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Safety evaluation of esperase
Fd Chem. Toxic. Vol. 31, No. 12, pp. 999-101 I, 1993 Printed in Great Britain. All rights reserved
0278-6915/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
SAFETY EVALUATION OF ESPERASE R. K. HJORTKJAER*, M. STAVNSBJERGt, P. B. PEDERSEN't, J. HEATHS, J. A. WILSON~, R. R. MARSHALL§ and J. CLEM~NTS~ *Danish Toxicology Centre, Agern All6 15, DK-2970 Horsholm, tNovo Nordisk A/S, Novo All& DK-2880 Bagsvaerd, Denmark, ~tlnveresk Research International, Tranent EH33 22NE, Scotland and §Hazleton Microtest, Otley Road, Harrogate, North Yorkshire HG3 1PY, UK (Accepted 24 June 1993)
Abstract--Esperase is a proteolytic enzyme preparation that can be used as a processing aid in the food industry. The following studies were performed to establish safety for the consumer: oral toxicity study (13 wk) in the rat; teratogenicity study in the rat; gene mutation assays in Salmonella typhimurium and mammalian cells in vitro, and chromosome aberration assay/n vitro. General toxicity was low; the effects seen were attributed to proteolytic activity and the loading with sodium chloride. Neither of these factors will be relevant to consumers of the processed food. There was no evidence of effects on pregnancy outcome or mutagenic potential. When these results are considered together with existing knowledge of the production organism and the chemical and microbiological characterization of the enzyme preparation, they indicate that Esperase will be safe for its intended application in food processing.
INTRODUCTION
The safety-in-use of microbial food enzymes can be established on a history of safe use and a safety evaluation based on scientific procedures (e.g. A M F E P , 1992; E E C Scientific Committee for F o o d (SCF), 1991; F o o d and D r u g Administration, 1983; IFBC, 1990; Jensen and Eigtved, 1990; Pariza and Foster, 1983). The safety review should include the following steps (Jensen and Eigtved, 1990): (1) safety assessment of the production strain; (2) identity and quality of raw materials used in the fermentation; (3) processing techniques used in recovery; (4) identity and quality of the stabilizers, diluents or formulation aids; (5) toxicology studies on enzyme concentrate, and (6) determination of safety margin. Esperase is a proteolytic enzyme (serine protease; EC 3.4.21.14) preparation with a broad catalytic activity. The enzyme is produced by Bacillus lentus, which is listed as a Class 1 Agent in the National Institute of Health Guidelines (NIH, 1986) and complies with the G o o d Industrial Large Scale Practice (GILSP) criteria suggested by O E C D (1986). A search has been carried out in the following databases: Biological Abstracts, Medline, Excerpta Medica, Toxline, Commonwealth Agricultural Bureau and Occupational Safety and Health. N o n e of the published literature related B. lentus to human or animal diseases or the formation of toxins, confirming that B. lentus can be considered a safe, non-pathogenic and non-toxinogenic microorganism (Babesgaard, 1991). The production method (i.e. raw materials, fermentation conditions, recovery techniques, etc.) complies with the requirements for microbial food enzymes. This paper summarizes a programme of studies that was
carried out to establish the consumer safety o f Esperase when used as a processing aid in the food industry. MATERIALS AND METHODS Test material
The chemical composition of batch PPA 3366 of the enzyme preparation used for the toxicity studies is given in Table I. The main source of the non-protein nitrogen is amino acids and polypeptides. Analyses of the antibiotic activity and microbial contamination showed that the batch complied with the Joint F A O / W H O Expert Committee on F o o d Additives ( J E C F A , 1984) and F o o d Chemical Codex (FCC, 1981) specifications for enzymes. The stability Table 1. Chemical composition (%) of Esperase batch PPA 3366 Component Percentage H20 59.3 Carbohydrate, total approx. 6 Ntota~x 6.25 9.19 Nprotei n X 6.25
Petroleum ether soluble residue Ash (600°C) Na K Mg Ca CI PO, SO4 CO3 Heavy metals As (ppm) Pb (ppm) Total organic solids (TOS) = 100% - (ash% + water% + diluents%) = 100% - (17.7% + 59.3% + 0%) 23% =
999
3.00
0.16 17.7 <5.1 0.7 0.06 1.5 7.8 0.05 0.14 <0.1 < 0.0040 <3 < 10
1000
R . K . HJORTKJAER et al.
Table 2. Esperase, 13 wk oral toxicity study in rats: methods, abbreviations and units used in laboratory investigations Parameters
Method
Units
Haematology Haemoglobin (Hb): Total red blood cell count (RBC): Haematocrit (Hct): Absolute values: Mean ceil volume (MCV): Mean cell haemoglobin (MCH): Mean cell haemoglobin concentration (MCHC): Total white blood cell count (WBC): Differential white cell count: Neutrophil (Neut): Lymphocyte (Lymp): Monocyte (Mono): Eosinophil (Eos): Platelet count (Plat): Hepato quick (Hep):
Coulter Counter, Coulter Electronics Limited
g/dl
Coulter Coulter Coulter Coulter
x 10~:/litre litre/litre
Counter, Counter, Counter, Counter,
Coulter Coulter Coulter Coulter
Electronics Electronics Electronics Electronics
Limited Limited Limited Limited
fl
Coulter Counter, Coulter Electronics Limited
pg
Coulter Counter, Coulter Electronics Limited
g/dl
Coulter Counter, Coulter Electronics Limited
x 109/litre
Visual appraisal of stained film (May Grunwald and Giemsa stain)
x 109/litre
Coulter Counter, Coulter Electronics Limited beck E. A. and Muschietti F., Schweiz Med. Wschr 35, 1218 (1974)
x 109/litre sec
Clinical Chemistry Blood urea nitrogen (BUN): Glucose (Glu): Aspartate aminotransferase (AST): Alanine aminotransferase (ALT): Lactate dehydrogenase (LDH): Sodium (Na): Potassium (K): Chloride (CI): Total Protein (TP): Albumin (Alb): Albumin/globulin ratio (AG-R): Cholesterol (Chol): Alkaline phosphatase (AP): Calcium (Ca): Creatinine (Crea): Inorganic phosphate (Phos): Total bilirubin (T.Bi):
Neumann U. and Zeigenhorn J., XVI. Nordiska Kongressen for Klinisk Kemi och Klinisk Fysiologi, Oulu, Finland (1977) Schmidt F. H. Klin. Wschr 39, 1244 (1961) Enzyme Commission of the German Society for Clinical Chemistry. Anon., Z. Klin. Chem. Klin. Biochem. 8, 658 (1970). Anon., ibid 10, 182 (1972) Enzyme Commission of the German Society for Clinical Chemistry. Anon., Z. Klin. Chem. Klin. Biochem. 8, 658 (1970). Anon., ibid 10, 182 (1972) Enzyme Commission of the German Society for Clinical Chemistry. Anon., Z. Klin. Chem. Kiln. Bioehem. 8, 658 (1970). Anon. ibid 10, 182 (1972) Ion Selective Electrode Ion Selective Electrode Ion Selective Electrode Weichselbaum T. E. Am. J. Clin. Path. 16, 40 (1964) Dounes B. T. et al., Clin. Chem. Acta 31, 87 (1971) Siedel J. et at, .I. Clin. Chem. Clin. Biochem. 19, 838 (1981). Stabler F. et al., Med. Lab 30, 29 (1977). Trinder P, Ann. Clin. Biochem. 6, 24 (1969) Anon., Z. Klin. Chem. Klin. Biochem. 8, 658 (1970). Anon., ibid I0, 182 (1972) Ray Sarker B. C. and Chauton U. P. S. Analyt. Biochem. 20, 155 (1967). Baginski E. et al., Clin. Chem. Acta 46, 46 (1973) Modified from Barkels H. et al., ClOt. Chem. Aeta 37, 193 (1972) Henry R. J., ed. Clinical Chemistry Principles and Techniques, p. 722 (1974) Wahlefeld A. W., Herz G. and Bernt E., Scand .1. Clin. Lab. Invest. 29 (Suppl 126), Abstract 11.12 (1972)
mmol/litre mmol/litre IU/litre IU/litre lU/litre mmol/litre mmol/litre mmol/litre g/litre g/litre mmol/litre IU/litre mmol/litre ~mol/litre mmol/litre /~mol/litre
Safety evaluation of Esperase of the batch during the conducting period of the toxicity studies was confirmed by determination of enzyme activity. Toxicity studies Subchronic oral toxicity in rats (Heath et al., 1991). Following dose range-finding studies, a 13 wk study was carried out with groups of 20 male and 20 female Sprague-Dawley rats. The animals were dosed once daily with the test material by means of a steel dosing cannula at a constant dose volume of 5 ml/kg body weight to give dose levels of 0, 1, 3 and 5 g/kg body weight/day. Routine clinical observations and determinations of body weights, food and water consumption were undertaken throughout the study period. Ophthalmoscopy was performed before the trial and during wk 13. Haematological and clinical chemical investigations were carried out on samples taken from ten males and ten females chosen at random from each group during wk 12/13. The parameters examined and investigation methods used are given in Table 2.
Table 3. Esperase, 13-wk oral toxicity study in rats: organs weighed (W), fixed (F) and examined histologically (H) Organ
W
F
H
Adrenals Aortic arch Any abnormal tissue Bladder Bone (sternum and rib) Brain Ears Eyes Heart Intestine: duodenum jejunum ileum caecum colon rectum Kidneys Stomach (glandular and non-glandular) Spinal cord (cervical, thoracic and lumbar portions) Submaxillary salivary gland Submandibular lymph node Liver Lungs Mammary gland Mesenteric lymph node Muscle (thigh) Nasal cavity Oesophagus Ovaries (with fallopian tubes) Optic nerve
X
X X X X X X X X X
X X X X
X X X X X
X X X X X
X X
X X
Pancreas Pituitary Prostate Sciatic nerve Seminal vesicles Skin Spleen Testes (plus epididymides)
X X
X
X X
X
X X
X X
X X X
X X X X X X X X X X X X
X X X
X X X X X X X X
X X X X X X X X
X X X X X X X
Thymus Thyroids Tongue Trachea
X
X X X X
X X X X
Uterus
X
X
X
FCT31/12--F
1001
At the end of the treatment period all animals were killed by asphyxiation with carbon dioxide followed by exsanguination, and were subjected to gross pathological examination and organ weight analyses. A comprehensive range of tissues was fixed, and examined microscopically from all animals in the control and high-dose groups (Table 3). Haematology, clinical chemistry, organ weight and body weight data were statistically analysed for homogeneity of variance using the F-max test. If the group variances appeared homogeneous a parametric ANOVA was used and pairwise comparisons made by Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log and square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, then a non-parametric test such as a Kruskal-Wallis ANOVA was used and pairwise comparisons made by Dunn Z test where considered appropriate. Organ weights were also analysed, conditional on body weight (i.e. analysis of covariance). Histology data were analysed using Fisher's exact probability test. There were five unscheduled deaths, none being related to treatment with the test material. A doserelated increase in incidence of red/brown staining around the muzzle and forepaws, and red extremities, were found in the intermediate and high-dose males and high-dose females. Body weight gain and food and water consumption are given in Table 4. The intermediate- and high-dose males showed a slight reduction in body weight gain (10 and 12%, respectively), whereas the intermediate-dose females had a slightly higher body weight gain (11%). There was an increase in total water consumption by low-dose males and the intermediate- and high-dose males and females. There were no other clinical observations. The haematological investigations revealed no changes (Table 5). A slight decrease was found in the alanine aminotransferase activities in the intermediate-dose males and the high-dose males and females, and in concentrations of sodium in the intermediate- and high-dose males and females (Table 6). There were no other notable intergroup differences in the other clinical chemical parameters. After adjustment for final body weight (covariance analysis), a decrease in prostate weight and an increase in kidney weight were seen in the intermediate- and high-dose males (Table 7). There were no macroscopic findings at autopsy that could be related to treatment. Focal hyperplasia of the keratinized epithelium in the stomach was seen in 2/20 high-dose males and 2/20 high-dose females compared with 0/20 in both male and female controls. The red/brown staining seen clinically is ascribed to the irritant potential of the test material and is an expected effect of treatment with a protease. The reduction in body weight gain in the intermediate and high dose males was not reflected in a concomitant change in food consumption and indicates an adverse
R . K . HJORTKJAER et al.
1002
Table 4. Esperase, 13-wk oral toxicity study in rats: body weight gains, food consumed and water consumed per rat Group/dose level (mg Esperase/kg/day) Time on study (wk)
IM (0)
Body weight gain (g) wk0-13
381
2M (1000)
3M (3000)
4M (5000)
IF (0)
2F (1000)
3F (3000)
4F (5000)
396 (96)*
344 (90)
334 (88)
176
186 (106)
196 (111)
I73 (98)
Total food consumed (g) wk 1-13
2915
2962 (102)
2812 (96)
2713 (93)
2060
2041 (99)
2124 (103)
2036 (99)
Total water consumed (ml) wk 7-13
1602
1811 (113)
2017 (126)
2221 (139)
1416
1433 (101)
1735 (123)
1820 (129)
M=male
F=female
*Percentages of control in parentheses.
