Document dQ3gvbqk0OyjerrxE91dLO5BR

AR226-3105 TNO-report V98.685 Augmented acute (4^iour) inhalation toxicity study witt^wo different batches of rats TNO Nutrition and Food Research Institute Utrechtseweg 48 P.O. Box 360 3700 AJ Zeist The Netherlands Phone +31 30 694 41 44 Fax+31 306957224 Date: November 1998 Authors): Dr H. Muijser Study director: Dr H. Muijser TNO Project number 480001/005 TNO Assay number 2034 All rights reserved No part of this publication may be reproduced and/or published by print, photoprint, microfilm or any other means without the previous written consent of TNO. In case thig report was drafted on instructions, the rights and obligations of contracting parties are subject to either the Standard Conditions for Research Instructions given to TNO or the relevant agreement concluded between the contracting parties Submitting the report for inspection to parties who have a direct interest is permitted At the request of: ELF Atochem S.A. Paris La Defense, France Status: Final Previous version: Unaudited draft, July 1998 Number of pages; 81 TNO Company Sanded. Does not c^fa.n TSCA CR| TNO Nutrition and Food Research Institute is dedicated to Ihe (inler)national food industry, pharmaceutical industry and chemical industry. Tha Institute's divisions are: Agrotechnology, Analytical Sciences, Biochemistry and Gene Technolonv Industrial &jfinrr>hirtlft/,i< rvr-iir^iHwat V98.685 November 1998 Page 2 Summary The acute inhalation toxicity of two batches of| fIVfvas studied by nose-only exposure for a single 4-hour period in rats. One group of 8 male and 8 female rats was exposed to the limit concentration of 20.7 g/m1 offlB----HHIjiuring a single period of four hours (Exposure A) and tojUBU^at another group of 8 male and 8 female rats was exposed a limit concentration of 20.9 g/m3(Exposure B). In each experiment half of the rats was necropsied the day after exposure, the remaining rats were kept for a.14-day obser vation period. Four additional, unexposed rats were also sacrificed for background histopathology at each exposure. The test atmosphere was generated by passing metered amounts of the test material using a roller pump to an airless ultrasound nebulizer. Upon entering the exposure equipment, the aerosolized material was mixed with humidified, pressurized air. The mean aerosol concentration of the non-volatile fraction based on gravimetrical forf analysis was 107 3 mg/m3 fopl|------------|ind1 329mg/m3 fffrhe MMAD of the paniculate matter was 5.4 urn and 4.9 um and the geometric standard deviation was 1.7 and 1.9 for batcher--------^ndft| respectively. During exposure, slight abnormalities in breathing pattern could be seen, consisting of slightly decreased breathing frequency and minimal irregular breathing during both exposure A and B complemented with clear restlessness during the second half of exposure A and slightly laboured breathing developing during exposure B. Shortly after both exposures, abnormalities consisted of piloerection, blepharospasm, sluggishness and slight irregular breathing. Mortality was the most important rinding in this study. As a result of exposure A , 6 out of 8 animals intended to be kept for an observation interval of 14 days died within 1-2 days after exposure while none of the 8 rats intended to be necropsied the day after exposure died before that time. The occurrence of late mortality suggests that late mortality could be expected to have occurred in the animals of the 1-day observation period if this group had been kept for a 14-day observation period as well, indicating that total mortality (6 out of 16) may be underestimated. The same holds for exposure B: 4 out of 8 animals of the 14-day observation group died within 1-3 days and none of the rats intended to be necropsied the day after exposure. Clinical signs observed on day 1 and 2 of the observation period included blepharospasm, sluggishnessand abdominal breathing. In surviving animals no clinical signs were observed after the second day. No abnormalities were seen in the animals of the control group. One day after each exposure, body weight loss was seen in all exposed animals. .Company Sanitized. Does not conta^ T!?^A f^ V98.685 November 1998 Page 3 During the second week of the 14-day observation period, body weight gain was seen in surviving animals. Normal weight gain was seen in all animais of the unexposed control groups. Macroscopic lung changes (viz. discolouration and hyalin appearance) were seen in rats necropsied one day after exposure and in deceased rats in both experiments. Animals that survived the 14-day observation interval still exhibited pulmonary gross lesions which are considered to be related to exposure. Mean absolute and relative lung weights were generally increased in surviving rats. Changes at the microscopic level in deceased rats and in rats scheduled for necropsy after one day, consisted of alveolar haemorrhages, increased septal cellularity and perivascular polymorphonuclear leucocytic infiltration. Similar histopathological changes were seen in animals found dead on day 1-3 after exposure. Animals that survived the 14-day observation period still exhibited histopathological changes which are considered to be related to exposure. It was concluded that a single 4-hour (acute) exposure of rats to a hu concentration (i.e. 20.7 g/m3 foipH------fqand 20.9 g/m3 forJ| ofH^HIHlll^^HHHHHHIBMresuIted in mortality and macroscopical and histopathologicariungchangeisrr,espective of the batch used. Therefore, from the results of the present study, it cannot be excluded that human exposure to high aerosol concentrations will result in serious and lasting lung changes. -f-^zc^- y^ i50 eonsain ('SCA lib; V98.685 November 1998 Page 4 Contents Summary .........:.............................................. 2 Statement ofGLP compliance ........................................ 6 Authentication by cooperating scientist................................. 7 Quality Assurance Statement......................................... 8 GLP compliance monitoring unit statement ............................. 9 Testing facility ................................................... 10 Contributors ..................................................... 10 1 Introduction ............................................. 11 2 . Experimental.............................................. 11 2.1 Test materials ...:................................ 11 2.2 Test animals Experimental ..................................... 12 13 2.3 conditions procedures ............................ 14 2.4 Experimental 2.5 Exposure chamber ............................ 14 2.6 Generation of the te.s.t .a.tm..o.s.p.h.e.re...................... 14 2.7 Analysis of the test atmosphere ..................... 15 2.8 Observations and measurements ...................... 16 2.9 Retention of records ...................... 17 2.10 Deviations from the pr.o.t.o.c.o.l ......................... 17 ........................ 3 Results Exposure A NflH^HHK^........................ 18 3.1 Analytical results ................................. 18 3.2 Behaviour, clinical signs and mortality ................. 18 3.3 Body weights 19 ..................................... 3.4 Lung function measurements ........................ 19 3.5 Lung weights ..................................... 19 3.6 Pathology........................................ 20 4 Results Exposure Bfi^U^U^^B^^B^^--H--IR^i 21 4.1 Analytical results ......................... 21 4.2 Behaviour, clinical .si.g.n.s.a.n.d..m.o.r.t.a.li.ty.................. 21 4.3 Body weights ................. 22 4.4 Lung function ............ measurements . . . . . . . . . . . . . . . . . . . . . . . . . 22 4.5 Lung weights ........................ 22 ..................................... Company Sanitized. Does not contain TSC& r.W V9B.685 November 1998 Page5 4.6 Pathology........................................ 22 5 Discussion and conclusion .................................. 24 6 References .............................................. 25 Figure 26 Tables 27 Appendices 62 Annexes .. 80 Company Sanitized. Dees no$ confai'n TSCA CR? V98.685 November 1998 Page 6 Statement of GLP compliance We, the undersigned, hereby declare that this report constitutes a true and complete representation of the procedures followed and of the results obtained in this study by TNO Nutrition and Food Research Institute, and that the study was carried out under our supervision. The study was carried out in accordance with the OECD Principles of Good Laboratory Practice. ^-' Dr H. Muijser (Study Director) Drs H.H. Emmen (Management) ^<V ^A^-L^ l^ S Date 3 v Date I / 0 ~ c> vye-^-^ ' ' '-i"^ \ 'Company Sanitized. Does not conta?n TSCA cnl V98.685 November 1998 Page? Authentication by cooperating scientist I, the undersigned, hereby declare that the pathology data presented in this report were compiled by me or under my supervision, and accurately reflect the raw data DrR.A.Woutersen (Pathologist) ^ A^^- ^ Date Company Sanitized. Doss rsof cor-ifn?" vfA ^a Company Sanitized. Does TTolconIaTn TSCA~bi V98.685 November 1998 PageS Quality Assurance Statement On: Report Number: Date: Augmented acute (4-hour) inhalation toxicity study with ter----^--TM--) fl^yvn rats '^V998^^85 November 1998 The protocol and the experimental phase of this study were inspected by the Quality Assurance Unit of TNO Nutrition and Food Research Institute as follows: Date of inspection: 17 March 1998 (protocol) 23 March 1998 Date of report: 17 March 1998 23 March 1998 This report was audited as follows: Dates of audit: 24-27 November 1998 Date of report: 27 November 1998 I, the undersigned, hereby declare that this report provides an accurate record of the procedures employed and the results obtained in this study; all inspections were reported to the study director and the management on the dates indicated. t ^ Ing. P.A. de Lang (Quality Assurance Officer) Date:3(? /I'c-Lt'-^^'i- 1'/'/^' ^^^s.a^ss""80^1 1 ifu repon V98.685 November 1998 Page 9 STAATSTOEZICHT OP DE VOLKSGE20NDHEID VETERINAIRE HOOFDINSPECTIE ENDORSEMENT OF COMPLIANCE WITH THE OECD PRINCIPLES OF GOOD LABORATORY PRACTICE Pursuant to the Netherlands GLP Compliance Monitoring Programme and according to Directive 88/320/EEC the conformity with the OECD Principles of GLP was assessed on 22-26 April, 15 May and 9 September 1996 at TNO Nutrition & Food Research Institute Toxicology Division Utrechtseweg 48, P.O. Box 360 3700 AJ Zeist, The Netherlands It is herewith confirmed that the afore-mentioned test facility is currently operating in compliance with the OECD Principles of Good Laboratory Practice in the following areas of expertise: Toxicity; Mutagenicity; Environmental Toxicity on aquatic and terrestrial organisms; Behaviour in water, soil and air; Residues; Effects on mesocosms and natural ecosystems; Analytical and clinical chemistry; Drug metabolism; Pharmacokinetics. ^B^ySarfiizea.DoesnolconlainTS^ C* /y/-^-^yT^R* I.