Document wgRK7yv6dNGamDN4XDx1V335o

R&S 006464 pu ^ civo institutes tno netherlands organization (or applied scientific research; division for nutrition and (bod research tno p.o. box 360 3700 AJ zeist netherlands Report No, V 82.276/291099 LIFE-SPAN ORAL CARCINOGENICITY STUDY WITH VINYL CHLORIDE IN RATS (seventh interim report: week 132 study termination) Authors : Dr H.P, Til H,,R. Immel At the request of" : Verband Runststofferzeugende Industrie E.V. Frankfurt am Main Approved by : Dr V.J. Feron Project number : B79-1099 Assay number : 125 Date : October, 1982 V 82.276 . 3 R&S 006465 summary' 1 The oral carcinogenicity of vinyl chloride monomer (VCM) vas examined in a life-span study with Vis tar ra-ts. VCM was ( administered by incorporating PVC--powder with a high VCM content into the diet. The test substance was fed at various dietary levels to provide oral exposure levels of approxi mately 0, 0.017, 0.17. or 1.7 mg VCM/kg body weight/day. Each of the dose levels was given to groups of 100 male and 100 female rats, except for the top dose which was given to 50 male and 50 female rats. An extra control group of 100 male and 100 female rats was housed in a separate room. The results obtained in the period of week 132 to termination of the study (week 149) are presented in this report. t c General health, affected by the behaviour, and test compound. body weight were The mortality in not the adversely top-dose group was relatively high during the final stage of the study, especially in females. 3. At gross pathological examination the number of liver nodules in males as well as in females of the 1.7 mg/kg group was found to be higher than in controls. 4. Preliminary histopathological examination revealed a clearly higher incidence of "foci of cellular alterations" in the liver of males and females of the 1.7 mg/kg group than in rats of the other groups including both control groups. More neoplastic liver nodules were found in females of the topdose group than in any of the other groups. V'/wyt OkoK/Ce/&**- Cc O--6 3k 0-r^'^pLhsd. -z? MS/, <&>/% Civo Institutes TNO Report No. V 83.285/291099 LIFESPAN ORAL CARCINOGENICITY STUDY OF VINYL CHLORIDE IN RATS (Final Report) R&S 006466 gjGj l / JJs-x. J\ trillvetx I R&S 006467 civo institutes tno .J *..* * netherlanos organization for applied scientific research ^/ dlvltlon lor nutrition and load research tno p o. box 360 3700 AJ zeist nethertands Report No. V 83.285/291099 LIFESPAN ORAL CARCINOGENICITY STUDY OF VINYL CHLORIDE IN RATS (Final report) Authors : Dr H.P. Til H.R. Immel Dr V.J. Feron At the request of Verband Kunststofferzeugende Industrie E.V., Frankfurt am Main, 6.R.D. Project number : B 79-1099 Start of the study : August 21, 1979 Termination of the study: June 29, 1982 Study director Dr H.P. Til Study supervisor : Dr V.J. Feron Approved by : Dr A.P. de Groot Date : September, 1983 Total or partial publication of this report without written assent is not allowed 23. to ooo-otai V 83.285 CONTENTS SUMMARY 1. INTRODUCTION 2. MATERIAL AND METHODS 2.1 Materials 2.2 Preparation and administrationof the diets 2.3 Animals and housing 2.4 Identification of the test system 2.5 Experimental conduct 2.6 Statistical analysis 2.7 Contributors 2.8 Deviations from the protocol 3. RESULTS 3.1 Analysis of VCM in the diets 3.2 Levels of VCM in the faeces 3.3 Actual oral exposure levels of VCM 3.4 Symptomatology and survival 3.5 Body weights 3.6 Food intake 3.7 Haematology 3.8 Glutathione levels of the liver 3.9 Pathology 4. DISCUSSION 5. CONCLUSIONS PaSe u 7 8 8 9 11 12 I7 18 18 19 19 19 19 20 27 21 22 22 22 25 27 R&S 006468 V 83.285 CONTENTS 6. REFERENCES 7. AUTHENTICATION 8. RETENTION OF RECORDS AND SPECIMENS STATEMENT OF CLP COMPLIANCE TABLES FIGURES ANNEXES page 28 30 31 32 33 70 71 3 R&S 006469 i I V 83.285 -k - SUMMARY 1. The oral carcinogenicity of vinyl chloride monomer (VCM) was examined in a lifespan study (149 weeks) with five groups of Wistar rats, each consisting of 100 males and 100 females, except for the top-dose group which comprised 50 males and 50 females. VCM was administered by in corporating polyvinyl chloride (PVC) powder with a high VCM content into the diet. The diet was provided daily for a period of 4 consecu tive hours, whereas food was withdrawn during the other 20 hours. The use of this way of oral VCM administration resulted in the following exposure levels: 0 (control), 0.014, 0.13 and 1.3 mg VCM/kg body weight/day. An extra control group of 100 rats/sex was housed in a separate room. Additional satellite groups of IS male and 10 female rats, each re ceiving the same treatment as the main groups were used for determi nations of glutathione levels in the liver after 9 and 18 months. Observations were made of general appearance mortality, growth, food Intake, thrombocyte count, prothrombin time, glutathione levels in the liver, gross pathology and microscopic pathology of the liver and of all grossly visible tumours or presumable tumours in the abdominal cavity, the glands of Zymbal and the mammary glands. 2. General health, behaviour, body weight and food intake were not ad versely affected by the test substance. 3. In the second half of the experimental period, mortality in the extra control group was higher than in all other groups. This was most prob ably due to a high incidence of chronic respiratory disease in the extra control group. In the final stage of the study, the mortality in the top-dose group was slightly higher than in the lower dose groups and the controls. 4. Thrombocyte count, prothrombin time and liver glutathione levels did not show treatment-related differences among the groups. R&S 006470 V 83.285 u 5 5. A clearly higher Incidence of grossly visible, tumourous, liver nod ules was found in both males and females of the top-dose group than in any of the other groups. Moreover, in females of the top-dose group the incidence of hepatic cysts was considerably higher than in con trols. 6. Microscopic examination of the liver revealed Increased incidences of liver-cell polymorphism, hepatic cysts, foci of cellular alteration, neoplastic nodules and hepatocellular carcinomas in the top-dose group as compared to the control group. Moreover, a hepatic angiosarcoma was found in one male and two females of the top-dose group, whereas no such tumours were encountered in any of the other groups. The number of animals bearing foci of cellular alteration in the liver was also statistically significantly increased in females of the mid-dose group as compared to controls. In addition, in females but not in males, the incidence of basophilic foci of cellular alteration in the liver was statistically significantly higher in both the lowand the mid-dose group than in the control group. 7. There was no evidence of VCM-feeding affecting the incidence of ab dominal mesotheliomas or the type and incidence of mammary gland tu mours. No Zymbal gland tumour was found, 8. It was concluded that under the conditions of the present experiment: - VCM at a level of 1.3 mg/kg body weight/day Induces neoplastic and non-neoplastic changes in the liver of rats, - VCM at a level of 0.13 mg/kg body weight/day may lead to more female rats bearing foci of cellular alteration in the liver, - VCM at levels of 0.014 or 0.13 mg/kg body weight/day may result in an increased incidence of basophilic foci of cellular alteration in the liver of female rats, " 0.13 mg VCM/kg body weight/day is a "no-observed-adverse-effectlevel" with respect to the induction of tumours in rats. V 83.285 ^ C JL *T -6- R&S 006472 9. Risk estimation based on the results of the present rat study and taking into account the prudence of the linear model applied and a lesser sensitivity of humans to the carcinogenic action of VCM in comparison with rats. Indicates that the cancer risk of a likely maximum oral daily Intake of 0.1 fig VCM per person per day can be practically neglected. V 83.285 -7- LIFE-SPAN ORAL CARCINOGENICITY STUDY OF VINYL CHLORIDE IN RATS R&S 006473 1. INTRODUCTION From a previous life-span oral toxicity study of vinyl chloride monomer (VCM) in rats with dose levels of 1.7, 5.0 and 14.1 mg/kg body welght/day (Feron et al., 1978; 1981), it appeared that hepatic angiosarcomas occurred at dose levels of 5.0 mg/kg body weight/day and above, while hepatocellular tumours (neoplastic nodules and carcinomas) were found at all dose levels. Zymbal gland tumours were found at VCM levels of 5.0 and 300 mg/kg body weight/day. Horeover, there was some evidence that VCM en hanced the formation of intra-abdominal mesotheliomas and adenocarcinomas of the mammary gland. In females of the low-dose group there was still a high incidence of liver-cell tumours, vis. 28/58. Thus, this study showed a) that VCM is a carcinogen in rats when administered orally, and b) that the "no observed-adverse-effect level" of VCM in rats with respect to the induction of tumours was lower than 1.7 mg/kg body weight/day. For extrapolating experimental data to man in a reliable way the infor mation obtained from such a test system ideally should Include both a "minimum tumour-level" and a "no tumour-level". Therefore, a similar life-span oral carcinogenicity study with VCM In rats was carried out, at lower dose levels, viz. nominally 0.017, 0.17 and 1.7 mg VCM/kg body welght/day (actual oral exposure levels 0.014, 0.13 and 1.3 mg VCM/ kg body weight/day) and two control groups. Based oh the pathological findings of the previous long-term study, histopathological examinations in the present experiment were focused on the detection of liver lesions, Zymbal gland tumours, mammary gland carcinomas and abdominal meso theliomas. A proposal for the present study - dated 19-02-1979 - was accepted by the sponsor. The present experiment and results are described in this report. V 83.285 2. MATERIAL AND METHODS -8- 2.1 Materials Vinyl chloride monomer (VCM), from Akzo Zouc Chemie, Rotterdam, The Netherlands. Physical chemical constants: MW - 62.50; m.p. - -153.8 "C; b.p. - -13.37 *C; density - 0.9106; - 1.3700. The product (colourless, clear, free of suspended matter) was delivered in pressurized stainless-steel cylinders, and was specified by the supplier as: Vinyl chloride monomer 99.97 wt 2 min.; acetylene 4 2 yl/1 (gas); mono-vinylacetylene ( 15 ^jl/1 (gas); 1,3-butandiene 4 10 ^1/1 (gas); methyl chloride ^ 75 fil/1 (gas); ethyl chloride ^ 50 fxl/1 (gas); ehloro- prene 4 1 jil/1 (gas); 1,1-dichloroethane Al pl/1 (gas); 1,2-dichloroeth- ane A 20 jil/1 (gas); acetaldehyde 4-5 mg/kg; hydrochloric acid ^ 1 mg/kg; iron 0.5 mg/kg; water 6 100 mg/kg; evaporation residue 4 10 mg/kg. PVC-powder. commercial name Carina S 65-02, was supplied by Shell Nederland Chemie, Pernis, The Netherlands, in closed steel barrels. The particle size distribution (by weight), specified by the supplier, was: 0.1 2 max. > 300 pm; A 2 max. > 200 pm; 90 2 max. > 88 pm; 95 2 max. AO pm. The remaining part of a batch of 500 kg PVC-powder (VCM content 3 ppm) received on.January 13, 1977 from Shell, which had been partly used In the previous study (Feron et al., 1978), was used. Part of this PVC-powder was freed from residual VCM by keeping it in layers of A to 6 cm thick in a vacuum oven at 60 "C for a period of 3 to A days. The so treated PVC-powder (VCM content less than 0.2 ppm) was used for control purposes. An amount of about 50 kg PVC powder was mixed with a calculated amount of the liquid VCM in a closed steel barrel to raise the VCM content of the PVC-powder to approximately A600 ppm. This PVC-powder was repacked and stored in tightly closed steel con tainers (containing about 10 kg PVC) in a freezer at -20 *C until a few minutes before mixing with each diet. R&S 006474 V 83.285 -9- 2.2 Preparation and administration of the diets Preparation and administration of the diets were done In the same way as in the previous life-span study (Feron et al., 1978). Only one distinction has to be made. In that study the dietary levels of VCM were obtained by Incorporating Into the diets 10 Z PVC-powder with varying proportions of VCM-containing and "VCM-free" powder. In the present study, the diets were prepared to a total concentration of PVC-powder of 1 Z only. The proportions of PVC-powder with or without VCM for each of the diets are specified below: group PVC-powder in the diet (Z) PVC-powder con PVC-powder taining about without 4600 ppm VCM VCM1) control group low-dose group mid-dose group top-dose group extra control group 0 0.01 0.1 1 0 1 0.99 0.9 0 1 ^ PVC-powder freed from VCM. The VCM-content was lower than 0.2 ppm Diets were prepared daily - by mixing appropriate amounts of PVC-powder (with or without VCM) with the Institute's rat stock diet - Just prior to offering the diets to the rats. The composition of the stock diet is presented in Annex 1. The level of nutrients and contaminants in the stock diet are determined twice a year. The contaminants determined are tabulated, together with detection limits and tentative maxima in Annex 2. Actual levels in batches produced on January 24, 1980, September 9, 1980, February 27, 1981, September 9, 1981 and February 9, 1982 are given in Annexes 3 to 7 (nutrients) and in Annexes 8 to 12 (contaminants). R&S 006475 V 83.285 - 10 - As in the previous study, the powdered diets were available to the rats each day for a period of four hours (generally between 10.00 a.m. and 02.00 p.m.), in such quantities that the animals did not consume all the feed. At the end of the four-hour feeding period the feeders were removed from the cages and the remainder of the diets was destroyed. The rats had constant access to bottled tap water. The contaminants in the drinking water which are determined twice a year, are given in Annex 13. The actual levels determined in November-December 1979, January--April 1980, September-November 1980, March-May 1981, September-November 1981 and January--March 1982 are given in Annexes 14 to 19. 2.3 Animals and housing Five hundred and twenty-five male and 525 female, SPF-bred, weanling rats (Cpb:WU; Uistar random) were obtained from the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands. The animals arrived on August 14, 1979, when they were about 25 days old. The body weight range was 38-71 g for males and 42-66 g for females. Upon arrival, the animals were checked for overt signs of ill health and anomalies. One rat showing hydrocephalus, one rat with closed eyes and two rats with a dirty yellow fur were discarded. The healthy animals were allocated to five main groups by a computer randomisation program. Four of the groups, viz. the control group, the low- and mid-dose group and the extra control group, each consisted of 100 males and 100 females. The top--dose group consisted of 50 males and 50 females. To each dietary group a satellite group, comprising 10 males and 10 females each, was attached. The rats of the satellite groups, which received the same diets as the rats of the corresponding main groups, were killed for determinations of glutathione levels in the liver, half of the animals when they had been on their diets for nine months and the remaining half after an experimental period of 18 months. The rats which were not allocated to the main or satellite groups were kept in reserve for a period of four weeks. One female rot of the extra control group showing epileptic fits had to be exchanged for a reserve two days after the start of the study. None of the other reserved were used. ^ 1 j r &S 006476 V 83.285 11 The rats were housed under conventional conditions, five rats per sex per cage, in suspended stainless steel cages (17 x 44 x 32 cm), fitted with wire mesh floors and fronts. Animals which were in bad condition were housed Individually In separate cages until they died or until they were killed because their condition was so bad that they were not expected to last out the night, (or, when observed on Friday, the weekend). If the condition of the singly housed rats improved they were returned to their original cage. The male rats of the control", low-, mid- and top-dose groups were housed in one room, the females of these groups in a second room and the males and females of the extra control group in a third room. The rooms were ventilated with about 10 air changes per hour. The temperature was kept at 23+1 *C, the relative humidity was 40-80 X and a 12 hour light/dark cycle was maintained (light from 06.00 a.m. till 06.00 p.m.). After an acclimatisation period of 7 days, the animals were placed on the test diets for four hours a day during the entire test period. During a period of 3 days, prior to the start of the experiment, the rats received stock diet without FVC for four to six hours each day. This was done to adapt the animals to the daily feeding-period of four hours. Drinking water was supplied in glass bottles, which were filled daily with fresh tap water, except for the weekend, and cleaned once weekly. 