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Petroleum hydrocarbon toxicity studies
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
Petroleum XIII.
Animal
and
Human
36.473-490
(1976)
Hydrocarbon Response
Toxicity to Vapors
Studies
of Toluene
Concentratelvz
C. P. CARPENTER, D. L. GEARY, JR., R. C. MYERS,D. J. NACHREINER, L. J. SULLIVAN,AND J. M. KINGS The Chemical Hygiene Carnegie-Mellon Received
Fellowship, University, June 2,1975;
Carnegie-Mellon Institute Pittsburgh, Pennsylvania accepted
December
of Research, 15213
22,1975
Petroleum Hydrocarbon Toxicity Studies. XIII, Animal and Human Responseto Vapors of Toluene Concentrate. CARPENTER, C. P., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., AND KING,
J. M. (1976).Toxicol. Appl. Pharmacol. 36,473-490.The suggested hygienic standardfor humaninhalation of TolueneConcentrate,basedupon results of theseinhalation studieswith rats and dogsand the sensoryresponseof human subjects,is 1.9 mg/liter (480 ppm). The LC50 for rats that inhaled Toluene Concentratefor 4 hr was 35 (31 to 38) mg/liter or 8800(7800to 9600)ppm. Rats tolerated 6.8 mg/liter (1700 ppm) for 4 hr and dogs3.0 mg/liter (760ppm) for 6 hr without any signsof discomfort. Four catsthat inhaled31mg/liter (7800ppm)for 6hr developedsignssuggestiveof central nervous systemeffects, including mydriasis,mild tremors, prostration in 80 min, and light anesthesiaafter 2 hr. One of four died as a result of pneumonicinfection within 14 days. Rats and dogsthat inhaled 3.9, 1.9, or 0.95 mg/liter of Toluene Concentratefor 13 weeks,6 hr/day, were not statistically significantly different from their air-exposedcontrols in any of the criteria of injury that weremonitored including hematology,clinical chemistry, and micropathology. The odor thresholdasdeterminedby a sniff test involving two trials with six humansubjectsis approximately 0.01 mg/liter (2.5 ppm). Six of six people indicated their willingnessto work in a concentration of 1.9 mg/liter (480 ppm) which is equivalent to 0.87 mg/liter or 220 ppm of contained toluene,the limiting concentrationrecommendedfor the hygienicstandard. Only the response of peoplein the workplacecandeterminewhetherthe toxic and irritative effectsof the componentsof this mixture are additive or less than additive, as the evidencepresentedsuggeststo us that they are not greaterthan additive. Investigation of animal and human response to the vapor inhalation of Toluene Concentrate paralleled the studies on Varnish Makers’ and Painters’ Naphtha, Stoddard Solvent, Rubber Solvent, Mixed Xylenes, “60 Solvent”, “70 Solvent”, “140” Flash Aliphatic Solvent”, “80 Thinner “, “50 Thinner”, Deodorized Kerosene, and “40 1 Supported by the American Petroleum Institute. 2 Urban0 C. Pozzani, before his death in July of 1970, was involved in the planning and execution of this series of inhalation studies of commercially available hydrocarbons. He was to have been the senior author of the formal publications as recognition for his devotion to this project. 3 Present address: New York State Veterinary College, Cornell University, Ithaca, New York. Copyright 0 1976 by Academic Press, Inc. 473 All rights of reproduction in any form reserved. Printed
in Great
Britain
474
CARPENTER
ET AL.
Thinner” (Carpenter et al., 1975-76b-1). The objectives of these studies have been presented by Carpenter et al. (1975a) in a report which contains the detailed protocol, compounds under investigation, and details of procedure common to the whole series. A literature review did not reveal mammalian toxicity studies on materials of similar composition. METHODS The details of the experimental design and the various procedures utilized are given in Carpenter et al. (1975a). Sample. The hydrocarbon mixture designated by the coined name, “Toluene Concentrate” was furnished by one of the major producers in the United States and was assigned the Chemical Hygiene Fellowship No. 35-l 54. Pertinent physicochemical properties are presented in Table 1 and composition in Table 2. TABLE 1 PHYSICOCHEMICAL
PROPERTIES
OF TOLUENE
CONCENTRATE
Boiling range” Percent
Temperature (“F)
Percent
Temperature 03
I.B. pt 5 10 20 30 40 50
203 208 209 210 211 212 213
60 70 80 90 95 DEP ASTM EP
214 216 218 221 224 226 231
Flash point Explosive limit Refractive index Specific gravity Mean b401Wtb At 25°C and 760 mm
25°F (tag closed cup) 1.Oto 7.0 %, by volume in air 1.4428 at 60°F 0.7780 at 60°F 97 1 mg/liter = 252 ppm 1 ppm = 0.003967 mg/liter
’ ASTM Method D-86. b Calculated from mass spectrography
data.
Analytical method for monitoring Toluene Concentrate. The analytical procedure developed was dependent upon measurement of peak height of three principal peaks with reasonable resolution and good symmetry characteristics. The conditions necessary for reliable analysis are given in Table 3. With but few exceptions, the vapor concentrations in the acute single inhalation and the human sensory studies were analyzed at least once per exposure and in the subacute (repeated) study, twice each day. The actual vapor concentrations are reported as measured concentrations as opposed to “metered” concentrations which are based upon empirical flow rates of dilution air and solvent. When reference is made to the number
VAPOR
INHALATION
OF TOLUENE
TABLE COMPOSITION
475
CONCENTRATE
2
OF TOLUENE
CONCENTRATE
Mass spectral analysis (volume percentage)
Component
&
Paraffins Butanes 2,2,3-Trimethylbutane
0.23
-
Pentanes 2,ZDimethylpentane 2,3-Dimethylpentane 2,CDimethylpentane 3,3-Dimethylpentane 3-Ethylpentane 2,2,4-Trimethylpentane 2,3,4-Trimethylpentane 2-Methyb3-ethylpentane
0.46 2.59 0.74 0.62 0.53 0.04 0.06 0.02
0.3 1.7 0.5 0.4 0.3 -
Hexanes n-Hexane 2-Methylhexane 3-Methylhexane 2,ZDimethylhexane 2,3-Dimethylhexane 2,CDimethylhexane 2,5-Dimethylhexane
0.02 6.63 6.65 0.87 0.48 I .68 1.41
5.8 5.7 0.7 0.5 1.4 1.2
13.16 1.20 0.88 0.37
11.8 1.3 0.7 0.5
Octanes n-Octane
0.05
-
Naphthenes Cyclopentanes Methylcyclopentane 1,1-Dimethylcyclopentane 1-trans-2-Dimethylcyclopentane 1-cis-2-Dimethylcyclopentane l-tram-3-Dimethylcyclopentane I-cis-3-Dimethylcyclopentane Ethylcyclopentane 1,1,2-Trimethylcyclopentane 1,1,3-Trimethylcyclopentane 1-tram-2-cis-4-trans-Trimethylcyclopentane I-trans-2-cis-2-trans-Trimethylcyclopentane 1,2,3-Trimethylcyclopentane
0.07 0.47 1.19 0.20 0.85 0.68 0.35 0.08 0.36 0.33 0.33 -
0.07 0.42
Heptanes n-Heptane 2-Methylheptane 3-Methylheptane 4-Methylheptane
1.29 1.32 0.33 0.35 0.34 0.33
476
CARPENTER ETAL.