Table 5. Esperase, 13-wk oral toxicity study in rats: haematology during wk 12/13~group mean values Group/dose level (mg Esperase/kg/day)
Hb
RBC
Hct
MCH
MCV MCHC WBC
Neut
Lymp Mono
Eos
Plat
Hep
8 3.2 1.5
8 13.3 4,0
8 0.4 0.4
8 0.2 0.1
8 687 165
9 24 I
1 (0)
Number Mean SD
8 15.4 0.6
8 7.28 0.42
8 0.401 0,015
Males 8 8 21.4 54 0.5 2
2 (1000)
Number Mean SD
l0 15.3 0.4
10 7.30 0.25
10 0.392 0.011
10 21.2 0.9
10 52 2
l0 39.6 0.7
l0 14.7 2.3
10 2.1 1.0
10 12.0 2.7
10 0.4 0.3
10 0.2 0.2
10 742 132
10 25 I
3 (3000)
Number Mean SD
9 15.4 1.0
9 7.34 0,58
9 0.393 0.025
9 21.2 0.5
9 52 2
9 39.7 1.0
9 14.6 4.5
9 3.1 1.4
9 1 1.0 3.2
9 0.4 03
9 0.1 0.1
9 726 257
8 25 3
4 (5000)
Number Mean SD
9 15.3 0.5
9 7,21 0,46
9 0.397 0.013
9 21.4 1.0
9 54 3
9 38.9 0.5
9 15.2 4.5
9 2.4 0.6
9 12.3 4.0
9 0.4 0.4
9 0.1 0.2
9 661 179
10 25 I
1 (0)
Number Mean SD
10 15.4 0.4
10 7.15 0.28
10 0.405 0.007
Femmies 10 10 21.9 56 0.5 2
10 38.6 0.5
10 9.8 3.0
10 1.2 1.0
10 8.3 2.2
10 0.2 0.2
10 0.2 0.1
l0 766 88
9 23 3
2 (1000)
Number Mean SD
7 15.7 0.4
7 7.25 0.19
7 0.413 0.014
7 22.0 0.4
7 57 I
7 38.5 0.5
7 11.2 1.8
7 1.3 0.3
7 9.6 1.5
7 0.2 0.1
7 0.1 0.1
7 797 78
10 22 3
3 (3000)
Number Mean SD
10 15.1 0.6
10 6.92 0.35
10 0.397 0.014
10 22.1 0.6
10 57 1
10 38.5 0.5
10 10.8 1.6
10 1.8 1.0
t0 8.6 1.5
10 0.3 0.2
10 0.1 0.2
10 682 195
10 24 1
4 (5000)
Number Mean SD
10 15.3 0.5
10 6.90 0.20
10 0.402 0.013
10 22.4 0,3
10 58 l
10 38.5 0.6
10 10.4 2.0
10 1,5 0.7
10 8.8 2.2
l0 0.1 0.1
10 0.1 0.1
10 750 I01
10 24 2
Abbreviations as in Table 2.
8 39.0 0.6
8 17.1 5.6
Safety evaluation of Esperase effect o f t r e a t m e n t - - a n effect t h a t is also s e e n w i t h other proteolytic enzymes. The increased water cons u m p t i o n m a y be a r e s u l t o f t h e a m o u n t o f a s h , especially s o d i u m a n d c h l o r i d e , g i v e n w i t h t h e test substance. In the high-dose animals the total sodium a n d c h l o r i d e i n t a k e s were a p p r o x i m a t e l y t h r e e t i m e s those of the control animals. An additional control group without NaC! might have confirmed that the N a C 1 l o a d w a s r e s p o n s i b l e f o r this i n c r e a s e d w a t e r i n t a k e . C h a n g e s in a l a n i n e a m i n o t r a n s f e r a s e activities, in c o n c e n t r a t i o n s o f s o d i u m in p l a s m a a n d in o r g a n w e i g h t s ( p r o s t a t e w e i g h t a n d k i d n e y w e i g h t in intermediate- and high-dose males) did not correlate w i t h a n y h i s t o l o g i c a l f i n d i n g s , a n d a r e c o n s i d e r e d to be o f n o t o x i c o l o g i c a l significance. T h e o c c u r r e n c e o f focal h y p e r p l a s i a o f t h e k e r a t i n i z e d e p i t h e l i u m in t h e s t o m a c h o f t h e h i g h - d o s e m a l e a n d f e m a l e is u n u s u a l in r a t s o f t h i s a g e a n d p r o b a b l y is a t t r i b u t a b l e to t h e i r r i t a n t p o t e n t i a l o f t h e test m a t e r i a l ; h o w e v e r , t h e i n c i d e n c e s p r o v i d e insufficient e v i d e n c e o f a t r e a t m e n t - r e l a t e d effect. O v e r a l l , it c a n be c o n c l u d e d t h a t n o a d v e r s e effects were s e e n f o l l o w i n g 1 g / k g b o d y w e i g h t , w h e r e a s 3 a n d 5 g / k g b o d y w e i g h t r e s u l t e d in m i n o r effects, w h i c h c o u l d be a s c r i b e d to t h e p r o t e o l y t i c
activity a n d preparation.
1003 the amount
o f s a l t s in t h e e n z y m e
Teratogenicity in rats (Wilson, 1991). T h e d o s e levels f o r this s t u d y were selected a f t e r e v a l u a t i o n o f t h e e x i s t i n g toxicity d a t a , a n d were t h e s a m e as t h o s e u s e d in t h e 1 3 w k s t u d y - - 0 , 1, 3 a n d 5 g / k g / d a y . T h e d o s e s were a d m i n i s t e r e d o n c e d a i l y by g a v a g e to g r o u p s o f 25 S p r a g u e - D a w l e y r a t s during days 6-16 of gestation. During the observ a t i o n p e r i o d ( d a y s 0 - 2 0 o f g e s t a t i o n ) clinical signs, b o d y w e i g h t s a n d f o o d a n d w a t e r c o n s u m p t i o n were r e c o r d e d . O n d a y 20 o f g e s t a t i o n t h e a n i m a l s were killed b y c a r b o n d i o x i d e a s p h y x i a t i o n , a n d t h e c o n t e n t s o f t h e t h o r a c i c a n d a b d o m i n a l cavities were e x a m i n e d m a c r o s c o p i c a l l y for a b n o r m a l i t i e s . T h e reproductive tract was removed and weighed, then o p e n e d a n d t h e c o n t e n t s were e x a m i n e d . T h e n u m b e r o f c o r p o r a l u t e a g r a v i d i t a t i s in e a c h o v a r y a n d t h e n u m b e r a n d s t a t u s o f all i m p l a n t a t i o n sites were r e c o r d e d . E a c h live f o e t u s w a s i n d i v i d u a l l y identified w i t h i n t h e litter a n d its w e i g h t w a s r e c o r d e d . T h e f o e t u s e s were e x a m i n e d f o r e x t e r n a l l y visible a b n o r m a l i t i e s b e f o r e fixation. A p p r o x i m a t e l y o n e - h a l f o f t h e f o e t u s e s f r o m e a c h u t e r u s were t h e n fixed in m e t h y l a t e d ethyl a l c o h o l , a n d t h e r e m a i n i n g h a l f in
Table 6. Esperase, 13-wk oral toxicity study in rats: clinical chemistry during wk 12-13--group mean values Group/dose level (mg Esperase/kg/day) BUN Glu AST ALT AP LDH Na K C1 TP Alb AG-R Chol Crea Ca Phos T.Bi Males
1 (0)
Number Mean SD
10 5.9 0.8
10 10 8.66 116 1.06 37
10 76 16
10 10 10 10 348 897 144 4.4 61 914 I 0.6
10 99 1
10 71 3
10 31 1
10 0.8 0.0
10 2.6 0.5
10 10 10 10 49 2.67 1.97 1.8 5 0.06 0.19 0.7
2 (1000)
Number Mean SD
10 5.4 0.9
10 10 8.12 106 0.56 24
10 65 14
10 10 10 10 307 474 144 4.3 69 309 1 0.3
10 98 2
10 70 2
10 31 1
10 0.8 0.1
10 2.4 0.4
10 10 10 10 48 2.70 1.85 1.7 4 0.07 0.21 0.4
3 (3000)
Number Mean SD
10 6.1 1.6
10 10 7.79 109 0.70 41
10 64 19
10 10 10 10 350 656 142 4.6 52 507 I 0.5
10 98 1
10 69 4
10 29 3
10 0.7 0.1
10 3.0 2.6
10 10 10 48 2.66 1.71 7 0.09 0.29
10 2.0 0.7
4 (5000)
Number Mean SD
9 6.5 0.7
9 9 7.87 101 0.97 22
9 55 7
9 9 389 556 106 492
9 98 2
9 68 2
9 29 1
9 0.8 0.1
9 2.0 0.3
9 47 3
9 1.7 1.2
1 (0)
Number Mean SD
10 6.2 0.6
10 8.65 0.81
10 87 17
10 71 20
10 10 10 10 307 243 138 3.5 79 232 1 0.3
10 101 2
10 72 4
10 33 3
10 0.9 0.1
10 2.2 0.4
10 10 10 10 52 2.61 1.70 1.5 2 0.09 0.29 0.4
2 (1000)
Number Mean SD
10 6.4 0.8
10 8.64 0.73
10 89 35
10 71 25
10 10 10 10 242 227 138 3.6 66 79 1 0.3
10 101 2
10 74 7
10 35 3
10 0.9 0.0
10 2.4 0.5
10 10 10 10 50 2.75 1.80 1.6 3 0.13 0.11 0.3
3 (3000)
Number Mean SD
10 6.9 1.3
10 8.58 0.81
10 84 18
10 63 21
10 10 10 10 257 212 137 3.7 56 115 1 0.3
10 100 2
10 73 3
10 33 2
10 0.8 0.1
10 2.4 0.5
10 10 10 10 53 2.66 1.67 1.7 4 0.08 0.23 0.7
4 (5000)
Number Mean SD
10 6.3 1.0
10 8.23 0.48
10 69 10
10 50 6
10 10 10 I0 237 162 137 3.7 63 20 1 0.3
I0 100 2
10 72 4
10 33 3
I0 0.9 0.1
I0 2.3 0.3
I0 10 10 10 50 2.70 1.78 1.6 3 0.09 0.24 0.4
9 9 143 4.4 1 0.4
*
9 9 2.69 1.70 0.06 0.20
Females
*Significantly different from control: P < 0.05. **Significantly different from control: P < 0.01. 2.
