Document O3x5j0nwZyjaE5VqQv5b1jp4v

TO: Distribution o FROM: DATE: Interoffice SUBJ: Communication T. G. Grumbles January 1~:--1900" <?-3o V7 MONSANTO FYI TSCA STUDY SUBMITTAL: VKIADODECYLBENZENE TOXICOLOGY Attached, is a copy of the paper that Monsanto submitted to EPA. Jim Mieure says their toxicologist has had no luck in trying to get more information regarding this report. T. G. Grumbles aj o .207 Attachment cc 0. C. Kerfoot A. M. Nielsen C. M. Starks VVV 000015032 PINAL REPORT ON THE CHARACTERIZATION OP THE CARCINOGENIC NATURE OP DODECYLBENZENE AS DELIVERED BY ESSOCHEM, IDENTIFICATION CODE NO. MRDE-7 A*********************************'****************************** The conditions of the test were formulated in a Research and Testing Services Contract between Essochem Europe and Universitetets Institutt for patologi, Rikshospitalet, Oslo, signed by R Wilson on behalf of Essochem on August 26th 1983, and by Olav Hilmar Iversen on behalf of the Pathology Institute September 13th 1983 Several interim reports have been submitted during the study. MATERIALS AND METHODS The idea was to test the carcinogenic potency of Dodecyl- benzene using skin applications on hairless mice. Dodecylbenzene in vehicle should be given in an amount of 50^ul twice a week. Some groups of mice consisted of 56 animals, 28 males/females, others consisted of 48 animals, 24 males/females (see below). Test animals used were hairless mice, hr/hr Oslo strain, pur chased from Gamle Bomholt Gaard, Aarhus, Denmark. DMBA was from Eastman Organic Chemicals, Rochester, N.Y., USA All acetone used was of reagent grade. The Dodecylbenzene was delivered by Essochem and was dissolved in reagent grade acetone. Both DMBA and Dodecylbenzene were given in various concentra tions with a graded pipetter. One negative control group (1.) (56 mice) was painted with 50^ul reagent grade acetone twice a week. One positive control group (4.) (48 mice) was given one single application of 51.2yUg DMBA in acetone. A third group (2.) (56 mice) was painted with 16% and another one (3.) (56 mice) with 80% Dodecyl benzene twice a week, and finally a group (6.) (48 mice) was first given 51.2^ug DMBA and thereafter continual treatment twice a week with 40% Dodecylbenzene. oooo^033 2 CRITERIA FOR HISTOLOGICAL ASSESSMENT By histological investigation it is easy to decide when there is an ulceration, a pronounced hyperplasia, excess pigmen tation, dermatitis, or whether it is a benign papilloma or a squamous cell carcinoma or other form of malignancy in the skin. Sometimes it may be difficult to distinguish between a spindle cell sarcoma and a spindle cell squamous carcinoma. In those cases where one could see clear connection between the epitheli um and the spindle cell tumor, these tumors were classified as spindle cell squamous cell carcinomas. In the cases where there was connective tissue between the tumor and the epithelium, the tumor was characterized as a sarcoma of the skin. The spleen was often enlarged, often hyperplastic, and some times changed by amyloidosis, which is a degenerative lesion with precipitation of a protein substance. Amyloidosis was some times also found in the kidneys and in the liver. Lung adenomas are known to be produced by many carcinogenic substances, especially urethan. In a control study at this labo ratory we have found a spontaneous frequency of about 2-3 adenomas per 56 mice. lung In swollen lymph nodes and swollen spleen we found some mal ignant lymphomas. As known from rodent pathology, collection of plasma cells in the spleen or lymph nodes should be registered as reactive changes and not as plasmocytomas. Sometimes lympho cytic infiltration was seen in the lung, in the liver and in the kidneys, indicating spread of lymphomas. When there was an obvious proliferative lesion in lymph nodes, but not an obvious malignant lymphoma, the lesion was classified as a reticulosis. The criteria for diagnosing malign ant lymphomas/leukemia in mice have been described by S. Wogan: "Tumors of the mouse hematopoietic system. Their diagnosis and interpretation in safety evaluation tests. Report of a study group. CRC Crit. Rev. Toxicol. 13 161-181, 1984". VVV 000015034 3 OBSERVATION The animals were painted twice a week and observed in con nection with the painting. In the DMBA alone group the animals were observed once a week. The mice were observed weekly for skin lesions or swollen lymph nodes. All mice that died or were killed were autopsied. Skin and inner organs were inspected. If any pathological lesion was found, lungs, liver, spleen, swollen lymph nodes and kidneys were embedded in paraffin. Histological sections were made and the slides were studied microscopically. DOCUMENTATION The following documentation was used: A list with a description of the experiment and the number of the experiment, and what was applied to the animals. Each mouse on these lists was given a number