (1.2%)
3 (3.5%) 2 (2.3%)
7 (8.1%) 1 (1.2%)
0 (0%) 2 (2.3%)
10 (2.9%) 6 (1.7%)
(3.7%) (3.0%)
BA BA
Male Female
0 (0%) 1 (1.2%)
3 (3.5%) 0 (0%)
5 (5.8%) 0 (0%)
3 (3.5%) 0 (0%)
I 1 (3.2%) 1 (0.3%)
(3.7%) (3.0%)
*Relative frequencies, given in parentheses, are based on 86 rats/group. t F r o m Sher (1982).
Plate I. Dorsomedial aspect of the nasal cavity of a control rat showing septum, intact olfactory epithelium, and the transitional region with the beginning of the respiratory epithelium (~7). Original magnification x 185.
~ ~ii~~!i!i~:~i:~i~!~ii~i~i~ii~i!!!i~i~i!i!~ii~ i~ i!i~
:
Plate 2. The same location after 24 months of exposure to 135 ppm BA vapour, showing a loss of columnar epithelium in the olfactory region with focal stratified reserve-cell hyperplasia and an intact respiratory epithelium (~7). Original magnification x 185.
Plate 3. The same location in a rat exposed to 135 ppm BA vapour for 24 months after a 6-month post-exposure period, showing respiratory epithelium (insert) in the olfactory region. Original mat,nification x 185.
MA and BA inhalation in rats
337
Table 8. Total incidence? of primary turnouts in rats after inhalation of MA vapour MA concn (ppm) Male rats 15 45
Female rats Tumour origin and type 0 135 0 15 45 135 Liver Adenoma, haemangioendothvlioma 2 1 0 0 2 5 3 5 Urinary bladder Papilloma 2 0 0 1 0 0 0 0 Carcinoma, sarcoma 1 2 0 3 0 0 I 0 Testis Leydig-cell adenoma, mesothelioma 3 6 7 6 . . . . Leydig-cell carcinoma 2 0 0 4 . . . . Ovary Thecoma, fibroma . . . . 0 1 0 3 Uterus Polyp . . . . 5 4 2 3 Carcinoma . . . . 0 0 0 1 Pituitary gland Adenoma 13" 2* 6* 6* 10" 21" 23* 9* Carcinoma 0 0 l 0 0 0 0 1 Thyroid Adenoma 0 2 3 1 0 2 2 I Carcinoma 3 1 0 2 l 0 0 0 Adrenal Adenoma, pheochromocytoma, unspecific benign tumour 5 6 4 3 1 0 0 0 Malignant pheochromocytoma 0 0 1 1 0 0 0 0 Soft tissue, unclassified Sarcoma 0* 4* 0* 6* l 1 1 0 Systemic, lymphoid tissue Lymphosarcoma, leukaemia 0 2 3 0 1 l 1 1 Non-lymphoid organ, parenchymal tissue Leukaemia, malignant lymphoma 0 0 3 0 0 0 0 0 Thymus, mesenchymal or epithelial tissue Thymoma 0 1 0 l 1 1 3 0 Mammary gland Adenoma, fibroadenoma l 0 0 0 14 19 II 17 Carcinoma 0 0 1 0 2 4 2 0 Abdominal cavity Lipoma I 3 0 2 5 1 2 2 Sarcoma, unspecific malignant tumour l 1 0 0 0 0 0 0 Other organs~ Benign tumour 9 6 4 4 2 2 1 1 Malignant tumour 5 4 3 6 2 3 1 3 ?86 Rats were autopsied and examined per sex and group. ~Cumulative incidence of tumours in organs with not more than two benign or two malignant tumours in any group. Those values marked with asterisks differ significantly(chi-square test, after 'at risk' correction) in the heterogeneous distribution of relative tumour rate between groups (*P < 0.05). heterogeneous s p o n t a n e o u s t u m o u r distribution a n d n o t a treatment-related incidence. DISCUSSION This 2 4 - m o n t h i n h a l a t i o n study in S p r a g u e Dawley rats revealed t h a t exposure to 15, 45 or 135 p p m M A or B A does n o t influence longevity or cause systemic toxicity detectable by haematological parameters, gross a n d histological examination, or urinalysis. This c o r r e s p o n d s to the results o f the subchronic ( 3 - m o n t h ) i n h a l a t i o n studies with the same c o m p o u n d s (H. H. Klimisch, K. Deckardt, K. O. Freisberg a n d D. Mirea, u n p u b l i s h e d data, 1978). In the nasal m u c o s a a n d c o r n e a b o t h test substances caused non-neoplastic changes, which were a t t r i b u t e d to the irritating effect o f acrylic esters. The changes were mainly restricted to a n a r r o w area o f the m o s t a n t e r i o r p a r t o f the olfactory epithelium. The sequence o f the observed changes p r o b a b l y begins with a t r o p h y of the neurogenic segment o f the epithelium, followed by progressive hyperplasia of the reserve cells a n d ultimately by a loss o f the u p p e r epithelial cell layer. A d v a n c e d changes are considered to be reversible pre-stages o f metaplasia.
