Document ZJadnnjN0k55zQj4vjBRkGjLO
AR226-3104
TNO-report
V98.337
aented acute (4-hour) inhalation toxicity study with ~ | p rats
TNO Nutrition and Food Research Institute
Utrechtseweg 48 P.0. Box 360
3700aj Zeist
The Netherlands
Phone +31 30 694 41 44 Fax +31 30 695 72 24
Date:
November 1998
Authors):
)r H. Muijser
Study director.
Dr H. Muijser
TNO Project number
470001/017
At the request of:
ELF Atochem S.A. Paris La Dfense, 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 lor 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
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TNO Nutrition and Food Research Institute is dedicated to the (interjnational 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
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Summary
The acute inhalation toxicity of by nose-only exposure of one groun of eidit male and p.irrht female rare m a tact atmosphere containing 21.2 0.2 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 pm 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 m 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,
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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 t h
a^
exposure of rats to a high
concentration
21.2 g/m3) resulted
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.
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Contents
Summary ................ ..................................................................
2
Statement of GLP compliance.............................................................
5
Authentication by cooperating scientist..........................................................
g
Quality Assurance Statement..................................................................
7
GLP compliance monitoring unit statement .............................................
3
Testing facility.............................................................................
C o n trib u to rs .........................................................................
g
1 Introduction .............................................................
jq
2 Experimental.................................................................
20
2.1 Test m aterial................................................................
Ig
2.2 Test animals .........................................................
jj
2.3 Experimental conditions...............................................
u
2.4 Experimental procedures............................................. 2.5 Exposure cham ber.......................................................
2 2
2.6 Generation of the test atmosphere......................................... 13
2.7 Analysis of the test atmosphere ....................................... [3
2.8 Observations and measurements....................................... 4
2.9 Statistical analysis.....................................................
jg
2.10 Retention of records .............................................
jg
2.11 Deviations from the protocol ........................................... jg
3 Results ..-.................................................................
jg
3.1 Analytical results . . . ___*.......................................
jg
3.2 Behaviour, clinicalsigns and mortality................................ \ g
3.3 Body weights...........................................................
jg
3.4 Lung functiopmeasurements .......................................... 20
3.5 Lung weights...............................................................
20
3.6 Pathology.........................................................
?q
4 Discussion and conclusion.......................................
0j
5 References ......................................................
?9
Appendices.........................................................
^
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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 earned out in accordance with the OECD Principles of Good Laboratory Practice.
Dr H. Muijser (Study Director)
Drs H.H. Emmen (Management)
Date Date
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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
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Quality Assurance Statement
On:
Report Number: Date :
A u g m e n te ^ c u t^ ^ o u ^ n h a la tio n toxicity study with
V98.337
lin rats
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: 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: / 3
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.
STAATSTOEZICHT OP DE VOLKSGEZONDHEID
VETERINAIRE HOOFDINSPECTIE
ENDORSEMENT OF COMPLIANCE
W IT H TH E OECD PRINCIPLES OF G O O D LA B O R A TO R Y PR A C TIC E
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 3 60 3 7 0 0 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.
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'-:Mim^iry;g.f health, Welfare and Sport
State Supervisory Public Health Service
Veterinary Public Health Inspectorate
r:i d
--
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Testing facility
The toxicity study was conducted by: TNO Nutrition and Food Research Institute Toxicology Division P.0. Box 360, 3700 AJ ZEIST, the Netherlands Telephone +31 30 69 44 144 Telefax+31 30 6960 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* ItJ.H.E. Arts Ms M.M. Andringa 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
* Toxicology Division, TNO Nutrition and Food Research Institute
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1 Introduction
At the request of ELF A to c h C T i^ ^ ^ ^ a ri^ ^ D 6 fe n g ^ ^ g c e ^ n acute (4-hour) inhalation toxicity study w i t l ^ l l ^ ^ m i m i l ^ l l l l l ^ l ^ M w ^ carried
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 jiugmentec^icute^4-hour) inhalation toxicity study w i t h ^ m ^ m ^ ^
flin rats", approvedbyth^tudy director on 18 November 1998 (see 2.1 and 2.11 for change f r o m ^ P | ^ f ^ i n 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
, T f e test material was supplied by the sponsor. Two plastic bottles, labelled
U I H I B H B H I P & o n t a i n i n g ca. 790 and 760 g test material, respectively, were received on 17 November 1997. As shown below the test material was actually i
_ _ _________ jO n request o ^ h ^ p o nsor the name of the test material in this report was therefore changed t o ^ l i l H I i H H H H H H H H H ^ V f c 'h e
TNO internal reference number was 970285. The test material was stored at ambi
ent temperatures.