2.4 Identification of the test system The individual animals were identified in the following way. Each group of rats (the control, low-dose, mid-dose, top-dose and extra control group) was fitted with a letter and colour code. A table showing the group letter, colour code, exposure level and number of animals is given on the next page. JJ fio W o o O) -Cx ^1 I V 83.285 i: R&S 006478 group colour letter code A white B blue C green D red E brown nominal actual exposure levels exposure levels (mg VCM/kg b.w./day) 00 0.017 0.014 0.17 0,13 1.7 1.3 00 number of rats main groups satellite groups study no. 125 males females 100 100 study no. 126 males females 10 10 100 100 10 10 100 100 10 10 50 so 10 10 100 100 10 10 Each rat was identified by a six digit computer reference number, which was even for males and odd for females. Within each subgroup of 10 rats, each rat was individually identified by sex and one out of 10 different V-shaped earmarks according to the following code: Z - zero R^L^ one right plus one left R^ " one right R1L2 one right plus two left R2 " two right RzH - two right plus one left - one left R2L2 " two right plus two left 2 two left - three right See also the cross reference listing (Annexes 20 and 21). Each cage was provided with a coloured card showing the computer refer ence number range, the earmark range, the cage number, the group letter and the study number. 2.5 Experimental conduct The experiment (main and satellite groups) was started on August 21, 1979. Half of the rats of the satellite groups (assay no. 126) was killed on May 20 or 22, 1980 and the remaining half on February 26 or 27, 1981 for determinations of glutathione in the liver. The final autopsy of the rats In the main groups (assay no. 125) was carried out on June 24 to 29, 1982, when about 80 X of the control-, low-dose and mid-dose rats had died. The following determinations were made: V 83.285 a) Analysis of VCM In the diets 13 - The VCM content of freshly prepared diets was generally determined once every fortnight. During the course of the study, 72 determinations for each dose level were carried out. At the same time the VCM content of the PVC-powder was determined. The rate of evaporation of VCM from the diets was determined by measuring the VCM content of the diets at the beginning of the feeding-period and after 1 hour and after 2, 3 and A hours. The diet samples to be analysed were taken at random from the feeders. In this way samples of each of the test diets were taken in week 3, 12, 27, 56, 79, 105 and 132, and were analysed for their VCM content. At the same time samples from the control diets were taken at the beginning of the feeding--period only and also analysed. The analyses were carried out by means of gas-liquid chromatography ac cording to a method described by Feron et al. (1975). b) Analysis of VCM in the faeces Freshly produced faeces from 5 rats of each dietary group were collected 2A hours after the start of the feeding-period at about 10.00 a.m. in week 6, 28, 52, 80 and 106. Fresh faeces was obtained by squeezing the lower part of a rat's abdomen. The droppings were weighed, submerged in 10 ml ethylacetate, and stored at A "C in a closed vessel until analysis of the supernatant liquid by gas chromatography. c) Calculation of the actual oral Intake and oral exposure levels of VCM R&S 006479 The amount of food eaten by the rats of each test group (g/kg body welght/day) was calculated for males and females separately from the grand averages of body weight (table 7) and food intake (table 9) of each test group over the whole experimental period. V 83.285 1A The VCM intake (mg/kg body weight/day) was calculated from the rate of evaporation of VCM from the diets (table 2) and the rate of overall food consumption over the four--hour feeding--period for males and females (table 10). Both the rate of evaporation and the rate of food intake were assumed t;o be linear within each hourof the feeding-period. The actual VCM Intake for each group during eachof these four periods was calculated by multiplying the amount of food eaten during a one-hour period by the average VCM content of the food eaten in that period. The total actual VCM Intake was obtained by adding the VCM Intakes during the four`one-hour periods of the feeding-period. To obtain the actual oral exposure levels of VCM, the actual VCM Intakes were corrected for the VCM excreted with the faeces. For that purpose, the faeces production (g/kg body weight/day) was calculated basedon an average of 36 g wet faeces/100 g foodconsumed. The amount of VCM in the wet faeces (mg/kg body weight/day) was calculated by multiplying the amount of faeces produced/kg body weight with the VCM content measured in the faeces (table 3). The amount of VCM excreted with the faeces, ex pressed In mg VCM/kg body weight/day, was substracted from the actual oral Intake of VCM (also expressed in mg VCM/kg body weight/day) to find the actual oral exposure level of VCM. d) Clinical signs (main groups) The animals were observed daily and carefully examined for signs of ill ness and tumours once every two weeks. All signs of ill-health or tox icity with any changes in behaviour were recorded, together with the pro gression or regression of such abnormalities, as well as mortality and the time of onset, dimensions and location of palpable tumours. e) Body weight (main and satellite groups) The weight of each animal was recorded when the administration of the test substance was started and further in week 2 and A and once every four weeks thereafter. From these individual figures the mean body welghl of the animals of each group was calculated. R&S 006480 R&S 006481 V 83.285 f) Food consumption; amount and rate (main groups) - 15 ~ The food consumption was measured in 20 animals/sex/group, in week 1, 2, 3 and 4, 11 and 12, 23 and 24, 35 and 36, 47 and 48, 59 and 60, 73 and 74, 85 and 86, 97 and 98, 109 and 110 and 121 and 122. The rate of food consumption was determined by weighing the feeders initially and after periods of 1 hour, 2 hours, 3 hours and 4 hours. This was done for each of the diet groups on one day in week 4, 12, 25, 38, 52, 64, 77, 90, 103, 116 and 129, The number of rats involved in these determinations was 20/sex/group. g) Haematology (satellite groups) Samples of blood were collected from the tip of the call of 10 rats/sex/ group on days 94 and 185 and of five rats/sex/group on day 367. All blood samples were examined for the following parameters. - thrombocyte count, by means of the Sysmex Platelet Counter PI 110 using the manual of Toa Medical Electronics Co., Ltd., Japan - prothrombin time using Normo-test reagents from Nyegaard & Co, As, Oslo, Norway (Owren, 1959). h) Glutathione in the liver (satellite group) In week 40 and 80, five rats/sex/group were killed by decapitation. The livers were quickly removed and cooled to 0 C and cut into two, about equal, pieces; both were weighed and stored in a freezer at -20 *C until analysis. After thawing, each sample was homogenised in ice-cold 0.1 M EDTA(Naj) solution. A part of the homogenates was centrifuged at 2800 rpm for 5 minutes. The non-protein bound sulfhydryl ('glutathione') con tent of the Bupematants was determined with 5,5'-dlthiobis-(2-nitrobenzoic acid) by means of an AutoAnalyser. V 83.285 1) Pathology - 16 - All males still alive in week 149 (June 24 and 25, 1982) and all females in week. 150 (June 28 and 29, 1982) were killed by decapitation, aucopsied and subjected to a careful gross examination. At that time a mortality of about 80 Z had been reached in the control, low- and mid-dose groups. A thorough autopsy was also performed on rats found dead or killed in ex tremis . All superficial tissues, including the urogenital orifices and tail, each pinna, eye and external auditory meatus, were examined visually and by palpation for distortion, swelling or evidence of tumour formation; simi lar attention was given to the mammary tracts and subcutaneous struc tures. The external nares, buccal cavity and tongue were then examined, and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflec tion, all subcutaneous tissues were examined, including regional lymph nodes, mammary and thyroid/parathyroid glands. The condition of the tho racic viscera was noted, and attention was given to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal. The lungs were removed and all pleural surfaces examined. The kidneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded. Any lesion suggestive of neoplasia was noted, including details of location, size and multiplicity. Any evidence of adhesion or possible Invasion to adjacent structures was noted. Samples of the following tissues and organs of all animals were preserved in 4 X aqueous neutral phosphate-buffered formaldehyde solution: adrenals aorta ovaries pancreas axillary lymph nodes brain caecum parotid salivary glands preputial glands pituitary cervix prostate coagulating glands seminal vesicles 33 (/) oo 05 0ro0 I V 83.285 17 colon skeletal muscle duodenum skin epididymides spinal cord exorbital lachrymal glands spleen eyes sternum (with bone marrow) sciatic nerve stomach heart submaxillary salivary glands ileum sublingual salivary glands jejunum testes kidneys thymus liver thyroid lungs trachea mammary glands urinary bladder mesenteric lymph nodes uterus nose Zymbal glands oesophagus all gross lesions Tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 pm and stained with haematoxylin and eosin. Histopqthology was restricted to the liver, all grossly visible tumours or presumable tumours in the abdominal cavity, the glands of Zymbal, and the mammary glands. As to the liver, of each rat three liver pieces, each taken from a different lobe, were studied; these pieces were always taken from the same three lobes at about the same sites; in addition sections were prepared from liver tissue showing gross changes. 2.6 Statistical analysis Data on body weights were subjected to one-way analysis of (co-)variance, followed by Dunnett's multiple comparison test. Thrombocyte counts and prothrombin time values were analysed by the Mann Whitney U-test. Levels of glutathione in the liver were evaluated by the Student t-test. Data on mortality and microscopical observations were analysed by the Fisher exact probability test. Grossly visible masses were evaluated by the Chi square test. ` i R&S 006483 V 83.285 2.7 Contributors Hajor contributions to this study were made by: Animal handling and : Ms. M.M. Andringa diet preparation J.M. Blom Ms. A. Dijkstra Ms. G.G.M. Fleer Ms. A.A. van Tuyl D.C. Veldhuysen Diet analysis : A. Schouten Clinical chemistry :: Dr H.E. Falke J.F. Catsburg Biochemistry : Dr D.C. Leegwater Histotechnique : Ms. M.T. v, Rijswijk. Ms. J. Wisman Ms. A.P.M. v. Tuyl**Fonville Ms* N* Hagemeyer Pathology : H.R. Xmmel Study director Dr V.J. Feron : Dr H.P. Til Study supervisor : Dr V.J. Feron 18 - 2.8 Deviations from the protocol " At the request of the sponsor, the glutathione determinations in the liver were carried out after 9 and 18 months Instead of after 6, 12 and 24 months. " Prothrombin time and thrombocyte count in rats of the satellite groups after 24 months could not be carried out, because all rats of the sat ellite group had already been killed before that time. 3J 99 CO o o a> > 00 I V 83.285 3. RESULTS 19 - 3.1 Analysis of VCM In the diets (tables 1 and 2) The average VCM content of freshly prepared test diets appeared to be 0.49, 4,49 and 44.1 ppm for the low-, mid- and top-dose group, respect ively (table 1). The VCM content In all three test diets decreased with time during the four-hour feeding-period (table 2). The greatest losses were observed during the first hour. Proportionally, the losses of VCM were virtually the same in all three test diets at the corresponding points of time dur ing the four--hour period. On average, the cumulative losses were 22, 30, 39 and 38 I after 1, 2, 3 and 4 hours, respectively. 3,2 Levels of VCM in the faeces (table 3) The VCM content of the faeces increased with increasing dietary levels of VCM. No appreciable differences in VCM content of the faeces were found within one particular test group. The average amount of VCM found in the faeces expressed as percentage of the actual oral VCM intake, was found ,to be 23.5, 25.9 and 23.9 Z for the low-, mid-, and top-dose groups, re spectively. R&S 006485 3.3 Actual oral exposure levels of VCM (table 4) The VCM content of the freshly prepared test diets (table 1) formed the basis for calculating the average VCM content of the various diets. Since the loss of VOl from the diets during the four-hour feeding-period (table 2), the rate of food Intake (table 10) and the VCM content of the faeces (table 3) were known, the actual oral exposure levels of VCM for each group could be calculated (see section 2.5), They were found to be 0,014, 0.13 and 1,3 mg VCM/kg body weight/day for the low-, mid-, and top-dose groups, respectively (table 4). V 83.285 20 - When the actual oral Intake of VCM was expressed as percentage of the theoretical Intake, the values found were 81.5, 81.2 and 81.3 Z for males, and 80.1, 79.8 and 79.8 Z for females of the low-, mid- and topdose groups, respectively. The overall average for males and females of the various test groups was calculated to be 80.6 Z. This percentage was in good agreement with the 80 Z found in the previous life-span study using much higher dietary levels of VCM (Feron et al., 1978; 1981). 3.A Symptomatology and survival (tables 5 and 6) There were no overt signs of reaction to treatment with VCM. The behav iour of the rats during the first 18 months of the experiment was unre markable. After 18 months ageing symptoms developed in all groups and the number of unthrifty rats Increased, more rapidly in males than in fe males, The poor condition started with a humpbacked position and slight emaciation, followed by dyspnoea, pale eyes, lethargy, filthiness and in many cases severe emaciation. Randomly distributed major abnormalities due to ageing included maloc clusion of incisors, stalred coats, a bloody discharge around nostrils and eyes, wet stools, focal alopecia, focal dermatitis, paresis of hind legs, loss of one or both eyes and white opaque cornea. Macroscopically visible or palpable masses occurred in all groups. The total number of rats which showed such a lesion at any time, together with the number of masses is given in table 5. In females both the inci dence and the total number of these gross lesions was considerably higher than in males. Taking into account that the top-dose group comprised only 50 rats of each sex, the total number of masses in females of the topdose group was higher than in the other groups. This remarkable differ ence appeared to be caused by the high number of top-dose females with liver cysts (see also tables 13 and lA), which are easily detected by palpation of the abdomen. In the extra control group the total number and the incidence of masses was relatively low as compared to the other groups, the control group included. The low figures for the extra control group are undoubtedly related to the relatively high and early mortality In this group, giving fewer animals of this extra control group the time to develop lesions associated with old age. R&S 006486 R&S 006487 V 83.285 - 21 - The cumulative mortality is given in table 6. Up to week 68 mortality was very low. Thereafter, mortality gradually increased, more quickly in the extra control group than in, the other groups. The extra control animals which were housed in a separate room - were much more severely affected by chronic respiratory disease than the animals in the other groups (see also 3.9.1). This phenomenon undoubtedly explains the higher and earlier mortality in the extra control group. 3.5 Body weights (tables 7 and 8; figures 1 and 2) In the three test groups, the mean body weights were generally comparable to those of the controls. Some isolated changes were observed, but there was no evidence of a dose-related response. In the extra control group mean body weights were statistically signifi cantly decreased throughout the study in males and from day 674 and on wards in females as compared to the standard control group. In males the lower body weights were accompanied by lower Intake of food (see 3.6). Since the extra controls were housed in a separate room, the lower body weights and food consumption might be due to unknown differences in en vironmental condition (see also 3.4), although this separate room was conditioned in the same way as the other rooms. 3.6 Food Intake (tables 9 and 10) Food Intake showed wide variations among the different weighings within one group. There was, however, no evidence that food Intake was affected by VCM-feeding (table 9). Males of the extra control group had a lower food intake than the other groups from week 3 onward. No appreciable variations were encountered in the rate of food consump tion in the various groups (table 10). Females consumed their food slightly less quickly than males. V 83.285 22 3.7 Haematology (table 11) Thrombocyte count did not show dose-related differences between the test groups and the controls. The slight, though statistically significant Increase in females of the top-dose group on day 366 was not apparent when compared to the extra controls. Prothrombin time of females of the top-dose group was slightly increased after 3 and 6 months. However, prothrombin time of females of the extra control group was even higher at the same stages. No differences between the groups occurred after a feeding-period of 12 months. 