^"R;:f^jg..wijk, 10 September 1996 ^i^--- |'2. We^^^::;^;^!^^i.: ' v '' './'' . . YMlnJstr.y of. Health, Th. Helder, Welfare and DVM Sport State Supervisory Public Health Service Veterinary Public Health Inspectorate V9S.685 November 1998 Page 10 Testing facility The toxicity study was conducted by: TNO Nutrition and Food Research Institute Toxicology Division P.O. Box 360, 3700 AJ ZEIST, the Netherlands Telephone+3130 69 44 144 Telefax+3130 69 60 264 Visitors address: Utrechtseweg 48, Zeist, the Netherlands Contributors Major contributions to this study were made by: Study Director Deputy Study Director: Senior Inhalation Technician Inhalation Technicians Biotechnicians Senior Biotechnician Pathologist Assistant pathologist Dr H. Muijser* Ir J.H.E. Arts Ing S.M. Spoor Mr F. Hendriksma / Mr W.G. Roverts Mr D.C. Veldhuysen / Mr G. de Kruijf Mr G. van Beek Dr R.A. Woutersen Mr J.P. Bruijntjes * Toxicology Division, TNO Nutrition and Food Research Institute ___________RmwmBf--s'--^--.--^.,. ,,,. ,,, '^oS'fpanytanmzeS.Soesnore-omaTnTSeA^eBi t\-' ICpUll V98.685 November 1998 Page 11 Introduction At the request ofELFAtochem S.A., Paris La Defense, France, an acute (4-hour) WU^BBqwas carried out in rats. In addition lung function measurements, lung weights and histopathology of the lungs were used as criteria for disclosing possible harmful effects in the present study. The study design was ~ 3 based on an augmented acute inhalation toxicity study (TNO reportjjB^, JJuunnie 1993). 2 Experimental The study was conducted according to a protocol, entitled: "Protocol for augmented acute (4-hourt inhalation toxicity study witljUB^^^^^g by the study director on 3 March 1998. The protocol had been drafted in accordance with the OECD Guideline for Testing of Chemicals no. 403, Acute inhalation toxicity, adopted 12 May 1981 and the OECD Principles of Good Laboratory Practice. 2.1 Test materials Three test materials were supplied by the sponsor: two batches o: and one batch o^jl^BM^BFj jeach containing according to the sponsor 1 (test material, gross "heights731, 747 and 746 grams were received on 13 March 1998. The TNO internal reference number was 980116. The test material was stored at ambient temperatures in the dark. . Company Sanitized. Does not contain TSCA CRF V98.685 November 1998 Page 12 i plastic bottles, labelled ^^^^^^^^^^^^^H^rfeBach 1-- - containing according to the sponsor 1 C test ----- material, gross weights 699, 724 and 749 grams, were received on 13 March 1998. The TNO internal reference number was 980117. The test material was stored at ambient temperatures in the dark. ; plastic bottles, labelled | ^^SySSSmSKy&ch containing according to the sponsor 1 Ctest material, gross weights 756, 775 and 779 grams, were received on 13 March 1998. The TNO internal reference number was 980118. The test material was stored at ambient temperatures in the dark. The animals used were male and female SPF-reared, Wistar derived (Crl:[WI]WU BR) rats obtained from Charles River Wiga, Suizfeld, Germany. The animals used for exposure A arrived on 11 March 1998, at an age of ca. 7-8 weeks. They were taken in their unopened shipping containers to animal room 15.07, were checked for overt signs of ill health and anomalies, and were kept in quarantine. After approval of the lot (negative liters to microorganisms tested), they were transferred on 13 March 1998 to animal room 6.0.04; the rats were randomly allocated to the cages, separated by sex and uniquely identified by ear tattoo. On 18 March 1998, 5 days before the start of exposure A, 8 male and 8 female rats were assigned to the ft study to be exposed to the present test material in two subgroups of 4 rats for each sex. In addition, 2 male and 2 female rats were assigned to the study as controls for --: ^eO, ffipany Sanitee-d. Qoes'no? corn-am T.^A V98.685 November 1998 Page 13 background histopathology. Three days before exposure, mean body weights of the male and female rats were 265 and 177 g, respectively. *The animals used for exposure B were similarly treated: arrival on 25 February 1998 at an age ofca. 5-6 weeks in room 15.07 and on 2 March 1998 in animal room 6.0.04, assigned to the study on 23 March 1998 (one day before exposure B), mean body weights at that time for male and female animals were 251 and 182 g, respectively. The groups of animals were identified by a letter (A or B) and a subcode (s 1, s2 and s3) and a colour code (white or blue). In this report, for the sake of clarity, the different (sub)groups are abbreviated as Al, A2, A3, B 1, B2 and B3, respectively. 2.3 Experimental conditions 2.3.1 Maintenance During exposure the animals had no access to feed or water and were housed individually in the holders. After an exposure, one subgroup (Al or B 1) was immediately returned to their living cages, held for an observation period of one day and sacrificed. The second subgroup (A2 or B2) was first subjected to lung function measurements before being returned to their living cages, subsequently held for an observation period of 14 days and sacrificed. The rats that entered the study as controls (subgroups A3 and B3) were sacrificed on day 14 for background lung histopathology. The animals were housed in animal room 6.0.04, 4 males or 4 females to a cage (Al, A2, B 1 and B2), or 2 males or 2 females to a cage (A3 and B3). The animals were housed under conventional conditions in suspended stainless steel cages fitted with wire-mesh floor and front. The number of air changes was about 10 per hour. The temperature was between 20.5 and 23.00C and relative humidity was between 50 and 70%. A 12-hour light and 12-hour dark cycle was maintained. - 2.32 Diet and drinking water All rats were fed a commercially available rodent diet (Rat & Mouse No. 3 Breeding Diet RM3) from SDS Special Diets Services, Witham, England. Each batch of this diet is analysed by SDS for nutrients and contaminants. The certifi cate of analysis pertaining to the batch used (Batch no. 4177) will be kept with the raw data in the Archives of the Institute. Tap water suitable for human consumption (quality guidelines according to Dutch legislation based on EEC Council Directive 80/778/EEC) was supplied by N.V. Waterleidingbedrijf Midden-Nederland (WMN). Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier are made available to TNO Nutrition and Food Research Institute. In addition, WMN periodically analyses water samples taken on the premises of TNO .Company Sanitized. Does'not contain TSCA C*^ V98.685 November 1998 Page 14 in Zeist for a limited number of variables. The results of these analyses will be kept with the raw data in the Archives of the Institute. 2.4 Experimental procedures Exposure was started on 23 March 1998 with group A consisting of 8 male and 8 female rats. This group was exposed by inhalation to the limit concentration of at single 4-hour period. One subgroup of 4 male and 4 female animals was necropsied the following day. The other subgroup of 4 male and 4 female rats was intended to be kept for an observation period of 14 days and to be subsequently sacrificed. Four additional rats were also sacrificed on that day for background lung histopathology. Group B was exposed on 24 March 1998 to ^HHIHIBBBlHHii^Brr1^ batch|m|| treate<^ similarly. Thereafter, no more animals were exposed. The study was finished with the necropsy of all remaining rats on 6 and 7 April 1998. 2.5 Exposure chamber ' " Animals were exposed to the test atmosphere in a nose-only inhalation chamber, a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts. SG4 SUB, United Kingdom (see Figure 1). The inhalation chamber consisted of a cylindrical aluminium column, surrounded by a transparent cylinder. The column had a volume of ca. 50 Cand consisted of a top assembly with two mixing chambers, underneath a rodent tube section and the exhaust section at the bottom. The rodent tube section had 20 ports for animal exposure. Several empty ports were used for test atmosphere sampling, particle size analysis, measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Male and female rats of each group were placed in alternating order. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. In our experience, the animal's body does not exactly fit in the animal holder which always results in some leak from high to tow pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose and dilution of test atmosphere at the nose of the animals is prevented. 