.
characteristics (as given by the sponsor) Composition:
id has the following
Composition: Colour: Boiling point:
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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, Sulzfeld, Germany. The animals used arrived on 5 November 1997, at an age of ca. 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 titers 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 fats were assigned to the study to be exposed to the present test material in two groups of 4 rats for each sex. In 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 (si, 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
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
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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 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 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/m3for 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 i 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 cur 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
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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 le 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 0/min.
All animals of group A1 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
- H2pressure:
0.5 bar
- air pressure:
0.8 bar
- back pressure:
200mbar
- sample line:
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 p l f r ^ ^ J pJ^Merck, p.a.), respectively!
^ ______ _
rie amount
considered to be negligible small. The resulting responses were
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used to construct a calibration curve which was needed to convert the responses in scale units of the TCA to concentrations (g/ra3). A quadratic relation was obtained: m m ^ o n c e n t r a t i o n = 0.0112 * X2- 1.477 * X + 64.28
in w h i c h Q ^ J ^ v a s 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 i test atmosphere at 5 i/min through 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 Nomina] 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 Mm. 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.
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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 sipial in the body chamber. Lung mechanical properties were obtained by determination of the transfer function as described in detail by Oostveen etal. (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 m the head chamber. Transfer impedance (ZJ, defined as applied pressure (P) dmded by the flow (v), was determined by means of a Fast Fourier transform and checked for internal coherence as described by Michaelson etal. (1975). Z,, is a complex function of the frequency and is described in a real (Z^) and imaginary part (ZjJ. Using an appropriate simplified parameter estimating program, the parameters were directly linked to the physiological variables resistance, inertance
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:
^tr --R - Bo>24-j [-1/coC + Ig) - E&)3]
in which R is total respiratory resistance, I is R and I were estimated from the Z,, data as:
total-inertance
and
C
is
compliance.
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R 4 ,(9 6 Hz) + 1/3 [4,(96 Hz) -4 ,(1 9 2 Hz) (ZRe = R - Beo2)
1= 4 ^ 1 6 0 Hz) / 1000
(2k 160 = 1005; Z,m= [-1/coC + Ieo - Eco2])
<he r e J L f l u e n c y T f " " 1 Md ^ = 0 < W m most cases Z*,> at 16 Hz and Z ^ 0 at 32 Hz. Because of t a k S " "'
above ,0 < . were c o n n e d 2.8.4 Pathology
js s s r ^ s s z ^ r ;s i,tr r ,
gross pathological changes.
Y` te ere exarmned for
n e lungs with trachea and larynx were removed and weighed Lungs were
2.9 Statistical analysis
ratios to detect possible sex d ifW n
female dam we ' i^ ^ ^ t exposure values.
rr.. V
A using post/pre-exposure
T P g pre' 30(1 Pst-
2.10 Retention of records
.
2.11 Deviations from the protocol : one f T ! ^ r 74 was
fJ inthis^ -
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- 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. Emmen as from that date.
These deviations were not considered to have influenced the validity of the study.
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3 Results
3.1 Analytical results
3.1.1 Total carbon analysis The actual concentration o; analysis of the volatile ffactio: 20.4 0.2 g/m3(N=48). Since] J H H f l R t h e concentration o 2 ^ ( U g / m 3.
;the exposure based on total carbon red at 5-min intervals was
3.1.2 Gravimetric analysis The results of the gravimetric analysis are given in Table L The calculated mean concentration of the solid fraction of1 j m ^ m i ^ ^ f ^ v a s ^22 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, indi cating a generation efficiency of 97%.
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 m. The Mass Median Aerodynamic Diameter (MMAD) was 3.8 /un 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.
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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 A1 (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 1lh), one male (no. 62) and one female animal (no. 85) were found dead in their home cage.
i
.. 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 A1 and 3 out of 8 in subgroup A2. Mortality in group A1 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 (A l) 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 weight attained was below that of the unexposed animals (A3), in which, as expected, normal weight gain was seen in both observation weeks.
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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 borne 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 (p<0.0001) 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 pre exposure 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 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 A1 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.