3.8 Glutathione levels of the liver (table 12) There were no significant differences in liver glutathione levels between the various test groups and the controls, either after 9 or 18 months. 3.9 Pathology 3.9.1 Gross examination (table 13) The incidence of liver nodules suspected of being tumours was higher in males and females of the top-dose groups than in controls or lower dose animals. Most of these tumourous masses were small (diameter up to 1 cm), solid and pale or had the same colour as the adjacent liver tissue; some were large (diameter 2 to A cm), soft or firm and occasionally slightly haemorrhagic. Cysts in the liver of old rats, particularly in females, Is a common finding in the strain of rats used. However, the incidence of such cysts was much higher in females of the top-dose group than in females of the other groups, the control group Included. The cysts varied widely in size, were often multiple and generally contained a turbid, watery liquid. There was no evidence that any of the other gross lesions observed and summarised in table 13 were related to the administration of VQi. V 83.285 23 - Severe chronic respiratory disease (CRD) often accompanied by focal emphysema (spongy lungs) was seen much more frequently in the extra control group than in the other groups. Many of the rats affected by ad vanced CRD lost weight and emaciated before they died or were killed in extremis. Other conspicuous findings in animals of the extra control group were the high Incidences of bilateral testicular atrophy and atrophy of the coagulating glands and seminal vesicles. Atrophy of the testes and of the secondary sexual glands were often seen in the same animals, but these lesions also occurred separately. Moreover, a relatively high number of females of this group showed protrusion of the eye balls; this was almost exclusively observed In severely emaciated rats. R&S 006489 3,9.2 Microscopic examination Liver (table 1*) Increased incidences of foci of cellular alteration, neoplastic nodules, hepatocellular carcinomas, liver-cell polymorphism and cysts have been observed in the top-dose group. Moreover, in the top-dose group two females and one male had developed a hepatic angiosarcoma, whereas such tumours have not been seen in any of the other groups. Since the morphology of the hepatocellular lesions was essentially the same as that of the liver lesions described in full detail in our previous report on the long-term effects of oral administration of VCM in rats (Feron et al., 1981), no further dlscription of these alterations is presented in this report. The morphology of VCM-induced hepatic angiosarcomas has also been described in a previous report (Spit et al., 1981). Table 14 also shows that in females, but not in males, of both the lowand mid-dose group the incidence of basophilic foci of cellular alter ation was significantly higher than in controls. In addition, in the mid-dose group the number of females bearing foci of cellular alteration was statistically significantly higher than In the control group. V 83.285 24 There was no evidence that any of the other hepatic lesions observed, was related to the feeding of VCM. There were a few statistically significant differences between test groups and the control group, but this concerned decreases in incidence in the test groups (e.g. slight cholanglofibrosis in males), or the differences were not statistically significant as com pared to the extra controls (e.g. single cell necrosis in females), or there was no dose-response relationship (e.g. foci of degenerated hepatocytes in males). As compared to controls lower incidences of several hepatic abnormalities were found in the extra control group. Examples of such lesions are cysts, bile duct proliferation, cholanglofIbrosis and vacuolisation of periportal hepatocytes in females, and liver cell polymorphism in males. Such changes might be associated with old age and their relatively low incidences in the extra control group is most probably due to the much lower average survival time of these animals (118 weeks) in comparison with that of the controls (129 weeks). Mammary glands (table 15) The number of females bearing a mammary gland tumour was considerably lower in the extra control group and in the low- and mid-dose groups than in the control group. In the same groups the number of females with a mammary fibroadenoma was also lower than in the control group. There was no indication that the incidence of mammary gland carcinomas increased with increasing VCM dose. Abdominal tumours (table 15) All intra-abdominal tumours found at autopsy were examined histologically. The numbers of mesotheliomas detected in rats of the different groups indicate that there was no evidence of a relationship between the administration of VCM and the development of this type of tumour. Zymbal gland tumours All grosB tumours in the head region were studied by light microscopy. No Zymbal gland tumour was found. R&S 006490 j j V 83.285 25 - A. DISCUSSION In the present study no effects attributable to VCM were observed other than liver changes, and a higher mortality In the top-dose group as com pared to the control group during the last 6 to 9 months of the exper imental period. The differences in mortality between the top-dose group and the control group were slight and attained a level of statistical significance only at a few points of time. This slightly increased mortality In the topdose group might be due to the Increased incidence of neoplastic and non-neoplastic liver changes in this group. A similar adverse effect has been observed in females, but not in males, of the low--dose group (1.7 mg VCM/kg body welght/day) used in the previous long-term oral rat study with VCM (Feron et al., 1981). From the previous study (Feron et al., 1981) it appeared that oral exposure to VCM at a level of 1.7 mg/kg body welght/day resulted in a series of changes in the hepatic parenchyma. These changes included an Increased incidence of cellular alteration and neoplastic nodules, a few hepatocellular carcinomas, an increased incidence and degree of liver-cell polymorphism, and an Increased Incidence of hepatic cysts. In the present study the same type of treatment-related liver changes were found in the top-dose group receiving 1.3 mg VCM/kg body welght/day. In addition, one male and two females of the top-dose group had developed a hepatic angiosarcoma; in the previous experiment hepatic angiosarcomas were observed at VQH levels of 5.0 mg/kg body welght/day and above but not at the lowest exposure level of 1,7 mg/kg body weight/day. R&S 006491 As compared to controls increased Incidences of basophilic foci of hepatocellular alteration occurred in males of the top-dose group and in females of each of the dose groups. In females the incidence of this type of foci of cellular alteration in the liver increased with Increasing dose levels. Moreover, the number of rats bearing foci of cellular alteration In the liver (all type of foci) was significantly higher In males of the top-dose group and in females of the mid- and top-dose group than in controls. These findings suggest a relationship between the oral exposure to VCM at levels of 0.01A and 0.13 mg/kg body weight/day and the increased occurrence of foci of cellular alteration In the liver of female rats. R&S 006492 V 83.285 - 26 - The incidence of basophilic foci of hepatocellular alterations in females of the control group (9/98 or 9 2) was clearly lower than the incidence of this type of foci in female controls used in the previous long-term oral rat study with VCM (10/57 or 18 2; Feron et al., 1981). Similar incidences were also found in females of control groups of three other long-term rat studies carried out in our Institute more or less simultaneously with the study of Feron et al. (1981); the incidences being 11/95 or 12 2, 3/20 or 15 Z and 17/99 or 17 Z. These data seem to indicate that the incidence of basophilic foci (9 2) in females of the control group of the present VCM study is unusually low, and, thus, would seem to throw doubt upon the toxicological significance of the slight increase in incidence of this type of foci of cellular alterations in the low-- and mid-dose groups. However, the incidences of basophilic foci of hepatocellular alterations in females of control groups of other long-term rat studies performed in our Institute in the same period as the present VCH-study were: 0/20 or 0 2, 1/20 or 5 2, 5/69 or 7 2 and 4/50 or 8 Z. Compared to these figures an Incidence of 9/98 or 9 2 as found in the present group of female controls is not unusually low, and, thus support the suggestion that the feeding of VCM at levels of 0.014 or 0.13 mg/kg body weight/day may have resulted in more basophilic foci of cellular alteration in the liver of female rats. The directive of the European Community (EC) of January 30, 1978 (78/142/EEC) imposes a maximum permitted limit of 1 ppm residual VCM in finished plastic materials or articles Intended for use in contact with foodstuffs. The same EC-directive stipulates that any migration of VCM to the food must not produce a concentration In the food of greater than 10 ppb VCM which is or is very close to the detection limit of VCM in foodstuffs. Calculations based on a survey of residual levels of VCM in PVC bottles, films and foods in the period of 1974-1977, suggest that the intake of VCM from the average diet will be less than 0.1 pg per person per day (Crosby, 1982; Ministry of Agriculture, Fisheries and Food, 1978). What does this maximum oral daily intake of VCM by humane mean in terms of cancer risk? V 83.285 - ?7 - Extrapolation of the results of the present rat study to humans using the linear extrapolation model and taking into account an acceptable cancer risk from oral VCM exposure for man of 10 ^, leads to an acceptable oral intake of 0.4 jig VCM per person per day. Since the estimated maximum oral daily Intake is only 0.1 fig VCM/person/day, the estimated cancer risk is at least a factor 4 lower. Moreover, the extrapolation model used is a conservative and prudent one, which Implies that the actual cancer risk might be (considerably) lower than that calculated by means of the linear model. In addition, comparison of the results of an epidemi ological study of vinyl chloride workers with the predicted cancer risk in the cohort of workers based on the results of VCM studies in rats suggested that humans are less sensitive to the carcinogenic action of VCM than rats (Gehrlng et al., 1979). Translated into practical terms this means that the cancer risk of a maximum oral dally intake of 0.1 pg VCM/person/day is small enough to be practically neglected. R&S 006493 5. CONCLUSIONS The results of the present oral study allow the following conclusions: - VCM at a level of 1.3 mg/kg body weight/day Induces neoplastic and non-neoplastic changes in the liver of male and female rats, - VCM at a level of 0.13 mg/kg body weight/day may lead to more female rats bearing foci of cellular alteration in the liver, - VCM at levels of 0.01A or -0.13 mg/kg body weight/day may result in an Increased incidence of basophilic foci of cellular alteration in the liver of female, but not of male, rats, and - 0.13 mg VCM/kg body weight/day is a "no-observed-adverse-effect-level" with respect to the induction of tumours in rats. V 83.285 28 - 6. REFERENCES Ministry of Agriculture, Fisheries and Food (1978). The Surveillance of Food Contamination in the United Kingdom: Survey of Vinyl Chloride Content of Polyvinyl Chloride for Food Contact and of Foods. The Second Report of the Steering Group on Food Surveillance - The Working Party on Vinyl Chloride. Food Surveillance Paper No. 2, p. 6. HMSO. London. Crosby, N.T. Analysis for residual monomer levels in plastic and in foods Anal. Proc. September 1982, 428-430 Feron, V.J,, et al. Life-span oral toxicity study of vinyl chloride in rats CIVO-Report No. R 5788 (1978) Feron, V.J., et al. Lifespan oral toxicity of vinyl chloride in rats Fd, Cosmet. Toxicol. 19 (1981) 317--333 Feron, V.J. , et al. Observations on the oral administration and toxicity of vinyl chloride in rats Fd. Cosmet. Toxicol. 13 (1975) 633-638 R&S 006494 Gerlng, P.J., P.G. Watanabe and C.N. Park Risk of angiosarcoma in workers exposed to vinyl chloride as predicted from studies in rats Toxicol. App. Pharmacol. 49 (1979) 15--21 Ouren, P.A. Thrombotest; a new method for controlling anticoagulant therapy Lancet, 1959, II, p. 754 Spit, B.J., V.J. Feron and C.F.M. Hendriksen Ultrastructure of hepatic angiosarcoma in rats induced by vinyl chloride Exp. Mol, Pathol. 35 (1981) 277-284 V 83.285 29 Squire, R.A. and M.H. Levitt Report of a workshop on classification of specific hepatocellular lesions in rats Cancer Res. 35 (1975) 3214-3223 R&S 006495 INST. CIVO-TOX. AND NUTR. TNO 83-09-01/AE r &S 006496 V 83.285 7. AUTHENTICATION This report was prepared by: - 30 - (Study director) date H.R. Imrael (Pathologist) date: C1- 3 (Study supervisor. Pathologist) ZP'Of- '$>3 and approved by: Dr A.P. de Groot date: (Head Dept. Biological Toxicology) 2j8 /<fd^ V 83.285 - 31 8. RETENTION OF RECORDS, SAMPLES AND SPECIMENS All records concerning the dietary formula, daily observations, body weights, food intake, haematology, clinical chemistry and gross- and microscopical examinations and all other Information relevant to the quality and Integrity of the study have been filed In the archives of the Department of Biological Toxicology together with the master copy of this report. Materials (samples of the test substance, wet specimens, blocks) have been stored, and will be retained for a period of eight years, i.e. till July 1990. The slides will be retained for a period of 15 years, l.e. till July 1997. 30 CO o o CT> (> i V 83.285 32 - QUALITY ASSURANCE UNIT TNO - I'.O. Box 360, 3700 AJ ZE1ST, Netherlands STATEMENT OF GLP COMPLIANCE On : Lifespan oral carcinogenicity study of vinyl chloride in rats Report no.: V 83.285/291099 Date : September, 1983 The study was carried out under conditions of good laboratory practice. Within reason there have been no circumstances that might have affected the quality and integrity of the results obtained. R&S 006498 Dates and number of inspections: 19 November 1979 22 and 25 February 1980 12 May 1980 12 November 1980 17 November 1980 23 January 1981 3-4 March 1981 14-15 May 1981 (1) (1) (1) CD (2) (1) (2) (3) 18 August 1981 27 October 1981 (1) (3) 22 January 1982 12-13 May 1982 (3) <2) 24 June 1982 8 July 1982 CD (3) 8-11 March 1983 (2) 10 March 1983 (D Draft report audit: 10-11 March 1983 Dates of reports to management: 19 November 1979 25 February 1980 12 May 1980 12 November 1980 18 November i960 26 January 1981 5 March 1981 18 May 1981 18 August 1981 27 October 1981 26 January 1982 13 May 1982 24 June 1982 8 July 1982 16 March 1983 24 March 1983 24 March 1983 Final report audit: 20 September 1983 (1) <c: y4 . yu- / Drs S, van Straten Quality Assurance Manager 20 September 1983 date: Z3 ^ V 83.285 CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIET 33 - TABLE 1 LEVELS OF VCM IN THE DIET IMMEDIATELY AFTER PREPARATION OF THE DIETST7 Group A B C D E Intended oral exposure levels of VCM (mg/kg b*v./day) 0 0.017 0.17 1.7 0 Dietary level of VCM (ppm) MEAN SEM N <0.1 0.487 4.487 44.07 < 0.1 0.013 0.097 0.926 72 72 72 72 65 ^ The VCM content of the PVC-powder with VCM was on average 4582 + 98 ppm. R&s 006499 CIVO/TNO STUDY NO 125 CARCINOCENICITY STUDY IN RATS WITH VCM IN THE DIET TABLE 2 LOSSES OF VCM FROM THE DIET DURING A PERIOD OF FOUR HOURS Group Intended oral exposui:e levels of VCM (mg/kg b.u,./day) VCM levels in the diet during the four-hour feeding-period at different points of time ^ expressed as ppm 2> expressed as percentage of tn ro t2 l3 *4 c2 c3 `4 A 0 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 B 0.017 0.48 0.37 0.32 0. 27 0.28 77 67 56 58 0.0373) 0.039 0.024 0.021 0.016 C 0.17 4.2 3.3 3.0 2.7 2.7 79 71 64 64 0.36 0.23 0.13 0.20 0.15 D 1.7 41.3 33.3 29.9 26.7 25.3 81 72 65 61 1.94 1.85 1.70 1.66 1.96 E Grand Mean 0 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 79 70 62 62 ^ Samples taken Immediately after preparation of the diets (eQ) and further from the feeders In the cages of the rats one hour (t^), two hours (tj). three hours (tj) and four hours (t^) after preparation ^ The values are the means of 7 different analyses. 