54, 431– 440 (2000) Copyright © 2000 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Prenatal Toxicity of Inhaled Polymeric Methylenediphenyl Diisocyanate (MDI) Aerosols in Pregnant Wistar Rats A. O. Gamer,* J. Hellwig,* J. E. Doe,† and R. W. Tyl‡ ,1 *BASF Department of Toxicology, Ludwigshafen/Rhein, Federal Republic of Germany; †Zeneca Central Toxicology Laboratory, Alderley Park, GB-Macclesfield, Cheshire, SK10 4TJ, United Kingdom; and ‡Center for Life Sciences and Toxicology, Research Triangle Institute, Research Triangle Park, North Carolina 27709 Received July 21, 1999; accepted December 3, 1999

Mated Wistar rats, 25/group, were exposed to polymeric methylenediphenyl diisocyanate (MDI) aerosol of respirable size for 6 h/day, on gestational days (gd) 6 through 15, at 0, 1, 4, and 12 mg/m 3. Maternal clinical signs, body weights, and feed and water consumption were measured throughout gestation. At scheduled sacrifice on gd 20, maternal body, gravid uterine, liver, and paired lung weights were documented. Corpora lutea were counted, implantation sites were identified: resorptions, dead and live fetuses, and placentas were weighed. All live fetuses were counted, sexed, weighed, and examined for external alterations; approximately 50% of the live fetuses/litter were preserved in Bouin’s fixative and examined for visceral alterations, and the remaining live fetuses/ litter were cleared and stained with alizarin red S and examined for ossified skeletal alterations. Maternal toxicity was observed at 12 mg/m 3, including mortality (2 of 24 pregnant), damage to the respiratory tract, reduced body weights and weight gain, reduced liver and increased lung weights, and reduced gravid uterine weight (the last not statistically significantly different from the control value). Developmental toxicity was also observed at 12 mg/m 3, including reduced placental and fetal body weights and an increased incidence of fetal skeletal variations and skeletal retardations. There was no evidence of maternal or developmental toxicity at 1 or 4 mg/m 3. The no observed adverse effect concentration for maternal and developmental toxicity was therefore 4 mg/m 3. There were no treatment-related teratogenic effects at any concentrations evaluated. Key Words: polymeric methylenediphenyl diisocyanate; developmental toxicity; maternal toxicity; fetal skeletal variations and retardations; whole-body exposure to aerosols.

Polymeric MDI is an industrial chemical that can react with a polyglycol to produce polyurethane plastics. Commercial polymeric MDI generally consists of approximately 40 – 60% monomeric MDI (4,4⬘-diphenylmethane diisocyanate), with a diminishing proportion of higher molecular weight oligomers. Vapor inhalation exposure is physically limited by the low 1

To whom correspondence should be addressed at Chemistry and Life Sciences, HLB-245, Center for Life Sciences and Toxicology, Research Triangle Institute, P.O. Box 12194, Research Triangle Park, NC 27709-2194. Fax: (919) 541–5956. E-mail: [email protected].

vapor pressure of polymeric MDI (about 10 –5 hPa, corresponding to 0.007 ppm or 0.07 mg/m 3). Certain conditions of spraying or heating could produce an aerosol, hence aerosol inhalation was selected for this study. Respirable size particles (mass median aerodynamic diameter ⱕ 2.8 ␮m) were used to maximize any potential effect. The primary toxicologic effect of MDI and polymeric MDI is respiratory sensitization or occupational asthma. Occupational exposure limits have been selected on the basis of a potential to cause respiratory sensitization and pulmonary function decrements. The 1998 ACGIH TLV (threshold limit value) is 0.005 ppm (approximately 0.05 mg/m 3), which is near the theoretical saturated vapor concentration (ACGIH, 1998). This study on the prenatal toxicity of polymeric MDI was undertaken to fill a data gap. Since this study was conducted, Buschmann et al. (1996) have reported an embryotoxicity evaluation of monomeric MDI in Wistar rats. Selection of target concentrations for this study was based on three previous studies with this test substance. A prenatal toxicity range-finding study (BASF, unpublished report) in rats was performed to assess the maternal toxicity of polymeric MDI aerosol and to establish concentrations for this full-scale study. In that study, pregnant female Wistar rats, eight/group, were exposed to HPLC-measured concentrations of 4.75, 10.4, and 15.0 mg/m 3 polymeric MDI aerosol, with a particle size below 2.8 ␮m MMAD during gd 6 through 15. At 15.0 mg/m 3, two out of eight dams died toward the end of the study. All animals showed marked respiratory distress, diminished feed and water consumption, reduced body weight gains, and increased lung weights. Changes in hematologic and clinicochemical parameters reflected the clinically detectable impairment of health. Increased postimplantation loss, which was predominantly caused by a high number of late resorptions, was also found at 15.0 mg/m 3. The other gestational parameters examined were not adversely affected. Exposure to concentrations of 10.4 or 4.75 mg/m 3 caused increased lung weights as a symptom of maternal toxicity, but no evidence of developmental toxicity. These results are also consistent with the results of another

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TABLE 1 Chamber Analyses Polymeric MDI aerosol, mg/m 3 Parameter

0.0

1.0

4.0

12.0

Target (T) concentration, mg/m 3 Analytical (A) concentration, mg/m 3 A/T ratio Nominal (N) concentration, mg/m 3 A/N ratio Particle size, MMAD b,e Mark III c Marple 298 d Conditioned air (m 3/h) Compressed air (m 3/h) Exhaust air (m 3/h) Chamber temperature (°C) Chamber relative humidity (%) Test substance flow (ml/h) Chamber pressure (Pa) f

0.0 — — — —

1.0 1.03 ⫾ 0.20 a 1.03 13.2 0.079

4.0 4.03 ⫾ 0.80 1.01 35.9 0.112

12.0 12.0 ⫾ 1.60 1.00 222.5 0.054

— — 28.1 ⫾ — 27.8 ⫾ 21.6 ⫾ 49.9 ⫾ — 10.1 ⫾

⬍ 2.8 1.6 (1.8) 26.2 ⫾ 1.0 1.39 ⫾ 0.07 28.6 ⫾ 0.9 22.2 ⫾ 0.4 42.3 ⫾ 1.3 0.27 –11.6 ⫾ 4.8