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V98.337 November 1998
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3.6.1 Macroscopy % At necropsy, several surviving animals of mum a 1
A fe w o f these rats showed sniffing and d i m j u deceased rats teveaied rad
P r health condition. observation o f
ta g s . Several s u rv iv in g **
discoloured and/or swollen lungs when*
T i b, od filled
a y after exPsure also showed
few othent. A few stnvivtag
Were < * - ' - a
red discoloured lungs. These macroscom'e oh ^
exposure also showed
treatment-related (Table
T C.nSideredt0
all groups, controls inclnded and were therein f0""d " or a few raIS of
treatment.
* " thercfore " ` considered to be related to
3.6.2 Microscopy
Microscopical examination of the lumrs nf
a
were characterized by moderate to severe alveolar'hTM* T "'" *'esions which
oedema and perivascular inflammatory cell a g g r e g ^ s T ^ ' perivascut a scheduled for necropsy one dav a fte r PYnTM g S lving ammals
lungs of surviving animals necropsied 14 davTafterTx SImjIarchanges-10 most multifocal pneumonia or accumulation of alvp 1 Posure sllght to severe
moderate increased septal cellularity could be d ^ maCrphages accompanied by baemorehag. were
3 on, o TM o " ;
" f r y cel, infl,dates were found in
only slight to ve; slight
MgK m `heSe
connol rats were
Discussion and conclusion
!
!"
!"l; '
inhalation toxicity of
rats was e x p o s e d T d T S ^ n S S o n o f 2 four hours. Half of the rats were necroosied th a
maJe and 8 female dUnng 3 SIngJe period of
cats were kept for a 14-iay observationperiodF o r n i r '``'T "' remai"lng
were also sacrificed for backgmund his,pathology.
"nexpose<l rats
animals groups
,, " t h i ^ days" ,?y' 5 " f `6 eXp0Sed
of late mortality i,, gr0,,p A2 (teended ^ d y fof " T TM ' The
days) suggests that late mortality could be e z n S ld
SeTM ' n " " r*al of 14
animals of grouD A i n do nk
pected t0 have occurred in the
14-day underestimate.
f " ,^
** * a
' S that total mortality may be an
S oes iid ecnfari TSiEKc m
V9B.337 November 1998
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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 conclude^thata_single 4-hour (acute) exposure of rats to a high
concentration 1-, ,
., 21.2 g/m3) resulted
lung ctoiges 311868 "
*** macroscoPical ^ histopathological
Therefore, from the results of the present study, it cannot be excluded that human
changes6 ^ ^ aer S01 COnCentrations wiI1 result in serious and lasting lung
5 References
Lee RJ. Jr., Science, 1972: 567-575
Michaelson E.D., Grassman EJD. and Peters W.R.,
J. Clin. Invest. 1975: 56, 1210-1230
'
Gostveen E., Zwart A., Peslin R. And Duvivier C., J. Appl. Physiol. 1992: 73,1598-1607
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Figure 1 - Schematic diagram of the generation and exposure system
Compressed Dry A ir
1 Mainvalve 2 Reducing valve 3 Flow meter 4 Valve 5 Humidifier 6 Thermal bath 7 US Nebuliser 8 Fluid pump 9 Supply vessel
10 Pressure gauge 11 Regulating valve
HNO Head/Nose Only Unit AH Animal holder EHD External Hood
Measurement: RH/T; PSize; G ravim etry; Total Carbon; Oxygen.
Exhaust to
filter
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ACUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research j stitute 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 (i) 50 50 50 50 50 50
Concentration (mg/m3) 121 120 119 118 123 129
122 4
Generation of test atmosphere: 9:30-16:30 h Exposure duration of the animals: 4 hours between 10:30 and 16:30 h
%
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November 1998
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i m m m m m * ) ,CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute
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Table 2 - Aerodynamic particle size distribution in test atmosphere
I Particle size (jim) | 4.2 1 * 3.8 | * 3.4
* 31 2.8 2.4 s 1.8 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 fj.m
mean geometric standard deviation (gsd): 1.1
SahitzeS. Does hot contain TSna
V98.337 November 1998
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%
. ..