2.6 Generation of the test atmosphere The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, metered amounts of the test material were passed with a roller pump (Gilson, Villiers Ie Bel, France) to an airless ultrasound nebulizer (Sono-Tek Corporation, Poughkeepsie, NY, USA). Upon entering the exposure equipment, the aerosolized material was mixed with ^^yS^?z.iryoi?ioc<3^" rSCACBl V98.685 November 1998 Page 15 humidified, pressurized air. The resulting test atmosphere was then directed downward through the mixing chamber towards the animals. At the bottom of the unit the test atmosphere was exhausted (see also Figure 1). The settings of the nebulizer and rotameter were recorded at regular intervals (ca. twice per hour). Total air flow through the exposure chamber was 25 (/min (exposure A and B). All animals of subgroup Al were placed in the exposure unit 88 min (Bl: 87 min) after the start of the generation of the test atmosphere. To allow lung function measurements to be performed just after the end of exposure in the animals of group A2 and B2, the animals were placed sequentially, one at a time (balanced with respect to sex), with an interval of 10 min in the exposure unit. 2.7 Analysis of the test atmosphere 2.7.1 Total carbon analysis The actual concentration of the test material in the test atmosphere was monitored with a total carbon analyser (Bemath Atomic, model 3005). The response of the analyser was recorded by an analogue recorder (Kipp & Zonen, Delft. The Netherlands). Test atmosphere samples were taken continuously from the exposure unit at the animals' breathing zone and were passed to the total carbon analyser. The mean response was calculated by averaging values read every 5 minutes. The response of the flame ionisation detector (FID) of the total carbon analyser was calibrated by injecting two PET bags rilled with 50 liters of air for each concentration with 1000, 1500 or 1750 p(?, respectively, using batchRBBHH construct a calibration curve which was needed to convert the responses in scale units of the TCA to concentrations (g/m3). A linear relation with a correlation coefficient of 0.99 was obtained: Y= 3.3851X+0.3837 and Y was the response in scale units of the TCA. After exposure A the stability of the calibration was checked by measuring 2 PET bags each filled with 50 liters of responses were within 1% of the value predicted by the relation above. Exposure B could be monitored using the same relation, since according to the sponsor, the same with respect to the solvents used. The validity of this was checked after exposure B bvmeasuring^PET bags each filled with 50 liters of air and 1500 pf and 2.5%, respectively, of the value predicted by the calibration relation, which is satisfactorily small. companySanHized. Does not contaFn TSCA rm V98.685 November 1998 Page 16 2.7.2 Gravimetric analysis The amount (by weight) of non-volatile particles present in the test atmospheres was determined approximately once each hour by means of gravimetric analysis. Representative test atmosphere samples were obtained by passing 100 Ctest atmosphere at 5 (/min through fiber glass filters (Sartorius). Samples were weighed before and after sampling. Forced drying with dry pressurized air did not influence the sampled weight. The concentration of the non-volatile particles was calculated by dividing the amount of test material present on each filter by the volume of the test atmosphere taken. 2.7.3 Nominal concentration The nominal concentrations were determined by dividing the total amount of test material used by the total volume of air passed through the exposure unit. 2.7.4 Particle size measurement * -v Particle size distribution measurement was carried out once during each exposure using a 10-stage Andersen cascade impactor, with the largest cut-off size of 32 ^m. The Mass Median Aerodynamic Diameter (MMAD) and the mean . geometric standard deviation (gsd) were calculated (Lee, 1972). 2.7.5 Measurement of temperature, relative humidity and oxygen concen tration The temperature and the relative humidity of the test atmospheres were recorded six times during exposure at intervals ranging between half an hour and one hour using a RH/T device (TESTO 610, GmbH & Co, Lenzkirch, Germany). The oxygen concentration was checked once during exposure (Beryl, Cosma, Igny, France). 2.8 Observations and measurements ^ 2.8.1 Behaviour, clinical signs and mortality The rats were visually inspected just before exposure, for reactions to treatment during the exposure, shortly after exposure, and at least once daily during the observation period. 2.8.2 Body weights Body weights of the animals were recorded before exposure (exposure A: day -3, exposure B: day -1), just prior to exposure (day 0) and in surviving animals on day 1 and on days 7 and 14 (subgroups A2, A3, B2 and B3) and at death. ^^paRy Sahiiize'd. Does not contain TSCA C6a TNO report V98.6B5 November 1998 Page 17 2.8.3 Lung function measurements Lung function measurements, consisting of determination of breathing frequency, tidal volume, mean ventilatory flow, and lung mechanical properties were carried out in spontaneously breathing rats. Four male and four female rats of the test group (subgroup A2 and B2, destined for autopsy 14 days after exposure) were measured before exposure (day-1, control value) and surviving animals directly after exposure (day 0). Since mortality occurred, lung function measurements were discontinued, the results obtained will be kept with the raw data, but will not be reported. 2.8.4 Pathology A group of 4 male and 4 female rats (subgroup Al) was killed one day after exposure by exsanguination from the abdominal aorta under ether anaesthesia. On day 14 of the observation period the remaining test rats of subgroup A2 together with the four unexposed rats (subgroup A3) were killed in the same way. All rats were examined for gross pathological changes. The same procedure was followed for animals of group B. The lungs with trachea and larynx were removed and weighed. Lungs were inflated with and preserved in a neutral, aqueous, phosphate-buffered, 4% solution of formaldehyde and embedded in paraffin wax. Sections were cut at 5 urn of each of the five lung lobes, stained with haematoxylm and eosin and then examined microscopically at one longitudinal level. 2.9 Retention of records Raw data, the master copy of the final report and all other information relevant to the quality and integrity of the study will be retained in the archives of TNO Nutrition and Food Research Institute for a period of at least 15 years after submission of the final report. 2.10 Deviations from the protocol o^lHIHHIHRHHH should have been tested first and followed by a test of batcr mortality occurred and by a test mortality occurred. Erroneously, batct^fffly/as tested first. Since mortality resulted after exposure tojUB^Ehe reverse sequence of testing did not matter. - Due to an organizational change within the Toxicology Division on the I" of July 1998, management responsibilities for this study have been assigned to Drs H.H. Emmen as from that date. These deviations were not considered to have influenced the validity of the study. Company San?tfzed. Does nol contain T.<^A ^B| V98.685 November 1998 Page 18 3 Results Exposure A 3.1 Analytical results 3.1.1 Total carbon analysis The actual concentration of^____ ff)|during A based total analysis of the , __^___ volatile fraction exposure on carbon averaged at 5-min intervals was 20.7 0.5 g/m3 (N=68). 3.1.2 Gravimetric analysis olfffiWfSt LI______ The results of the gravimetric analysis are given in Table The calculated mean concentration of the solid fraction of the actual concentration measured with total carbon analysis. 3.1.3 Nominal concentration t The nominal concentration was calculated to be 20.8 g/m3. The nominal concentration was comparable to the actual concentration of 20.7 g/m3, indicating a generation efficiency close to 100%. 3.1.4 Particle size measurement The particle size distribution is given in Table 2.1. It was shown that 45% of the particles present at the animals' breathing zone had an aerodynamic diameter equal to or smaller than 5 p.m. The Mass Median Aerodynamic Diameter (MMAD) was 5.4 p.m and the mean geometric standard deviation (gsd) of the particles was 1.7. 3.1.5 Measurement of temperature, relative humidity-and oxygen concen tration The mean temperature during exposure was 21.2 0.2 C (range 20.9-21.4 C) and the mean relative humidity was 44 4% (range 40-53%). The oxygen concentra tion during exposure was 21.5%. 3.2 Behaviour, clinical signs and mortality Data on behaviour and clinical signs are given in Table 3. During exposure, a visually slightly decreased breathing rate could be seen at almost all observation times, very slight irregular breathing could be seen during CompanySannTzed. Does not contain TSCA CBl V98.6B5 November 1998 Page 19 the last hour and clear restlessness during the last two hours of exposure (Tables 3.1.1 and 3.1.2). Shortly (see Tables 3.2.1 and 3.2.2) after exposure, abnormalities consisted of piloerection, blepharospasm, sluggishness and slight irregular breathing. In two male (nos. 34,36) and in two female animals (nos. 33, 35) of subgroup Al no exposure-related abnormalities could be observed on day 1 of the one-day observation period (the alopecia in one female animal was not considered to be exposure-related). In the other animals, clinical signs consisted of blepharospasm, sluggishness and abdominal breathing. Mortality was seen in subgroup A2 (intended to be kept for an observation period of 14 days): two male and one-' female animal died on day 1 and one male and two females died on day 2. Abnormalities seen on day 1 and day 2 were similar to those seen in subgroup A 1. Beyond day 2 no clinical signs were seen in surviving animals. No abnormalities were seen in animals of subgroup A3 (Tables 3.3.1-3.3.3). Mortality was 0 out of 8 in subgroup Al and 6 out of 8 in subgroup A2. Mortality in group Al may however be an underestimate, due to the necropsy on day 1. 3.3 Body weights Individual and mean body weights are indicated in Tables 4. One day after exposure, considerable body weight loss was seen in all exposed animals. The two surviving animals of subgroup A2 gained weight during the 14day observation period. As expected, normal weight gain was seen in all animals of the unexposed subgroup A3 (Tables 4.1-4.3), 3.4 Lung function measurements Since mortality occurred, lung function measurements are not reported (see protocol), results will be included in the raw data, however. 