and allocated to a cage with 8 mice in each. Altogether we had 33 cages with 8 animals* in each. Each cage was labelled with number of mice, sex, birth date, treatment type and cage number. Within each cage each, single mouse got an individual number, and in the laboratory books each mouse was given at least one page. On each page in the book two outlines of a mouse were stamped, one for the backside and one for the abdominal side. Tumors were charted and noted by text. As soon as a mouse got a lesion, it was given a particular number. Mice no. 1 in a cage was cut in the left ear, no..2 was cut in the right ear, no. 3 in both ears, and no. 4 remained" un touched. No. 5 was cut in the tail and in the_ left''fear, no. 6 in the tail and in the right ear, no. 7 in the tail and both ears, and finally mouse no. 8 in each cage had*'only its tail cut. In this way the mice were labelled individually and could be identi fied at each observation. We have found this way of identifying mice better than fixing a metal number to their ears, since such numbers can easily fall off. WV 000015035 4 In addition to the fir9t mentioned list and the laboratory ^ books, there was also for each experiment produced a big table on graph paper in which each single mouse had a horizontal line and each week was^designated on top of a vertical column. When skin lesions or swollen lymph nodes occurred this was drawn on the table, and from the start of the line for each tumor and onwards in time a line was drawn. When a mouse died this was marked with a cross, and at the end of the line the most import ant histology diagnoses were written. The whole experiment was performed under the responsibility of Professor Olav Hilmar Iversen, and the Head of the Laboratory for Experimental Animals, Or.med.vet. Leif Schierven. A great amount of the practical work was made by a student (Marit Berg) , and from October 1984 by a veterinary student (Inge Engeland). The personell in the Animal Laboratory also took care of the mice, changing in the cages, providing fresh water and food. STATISTICAL EVALUATION On the basis of the crude incidence of skin tumors, statis tical investigation was made. The results are presented as tumor rates (the percentage -of tumor-bearing animals in relation to the number of animals alive at the appearance of the first tumor related to time) and tumor yields (the cumulative occurrence of all skin tumors related to time) in all groups. To evaluate differences in tumor rate, we have used the meth od for "non-incidental" tumors described by Peto and elaborated with a computer-based test program by Peto et al. This program takes into account varying mortality rates among the experiment al groups, and assesses both the number of tumor-bearing animals and the time of the first tumor in each animal. To evaluate the differences in tumor yield, we have used the method of Gail et al. based on "multiple times to tumor". Method 3. This method assesses the number of tumors appearing, varying mortality between the groups, and the time of appearance of each tumor. Ol503b yvV 000 5 Finally the numbers of malignant skin tumors, lymphomas and lung adenomas in each group were analyzed with the Chi-square test. Since these variables may not be normally distributed, the Chi-square analysis is only a rough estimate. RESULTS Skin tumors These are shown in Table 1 and in Figs. 1 and 2. Statistical evaluation is shown in Table 2. By tumor rate is meant the per centage of tumor-bearing animals in relation to the number of animals alive at appearance of the first tumor in relation to time, and by tumor yield is meant the total number of tumors cal culated for 56 starting mice. Hence, the values found for the groups with 48 mice have been multiplied with 56/48 1.1667. These represent the values that would have appeared in a group of 56 mice with the same tumor yield as that observed in 48 mice. When the experiments are ranked as shown in Table 2, first* column, there is a very significant trend. As to the positive control groups shown (Groups 4 and 5), we have for statistical investigation used both the control group of this study, plus a control group comprising all mice which in this institute have been painted once with 51.2yUg DMBA, altogether 128 mice. It is obvious, as can be seen from Table 1 and the curves that there can be no statistically significant difference between the effects of acetone alone and those of 16% Dodecylbenzene. They have therefore not been statistically tested. There is, however (Table 2), a significant difference between acetone alone and 80% Dodecylbenzene, but no difference between 16% and 80% Dode cylbenzene, There is a very significant difference beteween acetone and DMBA initiation, but there is only a suggestive difference between DMBA alone and DMBA followed by 40% Dodecylben zene as regards tumor-bearing animals, and tumor yield when con current controls