- ,^-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
MALES 62,72,82,86
FEMALES 71,75,79,85
NO ABNORMALITIES NO ABNORMALITIES
Time of observation
11:30, 12:25, 13:25, 14:20
11:30, 12:25, 13:25, 14:20
exposure period of group A: 10h30 -14:30
tftinpsny Sanlfzrueest eetilain tsLm
Study: 470001/017
Table 3.1.2 - Clinical signs during exposure (group A2)
Animal number Clinical signs
MALES 66,68
74
78
NO ABNORMALITIES NO ABNORMALITIES NO ABNORMALITIES
FEMALES 73,77
81
89
NO ABNORMALITIES NO ABNORMALITIES NO ABNORMALITIES
exposure of animal 66: 10h45-14h45 exposure of animal 73: Ilh00-15h00 exposure of animal 68: Ilhl5-15hl5 exposure of animal 77: Ilh30-15h30 exposure of animal 74: Ilh45-15h45
exposure of animal 81: 12h00-16h00 exposure of animal 78: 12hl5-16hl5 exposure of animal 89: 12h30-16h30
Time of observation
11h45,12h45,13h45,14h45 . 12h45,13h45,14h45,15h40
12h45,13h45,14h45,15h40, 16hl0
11h45,12h45,13h45,14h45 12h45,13h45,14h45,15h40 12h45,13h45,14h45,15h40, 16hl0
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(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute
Study: 470001/017
Table 3.2.1 - Clinical signs shortly1after 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
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i n u repon
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I J a CUTE (4-HOUR) in h a l a t io n r a t TNO Nutrition and Food Research Institute Study: 470001/017
Table 3.2.2 - Clinical signs shortly1after 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
1time of observation 17h05; exposure ended between 14h45 and 16h30 /see nnfp
at the bottom of Table 3.1.2)
. TSCACB1
_ ^ s f f i z e d ; 1j s io V c o ^ a 1 n
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p ^ C U T E (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 A l)
Animal number
MALES 62
Clinical signs
Range*
GENERAL blepharospasm sluggish BREATHING laboured breathing FOUND DEAD just before necropsy
i i
l 1
72 GENERAL sluggish BREATHING laboured breathing
i l
82 GENERAL blepharospasm sluggish BREATHING laboured breathing
i i
l
86 FEMALES 71,79
NO ABNORMALITIES
GENERAL sluggish
1 i
75
GENERAL
'
sluggish
i
BREATHING
laboured'breathing
i
85* GENERAL
hunched appearance
j
sluggish
j
piloereciion
]
FOUND DEAD just before necropsy 1
range: 1 day (animals were sacrificed on day 1)
tsca cw
V98.337 November 1998
P a g e 31
^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
.O * ,* . -- " 6"
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(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute
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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
,, e d .0 0 " " " 4'0 "tmpaWS a
TNO report
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CUTE (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 day 1 - day 14
Range* 1-14 1-14
fvotc1 /*
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(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
Day 0
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
"TW '
tS C *
November 1998
Page 35
_ VCUTE (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
Animal No. | Day -1 Males (A2)
Day 0
Day 1
Day 7 Day 14 ||
66 68 74 78 mean sd
Females (A2)
239.6 257.9 260.7 248.3 251.6 9.6
243.7 262.3 264.8 253.8 256.2 + 9.5
226.0 224.6'd)
237.8
237.0
243.0 210.91(4)
236.2
251.0
235.8 + 244.02 7.1 1
- II 265.7
-I
277.0
271.42
73 77 81 89 mean sd
179.4 170.9 174.7 182.2 176.8 5.0
183.7 172.6 177.1 186.1
179.9 6.2
169.8 166.6'(2)
161.6
162.5
175.4
178.3
170.6
174.9
169.4 + 171.9 + 5.7 1 8.3
180.0
193.1
191.8 188.3 +
7.2
1no weight available on day 7, animal weight at death on day 1, day 2 or day 4
indicated as (1), (2) or (4), respectively.
..