A b b r e v i a t i o n s as in T a b l e
1004
R . K . HJORTKJAER el al. Table 7. Esperase, 13-wk oral toxicity study in rats: organ weight (covariance analysis}--group mean values
Group/dose level
Body weight (g) Adrenals Brain
(rag Esperase/kg/day)
Heart Kidneys Liver Lungs Pituitary Prostate Spleen Testes Thymus
Males 1 (0)
Number Mean SE
19 540 16
19 0.062 0.002
19 2.07 0.02
19 1.60 0.03
19 3.45 0.08
19 18.77 0.53
19 2.00 0.04
19 0.011 0.001
19 0.89 0.04
19 1.00 0.04
19 5.20 0. I I
19 0.42 0.02
2 (1000)
Number Mean SE
19 540 16
19 0.062 0.002
19 2.11 0.02
19 1.65 0.03
19 3.66 0.09
19 19.15 0.54
19 1.95 0.04
19 0.012 0.001
19 0.80 0.04
19 0.92 0.04
19 5.33 0.12
18 0.40 0.03
3 (3000)
Number Mean SE
19 540 16
19 0.063 0.002
19 2.09 0.02
19 1.65 0.03
19 3.79 0.08
19 19.08 0.53
19 1.95 0.04
19 0.012 0.001
19 0.72 0.04
19 0.93 0.04
19 5.23 0.11
19 0.42 0.02
4 (5000)
Number Mean SE
19 540 16
19 0.063 0.002
19 2.08 0.02
19 1.62 0.03
19 3.79 0.09
19 19.59 0.54
19 1.87 0.04
19 0.012 0.001
19 0.70 0.04
19 0.96 0.04
19 5.05 0.11
19 0.37 0.03
**Significantly different from control (P < 0.01). Group/dose level (mg Esperase/kg/day)
Body weight (g) Adrenals Brain Heart Kidneys Liver Lungs Ovaries Pituitary Spleen Thymus
Uterus
Females 1 (0)
Number Mean SE
20 312 8
20 0.075 0.002
20 1.92 0.02
20 1.07 0.03
20 2.18 0.04
20 20 10.42 1.46 0.29 0.03
20 0.097 0.004
20 0.013 0.001
20 0.60 0.02
20 0.35 0.02
20 0.67 0.05
2 (1000)
Number Mean SE
19 312 8
19 0.077 0.002
19 1.92 0.02
19 1.11 0.03
19 2.16 0.04
19 19 11.15 1.55 0.30 0.03
19 0.093 0.004
19 0.013 0.001
19 0.64 0.02
19 0.38 0.02
19 0.63 0.05
3 (3000)
Number Mean SE
20 312 8
20 0.076 0.002
20 1.93 0.02
20 1.12 0.03
20 2.24 0.04
20 20 10.28 1.54 0.30 0.03
20 0.108 0.004
20 0.013 0.001
20 0.60 0.02
20 0.39 0.02
20 0.74 0.05
4 (5000)
Number Mean SE
20 312 8
20 0.079 0.002
20 1.92 0.02
20 1.16 0.03
20 2.29 0.04
20 20 10.94 1 . 4 8 0.29 0.03
20 0.093 0.004
20 0.014 0.001
20 0.64 0.02
20 0.38 0.02
20 0.73 0.05
Bouin's fluid. Those foetuses fixed in alcohol were subsequently examined by open dissection for visceral abnormalities. The eviscerated carcasses were then cleared in potassium hydroxide and glycerol, and the skeletons were stained with Alizarin Red S. Skeletal structures in these foetuses were examined for abnormalities and variants, including state of ossification. Those foetuses fixed in Bouin's fluid were subsequently examined for visceral abnormalities and variants by means of a free-hand razor blade sectioning technique. During the treatment period a small dose-related proportion of the dams receiving 3 or 5g/kg body weight had brown staining around the muzzle, head and forepaws. A few incidences of excess salivation were also seen at 5 g/kg. Increased water consumption (10-15%) was observed at both 3 and 5g/kg. There was no effect on the conceptuses (Tables 8-11) at any of the doses and minor effects on the dams were consonant with those observed in the 13-wk toxicity study in rats.
Mutagenicity Gene mutation assay in Salmonella typhimurium (Pedersen, 1991). The test material was examined for
mutagenic activity using Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98. As the test material was a crude enzyme preparation containing free amino acids such as histidine, a liquid culture assay was used. The study consisted of two independent experiments in which the bacteria was exposed to six doses in a phosphate buffered nutrient broth. In the first experiment the concentrations were at half-log intervals from 21 to 6140 #g dry matter/ml incubation mixture, and in the second experiment at more narrow intervals from 192 to 6140/~g dry matter/ml incubation mixture. After incubation for 3 hr, all bacterial suspensions were washed three times by centrifugation at 3000rpm for 10min. After the first and second washings, the bacterial pellets were resuspended in 5 ml cold phosphate buffer and were finally resuspended in 2 ml phosphate buffer before plating. The numbers of revertants to prototrophy and viable cells were counted. The test was conducted in the presence and absence of metabolic activation, provided by a liver preparation from male rats, pre-treated with Aroclor 1254, and the co-factors required for mixed-function oxidase activity (S-9 mix). In all tests, inactivated test material (95°C for I hr) was used in the presence of
Safety evaluation of Esperase
1005
Table 8. Esperase, teratogenicity study in rats: pregnancy performance and foetal weight Group/dose level (mg Esperase/kg/day) 1 (0)
2 (1000)
3 (3000)
4 (5000)
No. of animals mated No. pregnant No. of premature decedents No. of decedents pregnant No. pregnant at day 20 autopsy Pregnancy frequency (%)
25 24 0 0 24 96
25 25 1 I 24 100
25 25 0 0 25 100
25 23 0 0 23 92
Total corpora lutea graviditatis Total number of implants Pre-implantation loss (%)
408 390 4
404* 387 4
442 423 4
376 361 4
367 (94) 23 (6) 20 (5) 2 (1) 1 (0.3)
368 (95) 19 (5) 17 (4) 2 (1) 0
403 (95) 20 (5) 19 (4) 1 (0.3) 0
351 (97) 10 (3) 9 (2) 1 (0.3) 0
16.8 ± 2.0 16.1 ± 2.3 15.3 ± 2.5 0.8 ± 0.7 0.7 ± 0.8 0.1 _+ 0.3 0
17.7±2.4 16.9±2.5 16.1±2.7 08±1.0 0.8±0.9 0.04±0.20 0
16.3±1.8 15.7±2.4 15.3±2.5 0.4±0.7 0.4±0.6 0.04±0.21 0
Total Total Total Total Total
live implants (%) dead implants (%) early embryonic deaths (%) late embryonic deaths (%) foetal deaths (%)
Mean Mean Mean Mean Mean Mean Mean
corpora lutea graviditatis implants live implants dead implants early embryonic deaths late embryonic deaths foetal deaths
17.0 ± 16.3 ± 15.3 ± 1.0 ± 0.8 ± 0.1 + 0.04 ±
1.8 2.0 2.0 0.9 0.8 0.3 0.20
Total live male foetuses (%) Total live female foetuses (%) Live foetal sex ratio
189 (51) 178 (49) 1:0.94
190 (52) 178 (48) 1:0.94
237 (59) 166(41) 1:0.70
183 (52) 168 (48) 1:0.92
Mean total uterus weight (g)
91.2 ± 12.5
91.0 ± 12.4
95.3 _+_14.8
90.5 ± 13.1
Mean litter mean foetal weight (g)
3.73 ± 0.25
3.69 ± 0.20
3.75 ± 0.25
3.71 + 0.28
Mean values are given ± SD. *Premature decedent excluded from this and all subsequent values.
Table 9. Esperase, teratogenicity study in rats: group incidence of major foetal abnormalities Group/dose level (mg Esperase/kg/day)
Abnormalities
1 (0)
2 (1000)
3 (3000)
4 (5000)
Oedematous; hindfeet malrotated; tail kinked
1 (1)
0
0
0
Interventricular septal defect; diaphragmatic hernia
0
0
0
1 (1)
Retro-oesophageal right subclavian artery
0
I (1)
0
0
Thoracic scoliosis; fusion of three thoracic vertebral laminae and two ribs, fusion and asymmetric insertion of costal cartilage elements onto sternum, asymmetric ossification of thoracic centra
0
0
1 (1)
0
Hem±vertebra and fused ribs
0
0
0
1 (I)
Umbilical hernia
0
I (1)
0
0
403 25
351 23
No. of foetuses examined No. of litters examined
367 24
368 24
Values represent incidence of abnormal foetuses; no. of affected litters in parentheses.
1006
R.K.
HJORTKJAER et al.
Table 10. Esperase, teratogenicity study in rats: g r o u p incidence of minor foetal abnormalities and variants G r o u p / d o s e level (mg Esperase/kg/day) Abnormalities Visceral Moderate distension of uterer(s) Hepatic h a e m o r r h a g e lntra-abdominal haemorrhage Increased cavitation of pelvis of right kidney Displaced testis/es Undescended testis/es Subdural h a e m o r r h a g e Subcutaneous h a e m o r r h a g e No. No. No. No.
of of of of
foetuses fixed in Bouin's fluid males foetuses examined viscerally litters examined viscerally
Skeletal Sutural bone in skull Right laminae of axis and 3rd cervical vertebra misshapen 'Vestigial rib-shaped' ossified body on the ventral surface of 5th cervical vertebra Vestigial rib(s) on the 7th cervical vertebra Asymmetric alignment of sternebral hemicentres One sternebra bipartite Asymmetric insertion of costal cartilage elements onto sternum W a v y ribs with curved humeri and scapulae Wavy or slightly wavy ribs Interrupted 13th rib Symmetric pelvic shift Asymmetric pelvic shift Hemicentric caudal vertebra Patchy ossification of: ~>four skull bones ~
1
2
3
4
(0)
(1000)
(3000)
(5000)
8 (4) 3 (3) 1 (1) 0 3 (3) 2 (2) 0 I (1)
3 (2) 1 (I) 0 0 8 (6) 0 0 0
0 1 (1) 4 (4) 0 5 (5) 0 2 (1) 0
6 (5) 2 (I) 1 (1) 1 (I) 8 (7) 0 0 I (1)
178 (24) 189 (24) 367 24
178 (24) 190 (24) 368 24
166 (25) 237 (25) 403 25
170 (23) 183 (23) 351 23
I (1)
0
0
3 (3)
0
I (1)
0
0
0 I (I) 0 0
I (1) 4 (2) 2 (2) 0
0 4 (1) 4 (4) 0
0 3 (3) 1 (1) I (1)
0 0 0 0 1 (I) 0 0
1 (1) 0 0 0 1 (I) 0 I (I)
0 I (1) 0 1 (I) I (1) 1 ( I) 0
0 0 I (I) 0 0 I (1) 0
5(3) 9(5) 1 ( I) 1 (I) 1 (1) 1 (1) 0 3 (3)
10(6) 17(9) I (I) I (1) 2 (2) 3 (3) 1 (1) 4 (3)
20(12) 25(11) 0 4(3) 7 (5) 8 (7) 2 (2) 10 (7)
I (I) 9(5} 1(I) I(I) 0 3 (2) 0 3 (2)
0 160 (24) 31 (14)
0 164 (24) 27(11)
2 (2) 168 (25) 39(13)
1 (1) 145 (23) 35(16)
191 24
191 24
209 25
18 I 23
Values represent incidence of a b n o r m a l foetuses; no. of affected litters in parentheses.
1007
Safety evaluation of Esperase Table I I. Esperase, teratogenicitystudy in rats: group incidenceof skeletal ossificationparameters Group/dose level(rag Esperase/kg/day) Abnormalities Overall slight skeletal ossification Cervical laminae retarded, metacarpals and 5th metatarsals unossified Any thoracic or lumbar vertebra retarded Pubes retarded 5th metacarpals unossified 5th metatarsal(s) unossified Anterior arch of atlas ossified o n e or two cervical centra ossified three or more cervical centra ossified Any phalangeal elements ossified
I
2
3
4
(0)
(1000)
(3000)
(5000)
0
0
I (1)
0
0 9 (6) 3 (3) 56 (18) 0 79 (21 ) 66 (21) 21 (8) 27 (1 I)
0 5 (5) 0 90 (20) 0 69 (20) 71 (20) 26(11) 9 (6)
0 9 (8) I (I) 105 (22) 1 (I) 73 (23) 88 (24) 31 (13) 20 (8)
1 (1) 10 (7) 3 (3) 73 (20) 0 64 (18) 77 (21) 22(10) 20 (8)
No. of sternebrae retarded: 0 1 2 >2
110 (22) 49 (20) 24 (12) 8 (4)
85 (23) 60 (22) 39 (17) 7 (5)
107 (24) 63 (25) 33 (14) 6 (6)
97 (20) 61 (22) 18 (7) 5 (5)
No. of caudal vertebral eentra ossified: <4 4 >4
28 (15) 140 (24) 23(11)
46(17) I 18 (24) 27(11)
3505) 136 (24) 38(15)
25(13) I 18 (23) 38(16)
209 25
181 23
No. of foetuses examined skeletally No. of litters examined skeletally
191 24
191 24
Values represent incidence of abnormal foetuses; no. of affected litters in parentheses.
S-9, since S-9 is destroyed by the proteotytic activity of Esperase. The sensitivity of the individual bacterial strains was confirmed by significant increases in the number of revertant colonies induced under similar conditions by diagnostic mutagens. Mean numbers obtained in the second experiment are given in Table 12. No dose-related and reproducible increases in revertants to prototrophy were obtained with any of the bacterial strains, either in the presence or absence of S-9 mix. It was concluded that there was no indication of mutagenic activity. Gene mutation assay in mammalian cells in vitro (Clements, 1991). The test material was assayed for mutation at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus (6-thioguanine resistance) in mouse lymphoma L5178Y cells using a fluctuation protocol. The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation by rat liver post-mitochondriai fraction induced by Aroclor 1254 (S-9). As in the gene mutation assay in S. typhimurium, inactivated test material (denaturation at pH 3.0 for 10min) was used in the presence of S-9. The exposure time was 2 hr. Following a wide range of treatments, separated by half-log intervals and reaching 5000/~g/ml, cells survived all doses of the test material, showing 91 and 128% relative survival in the absence and presence of S-9, respectively, at the top dose. This dose, together with the next four lower doses, was plated for viability and 6-thioguanine resistance 7 days
after treatment. In the second experiment a narrower dose range was used to maximize the chance of detecting any dose-related effects. The top close plated in this experiment was again 5000 #g/ml in the absence and presence of S-9, which yielded 86 and 99% survival, respectively. The validity of the test was confirmed by the results of negative (solvent) and positive control treatments. No test material treatment, either in the absence or presence of S-9, resulted in a statistically significant increase in mutation frequency (Table 13). It was concluded that there was no indication of mutagenic activity. Chromosome aberration assay in vitro (Marshall, 1991). The test material was examined in an in vitro cytogenetics assay using duplicate human lymphocyte cultures from a male and female donor in two independent experiments. The highest dose level used was 5000 #g/ml. In the main study, treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by S-9 from Aroclor 1254induced animals. As in the gene mutation assay in mammalian cells, inactivated test material was used in the presence of S-9. In the first experiment, treatment in the. absence of S-9 was continuous for 20 hr. Treatment in the presence of S-9 was for 3 hr only, followed by a 17-hr recovery period before harvest. The test material dose levels for chromosome analysis were selected by evaluating the effect of test material on the mitotic index. Chromosome
5 ,ug
100,ul 2#g
6140 3070 1535 767 384 192
15 ~2300
12 17 16 18 17 12
100 61
79 80 69 70 80 89
85
8
14 11 l0 10 10 11
*Corresponding to no. of viable cells × 106 on revertant plates. tWeak inhibition of the bacterial background lawn.