TABLE 2-con&ued _--Massspectral analysis(volume percentage)
Component
Naphthenes-continued Cyclohexanes Cyclohexane Methylcyclohexane 1-trans-4-Dimethylcyclohexane 1,1-Dimethylcyclohexane
1.74 8.48 0.23
glc
1.59 8.30 0.23 0.04
Aromatics Benzene Toluene
0.06 45.89
0.07 50.46
Totals Paraffins Naphthenes Aromatics
38.69 15.36 45.95
TABLE 3 GASCHROMATOGRAPHICPROGRAM:TOLUENECONCENTRATE'
Column Coating Support Conditions Solvent for calibration Carrier gas Burner Vapor samplesize Lower limit of detection
Coppertubing + in. o.d. x 25 ft. 15% Apiezon M Gas Chrom Q 80/100mesh 90 to 140°Cat 2.5”C/min Hexadecane Helium Beckman Hydrogen : 45 ml/min Air: 450ml/min l-10 ml 0.05 ,ug
’ A temperature-programmed gaschromatographequippedwith a flameionizationdetectorwasusedfor all analyses.
of days elapsed in the 13-week study, it refers to the actual number of days the animal inhaled the vapor and not calendar days. Any deviation from this practice will be noted. PrPcis of experimental design. A stepwise approach was undertaken to determine a no ill effect level by inhalation in rats and dogs. First, rats were subjected to a series of single 4-hr inhalation experiments to arrive at an LC50 and to detect cellular injury. Cats then were subjected to this LC50 concentration to detect any overt evidence of central nervous system effects that might occur. Based on these data, sublethal concentrations were set up to determine the earliest persistent signs of toxic stress as an aid in setting levels for the 6-hr-per-day, Sday-per-week, 66-day inhalation study. At the termination of the 66-day repeated study, some of the surviving rats were subjected
VAPOR
INHALATION
OF TOLUENE
CONCENTRATE
477
to a massive concentration of 1.3 times the 4-hr LC50 for 1.5 hr to demonstrate whether they had become more resistant, more susceptible, or neither as a result of their repeated inhalation of Toluene Concentrate. Respiratory irritation was studied in mice by a technique described by Alarie (1966). To complete the investigation, odor thresholds and sensory responses were determined through inhalation of Toluene Concentrate by human volunteers. RESULTS Acute Inhalation Toxicity Prior to determining the 4-hr acute response to the vapor it was determined empirically that fog formation (saturation) did not occur until a concentration in the neighborhood of 500 mg/liter (130,000 ppm) was generated. At a concentration of about 173 mg/liter (44,000 ppm), calculated on the dilution air-flow and weight of compound utilized, four of six rats succumbed in 30 min. Progressive signs of distress included: eye irritation in 2 min, loss of coordination in 5 to 6min, anesthesia in 8.5 min, one dead in 14 min, tonic convulsions in another rat in 16 min and a total of 3 deaths in 17 min, with a fourth dying in 30 min. Two of the four victims had slightly congested lungs but no other notable gross pathological changes. Acute 4-hr LC.50 rats. Groups of 10 young male albino Harlan-Wistar rats (130200 g) were subjected to measured concentrations of Toluene Concentrate for single 4-hr periods. At a measured 49 mgjliter (12,000 ppm) all succumbed following tremors (5 min) and prostration (15 min). The first death occurred within 1 hr and the remainder during the next 3.5 hr. Three of an additional group of six animals in each concentration were to be sacrificed immediately after the exposure and the other three, 2 days later. However, only one of these six survived the exposure and was sacrificed 2 days thereafter. At roughly one-half the above concentration, 25 mg/liter (6300 ppm), head tremors were seen in 1 hr and prostration occurred in 2 hr, but at 3-hr postexposure all appeared to be normal. Fourteen-day weight gains were normal for rats of this age. At 13 mg/liter (3300 ppm) the only sign was slight loss of coordination and at 6.8 mgjliter (1700 ppm) the rats were normal throughout the 4-hr exposure. The calculated LC50 is 35 (31 to 38) mg/liter or 8800 (7800 to 9600) ppm. The only micropathology noted other than the commonly found sporadic lesions was lung atelectasis in 4 of 8 rats examined that died in the 4-hr exposure to 49 mg/liter (12,000 ppm) of Toluene Concentrate. The sole survivor killed 2 days after the exposure had chronic tracheitis, slight bile duct proliferation, and slight tubular regeneration. Bone marrow smears on these rats were not remarkable. At the lower levels there were no micropathological changes related to the inhalation of this vapor. Dogs. Two female beagles that inhaled 6.0 mg/liter (1500 ppm) 6 hr per day successively for 3 days responded with slight lacrimation and head tremors. Thereupon the concentration was reduced to 4.1 mg/liter (1000 ppm) but the intermittent head tremors still persisted. Finally, another pair of female beagles were exposed 6 hr/day for 2 days at 3.0 mg/liter (760 ppm), rested for 4 days and then exposed for 3 successive days, One had a weight loss of 1.l kg after the fifth exposure and the other recovered unchanged. Both appeared normal throughout the ensuing 1Zday observation period. Cats. Four male cats of mixed breed inhaled a mean measured concentration of 31 mg/liter (7800 ppm) for 6 hr. These cats were carefully observed for signs of central.