⬍ 2.8 1.7 (1.8) 26.7 ⫾ 0.2 1.45 ⫾ 0.05 29.0 ⫾ 0.5 22.9 ⫾ 0.4 44.7 ⫾ 1.3 0.82 –9.8 ⫾ 0.1

⬍ 2.8 2.5 (1.6) 26.7 ⫾ 0.3 1.54 ⫾ 0.09 29.1 ⫾ 0.4 22.4 ⫾ 0.4 43.9 ⫾ 1.4 5.08 –9.8 ⫾ 0.1

0.1 a 0.1 a 0.5 a 1.6 a 0.2 a

Data are presented as mean ⫾ SD. Taken from the breathing zone of the animals for particle size determinations. c Mark III, stack sampler Mark III (Andersen). d Marple 298, stack sampler Marple 298 (Sierra). e MMAD, mass median aerodynamic diameter in microns with geometric standard deviation (GSD). f Pa, Pascal; pressure was set to positive in control chamber and was set to negative in MDI chambers. a b

prenatal inhalation toxicity range-finding study (TNO Nutrition and Food Research Institute, unpublished report), in which exposure to gravimetrically determined concentrations of MDI at 2.0, 7.9, and 12.0 mg/m 3 led mainly to an increased lung weight at the high concentration. Further corroborative evidence can be obtained from a 14-day inhalation study (TNO, unpublished report), in which exposure to about 15 mg/m 3 caused death and severe respiratory distress and growth retardation. Exposure to 2 or 5 mg/m 3 led to increased lung weights. Based on the results described above, which point towards a steep concentration-response curve in the range between 10 and 15 mg/m 3, the target concentrations selected for the prenatal inhalation toxicity study were 12, 4, and 1 mg/m 3. The high concentration was expected to cause overt maternal toxicity and possible prenatal toxicity; the mid concentration could have resulted in an increase in maternal lung weight, but no fetal effects should have been present; and the low concentration was anticipated to be a maternal and developmental no observed adverse effect concentration (NOAEC).

For each concentration, the test substance was supplied to a two-component atomizer with a continuous infusion pump (Perfusor, Braun) and heated with a circulating thermostat. By means of compressed air, the aerosol was generated into an aerosol mixing stage. In the mixing stage, the aerosol was mixed with conditioned air and passed through a cyclone separator into the inhalation chamber. The inhalation chamber at 0 mg/m 3 was not equipped with a generation system but was directly supplied with conditioned air. The exposure systems were also connected to an exhaust air system. Uniform distribution of the inhalation atmosphere (test compound in air) was assured by adequate measurement and technical design of the horizontal-flow chambers, along with establishment of appropriate airflow characteristics. The proportions of test substance were as follows: the 0 mg/m 3 chamber used fresh air (and no atomizer). The 1 mg/m 3 chamber used 0.27 ml/h of test chemical and an atomizer temperature of 45.0°C. The 4 mg/m 3 chamber used 0.82 ml/h of test chemical and an atomizer temperature of 44.3°C. The 12 mg/m 3 chamber used 5.08 ml/h of test chemical and an atomizer temperature of 45.4°C. Supply air (conditioned and compressed) and exhaust air, in m 3/h, for the four chambers are described in Table 1. For technical reasons, the study was carried out in two replicates, with each test group represented in each replicate. The treatment interval between the individual replicates was 1 day. Data are presented with the replicates combined. Exposure System

MATERIALS AND METHODS Test Material, Generation of Aerosol Atmospheres, and Chamber Parameters Polymeric MDI (isocyanic acid, polymethylene-polyphenylene ester; CAS No. 9016 – 87–9) was received from Bayer AG as technical product. It was stored in aluminum bottles blanketed with nitrogen in exhaust hoods at room temperature; there were no significant changes in purity or stability during the performance of this study.

The animals were kept singly in wire cages that were in a glass-steel inhalation chamber of 1.4 m 3 volume (manufacturer, BASF Aktiengesellschaft) for the whole-body exposures. The animals did not have access to water or feed during the exposure. Air flow rates, pressure conditions, relative humidities, and temperatures in the inhalation system were measured continuously by an automated measuring system and were monitored against preset limits and partially regulated. The generator parameter compressed air was also recorded by means of this system.

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PRENATAL TOXICITY OF MDI AEROSOLS IN RATS The temperatures in the generator systems were measured continuously (digital thermometer) and recorded one time/exposure. No surveillance of the oxygen content in the inhalation system was performed. The air change (about 20/h) within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals, and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

microbiologic contaminants. The drinking water was regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the Technical Services of BASF Aktiengesellschaft, as well as for the presence of germs by a contract laboratory. Both feed and water were suitable, based on the pertinent guidelines for animal feed and the German requirements for drinking water; their use has no effect on the design, conduct, or conclusions of this study.

Characterization of Inhalation Atmospheres

Experimental Procedures

The nominal concentrations were calculated from the mean of the supplied substance and the amount of the supply air. The concentrations of the inhalation atmospheres of the test groups were real-time monitored with noncalibrated, scattered-light photometers (RAM 1, MIE, USA) and quantified by HPLC after precolumn derivatization with 1-(2-pyridyl)-piperazin. For each concentration, samples were taken from the breathing zones of the animals in approximately hourly intervals. Amounts of 10 –90 l were sampled at a flow of 3 l/minute, with an array of a glass sampling probe with quartz wool plug and two absorption flasks filled with a 26-mM solution of 1-(2pyridyl)-piperazin in acetonitrile. MDI content of sampling probe, quartz wool plug, and first flask was determined after each sampling. The content of the second flask was analyzed at the end of daily exposure to control absorption efficiency of the array. After derivatization at 60°C for 20 min, the samples were prepared for analysis. High-performance liquid chromatography (HPLC; Hewlett Packard 1050) was used for analyses with a 250 mm (L) ⫻ 4 mm (ID) column; separation material was Nucleasil C8, 5 ␮m particle size. The detector was a photometric detector. The wavelength was 254 nm; injection volume 20 ␮l; column temperature was room temperature isocratic; the mobile phase 900 ml acetonitrile, 775 ml bidistilled water, 30 ml acetic acid glacial (100%), 18 g ammonium acetate; the flow rate was 1 ml/min.; and the retention time was 7.5 min. Mass values of polymeric MDI were obtained from the area integrals using the calibration point. The concentrations of the test groups were calculated from these mass values in relation to the injected volume and the sample volume of the inhalation atmosphere. The particle size analyses were carried out with Anderson Mark III and Sierra Marple 298 impactors. Aerosol volumes of 12–150 or 12–315 l were sampled from the breathing zones of the animals in the three MDI exposure chambers with the Mark III or Marple 298 impactor, respectively. The mass deposits inside the impactors were analyzed by the HPLC method described above. The calculation of particle size distributions from the masses deposited by the impactor stages was based on mathematical methods for evaluation of particle measurements (DIN 66141 and 66161).

