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AR226-3104 TNO-report V98.337 Augmented acute (4-hour) inhalation toxicity study with 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 Author(s): Dr H. Muijser Study director. DrH. Muijser TNO Project number 470001/017 At the request of: ELF Atochem S.A. Paris La Defense, France 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 this 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 Status: Final, November 1998 Previous version: Unaudited draft, March 1998 Number of pages: 58 TNO Company Sanitized. Does not conia'n TSCA CB1 TNO Nutrition and Food Research Institute is dedicated lo the (inlerjnational food industry, pharmaceutical industry and chemical industry. The Institute's divisions are: Agrotechnology, Analytical Sciences. Biochemistry and Gene Technology, Industrial Microbiology, Occupational Toxicology and Nutrition, Toxicology. Netherlands Organization for Applied Scientific Research V98.337 November 1998 Page 2 Summary by nose-only exposure of one group ofjsight male and eight female rats to a test atmosphere containing 21.2 0.2 g^irlJBi^^^lHiHI^HBiilBQ^a single 4-hour period. Half of the rats were necropsied the day after exposure, the remaining rats were kept for a 14-day observation period. Four additional, unexposed rats were also sacrificed for background histopathology. 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 analysis was 122 4 mg/m3. The MMAD of the particulate matter was 3.8 urn and the geometric standard deviation was 1.1. During exposure, abnormalities in breathing pattern or restlessness could not be seen. Shortly after exposure, abnormalities consisted of moderate piloerection in all animals, hunched appearance, laboured breathing, shallow breathing or irregular breathing. Slight dyspnea was also noticed. Mortality was the most important finding in this study, viz. 5 out of 16 animals died within 1-4 days after exposure. The occurrence of late mortality in the group intended to be kept for an observation interval of 14 days 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 may be underestimated. On day 1 of the observation period, sluggishness, laboured breathing, blepharospasm, hunched appearance, and piloerection were seen. With respect to breathing abnormalities, a biphasic pattern was observed. Initially, laboured breathing was seen in most surviving animals, which persisted for a number of days, but later, a visually increased breathing rate was noted in two female and in two male animals. No abnormalities' were seen in the animals of the control group. With respect to the measurement of breathing parameters, although statistical power was reduced due to mortality, breathing frequency was significantly increased just after exposure and 14 days after exposure, tidal volume was reduced just after exposure and one day and 4 days after exposure and ventilatory flow was significantly reduced just after exposure and one day after exposure. Changes in lung mechanical parameters could not be demonstrated. One day after exposure, body weight loss was seen in all exposed animals. On day 7 body weight in all but one of the surviving animals of group A2 was still below the pre-exposure value. During the second week of the 14-day observation period, Pompany Sanitized. Does no! contain TSCA CBI 'Company "8an?ze^. p"^ V98.337 November 1998 Page3 normal body weight gain was seen in the surviving animals of group A2, but of course the weight attained was below that of the unexposed animals (A3), in which, as expected, normal weight gain was seen in both observation weeks. Macroscopic lung changes, i.e. red discoloured and/or swollen lungs, were seen in deceased rats, in several rats necropsied one day after exposure and in a few rats necropsied 14 days after exposure. In addition, relative lung weights had increased. Changes at the microscopic level in deceased rats and in rats scheduled for necropsy after one day, consisted of moderate to severe alveolar haemorrhages, perivascular oedema and perivascular inflammatory cell aggregates, whereas surviving animals scheduled for necropsy 14 days after exposure showed slight to severe multifocal pneumonia or accumulation of alveolar macrophages accompanied by moderate increased septal cellularity. Alveolar haemorrhages were no longer present at this time point. Conclusion It was concluded, that asmgle^hom_[acutelexposure of rats to a high in mortality; changes in airway function, and macroscopical and histopathological lung changes. Therefore, from the results of the present study, it cannot be excluded that human exposure at high aerosol concentrations will result in serious and lasting lung changes. CempanySanitized. Does not contain TSCA CBI V98.337 November 1998 Page 4 Contents Summary ........................................................ 2 Statement of GLP compliance ........................................ 5 Authentication by cooperating scientist................................. 6 Quality Assurance Statement ......................................... 7 GLP compliance monitoring unit statement ............................. 8 Testing facility .................................................... 9 Contributors ...................................................... 9 1 Introduction ............................................. 10 2 Experimental............................................. 10 2.1 Test material ..................................... 10 2.2 Test animals Experimental ..................................... 11 11 2.3 conditions ............................ 2.4 Experimental procedures ............................ 12 2.5 Exposure chamber ................................. 12 2.6 Generation of the test atmosphere ..................... 13 2.7 Analysis of the test atmosphere ...................... 13 2.8 Observations and measurements ...................... 14 2.9 Statistical analysis ................................. 16 2.10 Retention of records ............................... 16 2.11 Deviations from the protocol ........................ 16 3 Results 18 3.1 .A.n.a.l.y.ti.c.a.l.r.es.u.l.ts.................................... 18 3.2 Behaviour, clinical .si.g.n.s.a.n.d..m.o.r.t.al.it.y.................. 18 3.3 Body weights ................. 19 3.4 L u n g function ...... measur . e ..... ments . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.5 Lung weights ........................ 20 3.6 ..................................... 20 Pathology........................................ 4 Discussion and conclusion .................................. 21 5 References .............................................. 22 Appendices ...................................................... 49 'pBTpany3anRtee3. >' Does not contain TSCA CBi V98.337 November 1998 Page5 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) ^ Mc^.