Sterile water MNNG 2-aminoanthracene 9-Aminoacridine 2-Nitrofluorene
Esperase Batch no. PPA 3366
Test substance
99
120
118 119 124 t18 149 115
TA 1535 Concn of test substance Without S-9 mix With S-9 mix (tJg/ml incubation Viable Viable mixture) Revertants cells* Revertants cells*
112
107
13 11 17 33 50 63
Viable cells*
99
6
7 7 7 7 3 6
Revertants
31
50
65 96 92 60 54 49
Viable cells*
With S-9 mix
~1550
17
17 18 18 16 15 22
Revertants
162
147
91 118 109 156 148 112
Viable cells*
763
25
26 32 23 33 19 22
122
128
59 127 218 152 164 131
Viable cells*
With S-9 mix
Revertants
TA98 Without S-9 mix
MNNG = N-methyI-N '-nitro-nitrosoguanidine
~2400
5
7t 6t 8 9 8 6
Revertants
Without S-9 mix
TA 1537
101 781
125 122 101 106 130 119
Revertants
58 14
67 54 74 75 66 52
Viable cells*
Without S-9 mix
~ 1025
155
152 152 149 143 154 141
74
122
138 169 153 98 147 137
Viable cells*
With S-9 mix
Revertants
TA 100
Table 12. Esperase, gene mutation assay in Salmonella typhimurium: number of revertant colonies per plate obtained with strains TA1535, TA 1537, TA98 and TA100 of Salmonella typhimurium following exposure to Esperase in the presence and absence of metabolic activation with Aroclor 1254-induced rat liver S-9 in a liquid culture assay (2nd experiment)
m
Safety evaluation of Esperase
1009
Table 13. Esperase, gene mutation assay at HGPRT locus in mouse lymphoma L51784 ceils m vitro (2nd experiment) Treatment (#g/ml)
Relative survival (%)
95% confidence limits Mutation frequency*
Upper
Results ( + or - )
4.3 5.1 4.6 5.2 4.5 3.7
7.7 8.8 8.0 9.0 7.8 6.8
------
19.1 24.9
32.0 40.6
+ +
Presence of 8-9 mix 5.5 4.2 4.7 3.4 5.9 4.5 7.2 5.5 5.6 4.2 5.2 3.9
7.3 6.4 7.7 9.'4 7~ 7.1
-
46.6 47.0
+ +
Lower
Absence of S-9 mix 0 312.5 625 1250 2500 5000
100 98 94 91 103 86
5.7 6.7 6.1 6.9 5.9 5.0
Positive control (4-nitroquinoline l-oxide) 0.1 0.15
0 312.5 625 1250 2500 5000
35 22
100 83 100 97 105 99
24.7 31.8
Positive control (benzo[a]pyrene ) 2 3
36 22
36.8 37.4
29.0 29.7
* = 6-thioguanine-resistant mutants/106 viable cells at least 7 days after treatment.
aberrations were analysed at three consecutive dose levels. The highest concentration chosen for analysis at this time, 5000 pg/ml, induced approximately 49 and 41% mitotic inhibition in the absence and presence of S-9, respectively. The second experiment (Table 14) included a delayed sampling time. Treatment in the absence of S-9 was continuous for 20 or 44 hr. Treatment in the presence of S-9 was for 3 hr followed by a 17 or 41 hr recovery period. The highest concentration chosen for analysis at 20 hr, 5000 #g/ml, induced approximately 70 and 54% mitotic inhibition in the absence and presence of S-9, respectively. The effects of single concentrations only, 1049 pg/ml without and 1638 #g/ml with S-9, were investigated at the delayed harvest, at which time 74 and 49% mitotic inhibition was induced, respectively. The validity of the test was confirmed by the lack of evidence of heterogenicity between replicate cultures, the sufficient number of cells analysed at each treatment level and the results of negative (vehicle) and positive controls. Treatment of cultures with the test material in the absence of S-9 resulted in proportions of aberrant cells at all sampling times in both experiments that were similar to, and not significantly different from, those in concurrent controls. Treatment of cultures with the test material in the presence of S-9 resulted in a small but statistically significant increase in frequencies of aberrant cells at 5000/zg/ml in the second experiment at the 20 hr sampling time. The result was not considered to be biologically significant as it was small and not reproducible. It was concluded that there was no evidence of clastogenic activity.
DISCUSSION
Calculation of human consumption of enzymes used as processing aids is based on the contents of total organic solids (TOS) in the preparation, as the rest of the preparation (i.e. ash, water and diluents) is fully characterized and is well known as safe for consumption in the quantities involved. However, it should be emphasized that the larger part of the enzyme-TOS is composed of proteins and carbohydrates from traditional food sources used in the fermentation. For the calculation of the safety margin, it is necessary to know the application of the enzyme, the concentrations of the enzyme-TOS in the final product(s), the human consumption of the product(s) and the no-adverse-effect level in the animal studies. Esperase can be used for cleaning from bones. Following this process, the meat fraction may contain 0.1% enzyme-TOS. This meat fraction can be used at a maximum of 10% in meat mixtures and sausages. The enzyme will be inactivated by heating during the process, and thu~ no active enzyme will be consumed. An average intake per day of these products is estimated to be less than 100 g per person or less than 2 g per kg body weight (US Department of Agriculture, 1983). A conservative calculation, assuming that all the meat mixtures and sausages consumed contain the (inactive) enzyme, gives a maximum daily intake of 0.2 mg enzyme-TOS per kg body weight from this source. No effects were seen in the animal studies at a dose level of 1 g of the test material per kg body weight. At the higher dose levels (i.e. 3 and 5 g of test material) only effects attributed to either the proteolytic activity or the load of sodium chloride were seen.
1010
R.K.
HJORTKJAER et al.
Table 14. Esperase, c h r o m o s o m e aberration assay using h u m a n lymphocytes in vitro (2nd experiment)* Treatment (,ug/ml)
Replicate
Cells scored
Cells with aberrations including gaps
Cells with aberrations excluding gaps
20 hr sampling time; --S-9; donor sex: female 100 6 3 100 3 0 200 9 3
Solvent
A B Totals
3200
A B Totals
100 100 200
0 0 0
0 0 0
A B Totals
100 100 200
3 2 5
1 0 1
A B Totals
100 73 173
2 4 6
1 3 4
A B Totals
25 25 50
22 19 4I
21 19 40
4000
5000
M M S , 50
A B Totals
3200
A B Totals
100 100 200
5 2 7
4 0 4
A B Totals
100 100 200
0 1 1
0 0 0
5000
A B Totals
100 100 200
5 6 11
C P A , 25
A B Totals
25 25 50
l0 14 24
Solvent
A B Totals
1049
A B Totals
Solvent
A B Totals
1638
A B Totals
NS
2.7 3.0 (2.9)
NS
2.3 2.4 (2.4)
NS
2.1 1.3 (1.7)
P ~< 0.001
4.4 6.0 (5.2)
NS
3.6 2.5 (3. I )
NS
2.7 2.6 (2.7)
3 5 8
P < 0.05
2.4 2.4 (2.4)
6 13 19
P ~< 0.001
44 hr sampling time; --S-9; donor sex: female 100 I 0 100 4 1 200 5 1 100 100 200
6 3 9
2 I 3
5.2 5.3 (5.3)
NS
44hr sampling time; +S-9; donor sex: female 100 2 2 100 3 3 200 5 5 100 100 200
4 0 4
M M S = methyl methane sulfonate N S = not significant * N u m b e r s in bold typeface exceed histological negative control ranges. t F i s h e r ' s exact test.
Mitotic index (mean) 5.3 6.0 (5.7)
20 hr sampling time; +S-9; donor sex: female 100 2 0 100 3 I 200 5 1
Solvent
4000
Statistical significancet
1 0 1
1.2 1.5 (1.4)
5.1 6.6 (5.9)
NS
C P A = cyclophosphamide
2.5 3.5 (3.0)
Safety evaluation of Esperase The application of the enzyme in food processing will lead to neither an intake of the active enzyme nor a high intake of salts, and thus these effects may be disregarded. The dose levels of 1 and 5 g test substance per kg body weight correspond to 230 and 1200mg TOS/kg body weight, respectively, or a safety margin of 1000-5000 in the food application described. According to the literature, the production organism B. lentus is non-pathogenic and nontoxinogenic. The test material was without antibacterial and mutagenic activity in the in vitro tests, and it had no effect on reproduction in the rat in vivo teratogenicity study. On the basis of these results and the low toxic potential in the subchronic toxicity, the application of Esperase in food processing should be considered safe for the consumer, with a reasonably high margin of safety. REFERENCES
AMFEP (Association of Microbial Food Enzyme Producers) (1992) Regulatory Aspects o f Microbial Food Enzymes. AMFEP Secretariat, Brussels. Babesgaard P. (1991 ) Bacillus lentus, Taxonomy and Patho genicity. Novo Nordisk A/S, F-910535, unpublished report. Clements J. (1991) Study to Determine the Ability o f E S P E R A S E to Induce Mutations to 6-Thioguanine Resist ance in Mouse Lymphoma L5178Y Cells using a Fluctuation Assay. Hazleton Microtest, 2MLRENOD.023,
unpublished report. FCC (Food Chemical Codex) (1981) National Academy o f Sciences~National Research Council, Food and Nutrition Board, Committee on Codex Specifications 107 110.
National Academy Press, Washington. Food and Drug Administration (1983) Direct food substances affirmed as GRAS, mixed carbohydrase and protease enzyme product. Federal Register 48, 239 240. Heath J., Mulhern M., Perry C. J. and Millar P. (1991) ESPERASE: 13 week toxicity study in rats. Inveresk Research International. Report No. 7680, unpublished report.
1011
IFBC (International Food Biotechnology Council) (1990) Safety evaluation of foods and food ingredients derived from microorganisms. Regulatory Toxicology and Pharmacology 12, 114-128. JECFA (Joint FAO/WHO Expert Committee on Food Additives) (1984) General Specifications for Enzyme Preparations used in Food Processing. FAO Food and Nutrition Paper 31/2, 129-131. Jensen B. F. and Eigtved P. (1990) Safety aspects of microbial enzyme technology, exemplified by the safety assessment of an immobilized lipase preparation, Lipozymetin. Food Biotechnology 4, 699-725. Marshall R. R. (1991) Study to Evaluate the Chromosome Damaging Potential o f E S P E R A S E '~t by its Effects on Cultured Human Lymphocytes using an in vivo Cytogenetics Assay. Hazleton Microtest Report: 2HLRENOD.023,
unpublished report. NIH (National Institutes of Health) (1986) Guidelines for research involving recombinant DNA molecules; notice. Federal Register 51, 16967 16968. OECD (1986) Recombinant DNA Safety Considerations. OECD, Paris. Pariza M. W. and Foster E. M. (1983) Determining the safety of enzymes used in food processing. Journal o f Food Protection 46, 453-468. Pedersen P. B. (1991) E S P E R A S E (Batch NO. PPA 3366): Testing for Mutagenic Activity with Salmonella typhimurium TA 1535, TA 1537, TA 98and TA lOOina Liquid Culture Assay. Study No. 91514. Novo Nordisk A/S,
F-911207, unpublished report. SCF (EEC Scientific Committee for Food) (1991) Guidelines for the presentation of data on food enzymes. Report o f the Scientific Committee for Food. 27th series.
Office for Official Publications of the EEC, Luxemburg. US Department of Agriculture (1983) Food Intakes: Individuals in 48 States, Year 197~78. Nationwide Food Consumption Survey 197~78. Report no. 1-1. Human Nutrition Information Service. United States Department of Agriculture. Wilson J. A. (1991) ESPERASE: Teratogenicity Study in Rats. Inveresk Research International. Report No. 7698, unpublished report.
[The unpublished reports cited in this list are available on request from Novo Nordisk A/S (M. Stavnsbjerg).]