478
CARPENTER
ET AL.
nervous system effect. Prior to inhalation, they had been examined and found normal in placing reactions, righting reflexes, aversion to foot pad pain, extensor thrust reflex, and pupillary contraction. Responses of cats included slight loss of coordination, mydriasis, and slight hypersensitivity to light within 20 min. These effects were progressive and were accompanied by ear twitching, sneezing, body jerks, and prostration all of which occurred within 80 min. Increasing tremors were noted in two of the cats. Within 2 hr all appeared to be under light anesthesia. This condition persisted throughout the remainder of the 6-hr exposure. All survived the exposure and only one succumbed during the ICday observation period. Gross autopsy revealed pneumonia to be the cause of death. One surviving cat had lost 0.59 kg the day following exposure, but body weights of the three survivors appeared normal prior to sacrifice at the conclusion of the 1Cday holding period. Sporadic lesions, common to cats, were reported but aside from the purulent bronchopneumonia and the attendant round cell foci in two of four animals, undoubtedly present before exposure, the other lesions were single occurrences or of no importance. None of these pathological changes were related to treatment. Short-term massive concentration. Groups of five male rats were subjected to a mean measured concentration of 180 mg/liter (45,000 ppm) for time-periods varied by a factor of 2 to determine the Lt50. This concentration is five times greater than the 4-hr LC50 for rats of 35 mg/liter (8800 ppm) and approximates substantial saturation of air with Toluene Concentrate vapor at room temperature. Signs of toxicity included lacrimation and loss of coordination within 2 min, and prostration and anesthesia within 5 min. The Lt50 was 11 (7.2 to 16) min. Microscopic examination of the entire lung, including the bronchi, trachea, bifurcation of the trachea, thyroid, parathyroids, and esophagus revealed no lesions related to treatment. Osmotic erythrocytefragility. No increase in erythrocyte fragility was observed among six rats that inhaled a metered concentration of 80 mg/liter (20,000 ppm) of Toluene Concentrate vapor for 45 min. The mean initial hemolysis was in 0.51% NaCl solution and final in 0.31%. These values were not statistically significantly different from those of five air-exposed controls, namely, 0.47 % initial and 0.31% final. Mouse respiratory tract irritation. The mouse respiratory irritation study was patterned after the method described by Alarie (1966). Two Charles River mice, 45 to 50 g, were tested simultaneously. A 50% or greater depression in respiratory rate was registered by four of six and three of six mice, which had inhaled measured concentrations of 60 mg/liter (15,000 ppm) and 34 mg/liter (8600 ppm), respectively. A similar decrease in rate did not occur when another group of six inhaled 20 mg/liter (5000 ppm). Subacute Inhalation Toxicity To determine the response of rats and dogs to repeated daily inhalation, in order to provide a basis to establish acceptable inhalation standards, groups of 25 male Harlan-Wistar rats and 4 male beagles were assigned randomly to each of three graded’ levels of Toluene Concentrate and to a solvent-free air-control. Three rats from each level were sacrificed for histopathological examination after 14- and 38-day intervals. After repeated inhalation for 6 hr/day, 5 days/week for 71 days, 10 randomly preselected
479
VAPORINHALATIONOFTOLUENECONCENTRATE
rats from each level were subjected to a challenge exposure to determine whether they had become more or less sensitive to the vapor. The remaining rats and all beagles were killed after 65 days and 66 days, respectively. The criteria of toxic response monitored for the subacute study included body weight change and analyses of blood and urine. Evaluation on blood included packed cell volume (hematocrit), total erythrocyte count, reticulocyte count, total and differential leukocyte counts, serum alkaline phosphatase activity (SAP), serum glutamic pyruvic transaminase activity (SGPT), serum glutamic oxalacetic transaminase activity @GOT), and blood urea nitrogen (BUN) on both species, with the addition of bilirubin and blood glucose on dogs. Preliminary or baseline values were established on blood drawn from the jugular vein of all dogs allocated to this subacute study prior to the first day of vapor inhalation. The dog blood values were determined again, prior to the sacrifice, after 61 days. Preliminary values were not established for the rats because not enough blood could be taken to accomplish the tests without debilitating the animals. The values for the treated rats were compared to the values of the air-controls concomitantly sacrificed at the 14-, 38-, and 65-day intervals. Rat blood was taken from the tip of the tail prior to sacrifice for the hematological determinations and from the severed cervical vessels after section of the cord, for clinical biochemistry. Procedures for blood determinations are outlined in Carpenter et al. (1975a). Food consumption was followed 1 day of each week, electrocardiograms were taken and urine analyses were performed on the dogs prior to and at termination of the study. At sacrifice, tissues taken from rats for microscopic examination included adrenal, brain, pituitary, trachea, thyroid, parathyroid, lung, heart, liver, kidney, spleen, stomach, duodenum, pancreas, ileum, jejunum, colon, skeletal muscle, sciatic nerve, and bone marrow impression smear. Similar tissues were collected from the dogs at sacrifice, with the addition of the pharynx, tonsil, bifurcation of the trachea, nasal mucosa, and bone marrow section. Gas chromatographic analyses of the nominal 6, 3, and 1.5 mg/liter vapor: air mixtures yielded mean measured concentrations of 3.9, 1.9, and 0.95 mg/liter corresponding to 980,480, and 240 ppm, as shown in Table 4. Rats. Two deaths occurred among rats during the repeated subacute inhalation. One moribund rat was killed after 27 and another died after 42 exposures to the 0.95-mg/ liter (240 ppm) concentration. Both rats had endemic pneumonia judging from the TABLE 4 GAS CHROMAT~GRAPHIC
ANALYSES OF TOLUENE CONCENTRATE VAPOR CONCENTRATIONS INHALEDBYRATSANDDOGSFOR 13 WEEKS
Number of analyses 85 Metered concentration (mg/liter) 6.0 Measured concentration (mg/liter) 3.9 95% Fiducial limits for measured concentrations (r&liter) 1J-5.94 Measured concentration (ppm) 980 Measured as percentages of metered concentration 65.0 Coefficient of variation 26.37
92 3.0 1.9
81 1.5 0.95
0.98-2.87 0.25-l .65 480 64.0 24.75
240 63.3 36.84
35 0.0 0.0
0.0 -
Hmtmmxx4t
6.99 7.45 7.94
6.83 7.83 7.36
3 8 13
3 8 13
0.95
a 0.01 > p > 0.001.