Cellular atypia a n d infiltrative g r o w t h were n o t observed. D u r i n g the post-exposure period, the altered olfactory epithelium was replaced by respiratory epithelium. T h e changes in the nasal m u c o s a were qualitatively similar for b o t h substances, b u t less p r o n o u n c e d in the BA-exposed groups. It was s h o w n t h a t only m i n i m a l effects are caused by exposure to 15 p p m M A , a n d t h a t 15 p p m is the no-effect level for exposure to BA. Similar nasal changes have been observed in rats a n d mice in i n h a l a t i o n studies with ethyl acrylate a n d acrylic acid (Miller et al., 1981a a n d 1985). H u m a n exposure to M A or B A does n o t seem to pose a considerable health risk provided t h a t regulatory limit c o n c e n t r a t i o n s (Deutsche Forschungsgemeinschaft, 1985) are n o t surpassed for p r o l o n g e d periods o f time; even c o n c e n t r a t i o n s t h a t cause only reversible changes are so irritative t h a t a p r o l o n g e d exposure is precluded. C h a n g e s o f the eye were restricted to the corneal p a r e n c h y m a , where cloudiness with varying degrees o f neovascularization was observed. T h e incidence was time- a n d c o n c e n t r a t i o n - d e p e n d e n t in b o t h male a n d female rats. T h e no-effect level for M A was below 15 ppm, the level at which only m i n o r changes
W. REININOHAUSet al.
338
Table 9. Total incidencet of primary tumours in rats after inhalation of BA vapour MA concn (ppm) Male rats Female rats Tumour origin and type Liver Adenoma, angioma, haemangioendothelioma Sarcoma Urinary bladder Papilloma Carcinoma Testis Leydig-cell adenoma Leydig-cell carcinoma Ovary Fibroma, thecoma Uterus Polyp Sarcoma, carcinoma Pituitary gland Adenoma Carcinoma Thyroid Adenoma Carcinoma Adrenal Adenoma, gangiioneuroma, pheochromocytoma Malignant pheochromocytoma Pancreas (endocrine) Adenoma Unspecific malignant tumour Soft tissue, fibrous connective Fibroma Sarcoma Soft tissue Neurofibrosareoma Systemic, lymphoid tissue Leukaemia Thymus, mesenchymal or epithelial tissue Thymoma Mammary gland Adenoma, fibroma, fibroadenoma Carcinoma Thoracic cavity Sarcoma Abdominal cavity Lipoma, mesothelioma Sarcoma Other organs~/ Benign tumour Malignant tumour
0
15
45
135
0
15
45
135
l 2
0 0
0 0
I 0
4 0
2 0
4 0
0 0
2 0
1 1
1 0
3 0
0 0
0 0
1 0
0 0
7 1
2 3
4 l
5 1
. .
. .
. .
. .
.
.
.
.
l
0
4
l
. .
. .
. .
. .