'
2no sd given for mean of two values
0$ ' 30^
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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
Day 0
247.7 257.2 252.4
257.1 261.6 259.4
189.5 170.4 180.0
190.2 176.8 183.5
Day 1
Day 7 Day 14
253.8 264.0 258.9
278.5 295.2 286.8
303.9 320.1 312.0
189.5 175.6 182.6
199.9 180.1 190.0
207.4 193.3 200.4
\SC^ C#` jtfW1 tv(Ac0
i icp o il
V98.337 November 1998
----------------------------------------------------------------- -------------------- .---------------- -------------- --------Page 37
- . . ____
_ ^ C U T E (4-HOUR) INHALATION RAT
TNO Nutrition and Food Research Institute
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Table 5.1 - Individual responses in breathing frequency (group A2)
Animal I il coae |--------- if 1 P r e
day 0
]| males I abs
66 I 3.20
68 3.17
1 74
3.65
I 78
3.33
I mean/ 3.34/
| sem | 0.11
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 I rel *
3.92 3.70 4.23 3.95/ 0.15
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 4.60V
rel 1 73 | 1 12 |
1females |
73 3.70
77 3.24 81 3.65 89 3.33
mean/ J 3.48/ sem 1 0.11
4.04 3.49 3.16 3.63 3.58V 0.18
1.09 2.36 1.08 3.14 0.86 4.25 1.09 4.22
3.49/ 0.46
0.64 * 0.97 4.82 1.16 3.36 1.26 5.10
4.43/ 0.54
*
1.49 3.43 0.92 5.19 1.53 5.36
4.66V 0.62
1 06 [ 1 42 | 161 |
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 ratios (no significant differences found)
I ^ Q h0e5d pair Ntest by " " Paring P- and post-exposure values (pooled sexes);
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(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017
Table 5.2 - Individual responses in tidal volume (group A2)
Anima code
males 66 68 74 78
mean/ sem
pre
abs
2.60 2.60 2.80 2.80 2.70/ 0.06
daly 0
abs rel
1.60 2.00 2.00 2.00 1.90V 0.10
0.62 0.77 0.71 0.71
Tidal volume (mf)
day 1
day 4
abs rel abs rel **
1.60 0.62 1.40 0.50
1.60 0.62 *
2.00 0.71 2.20 0.79
1.67V 0.18
1.90V
day 14
abs rel *
1.80 0.69 *
2.00 1.90/
0.71
females
73 2.00 1.40 0.70 0.80 0.40
*
*
77 2.00 1.00 0.50 0.80 0.40 1.00 0.50 2.80 1.40
81 2.00 1.80 0.90 1.20 0.60 2.00 1.00 1.40 0.70
89 2.20 1.40 0.64 1.20 0.55 1.20 0.55 1.60 0.73
mean/ 2.05/ 1.40V sem 0.05 0.16
l.ooV
0.12
1.40V 0.30
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
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) 5p<0.0001
1p<0.05
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(4-HOUR) INHALATION r a t TNO Nutrition and Food Research Institute Study: 470001/017
Table 5.3 - Individual responses in ventilatory flow (group A2)
IAnima 1 code
pre
males 66 68 74 78
mean/ sem
abs
8.32 8.25 10.23 9.33 9.03/ 0.47
dityO
abs rel
6.04 7.60 8.33 7.25 7.30V 0.48
0.73 0.92 0.81 0.78
Ventilatory flow (mf/s)
day 1
day 4
abs rel abs rel **
6.27 0.76 8.62
5.18 0.51
*
8.46 0.91 8.58
6.64V 0.96
8.60/
1.04 0.92
day U
abs *
9.87 *
7.45 8.66/
rel 1.20 0.80
females
73 7.40 5.65 0.76 1.89 0.26
*
77 6.47 3.49 0.54 2.51 0.39 4.82
81 7.31 5.68 0.78 5.09 0.70 6.73
89 7.33 5.08 0.69 5.6 0.69 6.12
mean/ 7.13/ 4.98V sem 0.22 0.51
3.64V 0.84
5.89/ 0.56
*
0.75 9.59 0.92 7.27 0.83 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 ratios (no significant differences found)
- Matched pair t-test by comparing pre- and post-exposure values
5: p<0.005
- , ' >V
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(4-HOUR) in h a l a t io n r a t TNO Nutrition and Food Research Institute
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Table 6.1 - Individual responses in airway resistance (group A2)
Anima Code
Males 66 68 74 78
mean sem
pre
abs
178 199 160 173 178 8
day 0
abs rel
163 0.91 169 0.85 189 1.18 163 0.94 171 0.97 6 0.07
Resistance (hPa.s/0)
day 1
day 4
abs rel abs rel **
177 0.89 196 0.98
219 1.37
*
181 1.05 188 1.09
192 1.10 192 13 0.14 -
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 sem _ 10
154 0.82 137 0.73 137 0.98 124 0.88 184 1.10 184 1.11 154 0.91 149 0.88
157 0.95 148 0.90 10 0.06 - 13 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).