33 Standard error of the me,A. 00S900 ssa CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIET TABLE 3 LEVELS OF VCM IN THE FAECES Group A B C D E Intended oral exposure levels of VCM (mg/kg b.w./day) 0 0.017 0.17 1.7 0 Level of VCM (ppm) found in the faeces 24 hours after the start of the feeding-period^ MEAN < 0.1 0.26 2.64 23.6 < 0.1 SEM 0.028 0.273 2.960 N 5 5 5 5 5 ^ Faeces were collected by squeezing the lower part of the rat's abdomen; they were Immediately submerged in 10 ml ethylacetate and then stored at 4 'C In a closed vessel until analysis for the VCM content. LJ U> 1.0S900 Stfy CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIET TABLE 4 DESICN AND ACTUAL DOSE LEVELS OF VCH IN RATS FED DIETS CONTAINING PVC POWDER Group A B C D E Intended oral exposure levels of VCH ppm mg/kg b.w./ In diet /day 00 0.34 0.017 3.4 0.17 34 1.7 00 Actual dietary VCH levels at the start of the feeding period ' ^ (ppm) 0 0.49 4.5 44.1 0 Theoretical oral Intake of VOi^ (mg/kg b.w./ day) 0 0.022 0.21 2.1 0 Actual oral intake of VCM3) (mg/kg b.w./ day) 0 0.018 0.17 1.7 0 Actual oral ex posure level of VCM*0 (mg/kg b.w./day) 0 0.014 0.13 1.3 0 .Average dietary VCH contents determined immediately after preparation of the diets* 21' Assuming no loss of VCH by evaporation from the diets (see also table 1). ^ Taking Into account the loss of VCH from the diets. M Oral intake of VCM diminished by the faecal VCM. The VCM excreted in the faeces was considered to be still enclosed In the PVC granules, and thus not to have been In contact with the body. 209900 S^H C1V0/TN0 STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCH IN THE DIET TABLE 5 CROSSLY VISIBLE OR PALPABLE SUPERFICIAL OR INTRA-ABDOMINAL MASSES OBSERVED IN THE COURSE OF THE STUDY Croup Intended exposure levels of VCM (tng/kg b.w./day) Total No. of rats with masses no. of rats at Total Non-survivors start HALES A0 B 0.017 C 0.17 D 1.7 E0 100 100 100 50 100 35 45 36 18 14*** 32 40 31 17 14** No , of masses Total d 1 sappea red In course of t line 55 10 55 10 42 7 23 6 16 1 FEMALES A0 100 68 59 B 0.017 100 80 69 C 0.17 100 63 49 D 1.7 50 38 36 z0 100 48** 48 112 3 119 8 115 6 87 2 79 4 STATISTICS: CHI-square test: ** P < O.Ol, *** P < 0.001 05900 S'SH TAPLE 6 fl 0 F: T A L I T Y HAY DAY DAY DAY DAY DAY DAY DAY DA i DAY DAY PAY DAY DAY DAY DAY DAY DAY DAY PAY DAY DAY DAY DAY DAY DAY DAY DAY 0 oo 56 S'! 112 1 -10 168 196 an ^ 252 280 30 S 33 1 A 392 ^20 lie 174 501 532 560 5B8 616 A .1 672 700 72 *- ?jr>' COHTROL INCIDENCES 0 03 A nv a o;; i ir; i 17. i 17. i 17. i 17. i 17. i 17. i 17. i 17. i 17. 2 2v a 27 2 2V, 3 27. A 17. 1 17. A AY A 17. 6 67. 6 67. 6 67. t bV 11 11 7. 13 1 27. 13 127. 0.017 MG/l.G INCIDENCES o or; 0 07. 0 03 1 17. 1 17. 1 i:: 1 13 1 13 1 13 1 13 1 13 1 13 1 13 I 13 1 13 1 13 1 13 1 13 A A3 A A3 1 A3 "7 73 B 83 B 83 12 pv 1 5 153 15 153 15 153 0.17 HG/KG IHCIDEHCES 0 03 A 03 a 03 a 03 0 03 A 03 A 03 a 03 0 03 0 03 A 03 l 13 l 13 i 13 A 23 a 2v 3 33 2 33 A A3 5 53 6 63 7 73 -i V 7 ? y. 8 83 o 1 3 1 33 1 A 1 A3 statistics: FISHER EXACT F'ROBAPILITY TEST ' P-;0,05 P<0.01 1,7 MG/NG IHCIDEHCES 0 03 A AV 0 07 0 03 0 03 0 03 0 03 0 03 0 03 0 03 0 03 0 03 0 03 0 03 0 03 0 03 0 03 0 03 1 23 1 23 2 63 2 4;; 2 4;; 3 63 3 63 A 83 7 16 3 10 203 F' \ 0,0 0 1 IIJO SIDED CONTROL INCIDENCE'. a r. ' 03 0 7. r. a r, o\ A0 r. 02 a 02 n 03 A 03 a O'. a0 a 02 A AV A O'2 A Av a 2': o o o c* c e* O' t7 17 1' 12' ->t> i 17 2 1 7'. a* 2- . t?0S900 S$b TABLE 6 HA L ES HAY DAY DAY DAY DAY El AY DAY DAY PAY DAY 78 4 O] 2 10 868 8?& 92 4 952 ^eo 1000 1036 MORTALITY (CONTINUED 1) CONTROL INCIDENCES 15 157. 21 21 28 2B7. 31 31% 38 38 /. 44 44% 53* 53;: 59 5?:: 72 722 BO 80% 0.017 MG/KG INCIDENCES 17 17% 25% 28 28% 34 34% 43 4 3% 51 51% 54 54% 43 4 3% 49 69% 80 80% 0.17 MG/KG INCIDENCES 14 16% 23 0 17 28 282 34 34% 1 O 38% 44 4 4% 53 53% 59 59% 72 72% 82 82% Ho ov statistics: FISHER EXACT PROBABILITY TEST P<0.05 11 P 1.7 MG/KG INCIDENCES 15 3(3 % 18 36% 21 4 2;; 24 48 % 24 29 7n 48 % 56 % 64 % 35' 70 % 39 ` * 78 % A2* * 1 34 % P70.001 TWO SIDED CONTROL incidence: 2?' - -i p C* o ' y 71 "A ' OO > 71 p 1 00' 1 00 * * 1 100' * i oo 1 00 100 100 100 S0S900 ssa TABLE 6 MORTALITY (CONTINUED 2) FEMALES PAY DAY PAY PAY DAY PAY PAY PAY DAY PAY PAY PAY PAY DAY DAY DAY DAY PAY DAY PAY DAY DAY DAY DAY DAY PAY PAY DAY n 30 53 86 114 142 170 198 226 25 A 282 310 338 366 394 4 22 150 178 506 531 562 590 610 616 671 702 730 758 CONTROL INCIDENCES 0 02 0 OX 0 07. 0 07. 0 07. 0 07. 0 07. 0 07. 0 07. 0 07. 0 07. 1 17. 1 17. 1 17. 1 17. 1 17. 1 17. 1 17. n 2v n 27. A A 7. 5 57. 6 67. 7 7% 10 1 27. 12 1 27. 15 15 7. 20 207. 0.017 MG/KG INCIDENCES 0 07. 0 07. 0 0 7. 0 07. 0 07. 0 07. 0 OX 0 07. 0 07. 0 07. 0 07. 0 07. 0 07. 0 07. 0 07. 0 OX 0 07. 1 17. n 27. n 27. A AX A AX A AX 5 57. e B2 8 87. 11 11 X 13 13 7. STATISTICS I rISHER EXACT PROBABILITY TEST 0.17 MG/KG INCIDENCES 0 OX 0 OX 0 OX 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 1 IX 1 IX 2 2X n 2X 3 3X 3 3X 3 3X 4 IX 4 IX 7 77. 10 10X 12 127. 1 A 14 X 19 19X 1 P<0.01 "n O O .<<1 "n A O oo 1.7 MG/KG INCIDENCES 0 OX 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 ox 0 07. 1 22 1 2X *1 AX. n Ay. 2 AX 5 10 X 6 12X 6 122 6 122 6 12X 12 24 2 11 28 2 TWO SIDED CONTROL INCIDENCES 0 OX n ox r, ox 0 ox 0 ox 0 rv v 0 CiL 0 ox 0 ox 0 ox 0 pv 00 0 07. 0 07. 0 07. 0 07. 0 07. A OX 0 OX 0 O'. 0 OX 11 IX 22 n *> 22 2 ~J V 7 ? V 1 4 147. 90S900 SSH TABLE 6 MORTALITY {CONTINUED 3) F E MALE S DAY PAY PAY DAY PAY DAY DAY PAY PAY PAY 706 81 4 942 370 B9B 926 954 982 1010 1038 CONTROL INCIDENCES 20 20% 26 267. 28 237 30 30% 35 35% 41 41% 47 47% 58 58% 66 66% 76 76% 0.017 HG/KG INCIDENCES 15 15% 25 25% 32 32% 36 36% 4 1 41% 51 51% 60 60% 66 66% 72 72% 77 77% 0.17 MG/KG INCIDENCES 19 19% 2_2 22% n t 27% 30 30% 35 35% 45 45% 52 52% 61 61% 67 67% 74 74% STATISTICS! FISHER EXACT 1PROBABILITY TEST P<0.05 > P-10.01 -1.7 MG/KG INCIDENCES 15 30% 16 32% 19 38% 21 62% 24 48% 28 56% 32* 64% 36'* 43" 72% 86% 45"* 90% . P<0.001 TWO SIDED CONTROL INCIDENCES 16 16% Te; 251. 37 7r; 50* * 50% 58' 1 921 * * 5 0% 92 v 100*" 100% 100'" 100% 100' 100% 100'* 100% zos9oo S'&a TABLE 7 MEAN BODY WEIGHTS <G) MALES PAY 0 PAY 1 4 PAY 2B PAY 56 PAY 84 PAY 112 PAY 1 40 DAY 168 PAY 196 PAY 224 PAY 252 PAY 280 PAY 308 DAY 336 PAY 364 PAY 392 PAY 420 PAY 44B PAY 4/6 PAY 504 PAY 532 JAY 560 JAY 588 -AY bib JAY 644 JAY 672 1 AY 700 if* Y 728 tAY 756 CONTROL MEAN SEM N 53.9 95.7 149.8 233.4 286.4 326.5 344.7 367.5 376.6 386.5 38B.5 403.4 411.2 416.5 413.3 410.8 404.2 404.2 405.9 400.4 415.6 419.4 414.5 419.4 428.8 431.1 427.8 425.4 419 .B 0.5 0.8 1.4 2,4 3.1 3.4 3.5 3.7 3.7 3,9 4,0 4.0 4.2 4.2 4.2 4.2 4.1 4.2 4.4 4.2 4.1 4.1 4, 1 4.2 4.3 4,2 4.0 4.0 4 .2 100 100 100 100 99 99 99 99 99 99 99 99 99 99 9B 98 98 97 96 96 96 96 94 94 94 94 89 BB 87 0.017 MEAN MG/KG SEM N 53.2 96.9' 147.8 223.O'1 271.8* * 309.2* * 340.2 357.0 ,370.8 377.7 3B1.2 400.0 403.3 406.5 409.1 403.6 397.3 399.0 403.3 395.2 407.5 414.9 412.5 415.9 425.9 427.5 424.8 421 .2 41B .5 0.5 1 .0 1 .6 2.4 3.0 3.3 3.6 3.7 3.9 4.0 4.0 4. 1 4.2 4.2 4.3 4.3 4.3 4.4 4 .4 4.3 4.4 4.3 4.5 4.5 4.5 4.7 4.8 4.8 4.7 100 1 00 100 100 99 99 99 99 99 99 99 99 99 99 99 99 99 99 99 96 96 96 93 92 92 89 86 85 85 0.17 MG/KG MEAN SEM N 53.2 95.8 148.9 232 * 0 283.3 323.8 343.0 364.7 372.8 385.7 387.8 405.2 407.7 415.0 411.7 407.4 402.8 404.7 410.9 404.0 416.1 422.6 418.4 425.2 431.5 437.8 431.4 428.0 423.3 0.5 0.8 1 .3 2.5 3.0 3.1 3.4 3.4 3.6 3.7 3.4 3.6 3.7 3.8 3.9 3.9 3.9 4,1 4.1 4.4 4.2 4.2 4.3 4.3 4.4 4.5 4.4 4. 4 4,6 100 100 100 100 100 100 100 100 100 100 100 100 99 99 99 98 9B 97 97 96 95 94 93 93 93 92 91 87 86 STATISTICS!! COVAR + PUNNETT TESTS * p<0 .05 *' F' <. 0 .01 TWO SI PEP I ,,7 MG/KG MEAN SEM N 54.3 98.2 149.2 224.5* 279.0 316.6 348.8 364.1 384.3 389.3 387.3 408.3 414.6 417,9 418.6 419.4 411.9 413.1 414.8 409.2 420.6 426.7 423.5 429.3 43B.5 446.5 440.4 428.8 423.9 0.6 1.1 1.8 2.9 3.6 3.9 4. 4 4.7 4.B 4.9 4.7 5.1 5.2 5.0 5.1 4.9 4.8 4.8 5.1 5.0 5.0 5.5 5.3 5.5 5.8 6. 1 5 .B 7.4 6.9 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 49 49 49 48 48 47 47 46 44 40 CONTROL HEAN SEM M 51.5 89.3* ' 141.7* 214.6** 251.1 * * 278.1 * * 297.5* * 314.5* * 318.5'* 339.8* * 351-.4* * 365.0* * 371.0'* 371.3* * 377.8* * 385.2* * 370.9* * 368.0* * 377,3* * 364.0* * 380.7* * 3B4.3* * 379.9* * 376*1* 379.7 * * 381.2'* 3 65 * 91' 357.2* * 344.8** 0.5 0.7 1 .1 1 .5 2.0 9. i 2.3 2*6 2 *8 2,6 nt 2*7 2.8 3*3 3*0 3.1 2*9 y. 9 3*0 3, 7 VI 3* 3 3* 7 1, 7 4. 1 4.7 j 4.9 c rJ 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 9? 92 92 92 92 92 90 87 33 7f) 71 (EXP. UNIT = Al [HAL ) 809900 S*U TABLE 7 MALES MEAN BODY WEIGHTS (G) (CONTINUED 1) CDNTROL MEAN SEM N 0.017 MEAN HG/KG SEM N DAY 784 PAY B12 DAY 840 DAY BAB DAY B96 DAY 924 DAY 952 DAY 980 PAY 1008 DAY1036 0 A Y1 0 4 3 408.6 394 . B 393.3 387.4 376.6 365.6 361.7 352.9 346.5 341.3 343.8 4.0 3,9 3.9 3.9 4,3 4.3 4.1 4.4 5,2 5.2 5.3 85 79 72 69 62 56 47 41 30 21 20 406.3 395.5 400.0 390.6 383.7 372.9 368.7 359.5 360.7 356.6 359.3 4 .B 4.9 4.7 5.0 5.6 5.B 6.4 6.1 6.5 5.5 5.6 B3 75 72 67 59 49 47 3B 31 20 20 0.17 MG/KG MEAN SEM N 409.4 402.9 400.6 394.8 386.3 371.2 363.4 360.2 358.6 356.3 358.1 4.6 4.5 4.7 4.7 4.5 4.4 4.7 4.9 6.3 6.2 6.3 84 77 72 66 62 56 47 43 2B 19 IB 1.7 MG/KG KEAN SEH H 414.9 403.5 398.3 393.7 3B2.5 3B0.0 36B.6 363.3 354.2 366.7 368.7 6.7 7.1 5.9 5.7 6.1 7.8 7.7 B.O 7.4 7.8 7.9 35 32 29 26 26 21 18 16 11 8 8 CONTROL KEAN SEH N 322.9'' 302.5'' 297.9?' 299.3*' 2BB. 1'' 329.8 -- -- --- -- 5.4 5,5 7.0 7.8 9.0 20.4 ----- -- 63 58 41 29 24 2 0 0 0 0 0 STATISTICS: COVAR 4 BUNNETT TESTS PI0.05 P'O.Ol TWO SIDED (EXF'.UNIT = ANIMAL) 60S900 S$y TABLE 7 F E HAL E S MEAN BODY WEIGHTS (G) (CONTINUED 2} PAY PAY PAY PAY PAY PAY PAY PAY PAY PAY PAY PAY PAY PAY 3AY .'AY JAY PAY 1A Y PAY PAY PAY 'AY 'AY JAY PAY .'AY AY 'AY 0 16 30 5B 84 114 142 170 190 226 254 282 310 330 364 394 450 4 /b b06 534 542 590 61 y 646 474 702 730 758 CONTROL MEAN SEH N 52 . S 92,7 118.9 151.4 144.7 17B.0 183.3 188,4 189.4 193.9 199.0 202 9 210.2 209,2 211 ,5 212.1 207.9 210.1 214.0 213.6 218.4 220.7 225.3 224.3 231.7 237.0 235 . B 232.5 232.9 0.5 0.B 0.9 1 .1 1.3 1.4 1.5 1.4 1.4 1.7 1.4 1.4 1.7 1.7 1 .8 1.9 1.9 1.9 2.1 2.0 2.3 2.5 2.8 2.5 2.7 2.7 2.7 nr 3.0 100 100 100 100 100 100 100 100 100 100 100 100 99 99 99 99 99 99 99 99 98 94 95 94 93 90 89 84 80 0,017 MEAN hG/KG SEH N 52.9 92.5 120.2 153.4 144.2 175.2 183.4 187.2 192.9 192.7 201.0 203.7 209.7 208.1 212.4 210.0 207.4 210.3 212.4 213.1 217.4 219.0 222 * 7 227.0 231.9 234.0 234.4 232.3 234.4 0.5 0.7 0,9 1.1 1.4 1 .5 1.7 1 .7 1 .B 1 .8 1 .7 1 .9 2.0 2.0 2.1 2.1 2.1 2.1 22 2.2 2.5 2.5 2.7 2.8 2.9 3.0 3.0 3.0 3.2 1 00 100 100 100 100 100 100 100 100 100 1 00 100 100 100 100 100 100 100 99 98 98 94 94 94 95 93 92 90 87 0.17 HG/KG MEAN SEH N 51.5 90.1 120.1 153.4 148.4* 178.5 184.0 190.4 190.8 195.4 201.8 205.9 210.B 210,8 211.4 212.9 209.0 213.4 215.4 214.0 219.4 221 . B 224.5 230 3 235. B 241.0 24 0.0 238.8 2 4 0.2 0,5 0.7 0.9 1.0 1.2 1 .3 1 .5 1 .5 1 .4 1.7 1.4 1 .4 1.7 1.9 1 .B 1 .9 1.9 1 .9 2.0 2.0 ?, 2*2 2.4 2.5 2.4 2.7 2.B 2.7 2.9 100 100 100 I 00 100 100 100 100 100 100 100 100 100 100 100 99 99 98 98 97 97 97 94 94 93 91 B9 B4 81 .TATISTICS I CO'PAR + DUHNE1T TESTS * R<0 . 05 F' < 0 .01 TWO SIDED 1 ,,7 HG/KG HEAN SEH N 51 . 2 SB, 5 * 117.9 152.0 147.8 178.8 187.B 194.3* 194.8 195.4 203.3 205.5 212.3 213.1 214.4 217.3 212.7 217.0 217.9 217.0 T'l'i n 223.3 228.0 232.9 240.8 245.1 242.8 237.0 237.4 0.7 1 .2 1.3 1 .5 I .8 2.0 2.1 n"n 2.4 2.3 2.4 2.4 2.9 2.8 3.1 3.1 3.0 3.2 3.3 7*7 3.4 3.7 3.5 3.8 4.0 4.2 4. 4 4.0 4.2 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 49 49 48 48 48 44 44 44 44 44 38 34 CONTROL HEAN SEH N 52.8 90.4* * 117.4 140.0* 1 147.3 174.3 182.B 188.9 193.0 195.8 205.2 204.7 212.3 210.0 211.5 213.2 209.7 211.2 215.9 210.8 21 9 1 219.3 221.9 223 * 2 22? . 7 226 * B* 224,0' 21^P * 219.0* * 0.4 0.8 0.9 1,2 100 100 100 100 1 .4 1.5 1.5 1 .4 1.4 1 .4 1 .0 1.8 1.9 100 100 100 too 100 100 100 1 00 100 i*e l .<* 1 .9 1.8 1 .9 too 1 00 1 00 100 100 2.0 2 . r. "> r, 2. 1 1 00 1 0^ 1 Ort 100 2*1 1 -i nn 99 OR OR * A 'i . f. T.A or op o3 2.5 84 (EXP . UNIT - ANIMAL 1 (HS900 S'Sd TABLE 7 MEAN BO BY HEIGHTS (G' (CONTINUED 3) FEMALES CONTROL MEAN SEM N 0.017 MEAN MG/KG SEM N DAY 786 DAY EM DAY 842 DAY 870 DAY 898 DAY 926 DAY 954 DAY 982 DAY1010 DAY103B DAY 10 42 230.7 224.0 224.2 222.7 217.9 216.6 212.3 211.1 212.7 212.4 214.9 2.8 2.6 2. 7 2.9 2.9 2. B 3.0 3.3 3.4 3.6 3.3 80 74 72 70 66 59 54 43 36 23 21 230.7 227.0 224.0 222.7 221.0 217.7 212.8 214.4 216.0 215.1 215.2 3.2 3.1 3.4 3.6 3.9 3.8 4,3 4.6 5.1 5.7 5.7 85 78 69 65 59 49 41 34 28 23 23 0.17 MG/KG MEAN SEM N 235.4 232.1 231. B 227.3 223.3 220.8 215.1 212.1 221.1 218.4 217.7 2.8 2.7 2. B 2.9 3,2 2,9 3,0 3, B 3,5 4.1 4,2 81 78 74 70 66 55 49 42 33 25 25 1 ,.7 MG/KG MEAN SEM N 232.9 227.9 227.4 227.1 227.0 224.6 217.2 194.2 208.6 214.2 3.7 3.7 4.4 4.2 4.9 5.4 5.5 4.8 3.7 3.1 3.8 35 34 31 29 26 22 19 14 7 4 4 CONTROL MEAN SEM N 209.B'' 207.7" 203.1*' 203.3* * 200.6* * 217.6 _ -- 2*8 2.6 3.A 3.7 4.0 4.3 -- 84 77 65 50 A2 8 o o o $ 0 STATISTICS! COOAR I DUNNETT TESTS * P4O.05 P <0.01 TWO SIDED (EXP.UNIT = ANIMAL > US900 SSU 1 ADLE 8 MALES MEAN BODY UE I GUTS { G ) . DAT DAT DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY DAY 0 14 26 56 64 112 140 166 196 224 252 280 308 336 364 392 420 448 476 504 532 CONTROL MEAN SEM N 51,4 09,4 141.6 229,2 276.4 312.1 336.1 360.4 373.5 383.4 382.6 417,1 424,6 430.3 413.0 419.7 416,5 413.7 422.1 413.5 426.6 2.0 2.5 3.1 4.6 4,8 5.1 5.4 7.1 6.2 7, 4 12.7 11.9 9,4 10.3 9.7 8.1 6.6 10.1 10. B 9,8 11.6 10 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5 5 c 5 C 5 0.017 MEAN MG/KG SEM N 53,4 94.0 146.3 232.8 2B8.7 325.9 354.7 374.5 384.2 3B5.5 395.4 425.5 434.3 439.9 440.0 436.0 432.6 434.0 421 .8 405,4 423.2 2.1 2.4 3.2 5.4 6.3 7.3 10.2 8.3 9.7 12.2 9.7 8.5 12 2 13.2 13.0 11,9 10.4 10.9 8.8 10.2 9.2 10 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5 5 5 5 5 5 0 MEAN 55 *5 96.9 139.5 230,4 289,4 325,7 350,7 372.3 377.5 38B, 4 394.2 393.1 392.4 400.1 395.6 391,0 379,2 382.4 381.5 374.9 395.1 HG/KG SEM N 1.5 2.0 9.t 4,3 6.5 8.8 9.6 10.6 9.5 B. 5 9.4 6.5 6.5 7.8 5.6 5.B 12.7 9.3 12.1 14.1 14.9 10 10 10 o 9 9 9 o 9 o 9 4 4 4 4 4 4 4 4 4 4 statistics: A TO A + DUNNET1 TEST C * P-'O, 05 P<0.01 TWO SIDED 1 .7 MG/KG MEAN SEM N 56,4 97,0 14 9.5 224.9 280.3 321.0 341.9 360.B 379.6 391.1 394,1 412.3 415.1 418.4 421.4 421 .B 413.2 417.9 421.8 408.7 422.7 1.0 1.0 2.0 3.2 5.0 7.2 6.3 6.1 4.9 5.5 7.0 10.1 10.0 8,2 8.9 B.4 8.8 8.1 7.5 4.8 6.0 10 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5 5 5 5 5 5 CONTROL MEAN SEM 54.9 96.4 14 7.? 218,1 246.0' * 270.3* * 286.9* * 306.5 * ' 314.6' ' 337.8* ' 340.6* 352.3* * 361.3* ' 364.1* ' 373.1 372.9* 359.2* * 356.0* * 359,6 ' ' 353.1' * 369.2* 1.5 2.3 4. ' 8.4 9.0 9.2 9.6 9.7 8.8 9.5 8.1 16.3 16.7 16.3 14.9 15.6 18. 1 16.9 16.5 15.7 17.0 (EXP,UNIT - ANIMAL' Si-9900 S9H -IUDY HU 1 jo 1 o"jiuttiu ^luiu lit mu ituti . I lit It JL IIILJ TABLE 8 HEAN BODY HEIGHTS CG), (CONTINUED) FEMALES DAY DAY DAY DAY DAY PAY DAY DAY DAY DAY DAY PAY DAY PAY PAY DAY PAY DAY PAY PAY DAY 0 16 30 SB 86 114 142 170 1?0 226 254 282 310 33B 366 394 422 450 478 506 534 CONTROL MEAN SEM N 53.3 95.4 119-8 150.3 161.7 172.3 177.8 lB-'.S 185.9 187.7 195.3 195.7 197.3 198.7 200.7 203.1 198.8 201.4 208.0 209 . B 214.5 1 .4 1.6 2.5 2.7 3.B 4.0 4 .9 5.1 5.4 5.5 5.5 3.2 4.5 3.0 5.2 5.3 5.5 5,9 6.4 5.5 7.0 10 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5 5 5 5 5 5 0. MEAN 54.3 92 .n 119.3 1 54 ; 5 168.9 179.5 185.3 193.3 193.9 195.9 204.8 209.8 214.3 213.2 220.3 216.5 215.6 217.1 219.1 225.2 226.6 MG/KG SEN N 1.7 2.7 2.8 3.7 3.6 3.4 3.4 3.9 3.4 3.6 3,2 3.0 5.1 4.4 5.5 6.1 6.4 6.8 7.8 7.9 9.1 10 10 10 9 9 9 9 9 9 9 9 z 5 5 5 5 5 5 5 5 5 0 . 17 MG/KG MEAN SEM N 54.7 95.2 123.1 154.4 166.3 178.5 185.9 190.7 191.0 196.7 201.3 195.1 202.2 199.5 198.2 198.7 199.6 201 .0 200.7 202.2 204.3 1.6 1.9 2.4 3.1 3.3 4. 1 4.6 4.9 4.2 5.0 4.3 7.2 8.3 6.9 8.1 8.4 8.1 7.5 8.1 0.8 9.0 10 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5 z 5 5 5 5 statistics: anova + punhett tests '0.05 P<0.01 TUO S1PEP i .7 MG/KG MEAN SEM N 52.5 87.2 116.2 147.9 162.7 175.6 182.1 195.9 188.9 191.0 199.4 19B.3 202.3 201.7 201.6 204.0 199.5 204.2 205.2 205.8 209.4 1 .6 2.9 3.3 4 .0 4.0 5.8 5.5 6.1 6.0 5.6 6.1 11.3 11.9 10.5 10.4 10.6 10.1 11,0 11.2 11.3 12.4 10 10 10 10 10 10 10 10 10 10 10 5 5 5 5 5 5 Z 5 5 CONTROL MEAN SEM 50.4 85.7' 111.8 152.2 160.9 173.6 179.6 184.8 186.2 190.4 198.3 204.7 211.4 211.6 215.4 214.0 210.7 21 ?+P 217.2 214.2 223.0 1.6 2.1 2.4 2.9 4. 4 4 .9 4.3 5.4 4.0 5.4 5.7 5.0 5.8 6.0 5.6 6. 4 5*3 5.7 6.4 5. 1 6.0 ! EXf'. UNIT = ANIMAL) 1.3900 SSU CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH1 VCM IN THE DIET TABLE 9 MEAN FOOD INTAKE Group A B C D E Intended Intake levels of VCM (mit/kg b.v./day HALES 0 0.017 0.17 1.7 0 Food intake (g/rat/day) in week 1 2 3 4 11 12 23 24 35 36 8.9 10.3 12.3 14.4 16.1 16.1 16.5 16.3 15.3 16.7 7.4 9.7 11.1 13.5 14.0 14.5 14.5 14.0 14.8 15.5 7.7 10.3 11.9 14.7 15.6 15.6 17.4 17.0 15.5 16.6 7.8 10.4 12.1 14.5 14.7 15.3 17.4 17.0 15.9 15.5 8.2 10.3 12.4 13.5 12.5 13.3 12.5 11.9 13.3 12.9 47 15.7 14.6 15.8 15.5 12.3 48 17.0 15.3 17.1 16.4 13.0 59 17.1 16.6 15.9 17.0 12.5 FEKALES A 0 8.9 10.0 9.9 10.7 10.4 10.2 9.2 9.1 9.3 9.8 10.6 10.2 9.9 B 0.017 8.3 9.8 10.4 11.0 10.2 10.0 9.0 9.2 10.3 10.5 9.4 9.8 9.6 C 0.17 8.5 9.7 11.5 12.0 11.0 10.8 10.8 10.4 10.3 11.3 10.3 10.6 10.0 D 1.7 8.3 9.4 10.4 10.6 11.0 10.7 9.3 9.6 10.8 11.1 9.3 10.4 10.3 E 0 8.3 9.3 10.2 9.9 9.4 10.3 9.1 9.2 9.9 9.7 9. B 9.9 10.0 ns900 s'sa CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIET TABLE 9 MEAN FOOD INTAKE (CONT.) Croup A B C D E Intended intake levels of VCM {mg/kg b.v./day KALES 0 0.017 0.17 1.7 0 Food intake (g/rat/day) in week 60 73 74 85 86 97 98 109 110 121 122 16.2 15.2 16.1 15.8 12.6 16.5 15.9 17.5 18.2 13.3 16.6 16.1 17.3 17.5 13.3 14.4 13.6 15.0 15.7 13.0 16.2 14.6 16.3 14.9 12.4 14.7 14.6 15.4 15.7 12.3 14.2 15.2 15.9 15.6 11.6 15.2 15.4 15. 