0278-6915/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd
SAFETY EVALUATION OF ESPERASE R. K. HJORTKJAER*, M. STAVNSBJERGt, P. B. PEDERSEN't, J. HEATHS, J. A. WILSON~, R. R. MARSHALL§ and J. CLEM~NTS~ *Danish Toxicology Centre, Agern All6 15, DK-2970 Horsholm, tNovo Nordisk A/S, Novo All& DK-2880 Bagsvaerd, Denmark, ~tlnveresk Research International, Tranent EH33 22NE, Scotland and §Hazleton Microtest, Otley Road, Harrogate, North Yorkshire HG3 1PY, UK (Accepted 24 June 1993)
Abstract--Esperase is a proteolytic enzyme preparation that can be used as a processing aid in the food industry. The following studies were performed to establish safety for the consumer: oral toxicity study (13 wk) in the rat; teratogenicity study in the rat; gene mutation assays in Salmonella typhimurium and mammalian cells in vitro, and chromosome aberration assay/n vitro. General toxicity was low; the effects seen were attributed to proteolytic activity and the loading with sodium chloride. Neither of these factors will be relevant to consumers of the processed food. There was no evidence of effects on pregnancy outcome or mutagenic potential. When these results are considered together with existing knowledge of the production organism and the chemical and microbiological characterization of the enzyme preparation, they indicate that Esperase will be safe for its intended application in food processing.
INTRODUCTION
The safety-in-use of microbial food enzymes can be established on a history of safe use and a safety evaluation based on scientific procedures (e.g. A M F E P , 1992; E E C Scientific Committee for F o o d (SCF), 1991; F o o d and D r u g Administration, 1983; IFBC, 1990; Jensen and Eigtved, 1990; Pariza and Foster, 1983). The safety review should include the following steps (Jensen and Eigtved, 1990): (1) safety assessment of the production strain; (2) identity and quality of raw materials used in the fermentation; (3) processing techniques used in recovery; (4) identity and quality of the stabilizers, diluents or formulation aids; (5) toxicology studies on enzyme concentrate, and (6) determination of safety margin. Esperase is a proteolytic enzyme (serine protease; EC 3.4.21.14) preparation with a broad catalytic activity. The enzyme is produced by Bacillus lentus, which is listed as a Class 1 Agent in the National Institute of Health Guidelines (NIH, 1986) and complies with the G o o d Industrial Large Scale Practice (GILSP) criteria suggested by O E C D (1986). A search has been carried out in the following databases: Biological Abstracts, Medline, Excerpta Medica, Toxline, Commonwealth Agricultural Bureau and Occupational Safety and Health. N o n e of the published literature related B. lentus to human or animal diseases or the formation of toxins, confirming that B. lentus can be considered a safe, non-pathogenic and non-toxinogenic microorganism (Babesgaard, 1991). The production method (i.e. raw materials, fermentation conditions, recovery techniques, etc.) complies with the requirements for microbial food enzymes. This paper summarizes a programme of studies that was
carried out to establish the consumer safety o f Esperase when used as a processing aid in the food industry. MATERIALS AND METHODS Test material
The chemical composition of batch PPA 3366 of the enzyme preparation used for the toxicity studies is given in Table I. The main source of the non-protein nitrogen is amino acids and polypeptides. Analyses of the antibiotic activity and microbial contamination showed that the batch complied with the Joint F A O / W H O Expert Committee on F o o d Additives ( J E C F A , 1984) and F o o d Chemical Codex (FCC, 1981) specifications for enzymes. The stability Table 1. Chemical composition (%) of Esperase batch PPA 3366 Component Percentage H20 59.3 Carbohydrate, total approx. 6 Ntota~x 6.25 9.19 Nprotei n X 6.25
Petroleum ether soluble residue Ash (600°C) Na K Mg Ca CI PO, SO4 CO3 Heavy metals As (ppm) Pb (ppm) Total organic solids (TOS) = 100% - (ash% + water% + diluents%) = 100% - (17.7% + 59.3% + 0%) 23% =
999
3.00
0.16 17.7 <5.1 0.7 0.06 1.5 7.8 0.05 0.14 <0.1 < 0.0040 <3 < 10
1000
R . K . HJORTKJAER et al.
Table 2. Esperase, 13 wk oral toxicity study in rats: methods, abbreviations and units used in laboratory investigations Parameters
Method
Units
Haematology Haemoglobin (Hb): Total red blood cell count (RBC): Haematocrit (Hct): Absolute values: Mean ceil volume (MCV): Mean cell haemoglobin (MCH): Mean cell haemoglobin concentration (MCHC): Total white blood cell count (WBC): Differential white cell count: Neutrophil (Neut): Lymphocyte (Lymp): Monocyte (Mono): Eosinophil (Eos): Platelet count (Plat): Hepato quick (Hep):
Coulter Counter, Coulter Electronics Limited
g/dl
Coulter Coulter Coulter Coulter
x 10~:/litre litre/litre
Counter, Counter, Counter, Counter,
Coulter Coulter Coulter Coulter
Electronics Electronics Electronics Electronics
Limited Limited Limited Limited
fl
Coulter Counter, Coulter Electronics Limited
pg
Coulter Counter, Coulter Electronics Limited
g/dl
Coulter Counter, Coulter Electronics Limited
x 109/litre
Visual appraisal of stained film (May Grunwald and Giemsa stain)
x 109/litre
Coulter Counter, Coulter Electronics Limited beck E. A. and Muschietti F., Schweiz Med. Wschr 35, 1218 (1974)
x 109/litre sec
Clinical Chemistry Blood urea nitrogen (BUN): Glucose (Glu): Aspartate aminotransferase (AST): Alanine aminotransferase (ALT): Lactate dehydrogenase (LDH): Sodium (Na): Potassium (K): Chloride (CI): Total Protein (TP): Albumin (Alb): Albumin/globulin ratio (AG-R): Cholesterol (Chol): Alkaline phosphatase (AP): Calcium (Ca): Creatinine (Crea): Inorganic phosphate (Phos): Total bilirubin (T.Bi):
Neumann U. and Zeigenhorn J., XVI. Nordiska Kongressen for Klinisk Kemi och Klinisk Fysiologi, Oulu, Finland (1977) Schmidt F. H. Klin. Wschr 39, 1244 (1961) Enzyme Commission of the German Society for Clinical Chemistry. Anon., Z. Klin. Chem. Klin. Biochem. 8, 658 (1970). Anon., ibid 10, 182 (1972) Enzyme Commission of the German Society for Clinical Chemistry. Anon., Z. Klin. Chem. Klin. Biochem. 8, 658 (1970). Anon., ibid 10, 182 (1972) Enzyme Commission of the German Society for Clinical Chemistry. Anon., Z. Klin. Chem. Kiln. Bioehem. 8, 658 (1970). Anon. ibid 10, 182 (1972) Ion Selective Electrode Ion Selective Electrode Ion Selective Electrode Weichselbaum T. E. Am. J. Clin. Path. 16, 40 (1964) Dounes B. T. et al., Clin. Chem. Acta 31, 87 (1971) Siedel J. et at, .I. Clin. Chem. Clin. Biochem. 19, 838 (1981). Stabler F. et al., Med. Lab 30, 29 (1977). Trinder P, Ann. Clin. Biochem. 6, 24 (1969) Anon., Z. Klin. Chem. Klin. Biochem. 8, 658 (1970). Anon., ibid I0, 182 (1972) Ray Sarker B. C. and Chauton U. P. S. Analyt. Biochem. 20, 155 (1967). Baginski E. et al., Clin. Chem. Acta 46, 46 (1973) Modified from Barkels H. et al., ClOt. Chem. Aeta 37, 193 (1972) Henry R. J., ed. Clinical Chemistry Principles and Techniques, p. 722 (1974) Wahlefeld A. W., Herz G. and Bernt E., Scand .1. Clin. Lab. Invest. 29 (Suppl 126), Abstract 11.12 (1972)
mmol/litre mmol/litre IU/litre IU/litre lU/litre mmol/litre mmol/litre mmol/litre g/litre g/litre mmol/litre IU/litre mmol/litre ~mol/litre mmol/litre /~mol/litre
Safety evaluation of Esperase of the batch during the conducting period of the toxicity studies was confirmed by determination of enzyme activity. Toxicity studies Subchronic oral toxicity in rats (Heath et al., 1991). Following dose range-finding studies, a 13 wk study was carried out with groups of 20 male and 20 female Sprague-Dawley rats. The animals were dosed once daily with the test material by means of a steel dosing cannula at a constant dose volume of 5 ml/kg body weight to give dose levels of 0, 1, 3 and 5 g/kg body weight/day. Routine clinical observations and determinations of body weights, food and water consumption were undertaken throughout the study period. Ophthalmoscopy was performed before the trial and during wk 13. Haematological and clinical chemical investigations were carried out on samples taken from ten males and ten females chosen at random from each group during wk 12/13. The parameters examined and investigation methods used are given in Table 2.
Table 3. Esperase, 13-wk oral toxicity study in rats: organs weighed (W), fixed (F) and examined histologically (H) Organ
W
F
H
Adrenals Aortic arch Any abnormal tissue Bladder Bone (sternum and rib) Brain Ears Eyes Heart Intestine: duodenum jejunum ileum caecum colon rectum Kidneys Stomach (glandular and non-glandular) Spinal cord (cervical, thoracic and lumbar portions) Submaxillary salivary gland Submandibular lymph node Liver Lungs Mammary gland Mesenteric lymph node Muscle (thigh) Nasal cavity Oesophagus Ovaries (with fallopian tubes) Optic nerve
X
X X X X X X X X X
X X X X
X X X X X
X X X X X
X X
X X
Pancreas Pituitary Prostate Sciatic nerve Seminal vesicles Skin Spleen Testes (plus epididymides)
X X
X
X X
X
X X
X X
X X X
X X X X X X X X X X X X
X X X
X X X X X X X X
X X X X X X X X
X X X X X X X
Thymus Thyroids Tongue Trachea
X
X X X X
X X X X
Uterus
X
X
X
FCT31/12--F
1001
At the end of the treatment period all animals were killed by asphyxiation with carbon dioxide followed by exsanguination, and were subjected to gross pathological examination and organ weight analyses. A comprehensive range of tissues was fixed, and examined microscopically from all animals in the control and high-dose groups (Table 3). Haematology, clinical chemistry, organ weight and body weight data were statistically analysed for homogeneity of variance using the F-max test. If the group variances appeared homogeneous a parametric ANOVA was used and pairwise comparisons made by Student's t-test using Fisher's F-protected LSD. If the variances were heterogeneous, log and square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, then a non-parametric test such as a Kruskal-Wallis ANOVA was used and pairwise comparisons made by Dunn Z test where considered appropriate. Organ weights were also analysed, conditional on body weight (i.e. analysis of covariance). Histology data were analysed using Fisher's exact probability test. There were five unscheduled deaths, none being related to treatment with the test material. A doserelated increase in incidence of red/brown staining around the muzzle and forepaws, and red extremities, were found in the intermediate and high-dose males and high-dose females. Body weight gain and food and water consumption are given in Table 4. The intermediate- and high-dose males showed a slight reduction in body weight gain (10 and 12%, respectively), whereas the intermediate-dose females had a slightly higher body weight gain (11%). There was an increase in total water consumption by low-dose males and the intermediate- and high-dose males and females. There were no other clinical observations. The haematological investigations revealed no changes (Table 5). A slight decrease was found in the alanine aminotransferase activities in the intermediate-dose males and the high-dose males and females, and in concentrations of sodium in the intermediate- and high-dose males and females (Table 6). There were no other notable intergroup differences in the other clinical chemical parameters. After adjustment for final body weight (covariance analysis), a decrease in prostate weight and an increase in kidney weight were seen in the intermediate- and high-dose males (Table 7). There were no macroscopic findings at autopsy that could be related to treatment. Focal hyperplasia of the keratinized epithelium in the stomach was seen in 2/20 high-dose males and 2/20 high-dose females compared with 0/20 in both male and female controls. The red/brown staining seen clinically is ascribed to the irritant potential of the test material and is an expected effect of treatment with a protease. The reduction in body weight gain in the intermediate and high dose males was not reflected in a concomitant change in food consumption and indicates an adverse
R . K . HJORTKJAER et al.
1002
Table 4. Esperase, 13-wk oral toxicity study in rats: body weight gains, food consumed and water consumed per rat Group/dose level (mg Esperase/kg/day) Time on study (wk)
IM (0)
Body weight gain (g) wk0-13
381
2M (1000)
3M (3000)
4M (5000)
IF (0)
2F (1000)
3F (3000)
4F (5000)
396 (96)*
344 (90)
334 (88)
176
186 (106)
196 (111)
I73 (98)
Total food consumed (g) wk 1-13
2915
2962 (102)
2812 (96)
2713 (93)
2060
2041 (99)
2124 (103)
2036 (99)
Total water consumed (ml) wk 7-13
1602
1811 (113)
2017 (126)
2221 (139)
1416
1433 (101)
1735 (123)
1820 (129)
M=male
F=female
*Percentages of control in parentheses.