Mating. After an acclimatization period of about 14 days, up to two untreated female rat(s) were mated with one untreated fertile male of the same breed. Mating took place from about 4 p.m. to about 7:30 a.m. of the following day. The day that sperm were detected microscopically in the vaginal smear in the morning was designated gd 0 (beginning of the study) and the following day “day 1” postcoitum (p.c.). From day 1 p.c. onward, the animals were placed in inhalation chambers for adaptation on 5 days over 6 h/day and were exposed to a stream of fresh air under similar conditions as during exposure.

Animals and Husbandry Sexually mature, virgin Wistar rats (Chbb:THOM [SPF]), supplied by Karl Thomae, Biberach an der Riss, FRG, that were free from clinical signs of disease were used for the investigations. This strain was selected as extensive experience is available on Wistar rats, and this strain has proved to be sensitive to substances with a teratogenic potential. The female rats were 61–70 days old when supplied. At the beginning of the study (gd 0, detection of sperm), the rats were 76 – 86 days old. Their mean weight was approximately 238.9 g. The animals were free from any clinically evident signs of disease prior to the beginning of the study. During the period when the rats were not exposed, they were housed singly in wire cages (type DK III of Becker & Co., Castrop-Rauxel, FRG). The animals were kept in air-conditioned rooms in which temperature was maintained in the range of 20 –24°C and relative humidity in the range of 30 –70%. A light/dark cycle of 12 h (light: 0600 –1800 h) was maintained. The animals were offered ground KLIBA laboratory diet rat/mouse/hamster maintenance GLP 343, Klingentalmuhle AG, Kaiseraugst, Switzerland, and tap water ad libitum, except during exposures when feed and water were withdrawn. The feed used in the study was assayed for chemical as well as for

Exposure and postexposure observations. The animals were exposed in the inhalation chambers, using wire cages of the above-mentioned type, for 6 h daily, from days 6 –15 p.c. From day 16 p.c. to the day of sacrifice (day 20 p.c.), the animals were subjected to a postexposure observation period under the housing conditions mentioned above. Maternal examinations. Dams were examined daily for mortality. In addition, the behavior and state of health of the test animals were checked at least three times on exposure days and, as a rule, once daily during the preflow period (chamber adaptation) and the postexposure observation period. During exposure, a groupwise examination was carried out. The body weights of the animals were recorded on day 0 (day of detection of sperm) and on days 2, 6, 9, 12, 15, 17, and 20 p.c., and body weight gains were calculated. Body weight gain was also calculated for the three different study periods: preflow period (chamber adaptation; days 0 – 6 p.c.), exposure period (days 6 –15 p.c.), and observation period (days 15–20 p.c.). These values were defined as body weight change. The corrected body weight gain was calculated after terminal sacrifice (terminal body weight on day 20 p.c. minus weight of the gravid uterus minus body weight on day 6 p.c.). With the exception of day 0, the consumption of feed and water was determined on the same days as body weight. Dams that died intercurrently (prior to scheduled sacrifice), as well as the contents of the uterus from these animals, were examined, if possible, in the same way as at terminal sacrifice (except that uterine weight was not recorded). On day 20 p.c., the surviving dams were sacrificed and necropsied in random order by cervical dislocation, and the fetuses were dissected from the uterus. Maternal head, liver, kidneys, larynx, and paired lungs were retained in 4% formaldehyde; no histopathology was performed. The uterus and the ovaries were removed and the following data were recorded: gravid uterine weight, liver and paired lung weights, number of ovarian corpora lutea, number and distribution of implantation sites classified as resorptions (early, late), dead fetuses, and live fetuses. Uteri from apparently nonpregnant females or from apparent single-horn pregnancies were stained by the method of Salewski (1964) to detect early resorption sites. Conception rate and pre- and postimplantation losses per dam, summarized within groups, were calculated as follows:

Conception rate:

Preimplantation loss:

number of pregnant animals ⫻ 100 number of fertilized animals

number of corpora lutea-number of implantations ⫻ 100 number of corpora lutea

Postimplantation loss:

number of implantations-number of live fetuses ⫻ 100 number of implantations

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Fetal examinations. At necropsy, each fetus was weighed, sexed, and examined macroscopically for any external findings. The sex was determined by anogenital distance and was later confirmed in all fetuses fixed in Bouin’s solution by internal examination. The viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes, and fluids were examined. Individual placental weights were recorded. After these examinations, approximately one-half of the fetuses per dam were placed in ethyl alcohol and the other half were placed in Bouin’s solution. After fixation in Bouin’s solution, the fetuses were examined for visceral alterations according to the method of Barrow and Taylor (1969). After these examinations, these fetuses were discarded. After fixation in ethyl alcohol, the skeletons of the remaining fetuses were stained with alizarin red S according to the modified method of Dawson (1926) and examined for skeletal alterations. These fetuses were retained by litter. Evaluation criteria for assessing the fetuses. There are differing opinions on the classification and assessment of changes in fetuses (Beltrame and Giavini, 1990). In the present investigations, the following terms (definitions) were used for describing a change: ● Malformations (concerning external, soft tissue and skeletal observations): permanent structural changes that may adversely affect survival, development, function, and/or occur spontaneously at a low frequency, were classified as malformations (e.g., exencephaly, atresia ani, hernia umbilicalis). ● Variations (concerning external, soft tissue and skeletal observations): divergences of the morphogenetic/organogenetic process that occur regularly also in control groups at a relatively high frequency and that may not adversely affect survival, development, or function were regarded as variations (e.g., dilated renal pelvis). ● Retardations (concerning skeletal observations only): transient delays in skeletal development that may not adversely affect survival, development, or function were considered to be retardations (e.g., sternebra (e) not ossified). Normally, skeletal retardations occur regularly also in control groups at a relatively high frequency. ● Unclassified observations (concerning external and soft tissue observations, only): external or soft tissue observations that could not be classified as malformations or variations (e.g., focal liver necrosis in fetuses).