COThP'
,t>o gatv&\td
fSC* C *' iicoo1
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CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017
Table 6.2 - Individual responses in airway inertance (group A2)
Anima Code
pre
Males 66 68 74 78
mean sem
abs
0.194 0.235 0.195 0.207 0.208 0.010
day 0
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.s2/0)
day 1
day 4
abs rel abs rel **
0.201 0.86 0.213 0.91
0.242 1.24
*
0.216 1.04 0.236 1.14
0.220 1.05 0.224 1.02
0.120 0.11
-
-
day 14
abs rel *
0.217 0.92 *
0.209 0.213
-
1.01 0.97
-
Females
73 0.226 0.133 0.59 0.120 0.53
*
*
77 0.145 0.119 0.82 0.094 0.65 0.203 1.40 0.146 1.01 81 0.203 0.190 0.94 0.226 1.11 0.153 0.75 0.135 0.67
89 0.155 0.169 1.09 0.134 0.86 0.171 1.10 0.202 1.30
mean 0.182 0.153 0.86 0.144 0.79 0.176 1.09 0.161 0.99 sem 0.019 0.016 0.11 0.029 0.13 0.015 0.19 0.021 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).
.ed. OoeS no
Z-*-
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__________________ _ 'k.CUTE (4-H O UR ) IN H A LA TIO N RAT
TNO Nutrition and Food Research Institute Study: 470001/017
Table 6.3 - Individual responses in airway compliance (group A2)
Anima Code
Males 66 68 74 78
mean sem
pre
abs
1.70 1.16
*
2.58 1.82 0.41
day 0 abs rel 1.30 0.76 1.45 1.25 1.88
1.54 1.00 0.18 0.24
Compliance (mi/kPa)
day 1
day 4
abs rel abs rel *** ***
1.12 0.96 1.12 0.96 0.75 ***
1.01 0.39 1.44 0.56
0.96 0.68 0.11 -
1.28 0.76
'-
-
day 14
abs rel ***
8.66 7.46 ***
1.18 0.46 4.92 3.96
-_
Females
73 1.13 2.49 2.20 3.63 3.21 ***
***
77 3.07 3.17 1.03 3.52 1.15 2.07 0.67 7.43 2.42
81 2.37 5.11 2.15 1.70 0.71 3.00 1.26 5.22 2.20
89 ** 2.16
3.15
1.57
1.22
mean 2.19 3.23 1.80 3.00 1.69 2.21 0.97 4.63 2.31 sem 0.57 0.66 0.38 0.44 0.77 0.42 - 1.82 -
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).
TSC* rW1
V98.337 November 1998
Page 43
. ..
________fJ^CU TE (4-HOUR) INHALATION RAT
TNO Nutrition and Food Research Institute
Study: 470001/017
Table 7.1 - Absolute and relative lung weights of exposed animals; individual and mean values
V9B.337 November 1998
Page 44
tC
(4-HOUR) INHALATION RAT
TNO Nutrition and Food Research Institute
Study: 470001/017
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/lOOg BW)
1.573 1.388
0.52 0.43
Females 83 91
1.220 1.005
0.59 0.52
:CompanvSmlOM-bomi' TSC*CW
V98.337 November 1998
Page 45
(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute
Study: 470001/017
Table 8 necropsy
Summary
of
macroscopic
lung
observations
of
male
and
female
rats
at
Company Sanitized. Doenot contain
TNO report
V98.337 November 1998
Page 46
(4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017
Table 8 (continued) - Summary of macroscopic lung observations o f male and female rats at necropsy
Company Sanltizad. Does noi coniala TSCft CBI
November 1998
Page 47
(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
V98.337 November 1998
Page 48
____ ____________
UTE (4-HOUR) INHALATION RAT
TNO Nutrition and Food Research Institute
Study: 470001/017
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 A1
MF
No examined 4
Very slight to slight diffuse/focal accumulation of alveolar macrophages
.4.