^1^ 4^'s Date 1-2. No/^fcer (^-T(P Date jCompanySanHIze'd. Does not contain TSCj V98.337 November 1998 Page6 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 fcA. Woutersen (Pathologist) \1 NQ\H^9<-r (<^? Date l&DtnpanSyanHIze3. Does ffol contain TSCA CBI V9B.337 November 1998 Page 7 Quality Assurance Statement On: Report Number: Date: Angmented.acute (4-hour) inhalation toxicity study with V98.337 November 1998 The protocol and the experimental phase of this study were inspected by the Quality Assurance Unit ofTNO Nutrition and Food Research Institute as follows: Date of inspection: 27 November 1998 Date of report: 28 November 1998 This report was audited as follows: Dates of audit: 2 and 10 November 1998 Date of report: "12 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. Ing. P.A. de Lang (Quality Assurance Unit) Date: )2) Wn^-^- /?fS V98.337 November 1998 Pages STAATSTOEZ1CHT OP DE VOLKSGEZONDHEID 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. U^'R--i],.vyi|k, 10 September 1996 ^-.D---------"""^061 Compaq ^. / Th. Holder, DVM ^Minfetry^.f-'Health, Welfare and Sport State Supervisory Public Health Service Veterinary Public Health Inspectorate ni D c^^<-:~-. V98.337 November 1998 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 +31 30 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: Study Assistant Senior Inhalation Technician Inhalation Technicians Biotechnicians Senior Biotechnician Pathologist Dr H. Muijser* Ir J.H.E. Arts Ms M.M. Andringa fag 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 * Toxicology Division, TNO Nutrition and Food Research Institute Company Sanitized. Does not contain TSC& ^w V98.337 November 1998 Page 10 Introduction At the request of ELF Atochem S.A., Paris La Defense^Erance, an acute (4-hour) out in rats. Although only mortality data are used for classifying acute toxicity, in the present study clinical observations, body weights, lung function measurements, lung weights, macroscopical observation at necropsy and histopathology of the lungs were additionally used as criteria for disclosing possible harmful effects. The study design was based on an augmented acute inhalation toxicity study (TNO report V93.181.June 1993). 2 Experimental The study was conducted according to a protocol, entitled: "Protocol for an ^BlIHIHUPQin rats", approved by the stady director on 18 November 1998 (see 2.1 and 2.11 for change froni^HHUB111 the name of the test material). 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 material The test material was supplied by the sponsor. Two plastic bottles, labelled respectively, were received on 17 November 1997. As shown below the test TNO internal reference number was 970285. The test material was stored at ambi ent temperatures. characteristics (as given by the sponsor): d has the following Composition: Composition: Colour: Boiling point: liompanySanitize, noeirroil contalfn TSCA CB; V98.337 November 1998 Page 11 Autoignition temperature: Explosive limits: Vapour pressure: Density: Batch number: 2.2 Test animals 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 arrived on 5 November 1997, at an age ofca. 6-7 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 10 November 1997 to animal room 06.05; the rats were randomly allocated to the cages, separated by sex and uniquely identified by ear tattoo. On 26 November 1997, one day before the start of exposure, 8 male and 8 female rats were assigned to the study to be exposed to the present test material in two groups of 4 rats for each sex. hi addition, 2 male and 2 female rats were assigned to the study as controls for background histopathology. At that time, mean body weights of the male and female rats were 252 and 177 g, respectively. The duration of the acclimatization period in the inhalation facilities was 16 days. The groups of animals were identified by a letter (A) and a subcode (sl, s2 and s3) and a colour code (white). In this report, for the sake of clarity, the different groups are abbreviated as Al, A2, and A3, respectively. The individual animals were identified by earmark (tat too). 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 exposure, one group (Al) was immediately returned to their living cages, held for an observation period of one day and sacrificed. The second group (A2) 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 (group A3) were sacrificed on day 14 of study for background lung histopathology. The animals were housed in animal room 06.05,4 males or 4 females to a cage (groups Al and A2), or 2 males or 2 females to a cage (group A3). 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 20.0 and 22.0C and relative humidity was between 46 and 68%. A 12-hour light and 12-hour dark cycle was jCSmpany:SanfHzeDdo. es not contain TSCA CBI V9B.337 November 1998 Page 12 maintained. 2.3.2 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. 3604) 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. WaterieidingbedrijfMidden-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 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 27 November 1997 with one group consisting of 8 male and 8 female rats. This group was exposed by inhalation to the limit concentration of at least 20 g/m3 for a single 4-hour period. One group of 4 male and 4 female animals was necropsied the following day. The other group 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. No more animals were exposed. The study was, therefore, finished with the necropsy of all remaining rats on 11 December 1997. 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 8UB, 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 (and 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 low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer ^^paLny^nnfeeg; Po^wrconiaTn TSCA C8? V98.337 November 1998 Page 13 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 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 24 e/min. All animals of group Al were placed in the exposure unit 60 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, the animals were placed sequentially, one at a time (balanced with respect to sex), with an interval of 15 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 (Ratfisch RS55, Munich, Germany). The response of the analyser was recorded by an analogue recorder (Kipp & Zonen, Delft, The Netherlands). The settings of the total carbon analyser were as follows: - oven temperature: 150 C - H; pressure: - air pressure: - back pressure: - sample line: 0.5 bar 0.8 bar 200 mbar 148 C 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 (FED) of the total carbon analyser was calibrated by injecting Teflon bags filled with 30 liters of air with 790, 790, 880, 880, 970 or 970 u(fabMfc}Merck,p.a.). respectively JSff--------k Jll^lH^^BIIBi^l^^H^HHBHBBIHBIHIHKr^e amount orj^^^^^Hri'as considered to be negligible small. The resulting responses were iWany Sanitized. Does not conh>? -^ ^, V9B.337 November 1998 Page 14 used to construct a calibration curve which was needed to convert the responses in scale units of the TCA to concentrations (g/m3). A quadratic relation was obtained: fl--------Jconcen= tr0a.0t1i1o2 n* X2 - 1.477 * X + 64.28 in whicwBUIrevas in g/m3, X was the response in scale units of the TCA and the correlation coefficient was 0.99. 2.7.2 Gravimetric analysis The amount (by weight) of non-volatile particles present in the test atmosphere was determined approximately once each hour by means of gravimetric analysis. Representative test atmosphere samples were obtained by passing 50 C test atmosphere at 5 <?/minthrough fiber glass filters (Sartorius). Samples were weighed before and after sampling. 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 concentration was 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 Particle size distribution measurement was carried out once during exposure using an 11-stage cascade impactor (Institute's design) with the largest cut-off size of 4.2 //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' twelve times during exposure at regular intervals (about twice per hour) using a wet and dry bulb thermometer. 