Statistics The data were evaluated statistically using the computer systems of the Department of Toxicology of BASF Aktiengesellschaft. The Dunnett Test (Dunnett, 1955, 1964) was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of the following parameters: maternal feed and water consumption, body weight, body weight change, corrected body weight gain (maternal gestational body weight change minus the weight of the gravid uterus), gravid uterine weight, number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions, and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight. For the parameter maternal feed and water consumption, the mean of means was calculated and is presented in the relevant summary tables. The mean of means values allow a rough estimation of the total feed and water consumption during the different time intervals (preflow, exposure, and postsobservation period); they are not exactly precise values, because the size of the intervals taken for calculation differed. For the mean of means values, no statistical analysis was performed. Fisher’s Exact Test (Siegel, 1956) was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice, and the number of litters with fetal findings. The Wilcoxon Test (Hettmansperger, 1984; Nijenhuis and Wilf, 1978) was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations, and/or unclassified obser-

vations in each litter. Statistically significant differences for the control group values are designated as * for p ⬍ 0.05, and ** for p ⬍ 0.01 on the summary tables.

RESULTS

Chamber Analyses The mean analytical concentrations for the 1.0, 4.0, and 12.0 mg/m 3 chambers were 1.03 ⫾ 0.20 (SD), 4.03 ⫾ 0.80, and 12.0 ⫾ 1.60 mg/m 3. The measurements with scattered light photometers showed acceptable constancy of concentrations over the daily exposure periods. The particle sizes (mass median aerodynamic diameter [MMAD] and geometric standard deviation [GSD]) were as follows: 1.6 ␮m (1.8) at 1.0 mg/m 3, 1.7 ␮m (1.8) at 4.0 mg/m 3, and 2.5 ␮m (1.6) at 12.0 mg/m 3 in the respirable range. The analytical to target concentration ratios (and analytical to nominal concentration ratios) were 1.03 (0.079) at 1.0 mg/m 3, 1.01 (0.112) at 4.0 mg/m 3, and 1.00 (0.054) at 12.0 mg/m 3. The loss of aerosol, as compared to the nominal concentration, is due to sedimentation of large particles shortly after spraying and impaction/sedimentation of aerosol during its path through the inhalation equipment. This phenomenon is routinely encountered in aerosol studies, especially if equipment for particle size reduction is used. Mean temperature in all four chambers ranged from 21.6 to 22.9°C; mean relative humidity ranged from 42.3 to 49.9% (Table 1). Maternal Toxicity The distribution and fate of all study females are presented in Table 2. Two females (of 24 pregnant) died at 12.0 mg/m 3 (one on gd 15 during exposures and one on gd 18 in the postexposure period). No females were removed from study or delivered early. Two females, one each at 4.0 and 12.0 mg/m 3, were not pregnant at scheduled sacrifice. Of the pregnant females, one each at 1.0 and 12.0 mg/m 3 carried a fully resorbed litter at termination; all remaining pregnant females had live litters (i.e., one or more live fetuses). The number of litters (and fetuses) examined were 25 (336 live fetuses), 24 (338 live fetuses), 24 (345 live fetuses), and 21 (274 live and 5 dead fetuses) at 0.0, 1.0, 4.0, and 12.0 mg/m 3, respectively (Table 2). Periodic maternal body weights are presented in Figure 1A. Values at 12.0 mg/m 3 were significantly reduced on gd 9, 12, 15, 17, and 20. Maternal feed consumption in grams/animal/ day was significantly reduced at 12.0 mg/m 3 for gd 6 –9, 9 –12, 12–15, 15–17, and 17–20, and at 4.0 mg/m 3 only for gd 6 –9 (first interval of the exposure period) (Figure 1B). Maternal water consumption in grams/animal/day was also significantly reduced at 12.0 mg/m 3 for gd 6 –9, 9 –12, 12–15, 15–17, and 17–20 (Figure 1C). Maternal body weight change was equivalent across all groups for gd 0 – 6 (prior to the start of the exposure period). The values at 12.0 mg/m 3 for gd 6 through 15 (exposure period), gd 15–20 (postexposure period), and gd

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TABLE 2 Maternal Toxicity Polymeric MDI aerosol, mg/m 3 Parameter No. mated females on study No. (%) dams died No. (%) pregnant at termination No. (%) fully resorbed litters No. (%) viable litters In-life Maternal body wt change (g) gd 0–6 (pre-exposure) gd 6–15 (exposure) gd 15–20 (post-exposure) gd 0–20 (gestation) Maternal feed consumption (g/animal/day) c gd 0–6 gd 6–15 gd 15–20 Maternal water consumption (g/animal/day) c gd 0–6 gd 6–15 gd 15–20 At necropsy Terminal body wt (g) Maternal liver wt (g) Maternal paired lung wt (g) Maternal relative liver wt (%) Maternal relative paired lung wt (%) Gravid uterine wt (g) Carcass wt (g) d Net wt change (gd 6–20) e

0.0

1.0

4.0

12.0

25 0 (0.0) 25 (100.0) 0 (0.0) 25 (100.0)

25 0 (0.0) 25 (100.0) 1 (4.0) 24 (96.0)

25 0 (0.0) 24 (96.0) 0 (0.0) 24 (100.0)

25 2 (8.0) a 22 (95.7) 1 (4.5) 21 (95.5)