(24’3
13
7.53 7.47 7.70
83
18.2 15.5 12.7
16.4 12.7 15.4
15.3 12.7 11.4
15.4 16.1 12.2
6.76 7.82 7.70
(i;890)
WBC (thousands/ mm3)
FOR GROUPS
RBC (millions/ mm3)
83 13
Period Wk)
FttmtNGs
$0)
Concn mg/liter (m-W
MEAN
41 .o 43.7 42.1
42.1 41.5 43.2
45.0 41.6 43.4
42.2 42.1 42.4
Ht (%)
OF THREE,
5
15.2 16.3 15.4
15.4 15.3 15.5
16.6 15.4 15.7
15.2 15.5 15.2
THAT
2.93 2.00 3.10
0.0 0.0 0.2
0.0 0.0 0.0
0.0 0.0 0.0
2.67 1.80 3.20 2.80 1.87 2.75
0.0 0.0 0.0
Baso
-
INHALED
2.53 1.73 3.10
Reticulocytes (%I
THREE, AND FOUR Rnrs 13 WEEKS,FCESPECTIVELY
TABLE
2.0 2.0 4.0
4.3 1.7 4.2
4.3 5.0 4.5
5.3 3.0 3.5
Eosino
-
CONCENTRATE
VAPOR
FOR
5.0 5.7 5.8
5.3 5.0 5.5
3.7 4.3 7.0
6.7 5.0 3.5
Immature neutro
56.3 53.7 49.5
50.0 54.0 51.5
54.3 55.0 50.2
51.0 51.7 49.5
Neutro
126.7 131.7 134.5
134.7 136.0 132.5
131.3 130.3 131.5
128.3 134.3 137.0
Lymph0
Based on a 200-cell count. To convert to percentage, divide by 2.
TOLUENE
8.7 6.7 5.2
5.7 2.7” 5.8
5.7 4.3 6.2
7.0 5.3 5.5
Mono
3, 8, AND
VAPOR
INHALATION
OF TOLUENE
481
CONCENTRATE
precipitate weight loss of 96 g in 7 days in the first case and 54-g loss by the second in the same time period. The only hematological finding that was determined to be statistically significant was not dosage-related, as it occurred only in the lowest level (0.95 mg/liter, 240 ppm) after approximately 8 weeks or 37 exposures. This was a reduction in the monocytes based upon evaluation of the differential white blood cell counts (Table 5). Although the serum glutamic oxalacetic transaminase (SGOT) values were high when compared to the controls at 13 weeks, they are all within our range of normal values (Carpenter et al., 1975a) and it is obvious that this was forced by the low control value. To determine whether the statistically significant values noted were primarily the result of unusual control values (Weil et al., 1969), the individual SGOT and SGPT values were correlated with the measured concentration the rats received. The correlations were run with and without the values of the control group. Coefficient of Correlation
Without control values With control values
SGOT
SGPT
-0.036 +O. 508
-0.519 -t-o.144
(r)
For SGOT, the “r” without the control value was practically zero; indicating no correlation between the amount of Toluene Concentrate received and SGOT. When the control values were added, the correlation became positive, indicating that the difference found when the means of each group were compared to the control mean was the result of an unusual control group. A slightly different situation was present with the SGPT values. In this case, without the control values, the correlation approached significance, but in a negative direction. Thus, the trend was that the higher the exposure concentration, the lower the SGPT values. The addition of control values, again, changed this to a positive, but not significant, correlation. Therefore, in summary, there is no deleterious, dosage-related, effect between the amount of Toluene Concentrate received and elevation of SGOT or SGPT values (Table 6). All other criteria of effect, including mean body weight and body weight change (Table 7) that were monitored showed no significant differences when compared to the controls. After 3 weeks of 6-hr per day inhalation of Toluene Concentrate vapor, there were no lesions in any of the treated or control groups among the three rats per group killed for examination. The incidence of slight kidney tubular regeneration was 0, 1, 1, 1 for the three concentrations and controls in descending order of concentration. At 8 weeks, after a similar sacrifice, the incidence of this condition was 3,1,3,0 while after 13 weeks this incidence was 6,6,4,6 in groups of nine rats killed. However, there were only two cases of moderate tubular regeneration at 13 weeks and both rats were from the middle level, 1.9 mg/liter (480 ppm). We have demonstrated that this rat stock normally has a better than 20 % incidence of slight or scattered tubular regeneration and for this reason the finding will be ignored (Carpenter et al., 1975b). The condition termed moderate regeneration is considered an effect only if dosage-related, and obviously it is not. Numerous sporadic lesions common to the rat were found in various tissues but none is considered by the pathologist as being related to the inhalation regimen. Furthermore, none differ in a statistically significant manner from the controls.
482
CARPENTER
TABLE MEAN
BLOOD INHALED
Concn mg/liter (ppm)
CHEMISTRY TOLUENE
Period VW
ET AL.
6
FINDINGS FOR GROUPS OF THREE, THREE, AND FOUR RATS CONCENTRATE VAPORS FOR 3,8, AND 13 WEEKS, RESPECTIVELY
BUN (mg/lOO ml)
SGOT (Sigma units/ml)
SGPT (Sigma units/ml)
Alkaline phosphatase (Sigma units/ml)
3 8 13
20.2 21.5 20.2
225.0 148.3 142.5”
41.7 33.7 27.5
6.35 6.51 4.66
3 8 13
20.7 22.3 19.4
305.0 228.3 I 50.0b
49.0 40.3 32.0”
7.55 5.25 4.18
0.95 (240)
3 8 13
20.5 20.8 18.9
240.0 251.7 142.5b
40.0 37.7 33.0b
8.17 6.02 6.08
(9;po)
3 8 13
21.5 21.7 19.9
230.0 303.3 96.2
36.3 44.0 23.0
9.00 5.76 4.18
$0)
THAT
a 0.05 >p > 0.01. D 0.01 >
p > 0.001.