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. ^^P^ySahWzea. Does not confa?n TftrA <oy V98.337 November 1998 Page 15 2.8.2 Body weights Body weights of the animals were recorded on the day before exposure (day -1), just prior to exposure (day 0), on day 1 and on days 7 and 14 (surviving animals of group A2 and group A3). 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 (group A2, destined for autopsy 14 days after exposure) were measured before exposure (day-1, control value) and surviving animals directly after exposure (day 0) and on days 1,4 and 14 of the observation period. Each rat was placed in a modified Battelle restraining tube with a water-wetted silicon diaphragm separating externally head and neck from thorax and abdomen. Each rat was subsequently placed in a double room plethysmograph (the so-called head and body chamber). Mouth flow was measured in the head chamber by means of a resistance and a pressure device and thoracic movement was determined with a pressure device (Honeywell) in the body chamber. Breathing frequency, tidal volume, and mean ventilatory flow were determined by means of recording the pressure signal in the body chamber. Lung mechanical properties were obtained by determination of the transfer function as described in detail by Oostveen et al. (1992). Hereto, small sinusoidal pressure fluctuations (around 1 hPa peak-peak) were applied around the thorax in the frequency range of 16-208 Hz. These pressure fluctuations yielded small sinusoidal changes in the ventilatory flow which were superimposed on the spontaneous breathing and which were measured in the head chamber. Transfer impedance (ZJ, defined as applied pressure (P) divided by the flow (v), was determined by means of a Fast Fourier transform and checked for internal coherence as described by Michaelson etaL (1975). Zy is a complex function of the frequency and is described in a real (ZR;) and imaginary part (Zim). Using an appropriate simplified parameter estimating program, the parameters were directly linked to the physiological variables resistance, inertan'ce and compliance. As described by Oostveen etal. (1992), the general description of the six-element Dubois model adequately describes the data above 60 Hz (beyond the influence of neck shunt). The Dubois model yields a descriptive equation of the form: Zy = R - Bo)2 + j [-I/G)C + Ifa) - Eco3] in which R is total respiratory resistance, I is total, inertance and C is compliance. R and I were estimated from the Zy data as: tempany Sanlllzea. Dees ffbl contain TSCA CB1 V98.337 November 1998 Page 16 R = Z^(96 Hz) + 1/3 {U% Hz) - 4e(192 Hz) (Z^=R- B&)2) I = Z^(160 Hz) / 1000 (2nl60 = 1005; Z,^ = [-1/coC +I&) - E&>3]) At low frequencies, the Z^ was determined by the terms C and I and if Z^ = 0 (at the resonance frequency &)o), then C= (I(>o2)"1T-he value of (>owas obtained by linear interpolation between the two adjacent frequencies at which Z^ has opposite signs, in most cases 7^<0 at 16 Hz and Z,,n>0 at 32 Hz. Because of insufficient coherence, sometimes the value ofZ,n,atl6Hz could not be determined. In that case the value ofZ;n,atl6Hz was linearly extrapolated from the values at 32 and 48 Hz using the formula: ZJ16Hz)=2*Zto(32Hz) - Z.^(48Hz). Finally, values above 10 m?/kPa were considered unrealistically high and left out. 2.8.4 Pathology A group of 4 male and 4 female rats (group 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 group A2 together with the four unexposed rats (group A3) were killed in the same way. All rats were examined for gross pathological changes. 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 haematoxylin and eosin and then examined microscopically at one longitudinal level. 2.9 Statistical analysis First, differences between pre- and post-treatment breathing parameters (mechan ical and ventilatory) were analysed by one-way ANOVA using post/pre-exposure ratios to detect possible sex differences. If there were no sex differences, male and female data were pooled and subjected to matched pair t-tests using pre- and post- exposure values. 2.10 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. 2.11 Deviations from the protocol - The lung weight of animal no. 74 was accidentally not measured. - On request of th^nonsor the name of the test material in this report was ^g^lizea^oii^^^nT^^ V98.337 Novsmber1998 Page 17 - Due to an organizational change within the Toxicology Division on the 1st of July 1998, management responsibilities for this study have been assigned to Drs H.H. Enunen as from that date. These deviations were not considered to have influenced the validity of the study. Company Sahltlzea. Odes Hel conlain TSCA CB V98.337 . November 1998 Page 18 3 Results 3.1 Analytical results 3.1.1 Total carbon analysis The actual concentration of^Wffglwmg, the exposure based on total carbon analysis of the volatile fractionflU^^Payeraged at 5-min intervals was 20.4 0.2 g/m3 (N=48). Sincel J------^Rthceoncentration ol 3.1.2 Gravimetric analysis The results of the gravimetric analysis are given in Table The calculated mean^ concentration of the solid fraction of| ^H^IHiHUf^ 122 4 mg/m3, which is around 0.6% of the actual concentration measured with total carbon analysis. 3.1.3 Nominal concentration The nominal concentration was calculated to be 21.9 g/m3. The nominal concentration was slightly higher than the actual concentration of 21.2 g/m3, cating a generation efficiency of 97%. indi 3.1.4 Particle size measurement The particle size distribution is given in Table 2. It was shown that 84% of the particles present at the animals' breathing zone had an aerodynamic diameter equal to or smaller than 4.2 /j,m. The Mass Median Aerodynamic Diameter (MMAD) was 3.8 //m and the mean geometric standard deviation (gsd) of the particles was 1.1. 3.1.5 Measurement of temperature, relative humidity and oxygen concen tration The mean temperature during exposure was 20.8 0.4 C (range 20.0-21.0 C) and the mean relative humidity was 43 1% (range 42-46%). The oxygen concentra tion during exposure was 21.7%. 3.2 Behaviour, clinical signs and mortality Data on behaviour and clinical signs are given in Tables 3. During exposure, abnormalities in breathing pattern or restlessness could not be seen, it must be kept in mind, however, that due to the stay in restraining tubes, observation of the rats was limited (Tables 3.1). Shortly (see Tables 3.2.1 and 3.2.2) after exposure, abnormalities consisted of moderate piloerection in all animals, hunched appearance (slight) in 3 male (nos. CSMPanygahifeea. DsesTOl contain TSCA Cl V98.337 November 1998 Page 18 62, 74, 78) and 7 female animals (nos. 73, 75, 77, 79, 81, 85, 89) and with respect to breathing, laboured breathing ranging in severity from slight to moderate was noted in 7 male (nos. 62, 68, 72,74, 78, 82, 86) and 6 female animals (nos. 71, 75, 79, 81, 85, 89), The other animals displayed shallow breathing (slight, no. 66) or irregular breathing (slight, nos. 73, 77). Finally, slight dyspnea was noticed in 5 male (nos. 62, 68, 74,78, 82) and in 2 female animals (nos. 81, 89). On day 1 of the one-day observation period of group Al (Table 3.3.1), all animals except one (no. 86) were sluggish. Laboured breathing was