30.0 ⫾ 5.6 b 48.2 ⫾ 7.3 71.7 ⫾ 12.2 150.0 ⫾ 19.7

28.9 ⫾ 4.9 47.4 ⫾ 4.8 69.8 ⫾ 16.1 146.1 ⫾ 19.0

27.6 ⫾ 5.6 47.7 ⫾ 6.4 73.9 ⫾ 11.5 149.2 ⫾ 18.7

23.4 ⫾ 1.8 27.1 ⫾ 1.1 30.6 ⫾ 0.6

23.4 ⫾ 1.9 26.3 ⫾ 1.5 30.3 ⫾ 1.0

22.9 ⫾ 1.2 25.4 ⫾ 1.4 29.2 ⫾ 1.3

23.0 ⫾ 0.9 22.0 ⫾ 0.5 22.9 ⫾ 2.4

26.5 ⫾ 2.7 31.9 ⫾ 3.3 39.7 ⫾ 1.0

27.1 ⫾ 2.7 31.8 ⫾ 3.1 40.6 ⫾ 1.8

25.9 ⫾ 2.4 30.8 ⫾ 3.0 39.0 ⫾ 2.3

25.3 ⫾ 1.4 26.6 ⫾ 1.3 30.7 ⫾ 3.6

384.38 ⫾ 27.10 16.86 ⫾ 1.71 1.27 ⫾ 0.15 4.38 ⫾ 0.26 0.33 ⫾ 0.04 77.6 ⫾ 15.4 312.2 ⫾ 20.5 42.4 ⫾ 6.0

381.82 ⫾ 27.54 16.75 ⫾ 1.59 1.40 ⫾ 0.30 4.26 ⫾ 0.35 0.37 ⫾ 0.08 78.7 ⫾ 20.3 308.6 ⫾ 21.1 38.6 ⫾ 7.7

376.11 ⫾ 37.90 15.97 ⫾ 2.12 1.40 ⫾ 0.26 4.24 ⫾ 0.24 0.37 ⫾ 0.07 81.3 ⫾ 13.3 305.6 ⫾ 19.0 40.4 ⫾ 9.7

26.2 ⫾ 6.1 23.9 ⫾ 33.0** 48.4 ⫾ 41.1** 106.2 ⫾ 64.0**

333.76 ⫾ 70.64** 13.82 ⫾ 3.90** 1.45 ⫾ 0.29* 4.06 ⫾ 0.48** 0.45 ⫾ 0.12** 67.3 ⫾ 23.5 278.3 ⫾ 46.5** 12.2 ⫾ 47.1**

a

Both females were pregnant (see text). Data are presented as mean ⫾ SD. c The values for these summary intervals were not examined statistically (see Statistics section of Materials and Methods). d Carcass weight ⫽ terminal body weight minus gravid uterine weight. e Net weight change (gd 6 –20) ⫽ carcass weight minus body weight on gd 6. * p ⬍ 0.05 versus control value; Dunnett’s test (two-sided). ** p ⬍ 0.01 versus control value; Dunnett’s test (two-sided). b

0 –20 (gestational period) were all significantly reduced. Maternal feed and water consumption for these same summarized intervals was clearly reduced at 12.0 mg/m 3 during and after exposures (but they were not statistically analyzed). At scheduled necropsy on gd 20, at 12.0 mg/m 3, maternal absolute and relative liver weights were significantly reduced, and maternal absolute and relative lung weights were significantly increased, and maternal terminal body weight, carcass weight (body weight minus gravid uterine weight) and net weight change (carcass weight on gd 20 minus body weight on gd 6) were all significantly reduced. Mean gravid uterine weight was clearly reduced at 12.0 mg/m 3 (67.3 ⫾ 23.4 g; 86.7% of the control value of 77.6 ⫾ 15.4 g) but the difference was not statistically significant, most likely due to the large variance term (Table 2). Treatment-related clinical signs of toxicity were limited to dams at 12.0 mg/m 3 and were observed on gd 12, 13, 14, and

15 (the last 4 days of the exposure period). These signs included predominantly alterations in respiration (2– 6 dams), bloody crust around nose (2–3 dams), and piloerection (a nonspecific indication of stress; 3–25 dams) (data not shown). Gestational parameters are presented in Table 3. There were no effects of treatment on preimplantation or postimplantation loss, although one dam at 12.0 mg/m 3 had 5 dead fetuses (and 10 live fetuses), the only dead fetuses in the study. Litter size and sex ratio (percent males/litter) were also unchanged across groups. Fetal body weight per litter and placental weight per litter (separately by sex or total) were significantly reduced at 12.0 mg/m 3 (Table 3). A summary of fetal observations in the present study (with historical control ranges from the performing laboratory) is presented in Table 4. There were no significant differences among groups for the incidence of fetal external, visceral,

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0.0 mg/m 3, and filiformed (threadlike) tail at 12.0 mg/m 3. The fetal visceral malformations, all at low incidence, included hydrocephaly at 0.0 mg/m 3, globular-shaped heart at 12.0 mg/m 3, and dilation of the left ventricle at 12.0 mg/m 3. The skeletal malformations observed in 6, 11, 9, and 10 fetuses at 0.0, 1.0, 4.0, and 12.0 mg/m 3, respectively, included asymmetric dumb-bell shaped or bipartite thoracic vertebral body(ies), absent thoracic, sacral or caudal vertebra(e), bipartite sternebra(e), cleft sternum, fused and bifurcated ribs (both of these rib effects in one fetus at 4.0 mg/m 3 only), and absent rib(s) at 1.0 and 4.0 mg/m 3 (data not shown). There were no fetal external variations observed in this study. The incidence of visceral variations (in terms of affected fetuses per litter) was significantly increased at 1.0 mg/m 3 but not at 4.0 or 12.0 mg/m 3. The incidences of skeletal variations and skeletal retardations (for affected fetuses per litter) were significantly increased at 12.0 mg/m 3. Specific skeletal variations/retardations, which exhibited significant changes in incidence, included irregularly shaped sternebra(e) increased at 12.0 mg/m 3 (for number of affected litters and affected fetuses/litter); bipartite sternebra(e) increased at 1.0 mg/m 3 (for number of affected litters and affected fetuses/litter) and at 12.0 mg/m 3 (for affected fetuses per litter); and incomplete ossified vertebral bodies increased at 12.0 mg/m 3 (for affected fetuses/litter). Total fetal skeletal retardations were also significantly increased at 12.0 mg/m 3 (for affected fetuses/litter). The incidences of total fetal variations (as affected fetuses per litter) were significantly increased in all polymeric MDI-exposed groups, 1.0, 4.0, and 12.0 mg/m 3 (Table 4). DISCUSSION