Dogs. There were no signs of distress noted among the beagles at any time during the 6-hr per day inhalation for 13 weeks and no statistically significant differences were found between the treated groups and the controls in any of the parameters monitored (Tables 7-10). Challenge
At the conclusion of the 70-day rat repeated inhalation study, 10 rats from each level, the air controls, and an additional 20 “naive controls” randomized from the same stock of Harlan-Wistar rats as those on study but not subjected to daily handling, were exposed to a challenge concentration of 47 mg/liter (12,000 ppm) until 25% had died. This concentration is 1.3 times the 4-hr LC50 of 35 mg/liter (8800 ppm). It was anticipated that this procedure might demonstrate whether the animals had been rendered more susceptible or more resistant by virtue of their prior treatment. The rat responses presented in Table 11 indicate that there were more deaths among the “naive controls” than any of the other groups including the air-exposed controls. It is our opinion that these rats, which had not become acclimated to daily handling and exposure, were much more inquisitive and active than the others that had been handled and consequently they breathed more rapidly and probably acquired higher blood concentration even though deaths occurred in relatively the same time interval. This conclusion was reached because no significant difference in time to recovery from prostration (regaining ability to move about) was noted when treated animals were compared to the exposed controls but the difference became significant for all treated
209.6 587.9
Body weight (g) 0 64
10.8 12.2
3.9
Body weight changes (kg) 2 -0.1 22 0.4 36 0.6 51 1.2 64 1.4
-3 64
Body weight (kg)
Body weight changes (g) 3 17.8 22 193.7 36 268.0 51 337.3 64 373.3
Mean
Days of exposure
0.3 0.7 0.4 0.7 1.0
1.3 2.1
6.6 27.5 36.9 43.1 45.7
23.3 62.3
SD
-0.2 0.3 0.4 0.7 0.8
10.7 11.5
20.2 187.2 253.3 303.9 331.6
223.4 548.0
Mean
1.9
Concentration
0.3 0.6 0.5 0.6 0.7
1.2 1.9
4.0 32.9 41.8 46.1 45.3
22.4 48.8
SD
(mg/liter) 0.95
-0.5 -0.2 0.0 0.4 0.6
10.1 10.6
Dogs
18.0 184.5 257.1 319.7 351.5
0.2 0.6 0.5 0.5 0.6
2.4 2.8
5.5 32.3 43.5 50.0 58.4
26.8 70.9
SD
Concentrate
Rats 202.9 551.8
Mean
Toluene
-0.4 0.0 0.4 0.6 0.8
10.1 10.9
16.2 190.8 263.5 323.2 354.5
197.9 553.7
Mean
0.0
0.3 0.6 0.8 1.2 1.1
1.9 2.6
6.0 28.9 36.4 47.6 45.3
26.0 56.5
..__ SD
TABLE 7 MEAN BODY WEIGHTS AND BODY WEIGHT CHANGES FOR RATS AND DOGS
0.28 0.08 0.95 2.54 1.35
1.68 0.55
5.95 1.46 1.21 -0.02 1.43
1.08 2.96
Homogeneity of variance (23
1.64 1.00 0.72 0.67 0.67
0.20 0.34
2.11 0.38 0.59 1.64 2.34
2.39 1.81
Analysis of variance
$ w
2 ;;I
z 8 3 ki?
% i: 2
8.02 7.32
7.63 8.00
7.50 7.30
7.61 6.66
Preexposure Plus 61 days
(&
0.95 Preexposure (240) Plus 61 days
0.0 Preexposure (0.0) PIUS61 days
RBC (millions/ m3)
HEMATOLOGICAL
3.9 Preexposure (980) Plus 61 days
Concn mg/liter bpm)
MEAN
9.6 9.9
9.6 9.2
12.9 10.7
12.3 11.2
51.6 44.4
51.6 50.2
52.6 54.7
56.2 49.8
Ht (%I
FOR GROUPS
WBC (thousands/ mm”)
FINDINGS
18.8 16.8
18.7 18.8
19.6 19.5
20.0 18.4
Hb (g/100 ml)
1.45 1.10
0.80 0.95
1.25 1.20
1.50
0.90
0
0
Baso
THAT INHALED
Reticulocytes (%>
DOGS
TABLE 8 OF FOUR
CONCENTRATE
VAPORS
7.2 10.8
5.2 7.5
7.2 8.2
9.2 8.5
Eosino
9.5 8.8
7.5 8.0
9.5
10.2 9.0
Immature neutro
100.2 102.5
108.5 109.8
106.2 112.5
111.8 114.2
Neutro
76.0 68.2
71.0 67.5
60.0 68.8
62.0 59.5
Lymph0
Based on a 200-cell count. To convert to percentage, divide by 2.
TOLUENE
6.8 8.5
7.2 6.8
7.5 8.5
6.2 7.8
Mono
14.4 14.9 15.1 15.4 14.8 14.1
1.9 Preexposure (480) Plus61 days
0.95 Preexposure (240) Plus61 days
0.0 Preexposure (0.0) Plus61 days
132.5 131.2
128.8 128.8
130.0 123.8
128.8 126.2
0.24 0.30
0.28 0.27
0.28 0.31
0.23 0.32
Bilirubin (mg/lOOml)
TABLE 10
Blood glucose (mg/lOOml)
24.0 21.8
19.2 18.2
22.0 18.2
27.2 20.0
29.5 21.2
26.2 20.0
27.5 19.8
33.8 23.5
Liver weights(g) Liver weights,percentageof body weight Kidney weights(g) Kidney weights,percentageof body weight
319.8 2.58 45.0 0.36
Mean
3.9
112.5 0.55 11.3 0.05
SD
344.8 3.11 43.8 0.38
Mean
1.9
37.4 0.89 5.7 0.04
SD
272.8 2.61 39.2 0.38
Mean
0.95
47.9 0.29 7.3 0.07
SD
Concentration (mg/liter) Toiuene Concentrate
264.8 2.38 40.5 0.38
Mean
0.0
91.0 0.29 5.9 0.04
SD
1.74 1.47
1.33 1.07
2.27 1.64
1.51 1.58
Alkaline SGOT SGPT phosphatase (Sigmaunits/ml) (Sigmaunits/ml) (Sigmaunits/ml)
MEAN ORGAN WEIGHTS OF GROINS OF FOUR BEAGLES AFTER THEY INHALED TOLUENE CONCENTRATE VAPOR FOR 66 DAYS
15.5 14.4
BUN (mg/lOOml)
3.9 Preexposure (980) Plus 61 days
Concn mg/liter (mm)
TABLE 9
MEAN BLEND CHEMISTRY FINDINGS OF GROUTS OF FOUR DOGS THAT INHALED TOLUENE CONCENTRATE VAPORS
2110 3/10 l/10 2110 1o/20
During exposureb 2/10 3110 l/10 2110 11120
Total within 7 days
Number of rats that died Number exposed
Time to death
70 83 59 76 78
Median time to death (min)
HAD SURVIVED GRADED CONCENTRATIONS 90-MIN CHALLENGE EXPOSURE TO 47
None None None None
vs Control
0.05 > p > 0.01 None p = 0.05 -
vs Naives
Statistical significance”
Time to prostration
18.5 18.5 20.0 22.0 35.0
Median time (min)
of naive rats is not statistically significantly different.
14.0 13.5 13.0 13.8 12.0
Median time (min)
-.
mg/liter
None None None None
0.05 >p > 0.01 0.05 > p > 0.01 0.05 > p > 0.01 None
Statistical significance’ ~ ~~ ~~ vs vs Control Naives
Time to recovery”
-
FOR 6 HR PER DAY, 5 DAYS PER WEEK FOR 70 DAYS TO A (12,000 ppm) OF TOLUENE CONCENTRATE
11
OF THIS SOLVENT
TABLE
a Regaining ability to move about. b Exposure was terminated after 25% of the animals had died. Mortality e Rank sum test of significance used.