3.5 Lung weights Lung weights and lung weights relative to body weight are given in Table 5. Several animals of subgroup Al (nos. 32, 38, 31 and 37) and one of the surviving animals of subgroup A2 (no. 43) showed distinctly increased absolute and relative lung weights. The relative lung weights of the other animals of subgroup Al (nos. 34, 36, 33, and 35) and the other survivor of subgroup A2 (no. 44) were considered to be only slightly higher than those of the unexposed controls (Tables 5.1 and 5.2). Cowpany Sanitized. Does not contain TSCA CB( V98.685 November 1998 Page 20 3.6 Pathology Summarized macroscopic and microscopic findings are given in Tables 6.1 and 7.1, respectively; individual findings are given in Appendices 1.1 and 2.1. 3.6.1 Macroscopy Three males and three females of subgroup A2 intended to be killed 14 days after exposure to the test substance were found dead within 2 days. The control rats (subgroup A3) revealed only some minor pulmonary gross changes such as a few spots and focal petechiation. Animals killed one day after exposure (subgroup A I): All animals killed one day after exposure demonstrated red discoloured lungs most frequently accompanied by a hyalin appearance. Besides these pulmonary changes several animals showed pink fluid in the trachea and one female (no. 37) had a haemothorax. These gross abnormalities are considered to be related to treatment. The other macroscopic changes observed are considered fortuitous findings not related to exposure. Animals scheduled to be killed 14 days after exposure (subgroup A2): The animals found dead one or two days after exposure demonstrated gross lesions of the lungs and trachea similar to those seen in animals killed one day after treatment. Two animals of subgroup A2 (nos. 44 and 43) survived the observation period of 14 days. These animals still exhibited pulmonary gross lesions (dark discolouration, black spots) which are considered to be related to the treatment. 3.6.2 Microscopy Microscopic examination of the lungs of the controls (subgroup A3) revealed only a minor change, viz. very slightly increased septal cellularity, which is occasionally found in control rats. Microscopic examination of the lungs of animals scheduled for necropsy one day after exposure (subgroup Al) revealed histopathological lesions characterized by alveolar haemorrhages, increased septal cellularity and perivascular polymorphonuclear leucocytic infiltration, which are considered to be related to exposure. Of subgroup A2, the animals found dead one or two days after exposure demonstrated similar pulmonary histopathological changes as those observed in animals scheduled killed one day after exposure. The lungs of the two surviving rats which were scheduled killed 14 days after exposure still exhibited histopathological changes such as increased septal cellularity, accumulation of alveolar macrophages and accumulation of pigment, which are considered to be related to the exposure. "BffipaBy SaniHzea. Uoesiot confaFn TSCA CRf V98.685 November 1998 Page 21 4 Results Exposure B 4.1 Analytical results 4.1.1 Total carbon anaj The actual concentration of^ during exposure A based on total carbon analysis of the volatile fraction averaged at 5-min intervals was 20.9 0.2 g/m-' (N=61). 4.1.2 Gravimetric analysis The results of the gravimetric analysis are given in Table L2. Thecalculated mean concentration of the solid fraction ogBHHmj^ the actual concentration measured with total carbon analysis. 4.1.3 Nominal concentration The nominal concentration was calculated to be 22.3,g/m3. The nominal concentration was slightly higher than the actual concentration of 20.9 g/m3, cating a generation efficiency of 94%, again close to 100%. indi 4.1.4 Particle size measurement The particle size distribution is given in Table 2.2. It was shown that 51% of the particles present at the animals' breathing zone had an aerodynamic diameter equal to or smaller than 5 p-m. The Mass Median Aerodynamic Diameter (MMAD) was 4.9 ^m and the mean geometric standard deviation (gsd) of the particles was 1.9. 4.1.5 Measurement of temperature, relative humidity and oxygen concen tration The mean temperature during exposure was 21.5 0.2 C (range 21.1-21.7C) and the mean relative humidity was 46 2% (range 42-48%). The oxygen concentra tion during exposure was 21.5%. 4.2 Behaviour, clinical signs and mortality Data on behaviour and clinical signs are given in Table 3. During exposure, a visually slightly decreased breathing rate and slightly irregular breathing could be seen at almost all observation times. Slightly laboured breathing also developed during the course of exposure (Tables 3.1.3 and 3.1.4). Shortly (see Table 3.2.3 and 3.2.4) after exposure, abnormalities consisted of piloerection, blepharospasm, sluggishness and irregular breathing (slight). Frequently noted clinical signs on day 1 of the one day observation period of group 'Pteo_m-'p'_a_ny Sanllizea. Does not contain TSCA CB V9S.685 November 1998 Page 22 B 1 included sluggishness, blepharospasm, abdominal breathing and soiled fur at the abdomen. One male animal (no. 400) was without clinical signs. Mortality was seen in animals of subgroup B2: one female animal died on day 1, one male and one female animal died on day 2 and one male animal died on day 3. Abnormalities seen on day 1 and day 2 were similar to those seen in subgroup B 1. Beyond day 2 no clinical signs were seen in surviving animals except for an increased breathing rate in the two females that survived the 14-day observation period. Mortality was 0 out of 8 in subgroup B 1 and 4 out of 8 in subgroup B2. Mortality in group B 1 may however be an underestimate, due to the necropsy on day 1. 4.3 Body weights Individual and mean body weights are indicated in Table 4. One day after exposure, considerable body weight loss was seen in all exposed animals. Weight gain was seen in surviving males during the 14-day observation period and in surviving females in the last week. As expected, normal weight gain was seen in all animals of the unexposed subgroup B3 (Tables 4.4-4.6). 4.4 Lung function measurements Since mortality occurred, lung function measurements are not reported (see protocol), results will be included in the raw data, however. 4.5 Lung weights Lung weights and lung weights relative to body weight are given in Table 5. Relative lung weights of one male animal of subgroup B 1 (no. 400) and two surviving males of subgroup B2 (nos. 408 and 410) were only slightly higher than those of the control group. The other animals of subgroup B 1, including the two surviving females of subgroup B2 (nos. 353 and 355) had distinctly increased absolute and relative lung weights (Tables 5.3 and 5.4). 4.6 Pathology Summarized macroscopic and microscopic findings are given in Tables 6.2 and 7:2, respectively; individual findings are given in Appendices 1.2 and 2.2. 4.6.1 Macroscopy Two males and two females of group A2 intended to be killed 14 days after exposure were found dead within 3 days. R^?Sffipany Sffli'iiizea Uoes TToTcWfaIn TgiCT P"? V98.685 November 1998 Page 23 The control rats. (subgroup B3) revealed only some minor pulmonary gross changes such as hyalin spots and petechiation. Animals killed one day after exposure (subgroup B I): All animals killed one day after exposure demonstrated red discoloured lungs accompanied by a hyaline appearance. Besides these pulmonary changes several animals showed pink fluid in the trachea. These gross abnormalities are considered to be related to treatment. The other macroscopic changes observed are considered fortuitous findings not related to exposure. Animals scheduled to be killed 14 days after exposure (subgroup B2): The animals found dead within three days after exposure demonstrated gross lesions of the lungs and trachea similar to those seen in animals killed one day after treatment. Four animals of subgroup B2 (nos. 408, 410, 353 and 355) survived the observation period of 14 days. These animals still exhibited gross lesions in the respiratory tract (petechiation, hyalin spots, discoloured spots, gray spongy tissue and foamy fluid in the trachea). Although hyalin .spots and petechiation were seen in control rats, the other changes were not and were therefore considered to be related to the exposure. 4.6.2 Microscopy Microcopic examination of the lungs of the controls (subgroup B3) revealed only a minor change: very slightly increased perivascular lymphoid aggregates and very slight to slight focal pneumonia, which is occasionally found in control rats. Microscopic examination of the lungs of animals scheduled for necropsy one day after exposure (subgroup B 1) revealed histopathological lesions characterized by alveolar haemorrhages, increased septal cellularity and perivascular polymorphonuclear leucocytic infiltration, which are considered to be related to exposure. Of subgroup B2, the animals found dead one, two or three days after exposure . demonstrated similar pulmonary histopathological changes as those observed in animals scheduled to be killed one day after exposure. The lungs of the rats which were scheduled killed 14 days after exposure still exhibited histopathological changes such as increased septal cellularity, accumulation of alveolar macrophages, alveolar haemorrhages and perivascular lymphoid aggregates, which are considered to be related to the exposure. T'^.^----'^^ """"' V9B.685 November! 