Moderate increased septal cellularity
Slight focal pleuritis
test animals, group A2 MF 44
3 2 1
controls, group A3
MF 2 .. 2
1
nol contain TSCA P o m p a i Sanitized. Does
TNO report
V98.337 November 1998
Page 49
%
fJiCUTE (4-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 A l)
Animal number
Macroscopic findings
MALES 062
072
082
086
FEMALES 071 075 079 085
Found dead on day 1
Lungs:
red discoloured, swollen
Trachea: filled with blood
Testes:
unilateral cryptorchism
Habitus: Lungs: Testes:
poor health condition, sniffing red spotted surface unilateral cryptorchism
Habitus: Lungs: Testes:
poor health condition, sniffing swollen, dark red discoloured cryptorchism
Habitus: Lungs: Testes:
no abnormalities red spots
unilateral cryptorchism
Habitus: Lungs:
poor health condition red discoloured
Habitus: Lungs:
poor health condition, diarrhoea red discoloured, swollen ' '
Habitus: Lungs:
poor health condition, diarrhoea red discoloured, swollen
Found dead on day 1
Habitus: Lungs:
haemorrhagic discharge from nasal cavity dark red discoloured, swollen
i. o es fiel ehtaln TSC CIB %
V98.337 November 1998
Page 50
r B H B H H B B H H r V c U T E (4-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:
dark red discoloured, swollen filled with blood
Thymus: spotted
068
Habitus:
no abnormalities
Lungs:
hyaline spot(s) and. local white discoloured
074 Found dead1on 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
077
081
Found dead on day 2
Habitus: Lungs:
haemorrhagic discharge from nasal cavity dark red discoloured, filled with blood
Habitus: Lungs: BLN:
no abnormalities
red discoloured, local white discoloured enlarged
Habitus: BLN:
no. abnormalities enlarged
089
Habitus:
no abnormalities
Lungs:
red discoloured
.
BLN:
enlarged
BLN = Bronchial Lymph Nodes
Cfflpny Sanitized. Does hot contain TSCA CBI
TNO report
V9B.337 November 1998
----------------------------------------------- -- -------------Page 51
% (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:
no abnormalities
Organs
no abnormalities detected
BLN = Bronchial Lymph Nodes
B a n n e d .T id e s W ot contain TSC* c
V98.337 November 1998
Page 52
A
_ . . -----
------ lACUTE (4-HOUR) INHALATION RAT
TNO Nutrition and Food Research Institute
Study: 470001/017
Appendix 2.1 - Individual microscopic observations at necropsy 1 day after exposure (group Al)
Animal number
Microscopic findings
MALES 062 Found 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 of polymorphonuclear 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
Company Sanitized. Does not Contain TSCi* QM
V98.337 November 1998
Page 53
. _ _________________ VCUTE (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 A l)
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
Satti oroP*^
ooes
November 1998
Page 54
^ ^ C U T E (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 infltrate
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 cell
' infiltrate
'
Slight increased number of polymorphonuclear inflammatory cells in the alveolar spaces
1
t ^ '* * * TM
^ ' "* * 'TM * "
I n u report
V98.337 November 1998
" -------- --------------------------------------------------- --------------------------------------------- -------------------------------------' ------- -- ---------Page 55
(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 msothlial proliferation
Scheduled kill on day 14
Lungs:
Moderate perivascular aggregates of mainly mononuclear inflammatory cells
Slight multifocal pneumonia
CCI
. CompanySanKl**-Bbsko*containtsca
%
November 1998
Page 56
CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research i istitute
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 alveolarmacrophages Slight focal pleuritis
089 Scheduled kill on day 14
Lungs:
Slight diffuse accumulation of alveolar macrophages Moderate diffuse increased septal cellularity (mainly macrophage-like cells)
TSCkCBl . 0 o n o c o rt> '"
Sani** Compaq
V98.337 November 1998
Page 57
{ m m m m y c m z (4-HOUR) in h a l a t io n r a t 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 multifocal 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
TSC ftc ^
TNO report
V98.337 November 1998
Page 58
( CUTE (4-HOUR) INHALATION RAT TNO Nutrition and Food Research Institute Study: 470001/017
CENTRE D'APPLICATION DE LEVALLOIS
Elf Atochem Centre d'application de Levallois Service SAI
Tlphone : 01 47 59 12 68 Tlcopie : 01 47 59 14 18
Composition: Colour Boiling point Autoignition temperature: Explosive limits Vapour pressure Density Batch number Storage conditions
Levallois, july 15, 1998
QiiVaia TSC * p o m p a i Sawiftlzed*0es no*c'
/ aNh n'o'N