3.9 mg/liter 1.9 mg/liter 0.95 n&liter 0.0 mg/liter
70 Days 70 Days 70 Days 70 Days Naives
x x x x
OF RATS THAT
RESPONSE
2 F
h
2 E
F z
VAPOR
INHALATION
OF TOLUENE
CONCENTRATE
487
groups versus the naive controls. The latter group recovered in 35 min as opposed to 22 for the air-exposed controls and 18.5 to 20 min for the treated groups. Human Sensory Response. Odor threshold. Vapor-air
concentrations for the odor threshold study were varied by a factor of 10. These concentrations were not analyzed by gas chromatography. Therefore, metered concentrations were reduced 36% to correspond with the mean percentage recovery in the repeated inhalation study. A group of six volunteers, between 23 and 50 years of age, inhaled a series of concentrations (each for approximately 10 set) in the following sequence: 0.64,0.00,0.0064,0.064,0.064,0.0064,0.64, and 0.00 mg/ liter. The responses are summarized in Table 12. A corrected concentration of 0.064 mg/liter (16 ppm) was readily perceived, as indicated by the 100% incidence of detection. Volunteers were able to detect 0.0064 mg/liter (1.6 ppm) at an incidence of 33 %. By the method of moving averages (Carpenter et al. 1975a) the most probable corrected concentration for the threshold lies between these limits or at 0.01 mg/liter (2.5 ppm). Sensory threshold. All concentrations were determined by gas chromatography. In order to determine the sensory threshold, six volunteers between 20 and 63 years of age inhaled measured vapor-air concentrations for 15-min periods in the following order: 1.9 mg/liter and 3.7 mg/liter, corresponding to 480 and 930 ppm, respectively. Exposures were limited to 1 per day to prevent any buildup of symptoms (Table 12). TABLE 12 ODOR DETECTION AND SENSORY THRESHOLD FOR TOLUENE CONCENTRATE(HUMAN
SUBJECTS)
Odor threshold Metered concentration (mg/liter) 1.0 0.1 0.01 0.0 Corrected concentration (mg/liter) 0.64 0.064 0.0064 0.0 Correctedconcentration (ppm) 160 16 1.6 0.0 Number detectingodor/number of subjects (two trials) 12/12 12/12 4112 o/12 Conclusion: The odor thresholdlies between0.0064and 0.064 mg/liter, the most probable concentration being 0.01mg/liter or 2.5 ppm. Sensorythresholds Measuredconcentration(mg/liter) 1.9 3.7 Measuredconcentration(ppm) 480 930 First Exposureorder for six subjects Number detectingodor 6 Number olfactory fatigue 0 Number throat irritation 0 Number eyeirritation 1 Number noseirritation 0 Number reporting “dizziness” or “lightheadedness” 0 Number tasting “something” 1 Number with effects1 hr after exposure 0 Conclusion: Number of subjectsconsideringconcentrationstolerable for 8-hr working day wassix of six at 1.9r&liter andthree of six at 3.7mg/liter.
488
CARPENTER
ET AL.
One volunteer reported a transitory mild drying sensation of the eyes after inhaling 1.9 mg/liter for 15 min. In the 3.7-mg/liter concentration one individual experienced transient nasal irritation and another transient throat irritation. The nasal irritation was described as a mild sensation of dryness experienced only during the final minute of inhalation. The throat irritation, reported as a mild burning sensation, persisted only from the third through the sixth min of the period. The same volunteer reported a cooling sensation in the throat for about 1 min immediately following the inhalation TABLE 13 RECAPITULATION
Acute 4-hr, no ill effect concn, rats Rat LC50 Acute 6-hr, no ill effect concn, dogs Acute 6-hr inhalation, cats Osmotic erythrocyte fragility, rats Short-term massive concn, rats Mouse respiratory tract irritation
13-Week subacute inhalation, and dogs
rats
Statistically significant finding Monocyte count, 8 weeks, rats SGOT, 13 weeks, rats SGPT, 13 weeks, rats
Challenge exposure to rats, 6 hr
Odor threshold, human subjects
Sensory threshold, human subjects
Suggested hygienic standard
6.8 mg/liter (1700 ppm) 35 (31 to 38) mg/liter or 8800 (7800 to 9600) pm 3.0 mg/liter (760 ppm) 31 mg/liter (7800 ppm); signs observed were suggestive of CNS depression Metered 80 mg/liter (20,000) ppm), 45 tin inhalation caused no statistically significant changes in hemolysis 180 mg/liter (45,000 ppm); lethal to five of five, in 20 min; Lt 50 = 11 (7.2 to 16) min Lowest concentration at which 50 % depression of respiratory rate occurred: 34 mg/liter (8600 ppm); no effect concentration: 20 mg/liter (5000 ppm) 3.9 mg/liter or 980 ppm; no ill effects
Concn (mg/liter) 0.95 3.9;1.9;0.95 1.9;0.95
Assessment Not dosage related, ignored Low control value; ignored Low control value and not dosage related; ignored
47 mg/liter (12,000 ppm); statistically significantly increased mortalities among “naive” controls. Assessment: due to increased activity and respiration of unacclimated animals 0.01 mg/liter or 2.5 ppm after application of the correction factor of 64% obtained from the measured to metered relationship in the subacute study 1.9 mg/liter or 480 ppm was considered as a tolerable concentration for an 8-hr working day. This concentration would contain 0.87 mgtliter or 220 ppm of toluene per se. 1.9 mg/liter (480 ppm) as a ceiling or limiting value based on response of human volunteers
VAPOR
INHALATION
OF TOLUENE
CONCENTRATE
489