998 Page 24 5 Discussion and conclusion The aim of the present study was to determine the acute inhalation toxicity of two 8 female rats was exposed to the limit concentration of 20.7 g/m3 of batchH^f ^Riuring a single period of four hours (Exposing A) and another group of 8 male ancfs female rats was exposed to batchjHH^t a limit concentration of 20.9 g/m3 (Exposure B). Li each experiment, half of the rats was necropsied the day after exposure, the remaining rats were kept for a 14-day observation period and four additional, unexposed rats were also sacrificed for background histopathology. Mortality was the most important finding in both experiments, viz. 6 out of 8 animals of the subgroup A2, intended to be kept for an observation interval of 14 days, died within 2 days after exposure A, while the 8 animals of group Al all survived exposure until the scheduled necropsy the next day. Similarly, 4 out of 8 animals of subgroup B2 died within 3 days after exposure B, while again none of the 8 rats of group B 1 died before scheduled necropsy the day after exposure. The occurrence of late mortality after both exposures suggests that late mortality could be expected to have occurred in the animals scheduled for necropsy the day after exposure if these groups (Al and B 1) had been kept for a 14-day observation period as well, indicating that total mortality may be an underestimate. Macroscopic lung changes, i.e. discoloured lungs almost always accompanied by a hyalin appearance, were seen in rats necropsied one day after exposure (subgroups Al and Bl). Besides these pulmonary changes, several animals showed pink fluid in the trachea. Similar changes could be seen in the animals of subgroups A2 and B2 found dead in the first 3 days of their 14-day observation period. Surviving animals of these groups still exhibited pulmonary gross lesions and occasionally foamy fluid in the trachea, which are considered to be related to the exposure. In addition, absolute and relative lung weights had generally increased in animals which were killed for necropsy. Changes at the microscopic level in deceased rats and in rats scheduled for necropsy after one day, consisted of alveolar haemorrhages, increased septal cellularity and perivascular polymorphonuclear leucocytic infiltration, which are considered to be related to exposure. Rats that survived the 14-day observation period still exhibited histopathological changes such as increased septal cellularity, accumulation of alveolar macrophages and focal pneumonia, which are considered to be related to the exposure to the test material. It was concluded that a single 4-hour (acute) exposure of rats to a high concentration (i.e. 20.7 g/m3 fot|HUI^BElind 20.9 g/m3 foij||| mortality and macroscopical and histopathological lung changes. Ppmpany Sanitized. Does not contain TSCA fRI V98.685 November 1998 Page 25 Therefore, from the results of the present study, it cannot be excluded that human exposure to high aerosol concentrations will result in serious and lasting lung changes. 6 References Lee R.J. Jr., Science, 1972: 567-575 ^^p^nyBa^^^Oo63^0160"131" T9CACW V98.685 November 1998 Page 26 Figure 1 - Schematic diagram of the generation and exposure system 1 Mainvalve 2 Reducing valve 3 Flow meter 4 Valve 5 Humidifier 6 Thermal bath 7 USNebuliser 8 Fluid pump 9 Supply vessel 10 Pressure gauge 11 Regulating valve HNO Head/Nose Only Unit AH Animal holder EHD External Hood 11 "t.-.'J1 '..^i..-'-^- .-umdilsl^L-At-fcli V98.6B5 November 1998 Page 27 >iCUTE (4-HOUR) INHALATION TNO Nutrition and Food Research Institute Study: 480001/005 RAT Table 1.1 - Concentration of solid fraction in the test atmosphere measured by gravimetry (Exposure A) Sample time (hh:mm) 11:44 12:50 14:08 15:10 16:02 mean sd Volume (C) 100 100 100 100 100 i < Concentration (g/m3) 0.103 0.105 0.107 0.110 0.110 0.107 0.003 Generation of test atmosphere: 9:30-16:40 h Exposure duration of the animals: 4 hours between 10:58 and 16:40 h 'Company SanHhed. Does not contain TSCA CBI V98.685 Iwernber 1998 Page 28 ICUTE (4-HOUR) INHALATION RAT ~TNO Nutrition and Food Research Institute Study: 480001/005 Table 1.2 - Concentration of solid fraction in the test atmosphere measured by gravimetry (Exposure B) Sample time (hlumm) 11:42 12:50 13:41 14:45 15:44 mean sd Volume (C) 100 100 100 100 100 Concentration (g/m3) 0.135 0.140 0.139 0.139 0.141 0.139 0.002 Generation of test atmosphere: 9; 10-16:20 h Exposure duration of the animals: 4 hours between 11:07 and 16:20 h mI^TSTpaWSsrnWfea Does not con?a?n TSCA CBT w^ V98.6B5 November 1998 Page 29 VCUTE (4-HOUR) INHALATION RAT 'TNO Nutrition and Food Research Institute Study: 480001/005 Table 2.1 - Aerodynamic particle size distribution in test atmosphere (Exposure A) Particle size (^m) $32 ^20.5 s8.2 &5.0 -.3.1 ' ^ 1.9 $ 1.1 s0.7 ^0.4 :s0.1* total Mass (mg) 0 0 0.6 1.83 1.11 0.44 0.35 0 0 ' ' 0.07 4.4 Cumulative (%) 100 100 86.4 44.8 19.5 9.5 1.6 1.6 1.6 1.6 *The amount sampled on this stage included back-up filter. The volume was 66.1 fi, sample flow was 2.2 C/min Mass Median Aerodynamic Diameter (MMAD): 5.4 /^m mean geometric standard deviation (gsd): 1.7 Company Sanitized. Does no contain TSCA C^ V98.685 November 1998 Page 30 tjYCUTE(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Table 2.2 - Aerodynamic particle size distribution in test atmosphere (Exposure B) Particle size (//m) $32 $20.5 $8.2 $5.0 $3.1 $ 1.9 $ 1.1 $0.7 $0.4 $0.1* total | Mass (mg) 0 0 1.13 2.07 1.7 1.21 0.15 0.1 0.08 0.04 6.48 - . ... Cumulative (%) 100 100 82.6 50.6 24.4 5.7 3.4 1.9 0.6 0.6 *The amount sampled on this stage included back-up filter. The volume was 72.6 C, sample flow was 2.4 C/min Mass Median Aerodynamic Diameter (MMAD): 4.9 ^m mean geometric standard deviation (gsd): 1.9 co^y^'^'05^''10"'3'"^"81 V98.6B5 November 1998 Page 31 ^CUTE (4-HOUR) INHALATION TNO Nutrition and Food Research Institute Study: 480001/005 RAT Table 3.1.1 - Clinical signs during exposure (subgroup Al) Animal number Clinical signs MALES 32,34,36,38 BREATHING visually decreased rate (slight) irregular breathing (very slight) BEHAVIOUR clear restlessness FEMALES 31,33,35,37 BREATHING visually decreased rate (slight) irregular breathing (very slight) BEHAVIOUR clear restlessness Time of observation 11:45, 12:44, 13:40, 14:41 14:41 13:40, 14:41 11:45, 12:44, 13:40, 14:41 14:41 13:40, 14:41 exposure of animals: 10:58- 14:58h [CompanySanitized. Does not contain TSCA cm V98.685 November 1998 Page 32 ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.1.2 - Clinical signs during exposure (subgroup A2) Animal number Clinical signs Time of observation MALES 40 42,44,46 NO ABNORMALITIES BREATHING visually decreased rate (slight) irregular breathing (very slight) BEHAVIOUR clear restlessness (slight) r BREATHING visually decreased rate (slight) irregular breathing (very slight) BEHAVIOUR clear restlessness (slight) 11:45 12:44, 13:40, 14:41, 15:50 13:40, 14:41 13:40, 14:41 12:44, 13:40, 14:41, 15:50 13:40, 14:41 13:40, 14:41, 15:50 FEMALES 39 NO ABNORMALITIES BREATHING visually decreased rate (slight) irregular breathing (veery slight) BEHAVIOUR clear restlessness (slight) 11:45 12:44, 13:40, 14:41, 15:50 13:40, 14:41 13:40, 14:41 41,43,45 BREATHING visually decreased rate .(slight) irregular breathing (very slight) BEHAVIOUR clear restlessness (slight) 12:44,-13:40, 14:41, 15:50 13:40, 14:41 13:40, 14:41, 15:50 exposure period of animals: no. 40- ll:30-15:30h no. 42- ll:50-15:50h no. 44- 12:10-16:10h no. 46- 12:30-16:30h no. 39- ll:40-15:40h no. 41 - !2:00-16:00h no. 43- 12:20-16:20h no. 45-12:40-l6:40h :GeffipanySaru{fzed. Does not contain TSCACai 1 NU repon V98.685 November 1998 Page 33 \.CUTE (4-HOUR) INHALATION _ TNO Nutrition and Food Research Institute Study: 480001/005 RAT Table 3.1.3 - Clinical signs during exposure (subgroupB 1) Animal number Clinical signs Time of observation MALES 400,402,404,406 BREATHING irregular breathing (slight) decreased breathing rate (slight) laboured breathing (slight) 11:50, 12:54, 13:40, 14:45 12:54, 13:40, 14:45 13:40,14:45 FEMALES 335,337,343,345 BREATHING ' irregular breathing (slight) decreased breathing rate (slight) laboured breathing (slight) 11:50, 12:54, 13:40, 14:45 12:54, 13:40, 14:45 13:40,14:45 exposure of animals: 11:07- 15:07h Company Sanitized. Does nol contain TSCA W V98.685 November 1998 Page 34 \.CUTE (4-HOUR) INHALATION RAT "TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.1.4 - Clinical signs during exposure (subgroup B2) Animal number Clinical signs Time of observation MALES 408,410 BREATHING irregular breathing (slight) decreased breathing rate (slight) laboured breathing (slight) 11:50, 12:54, 13:40, 14:45 12:54, 13:40, 14:45 13:40, 14:45 412,414 BREATHING irregular breathing (slight) decreased breathing rate (slight) laboured breathing (slight) 12:54, 13:40, 14:45, 15:40 12:54, 13:40, 14:45, 15:40 13:40, 14:45, 15:40 FEMALES 349,351 BREATHING irregular breathing (slight) decreased breathing rate (slight) laboured breathing (slight) 11:50, 12:54, 13:40, 14:45 12:54, 13:40, 14:45 13:40, 14:45 353,355 BREATHING irregular breathing (slight) decreased breathing rate (slight) laboured breathing (slight) 12:54, 13:40, 14:45, 15:40 12:54, 13:40, 14:45, 15:40 13:40. 