7 16.1 11.4 15.0 14.5 15.7 15.4 11.2 15.9 14.6 15.8 14.2 12.3 16.1 15.0 15.8 15.8 12.5 FEMALES A 0 9.7 10.4 9.0 8.9 9.7 9.7 9.6 10.1 10.2 10.8 10.8 B 0.017 10.0 10.9 10.5 9.9 9.3 10.4 11.8 10.5 10.4 11.7 12.1 C 0.17 9.9 12.0 11.0 9.8 10.8 10.4 10.9 11.0 10.4 11.4 11.6 D 1.7 9.7 11.1 10.6 10.1 9.9 10.8 10.9 11.4 10.5 11.3 13.3 E 0 9.5 9.8 10.3 9.2 9.2 8.8 8.7 9.7 9.2 10.0 11.0 SJ-S900 S^b CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIETS TABLE 10 FOOD INTAKE DUR1 G THE FOUR-HOUR FEEDING PERIOD Group A B C D E Intended oral intake levels of VCM (tng/kg b*v./day) MALES 0 0.017 0.17 1.7 0 Food Intake during the feeding-period over periods of one hour*^ expressed as g/rat expressed as I-tage of total food consumed per rat over the 4-hr period 0-1 1-2 2-3 3-4 TOTAL 0-1 1-2 2-3 3-4 11.62) 0.87 9.9 0.66 10.5 0.58 10.7 0.90 8.8 0.66 2.7 0.29 3.2 0.29 3.0 0.27 3.6 0.34 2.6 0.24 2.0 0.27 2.3 0.11 2.5 0.23 2.3 0.25 1.7 0.17 1.9 0.31 1.8 0.17 1.7 0.16 2.0 0.29 1.6 0.15 18.2 0.62 17.2 0.71 17.7 0.60 18.5 0.95 14.6 0.69 64 15 U 10 58 19 13 10 59 17 14 10 58 19 12 11 60 18 11 11 overall A B C D E FEMALES 0 0.017 0.17 1.7 0 6.6 0.32 6.5 0.44 6.5 0.30 7.1 0.46 6.2 0,36 2.1 0.19 2.4 0.23 2.3 0.20 2.3 0.21 2.1 0.13 1.7 0.10 1.6 0.19 1.9 0.18 1.8 0.16 1.5 0.14 1.7 0.26 1.5 0.18 1.6 0.24 1.6 0.17 1.6 0.18 12.1 0.40 12.0 0.53 12.3 0.30 12.8 0.62 11.4 0.31 60 18 12 10 55 17 14 14 54 20 13 13 53 19 15 13 55 18 14 13 54 19 13 14 overall Food Intake figures are the means of 11 different determinations. ^ Standard error of the mean 54 19 14 13 ' 9I-S900 S3H TABLE 11 MEAN HAEMATOLOGICAL FINDINGS RECORDED AT DAY 94.185 AND 367 MALES CONTROL MEAN SEN N 0.017 MG/KG MEAN SEM N 0.17 MG/KG MEAN SEM N 1.7 MG/KG MEAN SEM N CONTROL MEAN SEM N DAY 94 THRDMBOC f 10E9/L> 98?. ??. 10 1036. 33. 10 1008. 23. B 996. 20. 10 866. 50. 9 DAY 185 THRDMBOC (10E9/L) 898. 17. 10 915. A3. 8 860. AS. 9 825. 51 . 10 879. 31. 10 DAY 367 THRO) 1 DOC (10E9/L > 946. 23. 3 855. 43. 5 895. 21 . 4 930. 63. 3 872. 32. 3 DAY 94 F'TT (SEC) 55*0 1.7 10 52.9 1.5 10 53.0 2 2 9 50. B 1.3 10 54.2 2.3 8 DAY 185 F'TT (SEC) 36,8 0.8 10 35.2 0.4 8 37.2 0.6 9 37.0 0.7 8 39.0 0.5 10 DAY 367 FTT (SEC) 49.3 1.4 5 50.1 2.7 5 50 < 5 0.6 4 50.2 1.8 3 52.5 1.2 5 statistics: MANN 'WHITNEY U-TEST 1 F`< 0.05 1HR0MB0C = THROMBOCYTE F'T T = FRO THROHBIN TIME " F' 0.0 2 " F'i-0.002 TWO SIDED (EXF'.UNIT hH IM AL > I ( 1.5900 S*H slum nu TABLE 11 10-nun hi j i oli i JL I I MEAN HAEMATOLOGICAL FINDINGS RECORDED AT DAY 97r184 AND 366 FEMALE S CONTROL MEAN SEM N 0.017 MG/K'G MEAN SEM N 0.17 MG/KG MEAN SEM N 1.7 MG/KG MEAN SEM N CONTROL MEAN SEM N BAY 97 THROMBDC {10E9/L> 927. 19. 10 915. 27. 9 904 . 14 . 10 863. 34 . 10 943. 28. 10 DAY 184 THROMDOC <10E9/L > 972. 27. 10 1032. 34 . 9 871.`* 23. 9 1018. 22 * 9 1010. 29. 10 HAY 366 THROMDOC { 10E9/L) 879. 26. 5 930. 106 . 3 904 . 24 . 4 986 . ' 20. 4 929. 131 . 3 DAY 97 F'TT (SEC) 41,6 0.8 10 40.2 0.8 9 43.1 1.6 10 4 7,4 ' 2.1 8 51.3' ` * 1.9 10 DAY 1 E)4 PIT (SEC > 33.2 0.5 9 32.8 0.4 9 33.0 1.0 8 36.2 1.7 9 37,0'`` 0.7 9 DAY 366 F'TT ( SEC > 40.1 1.2 3 43.6 2.8 4 39.4 1.2 3 38.6 1.7 5 39.9 1.0 4 statistics: MANN/UHITNEY U-TEST * F`<,0.05 THROMBOC = thrombocyte: F'TT = PROTHROMBIN TIME P<0.02 1 ' F 0 002 TUO SIDED ANIMAL ) 81-9900 S$H CIVO/TNO STUDY NO 126 18-MONTH STUDY IN RATS WITH VCM IN THE DIETS TABLE 12 MEAN NON-PROTEIN BOUND SULFHYDRYL ("GLUTATHIONE'') CONTENT OF LIVERS OF RATS FED VCM FOR 9 AND 18 HONTHS Group A B C D E Intended exposure levels of VCH (mR/kg/day) 0 0.017 0.17 1.7 0 Non-protein bound liver SH in umol glutathione/g liver in months 9 18 9 18 MALES 7.58 7.52 0.34 0.38 7.32 7.57 0.38 0.47 7.38 7.33 (4) 0.29 7.30 0.28 7.63 0.22 0.22 7.31 7.44 0.36 0.38 FEMALES 6.05 5.58 0.34 0.63 5.66 (4) 5.69 0.18 0.14 5.95 5.95 0.29 0.31 6.57 6.13 0.36 0.43 6.52 6.30 0.58 0.29 Under each mean the standard error of the mean is given. In bracketB number of rats If not 5. I 6 (-9900 ssa p8tS 006520 JIVO/TNO 1TUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VQi IN THE DIET ABLE 13. MACROSCOPIC PATHOLOGY Site and type of observations initial number of animals Effective number of animals I0DE OF DEATH: a. found dead b. killed in extremis c. terminal sacrifice AUTOLYSIS/CANNIBALISM: a. slightly autolytic b. partially lost c. completely lost SKIN / SUBCUTIS 1. tumour or suspected of tumour 2. alopecia 3. edema 4. haemorrhage 5. injury AXILLARY LYMPH NODES ). tumour or suspected of tumour 2. enlarged 3. reddish PREPUTIAL -/CLITORAL GLANDS I. tumour or suspected of tumour 2. evidence of inflammation 3. enlarged A. small 3. cyst MAMMARY GLANDS 1. tumour or suspected of tumour 2. evidence of secretory activity 3 haemorrhage ABDOMINAL CAVITY 1 . tumour or suspected of tumour 2. ascites Incidence of observations MALES mg VCM/kg hI*v. /day 0 . O'! 7 6 .17 1.2 0 FEMALES mg VCM/k.g b.w. /day 0 .017 0.17 1 .7 0 100 100 100 50 100 100 100 100 50 100 99 100 99 49 100 98 100 96 49 100 26 26 25 16 10 54 54 57 27 90 20 20 18 7 0 8 15 9 5 8 71 62 67 41 92 21 23 24 4 0 10 15 13 8 9 11 4 4 9 1 101 I0 364 26 064 25 204 1 0 57 933 53323 4 1 7 2 3 10 9 9 4 8 232 1 0 1 1 1 00 00 1 00 00000 0020 1 0000 1 32000 2 2000 1 0000 000 1 0 0 1 20 1 00000 352 1 2 7 6 11 2 2 01 1 00 00 200 00000 3 1 020 55 1 35 00 ] 10 00000 0 1 000 8 14 7 3 3 39 25 28 18 23 12 6 8 2 6 53 35 41 21 29 00 1 00 0 0000 12 1 10 3 53 20 68 5 2 6 5554 6 cl'-'u/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIET TABU'. 13. MACROSCOPIC PATHOLOGY (CONTINUED I) R&S 006521 SiU' and type ul observations SPLEEN 1. tumour or suspected of tumour 2. splenomegaly 3. atrophy A. granular surface 5. small 6. small nodule 7. dark ADRENALS 1. tumour or suspected of tumour 2. small (unilateral) 3. haemorrhagic A. discoloured a. pale b. dark 5. spotted 6. enlarged a, unilateral b. bilateral KIDNEYS 1. tumour or suspected of tumour 2. granular surface 3. unilateral hydronephrosis A. discoloured a. pale b. dark c. greenish d. reddish 5. cyst(s) 6. enlarged 7. small 8. spotted STOMACH 1. enlarged/swollen 2. haemorrhage/erosion 3. rough inner surface A. thickened cardial/fundic wall 5. thin fundic wall 6. trichobezoar Incidence of observations __________ MAJ.F.S____________ EEHALES__________ mg VCM/kg h.w./d.iy mg VCM/kg h.w./dav 0 .017 0.17 I.7 0 0 .017 o,17 I.7 0 0 I0 I 0 8 1A 7 3 2 0 00 0 1 2 0 110 100 11 0 00 0 1 0 00 0 0 0 10 0 3 A2 A 32 0 0 0 10 0 0 I IC 0 0 2 00 00 0 00 0 10 0 0 7 52 0 1 10 10 1 0 00 0 0 1 0 I I 13 3 6 10 2 9 8 7 12 6 28 21 3 22 21 55 2 01 110 0 0 1110 0 2 0 12 1 A2 7 A7 26 25 27 1A 23 21 9 6 A7 1I 5 IA 0 23 II 10 9 1 10 5 27 10 2 112 0 00 5 AA 3 2A 0 00 10 0 00 I8 I0 23 01 00 01 2A 02 00 01 02 00 52 20 32 30 3 110 0 10 0 3 3 13 00 00 IA 30 112 0 0 10 0 00 00 0 0 0 0 1 0 0 1 0 0 1 AI 16 10 01 00 6 36 5 A1 000 0 00 10 0 IA 06 00 10 00 32 12 00 00 00 0 2 0 0 0 CJVO/TNO STUDY NO 125 CAKCI"UGKNICITY STUDY IN RATS WITH VCM IN THE DIET 1 A1:. ! II. MAi'KObCOI'l' PATHOLOGY (CONTINUED ?.) Site and type of observations SMALL INTESTINES 1. tumour or suspected of tumour 2. prominent Peyers's patches 3. enlarged/swollen A. haemorrhage 5. reddish 6. filled with watery fluid COECUM 1. tumour or suspected of tumour 2. enlarged/swollen 3. small A. filled with watery fluid COLON 1. tumour or suspected of tumour 2. enlarged/swollen 3. filled with watery fluid A. accreted to the cervix MESENTERY 1. tumour or suspected of tumour 2. edema 3. dilated blood-vessel A. thrombus in blood-vessel 5. haemorrhage PANCREAS 1. tumour or suspected of tumour 2. discoloured a. pale b. dark 3. edema A, spotted c. reddish 5. small haemorrhages MESENTERIC LYMPH NODES 1. tumour or suspected of tumour 2. enlarged 3. small A, edema 5. haemorrhagic Incidence of observations MALES mg VCM/kg b-w./day 0 .017 0.17 1.7 0 FEMALES mg VCM/kg b.w./dav 0 .0)7 0.17 1.7 0 02 000 0 00 0 0 3A 000 I 00 I 2 33 3 1 0 3 I 000 1 22 1 00 1 0 00 I 0 I 00 0 0000 00 1 0 1 0 00 00 00 1 00 0 85 753 3 0 1 000 0 0000 1 0 0000 30 00 0000 00 00 1 0000 I 0000 022 20 I 0000 0000 1 0 0000 00000 0 0 I 00 0 I 2002 00A 00 I AA3 I 05 00 1 00 0 00000 0 2A 03 0000 AI 13 0 0 00 0 I 00 2I I 00 AA2A5 2002 1 000 I 2 A030 1 I 2202 1 0 1 00 0 00 1 0 0I t 00 0 0000 30 000 0 0 0 00 00000 I 00 00 A 2 200 33 ] ] 1 01 10I 00 2 03 1 0000 A A 2 32 6 1 3 00 2- I 2 I 1 2 t I 03 0 000 I R&S 006522 \ \, ;no "0 125 CARC 1 NOGC NICITY STUD\' IN RATS WITH VCM IN THE DIET 13. MAUUISCOPU: PATHOLOGY (CONTINUED 3) .c and type of observations Iscoloured a* pale b. dark c. greenish d, reddish - Y BLADDER ^ur or suspected of tumour ' ..torrhage rged/swollen .tulus/concrements L VESICLES t- "Mr or suspected of tumour ;nce of inflammation rged phy TION GLANDS jt or suspected of tumour once of inflammation --ged - -pby ^ur or suspected of tumour dance of inflammation Larged 'rophy iur or suspected of tumour '-rophy a. unilateral b. bilateral -n'arged (unilateral) ptochismus -LYHIDES ,our or suspected of tumour -dcnce of inflammation ' rophy -limorrhage Incidence of observations MALES mg VCM/kg b-w ./day 0 . 017 0.17 1. 7 0 1 00 1 0 1 0 1 00 00000 00200 FEMALES mg VCM/kg. b . W. /day 0 . 017 0.17 1.7 0 0 000 0 0000 0 00 1 00 002 1 1 00 1 00 1 2000 1 2002 00 1 00 1 1 000 002 00 321 00 0000 0 0 1 000 2 1 1 00 1 0000 4 5 10 3 27 ----- ----- I 1 000 1 0000 00000 3 5 9 3 27 -- -- - -* -- -- -- ----- 0530 1 1 2000 1 1 00J 41216 __ --- -_ -_ ----- 3 04 14 10 18 10 9 6 202 1 0I 00 57 5 22 10 12 __ _ -- -- -- _ --___ _ __ 1 0000 00 1 00 -_ _,, _ 00 R&S 006523 jivo' :;o STUDY NO I2i CARCINOGENICITY STUDY IN RATS KITH VCM IN THE DIET Ai'l 13. MACROSCOPIC PATHOLOGY (CONTINUED 4) Site' and type of observations OVARIES 1. tumour or suspected of tumour 2. cyst(s) 3. enlarged 4. reddish UTERUS 1. tumour or suspected of tumour 2. hydrometra 3. evidence of inflammation 4. pyometra 3. enlarged/swollen LIVER 1. tumour or suspected of tumour 2. prominent lobular pattern 3. suollen/enlarged >, discoloured a. pale 3. cyst(s) b. dark ). sma11 ' small surface lesions (rosette, constriction) 3. spotted L granular surface ,'HORACIC CAVITY . tumour or suspected of tumour . hydrothorax haeroothorax 'HYMUS . tumour or suspected of tumour . enlarged . spotted EART . enlarged (atria) . thrombus . pale Incidence of observations HALES rag VCM/kg b< w. /day 0 . 017 0.17 1.7 0 FEMALES mg VCM/kg b.v. /day 0 . 017 0.17 1 .7 0 _ __ _ --- ---- --- - - - 64 2 8 1 1 15 S24 21 I 35 37 27 1 0 21 29 28 11 33 ---- - 21 1 32 ---- - 1 1 000 ----- 02 000 - - - - - 10 2 2 2 7 1 1 2 7 .1 2 1 380 1 4 2 00 01 3 1 0 245 1 0 3 2000 320 1 0 13121 3300 1 000 1 0 2 1 S 4 0 17 14 22 33 3 0 1 ] 02 00 2 00 1 01 01 5 68 2 3 2 002 0 3000 1 2 1 622 0. 1 2 0 0 00000 35830 48740 00000 262 1 0 S 1 000 1 1 000 0 1 000 1 0000 00020 1 000 1 0 0 0 00 14 19 16 5 3 3 7 650 00 1 00 6750 1 4 5 4 00 000 0 0 '1 I R&S 006524 R&S 006525 LVu/TNO TUNY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIET Ah!. 13. MACROSCOPIC PATHOLOGY (CONTINUED 5) - V) - ice and cype of observations 1EDIASTINAL LYMPH NODES . tumour or suspected of tumour 2". enlarged' JJNGS 1. tumour or suspected of tumour 2. evidence of CRD *) 3. spongy 4. spotted 5. haemorrhage 6. discoloured a. pale b. dark c. reddish 7. atelectasis (partially) 8. pleuritis TRACHEA 1. filled with blood AORTA 1 , dilated 2. calcified DIAPHRAGM 1. tumour or suspected of tumour 2. spotted surface CERVICAL LYMPH NODES l. tumour or suspected of tumour 2 enlarged SUBMAXILLARY SALIVARY GLANDS 1. tumour or suspected of tumour 2a edema 3. discoloured a. dark b. reddish 4. enlargtd/swollen Incidence of observations MALES mg VCM/kg b W. /day 0 .017 0.17 .7 0 FEMALES mg VCM/kg b V. /day 0 . 017 0.17 1 .7 0 31 I '2 010 2 " O' "1 0 0 00 0 0 2 f ' 2 ~o 010 4 64 6 8 11 18 18 26 1 04 1 20 1 00 13 1 1 24 000 01 5 72 1 50 96 01 42 00 00 00 02 000 4 3 10 5 54 20 17 9 00 1 564 000 0 20 1 03 1 00 01 9 67 3 36 64 01 02 00 00 22 01 0 I 0 00 0 0 00 0 599 1 0 1 0000 521 1 2 0 0000 1 0000 0 00 1 0 01 101 0 000 0 1 0000 0 1 1 00 00000 00000 1 1 1 00 12] 1 1 1 0000 00 1 00 I 1 000 0000 1 0001 0 00000 0 1 000 00000 CRD - chronic respiratory disease CIVi.'/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIET TAID u n. n/.n;osmi'ir pathology (continued 6) - 60 - Siu- ;iud i yj>c ol oh.MTvjt ions SUBLINGUAL SALIVARY GLANDS 1. tumour or suspected of tumour 2. enlarged/swollen 3. haemorrhage 4. edema 5. discoloured a. pale b. reddish ' PAROTID SALIVARY GLANDS 1. discoloured, pale 2. spotted 3. enlarged THYROID 1. tumour or suspected of tumour 2. enlarged 3. discoloured a, pale b. dark PARATHYROID c. reddish I. enlarged EYES 1. haemorrhagic scabs around eyes 2. evidence of inflammation 3. protruding 4. pale 5. opaque ERAIN 1. tumour or suspected of tumour 2. haemorrhage 3. a pale spot MENINGES I. haemorrhage Incidence of observations MALES mg VCM/kg b*w./duy 0 .017 0.17 1.7 0 FEMALES mg VCH/kg b.,w./dny 0 .017 0.17 11.7 0 1 00 00 0 10 0 0 10 0 0 0 0 110 0 10 0 0 0 0 00 0 0 0 0 0 00 0 0 0 00 0 0 0 00 0 0 0 00 0 0 0 00 0 10 0 0 3 0 0 10 0 0 0 13 0 10 0 0 00 00 10 0 00 0 00 00 0 2 6 3 10 3 0 110 10 0 0 0 2 13 0 1 00 00 0 120 00 00 0I 00 00 00 00 00 10 1 0 I 0 10 0 0 0 0 0 0 00 22 16 33 86 13 I 02 4 6 23 00 2 62 0 13 7 9 02 I 2 2 10 6 99 12 12 3 16 2 14 II I3 2 21 73 35 00 01 00 00 0 II 0 0 10 10 0 0 0 0 0 0 00 0 0 0 00 00 20 1 0 0 0 00 I R&S 006526 I Vn/TNIJ Jl'IlY NU I cakcinogknicity sruin in rats with vui in thk diet 1 r n. MACuascoi'ic pathology (CONTINUED 7) m- -.ili' .md type of observations PITUITARY 1. tumour or suspected of tumour 2. haemorrhagic (cyst) i. discoloured a. pale b. dark enlarged/svollen 10SE ! , tumour or suspected of tumour l. haemorrhagic scabs around nose JRAL CAVm . malocclusion of incisors 1X0RBITAL LACHRYMAL GLANDS . pale 1. granular appearence '.YMBAL's GLANDS . tumour or suspected of tumour :. enlarged i. evidence of inflammation .. haemorrhage iBDOMINAL/LUMBAL LYMPH NODES . tumour or suspected of tumour enlarged BDOMINAL wall . small cysts 'AIL . ulcer -. abscess .AR DUCT . compact debris . evidence of inflammation .IMBS . tumour or suspected of tumour . abscess . deformed Incidence of observations MALES mg VCM/kg b-w./day 0 .017 0.17 1.7 0 FEMALES mg VCM'/kg b.w./dnv 0.0170.171.7 0 8 9 10 5 3 13 13 17 10 5 ] 2 3 1 1 5 10 II 4 2 3 I 1 I 0 2 4000 I I 00 I 0 00C I 1 020 I I 1 00 1 00000 662 I I 0 03 00 9 32 00 53 2 1 1 2 1 00 1 18 12 10 696 8 13 17 00000 0 0000 0 1 0 1 00 0000 00000 1 0 1 00 0000 1 00000 000 0 00 I 000 1 1 00 00 2 I 00 0 0 0 1 00 00 0 0 00000 1 0000 2I 1 I00 00000 0 0000 0 1 00 0 1 000 0 0000 00 1 00 0 0000 20 0 0 0 0 0 0 0 0 0000 1 0 00 I 0 92 1 0 I 1 I 1 00 .... I i 1 R&S 006527 I I i IV t >/ I ;;i; MII.V Nit Id'- CAW' l:.UOLN I CITY STUitY IN RATS l.'ITII VCN IN THU HlIT 11 :v> :i> iv- riH'i.'uiY (continued 8i .it- .nut I |h` i-l till' rrv.il ions MISCELLANEOUS 1. emaciated 2. deformed spine 3. deformed dorsal vertebrae Incidence of observation:. MALES_____________ ________ ___ l-'IIMAUIS iiift Vt'H/kf. li.w.7*l:iy "IJ/^- A!,1i'*,J " o'.m 7~~6~.VrT.~7~5'.....(i -.mf q.37Y.o' J 15 19 17 1 30 00 I 7 48 00 00 32 22 26 14 55 0 I 000 0 0000 R&S 006528 CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIETS TABLE 14 TYPE AND INCIDENCE OF HISTOPATHOLOGICAL CHANCES IN THE LIVER Type of changes^ Number of animals examined Incidence of changes Hales Females mg VCH/kg b.u ./day mg VCH/kg b.u. /day 0 0.017 0.17 1.7 0 0 0.017 0.17 1.7 99 99 99 49 100 98 100 96 49 0 99 1. Foci of cellular alteration a. clear cell foci I one or a feu 12 II several 0 b. basophilic foci I one or a feu 4 II several 0 c. mixed cell foci I one or a feu 0 d. eosinophilic foci I one or a feu 1 All type of foci: Total 17 Number of foci** bearing animals 16 8 0 2 0 1 1 12 12 8 16** 0 3* 54 00 3 8* 00 09 00 22 06 21 00 15 30 5 19 15 23*** 5** 19 5 3 13*** 1 00 0 20* 26*** 102 4 0 6 4 8* 0 0 1 10*** 2 32 34 58 7 27 31* 32*** 7** 623900 S9d CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIETS TABLE 14 TYPE AND INCIDENCE OF HISTOPATHOLOGICAL CHANGES IN THE LIVER - CONTINUED 1 Type of changes 2. Neoplastic nodules a. one b. a few 3. Hepatocellular carcinoma 4. Angiosarcoma 5. Liver-cell polymorphism a. slight b. moderate c. severe 6. Prominent sinusoidal cells a. slight b* moderate/marked 7. Cysts a. one b. a few c. many 8. Bile duct proliferation a. slight b. moderate c. severe 0 0 0 0 0 27 4 1 1 1 1 4 0 37 9 2 Incidence of changes Kales Females rag VCM/kg b.w., /day mg VCM/kg b.w.. /day 0.017 0.17 1.7 0 0 0.017 0.17 1.7 J 00 00 00 00 10 20 3* 0 10 0 0 1 0 1 0 0 0 1 9*** 0i 13 02 0 0 0 0 0 23 26 19 12** 46 41 4 7 10** 1 14 13 1 1 3 02 3 49 23 38 8 15* 15 43 1 1 01 4 9 2 1 103 8 3 i6 5 20 01 00 3 2 4 3 4 1 11 11 0 0 00 3 4 4 11 12 7 2* * 9 24*** i 26 35 14 21** 27 23 9 8 3 J** 11 6 0 0 01 1 2 36 18 14* 10 2 3* 1 14 0SS900 ssu CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIETS TABLE 14 TYPE AND INCIDENCE OF HISTOPATHOLOCICAL CHANGES IN THE LIVER - CONTINUED 2 Type of changes 0 9. Cholangiofibrosls a. slight b. moderate 33 2 10. Periportal fibrosis a. slight 1 b. moderate 11. Perlportal/centrolobular 2 infiltrates of mononuclear cells a. slight 14 b* moderate c. severe 1 0 12. Foci of RES-cells occasionally accompanied by a few necrotic hepatocytes a. one or a few 18 b. several 5 13. Slight haematopoietic activity 2 Incidence of changes Males Females mg VCH/kg b.w,. /day rag VCH/kg b.w,./day 0.017 0.17 1.7 0 0 0,.017 0.17 1.7 19* 20* 15 23 16 6 8* 2 1 6 13 17 15 2 40 22 00 350 02 0 0 00 0 00 10 8 3 7 3 2 1 2 1 1 00 0 0 2 0 1 1 000 0 0 0 15 27 83 00 5 12 39 01 6 01 1 28 42 12* 3 50 0 30 0 2**: 0* 1 0 4 0 0 2 5* 3 0 LSS900 S'Sd CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCM IN THE DIETS TABLE 14 TYPE AND INCIDENCE OF HIST0PATH0L0C1CAL CHANCES IN THE LIVER - CONTINUED 3 Type of changes 