7 2
5 1
7 2
6 1
8 1
8 l
3 0
4 l
12 0
17 1
22 l
14 0
2 2
3 0
2 1
7 1
4 4
2 l
6 0
1 3
Il 1
8 0
13 0
6 0
1 0
3 0
2 0
2 0
2 1
1 0
0 0
3 0
0 0
0 0
I 0
0 0
0 1
l 1
1 3
0 0
0 4*
2 0*
0 0*
0 1*
3 3 0
0 0 3
0 0 4
2 2 2
2 2 1
0 0 0
0 0 0
2 2 0
0
0
0
0
2
0
3
l
3 1
2 1
1 0
0 0
32 2
31 4
20 4
22 4
0*
3*
0*
0*
0
0
l
0
0 I
0 I
2 I
0 0
0 0
3 0
0 I
2 0
4 5
5 5
7 9
10 6
5 4
2 l
0 3
2 2
t86 Rats were autopsied and examined per group. :~Cumulative incidence of tumours in organs with not more than two benign or two malignant tumours in any group. Those values marked with asterisks differ significantly (chi-square test, after 'at risk' correction) in the heterogeneous distribution of relative tumour rate between groups (*P < 0.05).
were o b s e r v e d . F o r BA, t h e no-effect level was l o w e r t h a n 135 p p m . N e o v a s c u l a r i z a t i o n is a k n o w n n o n specific r e a c t i o n to c h r o n i c i r r i t a t i o n o f the c o r n e a ( H e n k i n d , 1978). I n a p r e v i o u s study, after t o p i c a l a p p l i c a t i o n o f m e t h y l m e t h a c r y l a t e similar c h a n g e s were i n d u c e d ( H o l y k a n d Eifrig, 1979). H o w e v e r , they were n o t o b s e r v e d in 2 7 - m o n t h i n h a l a t i o n studies w i t h ethyl acrylate o n F i s c h e r - 3 4 4 rats (Miller et al., 1985). A p e e r review s u m m a r y ( H e n s c h l e r , 1985) c o n c l u d e d t h a t such c o r n e a l c h a n g e s in rats c a n n o t be directly e x t r a p o l a t e d to h u m a n s . T h e fact t h a t such findings h a v e never b e e n o b s e r v e d in employees of an MA manufacturing company (G. Dietrich, p e r s o n a l c o m m u n i c a t i o n , 1985) f u r t h e r s u b s t a n t i a t e s this assessment. T h e h i g h e r irritancy o f M A as c o m p a r e d w i t h B A , i n d i c a t e d b y t h e different degree a n d f r e q u e n c y o f c h a n g e s in t h e o l f a c t o r y e p i t h e l i u m a n d in the c o r n e a l p a r e n c h y m a , m a y p a r t l y be c a u s e d by differences in
diffusibility a n d w a t e r / l i p i d solubility o f the t w o test s u b s t a n c e s (Tanii a n d H a s h i m o t o , 1982). I n general, the toxicity o f h o m o l o g o u s acrylates is r e p o r t e d to decrease w i t h i n c r e a s i n g m o l e c u l a r w e i g h t ( A u t i a n , 1975). O t h e r i n v e s t i g a t i o n s p o i n t to t h e i m p o r t a n t influence o f epithelial m e t a b o l i s m o n t h e u p t a k e a n d effect o f irritants (Stott a n d M c K e n n a , 1984). U n d e r the test c o n d i t i o n s o f t h e p r e s e n t study, e x p o s u r e to M A o r B A v a p o u r d i d n o t lead to a n i n c r e a s e d f r e q u e n c y o f any t u m o u r t y p e in a n y o r g a n t h a t c o u l d be a t t r i b u t e d to a n o n c o g e n i c p o t e n t i a l o f the test s u b s t a n c e . This is c o n s i s t e n t w i t h t h e results published on the absence of a dermal tumorigenic p o t e n t i a l o f B A ( D e P a s s et al., 1984).
Acknowledgements--This
study was sponsored by the BASF Aktiengesellschaft. The authors gratefully acknowledge the contributions of Professor H.-K. Koch, for ophthalmological evaluation (Universit~its-Augenklinik, Bonn,
MA and BA inhalation in rats Germany), and Professor C. Thomas, for histopathological evaluation (Pathologisches Institut, Universit~t Marburg, Germany). REFERENCES
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