14:45, 15:40 exposure period of animals: no. 408- ll:'10-15:10h no.410-ll:30-15:30h no. 412 - ll:50-15:50h no. 414-12:10-16: lOh no. 349- ll:20-15:20h no. 351- ll:40-15:40h no. 353 - 12:00-16:00h no. 355- 12:20-16:20h ^m^ySanlHzeaboesffolc-oniaIn TSCA CBi V98.685 November 1998 ^ACUTE (4-HOUR) INHALATION RAT "TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.2.1 - Clinical signs shortly' after the end of exposure (subgroup Al) Animal number MALES 32,34,36,38 FEMALES 31,33,35,37 Clinical signs GENERAL piloerection, blepharospasm, sluggishness BREATHING irregular breathing (slight) GENERAL . ,, piloerection, blepharospasm, sluggishness BREATHING irregular breathing (slight) i Ti;me of observation l7hl0; exposure ended on 14h48 V98.685 November 1998 Page 36 (ACUTE(4-HOUR) INHALATION "TNO Nutrition and Food Research Institute Study: 480001/005 RAT Table 3.2.2 - Clinical signs shortly' after the end of exposure (subgroup A2) Animal number MALES 40,42,44,46 FEMALES 39,41.43,45 Clinical signs GENERAL piloerection, blepharospasm, sluggishness BREATHING irregular breathing (slight) GENERAL piloerection, blepharospasm^sluggishness BREATHING irregular breathing (slight) ' time of observation 17hl0; exposure ended between 15h30 and 16h40 (see note at the bottom of Table 3.1.2) Ppmpany Sanitized. Does not contain Tca ^ V98.685 November 1998 Page 37 ^.CUTE (4-HOUR) INHALATION RAT "TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.2.3 - Clinical signs shortly' after the end of exposure (subgroup B 1) Animal number MALES 400,406 402,404 FEMALES 335,337,343,345 Clinical signs GENERAL piloerection, sluggishness BREATHING irregular breathing (slight) GENERAL piloerection, blepharospasm, sluggishness BREATHING irregular breathing (slight) GENERAL piloerection, blepharospasm, sluggishness BREATHING irregular breathing (slight) Time of observation 16h20; exposure ended on 15h07 ^TOp^y^an^^^oeiffofeoniaIn TSCA CBI V98.685 November 1998 Page 38 nACUTE (4-HOUR) INHALATION. RAT ^0 Nutrition and Food Research Institute Study: 480001/005 Table 3.2.4 - Clinical signs shortly' after the end of exposure (subgroup B2) Animal number MALES 408,410,412,414 FEMALES 349,351,353,355 Clinical signs GENERAL piloerection, blepharospasm, sluggishness BREATHING irregular breathing (slight) GENERAL piloerection, blepharospasm, sluggishness BREATHING irregular breathing (slight) ' time of observation 16h30; exposure ended between 15hl0 and 16h20 (see note at the bottom of Table 3.1.4) Company SanWzea. Does Hoi contain TSCA C^l V9B fifi*^ November 1998 -- pa--c--)e--3--9 ILCUTE (4-HOUR) INHALATION 'TNO Nutrition and Food Research Institute Study: 480001/005 RAT Table 3.3.1 - Clinical signs during the 1-day observation period (subgroup Al) Animal number MALES 32,38 34,36 FEMALES 31 33 35 37 Clinical signs GENERAL blepharospasm sluggishness BREATHING abdominal breathing NO ABNORMALITIES GENERAL blepharospasm (right eye) sluggishness BREATHING abdominal breathing NO ABNORMALITIES GENERAL alopecia at head/neck GENERAL blepharospasm sluggishness BREATHING abdominal breathing Range' range: 1 day (animals were sacrificed on day 1) C,Bmpany Samlize'd. Does not tfoniain TSCA CRi V98.685 November 1998 page 40 ILCUTE (4-HOUR) INHALATION TNO Nutrition and Food Research Institute Study: 480001/005 RAT Table 3.3.2 - Clinical signs during the 14-day observation period (subgroup A2) Animal number MALES 40 42,46 44 Clinical signs GENERAL blepharospasm sluggishness soiled fur nasal encrustations BREATHING abdominal breathing FOUND DEAD on day 2 GENERAL blepharospasm sluggishness BREATHING abdominal breathing FOUND DEAD on day 1 GENERAL blepharospasm sluggishness BREATHING abdominal breathing NO ABNORMALITIES ' range day 1 - day 14 Range' 1 1,2 2 2 1,2 1 1 I I 1,2 I 3-14 Company Sanitized. Does not contam TSCA ^w 1 iiu repon V98.685 November 1998 Page 41 TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.3.2 (continued) - Clinical signs during the 14-day observation period (subgroup A2) Animal number FEMALES 39 41 43 45 Clinical signs GENERAL sluggishness blepharospasm BREATHING abdominal breathing FOUND DEAD on day 2 " GENERAL sluggishness blepharospasm nasal discharge BREATHING abdominal breathing FOUND DEAD on day 2 GENERAL sluggishness blepharospasm BREATHING abdominal breathing NO ABNORMALITIES GENERAL sluggishness blepharospasm BREATHING abdominal breathing FOUND DEAD on day 1 Range' 1 1 1 1,2 1 2 1,2 1 1 1 2-14 1 1 1 range day 1 - day 14 13n'ipany Sanitized. Does not contain TSCA CSi V98.685 November 1998 Page 42 JACUTE(4-HOUR) INHALATION RAT 'TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.3.3 - Clinical signs during the 14-day observation period (subgroup A3) Animal number MALES 48,50 FEMALES 47,49 Clinical signs NO ABNORMALITIES NO ABNORMALITIES range day 1 - day 14 Range' 1-14 1-14 Company Sanitized. Does not contain TSCA OW V98.685 November 1998 Page 43 ____^ ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.3.4 - Clinical signs during the 1-day observation period (subgroup B 1) Animal number MALES 400 402 404 406 Clinical signs NO ABNORMALITIES GENERAL blepharospasm BREATHING abdominal breathing irregular breathing GENERAL sluggish, blepharospasm GENERAL blepharospasm soiled fur at abdomen BREATHING abdominal breathing Range" company Sanitized. Does not contain TSCA CBi V98.6B5 November 1998 Page 44 TNO Nutrition and Food Research: Study: 480001/005 .CUTE (4-HOUR) INHALATION RAT stitute Table 3.3.4 (continued) - Clinical signs during the 1-day observation period subgroup Bl) Animal number FEMALES 335 337 343 345 Clinical signs GENERAL sluggishness soiled fur at abdomen nasal encrustations GENERAL blepharospasm sluggishness nasal encrustations BREATHING abdominal breathing GENERAL blepharospasm sluggishness soiled fur at abdomen BREATHING abdominal breathing GENERAL sluggishness soiled fur at abdomen BREATHING abdominal breathing Range* range: 1 day (animals were sacrificed on day 1) .Company Sanitized. Does nol confab TA fny V98.685 November 1998 Page 45 ICUTE (4-HOUR) INHALATION RAT ' TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.3.5 - Clinical signs during the 14-day observation period (subgroup B2) Animal number MALES 408,410 412 414 Clinical signs BREATHING increased breathing rate NO ABNORMALITIES GENERAL blepharospasm sluggishness ' encrustations at nose and eyes BREATHING laboured breathing FOUND DEAD on day 3 GENERAL blepharospasm (right eye) soiled fur at abdomen BREATHING abdominal breathing FOUND DEAD on day 2 Range* 1 2-14 1.2 'range day 1 -day 14 9ySaiin2ea.'D'o^Hoclontain TSCA CB1 V98.685 November 1998 ____ J ACUTE (4-HOUR) INHALATION RAT "TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.3.5 (continued) - Clinical signs during the 14-day observation period (subgroup B2) Animal number FEMALES 349 351 353,355 Clinical signs GENERAL sluggishness soiled fur at abdomen BREATHING abdominal breathing FOUND DEAD on day 1 , GENERAL sluggishness soiled fur at abdomen BREATHING laboured breathing FOUND DEAD on day 2 GENERAL sluggishness BREATHING laboured and irregular breathing increased breathing rate ' range day 1 - day 14 Range* 1 1 1 1,2 1 1,2 I 1 2-14 -:,.,;';' .'XrtHti'i'.". SwAi-.fci. V98.685 November 1998 Page 47 ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Table 3.3.6 - Clinical signs during the 14-day observation period (subgroup B3) Animal number MALES 416,418 FEMALES 333,341 Clinical signs NO ABNORMALITIES NO ABNORMALITIES * range day 1 - day 14 Range' 1-14 1-14 .Company Sanitized. Does not contain TSCA '"w V98.685 November 1998 Page 48 ^CUTE (4-HOUR) INHALATION JO Nutrition and Food Research Institute Study: 480001/005 RAT Table 4.1 - Individual and mean body weights (g) of male and female rats (subgroup A 1) Animal No. Males (Al) 32 34 36 38 mean sd Females (Al) 31 33 35 37 mean Day-3 257.8 278.5 263.1 265.8 266.3 8.8 185.1 182.3 171.0 159.8 174.6 + 1.6 DayO 261.6 290.9 276.2 279.2 277.0 12.1 188.4 187.1 174.0 162.0 177.9 Day 1 243.8 284.8 259.3 249.5 259.4 18.1 173.0 177.0 158.4 148.7 164.3 yco, mpany Sanitized. Does not c.onham T3CA Cat V98.685 November 1998 Page 49 ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Table 4.2 - Individual and mean body weights (g) of male and female rats (subgroup A2) Animal No. Males (A2) 40 42 44 46 mean sd Females (A2) 39 41 43 45 mean +sd Day-3 258.6 259.8 270.7 264.2 263.3 5.5 171.7 191.5 181.0 165.9 177.5 +11.2 DayO 265.6 272.3 280.6 273.2 272.9 6.1 184.8 195.3 183.4 177.4 185.2 +7.4 Dayl Day 7 Day 14 245.4 250.2 255.5 251.5 250.7 4.2 230.3 (2) 248.6(1) 270.8 249.3 (1) i - 311.3 - 2 166.4 177.9 164.7 157.4 166.6 +8.5 157.2 (2) 167.0(2) 168.7 158.9(1) i - 187.8 - 2 ' no weight available on day 7, animal weight at death on day () 2 no mean and sd given for mean of less than two or three values, respectively . - Company Sanitized. Does not contain TSCA CBI V98.6B5 November 1998 Page 50 CUTE (4-HOUR) INHALATION TNO Nutrition and Food Research Institute Study: 480001/005 RAT Table 4.3 - Individual weights (g) of male and female rats (subgroup A3) Animal No. Males (A3) 48 50 mean Females (A3) 47 49 mean Day-3 259.3 272.1 265.7 184.6 181.4 183.0 DayO 266.4 291 278.7 188.9 185.7 187.3 Day 1 268.6 293.1 280.9 187:5 185.5 186.5 Day 7 Day 14 281.9 315.7 298.8 303.1 338.8 321.0 202.6 196.9 199.8 213.0 199.1 206.1 ^aRRizea. Dtfegffiof contain TSCA C8t V98.685 November 1998 BMHBHlBl^[)\CUTE TNO Nutrition and Food Research Institute Study: 480001/005 (4-HOUR) INHALATION RAT (Tsaubblger4o.u4p- BInl)dividual and mean body weights (g) of male and female rats V98.685 November 1998 Page 52 JACUTE(4-HOUR) INHALATION RAT TO Nutrition and Food Research Institute Study: 480001/005 Table 4.5 - Individual and mean body weights (g) of male and female rats (subgroup B2) Animal No. Males (B2) 408 410 412 414 Day-1 264.7 241.2 231.4 262.4 DayO 280.4 247.3 241.4 269.4 Dayl Day 7 268.0 242.6 217.7 249.7 293.1 258.7 204.0 (2) 238.5. (2) mean sd Females (B2) 349 351 353 355 mean +sd 249.9 16.3 190.3 190.1 176.6 171.2 182.1 +9.7 259.6 18.4 192.3 193.9 177.9 177.6 185.4 +8.9 244.5 20.8 275.92 179.7 177.7 166.3 164.9 172.2 +7.6 176.7(1) 169.4 (2) 168.8 163.8 166.32 1 no weight available on day 7, animal weight at death on day () 2 no sd given for mean of less than three values Day 14 315.8 280.4 - 298.12 - 190.9 182.8 186.92 Company Sanitized. OsesHbt conta'n TSCA "w V98.6B5 November 1998 Page 53 t^CUTE(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Table 4.6 - Individual weights (g) of male and female rats (subgroup B3) Animal No. Males (B3) 416 418 mean Females (B3) 333 341 mean Day-1 DayO 226,4 282.0 254.2 239.3 300.2 269.8 183.7 199.7 -.19LL -, 190.8 204.8 197.8 Dayl 240.7 297.6 269.2 193.t 210.2 201.7 Day 7 Day 14 261.4 310.8 286.1 274.8 330.1 302.5 203.0 222.4 212.7 208.0 230.7 219.4 Ib.owpanySanMzeiJ, Does not contain TSC& "e" V98.685 November 1998 Page 54 1?AC (ACUTE (4-HOUR) INHALATION TNO Nutrition and Food Research.. GInissttiitute Study: 480001/005 RAT Table 5.1 - Individual and mean lung weights of exposed animals (subgroup A 1 recorded on day 1 and subgroup A2 recorded on day 14) Animal no. Males 32 34 36 38 40 42 44 46 mean sd Subgroup Al lung weight (g) rel lung weight (g/lOOgBW) Subgroup A2 lung weight (g) rel lung weight (g/lOOgBW) 2.707 1.573 1.469 2.693 2.111 0.682 1.11 0.55 0.57 1.08 0.83 0.31 c 4.420* 3.320* 1:634 4.633* - - 1.92* 1.34* 0.52 1.86* - - Females 31 33 35 37 39 41 1.650 - 1.121 1.067 1.841 43 45 mean 1.420 sd 0.385 * animal found dead 0.95 0.63. 