14, Single cell necrosis a. slight b. moderate/severe 15, Focal necrosis a. alight b. moderate c* severe 16, Centrolobolar liver-cell degenera tIon 17. One or a few foci of degenerated hepatocytes 18. Haemorrhagic areas 19, Peliosls-1ike changes 20. Small granuloma 21. Vacuolization of hepatocytes mainly a. periportal 1 slight II moderate III severe Incidence of changes Males Females rag VCH/kg b.w./d ay mg VCM/kg b.w,./day 0 0.017 0.17 1.7 0 0 0,.017 0.17 1.7 23 1 210 04 2 001 3 13** 5* 3 42 11 3* 8 2 4* 4 4 6 6 1 2 1 003 1 2 0 1 0 0 203 0 1 0 1 3 3 103 2 2 0 1' 0 10 01 00 7* 3 0 1 1 000 0 201 0 002 2 00 0 10 1 00 0 00 3 6 n* 3 4 24 22 29 4* 0 2 5* 0 0 3 2 6 0 0 1 1 00 0 0 00 0 9* 2 3 3 1 0 0 0 O 0 6**' i 0 ZSS900 SSH CIVO/TNO STUDY NO 125 CARCINOGENICITY STUDY IN RATS WITH VCH IN THE DIETS TABLE 14 TYPE AND INCIDENCE OF HISTOPATH0LOGICAL CHANCES IN THE LIVER - CONTINUED 4 Type of changes b. diffuse 1 slight II moderate III severe c. centrolobular I slight Incidence of changes Males Females mg VCH/kg b.w,. /day mg VCM/kg b.w.. /day 0 0.017 0.17 1.7 0 0 0.017 0.17 1.7 I 0 1 00 0 0 00 1 0 2 00 2 0 00 0 2 0 00 1 0 00 0 1 0 00 0 0 00 0 0 0 2 0 Specific hepatocellular lesions were classified according to Squire and Levitt (1975). * P C 0.05; ** P K 0.01; *** P <0.001, according to the Fishers' exact test (one tailed). ees900 ssu CIVO/TNO STUDY NO 125 CARCINOCEN1CITY STUDY IN RATS WITH VCM IN THE DIET TABLE 15 TYPE AND INCIDENCE OF TUMOURS OF THE MAMMARY GLANDS AND INCIDENCES OF ABDOMINAL MESOTHELIOMAS Site and type of tumours Incidence of tumours Males Females mg VCM/kg b.w,./day mg VCM/kg b.w./day 0 0.017 0.17 1*7 0 0 0.017 0.17 1.7 0 Number of animals examined 99 99 99 49 100 98 100 96 48 98 Mammary glands - No of tumour-bearing animals 1* Adenoma a. single b. two 2. Intraductal papilloma 3. Fibroadenoma a. single b. two c. multiple 58 00 00 00 01 00 00 3 0 3 41 21*** 28* 21 20*' 0 0 06 4 2 52 0 0 01 1 1 00 0 0 00 0 0 10 00 0 25 10** 15 14 11* 0 0 02 3 2 24 0 0 03 1 1 11 I ^9900 ssa CIVO/TNO STUDY NO 125 CARCINOCENICITY STUDY IN RATS WITH VCM IN THE DIET TABLE 15 TYPE AND INCIDENCE OF TUMOURS OF THE MAMMARY CLANDS AND INCIDENCES OF ABDOMINAL MESOTHELIOMAS CONTINUED Site and type of tumours 4. Fibroma a. single b. two c. multiple 5. Adenocarcinoma a. single b. two Incidence of tumours Males Females mg VCM/kg b.w. /day mg VCM/kg b.w./day 0 0.017 0.17 1.7 0 0 0..017 0.17 1.7 0 46 00 00 2 0 31 4 1 22 1 0 00 0 0 00 0 0 01 0 0 00 01 10 0 0 03 0 5 10 0 0 00 0 2 01 Abdomen 1. Mesothelioma 01 2 0 03 2 2 11 * P < 0.05; ** P < 0.01; *** P < 0.001, according to the Fishers' exact test (one tailed) SSS900 SSH 70 i,~/ --1^. * N V \ \. non i. m* *o** Hian mU i*T1 (hU Mil R&S 006536 Mtf H Hr It* It) 7*4 H) It] 4*1 4t* tit M* *17 CIVt/fM <Tw*Y UH(I * in tMCICI Ml * HCIK HW ICMH *9Uf l(l(u: C*t0L *'*.*17 M/Cl C# 1.17 H/l| ** 1,7 */< ( (MHL 711 71* III FIGURE 1 94*, 949. 37314*.. 91*. 99*. 939. 9*1)1t,, 911. 9*7. 9*3. I**. IT1. 1*1. 1Il*l7,. I1I>**.. 172. 141, 1*4. 14*. 13*. 113, 140. 113317,. 13*. 1*1. 71. riitm ** M 147 |f 343 ;t4 HI 3*3 441 4*0 334 1*0 *J7 lit 711 7*4 *11 CIV0/1M MatlHAl MU t" THf fVU04 Uvn MMMI 133 C0lClKt*l M*Wf iCoflf If(C 1(1 0*4 * L(l(4l *' C*W1M *'*.*17 M/( (* * * 17 Mfl| I' |.7 M/K* t* (MTI^ FIGURE 2 ANNEX I PERCENTAGE COMPOSITION OF BASAL DIET FOR RATS AND MICE soya bean oil meal fish meal meat and bone scraps wheat (whole ground) maize (whole ground) brewer's yeast grass meal whey powder defatted bone meal salt with trace elements*) 2 B-vitamin mixture ) vitamin ADEK mixture*) soya bean oil 11 7 A 36 29.7 3 3 2 0.4 0.5 0.1 0.3 3 100.0 ) Trace elements in salt MnSO^-HjO ZnCl, KJ FeSO^.7H20 CoC12.6H20 CuS04.5H20 NaCl B^vitamin mixture Thiamin-HCl Riboflavin Pyridoxin-HCl Niacin Ca '`'pantothenate Biotin Folic acid Vitamin B32"Prep. Ground sucrose (0.1 Z) Vitamin ADEK mixture Vit. AD3-prills (Farmix): 2250 IU vit. A, 750 IU vit. D3/g Vit. E-dry powder (Merck) 501 Menadion-Na-bisulphite (Vit. k3) Wheat starch Batch 2380 250 20 1250 20 400 45680 8 8 g g g g g 50000 g Batch 25 g 30 g 100 g 125 g 75 8 1.5 g 5 50 9588.5 g 10000 g Batch 25000 800 27 800 26627 g g g S added to 1 kg diet 41 mg Mn 12 mg Zn 1. 5 mg J 25 mg Fe 0. 7 mg Co 8 mg Cu added to 1 kg diet 2.5 mg 3.0 mg 10.0 mg 12.5 mg 7.5 mg 0.15 mg 0.5 mg 5.0 mg (- 5 added to 1 kg diet 6339 IU vit. A 2112 IU vit. D3 45 mg vit. E 3 mg vit, K3 71 R&S 006537 ANNEX 2 CONTAMINANTS REGULARLY DETERMINED IN BASAL DIET 1. Contaminants in basal diet contaminant - inorganic substances_|mg/)cg^ arsenic cadmium lead mercury selenium tin potassium nitrate sodium nitrite - organochlorine_comgounds (mg/kg) HCB lindane (y-HCH) tj-HCH B-HCH heptachlor heptachlorepoxide a-chlordane y-chlordane aldrin dieldrin endrin methoxychlor p,p'-DDE p,p'-TDE o rp*-DDT p, p* '-DDT PCBs organophosphorus compounds (mg/kg) chlorpyrifos diazinon dichlorvos fenitrothion malathion mevinfos parathion sulfotep dithiocarbamates, in total, determined as carbondisulfide (mg/kg) ferbam nancozeb maneb nabam thiram zineb ziram detection limit 0.01 0.005 0.05 0.005 0.01 1 25 5 0.005 0.01 0.01 0.02 0.01 0.02 0.02 0.02 0.01 0.01 0.02 0.05 0.02 0.03 0.03 0.04 0.3 0.01 0,01 0.01 0.01 0.01 0.01 0.01 0.01 0.5 72 tentativimaximum 0.25 0.05 1.0 0.05 0.5 25 500 10 0.02 0.1 0. 1 0.02 0.02 0.02 0.02 0.05 0.01 0.01 0.02 0.5 0.02 0.03 0.05 0.05 0.5 0.05 0.05 0.05 0.05 0. 5 0.05 0.05 0.05 1.0 R&S 006538 ANNEX 2 (CONI.) Contaminants in basal diet (cont.) contaminant - polycyclic aromatic hydrocarbons (ug/kg) benzo<b)fluoranthene benzo(k)fluoranthene benzo(a)pyrene fluoranthene indeno(1,2,3-cd)pyrene - nitroso compounds (lig/kg) nitrosodiethylamine nitrosodimethylamine nitrosodipropylamine nitrosomethylbutylamine nitrosomethylpropylamine nitrosomorpholine nitrosopiperidine nitrosopyrrolidine - mycotoxins (mg/kg) aflatoxin Bj - miscellaneous determinations oestrogenic activity (Tiecco test) urease activity (ApH) trypsin inhibitors (mg trypsin/g food) haeraagglutinins negative reaction at dilution detection limit 0.2 0.4 0.2 1.0 0.4 0.5-1.5 0.3-0.9 1.5-4.5 1.5-4.S 1.0-3.0 3.0-9 l.S-4.5 1.5-4.5 0.01 tentative maximum 10 10 10 50 10 10 10 10 10 10 10 10 10 0.05 negative 0.3 8.0 1:200 R&S 006539 , ' 1t ANNEX 3 - 74 - NUTRIENT COhPOSITION CF BASAL DIET FOR RATS Analyses of a 1000 kg batch produced January 24, 1980 moisture 12.6 % crude protein (N x 6.25) 20.9 % crude fat 8.9 % crude fibre 3.6 % total ash 5.6 Si calcium 0.89 % phosphorus 0.72 % magnesium 0.14 X potassium 0.75 % sodium 0.28 S Iron manganese copper zinc cobalt chromiun selenium 175 ppm 95 ppm 15 ppm 50 ppm 2 ppm 3 ppm 0.24 ppm vitamin A vitamin D vitamin E carotene vitamin K3 5200 I.U./kg vitamin B^ 1380 I.U./kg vitamin Bj 44 mg/kg vitamin 2.0 mg/kg niacin 1.6 mg/kg pantothenic acid folic acid choline biotin vitamin B^ 4.7 5.8 12.0 66 mg/kg mg/kg mg/kg mg/kg 15.5 mg/kg 1.25 mg/kg 1600 mg/kg 0.22 mg/kg 8 vg/kg isoleucine leucine lysine methionine cystine phenylalanine tyrosine threonine tryptophan g/100 g g/16 g N 0.85 4.1 1.60 7.7 1.03 5.0 0.39 1.89 0.36 1.75 0.89 4.3 0.64 3.1 0.81 3.9 0.21 1.01 valine arginine histidine alanine aspartic acid glutamic acid glycine proline serine g/100 g g/16 g N 1.00 4.8 1.16 5.6 0.45 2.2 1.09 5.3 1.74 8.4 3.69 17.9 1.11 5.4 1.40 6.8 1.10 5.3 J3 fi C/> 0 OS 01 0 ANNEX 4 NUTRIENT COMPOSITION OF BASAL DIET FOR RATS 75 Analyses of a 1000 kg batch produced September 9, 1980. moisture 11.9 Z crude protein (N x 6.25) 22.2 Z crude fat 6.4 Z crude fibre 3.1 Z total ash 5.4 Z calcium 0.90 Z phosphorus 0.78 Z magnesium 0.15 Z potassium 0.82 Z sodium 0.29 Z iron manganese copper tine cobalt chromium selenium iodine 135 ppm 95 ppm 16 ppm 55 ppm 2 ppm 2 ppm 0.40 ppm 2.0 ppm vitamin A vitamin D vitamin E carotene vitamin isoleucine leucine lysine methionine cystine phenylalanine tyrosine threonine tryptophan 7000 I.U./kg I.U./kg 55 mg/kg mg/kg 1.8 mg/kg vitamin B1 vitamin Bj vitamin B, 6 niacin pantothenic ac id folic acid choline biotin vitamin 6.2 mg/kg 5.4 mg/kg 11.8 mg/kg 74 mg/kg 11.5 mg/kg 2.4 mg/kg 1600 mg/kg 0.20 mg/kg 16.5 yu,g/kg g/100 g g/16 g N 0.9B 4.4 1.78 8.0 1. 13 5.1 0.41 1.8 0.38 1.7 0.98 4.4 0.69 3.1 0.85 3.9 0.23 1.0 valine arginine histidine alanine aspartic acid glutamic acid glycine pro line serine g/100 g 1.14 1.21 0.44 1.19 1.93 4.20 1.12 1.42 1.17 g/16 g N 5.2 5.4 2.0 5.4 8.7 18.9 5.1 6.4 5.3 R&S 006541 ANNEX 5 Nt/TRIENT COMPOSITION OF BASAL DIET FOR RATS Analyses of a 1000 batch produced February 27, 1981 moisture crude protein (N x 6.25) crude fat crudefiber total ash calcium phosphorus magnesium potassium sodium 12.6 X 20.6 t 6.1 Z 3.8 X 5.6 X 1.05 X 0.74 X 0.13 X 0.75 X 0.32 X iron manganese copper zinc cobalt chromium selenium iodine 150 90 17 45 0.6 3 0.4 1.5 ppm PPm ppm ppm ppm ppm ppm ppm vitamin A vitamin D vitamin E carotene vitamin 1C^ isoleucine leucine lysine methionine cystine phenylalanine tyrosine threonine tryptophan 6600 _1) 57 1.6 I.U./kg I.U./kg mg/kg mg/kg mg/kg vitamin Bj vitamin vitamin niacin pantothenic acid folic acid choline biotin vitamin B^ 7.5 5.7 12.1 55 14.7 5.1 1500 0.25 15.5 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg Ug/kg g/100 g g/16 !B N 0.81 4.0 1.52 7.5 1.03 0.38 5.1 1.9 0.33 1.6 0.88 4.3 0.63 3.1 0.78 3.8 0.19 0.9 valine arginine histidine alanine aspartic acid glutamic acid glycine proline serine g/100 g g/16 g 0.95 4.7 1.13 5.6 0.46 1.08 2.3 5.3 1.74 3.70 8.6 18.2 1.13 5.6 1.46 7.2 1.06 5-2 ^not analysed CIV0-TN0, June 1981 R&S 006542 R8tS 006543 ANNEX b NUTRIENT COMPOSITION OF CIVO-BASAL DIET FOR RATS Analyses of a 4000 kg batch produced by Hope Farms on Sept. 9, 1981 moisture crude protein (N x 6.25) crude fat crude fiber total ash calcium phosphorus magnesium potassium sodium 11.3 Z 20.2 Z 6.9 z 3.9 z 5.3 z 0.65 z 0.7 z 0.21 z 0.8 z 0.32 z iron manganese copper rinc cobalt chromium selenium iodine 220 105 18 55 1 1 0.25 3.0 ppm ppm ppm ppm ppm ppm ppm ppm vitsmin A vitamin D vitamin E carotene vitamin isoleucine leucine lysine methionine cystine phenylalanine tyrosine threonine tryptophan 5700 _1) 56 13 <3 I.U./kg I.U./kg mg/kg mg/kg mg/kg vitamin vitamin vitamin Bg niacin pantothenic acid folic acid choline biotin vitamin 9.0 7 12 70 18.6 3.3 1700 0.24 13 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg g/kg g/100 g g/16 g N 0.81 4.0 1.60 1.07 7.9 5.3 0.40 2.0 0,34 1.69 0.89 4.4 0.65 3.2 0.78 3.9 0.23 1.14 valine arginine histidine alanine aspartic acid glutamic acid glycine proline serine g/100 g g/16 g 1.05 5.2 1.13 5.6 0.49 2.4 1.14 5.6 1.73 8.6 3.51 17.4 1.07 5.3 1.27 1.01 6.3 5.0 ^not analysed CIVO-TNO, June 1981 R&S 006544 ANNEX 7 NUTRIENT COMPOSITION OF CIV0-8ASAL DIET FOR RATS Analyses of a 4000 kg batch produced by van Eck, Cothen,, on Febr. 9, 1982 78 moisture crude protein (N x 6.25) crude fat crude fiber total ash calcium phosphorus magnesium potassium sodium 11.3 % 19.8 Z 6.2 X 4.1 I 5.5 z 0.95 z 0.66 z 0.16 z 0.73 z 0.32 z iron manganese copper sine cobalt chromium selenium iodine 315 95 19 65 0.5 1.0 0.3 0.9 ppm ppm ppm ppm ppm ppm ppm ppm vitamin A vitamin D vitamin E Carotene vitamin 8000 _1) 56 12.5 <2 I.u./kg I.U./kg mg/kg mg/kg mg/kg vitamin B^ vitamin B2 vitamin B niacin pantothenic acid folic acid choline biotin vitamin BJ2 7 5 12 55 15 4 1600 0,21 26.8 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg rag/kg mg/kg Mg/kg isoleucine leucine lysine me thionine cystine phenylalanine tyrosine threonine tryptophan g/100 8 g/16 ;8 N 0.79 4.0 1.52 7.7 0.99 5.0 0.40 0.32 2.0 1.62 - 0.84 4.3 0.51 2.6 0.77 3.9 0.22 1.11 valine arginine histidine alanine aspartic acid glutamic acid glycine proline serine g/100 g g/16 g N 1.00 1.08 5.1 5.5 0.43 2.2 1.02 5.2 1.65 8.4 3.48 17.6 1.06 1.22 5.4 6.2 0.94 4.8 not analysed CIVO-TNO, June 1982 I ANNEX 8 79 - R&S 006545 CONTAMINANTS in basal diet for rats and mice Sample of a 1000 kg batch produced on January 24, 1980 lead cadmium mercury tin arsenic selenium 0.95 0.06 0.02 2 0.4 0.24 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg organo-P-compounds organo-Cl-compounds ND NO dithiocarbamates (as CS^) 0.5 mg/kg aflatoxin B^'B2'Gi'G2 oestrogenic activity(Tleceo test) 4 5 pgAg ND benzo( a) pyrene benzo(b)fluoranthene indeno (l,2r3-c,d) pyrene benzo(k)fluoranthene fluoranthene K-nitrate Na~nitrite 2.5 4.4 2.8 2.6 12.0 pgAg ug/kg ug/kg pg/kg pg/kg 163 mg/kg 1 mg/kg nltrosodimethylamine nitrosopyrrolldine other nltrosamines urease activity trypsin inhibitor haemagglutlnins positive dilution < 0.4 <2 ND 0.15 < 0.6 none ug/kg pg/kg A pH mg tryps/g sample ND not detectable CIVO-TNO-May, 1980 ANNEX 9 CONTAMINANTS IN BASAL DIET FOR RATS (1000 kg batch produced 27 February 1981) lead cadmium mercury tin arsenic selenium organo-P-compounds organo-Cl-compounds PCB'a dithiocarbamates 0.3 0.07 0.25 < 1.0 0.35 0.4 mg/kg mg/kg mg/kg mg/kg rag/kg mg/kg ND NO ND ND - 80 - aflatoxin 81.B2,G1,G2 oestrogenic activity (Tiecco test) bemo(a)pyrene benzoCb)fluoranthene indeno (l,2,3-e,d) pyrene benzoC k)fluoranthene fluoranthene K-nitrate Na-nitrite nitrosodimethylamine nitrosopyrrolidine other nitrosamines urease activity trypsin inhibitor haemagglucinins positive dilution negative dilution <5 bg/kg ND 0.8 0.9 0.6 0.3 5.7 bg/kg bg/kg bg/kg bg/kg bg/kg 77 mg/kg 1 rag/kg 1.2 bS/^ 1.7 bg/kg ND 0.05 A pH < 0.6 mg tryps/g sample 1 : 10 1 : 20 R&S 006546 ND " not detectable CIVO-TNO, June 1981 I'A i U CONTAMINANTS IN BASAL DIETS FOR RATS C1000 kg batch, produced 9 September 1980) lead cadmium mercury tin arsenic selenium organo-P-compounds organo-Cl-compound dithiocarbamates 0.25 mg/kg 0.045 mg/kg 0.025 mg/kg < 1.0 mg/kg 0.45 mg/kg 0.40 mg/kg ND ND ND af la toxin and G2 <5 pg/kg oestrogenic activity (Tiecco test) ND benzo(a)pyrene benzo(b)fluoranthene indeno (1,2,3-C.d) pyrene benzo(k)fluoranthene fluoranthene 2.0 2.7 2.0 1.0 16.0 pg/kg Pg/kg pg/kg pg/kg pg/kg K-nitrate Na-nitrite 80 mg/kg 1 mg/kg nit ro sodimethylamine nitrosopyrrolidine other nitrosamines 0.5 1.5 ND pg/kg pg/kg urease activity trypsin inhibitor haemagglutinins positive dilution negative dilution 0.05 <0.6 none A pH mg tryps/g sample ND not detectabli CIVO-TNO december 1980 / R&S 006547 ANNEX II CONTAMINANTS IN CIVQ BASAL DIET FOR RATS (4000 kg batch, produced by Hope Farms on September 9, 1981). . s; - lead cadmium mercury tin arsenic selenium 0.9 mg/kg 0.07 mg/kg 0.005 mg/kg < 1.0 mg/kg 0.45 mg/kg 0.25 mg/kg organo-P-eompounds malathion organo-Cl-compounds PCB's dithiocarbamates (as CSj) ND 0.08 ND ND < 0.5 mg/kg mg/kg aflatoxin <5 ug/kg oestrogenic activity (Tiecco test) ND benzo(a)pyrene benzo(b)fluoranthene indeno (l,2,3-c,d) pyrene benzo(k)fluoranthene fluoranthene benz(ghi) perylene 0.4 0.7 < 0.5 0.1 0.9 0.3 ug/kg Ug/kg ug/kg ug/kg ug/kg ug/kg K-nitrate Na-nitrite 83 *1 mg/kg mg/kg nitrosodimethy 1 amine nitrosopyrrolidine other nitrosamines urease activity trypsin inhibitor haemagglutinins positive dilu ion negative dilu ion 0.9 1.0 ND ug/kg ug/kg 0.05 A pH 0.51 mg trypa/g sample 1 : 50 t 100 ND " not detectable CIV0-TN0. November 1981 33 fio (/) o o cn CJI 03 ANNEX 12 CONTAMINANTS IN Cl VO BASAL DIET FOR RATS (4000 kg batch, produced by van Eck, Cothen, on Febr. 9, 1982) lead cadmium mercury tin arsenic selenium 1.4 0.09 0.005 < 1.0 0.3 0.3 ng/kg mg/kg mg/kg mg/kg mg/kg mg/kg organo-P-eompounds organo**Cl--compounds PCB's dithiocarbamates ND ND ND < 0.5 mg/kg aflatoxin B1.B2'G1'G2 <5 oestrogenic activity (Tiecco teat) ND benzo( a) pyrene 0.5 benzol b) fluoranthene 0.1 indeno (l,2,3~c,d) pyrene < 0.1 benzo(k)fluoranthene < 0.2 fluoranthene 0.2 benz(ghi) perylene < 0.3 K-nitrate Na-nitrite 34 ND dg/kg dg/kg dg/kg dg/kg dg/kg dg/kg dg/kg mg/kg nitrosodimethy1amine nitrosopyrrolidine other nitrosamines ND 0.7 dg/kg ND urease activity trypsin inhibitor haemagglutinina 0.00 A pH 1.4 mg trypa/g sample positive dilution none negative dilution ND * not detectable CIVO-TNO,June 1982 83 - R&S 006549 ANNEX 13 Contaminants in drinking water contaminant - inorganic substances^ug/U arsenic cadmium chromium copper cyanide lead mercury selenium zinc potassium nitrate sodium nitrite ammonia - organochlorlne compounds (ug/1) 1,1-dichloroethane dichloromethane tetrachloroethene tetrachloromethane 1,1,1-trichloroethane trichloroethene trichloromethane HCB lindane (y-HCH) a-HCH 6-HCH heptachlor heptachlorepoxide a-chlordane y-chlordane aldrin dieldrin endrin methoxychlor p,P1-DDE p,p`-TDE o,p'-DDT p, p'-DDT PCBs - organophosphorus compounds (yg/1) chlorpyrifos diazinon dichlorvos fenitrothlon malathion mevinfos parathion sulfotep detection limit 10 1 10 1 100 1 1 1 1 1,000 1,000 soo 84 tentative maximum 50 5 50 SO 100 SO 1 10 100 50,000 1,000 1,000 1 5 1 0.1 0.1 0.5 0.3 0.01 0.01 0.01 0.03 0.01 0.02 0.03 0.03 0.02 0.03 0.05 0.15 0.03 0.06 0.06 0.1 0.B 0.1 0.1 0.1 0.2 0.2 0.1 0.2 0.1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 R&S 006550 ANNEX 13 (CONT.) Contaminants i,n drinking water (cont.) contaminant - polycyclic aroraatlc_hydrocarbons (ng/1) benzo(b)fluoranthene benzo(k)fluoranthene benzo(ghi)perylene benzo(a)pyrene fluoranthene indeno(1,2,3-cd)pyrene detection limit ~ Pb - tentative maximum 5 200 5 200 5 200 5 200 10 200 5 200 3 99 (/) o o O) CtnJ1 1 Nov, 1979 ANNEX 14 86 CONTAMINANTS IN TAP WATER (determined in period Nov. -Dec. 1979 lead cadmium mercury arsenic selenium copper chromium sine cyanide PPb <1 <1 <5 2 <1 50 <1 - 5 3 ammonia nitrate nitrite chloride sodium calcium potassium iron EE2 < 0.01 9.6 < 0.01 23 14.5 31 1.2 0.04 R&S 006552 EEb dlchlorosethane <5 HOT 1/1-dlchloroethane chloroform absent 0.8 a-HCH lindane * 1,1,1-trlchloroethane 0.2 heptachlor tetrachlorooothane 0.5 heptachl. epoxide trichloroethene absent aldrin tetrachloroetheno absent EE! dieldrin a-chlordano bemo(a)pyrene benzo(b)fluoranthene <5 <S y-chlordone endrln indeno (l,2,3,-c,d) pyrene< 5 p,p'-DDE hento (JO fluoranthene fluoranthene benzo (g,h,i) perylene <5 < 10 <5 o,p'-0DT p, p' -TDE p.p'-DOT 6-hch PCB's DDVP mevlnfos dlazlnon taalathlon parathlon chlorpyrlfos sulfotep fenitrothlon EE! < 0.01 < 0.01 < 0.01 < 0.01 < 0.02 < 0.02 < 0.03 ' < 0.03 < 0.03 < 0.05 < 0.03 < 0.06 < 0.06 < 0.10 < 0.03 <0.8 < 0. 