FIG. 1. (A) Maternal body weight versus gestational days. (B) Maternal feed consumption versus gestational days. (C) Maternal water consumption versus gestational days.

skeletal, or total malformations. The fetal external malformations, all at low incidence, included cleft palate at 1.0 mg/m 3, anasarca (whole body edema) at 12.0 mg/m 3, anophthalmia at

In this study, exposure to 12 mg/m 3 of respirable polymeric MDI aerosol (but not to 4 or 1 mg/m 3) resulted in maternal toxicity, including mortality of 2 out of 25 dams. Lung damage, characterized by increased lung weights and respiratory symptoms, most likely was the cause of decreased feed and water consumption and reduced body weight gain, and was probably also responsible for the mortality. The increased absolute (and therefore relative) lung weights at 1.0 and 4.0 mg/m 3 were not statistically different for the control values, were very small (both 10.2% increased), did not exhibit a concentration-related pattern, and were not accompanied by respiratory symptoms. Maternal pulmonary histopathology was not performed in this study, nor in the range-finding study for this test substance, nor in another range-finding study (TNO, unpublished report) in the presence of increased maternal lung weights at 15.0, 10.4, and 4.75 mg/m 3 in the first study and at 12.0 mg/m 3 in the second study. In a 14-day study (TNO, unpublished report), increased lung weights were also observed at 15.0 mg/m 3 (along with mortality), 5.0, and 2.0 mg/m 3 (the performing laboratory has no historical control data based on lung weights). Maternal feed consumption was significantly reduced at 4 mg/m 3 only for gd 6 –9 (the first interval

437

PRENATAL TOXICITY OF MDI AEROSOLS IN RATS

TABLE 3 Gestational Parameters Polymeric MDI aerosol, mg/m 3 Parameter No. pregnant dams at terminal sacrifice All litters No. corpora lutea/dam No. implantation sites/litter Preimplantation loss/litter, % Postimplantation loss/litter, % Resorptions/litter Total Early Late Dead fetuses Live litters No. live fetuses/litter Total Females Males Percent males/litter Fetal body wt/litter (g) Total Females Males Placental wt/litter (g) Total Females Males

0.0

1.0

4.0

12.0

25

25

24

22

15.8 ⫾ 1.6 a 15.0 ⫾ 2.0 a 4.9 ⫾ 9.5 a 10.0 ⫾ 15.4 a

16.0 ⫾ 1.8 15.2 ⫾ 2.4 4.5 ⫾ 12.0 12.7 ⫾ 20.7

16.0 ⫾ 2.0 15.2 ⫾ 2.6 4.9 ⫾ 11.8 5.1 ⫾ 6.0

15.9 ⫾ 1.9 14.3 ⫾ 3.8 10.9 ⫾ 20.6 11.8 ⫾ 21.9

1.5 ⫾ 2.4 a 1.2 ⫾ 1.7 a 0.3 ⫾ 0.8 a 0 25

1.7 ⫾ 2.1 1.2 ⫾ 1.6 0.5 ⫾ 1.8 0 24 c

0.8 ⫾ 1.0 0.6 ⫾ 0.9 0.2 ⫾ 0.6 0 24

1.6 ⫾ 3.1 0.8 ⫾ 0.9 0.8 ⫾ 3.2 5b 21 c

13.4 ⫾ 2.9 d 7.0 ⫾ 2.6 d 6.5 ⫾ 2.5 d 48.2

14.1 ⫾ 2.4 6.8 ⫾ 2.2 7.3 ⫾ 2.7 51.5

14.4 ⫾ 2.5 7.4 ⫾ 2.5 7.0 ⫾ 2.1 48.7

13.0 ⫾ 3.6 6.3 ⫾ 2.4 6.8 ⫾ 2.6 51.8

4.0 ⫾ 0.2 d 3.8 ⫾ 0.2 d 4.1 ⫾ 0.2 d

4.0 ⫾ 0.2 3.9 ⫾ 0.2 4.1 ⫾ 0.2

3.9 ⫾ 0.2 3.8 ⫾ 0.2 3.9 ⫾ 0.2

3.6 ⫾ 0.7** 3.5 ⫾ 0.7** 3.6 ⫾ 0.8**

0.45 ⫾ 0.04 b 0.44 ⫾ 0.04 b 0.46 ⫾ 0.04 b

0.44 ⫾ 0.03 0.43 ⫾ 0.04 0.44 ⫾ 0.04

0.44 ⫾ 0.04 0.43 ⫾ 0.04 0.45 ⫾ 0.04

0.42 ⫾ 0.03* 0.42 ⫾ 0.04 0.43 ⫾ 0.04*

Data are presented as mean ⫾ SD, based on all pregnant females. The five dead fetuses were from one litter, which also contained 10 live fetuses. c One litter in each designated group was fully resorbed at scheduled termination. d Data are presented as mean ⫾ SD, based on litters with live fetuses. * p ⱕ 0.05 versus control value; Dunnett’s Test (two-sided). ** p ⱕ 0.01 versus control value; Dunnett’s Test (two-sided). a b

of the exposure period); this finding was considered incidental (Gamer, unpublished report). The designation of 4 mg/m 3 as a NOAEC for maternal toxicity is supported by the data of Reuzel et al. (1994), who concluded from subchronic studies (with exposure duration about 6-fold longer than in prenatal toxicity studies) that “the dose response curve for repeated exposures of rats to respirable polymeric MDI is very steep, and that the “no-observed-adverse-effect level” of polymeric MDI was 1.4 mg/m 3, the actual no-adverse-effect level being lower than but most probably very close to 4.1 mg/m 3.” Their conclusions are in close agreement with the 4.0 mg/m 3 NOAEC identified in this study. The obvious compromised status of the dams at 12 mg/m 3 may have resulted in reduced gravid uterus weight. The observed developmental toxicity, including reduced fetal body weights (and related reduced placental weights) and an increased incidence of fetuses/litters with skeletal variations and skeletal retardations may have been due, at least in large part, to the reduced fetal body weights (Hood and Miller, 1997). No