0.67 1.24 0.87 0.28 3.551* 3.360* 2.069 3.109* - - 2.26* 2.01* 1.10 1.96* - Company Sanitized. Does not contam TSCA C'3 V9B.685. November 1998 Page 55 TNO Nutrition and Food Research Institute Study: 480001/005 Table 5.2 - Individual lung weights of non-exposed animals (subgroup A3) Animal no. Males 48 50 Day 14 lung weight (g) rel lung weight (g/lOOgBW) 1.275 1.334 0.42 0.39 Females 47 49 1.042 0.94 0.49 0.47 IgoWpanySaMnzea.'Does ffot contain TSCA C'Bi V98.685 November 1998 Page 56 TNO Nutrition and Food Research institute Study: 480001/005 Table 5.3 - Individual and mean lung weights of exposed animals (subgroup Bl recorded on day 1 and subgroup B2 recorded on day 14) Animal no. Males 400 402 404 406 408 410 412 414 mean sd Subgroup Bl lung weight (g) rel lung weight (g/lOOgBW) Subgroup B2 lung weight (g) rel lung weight (g/lOOgBW) 1.271 2.161 2.610 2.511 2.138 0.609 0.51 0.90 1.17 1.01 0.90 0.28 ( 1.514 1.285 4.053* 3.912* 1.400 - 0.48 0.46 1.99* 1.64* 0.47 - Females abs rel (%) abs rel (%) 335 1.578 0.99 337 2.093 1.29 343 1.949 1.15 - 345 1.881 1.13 349 2.667* 1.51* 351 3.273* 1.93* 353 2.208 1.16 355 2.273 1.24 mean 1.875 1.14 2.240 1.20 sd 0.217 0.12 - - * animal found dead; no sd given for mean of less than three values (dead animals not included) Company Sanitized. Does not confqtn TRCA rw V9B.685 November 1998 Page 57 TNO Nutrition and Food Research Institute Study: 480001/005 Table 5.4 - Individual lung weights of non-exposed animals (subgroup B3) Animal no. Males 416 418 Day 14 lung weight (g) rel lung weight (g/lOOgBW) 1.125 1.372 0.41 0.42 Females 333 341 1.047 1.081 0.50 0.47 company Sanitized. Doss not contain TSCA CBt V98.685 November 1998 Page 58 |ACUTE (4-HOUR) INHALATION RAT ' TNO Nutrition and Food Research Institute Study: 480001/005 Table 6.1 - Summary of macroscopic observations of male and female rats at necropsy (subgroups A) Changes Habitus Crusts (around nose/mouth/eyes) Blood from/around nose/mouth Dirty fur Lungs Hyalin appearance Discoloured Spotted Bloody content Spongy tissue Petechiation Trachea Fluid/bloody content rhoracic cavity Hydrothorax rhymus Spotted ^estes Cryptorchism Skin Focal alopecia Incidence of changes (numeric) M(aIe)/F(emaIe) test animals Al M F test animals A2 M F controls A3 M F Number examined 4 4 4 4 2 2 Found dead 3 3 f 1 1 2 1 4 4 1 2 4 4 3 4 1 2 1 1 1 1 2 2 2 1 2 1 1 1 1 1 Company Sanitized. Does not contain TSCA C.W V98.685 November 1998 Page 59 YCUTE (4-HOUR) INHALATION TNO Nutrition and Food Research Institute Study: 480001/005 RAT Table 6.2 - Summary of macroscopic observations of male and female rats at necropsy (subgroups B) Incidence of changes (numeric) Changes M(aIe)/F(emale) test animals BI M F test animals B2 M F controls B3 M F Habitus Crusts (around nose/mouth/eyes) Dirty fur Number examined 4 4 4 4 2 2 Found dead 2 2 ] "2 2 3 1 2 Lungs Hyalin appearance/spots Spotted "Discoloured Petechiation Spongy tissue 4 4 3 1 2 1 1 1 1 4 4 2 3 2 2 2 1 2 Thymus Discoloured Spotted 1 2 2 2 1 Trachea Filled with blood/pink fluid/foamy fluid Skin Focal alopecia 1 2 2 4 1 1 VIediastinal lymph nodes Enlarged 1 *arathymic lymph nodes Enlarged 2 .Company Sanitized. Does not contain TSCA C ;:^ .JNO report V98.685 November 1998 'K^sSQiy:"','.-. -. TNO_ Nutrition and Food Resea_rc_hJIAnsCtitUutTe E (4-HOUR) INHALATION RAT Study: 480001/005 (Tsaubblgero7u.p1s- SAu) mmary of microscopic lung observations of male and female rats V9S.685 November 1998 Page 61 IICUTE (4-HOUR) INHALATION RAT < TNO Nutrition and Food Research Institute Study: 480001/005 Table 7.2 - Summary of microscopic lung observations of male and female rats (subgroups B)_________________________________ Incidence of changes (numeric) Changes M(ale)/F(emale) test animals (1 day) M F test animals (14 days) M F controls (14 days) M F Alveolar haemorrhages very slight slight moderate Alveolar oedema very slight slight No examined. 4 4 4 4 2 2 2 2 2 2 2 1 2 t < 1 1 Perivascular oedema very slight 1 Perivascular polymorphonuclear leukocytic infiltration slight 1 'erivascular lymphoid aggregates very slight slight 1 1 1 1 1 ncreased septal cellularity very slight slight moderate 1 2 1 ''ocal increased number of alveolar macrophages slight - 2 2 ''ocal pneumonia very slight slight 1 1 1 1 2 1 [ncreased number of intra-alveoiar )olymorphonuclear inflammatory cells very slight 1 Company Sann?zed. Does not cw(an rs^fi /"?! V98.685 November 1998 Page 62 ICUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Appendix I.I.I - Individual macroscopic observations at necropsy 1 day after exposure A (subgroup Al) Animal number Macroscopic findings MALES 32 Survivor, killed on day 1 Lungs: Hyalin appearance Red discoloured Trachea: Pink fluid 34 Survivor, killed on day 1 ' Lungs: Dark red margins Hyalin appearance 36 Survivor, killed on day 1 Lungs: Dark red margins Hyalin appearance 38 Survivor, killed on day 1 Lungs: Hyalin appearance Dark red discoloured Trachea: Pink fluid Thymus: Red spotted Testes: Uni-lateral cryptorchism ^o^pany^W^ar.^oesTfo^-on^^T^X^T V98.685 November 1998 page 63 ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Appendix I.I.I (continued) - Individual macroscopic observations at necropsy I day after exposure A (subgroup Al) Animal number Macroscopic findings FEMALES 31 Survivor, killed on day 1 Lungs: Dark red discoloured Hyalin appearance Trachea: Fluid 33 Survivor, killed on day 1 Lungs: Dark red margins Hyalin appearance 35 Survivor, killed on day 1 Skin: Focal alopecia Lungs: Dark red margins Hyalin appearance 37 Survivor, killed on day 1 Lungs: Dark red discoloured Hyalin appearance Thoracic cavity: Hydrothorax Trachea: Pink fluid. P&mpany Sanitized. Does not contain TSCA CW V98.685 November 1998 Page 64 ACUTE (4-HOUR) INHALATION RAT 'T_N_O_N_u_tr_it_io_n _an_d _Fo_o_d _R_es_ea_rcJhllnj stitute Study: 480001/005 Appendix 1.1.2- Individual macroscopic observations at necropsy 14 days after exposure A or at death (subgroup A2) Animal number Macroscopic findings MALES 40 Found dead 2 days after exposure Habitus: Crusts around nose/mouth Dirty fur in urogenital area Lungs: Dark red discoloured Hyalin appearance Bloody content Testes: Cryptorchism Trachea: Filled with blood Thymus: Spots 42 Found dead 1 day after exposure Lungs: Red discoloured 44 Survivor, killed on day 14 Lungs: Black spots on one lobe 46 Found dead 1 day after exposure Lungs: Red discoloured ySw Sa^S."^0'COTIi"" TSCA CBI V98.685 November 1998 page 65 [ LCUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Appendix 1.1.2 (continued) - Individual macroscopic observations at necropsy 14 days after exposure A or at death (subgroup A2) Animal number Macroscopic findings FEMALES 39 41 Found dead 2 days after exposure Habitus: Crusts around nose/mouth Lungs: Dark red discoloured Hyalin appearance Trachea: Filled with red fluid f * Found dead 2 days after exposure Habitus: Blood around nose/mouth Lungs: Dark red discoloured Hyalin appearance Bloody content Trachea: Filled with blood Thymus: Spotted 43 Survivor, killed on day 14 Lungs: Dark discoloured Spongy tissue 45 Found dead 1 day after exposure Habitus: Haemorrhagic nasal discharge Lungs; Red discoloured ',;d 'W-'- '"' '" V98.685 November 1998 Page 66 |ACUTE (4-HOUR) INHALATION ' TNO Nutrition and Food Research Institute Study: 480001/005 RAT Appendix 1.1.3 - Individual macroscopic observations at necropsy in unexposed animals (subgroup A3) Animal number Macroscopic findings MALES 48 Unexposed control, killed on day 14 Lungs: Dark spot on one lobe 50 Unexposed control, killed on day 14 Lungs: Dark spots on one lobe FEMALES 47 49 Unexposed control, killed on day 14 Lungs: Pale spot on one lobe Petechiation on one lobe Unexposed control, killed on day 14 Lungs: Petechiation on one lobe Company Sanitized. Does not contain TSCA CF V98.685 November 1998 ...... Page 67 IICUTE (4-HOUR) INHALATION RAT ^0 Nutrition and Food Research Institute Study: 480001/005 Appendix 1.2.1 - Individual macroscopic observations at necropsy 1 day after exposure B (subgroup Bl) Animal number Macroscopic findings MALES 400 Survivor, killed on day 1 Lungs: Hyalin appearance Red margins 402 Survivor, killed on day 1 Lungs: Red discoloured Thymus: Hyalin appearance (Diffuse) red discoloured 404 Survivor, killed on day 1 Lungs: Red discoloured Hyalin appearance Thymus: Red spots Trachea: Pink fluid 406 Survivor, killed on day 1 Habitus: Lungs: Crusts around one eye Red discoloured Thymus: Hyalin appearance Red spots Wny Sanll^. Doe.not^n TSCA CBI V98.685 November 1998 Page 68 ^ CUTE (4-HOUR) INHALATION 0 Nutrition and Food Research Institute Study: 480001/005 RAT Appendix 1.2.1 (continued) - Individual macroscopic observations at necropsy 1 day after exposure B (subgroup Bl) Animal number Macroscopic findings FEMALES 335 Survivor, killed on day 1 Habitus: Red crusts around nose Dirty fur in urogenital area Lungs: Red discoloured Hyalin appearance Skin: Focal alopecia 337 Survivor, killed on day 1 Habitus: Crusts around nose Lungs: Thymus: Red discoloured Hyalin appearance Red spots Trachea: Pink fluid 343 Survivor, killed on day 1 Habitus: Dirty fur in urogenital area Lungs: Red discoloured Thymus: Hyalin appearance Red spots Trachea: Pink fluid 345 Survivor, killed on day 1 Habitus: Dirty fur in urogenital area Lungs: Red discoloured Hyalin appearance Company Sanitized. Does not cnn^" r ^ ^ V98.685 November 1998 Page 69 ^.CUTE (4-HOUR) INHALATION ' Nutrition and Food Research Institute Study: 480001/005 RAT Appendix 1.2.2 - Individual macroscopic observations at necropsy 14 days after exposure B or at death (subgroup B2) Animal number Macroscopic findings MALES 408 410 Survivor, killed on day 14 Lungs: Petechiation Hyalin spots Mediastinal lymph nodes: Uni-lateral enlarged V -V Survivor, killed on day 14 Lungs: Petechiation Hyalin spot on lobe E ' Dark spots 412 Found dead 3 days after exposure Habitus: Red crusts