1 < 0.1 < 0.1 < 0.2 <0.2 < 0. 1 < 0. 1 < 0.2 ANNEX 15 87 CONTAMINANTS IN TAP WATER (determined in period Jan.-April 1980) lead cadmium mercury arsenic selenium coppe r chromium zinc cyanide EE* 1 < 0. 1 0.1 7 '1 55 <1 3 <2 ammonia nitrate nitrite chloride sodiurn calcium potassium iron PP 0.01 11 < 0.01 30 18.5 31.0 1.3 0.08 EE* dichloromethane <s 1,1-dichloroe thane chloroform c1 0.3 1.1,1-trichloroethane tetrachlororoethane < 0.1 < 0. 1 trichloroethene tetrachloroethene 0.3 < 0.2 EE! benzolalpyrene benzo(b)fluoranthene <^ <$ indeno (l,2,3,-c,d) pyrene < 5 benzo (k) fluoranthene <S fluoranthene < 10 benzo (g,h,i) perylene < '5 HCB q-HCH lindane heptachlor heptachl. epoxide aldnn dieldrin a-chlordane y-chlordane endrin p,p'-DDE o,p'"DDT p,p`-TDE p,p'-DDT P- T'CH EE* < 0.01 < 0.01 < 0.01 < 0.01 < 0.02 < 0.02 < 0.03 < 0.03 < 0.03 < 0.05 < 0.03 < 0.06 < 0.06 < 0.10 < 0.03 PCB's < 0.8 DDVP mevinfos diazinon malathion parathion chlorpyrifos sulfotep fenitrothion < 0.1 < 0. 1 < 0.1 < 0.2 < ,. 0.2 < 0. 1 < 0.1 < n? R&S 006553 I ANNEX 16 CONTAMINANTS IN TAP HATER (determined in period Sept.- Nov. 1980) - 83 leed cadmium mercury arsenic selenium copper ch romium zinc cyanide EPk <1 < 0.1 0.1 1 <1 50 <1 10 <1 Anmon ia nitrate nitrite chloride sodium calcium potassium iron diehloromethane 1,1-dichloroethane chloroform 1,1,1-trichloroethane te trachlorome thane trichloroethene te trachloroe thene b <5 0.3 0.3 0.1 < 0.1 0.4 < 0.2 benzo(a)pyrene <5 benzo(b)fluoranthene <5 indeno (1,2,3 ,-c,d)pyrene < 5 benzo(k)fluoranthene <5 fluoranthene < 10 benzolg,h, i)perylene <5 HCB o-HCH lindane heptachlor heptachl. epoxide aldrin dieldrin o-chlordane Y -chlordane endrin p,p'-DDE o.p'-DDT p.p'-TDE p.p'-DDT 6 -HCH tsethoxychlor PCB's PPrc < 0.01 8.0 < 0.01 25 15.0 28.5 1.2 0.01 b < 0.01 e 0.01 < 0.01 < 0.01 < 0.02 < 0.02 < 0.03 < 0.03 < 0.03 < 0.05 < 0.03 < 0.06 < 0.06 < 0.1 < 0.03 < 0.8 R&S 006554 m O V CIVO-TNO December, 1980 DDVP mevinfos diazinon malathion parathion chlorpyrifos sulfotep fenitrothion < 0.1 < 0.1 < 0.1 < 0.2 < 0.2 < 0.1 < 0.1 < 0.2 R&S 006555 ANNEX !7 CONTAMINANTS IN TAP WATER (determined in period March - May 1981) 89 - lead cadmium mercury arsenic selenium copper chromium zinc cyanide k 1 < 0.1 < 0.1 2 <1 30 <1 2 6 ammonia nitrate nitrite chloride aodium calcium potassium iron EE < 0.01 lb < 0.01 25 14.0 30.0 l.l 0.03 diehlorontethane 1,1-dichloroethane Ek <5 <1 chloroform 1,1,1-trichloroe thane < 0.1 < 0.1 tetrachloromethane < 0.05 tricnloroethene Ce trach1oroe thene 1.0 <0.4 C benzo(a)pyrene benzo(b)fluoranthene <5 <5 indeno (1,2,3,*c,d)pyrene < 5 benzo(k)fluoranthene <5 fluoranthene <10 benzo(g,h,i)perylene <5 HCB o-HCH lindane heptachlor heptachl. epoxide aliirin dieldrin a-chlordane Y-chlordane endrin p,p1-DDE o, p'-DDT p,p'-TDE p, p'-DDT 0-HCH methoxychlor O V < 0.01 < 0.01 < 0.01 < 0.01 < 0.02 < 0.02 < 0.03 < 0.03 < 0.03 < 0.05 < 0.03 < 0.06 < 0.06 < 0.1 < 0.03 CIVO-TNO May 1981 PCB's DDVP mevinfoa diazinon malathion parathion chlorpyrifoa sulfotep fenitrothion < 0.8 < 0.1 < 0.1 < 0.1 < 0.2 < 0.2 < 0.1 < 0.1 < 0.2 ANNEX 18 - 90 - CONTAMINANTS IN CIVO TAP WATER (determined in period Sept. - Nov. 1981) 1 ead cadmium mercury arsenic selenium copper chromium zinc cyanide b <1 <1 <5 < 10 <1 50 < 10 5 < 10 ammonia nitrate nitrite chioride sodium calcium potassium iron PPm < 0.01 9.3 < 0.01 30 17.5 30.0 1.2 0.03 R&S 006556 ppb dichloromethane <5 1,1-dichloroethane <1 chloroform < 0.1 1,1,1-trichloroethane 0.2 te tra ch1orome thane 0.2 trichloroethene 1.5 te trachloroe thane < 0.5 PPt benzoCa)pyrene <2 benzo(b)fluoranthene <2 indeno (1,2,3,-c,d)pyrene <10 benzo(k)fluoranthene <2 fluoranthene <1 benzofg ,h , i)pery lene <3 HCB a-HCH lindane heptachlor heptachl. epoxide aldrin dieldrin ct-chlordane y-chlordane endrin p,p'-DDE o,p'-DDT p,p1-TDE P , P' -DDT 8-HCH methoxychlor Eb < 0.01 < 0.01 < 0.01 < 0.01 < 0.02 < 0.02 < 0.03 < 0.03 < 0.03 < 0.05 < 0.03 < 0.06 < 0.06 < 0.1 < 0.03 < 0.15 PCB's < 0.8 CIVO-TNO May 1982 DDVP mevi nfos diazinon mala thion para thion chlorpyrifos sulfotep fenitrothion < 0.1 < 0.1 < 0.1 < 0.2 < 0.2 < 0.1 < 0.1 < 0.2 ANNEX 19 CONTAMINANTS IN TAP WATER (determined in period Jan - March 1982) - 91 1 ead cadmium mercury arsenic selenium copper ch romium zinc cyanide Ek <1 <1 <5 < 10 <1 40 < 10 10 < 10 ammonia nitrate nitrite chloride sodium calcium potassium iron PPm < 0.01 8.3 < 0.01 2-3 13.0 30.0 1.1 0.02 dich1oromethane 1,1-dichloroethane chioroform 1,1,1-trichloroethane te trachlorome thane tri ch loroe the ne te trachloroe thene 2^ <5 <5 < 0.5 < 0.2 < 0.1 0.4 < 0.2 benzola)pyrene < 1.3 benzo(b)fluoranthene < 0.5 indeno (1,2,3,-c,d)pyrene < 1.3 benzo(k)fluoranthene < 0.7 fluoranthene < 0.5 bemo(g,h,i) pery lene < 5.4 HCB o-HCH lindane heptachlor heptachl. epoxide aldrin dieldrin a -ch lordane Y -chlordane endrin p, p '-DDE o, p'-DDT p,p'-TOE p,p'-DDT B-HCH methoxychlor PCB' s 2fe < 0.01 < 0.01 < 0.01 < 0.01 < 0.02 < 0.02 < 0.03 < 0.03 < 0.03 < 0.05 < 0.03 < 0.06 < 0.06 < 0.1 < 0.03 < 0.15 < 0.8 CIV0-TN0 May 1982 DDVP mevinfos diazinon malathion parathion chlorpyrifos aulfotep fenitro thion < 0.1 < 0.1 < 0.1 <0.2 < 0.2 < 0.1 < 0.1 <0.2 R&S 006557 i I ANNEX 20 l/ING 9L' OSS REFERENCE LISTING nci. i;*fi ii /FRO REFFKkNCt HO It - 21/ M-.v MAI t 8 FFMALES Jhk 300/ K 0004K OOO6I1 0008K 0010U 0012K 0014D 0 016 Li 00181 0020D 00220 00240 0026D 00280 00300 0032D 00340 0036K 0038D 00400 00421 00440 00460 00480 00500 00520 00540 00560 00580 00600 00620 00640 0066K 00680 00700 '00720 0074 0 '0076K 007BK 00800 '00820 '00840 '00860 '00880 '00900 '00920 '00940 'OOV6K '00980 EXPR A ',1 A4 A6 A8 A 10 A 12 A 14 A 16 A 18 A 20 A V'? A 24 A 26 A 28 A 30 A 32 A 34 A 36 A 38 A 40 A 42 A 44 A 46 A 40 A 50 A 52 A 54 A 56 A 58 A 60 A 62 A 64 A 66 A 48 A 70 A 72 A 74 A 76 A 78 A HO A 82 A 84 A 86 A 88 A VO A 92 A 94 A 96 A 98 SGP CO) (0) (0) (0) <<>) (0) (0) (0) CO) (0) CO) CO) CO) CO ) CO) CO) CO) CO) CO) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) CO) CO) 0) sO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) (0) CAGE 2 *7 V 2 9 4 4 4 4 4 6 6 6 6 6 8 8 8 8 8 10 )0 10 10 10 12 )2 12 12 12 14 14 .1 4 14 14 16 16 16 16 16 18 18 18 18 18 20 20 20 20 EARR 7 K1 R2 LI L2 R1L1 R1L2 R2I..1 R2L2 R3 7 Fcl R7 LI L2 R1L1 R1L.2 R2I..1 K2L2 R3 7 R1 R2 LI L2 R1L1 KM 1.2 R21..1 R2L2 R3 7 K1 R2 LI L2 R1L1 R1L.2 R2L 1 R2L2 R3 7 K1 R2 LI L2 R1L1 R1L 2 R2I. 1 R2L2 COMR 7700010 7700030 7700050 7700070 7700090 77001 IK 7700130 7700150 770017K 7700190 7700210 7700230 770025K 7700270 7700290 7700310 770033K 7700350 770037k 7700390 77004 IK 7700430 770045K 770047K 7700490 770051K 7700530 7700550 7700570 7700590 7700610 7700631 770065K 7700671 7700690 7700710 7700730 7700750 7700770 7700791 7700810 7700H3K 7700850 7700870 7700890 7700910 7700930 7700950 7700970 EXPR A1 A3 A5 A7 A9 A 11 A 13 A 15 A 17 A 19 A 21 A 23 A 25 A 27 A 2V A 31 A 33 A 35 A 37 A 3V A 41 A 43 A 45 A 47 A 49 A 51 A 53 A 55 A 57 A 59 A 61 A 63 A 65 A 67 A 6V .1 71 A 73 A 75 A 77 A 79 A HI A 83 A 85 A R7 A 89 A 91 A 93 A 95 A 97 SGP ('.AGE FARR CO) CO) (0) (0) CO) (0) CO) CO) CO) (0) CO) CO) CO) CO) CO) CO) (0) CO) (0) CO) CO) CO) CO) CO) CO) (0) CO) CO) CO) CO) CO) (0) CO) CO) CO) CO) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) (0) CO) 17 1 Kl 1 R3 1U 1 L2 3 R1L. 1 3 R.U.2 3 R2I. 1 3 R2L2 3 R3 57 2, KM 5 R2 5 L1 5 L2 7 R1L1 7 R.1L2 V K2I..1 7 K2L2 7 R3 9 7. 9 R1 9 R2 9 LI 9 L2 11 R1L I 11 R11. 2 11 R2I. 1 11 K2L2 11 R3 13 7. 13 KM 13 R2 13 LI 13 l 2 15 R1LI 15 R11. 2 15 R21.1 15 R2L2 15 R3 17 7 1 7 KM 17 R2 17 LI 17 1.2 19 R1L 1 19 Kit 8 19 K21.1 19 R2L2 K - killed D - dead I ill - (dead) R&S 006558 ANNEX 20 (CONT. I) ),' 1 Nit " 93 OSS REF E R E N C E LI STING 35 NCJ. 125 0 it- Rl) KKt-hKH'il.F. DAtL - 21 MALES FFMALES OMR EXF'R 0100LI A100 010211 A102 010 'l 0 A1 04 01060 A106 01080 A1 08 01100 A110 01120 A112 '01140 All 4 01160 A1 3 6 '01180 A118 '01200 A120 01220 A122 '01240 A3 24 '01260 A126 '01280 A1 28 *01300 A130 '01320 A132 *01340 A134 '01360 A136 70138K A138 >0140K A140 '01420 A142 >01440 A1 44 >01460 A146 701480 A148 701500 A150 701520 A152 701541 A1 54 701560 AJ 56 701580 A158 701600 A160 70162K A162 70164K A3 64 70166K A166 70168K A168 70170K A170 701720 A17 2 701740 A174 701760 A176 7017BK A178 70180K A180 701820 A 3.8 2 701840 A184 '70186)0 A106 701880 A188 '70190K A190 '701920 A192 '70194K A194 '701960 A) 96 '701980 A198 '702000 A200 SGF' CAGE EARR CC) MR <0) 20 to > V R3 7700990 2 770101D (0) '? (0) oo (0) o " CO) ',) R1 7701031.' R2 770105H L. 3 7703 07H 1. 2. 7701091.1 (0) 24 R1L3 7701111 (0) 2.4 R1L2 7701131 (0) 24 R21.1 770115k (0) 24 R2L.2 77013 7H CO) 24 R3 770119H CO) 26 7. 7701211 CO) 26 R1 7701231 CO) 26 R2 770125K CO) 26 1.1 7701270 CO) 26 1.2 770129D CO) 28 R1L1 7701310 CO) 2.8 R1L2 770133K CO) 28 R21.3 7703350 CO) 28 R21..2 7701370 CO) 28 R3 7701390 CO) 30 Z 77014 IK CO) 30 R1 7701430 CO) 30 R2 770145(1 (0) 30 1.1 7701470 CO) 30 1.2 7701490 CO) 32 R1L1 7701510 CO) 32 R1L2 7701530 CO) 32 R2L.1 7703.550 (0) 32 K21.2 7701570 CO) 32 CO) 34 R3 770 3.59K Z 7701610 CO) 34 R1 7701630 CO) 34 R2. 7701650 cot 34 1.1 770167k < 34 1.2 7703690 C v l 36 R1L1 7701710 CO) 36 R1L2 770173k CO) 36 R2I. 3 7701750 CO) 36 R2I.2 7701770 CO) 36 R3 770 3 790 CO) 38 Z 7701810 CO) 38 R1 7701830 CO) 38 R2 7701850 CO) 38 I..1 7701870 CO) 3B L2 770189K CO) 40 R1L3 77019IK CO > 40 R1L2 7701930 CO) 40 R2L 1 7701V50 CO) 40 R2L2 7701970 CO) 40 R3 . .7703 99K FXPR A 99 A101 A103 At 05 A107 A109 Alll All 3 A115 All 7 All? A121 A123 A125 A 12.7 A129 A131 A 133 A13 5 A3 37 A139 A] 41 A143 Aj 45 A3 47 A149 A15.3 A153 A i 55 A157 A159 A3 61 A163 A165 A167 A169 A3 7 3 A173 A175 A177 A179 A3 81 A3.83 A185 A187 A1B9 A3 93 A CV3 A195 A197 A19V SGF' CAGE EARR CO) IV R3 CO) 21 /. CO) 23 Rl CO) 21 R? CO) 21 1. 1 CO) 21 1. 2 CO) 23 K 31. 3 CO) 23 R1L2. CO) 23 R21 3 CO) 23 K2I. 3 (0) 23 R3 CO) 25 7 CO) 25 Rl CO) 25 R2 CO) 25 I..1 CO) 25 1.2 CO) 27 Rl LI CO) 27 R1L2 (0) 27 R2L1 CO) 27 R7I. 2 CO) 27 R3 CO) 29 Z CO) 29 Rl (0) 29 K3 CO) 29 1.1 CO) 2? 1.7 (0) 31 R3L1 CO) 31 R1L2 CO) 31 R2L.3 CO) 31 R21. 2 CO) 31 K3 CO) 33 Z CO) 33 Rl CO) 33 R2 CO) 33 (. i (0) 33 1.2 CO) 35 klLi CO) 35 R1L2 (0) 35 R2L.3 CO) 35 R2I. 3 CO) 35 K3 CO) 37 Z CO) 37 Rl CO) 37 R3 CO) 37 1. t CO) 37 13 CO) 39 Rl L1 CO) 39 R1L2 (0) 39 R2L.1 (0) 39 R2L2 CO) 39 R3 33 CO o o 05 cn cn (> I R&S 006560 ANNEX 20 (CONT. 2) / I NI.J OSS REFERENCE LISTING M NO. 125 0 ZERO REFt KL.NRI HAH 7 1 . MALES )MR EXPR j>20?o J2040 .`2060 02osn j:>i on 02120 021 40 02160 021SD 0220k 02221* 022/Ul Q226K 022BH 02301* 02320 0234K ' 0 2 3 6 Li 02380 0240D '0242k '02440 0246K '02480 '0250D 70257l\ ''0224 0 '02560 70258k /02600 702620 702640 702660 702680 702701! 7027211 70274k 70276K 702780 702800 702820 702840 702860 702881 '70290k '707920 '70294k 8 '7 B4 B6 H8 B 10 H 12 0 14 0 16 P 18 P 2.0 B **'? B 24 P 26 P 28 B 30 H 32 B 34 P 36 P 88 B 40 P 42 B 44 P 46 P 48 B 50 P 52 P 54 B 56 P 58 P 60 B 62 B 64 P 66 B 68 B 70 P 72 B 74 P 76 P 78 B 80 B 82 P 84 B 86 B 88 B 90 B y? B 94 Slip CAGE H ARK (0) 4 2 / (0) 42 Ri <0> 47 R7 (0) 47 LI (0) 42 L2 (0) 44 R1U (0) 44 K1I. 2 (0) 44 K2L1 (0) 44 R2L2 (0) 44 R3 (0) 46 7 (0) 46 K1 (0) 46 R2 (0) 46 LI (0) 46 L2 (0) 48 R1L. 1 (0) 48 R11. 2 (0) 48 R2L 1 (0) 48 R2L2 (0) 48 R3 (0) 50 Z (0) 50 R1 (0) 50 R2 (0) 50 11 (0) 50 l2 (0) 5? R1L.1 (0) 52 Rll.,2 (0) 5:.* R2L1 (0) 52 R2L2 10) 52 R3 (0) 54 Z (0) 54 ki (0) 54 R2 (0) 54 11 (0) 54 1-2 (0) 56 Kll. 1 (0) 56 R11. 2 (0) 56 R2L1 (0) 56 R2L 2 CO) 56 N.4 CO) 58 Z (0) 58 KM <0> 58 R2 CO) 58 l1 CO) 58 L.2 CO) 60 R1I..J CO) 60 R1I.2 femaus CDMR EXF'R SGR LAGF E AKK 77020IK 770203k 7702050 7702070 7702090 7702110 7702130 7702150 770217K 7702190 77022 !J! 7702231 7702250 7702270 7702290 7702310 7702330 7702350 7702370 770239K 7702410 7702430 7702450 770247K 7702490 7702510 7702530 7702550 770257K 7702590 770261k 770263K 7702650 770267k 7702690 77027IK 7702730 770275k 7702770 7702/90 '77028.10 7702830 7702850 7702B7K 7702890 7702910 7702930 B1 B3 B5 B7 B9 B 11 B 13 B 15 B 17 B 19 B 21 B 23 B 25 B 27 B 29 B 31 B 33 P 35 B 37 B 39 B 41 B 43 B 45 B 4/ B 49 B 51 B 53 B 55 B 57 B 59 B 61 B 63 B 65 B 67 B 69 B 71 B 73 B 75 B 77 B 79 B 81 B 83 B 85 B 87 B 89 B 91 B 93 40) CO) CO) CO) (0) CO) (0) CO) CO) CO) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) cO) CO) CO) CO) CO) CO) CO) CO) CO) (0) CO) CO) CO) (0) CO) CO) CO) CO) CO) CO) CO) (0) CO) CO) CO) CO) 41 4) 41 4) 41 43 43 43 43 43 45 45 45 45 45 47 47 47 47 47 49 4V ' 49 49 49 51 51 51 51 51 53 53 53 53 53 55 55 55 55 55 57 5/ 57 57 57 59 59 7 1C) K2 LI L2 Rll.l Fell. 2 R2I. 1 R2L2 R3 Z R1 R2 LI L2 Kll. 1 R1I..2 R2L1 R2L2 R3 7 KM R2 L.l L2 K1L. 1 Rll.,2 R2L1 R2L2 R3 / K1 R2 L1 L.2 Kll 1 Kll .2 R2L1 R2L2 K3 7 KM R2 L1 1.7 KM 1, l Kll 7 I ANNEX 20 (CONT. 3) 6TNO 95 - OSS REF ERE N C E L I ST I NG .Y Nil. 125 n i I. k'U RI.H. Kl N( 1 DAI L . I MALES E H HAL K S JMR E xrk SUP CAGE EARR COMR EXRR SGI' (.AGE EAR K 0296D 8 96 (0) 60 R2L.1 029BD 8 98 (0) 60 R21 2 0300H 8100 (0) 60 R3 0302D 8102 <0> 62 2 03048 8104 CO) 62 Rl 0306D 8106 CO) 62 K2 0308K B108 CO) 62 I..1 0310D 8110 CO) 62 12 0312D 81 12 CO) 64 R1L1 ' 0 31 4 D 8114 (0) 64 R1L2 03161 8116 CO) 64 R2I 1 '0318D 8118 (0) 64 K2I.2 '0320D 8120 CO) 64 K3 '0322D 8122 CO) 66 Z -'0324 8 8124 CO) 66 Rl '0326D 8126 CO) 66 K2 '0328D 8128 (0) 66) 1.1 ''0330D 8130 CO) 66 L2 60 3 3 2 D 8137 CO) 68 ftlLl 7033411 8134 CO) 68 Rl L 2 70336D 8136 CO) 68 K21.1 70338D B13B CO) AH R2I.2 703401 8140 CO) AH R3 70342D 814 7 CO) 70 Z 70344D 8144 CO) 70 Rl 70346D 8146 CO) 70 K2 70348D 8148 CO) 70 11 70350k 8150 CO) 70 L2 70352D 815? CO) 72 R1L 1 70354K 8154 CO) 72 R1L 2 70356D 8156 CO) 72 R2I. 1 60358D 8158 (0) 72 R2I. 2 70360K 8160 CO) 72 R3 70362D 8.1 63 CO) 74 Z 70364K 8.164 CO) 74 K1 7036AD 8166 CO) 74 R2 '703&SD 8168 CO) 74 11 70370D 8170 CO) 74 L2 '70372K 8172 CO) 76 R1L1 '70374D 8174 (0) 76 Kll 2 '70376D 8176 (0) 76 R21.1 77037BK 8178 CO) 76 R3L.3 '70380k 81 WO CO) 76 R3 770382D 8182 CO) 78 Z 7703840 8184 CO) 78 Rl 770386D 8186 CO) 78 R2 /70388D 8188 CO) 78 11 7703901 8190 CO) 78 L2 7703V2D 8193 CO) 80 R1L1 7703948 8194 CO) DC) RIL2 7703V6D 8196 CO) HO R2L1 770398k 8198 CO) 80 R2L2 770400D 8200 CO) 80 K3 770295k 8 y5 CO) 59 K2I..1 7702978 8 97 CO) 59 k:.'I 770299D 8 99 (0) 59 ft 3 770301D 81 01 CO) 61 7703031 8103 CO) 61 KI 770305K 8105 CO) 61 R2 7703078 8107 CO) 61 I. 1 770309k 8109 CO) 61 I2 77031ID 8111 CO) 63 R1L1 770313). 8113 CO) 63 R1L? 770315D 8115 (0) 63 R2I 1 770317D 8117 CO) 63 K21..2 770319D 8119 CO) 6*3 r3 770323D 83.21 (0) 65 Z 7703231 8123 (0) 65 Rl 7703258 8175 CO) 65 k;- 770327K 8127 CO) 65 i. i 770329D 8129 CO) 65 Li 770331D 8131 (0) 67 R1L1 770333D 8133 CO) 67 Rl L.: 7703358 8135 (0) 67 k:/i ,i 770337D 8137 (0) 67 R2i : 7702291 8139 CO) 67 R5 77034 ID 8141 CO) 69 770343D 8143 CO) 69 Rl 77034 5)1 8145 co> 69 K2 770347K 8147 CO) 69 I. ,1 770349D 8149 CO) 69 Li 770351K 8151 CO) 71 R1L1 770353K 8153 (0) 71 KM l 770355D 8155 CO) 71 R2i i 770357D 8157 CO) 71 k:?i. ; 770359D 8159 CO) 71 R3 770361D 8161 )(>) 73 z 7703631 8163 CO) 73 Rl 770365k 8165 (0) 73 R2 770367D 8167 CO) 73 IJ 770369K 8169 CO) 73 L2 770371D 8171 CO) 75 R1L 1 7703738 8173 (0) 75 Rl L 2 7703758 8175 CO) 75 r:.'I i 770377D 8177 CO) 75 r:.'|. : 770379D 8179 CO) 75 R3 770381D 83.H1 CO) 77 i 7703H38 8183 CO) 77 Rl 7703H5D 8185 CO) 7/ k 770387K 8187 CO) 7 7 ii 770389K 8189 CO) 77 L.2 77039 ID 8) 91 CO) 79 Rl L I 770393D 81 93 CO) 79 K.U . 770395D 81 VC) CO) 69 R2I 1 7703971' 8 i 9/ CO) 79 ft 21 770399) 8199 CO > 79 R > 06567 23 ffo C/3 ANNEX 20 (CONT. 4) 1/7 NU OSS REFERENCE L I ST I NG 3Y Nil. 125 0 7t.R0 KFFLKFNl.l Ini I h MALES OMR '04020 0404K 04060 '04080 '041 on '0412K '0414K '0416K '0418K '04200 .704220 704240 '04260 704230 704300 /0432D 704340 704360 704380 704400 70442D 7044411 704460 704480 70450k 704520 704540 704560 704580 704600 70462K 704640 70466K 70468k 70470k 704720 70474k 704760 704780 704800 `704820 '704840 704860 '704880 '704900 EXFR C2 L4 C6 cH c: 10 c 12 c 14 c 16 i; 1H c 20 c 22 c 24 c 26 <: 28 c: 30 c 32 c. 34 (; 36 (; 38 c 40 C 42 t 44 c 46 (; 48 C 50 c 52 C 54 L 56 i; 58 c 60 c 62 (_: 64 (; 66 C 68 C 70 c 72 (. 74 <; 76 c 78 c 80 c 82 c 84 i; H6 c 88 c 90 SGP (0) (0) (0) <0> (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) UAGF. 82 82 82 82 82 84 84 84 84 84 86 86 86 86 86 88 88 88 88 88 90 90 90 90 90 92 92 92 92 92 94 y4 94 94 94 96 96 96 96 96 98 98 98 98 98 F ARK 7 R1 R2 L1 L.2 R1I.1 K1L2 R2L1 R2L2 K3 Z R1 R2 LI 1.2 kll. 1 R1L2 R2L1 R2L2 K3 2 R1 R2 1. 1 1.2 R1L 1 R1L2 R2L1 R2.L.2 R3 7 R1 R2 1.1 1.2 klL 1 R1L2 R2L1 R2I. 2 R3 2 R1 R2 11 1. 