seen in 3 male (62, 72, 82) and in one female animal (no. 75). Less frequently observed effects were blepharospasm in two male animals (nos. 62, 82) and a hunched appearance combined with piloerection in one female animal (no. 85). More importantly, mortality occurred: later that day (at 1 Ih), one male (no. 62) and one female animal (no. 85) were found dead in their home cage. i During day 1 of the 14-day observation period of the animals of group A2 (Tables 3.3.2), similar signs were found. Also in this subgroup mortality occurred: on day 1, 2 and 4, respectively, a male (no. 66), a female (no. 73) and another male animal (no. 74) died. With respect to breathing abnormalities, the data of group A2 suggested a biphasic pattern. Initially, laboured breathing was seen in most animals, which persisted for a number of days, but later, an increased breathing rate was found in two surviving female (nos. 77, 89) and in 2 male (nos. 68,74) animals of which one died on day 4. One male (no. 81) and one female animal (no.78) were without signs from day 1 on. No abnormalities were seen in the animals of the control group (Table 3.3.3). Mortality was 2 out of 8 in subgroup Al and 3 out of 8 in subgroup A2. Mortality in group Al may however be an underestimate, because the occurrence of late mortality in the group intended to be kept for an observation interval of 14 days (A2) suggested that late mortality could have been expected to have occurred in the animals of the 1-day observation period (Al) if this group had been kept for a 14-day observation period as well. 3.3 Body weights Individual and mean body weights are indicated in Table 4. One day after exposure, body weight loss was seen in all exposed animals. On day 7 body weight in all but one of the surviving animals of group A2 was still below the pre-exposure value. During the second week of the 14-day observation period, normal body weight gain was seen in the surviving animals of group A2, but of course the v/eight attained was below that of the unexposed animals (A3), in which, as expected, normal weight gain was seen in both observation weeks. red. Does ffot contain TSCA rw V98.337 November 1998 :s^ Page 20 3.4 Lung function measurements 3.4.1 Ventilatory variables Data on breathing frequency, tidal volume and mean ventilatory flow are given in Table 5. Just after exposure all but one female animal showed an increase in breathing frequency compared to pre-exposure values. The difference was statistically significant as well as the difference at day 14 (p<0.05). Although the values in the males remained higher at day 1 and day 4, this was not statistically significant (Table 5.1). It must be bome in mind that, due to mortality, the statistical power was seriously reduced. Compared to the pre-exposure level, tidal volume was significantly reduced just after exposure, and 1 day (psO.OOOl) and 4 days (p<0.05) after exposure. The decrease on day 14 was not significant (Table 5.2). Ventilatory flow, the product of breathing frequency and tidal volume, was significantly reduced (p<0.005)just after exposure and on day 1 compared to preexposure values. This reduction did not persist four or fourteen days after exposure (Table 5.3). 3.4.2 Lung mechanical measurements Individual and mean responses in lung function measurements are given in Table 6. Changes in the lung mechanical variables, airway resistance, inertance and compliance were not detected. Data on airway compliance were more variable (Table 6.3), which was explained by a loss of some of the 16 Hz data (the lowest frequency tested) due to a too low internal coherence at that frequency. As such, the points where Zin, was zero (which is necessary to determine the compliance) occasionally had to be extrapolated which resulted in less reliable estimates. 3.5 Lung weights Absolute and relative (to body weight) lung weights are given in Table 7. Mean relative lung weights recorded one day after exposure in group Al were comparable to those observed 14'days after exposure in group A2 (Table 7.1). However, these values were high compared to mean relative lung weights of unexposed rats (Table 7.2). 3.6 Pathology Summarized macroscopic and microscopic findings are given in Table 8 and 9, respectively; individual findings are given in Appendices 1 and 2. f SahHIzeff. Uses not contain TSCA cw V98.337 November 1998 Page 21 3.6.1 Macroscopy At necropsy, several surviving animals of group Al were in poor health condition. A few of these rats showed sniffing and diarrhoea. Macroscopic observation of deceased rats revealed red discoloured and/or swollen, occasionally blood filled lungs. Several surviving rats necropsied one day after exposure also showed discoloured and/or swollen lungs whereas red spotted lungs were observed in a few others. A few surviving rats necropsied 14 days after exposure also showed red discoloured lungs. These macroscopic changes were considered to be treatment-related (Table 8). The other changes were found in one or a few rats of all groups, controls included, and were therefore not considered to be related to treatment. 3.6.2 Microscopy Microscopical examination of the lungs of deceased rats revealed lesions which were characterized by moderate to severe alveolar haemorrhages, perivascular oedema and perivascular inflammatory cell aggregates. Surviving animals scheduled for necropsy one day after exposure showed similar changes. In most lungs of surviving animals necropsied 14 days after exposure slight to severe multifocal pneumonia or accumulation of alveolar macrophages accompanied by moderate increased septal cellularity could be demonstrated. Alveolar haemorrhages were no longer present at this time point. In unexposed control rats, perivascular inflammatory cell infiltrates were found in 3 out of 4 rats, pneumonia in 1 out of 4 rats, and accumulation of alveolar macrophages in 1 out of 4 animals. Only one rat showed no abnormalities. It should, however, be noted that the changes in these unexposed control rats were only slight to very slight. 4 Discussion and conclusion The aim of the presen^study was to determine the acute inhalation toxicity of rats was exposed to the limit concentration of 21.2 g/m3 during a single period of four hours. Half of the rats were necropsied the day after exposure, the remaining rats were kept for a 14-day observation period. Four additional, unexposed rats were also sacrificed for background histopathology. Mortality was the most important finding in this study, viz. 5 out of 16 exposed animals (groups Al and A2) died within 1-4 days after exposure. The occurrence of late mortality in group A2 (intended to be kept for an observation interval of 14 days) suggests that late mortality could be expected to have occurred in the animals of group Al (1-day observation period) if this group had been kept for a 14-day observation period as well, indicating that total mortality may be an underestimate. 