Table 5. Esperase, 13-wk oral toxicity study in rats: haematology during wk 12/13~group mean values Group/dose level (mg Esperase/kg/day)
Hb
RBC
Hct
MCH
MCV MCHC WBC
Neut
Lymp Mono
Eos
Plat
Hep
8 3.2 1.5
8 13.3 4,0
8 0.4 0.4
8 0.2 0.1
8 687 165
9 24 I
1 (0)
Number Mean SD
8 15.4 0.6
8 7.28 0.42
8 0.401 0,015
Males 8 8 21.4 54 0.5 2
2 (1000)
Number Mean SD
l0 15.3 0.4
10 7.30 0.25
10 0.392 0.011
10 21.2 0.9
10 52 2
l0 39.6 0.7
l0 14.7 2.3
10 2.1 1.0
10 12.0 2.7
10 0.4 0.3
10 0.2 0.2
10 742 132
10 25 I
3 (3000)
Number Mean SD
9 15.4 1.0
9 7.34 0,58
9 0.393 0.025
9 21.2 0.5
9 52 2
9 39.7 1.0
9 14.6 4.5
9 3.1 1.4
9 1 1.0 3.2
9 0.4 03
9 0.1 0.1
9 726 257
8 25 3
4 (5000)
Number Mean SD
9 15.3 0.5
9 7,21 0,46
9 0.397 0.013
9 21.4 1.0
9 54 3
9 38.9 0.5
9 15.2 4.5
9 2.4 0.6
9 12.3 4.0
9 0.4 0.4
9 0.1 0.2
9 661 179
10 25 I
1 (0)
Number Mean SD
10 15.4 0.4
10 7.15 0.28
10 0.405 0.007
Femmies 10 10 21.9 56 0.5 2
10 38.6 0.5
10 9.8 3.0
10 1.2 1.0
10 8.3 2.2
10 0.2 0.2
10 0.2 0.1
l0 766 88
9 23 3
2 (1000)
Number Mean SD
7 15.7 0.4
7 7.25 0.19
7 0.413 0.014
7 22.0 0.4
7 57 I
7 38.5 0.5
7 11.2 1.8
7 1.3 0.3
7 9.6 1.5
7 0.2 0.1
7 0.1 0.1
7 797 78
10 22 3
3 (3000)
Number Mean SD
10 15.1 0.6
10 6.92 0.35
10 0.397 0.014
10 22.1 0.6
10 57 1
10 38.5 0.5
10 10.8 1.6
10 1.8 1.0
t0 8.6 1.5
10 0.3 0.2
10 0.1 0.2
10 682 195
10 24 1
4 (5000)
Number Mean SD
10 15.3 0.5
10 6.90 0.20
10 0.402 0.013
10 22.4 0,3
10 58 l
10 38.5 0.6
10 10.4 2.0
10 1,5 0.7
10 8.8 2.2
l0 0.1 0.1
10 0.1 0.1
10 750 I01
10 24 2
Abbreviations as in Table 2.
8 39.0 0.6
8 17.1 5.6
Safety evaluation of Esperase effect o f t r e a t m e n t - - a n effect t h a t is also s e e n w i t h other proteolytic enzymes. The increased water cons u m p t i o n m a y be a r e s u l t o f t h e a m o u n t o f a s h , especially s o d i u m a n d c h l o r i d e , g i v e n w i t h t h e test substance. In the high-dose animals the total sodium a n d c h l o r i d e i n t a k e s were a p p r o x i m a t e l y t h r e e t i m e s those of the control animals. An additional control group without NaC! might have confirmed that the N a C 1 l o a d w a s r e s p o n s i b l e f o r this i n c r e a s e d w a t e r i n t a k e . C h a n g e s in a l a n i n e a m i n o t r a n s f e r a s e activities, in c o n c e n t r a t i o n s o f s o d i u m in p l a s m a a n d in o r g a n w e i g h t s ( p r o s t a t e w e i g h t a n d k i d n e y w e i g h t in intermediate- and high-dose males) did not correlate w i t h a n y h i s t o l o g i c a l f i n d i n g s , a n d a r e c o n s i d e r e d to be o f n o t o x i c o l o g i c a l significance. T h e o c c u r r e n c e o f focal h y p e r p l a s i a o f t h e k e r a t i n i z e d e p i t h e l i u m in t h e s t o m a c h o f t h e h i g h - d o s e m a l e a n d f e m a l e is u n u s u a l in r a t s o f t h i s a g e a n d p r o b a b l y is a t t r i b u t a b l e to t h e i r r i t a n t p o t e n t i a l o f t h e test m a t e r i a l ; h o w e v e r , t h e i n c i d e n c e s p r o v i d e insufficient e v i d e n c e o f a t r e a t m e n t - r e l a t e d effect. O v e r a l l , it c a n be c o n c l u d e d t h a t n o a d v e r s e effects were s e e n f o l l o w i n g 1 g / k g b o d y w e i g h t , w h e r e a s 3 a n d 5 g / k g b o d y w e i g h t r e s u l t e d in m i n o r effects, w h i c h c o u l d be a s c r i b e d to t h e p r o t e o l y t i c
activity a n d preparation.
1003 the amount
o f s a l t s in t h e e n z y m e
Teratogenicity in rats (Wilson, 1991). T h e d o s e levels f o r this s t u d y were selected a f t e r e v a l u a t i o n o f t h e e x i s t i n g toxicity d a t a , a n d were t h e s a m e as t h o s e u s e d in t h e 1 3 w k s t u d y - - 0 , 1, 3 a n d 5 g / k g / d a y . T h e d o s e s were a d m i n i s t e r e d o n c e d a i l y by g a v a g e to g r o u p s o f 25 S p r a g u e - D a w l e y r a t s during days 6-16 of gestation. During the observ a t i o n p e r i o d ( d a y s 0 - 2 0 o f g e s t a t i o n ) clinical signs, b o d y w e i g h t s a n d f o o d a n d w a t e r c o n s u m p t i o n were r e c o r d e d . O n d a y 20 o f g e s t a t i o n t h e a n i m a l s were killed b y c a r b o n d i o x i d e a s p h y x i a t i o n , a n d t h e c o n t e n t s o f t h e t h o r a c i c a n d a b d o m i n a l cavities were e x a m i n e d m a c r o s c o p i c a l l y for a b n o r m a l i t i e s . T h e reproductive tract was removed and weighed, then o p e n e d a n d t h e c o n t e n t s were e x a m i n e d . T h e n u m b e r o f c o r p o r a l u t e a g r a v i d i t a t i s in e a c h o v a r y a n d t h e n u m b e r a n d s t a t u s o f all i m p l a n t a t i o n sites were r e c o r d e d . E a c h live f o e t u s w a s i n d i v i d u a l l y identified w i t h i n t h e litter a n d its w e i g h t w a s r e c o r d e d . T h e f o e t u s e s were e x a m i n e d f o r e x t e r n a l l y visible a b n o r m a l i t i e s b e f o r e fixation. A p p r o x i m a t e l y o n e - h a l f o f t h e f o e t u s e s f r o m e a c h u t e r u s were t h e n fixed in m e t h y l a t e d ethyl a l c o h o l , a n d t h e r e m a i n i n g h a l f in
Table 6. Esperase, 13-wk oral toxicity study in rats: clinical chemistry during wk 12-13--group mean values Group/dose level (mg Esperase/kg/day) BUN Glu AST ALT AP LDH Na K C1 TP Alb AG-R Chol Crea Ca Phos T.Bi Males
1 (0)
Number Mean SD
10 5.9 0.8
10 10 8.66 116 1.06 37
10 76 16
10 10 10 10 348 897 144 4.4 61 914 I 0.6
10 99 1
10 71 3
10 31 1
10 0.8 0.0
10 2.6 0.5
10 10 10 10 49 2.67 1.97 1.8 5 0.06 0.19 0.7
2 (1000)
Number Mean SD
10 5.4 0.9
10 10 8.12 106 0.56 24
10 65 14
10 10 10 10 307 474 144 4.3 69 309 1 0.3
10 98 2
10 70 2
10 31 1
10 0.8 0.1
10 2.4 0.4
10 10 10 10 48 2.70 1.85 1.7 4 0.07 0.21 0.4
3 (3000)
Number Mean SD
10 6.1 1.6
10 10 7.79 109 0.70 41
10 64 19
10 10 10 10 350 656 142 4.6 52 507 I 0.5
10 98 1
10 69 4
10 29 3
10 0.7 0.1
10 3.0 2.6
10 10 10 48 2.66 1.71 7 0.09 0.29
10 2.0 0.7
4 (5000)
Number Mean SD
9 6.5 0.7
9 9 7.87 101 0.97 22
9 55 7
9 9 389 556 106 492
9 98 2
9 68 2
9 29 1
9 0.8 0.1
9 2.0 0.3
9 47 3
9 1.7 1.2
1 (0)
Number Mean SD
10 6.2 0.6
10 8.65 0.81
10 87 17
10 71 20
10 10 10 10 307 243 138 3.5 79 232 1 0.3
10 101 2
10 72 4
10 33 3
10 0.9 0.1
10 2.2 0.4
10 10 10 10 52 2.61 1.70 1.5 2 0.09 0.29 0.4
2 (1000)
Number Mean SD
10 6.4 0.8
10 8.64 0.73
10 89 35
10 71 25
10 10 10 10 242 227 138 3.6 66 79 1 0.3
10 101 2
10 74 7
10 35 3
10 0.9 0.0
10 2.4 0.5
10 10 10 10 50 2.75 1.80 1.6 3 0.13 0.11 0.3
3 (3000)
Number Mean SD
10 6.9 1.3
10 8.58 0.81
10 84 18
10 63 21
10 10 10 10 257 212 137 3.7 56 115 1 0.3
10 100 2
10 73 3
10 33 2
10 0.8 0.1
10 2.4 0.5
10 10 10 10 53 2.66 1.67 1.7 4 0.08 0.23 0.7
4 (5000)
Number Mean SD
10 6.3 1.0
10 8.23 0.48
10 69 10
10 50 6
10 10 10 I0 237 162 137 3.7 63 20 1 0.3
I0 100 2
10 72 4
10 33 3
I0 0.9 0.1
I0 2.3 0.3
I0 10 10 10 50 2.70 1.78 1.6 3 0.09 0.24 0.4
9 9 143 4.4 1 0.4
*
9 9 2.69 1.70 0.06 0.20
Females
*Significantly different from control: P < 0.05. **Significantly different from control: P < 0.01. 2.
A b b r e v i a t i o n s as in T a b l e
1004
R . K . HJORTKJAER el al. Table 7. Esperase, 13-wk oral toxicity study in rats: organ weight (covariance analysis}--group mean values
Group/dose level
Body weight (g) Adrenals Brain
(rag Esperase/kg/day)
Heart Kidneys Liver Lungs Pituitary Prostate Spleen Testes Thymus
Males 1 (0)
Number Mean SE
19 540 16
19 0.062 0.002
19 2.07 0.02
19 1.60 0.03
19 3.45 0.08
19 18.77 0.53
19 2.00 0.04
19 0.011 0.001
19 0.89 0.04
19 1.00 0.04
19 5.20 0. I I
19 0.42 0.02
2 (1000)
Number Mean SE
19 540 16
19 0.062 0.002
19 2.11 0.02
19 1.65 0.03
19 3.66 0.09
19 19.15 0.54
19 1.95 0.04
19 0.012 0.001
19 0.80 0.04
19 0.92 0.04
19 5.33 0.12
18 0.40 0.03
3 (3000)
Number Mean SE
19 540 16
19 0.063 0.002
19 2.09 0.02
19 1.65 0.03
19 3.79 0.08
19 19.08 0.53
19 1.95 0.04
19 0.012 0.001
19 0.72 0.04
19 0.93 0.04
19 5.23 0.11
19 0.42 0.02
4 (5000)
Number Mean SE
19 540 16
19 0.063 0.002
19 2.08 0.02
19 1.62 0.03
19 3.79 0.09
19 19.59 0.54
19 1.87 0.04
19 0.012 0.001
19 0.70 0.04
19 0.96 0.04
19 5.05 0.11
19 0.37 0.03
**Significantly different from control (P < 0.01). Group/dose level (mg Esperase/kg/day)
Body weight (g) Adrenals Brain Heart Kidneys Liver Lungs Ovaries Pituitary Spleen Thymus
Uterus
Females 1 (0)
Number Mean SE
20 312 8
20 0.075 0.002
20 1.92 0.02
20 1.07 0.03
20 2.18 0.04
20 20 10.42 1.46 0.29 0.03
20 0.097 0.004
20 0.013 0.001
20 0.60 0.02
20 0.35 0.02
20 0.67 0.05
2 (1000)
Number Mean SE
19 312 8
19 0.077 0.002
19 1.92 0.02
19 1.11 0.03
19 2.16 0.04
19 19 11.15 1.55 0.30 0.03
19 0.093 0.004
19 0.013 0.001
19 0.64 0.02
19 0.38 0.02
19 0.63 0.05
3 (3000)
Number Mean SE
20 312 8
20 0.076 0.002
20 1.93 0.02
20 1.12 0.03
20 2.24 0.04
20 20 10.28 1.54 0.30 0.03
20 0.108 0.004
20 0.013 0.001
20 0.60 0.02
20 0.39 0.02
20 0.74 0.05
4 (5000)
Number Mean SE
20 312 8
20 0.079 0.002
20 1.92 0.02
20 1.16 0.03
20 2.29 0.04
20 20 10.94 1 . 4 8 0.29 0.03
20 0.093 0.004
20 0.014 0.001
20 0.64 0.02
20 0.38 0.02
20 0.73 0.05
Bouin's fluid. Those foetuses fixed in alcohol were subsequently examined by open dissection for visceral abnormalities. The eviscerated carcasses were then cleared in potassium hydroxide and glycerol, and the skeletons were stained with Alizarin Red S. Skeletal structures in these foetuses were examined for abnormalities and variants, including state of ossification. Those foetuses fixed in Bouin's fluid were subsequently examined for visceral abnormalities and variants by means of a free-hand razor blade sectioning technique. During the treatment period a small dose-related proportion of the dams receiving 3 or 5g/kg body weight had brown staining around the muzzle, head and forepaws. A few incidences of excess salivation were also seen at 5 g/kg. Increased water consumption (10-15%) was observed at both 3 and 5g/kg. There was no effect on the conceptuses (Tables 8-11) at any of the doses and minor effects on the dams were consonant with those observed in the 13-wk toxicity study in rats.