2 OOMk 770401K 770403D 7704050 7704070 770409k 7704110 7704130 770415K 7704170 7704190 7704210 7704230 770425) 7704270 7704290 770431k 7704330 7704351 770437K 770439K 7704410 770443H 7704450 7704470 770449K 7704510 7704530 7704550 7704570 7704590 770461K 770463K 7704651 770467k 7704690 7704710 7704730 7704/5K 77047/0 77O47y0 7704810 7704830 770485k 7704870 7704890 FEMALtfa EXRR C1 <_: 1$ i: 5 t; 7 c, 9 c 11 r. 13 c; 15 c 17 8 19 c 21 l: 23 c; 25 <; 27 r. 29 c 31 c 33 c 35 c; 37 c 39 c 41 c 43 i; 45 8 47 c 49 c 51 8 53 8 55 8 57 C 59 C 61 8 63 8 65 8 67 C 69 C 71 8 73 8 75 8 77 C ?y C 81 8 83 8 85 0 87 C 89 SGR (0) CO) (0) CO) CO) CO) CO) (0) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) CO) CO) CO ) CO) CO) (0) (0) (0) CO) LAkF 8( Hi 81 81 81 H3 83 83 83 83 85 85 85 85 85 8/ 8/ 87 87 87 89 8V 89 89 89 9.1 91 91 91 91 93 93 93 93 93 V5 95 95 95 95 97 97 97 97 y7 K1 R? L1 I ' kll. y K ] L2 R2L_ 1 R21 2 kv, 7 R1 k? I. 1 I. k :u. \ R1L2 R2L1 r:>l? k3 7 R1 r? i1 i.2 kill R1L2 R2L 1 k'2l 2 k,*. / R1 R2 - 1.1 l. 7 K1L1 R1L2 R7I 1 k2i. : K3 R1 K7 Ii I. 7 r &S 006562 ; ! I ; ! j j i ANNEX 20 (CONT. 5) U.'TNU OSS REF E R E N C E L AY NU. 125 Cl MALMS OMR FXF'R Slip CABF EARR 0492D 04948 0496K 04981 0500K 05021 0504 0 0506K 05080 05100 05120 05140 05160 05180 '05200 05220 '05240 '05260 05280 '0530K '05320 '05340 '05360 05380 05400 05420 '0544K 05460 05480 '05500 '0552K '05540 '05560 '05580 05600 '05620 '05640 '05660 '05681 05700 '05720 '05740 '05760 '05780 '05800 '0582K '05840 '05861 05880 '05900 '05920 '05940 '05960 '05980 '06000 (I 92 i; 94 C 96 C 98 C100 Cl 02 C104 C106 C108 cno Cl 1.2 Cl 14 C116 CUB Cl 20 Cl 22 Cl 24 C126 Cl 28 Cl 30 Cl 3? C134 C136 Cl 38 Cl 40 C147 Cl 44 C146 Cl 48 Cl 50 Cl 52 Cl 54 C156 C J 58 Cl 60 Cl 62 C164 C166 C16B Cl 70 Cl 72 C174 C176 C1.7H Cl 80 Cl 82 C184 Cl 86 i; 188 C190 Cl 92 (; 194 Cl 96 Cl 98 C200 (0) CO) (0) CO) CO) (0) CO) CO) (0) CO) (0) CO) (0) CO) CO) (0) CO) CO) CO) (0) CO) (0) CO) CO) (0) CO) (0) CO) CO) (0) (0) (0) CO) (CD CO) CO) (0) CCD CO) (0) (0) (0) CO) CO) (0) CO) CO) CO ) CO) CO) CO) CO) (0) (0) CO) 100 100 100 100 too 102 102 1 02 102 102 104 104 104 104 104 106 106 106 106 106 108 108 108 108 108 110 110 U0 110 no n? 1 12 Ti 11' .11 2 112 1 14 )14 1 14 11 4 114 1 1.6 1.1 6 i i. 6 116 116 118 11B 1 IB 1 18 1 18 l 20 120 120 J 30 130 Rll 1 Kit. 2 K21.1 K2L2 R3 7. KM K3 Ll L2 R1L.1 KM 1.2 R21.1 R2L2 R3 7 R1 R2 Ll L2 R1U Rll.. 3 K31.1 R2L2 R3 7. R1 k? Ll L2 RJL.1 KMI..2 K2I. 1 R2L2 R3 7 I.M R2 Ll L2 K1L 1 R11.2 R2L1 R2L2 R3 7 R1 R2 L1 12 R1L1 R1.L2 R3I 1 K2I.2 K3 - 9^ - STING 7 b K LJ kfcPERENCfc BA t fc. => 71 bbMAl.hS COMR IX PR Blip (.A fib' EARR 770491)1 7/0493D 770495K 770497K 770499K 770501)1 770503K 770505H 770507K 770509D 77051 IK 770513)1 7705150 770517K 7705190 770521)1 770523K 770525K 7705270 770529] 770531)1 770533)1 7705350 7705370 770539JI 770541)1 770543K 7705451 7705470 7705490 770551)1 7705530 7705550 7705570 7705591 7705611.1 7705630 7705650 7705670 7705690 7 70571)1 7705730 7705750 7705770 7705790 '77051! 1)1 7 705831) 7705B5K 7705870 7705890 77059IK 77059311 7 70595)1 77059/K 7705990 C 91 C 93 C 95 C 97 C 99 (,'101 Cl 03 C105 Cl 07 Cl 09 cm C113 C115 Cl 17 CUV Cl 21 C123 C125 Cl 27 Cl 29 Cl 3.1 C133 C135 CM 37 Cl 39 Cl 41 Cl 43 Cl 45 Cl, 4 7 C>49 (; 151 C153 C155 Cl 57 Cl 59 Cl 61 C163 C.165 (.167 C169 C171 C173 Cl 75 Cl 77 Cl 7 V Cl 81 C183 Cl 85 (-1H7 C1B9 C191 (-193 C1V5 C197 Cl 99 CO) (0) (0) CO) CO) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) CO) (0) (0) (0) CO) (0) CO) (0) (0) CO) CO) CO) (0) CO) CO) CCD CO) CO) CO) CO) CO) CO) CO) (0) CO) CCD CO) CO) CO ) (0) (0) CO) (0) CO) CO) (0) < (11 ' 99 99 vv 99 99 101 101 10] A01 101 103 103 1 03 103 103 105 105 1 05 105 105 107 1 07 10/ 107 107 109 ioy .109 ioy 109 111 111 in in in 113 1 .1 3 113 113 113 115 1 15 1.15 1 15 115 1.17 ,1! 7 .1 ' 7 117 1 i7 1 iV 1IV 119 119 1 11.' K1 L .1 Rtl. 7 k:..m. 1 K?L2 R3 7 kl K3 Ll L2 R1L1 Kll.2 K2U K2L2 R3 7 KM R2 Ll L2 RI L 1 K1I..2 K21.1 R2L2 R3 7. KM R2 Ll L2 R1L1 IMI, 2 K21.1 R2L2 R3 7 IM R2 Ll 12 kll.l K11 2 K?L1 R2L2 R3 7 IM R2 Ll 12 R1L1 R1L 2 K7I 1 R2I 2 L" J 8/ /V R&S 006563 ANNEX 20 (CONT 6) 0/1 NO - 9S - OSS REFERENCE LISTING AY Nil. 12b (I ZE:RO k'E I-h kh k'l> 1'AIl 21/ 8/ MALL'S FEMALES Ohk 06020 060-10 06060 '06080 06100 '06120 061-10 06160 '0618k >06200 >06220 >06240 >06260 70628K >06300 706321' >06340 706360 706380 >06400 706420 70644k 706460 706480 706500 70652k 706540 706560 706580 706600 706620 70664k 706660 70668k 70670k 706720 70674k 706760 706780 706800 706820 706840 706861 706880 706900 706920 706940 LXPR 0 */ 04 06 08 0 10 0 12 0 14 0 16 0 18 0 20 0 ir! 0 24 0 26 0 28 0 30 0 32 0 34 0 36 0 38 0 40 0 42 0 44 0 46 0 48 0 50 0 52 0 54 0 56 0 58 0 60 0 62 0 64 0 66 0 68 n 70 0 72 0 74 0 76 0 78 0 HO 0 87 0 84 0 86 0 HH 0 90 0 9o 0 94 SGP CAGF b Akk C0 > (0) CO) (0) (0) CO) (0) (0 ) (0) (0) (0) CO) CO) CO) CO) CO) CO) (0) (0) CO) (0) CO) (0) CO) CO) CO) CO) (0) (0) CO) CO) CO) CO 1 C\ CO) CO) CO) CO) CO) CO) CO 1 CO) CO) CO) CO) CO) CO) 1 22 122 1 22 172 124 124 124 174 .174 126 126 126 126 176 128 128 128 178 128 130 130 130 130 1 30 132 132 137 137 137 134 134 134 134 1 34 136 136 1 36 136 136 138 138 138 138 138 140 140 Z R1 K2 1. .1 1.2 klLl R1L2 R2L.1 k'7L2 R3 Z R1 R7 1.1 L2 R1L1 R1L2 K2I..1 R2I. 2 R3 Z R1 R2 1. 1 L2 R1L1 R1L7 K2I. 1 R7L 2 R3 Z K.1 R7 1, 1 L2 R1L1 RI L.2 R21. 1 R2L 2 R3 Z Rl R7 LI 1.2 kli. i R1L 2 CIJMR 770601H 770603)1 7706051' 7'7C)6C)71l 770609K 7706118 770613)1 7 7061 5)1 7706171 7706191' 770621D 7706231! 770625K 770627D 770629D 7706311' 770633).' 7706351' 770637D 7706391' 77064 ID 77064 3).' 7 7064 5 J! 770647D 770649D 770651D 7706531.' 770655P /70657D 770659D 7706611 770663K 7706651 770667L' >0669K 77067 ID 7 706731! 7706751! 7706771 7706791! 770681H 7706831! 7706851' 7706B7P / 706891! 7706911' 7706931' 1 XPR H1 )' 3 ll 5 D7 D9 I' 1 1 1! 13 n 1.5 p 17 D 1.9 n 21 l! 23 Li 25 D 27 l' 29 Ii 31 )! 33 )' 35 D 37 D 39 li 4 1 li 43 )' 45 D 47 I' 49 H 51 11 53 D 55 D 57 I' 59 )' 61 D 63 D 65 D 67 H 69 Li 73 )! 73 D 75 D 77 H 79 H 8) I! 83 D 85 )' 87 n 89 D 91 D 93 SGP CO) ',0) CO) CO) CO) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) (0) CO) CO) CO) CO) CO) CO) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) CO) (0) CO) CO) CO) (0) CAGE 121 121 121 121 i. 21 123 123 123 1 23 123 125 125 125 125 125 127 127 127 12 7 1 27 129 129 129 1 2V 129 131 131 131 133 131 133 133 133 1 33 133 135 135 I 35 135 ,i 35 137 137 3 37 137 137 3 39 139 FARR Z ki K2 1! 1,3 R1L1 R1L2 k3l .1 k'2l. 2 R3 `7 k'3 IC2 1. 1 L2 R1L1 R1L2 K2I. 1 K2I. 2 R3 Z R3 R2 1. 1 L2 R1L1 R3 L2 K2I. 1 K2I. 2 R3 Z K3 K2 13 L2 1 Ll R3L.2 R2I. .1 K3I. 2 R3 J k'3 k2 Ll 13 k' 1 1 , 1 k 1L2 r &S 006564 ANNEX 20 CCONT. 7) U/TNO OSS REF E R E N C E AY NO. 122 (J L MAL F5 :omr EXkk 888 CAGE EARR /0696L' 7069BM 70700H 70702k 70/04K /0706k 7070BK 7071 OK 70712K 70714k 70716K 7071BK 70720K 70722N 70724k 70726K 7072BK 70730K 70732K 70734K 70736k 70738K 70740k 70742k '70744k '70746k '70748K '/0750K 70752K 1 70754k "70756K '70758K ' 70760K '70762k '70764k 70766K "707681% '70770k '70772K '70774k 770776k 77077HK /70780k /70782k 770784k 770786k /70788k 770790k 770792k 770794K 770796K 770798K 770800K )i 96 II 98 m 00 D102 D104 Ml 06 Ml 08 M110 Ml 12 Ml 14 M11 6 M11 8 Ml 20 Ml 22 Ml 24 Ml 76 M178 Ml 30 Ml 32 M13 4 Ml 36 Ml 38 Ml 40 Ml 42 n1 44 Ml 46 Ml 48 Ml 50 D152 Ml'..4 M.I .7. Ml 08 Ml 60 M16 3 Ml 64 Ml 66 Ml 68 Ml 70 M177 Ml. 7 4 Ml 76 Ml 78 Ml UO Ml 82 MI 84 Ml 86 Ml 88 Ml 90 M l 92 M194 Ml 96 Ml 98 M200 (0) (0) (0) (0) (0) (0) (0) CO) (0) (0) <(>) (0) (0) (0) <0> (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) CO) (0) (0) (0) <0> (0) (0) (0) (0) (0) 10) (0) (0) (0) (0) (0) (0) <0) (0) <0> (0) CO) (0) 140 140 140 14 2 147 14? 142 142 144 144 144 144 144 146 146 146 146 146 148 148 148 148 148 150 150 150 150 1 50 152 152 152 152 lS 154 154 154 154 154 156 156 156 156 156 158 158 158 158 158 160 160 160 160 160 R2L1 R2L2 K3 7 R1 R2 LI L2 R1L 1 R1L2 R2L1 R2L2 R3 7. R1 R2 11 1.2 Rll. 1 R1L2 R2L1 R21 2 R3 2 R1 R2 11 1.2 Kll 1 R1L2 R2L1 R2I. 2 K3 7 R1 R2 1. 1 1.2 K1L1 R1L2 R2L. I K2I. 2 R3 Z R1 R2 LI L2 kll 1 Kll. ? R2L 1 R2L2 R3 - 99 TING xh ku reference matf |i/ 7v FhMnl.I.S CQriR 1- XPR Sbk C AGE EARR 770695M 770697H 77069VM 7/O/Ol.k 770703k 77G705K 770707k 770709K 770711k 770713k 770715k 770717K 770/1Vk 770721K 770723K 770725k 770727k 77072VK 770731k 770733k 770735k 77073/K 77073VK 77074 IK 770743K 770745k 770747k 77074yk 770751k 770753k 770755k 770757k 7/0759K 770761k 770763k 7707651 770767k 770769k 770771k 770773k 770775k 770777k 770779k 770781k 770783K 770785K 770787k 770789k 7707VIk 770793k 770795k 770797k 77079VK M 95 M 97 M 99 Ml 01 Ml 03 Ml 05 Ml 07 Ml 09 Mill Ml 13 Mil 5 M.1.17 Ml 19 Ml 21 Ml 23 M125 Mi 27 M129 Ml 31 Ml 33 Mi 35 Mi 37 Ml 39 Ml 41 Ml 43 Mi 45 Mi 4 7 M149 M151 MI 53 MI 5 5 Ml 57 Ml 59 Ml 61 M.163 Ml 6 5 Ml 6 7 Ml 69 Ml 71 Mi 73 Ml 75 Ml 77 Ml 79 M181 Ml 83 Ml 85 Ml 87 Ml 89 M i 91 Ml 93 Ll 195 Ml 9 7 Ml 99 (0) (0) (0) (0) (0) (0) (0) (0) <0> (0) CO) (0) (0) (0) (0) (0) (0) CO) CO) CO) CO) SO) CO) CO) CO) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) CO) (0) CO) CO) CO) CO) CO) CO) CO) CO) CO) CO) (0) CO) CO) CO) 139 139 139 J 41. 14i .141 141 141 143 143 143 143 143 145 145 1 45 145 1.45 147 147 147 147 147 149 149 149 149 149 151 151 151 151 151 153 153 153 153 1 53 155 155 155 155 155 15/ 157 157 157 157 159 159 159 159 159 R2L1 K2L2 R3 / R1 R2 LI L2 k:.U..i RILE R2L1 R2L2 K3 7 R1 R2 1. 1 1.7 Kll. J R1L2 R2L1 R21 7 k3 2 R1 R2 1.1 1. 7 K.U.1 R1L 2 R2L1 K7I. 7 k'3 I R1 K2 1, 1 1. KJLl R1L2 R 2 L1 k2l. 7 k3 l R1 R2 Ll L2 R1L. .1 kil.7 k`21_ 1 R2L2 R3 03 999900 R&s 006566 ANNEX 20 (CONT. 8) i/TNd OSS REFERENCE L ISTING 100 -,Y NO, 125 0 .'fckO Kt- Fh Kl- NfcJ HA J't " 21 0.' ' - MALES FEMALES :jmr 08020 08040 08060 080BP 08100 0812D '08140 '08160 '0818D '0820D '08220 '08240 '08260 '0828D '0830D >08320 >08340 70836D 2083BD 2084011 70842D 70844D 2084 6D 70848D 708500 708520 708540 708560 708580 7(386(30 7(38620 70864D 70866D 708680 708700 708720 708740 '708760 '7087BU '708800 '708820 '708840 '708860 '708880 '708900 '708920 770H940 EX PR t E4 E6 k8 F 10 F 12 E 14 E 16 k 18 fc 20 E. nn E 24 E 26 E 2.8 E 30 E 32 E 34 E 36 E 38 E 40 E 42 E 44 E 46 a 48 E 50 E 52 k 54 E 56 K 58 E 60 E 6? fc. 64 E 66 fc 68 t 70 E. 72 fc 74 fc 76 fc 78 F 80 t 82 t 84 t: 86 F 88 t 90 i- 92 F 94 SGfc' CAGE fc AKK (0) (0) (0) (0) (0) (03 (0) (0) (03 (0) (0> <03 (0) (0) (0) (0) <0> (0) (0) (0) (0) (0) (0) (0) (0) (0) (0) <0> (0) (0) (0) (0) (0) (0) <03 (0 <0; <0> (0) (0) (0) (0) (0) (0) (0) (0) (0) 162 1 62 162 162 162 1 64 164 164 164 164 166 166 166 166 166 168 168 16B 16B 168 170 170 170 170 170 172 172 177 1 72 1 72 174 174 1 74 174 1 74 176 1 76 176 1 76 3 76 17B 1 7H 178 178 178 I bO 180 7 R1 K2 LI L2 Kll.l R1I..7 R2L1 R2L2 R3 7 R1 R2 LI L2 R1L1 R1I..2 R2L1 R2L2 R3 2 R1 R2 L.1 L2 R1L1 R1L.2 R2L 1 R2L2 R3 7 R1 R2 L1 1.7 K1L.1 R1L 2 R2L1 R2L 2 K3 7. kl R2 L1 L2 R1L1 R1I..2 (,()MK 7708011.1 7708030 7708050 7708070 7708090 7708110 7708130 7708150 7708170 7708190 7708210 7708230 7708250 7708270 7708290 7708310 7708330 7708350 770B370 7708390 770841D 7708430 7708450 7708470 7708490 7708510 770B530 7708550 7708570 7708590 7708610 7708630 7708650 7708670 7708690 7708710 7708730 7708750 7708770 7708790 770B810 7708830 7708850 7708870 7708890 7708910 7708930 fc XPR SGP E1 E3 E5 fc 7 fc 9 E. 11 E 13 E 15 E 17 E 19 E. 21 E 23 E 25 E. 27 e: 29 E 31 E 33 E 35 fc. 37 E 39 E 41 E 43 E 45 t; 47 E 49 E' 51 E S3 E E, 57 t 59 F 61 . t. 63 F 65 F. 67 E 69 t 73 t 73 F 75 fc 77 fc 79 fc 81 fc 83 t 85 fc 87 fc B9 i- 91 F 93 (0) (0) (0) (0) (0) (0) (0) (()> (0) (0) (0) <03 (0) (0) <0> (0) (0) (0) (03 (03 (0) (03 (03 (03 (03 (03 (0) (03 (03 (0) (03 (03 (03 . (03 (03 (0) (0) (03 (03 (03 (03 (0) (0) (0 3 (0) (03 (03 CAGF 161 161 161 .161 161 163 163 163 163 163 165 .165 165 165 165 167 .167 167 167 167 169 169 169 169 169 171 171 171 171 1 71 1 73 173 173 173 173 1 75 1 75 175 175 1 75 177 1 77 1 7/ 177 1 77 179 1 79 fc AKk 7 R1 R2 LI L2 K1L1 R1I.2 R2H R2L2 R3 2 R1 R2 LI L2 R1L 1 Rll. 2 R2.L1 R2L.2 R3 2 R1 R2 LI L2 R.1L1 R1L2 R2.L1 R2L.2 R3 / RJ R7 1.1 I..2 Rll.1 R1.L2 R2L1 R21 2 K,> / Kl Kl* L1 L 1' KH 1 KU i ANNEX 20 (CONT. 9) 1 Wil 3 s s reference listing NO. 1 0 ZERO REF ERF NOT in'll! - 7 i .' 8/7\ R&S 006567 MALES MR EXFR SGR CAGE EARR 8960 8980 9000 9020 904I'i 90611 9080 9100 i91.20 >9140 >9160 >9181 >9200 192.20 19240 >9261 19281 19301 19320 9934 0 09360 09381 09401 09420 09440 094 60 09480 09500 09521 09540 09560 09580 09600 09620 09640 09660 09680 '09700 09720 '09740 '09761 '09780 '09801 '09820 '09840 '09860 '09880 '09901 '09920 '09940 '09960 ,'09980 '10000 t 96 F 98 F 1 0(1 1102 E104 t 106 F 1 08 F 1 10 FI 1.2 El 14 F`. 11 6 F 1 18 F.120 F. 1 22 E124 F" 126 Fine F I 30 El 32 E134 El 36 F. 138 F 1 40 FI 42 E14 4 F 1 46 F 148 F 3 50 E1.52 F. 154 F 156 F 158 F 160 FI 62 E.1 64 F 1 66 F 1 68 El 70 El 72 1 1 74 F 1 76 F 1 78 E1 SO F 182 F 1.84 E186 t 188 F 1 VO F I 92 F 194 FI 96 F I 98 F 200 (01 (0 > (01 (01 (0) (0) (0) (0) (0) (0) (0) (0) (01 (01 (01 (01 (0) (0) (0) (01 (01 (01 (0) (0) (01 (0) (01 (0) (01 (01 (01 (0) (01 (01 (01 (0) (0) (01 (01 (0) (0) (0) (01 (0) (0) (0) (01 (01 (0) (0) (01 (0) (01 180 .1.80 iso 182 182 182 182 182 1 84 184 184 184 3 84 186 186 186 3 86 1 36 1 83 188 188 1 88 188 190 .190 i yo 1 90 1 90 1 92 192 1 V? 1 92 192 194 194 194 1 94 1 94 1 96 196 196 1 96 1 VA 198 1 * `ii .1 V8 1 98 3 98 200 70<> 70(1 200 20(1 K2L1 R2L2 K3 7 Kl R2 LI L2 K1L1 Rlt.2 R2L1 R2L2 R3 7. R1 R2 t.l 12 Rill RH.2 R2L1 R2L2 ft 3 7 R1 R2 LI 1.2 Kit 1 R1L2 R2L1 R2L2 R3 7 R1 R2 11 l2 Rll 1 R1L2 R2L1 ft 21 2 K3 7. KI R2 LI l2 ft 1L 1 Rll 2 K2L 1 R2L2 R3 FFMALES CUMR F XF'R SGR CAGE FARR 770895D 770897D 7708998 77090111 770903H 770905D 770907D 77090911 770911H 77093 3D 7709158 7709178 7709198 7709218 7709238 7709258 77092.78 /709298 7709318 7709338 7709358 7709378 7709398 7709418 7709438 7709458 7709478 7709498 7709318 7709538 7709558 /709378 7709598 770963 8 7709638 7709658 7709678 7709698 7709718 7709738 7709738 7709778 7709798 7709818 7709838 7709858 7709878 7709898 7709918 7709938 7709958 7709978 7709998 F 95 F. 97 F 9V E 103 E103 El 05 F 107 E 109 E.l 11 El 13 El 15 E 11 7 F l 39 F.121 1123 F 3 23 k 127 E129 F.3 31 El 33 E.l 35 E 137 F 139 El 41 El 43 E.l 45 E 14 V E 1 49 F 1 51 El 33 F. 3 53 E 137 E 1 5y E 161 E.l 63 E 163 E 1 67 E169 El 71 E 173 E 173 E 17 7 El 79 F 1H1 F 183 El 83 F. 187 F 189 F 191 1 1 93 El 93 El 9 7 fc 1. V V (0) 17V (0) 1 (Ol 179 (0) 181 (0) 181 (0) 3 8.1 (0) 181 (0) 181 (0) 183 (0) 183 (0) 1 83 (0) 183 (0) 183 (01 1C5 <0> 3 83 (0) 1 85 (0) 185 (0) 185 (0) 187 (0) 187 l(>) 187 (0) 137 (0) 187 (0) 1 89 (0) 189 (0) 189 (0 i 1 89 (0) 1 89 (0) 193 (0) 191 (0) 191 (0) 191 (0) 3 VI (0) 1 93 (0) 1 93 (01 193 (01 193 ((11 J V3 (0) 1 9'.. (01 193 (0) 195 (0) 195 (0) 1 95 (01 197 (0! 197 (0) 19/ (0) 1 9 7 (0) 197 (0) 1VV (0) 1 99 <01 1 99 (01 1 99 (0) 1 99 K2I 1 R.'l 2 K3 '/ R.t ft'7 LI 12 Rll, 1 RU 2 R2L 1 R2L2 R3 1 K'l R2. 1. 1 L7 Rll 1 Kill' R2I.1 R21 2 K3 7 Kl R2 l1 1.2 EMI 3 ft' 1L. 2 ft'2L 1 ft 21 2 ft`3 7 ft'1 K2 1. 1 17 ftll 1 ft 3 L'2 R2L.1 R2L.2 ft 3 / ft! R7 LI 12 ftll J ft ,11 7 K7I 1 R7L:: ft 3 J1AL MALES 500 [CJTAI FF.KAI.tb 300 ANNEX 21 VO'' 1 ND It'. - ROSS REFERENCE LI S'T I N G .sat no. 126 o MALES FEMALES CO MR ' EXRF: SGF' CAGE EARR COMR EXRR SGF' C AGL E ark R&S 006568 7B0002 -'80004 780006 780008 780010 ' 780012 780014 780016 780018 780020, 780022 780024 780026 780028 780030. 780032 780034 780036 780038 780040 7S0041T 780044 780046 780048 780050_ 780052 780054 780056 780058 780060 780062 780064 780066 780068 78007& 780072 780074 780076 780078 780080. 7S00B2 780084 7800B6 780088 78009&. 780092 780094 780096 780098 780100 An A4 A6 A8 A 10 A 12 A 14 A 16 A 18 A 20 nT 84 B6 Es 8 B 10 B 12 B 14 B 16 .B 18 B 20 C2 C4 c6 c8 c 10 c 12 c 14 c 16 c 18 c 20 El *? B4 D6 B8 D 10 D 12 D 14 n If I' 18 D 20 En E4 E6 E8 E 10 E 12 E 14 E 16 E 18 F 20 (0) (0) <0> (0) (01 <01 (0) (01 (0) (01 (01 <01 (01 (0) (01 (01 (0) (01 <0> (0) (0) (01 (01 <01 (01 (0) (0) (01 <01 (0) (01 (0) (01 (01 (0) <01 (0) <01 (01 (0) (0) (0) <01 (01 <01 (01 (0) (0) . <0) (01 Z n RJ 2 R2 2 LI *7 L2 4 R1L1 4 R1 L 2 4 K2L1 4 R2L2 4 52- 6z 6 R1 6 R2 6 LI 6 L2, 8 R1L1 8 R1L2 8 R2L1 . 8 R2L2 8 FJL 10 Z 10 R1 10 R2 10 LI 10 k312 R1L1 12 R1L2 12 R2L1 12 R2L2 12 5314 z 14 R1 14 R2 14 LI 14 L2 16 R1L1 16 R1L2 16 R2L1 16 R2L2 16 R3, 18 Z 18 R1 18 R2 18 LI 18 L2 20 R1L1 20 R1L2 20 R2L1 20 R2L2 20 R3 780001 780003 780005 780007 780009 780011* 780013 780015 780017 780019_ 780021 780023 780025 ' 780027 780029 780031 780033 780035 780037 780039 780041 780043 780045 780047 780049 780051 780053 780055 780057 780059 78006T 780063 780065 780067 78006%. 780071 780073 780075 780077 780079_ 780081 780083 780085 780087 780089_ 780091 780093 780095 780097 780099 A1 A .3 A5 A7 A9 A 11 A 13 A 15 A 17 A 19 B1 B3 B5 B7 B9 B 11 B 13 B 15 B 17 B 19 C1 C3 C5 C7 C9 C 11 C 13 C 15 C 17 C 19 n1 D3 Ii 5 n7 D9 EI 11 D 13 D 15 I> 17 D 19 E1 E3 E5 E7 E9 E 11 E 13 E 15 E 17 E 19 (0) 1 (0) 1 (0) 1 (0) 1 (0) 1 <0> 3 (0) 3 (0) 3 (0) 3 (0) 3 (0) 5 (0) 5 (0) 5 (0) 5 (0) 5 (0) 7 (0) 7 (0) 7 (0) 7 (0) 7 (0) 9 (0) 9 (0) 9 (0) 9 (0) 9 (0) 11 (0) 11 (0) 11 (0) 11 (0) 11 (0> 13 (0) 13 (0) 13 (0) 13 <0> 13 (0) 15 (0) 15 (0) ' IS (0) 15 (0> 15 (0) 17 (0) 17 (0> 17 <0> 17 (0> 17 (0) 19 (0) 19 (0) 19 (0) 19 (0) 19 z R1 R2 LI L2. R1L1 R1L2 R2L 1 R2L2 R3 . Z R1 R2 LI LiL K1L1 R1L2 R2L1 R2L2 z R1 R2 LI Lg_ R1L1 R1L2 R2L1 R2L2 R3 z Rl R2 LI LJL R1L1 R1L2 R2L1 R2L2 R3 Z Rl R2 LI L2_ R1L1 R1L2 R2L1 R2L2 R3 TtVlAI. males 50 TOTAL FEMALES = 50