1Snp:%Sanitized, Soes^liofflaTri TS'CA 'CSi V98.337 November 1998 Page 22 With respect to breathing parameters, although statistical power was reduced due to mortality, breathing frequency was significantly increased just after exposure and 14 days after exposure, tidal volume was reduced just after exposure and one day and 4 days after exposure and ventilatory flow was significantly reduced just after exposure and one day after exposure. Changes in lung mechanical parameters could not be demonstrated. Macroscopic lung changes, i.e. red discoloured and/or swollen lungs, were seen in deceased rats, in several rats necropsied one day after exposure and in a few rats necropsied 14 days after exposure. In addition, relative lung weights had increased. Changes at the microscopic level in deceased rats and in rats scheduled for necropsy after one day, consisted of moderate to severe alveolar haemorrhages, perivascular oedema and perivascular inflammatory cell aggregates, whereas surviving animals scheduled for necropsy 14 days after exposure showed slight to severe multifocal pneumonia or accumulation of alveolar macrophages accompanied by moderate increased septal cellularity. Alveolar haemorrhages were no longer present at this time point. It was concludedjhat a single 4-hour (acute) exposure of rats to a high in mortality, changes in airway function, and macroscopical and histopathological lung changes. Therefore, from the results of the present study, it cannot be excluded that human exposure at high aerosol concentrations will result in serious and lasting lung changes. 5 References Lee R.J. Jr., Science, 1972: 567-575 Michaelson E.D., Grassman EJ). and Peters W.R., J. Clin. Invest. 1975: 56, 1210'1230 Oostveen E., Zwart A., Peslin R. And Duvivier C., J. Appl. Physiol. 1992: 73,1598-1607 CwipanySahIfcea.Boesnot^aInTSCAeBI V98.337 November 1998 Page 23 Figure 1 - Schematic diagram of the generation and exposure system Compressed Dry Air Mainvalve Reducing valve Flow meter Valve Humidifier Thermal bath US Nebuliser Fluid pump Supply vessel 10 Pressure gauge 11 Regulating valve HNO Head/Nose Only Unit AH Animal holder EHD External Hood penipanySanfflzea Does Hbl contain TSCA CW V98.337 November 1998 Page 24 I ACUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 1 - Concentration of solid fraction in the test atmosphere measured by gravimetry Sample time 11:10 12:05 13:15 14:15 15:20 16:10 mean sd Volume (t) 50 50 50 50 50 50 Concentration (mg/m3) 121 120 119 118 123 129 1224 Generation of test atmosphere: 9:30-16:30 h Exposure duration of the animals: 4 hours between 10:30 and 16:30 h RWBpSBySaifiWzea. Does^nof conta'n Tfica fn V98.337 November 1998 Page 25 TNO Nutrition and Food Research Institute Study: 470001/017 Table 2 - Aerodynamic particle size distribution in test atmosphere Particle size (/an) <4.2 $3.8 ^3.4 ^3.1 ^2.8 ^2.4 ^ 1.8 s 1.4 $ 1.0 ^0.8 ;0.1* total Mass (mg) 1.04 2.20 1.70 0.50 0.40 0.23 0.18 0.11 0.00 0.00 0.00 6.36 Cumulative (%) 83.6 49.1 22.3 14.5 8.2 4.6 1.7 0.0 0.0 0.0 0.0 *The amount sampled on this stage included back-up filter. The volume was 75 t, sample flow was 5.0 C/min Mass Median Aerodynamic Diameter (MMAD): 3.-8 ^m mean geometric standard deviation (gsd): 1.1 Jpr.ySahlSizea.Doesiiol c<fn1iain TSRo c1"- V98.337 November 1998 Page 26 ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study:470001/017 Table 3.1.1 - Clinical signs during exposure (group Al) Animal number Clinical signs Time of observation MALES 62,72,82,86 FEMALES 71,75,79,85 NO ABNORMALITIES NO ABNORMALITIES 11:30,12:25, 13:25, 14:20 11:30, 12:25, 13:25, 14:20 exposure period of group A: 10h30 -14:30 ICompSRSyanTlTzea.'Ue&STOlCCTlaIn TSC.M < ;si< V98.337 November 1998 Page 27 ^.CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study:470001/017 Table 3.1.2 - Clinical signs during exposure (group A2) Animal number Clinical signs MALES 66,68 NO ABNORMALITIES 74 NO ABNORMALITIES 78 NO ABNORMALITIES FEMALES 73,77 81 89 NO ABNORMALITIES NO ABNORMALITIES NO ABNORMALITIES Time of observation llh45,12h45,13h45,14h45 12h45.13h45,14h45,15h40 12h45,13h45,14h45,15h40, 16hl0 llh45,12h45,13h45,14h45 12h45,13h45,14h45,15h40 12h45,13h45,14h45,15h40, 16hl0 exposure of animal 66: 10h45-14h45 exposure of animal 73: llh00-15h00 exposure of animal 68: llhl5-15hl5 exposure of animal 77: llh30-15h30 exposure of animal 74: llh45-15h45 exposure of animal 81: 12h00-16h00 exposure of animal 78: 12hl5-16hl5 exposure of animal 89: 12h30-16h30 'gwnpaiySannfeea. noesffoi contain TSRA i V98.337 November 1998 Page 28 U\CUTE(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 3.2.1 - Clinical signs shortly' after the end of exposure (group Al) Animal number MALES 62 72,86 82 FEMALES 71 75,79,85 Clinical signs GENERAL piloerection hunched appearance BREATHING laboured breathing dyspnea GENERAL piloerection BREATHING laboured breathing GENERAL piloerection BREATHING laboured breathing dyspnea GENERAL piloerection BREATHING laboured breathing. GENERAL piloerection hunched appearance BREATHING laboured breathing Grade moderate slight slight slight moderate slight moderate slight slight moderate slight moderate slight slight Time of observation 16h40; exposure ended on 14h30 ^iii^SOteea.^""""""TM*"' Mkk 1iiu rspon V98.337 November 1998 Page 29 TNO Nutrition and Food Research Institute Study: 470001/017 Table 3.2.2 - Clinical signs shortly' after the end of exposure (group A2) Animal number MALES 66 68 74,78 FEMALES 73,77 81,89 Clinical signs GENERAL piloerection BREATHING shallow breathing GENERAL piloerection BREATHING laboured breathing dyspnea GENERAL piloerection hunched appearance BREATHING laboured breathing dyspnea GENERAL piloerection hunched appearance BREATHING irregular breathing GENERAL piloerection hunched appearance BREATHING laboured breathing dyspnea Grade moderate slight moderate moderate slight moderate slight slight slight moderate slight slight moderate slight moderate slight ' time of observation 17h05; exposure ended between 14h45 and 16h30 (see note at the bottom of Table 3.1.2) , -cr&CBl --------^M.W"'"^"^ V98.337 November 1998 Page 30 ^CUTE (4-HOUR) INHALATION RAT 'TNO Nutrition and Food Research Institute Study: 470001/017 Table 3.3.1 - Clinical signs during the 1-day observation period (group Al) Animal number MALES 62 Clinical signs Range* GENERAL blepharospasm 1 sluggish 1 BREATHING laboured breathing 1 FOUND DEAD just before necropsy 1 72 GENERAL sluggish 1 BREATHING laboured breathing 1 82 GENERAL blepharospasm 1 sluggish 1 BREATHING laboured breathing 1 86 NO ABNORMALITIES 1 FEMALES 71,79 GENERAL sluggish 1 75 GENERAL sluggish BREATHING laboured'breathing 85- GENERAL hunched appearance sluggish piloerection FOUND DEAD just before necropsy range: 1 day (animals were sacrificed on day 1) .WW^663^ V98.337 November 1998 ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 3.3.2 - Clinical signs during the 14-day observation period (group A2) Animal number MALES 66 68 74 78 Clinical signs GENERAL blepharospasm sluggish BREATHING laboured breathing FOUND DEAD on day 1 GENERAL sluggish BREATHING laboured breathing shallow breathing increased breathing rate NO ABNORMALITIES GENERAL sluggish nasal encrustations BREATHING laboured breathing shallow breathing increased breathing rate FOUND DEAD on day 4 NO ABNORMALITIES Range* 1 1 1 1-7 1 2-7 2-8 9-14 1-3 2-3 1 2-3 2-3 1-14 * range day 1 - day 14 V98.337 November 1998 y]\TUT(E4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 3.3-2 (continued) - Clinical signs during the 14-day observation period (group A2) Animal number FEMALES 73 77 81 89 Clinical signs GENERAL sluggish eyes closed BREATHING laboured breathing FOUND DEAD on day 2 GENERAL sluggish hunched appearance meager/slender appearance BREATHING laboured breathing increased breathing rate NO ABNORMALITIES NO ABNORMALITIES GENERAL sluggish hunched appearance meager/slender appearance BREATHING increased breathing rate NO ABNORMALITIES range day 1 - day 14 Range' 1 1 1 1-3 1-3 6 1-3 6-8 4-5,9-14 1-14 1-2 1-2 6 . 