period. Two volunteers reported feeling “slightly light-headed” after 15 min in 3.7 mg/liter and another reported a “stuffy, drowsy feeling.” All volunteers detected the odor of the Toluene Concentrate at both concentrations and none reported complete olfactory fatigue. Following each inhalation period, the volunteers were asked if they thought they could work for 8 hr in that particular concentration. Six gave an affirmative response after inhaling 1.9 mg/liter, while only three thought they could work in the 3.7-mg/liter concentration. Based on the foregoing data, 1.9 mg/liter (480 ppm) of Toluene Concentrate vapor would seem to be an acceptable level for an 8-hr daily inhalation by normal healthy individuals of working age. This concentration corresponds to 0.87 mg/liter (220 ppm) of toluene based on the 46 % aromatic content of the Toluene Concentrate. Table 13 summarizes the findings. DISCUSSION
There is no evidence that either rats or beagles were harmed as a result of inhaling 3.9,1.9, or 0.95 mg/liter of Toluene Concentrate for 13 weeks, 6 hr/day. A concentration of 1.9 mg/liter (480 ppm) was considered as acceptable for 8-hr continuous inhalation by a panel of six human subjects but 3.7 mg/liter (930 ppm) was not. Based upon these findings, the hygienic standard should be set at 1.9 mg/liter (480 ppm) which we realize is slightly over twice the current (1975) TLV recommended by the American Conference of Governmental Industrial Hygienists and the National Institute for Occupational Safety and Health for toluene alone. The fact that Toluene Concentrate contains 46 % aromatics (only 0.06 % benzene) accompanied by some 15 % naphthenics and 39 % paraffinics raises the legitimate question as to whether the net effect ofall three is additive, less than additive, or greater than additive as regards both toxicity and irritation (Pozzani et al. 1959). The evidence at hand denies the latter and we suggest that whether the final effects are simply additive or less than additive (antagonistic) will rest on future monitoring of airborne concentrations coincident with medical surveillance of those exposed. Therefore, our interpretations of the findings presented may or may not be justified by further observations. In any case, it is evident that any claim of human discomfort will result in the prompt reduction of the TLV. ACKNOWLEDGMENTS
Practically the entire staff of the Chemical Hygiene Fellowship has been involved in this project and the authors wish to extend sincere appreciation and thanks for their faithful attention and cooperation. Analytical Procedures: J. M. Eldridge and S. J. Kozbelt; Hematology and Histopathology: P. A. Baker, P. A. Crawford, and M. A. McGee; Experimental Assistance: C. S. Weil, and M. D. Woodside. REFERENCES ALARIE, Y. (1966). Irritating properties of airborne materials to the upper respiratory tract. Arch. Environ. Health 13,433+9. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975a). Petroleum hydrocarbon toxicity studies. I. Methodology. Toxicol. Appl. Pharmacol. 32,246-262.
490 CARPENTER,
CARPENTER C. P., KINKEAD,
E. R.,
GEARY,
ET AL. D.
L.,
JR., SULLIVAN,
L. J., AND
KING,
J. M.
(1975b). Petroleum hydrocarbon toxicity studies. II. Animal and human response to vapors of Varnish Makers’ and Painters’ Naphtha. Toxicol. Appl. Pharmacol. 32, 263-281. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975c). Petroleum hydrocarbon toxicity studies.III. Animal and human responseto vapors of Stoddard Solvent. Toxicol. Appl. Pharmacol. 32,282-297. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975d). Petroleum hydrocarbon toxicity studies.IV. Animal and human responseto vapors of Rubber Solvent. Toxicol. Appl. Pharmacol. 33,526542. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975e).Petroleumhydrocarbon toxicity studies.V. Animal and humanresponseto vapors. of Mixed Xylenes. Toxicol. Appl. Pharmacol. 33,543-558. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975f).Petroleumhydrocarbon toxicity studies.VI. Animal and humanresponseto vapors of “60 Solvent”. Toxicol. Appl. Pharmacol. 34, 374-394. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975g).Petroleum hydrocarbon toxicity studies.VII. Animal and human responseto vapors of “70 Solvent”. Toxicol. Appl. Pharmacol. 34, 395-412. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975h).Petroleumhydrocarbon toxicity studies, VIII. Animal and human responseto vapors of “140” Flash Aliphatic Solvent”. Toxicol. Appl. Pharmacol. 34,413429. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., AND KING, J. M. (1976i). Petroleum hydrocarbon toxicity studies.IX. Animal and human responseto vapors of “80 Thinner.” Toxicol. Appl. Pharmacol. 36, 409425. CARPENTER, C. P., GEARY, D. L., JR., MYERS, R. C., NACHREINER, KING, J. M. (1976j).Petroleumhydrocarbontoxicity studies.X.
D. J., SULLIVAN, L. J., AND Animal and humanresponse
to vapors of “50 Thinner”. Toxicol. Appl. Pharmacol. 36,427-442. C. P., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., AND M. (1976k). Petroleum hydrocarbon toxicity studies.XI. Animal and human responseto vapors of Deodorized Kerosene.Toxicol. Appl. Pharmacol. 36,443-456. CARPENTER, C. P., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., AND KING, J. M. (19761).Petroleum hydrocarbon toxicity studies.XII. Animal and human responseto vapors of “40 Thinner”. Toxicol. Appl. Pharmacol. 36,457472. POZZANI, U. C., WEIL, C. S., AND CARPENTER, C. P. (1959).The toxicological basisof threshold limit values.5. The experimentalinhalation of vapor mixtures by rats, with notesupon the relationship betweensingledoseinhalation and single doseoral data. Amer. Znd. Hyg. CARPENTER, KING, J.
Assoc. J. 20, 364369. C. S., AND CARPENTER, C. P. (1969).Abnormal valuesin control groupsduring repeated dosetoxicologic studies.Toxicol. Appl. Pharmacol. 14, 335-339.
WEIL,
AND
APPLIED
PHARMACOLOGY
Petroleum XIII.