substance-induced teratogenic effects were observed up to and including the highest concentration (12 mg/m 3). Concerning fetal morphology, the historical control data shown in Table 4 for total malformations indicate that the 2-fold increase in the percent of litters with malformations in the high exposure group (38.1% vs. 20.0% in the control) is still within the historical control range (0.0 – 43.0%). The same applies to the 2.5-fold increase in the percentage of fetuses with malformations (5.4% vs. 2.1% in the control; historical control range is 0.0 –5.7%). The incidences and the types of fetal external visceral and skeletal malformations observed (see Results section) were scattered across groups and at low incidence. There was a significant increase in the incidence of fetal visceral variations (particularly dilated renal pelvis) on a fetus per litter basis at 1 mg/m 3 (but not at 4 or 12 mg/m 3); in the incidence of bipartite sternebra(e), in terms of litters with one or more affected fetuses per litter at 1 mg/m 3 (but not at 4 or 12 mg/m 3) and on a fetus per litter basis at 1 and 12 mg/m 3 (but not at 4 mg/m 3); and of irregular-shaped sternebra(e) in terms

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TABLE 4 Summary of Fetal Observations Polymeric MDI aerosol, mg/m 3 Parameter External findings a No. litters evaluated No. fetuses evaluated Total Live Dead External malformations Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b External variations Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b Visceral findings c No. litters evaluated No. fetuses evaluated Total Live Dead Visceral malformations Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b Visceral variations Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b Skeletal findings d No. litters evaluated No. fetuses evaluated Total Live Dead Skeletal malformations Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b Skeletal variations Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b Skeletal retardations e Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b Specific skeletal variations/retardations Sternebra(e) of irregular shape Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b Sternebra(e) bipartite Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b

0.0

1.0

4.0

12.0

25

24

24

21

336 336 0

338 338 0

345 345 0

279 274 5

1 (0.3) 1 (4.0) 0.3 ⫾ 1.5

2 (0.6) 2 (8.3) 0.5 ⫾ 1.8

0 (0.0) 0 (0.0) 0.0

2 (0.7) 2 (9.5) 0.6 ⫾ 1.8

0 (0.0) 0 (0.0) 0.0

0 (0.0) 0 (0.0) 0.0

0 (0.0) 0 (0.0) 0.0

0 (0.0) 0 (0.0) 0.0

25

24

24

21

161 161 0

164 164 0

168 168 0

133 130 3

1 (0.6) 1 (4.0) 0.7 ⫾ 3.3

0 (0.0) 0 (0.0) 0.0

0 (0.0) 0 (0.0) 0.0

15 (9.3) 11 (44.0) 9.4 ⫾ 12.7

34 (20.7) 15 (62.5) 20.4 ⫾ 21.5*

31 (18.4) 12 (50.0) 18.4 ⫾ 23.5

25

24

24

21

175 175 0

173 173 0

177 177 0

146 144 2

Historical control range (%)

0.0–0.6 0.0–9.1 0.0–0.6

5 (3.8) 2 (9.5) 3.4 ⫾ 12.7

0.0–2.2 0.0–12.5 0.0–1.9

17 (12.8) 12 (57.1) 13.0 ⫾ 14.3

7.1–36.9 30.4–95.8 7.5–38.5

6 (3.4) 4 (16.0) 3.7 ⫾ 10.7

11 (6.4) 7 (29.2) 6.2 ⫾ 12.4

9 (5.1) 8 (33.3) 5.1 ⫾ 8.0

10 (6.8) 7 (33.3) 7.9 ⫾ 14.2

0.0–0.1 0.0–43.5 0.0–8.7

69 (39.4) 22 (88.0) 38.9 ⫾ 27.1

84 (48.6) 24 (100.0) 48.7 ⫾ 23.7

85 (48.0) 23 (95.8) 47.8 ⫾ 20.6

92 (63.0) 21 (100.0) 63.2 ⫾ 24.0**

38.1–53.7 83.3–100.0 37.5–52.4

94 (53.7) 24 (96.0) 55.5 ⫾ 27.7

79 (45.7) 22 (91.7) 46.0 ⫾ 28.2

110 (62.1) 24 (100.0) 61.4 ⫾ 25.6

105 (71.9) 21 (100.0) 73.6 ⫾ 26.4*

27.2–72.0 79.2–100.0 27.3–71.5

61 (34.9) 20 (80.0) 34.6 ⫾ 26.0

54 (31.2) 23 (96.0) 32.5 ⫾ 17.6

69 (39.0) 23 (95.8) 38.6 ⫾ 15.2

65 (44.5) 21 (100.0)* 47.1 ⫾ 24.4

17.1–48.5 60.0–100.0 15.3–48.2

2 (1.1) 2 (8.3) 1.0 ⫾ 3.3

6 (4.1) 3 (14.3) 3.6 ⫾ 11.3*

0.0–5.4 0.0–29.2 0.0–5.2

0 (0.0) 0 (0.0) 0.0

6 (3.5) 5 (20.8)* 3.3 ⫾ 6.9*

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PRENATAL TOXICITY OF MDI AEROSOLS IN RATS

TABLE 4—Continued Polymeric MDI aerosol, mg/m 3 Parameter Thoracic vertebral body/bodies incompletely ossified Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b Total malformations Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b Total variations Fetuses: no. (%) Litters: no. (%) Affected fetuses/litter b

0.0

1.0

4.0

12.0

Historical control range (%)