around nose/eyes Lungs: Dark red discoloured Hyalin appearance Thymus: Spotted Trachea: Filled with blood 414 Found dead 2 days after exposure Habitus: Dirty fur in urogenital area Crusts around nose Lungs: Dark red discoloured Thymus: Red spots Trachea: Filled with blood ^5Bp^SOTTlTza3ri?oesffi?ionTtaSSCnACBi V98.685 November 1998 Page 70 QACUTE(4-HOUR) INHALATION RAT S-N0 Nutrition and Food Research Institute Study: 480001/005 Appendix 1.2.2 (continued) - Individual macroscopic observations at necropsy 14 days after exposure B or at death (subgroup B2) Animal number Macroscopic findings FEMALES 349 Found dead 1 days after exposure Habitus: Dirty/wet fur on belly Lungs: Hyalin appearance Red discoloured Trachea: Filled with blood Thymus: Spotted 351 Found dead 2 days after exposure Habitus: Dirty fur in urogenital area Lungs: Dark red discoloured Trachea: Filled with blood 353 Survivor, killed on day 14 Lungs: Gray spongy tissue White margins Petechiation White spots Trachea: Parathymic Foamy fluid lymph nodes: Enlarged 355 Survivor, killed on day 14 Lungs: Gray spongy tissue Petechiation Trachea: Parathymic . Foamy fluid lymph nodes: Enlarged Company Sanitized. Does not contain TSCA CW V98.685 November 1998 Page 71 IJLCUT(E4-HOUR) INHALATION RAT 'TNO Nutrition and Food Research Institute Study: 480001/005 Appendix 1.2.3 - Individual macroscopic observations at necropsy in unexposed animals (subgroup B3) Animal number Macroscopic findings MALES 416 Unexposed control, killed on day 14 Lungs: Hyalin spots Petechiation 418 Unexposed control, killed on day 14 r -V Lungs: Hyalin spots Petechiation FEMALES 333 Unexposed control, killed on day 14 Lungs: White spots Skin: Focal alopecia 341 Unexposed control, killed on day 14 Lungs: Petechiation on one lobe Hyalin spots ..p..^-^"' V98.685 November 1998 Page 72 ICUTE (4-HOUR) INHALATION "TNO Nutrition and Food Research Institute Study: 480001/005 RAT Appendix 2.1.1 Individual microscopic observations in lungs of animals necropsied 1 day after exposure (subgroup Al) Animal number Microscopic findings in lungs MALES 32 Survivor, killed on day 1 Moderate alveolar haemorrhages 34 Survivor, killed on day 1 Slight alveolar haemorrhages (fibrinoid material within the alveolar spaces) Slight alveolar oedema Slight peri vascular oedema Slight perivascular polymorphonuclear leukocytic infiltration Slight focal increased septal cellularity 36 Survivor, killed on day 1 Slight alveolar haemorrhages (fibrinoid material within the alveolar spaces) Slight perivascular polymorphonuclear leukocytic infiltration Very slight focal increased number of alveolar macrophages Slight focal increased septal cellularity 38 Survivor, killed on day 1 Slight alveolar haemorrhages Very slight perivascular polymorponuclear leukocytic infiltration Very slight increased septal cellularity (Company Sanitized. Does not con^n TSCA CB| V98.685 November 1998 Page 73 ACUTE (4-HOUR) INHALATION RAT 'TNO Nutrition and Food Research Institute Study: 480001/005 Appendix 2.1.1 (continued) - Individual microscopic observations in lungs of animals necropsied 1 day after exposure (subgroup Al) Animal number FEMALES 31 33 35 37 Microscopic findings in lungs Survivor, killed on day 1 Slight alveolar haemorrhages Survivor, killed on day 1 Slight alveolar haemorrhages Slight perivascular polymorphonuclear leukocytic infiltration Very slight perivascular oedema Slight focal increased septal cellularity Survivor, killed on day 1 Slight alveolar haemorrhages (fibrinoid material within alveolar spaces) Very slight perivascular polymorphonuclear leukocytic infiltration Slight focal increased septal cellularity Survivor, killed on day 1 Slight alveolar haemorrhages .poffipany Sanitkt-te?. Does not contain TS^i* ';**" V98.685 November 1998 (ACUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 480001/005 Appendix 2.1.2 - Individual microscopic observations in lungs of animals necropsied 14 days after exposure or at death (subgroup A2) Animal number 40 42 44 46 FEMALES 39 41 43 45 Microscopic findings in lungs Found dead 2 days after exposure Moderate alveolar haemorrhages (fibrinoid material within alveolar spaces) Moderate focal increased septal cellularity Slight focal alveolar oedema Found dead 1 day after exposure Slight alveolar haemorrhages Survivor, killed on day 14 Very slight focal brown pigment accumulation Found dead 1 day after exposure Slight alveolar haemorrhages Found dead 2 days after exposure Slight alveolar haemorrhages Slight perivascular oedema Very slight focal pneumonia Found dead 2 days after exposure Moderate alveolar haemorrhages (fibrinoid material within alveolar spaces) Very slight alveolar oedema Survivor, killed on day 14 Slight increased number of alveolar macrophages Slight focal increased septal cellularity Found dead 1 day after exposure Slight alveolar haemorrhages Very slight alveolar oedema V98.685 November 1998 Page 75 VCUTE (4-HOUR) INHALATION RAT ^TNO Nutrition and Food Research Institute Study: 480001/005 Appendix 2.1.3 - Individual microscopic observations in lungs ofunexposed animals (subgroup A3) Animal number MALES 48 50 FEMALES 47 49 Microscopic findings in lungs Unexposed control, killed on day 14 No abnormalities detected, hence no microscopic evidence of macroscopic finding Unexposed control, killed on day 14 Very slight focal increased* sep'tal cellularity Unexposed control, killed on day 14 No abnormalities detected, hence no microscopic evidence of macroscopic finding Unexposed control, killed on day 14 No abnormalities detected, hence no microscopic evidence of macroscopic finding Company Sanitized. Does not con^n T.CA C. TNO report V98.685 November 1998 Page 76 TNO Nutrition and Food Research Study: 480001/005 ACUTE (4-HOUR) INHALATION RAT istitute Appendix 2.2.1 - Individual microscopic observations in lungs of animals necropsied 1 day after exposure (subgroup B 1) Animal number MALES 400 402 404 406 FEMALES 335 337 343 345 Microscopic findings in lungs Survivor, killed on day 1 Slight alveolar haemorrhages Very slight focal increased septal cellularity Slight perivascular polymorphonuclear leukocytic infiltration Survivor, killed on day 1 Moderate alveolar haemorrhages (fibrinoid material within alveolar spaces) Survivor, killed on day 1 Slight perivascular lymphoid aggregates Slight alveolar haemorrhages Slight focal increased septal cellularity Very slight perivascular oedema Survivor, killed on day 1 Moderate alveolar haemorrhages Survivor, killed on day 1 Slight alveolar haemorrhages Survivor, killed on day 1 Very slight alveolar haemorrhages Very slight focal increased septal cellularity Survivor, killed on day 1 Slight alveolar haemorrhages Very slight focal increased septal cellularity Survivor, killed on day 1 Very slight alveolar haemorrhages tenp.nyS..,tl,ed.Do.so.conlal.TSC V98.685 November 1998 ICUTE (4-HOUR) INHALATION RAT FNO Nutrition and Food Research'Institute Study: 480001/005 Appendix 2.2.2 - Individual microscopic observations in lungs of animals necropsied 14 days after exposure or at death (subgroup B2) Animal number MALES 408 410 412 414 Microscopic findings in lungs Survivor, killed on day 14 Slight focal pneumonia Slight perivascular lymphoid aggregates Survivor, killed on day 14 Slight focal pneumonia Found dead 3 days after exposure Slight alveolar haemorrhages (fibrinoid materialwithin alveolar spaces) Very slight alveolar oedema Very slight focal pneumonia Found dead 2 days after exposure Slight alveolar haemorrhages (fibrinoid material within alveolar spaces) Ooesnol Company ganged. V98.685 November 1998 Page 78 [ACUTE (4-HOUR) INHALATION RAT 'TNO Nutrition and Food ResearchTnstitute Study: 480001/005 Appendix 2.2.2 (continued) - Individual microscopic observations in lungs of animals necropsied 14 days after exposure or at death (subgroup B2) Animal number FEMALES 349 351 353 355 Microscopic findings in lungs Found dead 1 day after exposure Very slight alveolar haemorrhages Very slight increased number of intra-alveolar polymorphonuclear inflammatory cells Found dead 2 days after exposure Slight alveolar haemorrhages Slight alveolar oedema Very slight focal pneumonia Survivor, killed on day 14 Moderate increased septal cellularity Slight accumulation of alveolar macrophages (inci some multinucleated giant cells) Very slight alveolar haemorrhages Survivor, killed on day 14 Moderate increased septal cellularity Slight accumulation of alveolar macrophages (inci some multinucleated giant cells) contain TSCACBt Company Sanded. Does not V98.685 November 1998 Page 79 ILCUTE (4-HOUR) INHALATION TNO Nutrition and Food Research Institute Study: 480001/005 RAT Appendix 2.2.3 - Individual microscopic observations in lungs ofunexposed animals (subgroup B3) Animal number MALES 416 418 FEMALES 333 341 Microscopic findings in lungs Unexposed control, killed on day 14 Very slight increased perivascular lymphoid aggregates Very slight focal pneumonia Unexposed control, killed on day 14 Slight perivascular lymphoid aggregates Slight focal pneumonia Unexposed control, killed on day 14 No abnormalities detected, hence no microscopic evidence of macroscopic finding Unexposed control, killed on day 14 Very slight perivascular lymphoid aggregates Very slight focal pneumonia . _,c^f <-"< Company S^.5.--0.ri-0Does not c"'^" V98.685 November 1998 Annex 1.1: Certificate of analysis o; Name of the test materia, Appearance: Composition: Composition: Colour Boiling point Autoignition temperatui Explosive limits Vapour pressure Density Batch number Storage conditions Page 80 Service Agents d'lnterfaces Ie 29/0.5/98 f^S^" -^^.o.^f ^- '"- - -.0 '{'-.'-- , ,;,d. i.^ efiF atochem TNO report V98.685 November 1998 Annex 1.2: Certificate of analysis o Name of the test material Appearance: Composition; Composition: Colour Boiling point Autoignition temperature, Explosive limits Vapour pressure Density Batch number Storage conditions Sen/ice Agents d'lnterfaces Ie 29/05/98 38&ffltanyanWzetf. Soes no! confaTn TSC C^^ elF atochem