Mutagenicity Gene mutation assay in Salmonella typhimurium (Pedersen, 1991). The test material was examined for
mutagenic activity using Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98. As the test material was a crude enzyme preparation containing free amino acids such as histidine, a liquid culture assay was used. The study consisted of two independent experiments in which the bacteria was exposed to six doses in a phosphate buffered nutrient broth. In the first experiment the concentrations were at half-log intervals from 21 to 6140 #g dry matter/ml incubation mixture, and in the second experiment at more narrow intervals from 192 to 6140/~g dry matter/ml incubation mixture. After incubation for 3 hr, all bacterial suspensions were washed three times by centrifugation at 3000rpm for 10min. After the first and second washings, the bacterial pellets were resuspended in 5 ml cold phosphate buffer and were finally resuspended in 2 ml phosphate buffer before plating. The numbers of revertants to prototrophy and viable cells were counted. The test was conducted in the presence and absence of metabolic activation, provided by a liver preparation from male rats, pre-treated with Aroclor 1254, and the co-factors required for mixed-function oxidase activity (S-9 mix). In all tests, inactivated test material (95°C for I hr) was used in the presence of
Safety evaluation of Esperase
1005
Table 8. Esperase, teratogenicity study in rats: pregnancy performance and foetal weight Group/dose level (mg Esperase/kg/day) 1 (0)
2 (1000)
3 (3000)
4 (5000)
No. of animals mated No. pregnant No. of premature decedents No. of decedents pregnant No. pregnant at day 20 autopsy Pregnancy frequency (%)
25 24 0 0 24 96
25 25 1 I 24 100
25 25 0 0 25 100
25 23 0 0 23 92
Total corpora lutea graviditatis Total number of implants Pre-implantation loss (%)
408 390 4
404* 387 4
442 423 4
376 361 4
367 (94) 23 (6) 20 (5) 2 (1) 1 (0.3)
368 (95) 19 (5) 17 (4) 2 (1) 0
403 (95) 20 (5) 19 (4) 1 (0.3) 0
351 (97) 10 (3) 9 (2) 1 (0.3) 0
16.8 ± 2.0 16.1 ± 2.3 15.3 ± 2.5 0.8 ± 0.7 0.7 ± 0.8 0.1 _+ 0.3 0
17.7±2.4 16.9±2.5 16.1±2.7 08±1.0 0.8±0.9 0.04±0.20 0
16.3±1.8 15.7±2.4 15.3±2.5 0.4±0.7 0.4±0.6 0.04±0.21 0
Total Total Total Total Total
live implants (%) dead implants (%) early embryonic deaths (%) late embryonic deaths (%) foetal deaths (%)
Mean Mean Mean Mean Mean Mean Mean
corpora lutea graviditatis implants live implants dead implants early embryonic deaths late embryonic deaths foetal deaths
17.0 ± 16.3 ± 15.3 ± 1.0 ± 0.8 ± 0.1 + 0.04 ±
1.8 2.0 2.0 0.9 0.8 0.3 0.20
Total live male foetuses (%) Total live female foetuses (%) Live foetal sex ratio
189 (51) 178 (49) 1:0.94
190 (52) 178 (48) 1:0.94
237 (59) 166(41) 1:0.70
183 (52) 168 (48) 1:0.92
Mean total uterus weight (g)
91.2 ± 12.5
91.0 ± 12.4
95.3 _+_14.8
90.5 ± 13.1
Mean litter mean foetal weight (g)
3.73 ± 0.25
3.69 ± 0.20
3.75 ± 0.25
3.71 + 0.28
Mean values are given ± SD. *Premature decedent excluded from this and all subsequent values.
Table 9. Esperase, teratogenicity study in rats: group incidence of major foetal abnormalities Group/dose level (mg Esperase/kg/day)
Abnormalities
1 (0)
2 (1000)
3 (3000)
4 (5000)
Oedematous; hindfeet malrotated; tail kinked
1 (1)
0
0
0
Interventricular septal defect; diaphragmatic hernia
0
0
0
1 (1)
Retro-oesophageal right subclavian artery
0
I (1)
0
0
Thoracic scoliosis; fusion of three thoracic vertebral laminae and two ribs, fusion and asymmetric insertion of costal cartilage elements onto sternum, asymmetric ossification of thoracic centra
0
0
1 (1)
0
Hem±vertebra and fused ribs
0
0
0
1 (I)
Umbilical hernia
0
I (1)
0
0
403 25
351 23
No. of foetuses examined No. of litters examined
367 24
368 24
Values represent incidence of abnormal foetuses; no. of affected litters in parentheses.
1006
R.K.
HJORTKJAER et al.
Table 10. Esperase, teratogenicity study in rats: g r o u p incidence of minor foetal abnormalities and variants G r o u p / d o s e level (mg Esperase/kg/day) Abnormalities Visceral Moderate distension of uterer(s) Hepatic h a e m o r r h a g e lntra-abdominal haemorrhage Increased cavitation of pelvis of right kidney Displaced testis/es Undescended testis/es Subdural h a e m o r r h a g e Subcutaneous h a e m o r r h a g e No. No. No. No.
of of of of
foetuses fixed in Bouin's fluid males foetuses examined viscerally litters examined viscerally
Skeletal Sutural bone in skull Right laminae of axis and 3rd cervical vertebra misshapen 'Vestigial rib-shaped' ossified body on the ventral surface of 5th cervical vertebra Vestigial rib(s) on the 7th cervical vertebra Asymmetric alignment of sternebral hemicentres One sternebra bipartite Asymmetric insertion of costal cartilage elements onto sternum W a v y ribs with curved humeri and scapulae Wavy or slightly wavy ribs Interrupted 13th rib Symmetric pelvic shift Asymmetric pelvic shift Hemicentric caudal vertebra Patchy ossification of: ~>four skull bones ~
1
2
3
4
(0)
(1000)
(3000)
(5000)
8 (4) 3 (3) 1 (1) 0 3 (3) 2 (2) 0 I (1)
3 (2) 1 (I) 0 0 8 (6) 0 0 0
0 1 (1) 4 (4) 0 5 (5) 0 2 (1) 0
6 (5) 2 (I) 1 (1) 1 (I) 8 (7) 0 0 I (1)
178 (24) 189 (24) 367 24
178 (24) 190 (24) 368 24
166 (25) 237 (25) 403 25
170 (23) 183 (23) 351 23
I (1)
0
0
3 (3)
0
I (1)
0
0
0 I (I) 0 0
I (1) 4 (2) 2 (2) 0
0 4 (1) 4 (4) 0
0 3 (3) 1 (1) I (1)
0 0 0 0 1 (I) 0 0
1 (1) 0 0 0 1 (I) 0 I (I)
0 I (1) 0 1 (I) I (1) 1 ( I) 0
0 0 I (I) 0 0 I (1) 0
5(3) 9(5) 1 ( I) 1 (I) 1 (1) 1 (1) 0 3 (3)
10(6) 17(9) I (I) I (1) 2 (2) 3 (3) 1 (1) 4 (3)
20(12) 25(11) 0 4(3) 7 (5) 8 (7) 2 (2) 10 (7)
I (I) 9(5} 1(I) I(I) 0 3 (2) 0 3 (2)
0 160 (24) 31 (14)
0 164 (24) 27(11)
2 (2) 168 (25) 39(13)
1 (1) 145 (23) 35(16)
191 24
191 24
209 25
18 I 23
Values represent incidence of a b n o r m a l foetuses; no. of affected litters in parentheses.
1007
Safety evaluation of Esperase Table I I. Esperase, teratogenicitystudy in rats: group incidenceof skeletal ossificationparameters Group/dose level(rag Esperase/kg/day) Abnormalities Overall slight skeletal ossification Cervical laminae retarded, metacarpals and 5th metatarsals unossified Any thoracic or lumbar vertebra retarded Pubes retarded 5th metacarpals unossified 5th metatarsal(s) unossified Anterior arch of atlas ossified o n e or two cervical centra ossified three or more cervical centra ossified Any phalangeal elements ossified
I
2
3
4
(0)
(1000)
(3000)
(5000)
0
0
I (1)
0
0 9 (6) 3 (3) 56 (18) 0 79 (21 ) 66 (21) 21 (8) 27 (1 I)
0 5 (5) 0 90 (20) 0 69 (20) 71 (20) 26(11) 9 (6)
0 9 (8) I (I) 105 (22) 1 (I) 73 (23) 88 (24) 31 (13) 20 (8)
1 (1) 10 (7) 3 (3) 73 (20) 0 64 (18) 77 (21) 22(10) 20 (8)
No. of sternebrae retarded: 0 1 2 >2
110 (22) 49 (20) 24 (12) 8 (4)
85 (23) 60 (22) 39 (17) 7 (5)
107 (24) 63 (25) 33 (14) 6 (6)
97 (20) 61 (22) 18 (7) 5 (5)
No. of caudal vertebral eentra ossified: <4 4 >4
28 (15) 140 (24) 23(11)
46(17) I 18 (24) 27(11)
3505) 136 (24) 38(15)
25(13) I 18 (23) 38(16)
209 25
181 23
No. of foetuses examined skeletally No. of litters examined skeletally
191 24
191 24
Values represent incidence of abnormal foetuses; no. of affected litters in parentheses.
S-9, since S-9 is destroyed by the proteotytic activity of Esperase. The sensitivity of the individual bacterial strains was confirmed by significant increases in the number of revertant colonies induced under similar conditions by diagnostic mutagens. Mean numbers obtained in the second experiment are given in Table 12. No dose-related and reproducible increases in revertants to prototrophy were obtained with any of the bacterial strains, either in the presence or absence of S-9 mix. It was concluded that there was no indication of mutagenic activity. Gene mutation assay in mammalian cells in vitro (Clements, 1991). The test material was assayed for mutation at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus (6-thioguanine resistance) in mouse lymphoma L5178Y cells using a fluctuation protocol. The study consisted of two independent experiments, each conducted in the absence and presence of metabolic activation by rat liver post-mitochondriai fraction induced by Aroclor 1254 (S-9). As in the gene mutation assay in S. typhimurium, inactivated test material (denaturation at pH 3.0 for 10min) was used in the presence of S-9. The exposure time was 2 hr. Following a wide range of treatments, separated by half-log intervals and reaching 5000/~g/ml, cells survived all doses of the test material, showing 91 and 128% relative survival in the absence and presence of S-9, respectively, at the top dose. This dose, together with the next four lower doses, was plated for viability and 6-thioguanine resistance 7 days
after treatment. In the second experiment a narrower dose range was used to maximize the chance of detecting any dose-related effects. The top close plated in this experiment was again 5000 #g/ml in the absence and presence of S-9, which yielded 86 and 99% survival, respectively. The validity of the test was confirmed by the results of negative (solvent) and positive control treatments. No test material treatment, either in the absence or presence of S-9, resulted in a statistically significant increase in mutation frequency (Table 13). It was concluded that there was no indication of mutagenic activity. Chromosome aberration assay in vitro (Marshall, 1991). The test material was examined in an in vitro cytogenetics assay using duplicate human lymphocyte cultures from a male and female donor in two independent experiments. The highest dose level used was 5000 #g/ml. In the main study, treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by S-9 from Aroclor 1254induced animals. As in the gene mutation assay in mammalian cells, inactivated test material was used in the presence of S-9. In the first experiment, treatment in the. absence of S-9 was continuous for 20 hr. Treatment in the presence of S-9 was for 3 hr only, followed by a 17-hr recovery period before harvest. The test material dose levels for chromosome analysis were selected by evaluating the effect of test material on the mitotic index. Chromosome
5 ,ug
100,ul 2#g
6140 3070 1535 767 384 192
15 ~2300
12 17 16 18 17 12
100 61
79 80 69 70 80 89
85
8
14 11 l0 10 10 11
*Corresponding to no. of viable cells × 106 on revertant plates. tWeak inhibition of the bacterial background lawn.