47 (26.9) 20 (80.0) 29.9 ⫾ 29.3

36 (20.8) 15 (62.5) 20.9 ⫾ 24.7

51 (28.8) 21 (87.5) 28.8 ⫾ 19.3

68 (46.8) 17 (81.0) 49.8 ⫾ 33.4*

0.0–43.5 0.0–82.6 0.0–39.7

7 (2.1) 5 (20.0) 2.2 ⫾ 5.5

12 (3.6) 7 (29.2) 3.4 ⫾ 6.7

9 (2.6) 8 (33.3) 2.6 ⫾ 4.0

15 (5.4) 8 (38.1) 6.0 ⫾ 12.1

0.0–5.7 0.0–43.0 0.0–4.8

84 (25.0) 23 (92.0) 24.6 ⫾ 15.7

118 (35.0) 24 (100.0) 34.7 ⫾ 15.6*

116 (33.6) 23 (95.8) 33.4 ⫾ 16.7*

109 (39.1) 21 (100.0) 40.0 ⫾ 12.1**

21.0–44.0 88.0–100.0 20.7–35.2

a

All fetuses per litter were examined externally. Affected fetuses per litter are presented as mean ⫾ SD. c Approximately 50% of the fetuses per litter were examined viscerally. d Approximately 50% of the fetuses per litter were examined skeletally. e Retardations were assessed for skeletal findings only. * p ⬍ 0.05 versus control, Fisher’s exact test (one-sided) or Wilcoxon test (one-sided). ** p ⬍ 0.01 versus control, Fisher’s exact test (one-sided) or Wilcoxon test (one-sided). b

of litter incidence at 12 mg/m 3 (but not at 1 or 4 mg/m 3). The incidences of total fetal variations, in terms of affected fetuses per litter, were significantly increased in all polymeric MDIexposed groups at 1, 4, and 12 mg/m 3. These fetal effects at exposure concentrations other than at 12 mg/m 3 were most likely due to the unexpectedly low number of fetal variations in the concurrent control group and occurred in the absence of a concentration relationship; they were therefore considered spontaneous in nature. The fetal effects observed at 12 mg/m 3, involving exclusively increases in fetal skeletal variations (mostly increases in indications of delayed ossification) and therefore increases in total fetal variations, exceeded the historical control range in the performing laboratory (Table 4), were associated with compromised maternal status and reduced fetal body weights, and were therefore considered treatment related (Gamer, unpublished report). A previous developmental toxicity study in Wistar rats exposed to monomeric MDI as the aerosol, 6 h/day, gd 6 through 15 at 0, 1, 3, or 9 mg/m 3 resulted in concentration-dependent reductions in maternal feed consumption during the exposure period in all MDI-exposed groups, and increased maternal lung weights only at 9 mg/m 3. There were no effects on any other parameters of maternal toxicity and no effects on gestational or fetal parameters (including no effects on pre- or postimplantation loss, litter size, fetal body or placental weights, or on the incidences of fetal external or visceral anomalies or of the extend of fetal ossification). There was a slight but significant increase in litters with one or more fetuses at 9 mg/m 3 with asymmetric sternebra(e); the incidence was considered within the limits of biological variability (Buschmann, 1994; Buschmann et al., 1996). As the current study evaluated poly-

meric MDI at 0, 1, 4, and 12 mg/m 3 with effects only at 12 mg/m 3, the results of the two studies are consistent. The results of the present study are also consistent with the results from the two developmental toxicity range-finding studies on polymeric MDI (BASF, TNO, unpublished reports) and with results of a 14-day study on polymeric MDI (TNO, unpublished report), all of which indicated increased lung weights at exposure concentrations ⱖ 9 mg/m 3, adult mortality at ⱖ 12 mg/m 3, and increased postimplantation loss only at 15 mg/m 3. The NOAEC for maternal and developmental toxicity therefore is 4 mg/m 3, and the NOAEC for teratogenic effects is at or above 12 mg/m 3 in rats under the conditions of this study. ACKNOWLEDGMENTS This study was sponsored by the International Isocyanate Institute Inc., Parsippany, NJ, USA. The Sponsor’s Monitoring Scientist was Dr. J. E. Doe, Zeneca Central Toxicology Laboratory, Cheshire, United Kingdom. The study was performed according to the OECD Guidelines (1981) under OECD and U.S. EPA TSCA Good Laboratory Practice Standards (U.S. EPA, 1983). The authors wish to thank the following BASF personnel: Prof. Dr. med. Dr. rer. Nat. H.-P. Gelbke (Head), Dr. med. vet. K. Ku¨ttler (Pathology) and Dr. med. vet. B. Hildebrand (Head of Experimental Toxicology). Dr. med. vet. A. O. Gamer of BASF was Study Director (and was responsible for the generation and analyses of test atmospheres), and Dr. med. vet. J. Hellwig of BASF was responsible for the toxicology assessments. Dr. R. W. Tyl wrote this manuscript from the Final Report (Gamer, unpublished report).

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Barrow, M. V., and Taylor, W. J. (1969). A rapid method for detecting malformations in rat fetuses. J. Morphol. 127, 291–305. Beltrame, D., and Giavini, E. (1990). Morphological abnormalities in experimental teratology: Need for a standardization of current terminology? Cong. Anom. 30(3), 187–195. Buschmann, J., Koch, W., Fuhst, R., and Heinrich, U. (1996). Embryotoxicity study of monomeric 4,4⬘-methylene diphenyl diisocyanate (MDI) aerosol after inhalation exposure in Wistar rats. Fundam. Appl. Toxicol. 32, 96 –101. Buschmann, J. (1994). Teratogenicity study of monomeric 4,4⬘-methylene-diphenyl diisocyanate (MDI) aerosol after inhalation exposure in Wistar rats. Poster presented at the 22nd Annual Conference of the European Teratology Society, Prague, Czechoslovakia, September 12– 15, 1994. Dawson, A. B. (1926). A note on the staining of the skeleton of cleared specimens with Alizarin red S. Stain Technol. 1, 123. DIN 66141: Darstellung von Korngro¨␤enverteilungen. DIN 66161: Partikelgro¨␤enanalyse (Beuth-Vertrieb GmbH, Berlin und Ko¨ln). Dunnett, C. W. (1955). A multiple comparison procedure for comparing several treatments with a control J. Amer. Statist. Assoc. 50, 1096 –1121. Dunnett, C. W. (1964). New tables for multiple comparisons with a control. Biometrics 20, 482– 491.

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