6-8 3-5, 9-14 ^.n^"01^'1' iSanw11", V98.337 . November 1998 Page 33 ICUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 3.3.3 - Clinical signs during the 14-day observation period (group A3) Animal number MALES 64,70 FEMALES 83,91 Clinical signs NO ABNORMALITIES NO ABNORMALITIES Range* 1-14 1-14 * range day 1 - day 14 .......<-'"" tSi**,^ V98.337 November 1998 Page 34 iiCUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study:470001/017 Table 4.1 - Individual and mean body weights (g) of male and female rats (group Al) Animal No. Males (Al) 62 72 82 86 mean sd Females (Al) 71 75 79 85 mean sd Day-1 245.4 246.3 254.5 260.9 251.8 7.3 180.2 180.1 177.1 168.3 176.4 5.6 DayO 249.1 249.6 260.9 264.9 256.1 8.0 184.6 189.6 183.5 175.1 183.2 6.0 Day 1 229.4 228.6 239.8 255.5 238.3 12.5 171.7 171.4 167.5 160.9 167.9 5.0 ^ ^^^ S^'1 <5Ct^ Aft*- V98.337 November 1998 Page 35 ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 4.2 - Individual and mean body weights (g) of male and female rats (group A2) Animal No. Males (A2) 66 68 74 78 mean sd Females (A2) 73 77 81 89 mean sd Day-1 239.6 257.9 260.7 248.3 251.6 9.6 179.4 170.9 174.7 182.2 176.8 5.0 DayO 243.7 262.3 264.8 253.8 256.2 9.5 183.7 172.6 177.1 186.1 179.9 6.2 Dayl 226.0 237.8 243.0 236.2 235.8 7.1 169.8 161.6 175.4 170.6 169.4 5.7 Day 7 Day 14 224.6'd) 237.0 210.9'(4) 251.0 244.02 - 265.7 . 277.0 271.42 166.6'(2) 162.5 178.3 174.9 171.9 8.3 .. 180.0 193.1 191.8 188.3 7.2 ' no weight available on day 7, animal weight at death on day 1, day 2 or day 4, indicated as (1), (2) or (4), respectively. 2 no sd given for mean of two values 15C^ ^ ^ {^w -A^689 ^ ^ ^.8' V98.337 November 1998 Page 36 ^.CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 4.3 - Individual weights (g) of male and female rats (group A3) Animal No. Males (A3) 64 70 mean Females (A3) 83 91 mean Day-1 247.7 257.2 252.4 189.5 170.4 180.0 DayO 257.1 261.6 259.4 190.2 176.8 183.5 Dayl 253.8 264.0 258.9 189.5 175.6 182.6 Day 7 Day 14 278.5 295.2 286.8 303.9 320.1 312.0 199.9 180.1 190.0 207.4 193.3 200.4 V98.337 November 1998 Page 37 ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 5.1 - Individual responses in breathing frequency (group A2) Animai code males 66 68 74 78 mean/ sem pre abs 3.20 3.17 3.65 3.33 3.34/ 0.11 dayO abs 3.77 3.80 4.17 3.63 3.845/ 0.12 rel 1.18 1.20 1.14 1.09 Breathing frequency (1/s) day 1 day 4 abs * 3.92 3.70 4.23 3.95/ 0.15 rel 1.24 1.01 1.27 abs * 5.38 * 3.90 4.64/ rel 1.70 1.17 day 14 abs * 5.48 * 3.73 4.60s/ rel 1.73 1.12 'emales 73 77 81 89 mean/ sem 3.70 3.24 3.65 3.33 3.48/ 0.11 4.04 3.49 3.16 3.63 3.58s/ 0.18 1.09 1.08 0.86 1.09 2.36 3.14 4.25 4.22 3.49/ 0.46 0.64 0.97 1.16 1.26 * 4.82 3.36 5.10 4.43/ 0.54 1.49 0.92 1.53 * 3.43 5.19 5.36 4.66s/ 0.62 abs: absolute value; rel: value in % relative to pre-exposure value - animal died, hence no values available - no sem given for mean of two values 1.06 1.42 1.61 Statistics: - - - One way ANOVA for sex differences using post/pre-exposure ratios (no significant differences found) - Matched pair t-test by comparing pre- and post-exposure values (pooled sexes); s: p<0.05, ^^ ^ r-^^ V98.337 November 1998 Page 38 TNO Nutrition and Food Research Institute Study: 470001/017 Table 5.2 - Individual responses in tidal volume (group A2) Animal code males 66 68 74 78 mean/ sem pre abs 2.60 2.60 2.80 2.80 2.70/ 0.06 dayO abs rel 1.60 2.00 2.00 2.00 1.905/ 0.10 0.62 0.77 0.71 0.71 Tidal volume (mC) day 1 day 4 abs rel abs rel * * 1.60 1.40 2.00 1.675/ 0.18 0.62 0.50 0.71 1.60 * 2.20 1.901/ 0.62 0.79 day 14 abs rel * 1.80 * 0.69 2.00 0.71 1.90/ emales 73 77 81 89 mean/ sem 2.00 2.00 2.00 2.20 2.05/ 0.05 1.40 1.00 1.80 1.40 1.405/ 0.16 0.70 0.50 0.90 0.64 0.80 0.80 1.20 1.20 1.005/ 0.12 0.40 0.40 0.60 0.55 * 1.00 2.00 1.20 1.401/ 0.30 0.50 1.00 0.55 * 2.80 1.40 1.60 1.93/ 0.44 abs: absolute value; rel: value in % relative to pre-exposure value - animal died, hence no values available - no sem given for mean of two values 1.40 0.70 0.73 Statistics - One way ANOVA for sex differences using post/pre-exposure ratios (no significant differences found) - Matched pair t-test by comparing pre- and post-exposure values (pooled sexes) ^sO.OOOl 'p<0.05 ^ ^89' V98.337 November 1998 Page 39 \.CUTE (4-HOUR) INHALATION RAT "TNO Nutrition and Food Research Institute Study: 470001/017 Table 5.3 - Individual responses in ventilatory flow (group A2) Animal code males 66 68 74 78 mean/ sem pre abs 8.32 8.25 10.23 9.33 9.031 0.47 dayO abs rel 6.04 7.60 8.33 7.25 7.305/ 0.48 0.73 0.92 0.81 0.78 Ventilatory flow (mC/s) day 1 day 4 abs rel abs rel * * 6.27 5.18 8.46 6.64s/ 0.96 0.76 0.51 0.91 8.62 * 8.58 8.60/ 1.04 0.92 day 14 abs * 9.87 * 7.45 8.66/ rel 1.20 0.80 emales 73 77 81 89 mean/ sem 7.40 6.47 7.31 7.33 7.13/ 0.22 5.65 3.49 5.68 5.08 4.93s/ 0.51 0.76 0.54 0.78 0.69 1.89 2.51 5.09 5.06 3.648/ 0.84 0.26 0.39 0.70 0.69 * 4.82 6.73 6.12 5.89/ 0.56 0.75 0.92 0.83 * 9.59 7.27 8.58 8.48/ 0.67 1.48 0.99 1.17 abs: absolute value; rel: value in % relative to pre-exposure value - animal died, hence no values available - no sem given for mean of two values Statistics - One way ANOVA for sex differences using post/pre-exposure ratio? (no significant differences found) - Matched pair t-test by comparing pre- and post-exposure values ^p^^ g> V98.337 Novembsr1998 Page 40 I^CUTE(4-HOUR) INHALATION TNO Nutrition and Food Research Institute Study: 470001/017 RAT Table 6.1 - Individual responses in airway resistance (group A2) Anima] Code pre Males abs 66 178 68 199 74 160 78 173 mean 178 sem 8 dayO abs rel 163 0.91 169 0.85 189 1.18 163 0.94 171 0.97 6 0.07 Resistance (hPa.s/?) day 1 day 4 abs rel abs rel * * 177 0.89 196 219 1.37 * 181 1.05 188 192 1.10 192 13 0.14 0.98 1.09 1.04 day 14 abs rel * 178 0.90 * 179 1.03 179 0.97 Females 73 187 77 140 81 167 89 169 mean 166 154 0.82 137 0.98 184 1.10 154 0.91 157 0.95 +10 +0.06 137 0.73 124 0.88 184 1.11 149 0.88 148 0.90 +0.08 * 166 1.19 150 0.90 159 0.94 158 1.01 +5 +0.09 * 167 1.19 144 0.87 175 1.03 162 1.03 +9 +0.09 abs: absolute value; rel: value relative to pre-exposure value - animal died, hence no values available; sem - no sem given for mean of two values Statistics - One way ANOVA for sex differences using post/pre-exposure ratios; (no significant differences found) - Matched pair t-test comparing pre- and post-exposure values (pooled sexes; no significant differences found). ^OlftPitf^f S. t f ^ ISO' *^aW ^ ACO V98.337 November 1998 Page 41 PjiCUT(4E-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 6.2 - Individual responses in airway inertance (group A2) Animal Code Males 66 68 74 78 mean sem pre abs 0.194 0.235 0.195 0.207 0.208 0.010 dayO abs rel 0.221 0.201 0.199 0.206 0.207 0.005 1.14 0.86 1.02 1.00 1.00 0.06 Inertance (hPa-s2/?) day 1 day 4 abs rel abs rel * * 0.201 0.242 0.216 0.220 0.120 0.86 1.24 1.04 1.05 0.11 0.213 * 0.236 0.224 0.91 1.14 1.02 day 14 abs rel * 0.217 * 0.92 0.209 0.213 1.01 0.97 ^males 73 77 81 89 mean sem 0.226 0.145 0.203 0.155 0.182 0.019 0.133 0.119 0.190 0.169 0.153 0.016 0.59 0.82 0.94 1.09 0.86 0.11 0.120 0.094 0.226 0.134 0.144 0.029 0.53 0.65 1.11 0.86 0.79 0.13 * 0.203 0.153 0.171 0.176 0.015 1.40 0.75 1.10 1.09 0.19 * 0.146 0.135 0.202 0.161 0.021 1.01 0.67 1.30 0.99 0.18 abs: absolute value; rel: value relative to pre-exposure value - animal died, hence no values available - no sem given for mean of two values Statistics - One way ANOVA for sex differences using post/pre-exposure ratios; (no significant differences found) - Matched pair t-test comparing pre- and post-exposure values (pooled sexes; no significant differences found). . ^ >1Co^8' ^d-001s^ ^ t;t1 V98.337 November 1998 Page 42 ^CUTE(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 6.3 - Individual responses in airway compliance (group A2) Animal Code Males 66 68 74 78 mean sem pre abs 1.70 1.16 * 2.58 1.82 0.41 dayO abs rel 1.30 1.45 1.88 0.76 1.25 1.54 0.18 1.00 0.24 Compliance (mMdPa) day 1 day 4 abs *** 1.12 0.75 1.01 0.96 0.11 rel 0.96 0.39 0.68 abs *** 1.12 *** 1.44 1.28 rel 0.96 0.56 0.76 day 14 abs *** 8.66 *** 1.18 4.92 rel 7.46 0.46 3.96 ^emales 73 77 81 89 mean sem 1.13 3.07 2.37 ** 2.19 0.57 2.49 3.17 5.11 2.16 3.23 0.66 2.20 1.03 2.15 1.80 0.38 3.63 3.52 1.70 3.15 3.00 0.44 3.21 1.15 0.71 1.69 0.77 *** 2.07 3.00 1.57 2.21 0.42 0.67 1.26 0.97 *** 7.43 5.22 1.22 4.63 1.82 2.42 2.20 2.31 abs: absolute value; rel: value relative to pre-exposure value * value outside realistic range (> 10) ** could not be determined from the data *** animal died, hence no values available - no sem given for mean of two values Statistics - One way ANOVA for sex differences using post/pre-exposure ratios; (no significant differences found) - Matched pair t-test comparing pre- and post-exposure values (pooled sexes; no significant differences found). ftOlC0"1.'nTSC^ COWP^^ ^'^ V98.337 November 1998 Page 43 TNO Nutrition and Food Research Institute Study: 470001/017 Table 7.1 - Absolute and relative lung weights of exposed animals; individual and mean values Animal no. Group Al absolute lung weight (g) relative lung weight (g/lOOgBW) Group A2 absolute lung weight (g) relative lung weight (g/lOOgBW) Males 62 3.202 1.40 72 1.672 0.73 82 2.782 1.16 86 1.381 0.54 66 3.247 1.37 68 2.723 1.02 74 -* -* 78 1.446 0.52 mean sd 2.259 0.871 0.957 0.391 2.472 0.926 0.972 0.426 Females 71 1.118 75 2.05 0.65 1.20 79 1.681 ' 1.00 85 2.720 1.69 73 3.012 1.67 77 2.046 1.06 81 1.237 0.64 89 2.398 1.44 mean 1.892 sd 0.672 * accidently not determined 1.135 0.433 2.173 0.741 1.20 0.45 gompan^ Sanitized. Does not contain TSCA CBi V98.337 November 1998 Page 44 1'^CUT(4E-HOUR) INHALATION TNO Nutrition and Food Research Institute Study: 470001/017 RAT Table 7.2 - Individual lung weights of non-exposed animals (group A3) Animal no. Males 64 70 Day 14 absolute lung weight (g) relative lung weight (g/lOOgBW) 1.573 1.388 0.52 0.43 Females 83 91 1.220 1.005 0.59 0.52 V98.337 November 1998 Page 45 TNO Nutrition and Food Research Institute Study: 470001/017 Table 8 - Summary of macroscopic lung observations of male and female rats at necropsy Incidence of changes (numeric) Changes M(ale)/F(emale) test animals, group A 1 M F test animals, group A2 M F controls, group A3 M F Habitus Poor health condition Sniffing Number examined 4 4 4 4 2 2 Found dead: 1 1 2 1 2 3 2 Diarrhoea 2 Haemorrhagic discharge (nasal cavity and/or oral cavity) Crusts on nose and fore paws 1 1 1 1 Lungs Red discoloured 2 4 2 3 Swollen 2 3 1 Hyalin spot(s) 1 1 1 Red spotted surface 2 Petechia 2 Bloody content White area(s) 1 1 1 1 1 Yachea Bloody content 1 1 Company Sanfflzed. Does not confo'n T^< ^ TNO report V98.337 November 1998 Page 46 J^.CUTE(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Table 8 (continued) -Summary of macroscopic lung observations of male and female rats at necropsy Changes Testes (Unilateral) cryptorchism Thymus Spotted Bronchial lymph nodes Enlarged Incidence of changes (numeric) M(ale)/F(emale) test animals, group Al M F Number examined 4 4 test animals, group A2 M F 4 4 controls, group A3 M F 2 2 4 1 1 1 3 2 1 Sanitized. Does not conlatn TSCA CB1 Company TNO report V98.337 November 1998 Page 47 ^CUTE (4-HOUR) INHALATION RAT "TNO Nutrition and Food Research Institute Study: 470001/017 Table 9 - Summary of microscopic lung observations of male and female rats Incidence of changes (numeric) Changes M(ale)/F(emale) test animals, group Al M IF test animals, group A2 M IF controls, group A3 M IF Diffuse alveolar haemorrhages Slight Moderate Severe No examined 4 4 4 4 2 2 Perivascular oedema Very slight Slight Perivascular mononuclear and/or polymorphonuclear inflammatory cell infiltrate Very slight Slight Moderate 2 2 1 Increased number ofpolymorpho-nuclear .nflammatory cells in the alveolar spaces Very slight Slight' Moderate Hypertrophic respiratory epithelium Multifocal) pneumonia Very slight Slight Severe . Very severe ^^^^^?^g^^^?^^ain'iSGA CBI c:^' ' TNO report V98.337 November 1998 Page 48 P CUTE (4-HOUR) INHALATION TNO Nutrition and Food Research Institute Study: 470001/017 RAT Table 9 (continued) - Summary of microscopic lung observations of male and female rats Incidence of changes (numeric) Changes M(ale)/F(emale) test animals, group Al M P test animals, group A2 M F controls, group A3 M F No examined 4 Very slight to slight diffuse/focal accumulation of alveolar macrophages .4. 4 4 3 2 2 ., 1 Moderate increased septal cellularity 2 Slight focal pleuritis 1 ^,,^.^9swc"a">T8<;lffl' pompww TNO report V98.337 November 1998 Page 49 fJiCUT(4E-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Appendix 1.1 - Individual macroscopic observations at necropsy 1 day after exposure (group Al) Animal number Macroscopic findings MALES 062 Found dead on day 1 Lungs: red discoloured, swollen Trachea: rilled with blood Testes: unilateral cryptorchism 072 Habitus: poor health condition, sniffing Lungs: red spotted surface Testes: unilateral cryptorchism 082 Habitus: poor health condition, sniffing Lungs: swollen, dark red discoloured Testes: cryptorchism 086 Habitus: no abnormalities Lungs: red spots Testes: unilateral cryptorchism FEMALES 071 Habitus: Lungs: poor health condition red discoloured 075 Habitus: poor health condition, diarrhoea Lungs: red discoloured, swollen 079 Habitus: poor health condition, diarrhoea Lungs: red discoloured, swollen 085 Found dead on day 1 Habitus: haemorrhagic discharge from nasal cavity Lungs: dark red discoloured, swollen Bpany ganMzeflf. Soes HSl BoMaIn TSCA CBi V98.337 November 1998 Page 50 fT^CUT(E4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Appendix 1.2 - Individual macroscopic observations at necropsy 14 days after exposure or earlier in animals that were found dead (group A2) Animal number Macroscopic findings MALES 066 Found dead on day 1 Habitus: haemorrhagic discharge from nasal and oral cavity Lungs: Trachea: Thymus: dark red discoloured, swollen filled with blood spotted 068 Habitus: no abnormalities Lungs: hyaline spot(s) and. local white discoloured 074 Found dead on day 4 Habitus: crusts on nose and fore paws Testes: cryptorchism Lungs: dark red discoloured, filled with blood 078 Habitus: no abnormalities BLN: enlarged FEMALES 073 Found dead on day 2 Habitus: haemorrhagic discharge from nasal cavity Lungs: dark red discoloured, filled with blood 077 Habitus: no abnormalities Lungs: red discoloured, local white discoloured BLN: enlarged 081 Habitus: no abnormalities BLN: enlarged 089 Habitus: no abnormalities Lungs: red discoloured BLN: enlarged BLN = Bronchial Lymph Nodes 'CW"ySahffl(L DoesWt WMaIn TSCA CBI TNO report V9B.337 November 1998 Page 51 I^CUTE(4-HOUR) INHALATION RAT "TNO Nutrition and Food Research Institute