Sterile water MNNG 2-aminoanthracene 9-Aminoacridine 2-Nitrofluorene
Esperase Batch no. PPA 3366
Test substance
99
120
118 119 124 t18 149 115
TA 1535 Concn of test substance Without S-9 mix With S-9 mix (tJg/ml incubation Viable Viable mixture) Revertants cells* Revertants cells*
112
107
13 11 17 33 50 63
Viable cells*
99
6
7 7 7 7 3 6
Revertants
31
50
65 96 92 60 54 49
Viable cells*
With S-9 mix
~1550
17
17 18 18 16 15 22
Revertants
162
147
91 118 109 156 148 112
Viable cells*
763
25
26 32 23 33 19 22
122
128
59 127 218 152 164 131
Viable cells*
With S-9 mix
Revertants
TA98 Without S-9 mix
MNNG = N-methyI-N '-nitro-nitrosoguanidine
~2400
5
7t 6t 8 9 8 6
Revertants
Without S-9 mix
TA 1537
101 781
125 122 101 106 130 119
Revertants
58 14
67 54 74 75 66 52
Viable cells*
Without S-9 mix
~ 1025
155
152 152 149 143 154 141
74
122
138 169 153 98 147 137
Viable cells*
With S-9 mix
Revertants
TA 100
Table 12. Esperase, gene mutation assay in Salmonella typhimurium: number of revertant colonies per plate obtained with strains TA1535, TA 1537, TA98 and TA100 of Salmonella typhimurium following exposure to Esperase in the presence and absence of metabolic activation with Aroclor 1254-induced rat liver S-9 in a liquid culture assay (2nd experiment)
m
Safety evaluation of Esperase
1009
Table 13. Esperase, gene mutation assay at HGPRT locus in mouse lymphoma L51784 ceils m vitro (2nd experiment) Treatment (#g/ml)
Relative survival (%)
95% confidence limits Mutation frequency*
Upper
Results ( + or - )
4.3 5.1 4.6 5.2 4.5 3.7
7.7 8.8 8.0 9.0 7.8 6.8
------
19.1 24.9
32.0 40.6
+ +
Presence of 8-9 mix 5.5 4.2 4.7 3.4 5.9 4.5 7.2 5.5 5.6 4.2 5.2 3.9
7.3 6.4 7.7 9.'4 7~ 7.1
-
46.6 47.0
+ +
Lower
Absence of S-9 mix 0 312.5 625 1250 2500 5000
100 98 94 91 103 86
5.7 6.7 6.1 6.9 5.9 5.0
Positive control (4-nitroquinoline l-oxide) 0.1 0.15
0 312.5 625 1250 2500 5000
35 22
100 83 100 97 105 99
24.7 31.8
Positive control (benzo[a]pyrene ) 2 3
36 22
36.8 37.4
29.0 29.7
* = 6-thioguanine-resistant mutants/106 viable cells at least 7 days after treatment.
aberrations were analysed at three consecutive dose levels. The highest concentration chosen for analysis at this time, 5000 pg/ml, induced approximately 49 and 41% mitotic inhibition in the absence and presence of S-9, respectively. The second experiment (Table 14) included a delayed sampling time. Treatment in the absence of S-9 was continuous for 20 or 44 hr. Treatment in the presence of S-9 was for 3 hr followed by a 17 or 41 hr recovery period. The highest concentration chosen for analysis at 20 hr, 5000 #g/ml, induced approximately 70 and 54% mitotic inhibition in the absence and presence of S-9, respectively. The effects of single concentrations only, 1049 pg/ml without and 1638 #g/ml with S-9, were investigated at the delayed harvest, at which time 74 and 49% mitotic inhibition was induced, respectively. The validity of the test was confirmed by the lack of evidence of heterogenicity between replicate cultures, the sufficient number of cells analysed at each treatment level and the results of negative (vehicle) and positive controls. Treatment of cultures with the test material in the absence of S-9 resulted in proportions of aberrant cells at all sampling times in both experiments that were similar to, and not significantly different from, those in concurrent controls. Treatment of cultures with the test material in the presence of S-9 resulted in a small but statistically significant increase in frequencies of aberrant cells at 5000/zg/ml in the second experiment at the 20 hr sampling time. The result was not considered to be biologically significant as it was small and not reproducible. It was concluded that there was no evidence of clastogenic activity.
DISCUSSION
Calculation of human consumption of enzymes used as processing aids is based on the contents of total organic solids (TOS) in the preparation, as the rest of the preparation (i.e. ash, water and diluents) is fully characterized and is well known as safe for consumption in the quantities involved. However, it should be emphasized that the larger part of the enzyme-TOS is composed of proteins and carbohydrates from traditional food sources used in the fermentation. For the calculation of the safety margin, it is necessary to know the application of the enzyme, the concentrations of the enzyme-TOS in the final product(s), the human consumption of the product(s) and the no-adverse-effect level in the animal studies. Esperase can be used for cleaning from bones. Following this process, the meat fraction may contain 0.1% enzyme-TOS. This meat fraction can be used at a maximum of 10% in meat mixtures and sausages. The enzyme will be inactivated by heating during the process, and thu~ no active enzyme will be consumed. An average intake per day of these products is estimated to be less than 100 g per person or less than 2 g per kg body weight (US Department of Agriculture, 1983). A conservative calculation, assuming that all the meat mixtures and sausages consumed contain the (inactive) enzyme, gives a maximum daily intake of 0.2 mg enzyme-TOS per kg body weight from this source. No effects were seen in the animal studies at a dose level of 1 g of the test material per kg body weight. At the higher dose levels (i.e. 3 and 5 g of test material) only effects attributed to either the proteolytic activity or the load of sodium chloride were seen.
1010
R.K.
HJORTKJAER et al.
Table 14. Esperase, c h r o m o s o m e aberration assay using h u m a n lymphocytes in vitro (2nd experiment)* Treatment (,ug/ml)
Replicate
Cells scored
Cells with aberrations including gaps
Cells with aberrations excluding gaps
20 hr sampling time; --S-9; donor sex: female 100 6 3 100 3 0 200 9 3
Solvent
A B Totals
3200
A B Totals
100 100 200
0 0 0
0 0 0
A B Totals
100 100 200
3 2 5
1 0 1
A B Totals
100 73 173
2 4 6
1 3 4
A B Totals
25 25 50
22 19 4I
21 19 40
4000
5000
M M S , 50
A B Totals
3200
A B Totals
100 100 200
5 2 7
4 0 4
A B Totals
100 100 200
0 1 1
0 0 0
5000
A B Totals
100 100 200
5 6 11
C P A , 25
A B Totals
25 25 50
l0 14 24
Solvent
A B Totals
1049
A B Totals
Solvent
A B Totals
1638
A B Totals
NS
2.7 3.0 (2.9)
NS
2.3 2.4 (2.4)
NS
2.1 1.3 (1.7)
P ~< 0.001
4.4 6.0 (5.2)
NS
3.6 2.5 (3. I )
NS
2.7 2.6 (2.7)
3 5 8
P < 0.05
2.4 2.4 (2.4)
6 13 19
P ~< 0.001
44 hr sampling time; --S-9; donor sex: female 100 I 0 100 4 1 200 5 1 100 100 200
6 3 9
2 I 3
5.2 5.3 (5.3)
NS
44hr sampling time; +S-9; donor sex: female 100 2 2 100 3 3 200 5 5 100 100 200
4 0 4
M M S = methyl methane sulfonate N S = not significant * N u m b e r s in bold typeface exceed histological negative control ranges. t F i s h e r ' s exact test.
Mitotic index (mean) 5.3 6.0 (5.7)
20 hr sampling time; +S-9; donor sex: female 100 2 0 100 3 I 200 5 1
Solvent
4000
Statistical significancet
1 0 1
1.2 1.5 (1.4)
5.1 6.6 (5.9)
NS
C P A = cyclophosphamide
2.5 3.5 (3.0)
Safety evaluation of Esperase The application of the enzyme in food processing will lead to neither an intake of the active enzyme nor a high intake of salts, and thus these effects may be disregarded. The dose levels of 1 and 5 g test substance per kg body weight correspond to 230 and 1200mg TOS/kg body weight, respectively, or a safety margin of 1000-5000 in the food application described. According to the literature, the production organism B. lentus is non-pathogenic and nontoxinogenic. The test material was without antibacterial and mutagenic activity in the in vitro tests, and it had no effect on reproduction in the rat in vivo teratogenicity study. On the basis of these results and the low toxic potential in the subchronic toxicity, the application of Esperase in food processing should be considered safe for the consumer, with a reasonably high margin of safety. REFERENCES
AMFEP (Association of Microbial Food Enzyme Producers) (1992) Regulatory Aspects o f Microbial Food Enzymes. AMFEP Secretariat, Brussels. Babesgaard P. (1991 ) Bacillus lentus, Taxonomy and Patho genicity. Novo Nordisk A/S, F-910535, unpublished report. Clements J. (1991) Study to Determine the Ability o f E S P E R A S E to Induce Mutations to 6-Thioguanine Resist ance in Mouse Lymphoma L5178Y Cells using a Fluctuation Assay. Hazleton Microtest, 2MLRENOD.023,
unpublished report. FCC (Food Chemical Codex) (1981) National Academy o f Sciences~National Research Council, Food and Nutrition Board, Committee on Codex Specifications 107 110.
National Academy Press, Washington. Food and Drug Administration (1983) Direct food substances affirmed as GRAS, mixed carbohydrase and protease enzyme product. Federal Register 48, 239 240. Heath J., Mulhern M., Perry C. J. and Millar P. (1991) ESPERASE: 13 week toxicity study in rats. Inveresk Research International. Report No. 7680, unpublished report.
1011
IFBC (International Food Biotechnology Council) (1990) Safety evaluation of foods and food ingredients derived from microorganisms. Regulatory Toxicology and Pharmacology 12, 114-128. JECFA (Joint FAO/WHO Expert Committee on Food Additives) (1984) General Specifications for Enzyme Preparations used in Food Processing. FAO Food and Nutrition Paper 31/2, 129-131. Jensen B. F. and Eigtved P. (1990) Safety aspects of microbial enzyme technology, exemplified by the safety assessment of an immobilized lipase preparation, Lipozymetin. Food Biotechnology 4, 699-725. Marshall R. R. (1991) Study to Evaluate the Chromosome Damaging Potential o f E S P E R A S E '~t by its Effects on Cultured Human Lymphocytes using an in vivo Cytogenetics Assay. Hazleton Microtest Report: 2HLRENOD.023,
unpublished report. NIH (National Institutes of Health) (1986) Guidelines for research involving recombinant DNA molecules; notice. Federal Register 51, 16967 16968. OECD (1986) Recombinant DNA Safety Considerations. OECD, Paris. Pariza M. W. and Foster E. M. (1983) Determining the safety of enzymes used in food processing. Journal o f Food Protection 46, 453-468. Pedersen P. B. (1991) E S P E R A S E (Batch NO. PPA 3366): Testing for Mutagenic Activity with Salmonella typhimurium TA 1535, TA 1537, TA 98and TA lOOina Liquid Culture Assay. Study No. 91514. Novo Nordisk A/S,
F-911207, unpublished report. SCF (EEC Scientific Committee for Food) (1991) Guidelines for the presentation of data on food enzymes. Report o f the Scientific Committee for Food. 27th series.
Office for Official Publications of the EEC, Luxemburg. US Department of Agriculture (1983) Food Intakes: Individuals in 48 States, Year 197~78. Nationwide Food Consumption Survey 197~78. Report no. 1-1. Human Nutrition Information Service. United States Department of Agriculture. Wilson J. A. (1991) ESPERASE: Teratogenicity Study in Rats. Inveresk Research International. Report No. 7698, unpublished report.
[The unpublished reports cited in this list are available on request from Novo Nordisk A/S (M. Stavnsbjerg).]