are used. There is a significant difference in total tumor yield for DMBA plus 40% Dodecylbenzene when compared with historical controls VW 000015037 6 Hence, as regards skin tumor it can be concluded that 80% Dodecylbenzene alone is a weak, complete tumorigen, whereas if 16% Dodecyibenzene has a carcinogenic potency, it is below mea surable level. As regards 40% Dodecylbenzene asan enhancer, there is a weak promoting activity. Lymphoid tumors Eighty per cent Dodecylbenzene gave 6 lymphomas in 6 ani mals, whereas the acetone group gave only 2. DMBA alone and DMBA followed by Dodecylbenzene gave 5 and 4, respectively- If reticu loses are also counted, there seems to be no significant in- J crease in lymphoma incidence. Lung adenomas For lung adenomas there is no significant difference between controls and Dodecylben2ene-painted animals, and the same refers to other tumors occurring. Amyloidosis As to the degenerative lesion amyloidosis, there is a sig nificant increase in amyl-oidosis after 80% Dodecylbenzene. The explanation for this is difficult to express, since the mecha nisms behind amyloidosis development are partly unknown. How ever, 80% Dodecylbenzene seems to be generally somewhat toxic to the animals. Skin toxicity As to skin toxicity, the most pronounced hyperplasia and some ulcerations were seen after 80% Dodecylbenzene and after DMBA followed by 40% Dodecylbenzene. So it can be concluded that there is some skin toxicity for hairless mice exerted by Dodecylbenzene. yVV 000015038 7 FINAL CONCLUSION It can be concluded that Dodecylbenzene has a weak skin tumorigenicity on mice, both as a complete carcinogen and as re gards enhancement of DMBA carcinogenesis. There may also be a weak effect on the lymphoid system, but this is not really signi ficant . It is difficult to say whether such results in animals are of significance for the human situation. It is traditional to accept tumorigenicity in animals as a sign of possible carcino genic hazard for humans. If the result of this test should be of any significance, one would say that Dodecylbenzene may be a weak, complete carcinogen and a weak promoter. Since humans are usually more resistent than mice, and the latency time is much longer, one might venture the opinion that the hazard is prob ably small and of littlev practical significance. Generally. good hygiene at the working place with reduction of exposure as much as possible, seems to me to be the only necessary precautions. Oslo, 23rd September 1985 Olav Hilmar Iversen Professor of Pathology WV 000015039 TABI 1 - CARCINOGQ'IICITY CF DCCECYLBDJZENE - TTJMCR ANALYSIS AM) PATHOLOGICAL FINDINGS Group no.: 1 2J 4 & Treatment: Type of turor: Acetone Dodecyibenzene 16% 80% dmER alcne E*MT+ 40% dcdecylbenz. Skin tumors: Papillomas Carcinomas Saroanas Sun skin tumors: 3/2 0 0 3/2 Lymphoid tumors: B-cell l^mphana Lymphoma NOS Sum lymphomas: Reticuloses Sum lymphoid lesions: 0 2/2 2/2 3/3 5/5 Lunq adenomas: Per oent of autopsied 4/3 10 Other tuners: Abdominal sarcoma Angiosarcoma of liver Hepatana 1/1 0 0 Amyloidosis: Per cent of autopsied 2/2 5 Skin tcocicity: Pronounced hyperplasia Ulcerations 0 0 4/4 13/9 1/1 1/1 1/1 0 6/6 14/9 56/27 6/6 0 62/27 68/26 6/6 7/7 81/26 00 2/2 6/6 2/2 6/6 4/4 1/1 6/6 7/7 8/6 6/6 20 11 1/1 4/4 5/5 2/2 7/7 16/12 27 0 4/4 4/4 2/2 6/6 7/5 13 1/1 0 0 1/1 0 1/1 0 0 0 9/9 22/22 14/14 23 39 23 1/1 1/1 0 6/6 n 4- 0 4"4 0 4- 0 VVV 000015040 FJcg . 1 Skin carcinogenicity of Oodacylb tumor* rata* ( % t.umo r--bearing an versus tlms ooooi'0*1 <9 2 Skin carcinogenicity ot Oodecy lfc>r- - tumor- yields (total number or tumor's) ver sus time ivi m a to o m c c j 90 Group No I BO ,,a. 6(13 malignant) TO 60 /* 4 (6 malignant) 60 404 -) 30' * 204 S > 10* < < < o o oo-- ui o * N Ls / y ^-----a 3(1 malignant) 2 (2 malignant) 1 (no malignant) f 'I 1 11 i Bir if t i `pi i iMir 14 16 22 26 30 34 36 42 46 60 54 66 62 70 74 78 0 Tima in waaka TABU 2 - SKIN CMCIMOGENICITf Of DOOBCYLBOCn* - STATISTIOL PNOKTICN 7\jc*- rates -- Ovenl 1 1 v 5 1 v4 1 v5 6v4 6 v5 ofcs/exp cba7enp ctos7ep cfcs?eKp ctoa7exp cba7exp Ty7 odda TVT odds Timor yields 1 V5 Odds Tv~4 Tvl> odd* 1. Acetone alone 0 00 0.26 0.08 0.06 1 00 0.0) 0.04 2. 16% Oodecylberoene alone 0.30 3. 80% Dodecylbenzene alone 0.56 4 S1.2^ug nek once ItMs study) 2.27 5 51.2^ ere* cnee (128 alee) - 6- 5i.2^u? OCA one and 40% dodc cyl benzene 2.65 T 39.01 1.94 2.91 2.39 1.12 1.49 1.37 11.64 15.70 0.89 2.43 0.89 6 18 8.69 ueek 57) 1.00 1.00 1.56 (week 38) 1.55 (week 39) 1.00 1.00 One-tailed P value for positive trend 0.0001 0.01)0 <0.0001 <0.0001 0 2274 0.0406 CM KpUV 64.40 4.96 29.26 23 97 0.56 3 04 Degree of freedom 4 1 1 l1 1 P value for heterogeneity <0.0001 0.0260 <0.0001 <0.0001 0.4547 0.0811 545 28.82 2522 3.34 till 4.52 1 VVV 0 0 0 0 1 5 0 4 3 Best P value for tumor yield Conclusion v.a 0.025>P>0.010 <0 0005 a. v.a. v.a. n.a. augg. v .a. <0.0005 v.a. 0 10>P>0.05 0.05>P>0 025 *jgq.