Study: 470001/017 Appendix 1.3 - Individual macroscopic observations at necropsy in unexposed animals (group A3) Animal number Macroscopic findings MALES 064 Habitus: Lungs: BLN: no abnormalities hyalin spot(s), petechia enlarged 070 Habitus: no abnormalities Lungs: petechia; pronounced white spot BLN: enlarged FEMALES 083 Habitus: Lungs: BLN: no abnormalities hyalin spots enlarged 091 Habitus: Organs BLN = Bronchial Lymph Nodes no abnormalities no abnormalities detected ^^..n^,^---------'"-"0" V98.337 November 1998 . . -.. . . Page 52 B Study: 470001/017 ACUTE (4-HOUR) INHALATION RAT stitute Appendix 2.1 - Individual microscopic observations at necropsy 1 day after exposure (group Al) Animal number Microscopic findings MALES 062 Pound dead on day 1 Lungs: Severe diffuse alveolar haemorrhages (fibrinoid material within the alveolar spaces) Slight perivascular oedema Moderate perivascular polymorphonuclear inflammatory cell infiltrate Hypertrophic respiratory epithelium Moderate increased number of polymorphonuclear inflammatory cells in the alveolar spaces 072 Scheduled kill on day 1 Lungs: Severe diffuse alveolar haemorrhages (fibrinoid material within the alveolar spaces) Slight perivascular oedema Moderate perivascular polymorphonuclear inflammatory cell infiltrate Hypertrophic respiratory epithelium Moderate increased number ofpolyroorphonuclear inflammatory cells in the alveolar spaces 082 Scheduled kill on day 1 Lungs: Severe diffuse alveolar haemorrhages (fibrinoid material within the alveolar spaces) Slight perivascular oedema Moderate perivascular polymorphonuclear inflammatory cell infiltrate Hypertrophic respiratory epithelium Moderate increased number of polymorphonuclear inflammatory cells in the alveolar spaces Ppmpany Sanftlzea. Bow ffel eonlain TSd C^ V98.337 November 1998 Page 53 ^.CUTE (4-HOUR) INHALATION RAT ' TNO Nutrition and Food Research Institute Study: 470001/017 Appendix 2.1 (continued) - Individual microscopic observations at necropsy 1 day after exposure (group Al) Animal number Microscopic findings MALES 086 Scheduled kill on day 1 Lungs: Slight diffuse alveolar haemorrhages (fibrinoid material within the alveolar spaces) Very slight perivascular oedema Moderate perivascular polymorphonuclear inflammatory cell infiltrate Slight increased number of polymorphonuclear inflammatory cells in the alveolar spaces -- - - " " ' canW*6' toWpa^58" V98.337 November 1998 Page 54 ACUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Appendix 2.1 (continued) - Individual microscopic observations at necropsy 1 day after exposure (group Al) Animal number Microscopic findings FEMALES 071 Scheduled kill on day 1 Lungs: Moderate diffuse alveolar haemorrhages (alveolar oedema and fibrinoid material within the alveolar spaces) Slight perivascular oedema Moderate perivascular polymorphonuclear inflammatory cell infiltrate Moderate increased number of polymorphonuclear inflammatory cells in the alveolar spaces 075 Scheduled kill on day 1 Lungs: Moderate diffuse alveolar haemorrhages (fibrinoid material within the alveolar spaces) Slight perivascular oedema Slight perivascular polymorphonuclear inflammatory cell infiltrate Slight increased number of polymorphonuclear inflammatory cells in the alveolar spaces 079 Scheduled kill on day 1 Lungs: Moderate diffuse alveolar haemorrhages (fibrinoid material within the alveolar spaces) Slight perivascular oedema Slight perivascular polymorphonuclear inflammatory cell infiltrate Slight increased number of polymorphonuclear inflammatory cells in the alveolar spaces 085 Found dead on day 1 Lungs: Severe diffuse alveolar haemorrhages (fibrinoid material within the alveolar spaces) Slight perivascular oedema Slight perivascular polymorphonuclear inflammatory ceil infiltrate Slight increased number of polymorphonuclear inflammatory cells in the alveolar spaces gSMnpafiy^ ^ . ^ w ^ w ' k c w V98.337 November 1998 Page 55 (ACUTE(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Appendix 2.2 - Individual microscopic observations at necropsy 14 days after exposure or earlier in animals that were found dead (group A2) Animal number Microscopic findings MALES 066 Found dead on day 1 Lungs: Moderate alveolar haemorrhages (fibrinoid material within the alveolar spaces) Slight perivascular oedema Very slight perivascular infiltrates of mononuclear and polymorphonuclear inflammatory ceils Very slight increased number of polymorphonuclear inflammatory cells in the alveolar spaces 068 Scheduled kill on day 14 Lungs: Very severe pneumonia seen as diffuse accumulation of alveolar macrophages. increased septal cellularity (mainly macrophages), septal fibrosis. 074 078 Found dead on day 4 Lungs: Moderate alveolar haemorrhages (with fibrinoid material within the alveolar spaces) Severe pneumonia seen as alveolar oedema, accumulation of alveolar macrophages, alveolar and interstitial aggregates of inflammatory cells, perivascular aggregates of macropahges and fibroblasts and a diffuse mesothelial proliferation Scheduled kill on day 14 Lungs: Moderate perivascular aggregates of mainly mononuclear inflammatory cells Slight multifocal pneumonia ,,^..'wrt"BT8CAC'" - pompany TNO report V98.337 November 1998 ^CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017 Appendix 2.2 (continued) - Individual microscopic observations at necropsy 14 days after exposure or earlier in animals that were found dead (group A2) Animal number Microscopic findings FEMALES 073 Found dead on day 2 Lungs: Slight alveolar haemorrhages (with fibrinoid material in the alveolar spaces) Slight perivascular aggegregates of mainly polymorpho- nuclear inflammatory cells 077 Scheduled kill on day 14 Lungs: Slight diffuse accumulation of alveolar macrophages Moderate increased septal cellularity (mainly macrophages) 081 Scheduled kill on day 14 Lungs: Slight interstitial pneumonia Slight perivascular aggregates of mononuclear and polymorphonuclear inflammatory cells Slight focal accumulation of alveolar macrophages Slight focal pleuritis 089 Scheduled kill on day 14 Lungs: Slight diffuse accumulation of alveolar macrophages Moderate diffuse increased septal cellularity (mainly macrophage-Iike cells) - ^ ^ s ^ C.n^n,^"^-008 V98.337 November 1998 TNO Nutrition and Food Research Institute Study: 470001/017 Appendix 2.3 - Individual microscopic observations at necropsy of unexposed animals (group A3) Animal number Microscopic findings MALES 064 Scheduled kill on day 14 Lungs: Slight increased perivascular mononuclear inflammatory cell infiltrate Very slight multifbcal pneumonia 070 Scheduled kill on day 14 Lungs: Slight lobar perivascular mononuclear and polymorphonuclear inflammatory cell aggregates FEMALES 083 Scheduled kill on day 14 Lungs: Slight increased perivascular inflammatory cell infiltrate Very slight focal accumulation of alveolar macrophages 091 Scheduled kill on day 14 Lungs: No abnormalities detected SB^CS V98.337 November 1998 Fj\CUT(E4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study:470001/017 CENTRE DUPLICATION DE LEVALLOIS. Elf Atochem Centre d'application de Levallois Service SAI Telephone : 01 47 59 1268 Telecopie : 01 47 59 14 18 Name of the test material Appearance: Composition: Composition: Colour Boiling point Autoignition temperature: Explosive limits Vapour pressure Density Batch number Storage conditions Levallois, July15, 1998 ^ - ^ f noico"' flUsd-0065 Company S ^ /An n/-^\