Animal
and
Human
36.473-490
(1976)
Hydrocarbon Response
Toxicity to Vapors
Studies
of Toluene
Concentratelvz
C. P. CARPENTER, D. L. GEARY, JR., R. C. MYERS,D. J. NACHREINER, L. J. SULLIVAN,AND J. M. KINGS The Chemical Hygiene Carnegie-Mellon Received
Fellowship, University, June 2,1975;
Carnegie-Mellon Institute Pittsburgh, Pennsylvania accepted
December
of Research, 15213
22,1975
Petroleum Hydrocarbon Toxicity Studies. XIII, Animal and Human Responseto Vapors of Toluene Concentrate. CARPENTER, C. P., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., AND KING,
J. M. (1976).Toxicol. Appl. Pharmacol. 36,473-490.The suggested hygienic standardfor humaninhalation of TolueneConcentrate,basedupon results of theseinhalation studieswith rats and dogsand the sensoryresponseof human subjects,is 1.9 mg/liter (480 ppm). The LC50 for rats that inhaled Toluene Concentratefor 4 hr was 35 (31 to 38) mg/liter or 8800(7800to 9600)ppm. Rats tolerated 6.8 mg/liter (1700 ppm) for 4 hr and dogs3.0 mg/liter (760ppm) for 6 hr without any signsof discomfort. Four catsthat inhaled31mg/liter (7800ppm)for 6hr developedsignssuggestiveof central nervous systemeffects, including mydriasis,mild tremors, prostration in 80 min, and light anesthesiaafter 2 hr. One of four died as a result of pneumonicinfection within 14 days. Rats and dogsthat inhaled 3.9, 1.9, or 0.95 mg/liter of Toluene Concentratefor 13 weeks,6 hr/day, were not statistically significantly different from their air-exposedcontrols in any of the criteria of injury that weremonitored including hematology,clinical chemistry, and micropathology. The odor thresholdasdeterminedby a sniff test involving two trials with six humansubjectsis approximately 0.01 mg/liter (2.5 ppm). Six of six people indicated their willingnessto work in a concentration of 1.9 mg/liter (480 ppm) which is equivalent to 0.87 mg/liter or 220 ppm of contained toluene,the limiting concentrationrecommendedfor the hygienicstandard. Only the response of peoplein the workplacecandeterminewhetherthe toxic and irritative effectsof the componentsof this mixture are additive or less than additive, as the evidencepresentedsuggeststo us that they are not greaterthan additive. Investigation of animal and human response to the vapor inhalation of Toluene Concentrate paralleled the studies on Varnish Makers’ and Painters’ Naphtha, Stoddard Solvent, Rubber Solvent, Mixed Xylenes, “60 Solvent”, “70 Solvent”, “140” Flash Aliphatic Solvent”, “80 Thinner “, “50 Thinner”, Deodorized Kerosene, and “40 1 Supported by the American Petroleum Institute. 2 Urban0 C. Pozzani, before his death in July of 1970, was involved in the planning and execution of this series of inhalation studies of commercially available hydrocarbons. He was to have been the senior author of the formal publications as recognition for his devotion to this project. 3 Present address: New York State Veterinary College, Cornell University, Ithaca, New York. Copyright 0 1976 by Academic Press, Inc. 473 All rights of reproduction in any form reserved. Printed
in Great
Britain
474
CARPENTER
ET AL.
Thinner” (Carpenter et al., 1975-76b-1). The objectives of these studies have been presented by Carpenter et al. (1975a) in a report which contains the detailed protocol, compounds under investigation, and details of procedure common to the whole series. A literature review did not reveal mammalian toxicity studies on materials of similar composition. METHODS The details of the experimental design and the various procedures utilized are given in Carpenter et al. (1975a). Sample. The hydrocarbon mixture designated by the coined name, “Toluene Concentrate” was furnished by one of the major producers in the United States and was assigned the Chemical Hygiene Fellowship No. 35-l 54. Pertinent physicochemical properties are presented in Table 1 and composition in Table 2. TABLE 1 PHYSICOCHEMICAL
PROPERTIES
OF TOLUENE
CONCENTRATE
Boiling range” Percent
Temperature (“F)
Percent
Temperature 03
I.B. pt 5 10 20 30 40 50
203 208 209 210 211 212 213
60 70 80 90 95 DEP ASTM EP
214 216 218 221 224 226 231
Flash point Explosive limit Refractive index Specific gravity Mean b401Wtb At 25°C and 760 mm
25°F (tag closed cup) 1.Oto 7.0 %, by volume in air 1.4428 at 60°F 0.7780 at 60°F 97 1 mg/liter = 252 ppm 1 ppm = 0.003967 mg/liter
’ ASTM Method D-86. b Calculated from mass spectrography
data.
Analytical method for monitoring Toluene Concentrate. The analytical procedure developed was dependent upon measurement of peak height of three principal peaks with reasonable resolution and good symmetry characteristics. The conditions necessary for reliable analysis are given in Table 3. With but few exceptions, the vapor concentrations in the acute single inhalation and the human sensory studies were analyzed at least once per exposure and in the subacute (repeated) study, twice each day. The actual vapor concentrations are reported as measured concentrations as opposed to “metered” concentrations which are based upon empirical flow rates of dilution air and solvent. When reference is made to the number
VAPOR
INHALATION
OF TOLUENE
TABLE COMPOSITION
475
CONCENTRATE
2
OF TOLUENE
CONCENTRATE
Mass spectral analysis (volume percentage)
Component
&
Paraffins Butanes 2,2,3-Trimethylbutane
0.23
-
Pentanes 2,ZDimethylpentane 2,3-Dimethylpentane 2,CDimethylpentane 3,3-Dimethylpentane 3-Ethylpentane 2,2,4-Trimethylpentane 2,3,4-Trimethylpentane 2-Methyb3-ethylpentane
0.46 2.59 0.74 0.62 0.53 0.04 0.06 0.02
0.3 1.7 0.5 0.4 0.3 -
Hexanes n-Hexane 2-Methylhexane 3-Methylhexane 2,ZDimethylhexane 2,3-Dimethylhexane 2,CDimethylhexane 2,5-Dimethylhexane
0.02 6.63 6.65 0.87 0.48 I .68 1.41
5.8 5.7 0.7 0.5 1.4 1.2
13.16 1.20 0.88 0.37
11.8 1.3 0.7 0.5
Octanes n-Octane
0.05
-
Naphthenes Cyclopentanes Methylcyclopentane 1,1-Dimethylcyclopentane 1-trans-2-Dimethylcyclopentane 1-cis-2-Dimethylcyclopentane l-tram-3-Dimethylcyclopentane I-cis-3-Dimethylcyclopentane Ethylcyclopentane 1,1,2-Trimethylcyclopentane 1,1,3-Trimethylcyclopentane 1-tram-2-cis-4-trans-Trimethylcyclopentane I-trans-2-cis-2-trans-Trimethylcyclopentane 1,2,3-Trimethylcyclopentane
0.07 0.47 1.19 0.20 0.85 0.68 0.35 0.08 0.36 0.33 0.33 -
0.07 0.42
Heptanes n-Heptane 2-Methylheptane 3-Methylheptane 4-Methylheptane
1.29 1.32 0.33 0.35 0.34 0.33
476
CARPENTER ETAL.
TABLE 2-con&ued _--Massspectral analysis(volume percentage)
Component
Naphthenes-continued Cyclohexanes Cyclohexane Methylcyclohexane 1-trans-4-Dimethylcyclohexane 1,1-Dimethylcyclohexane
1.74 8.48 0.23
glc
1.59 8.30 0.23 0.04
Aromatics Benzene Toluene
0.06 45.89
0.07 50.46
Totals Paraffins Naphthenes Aromatics
38.69 15.36 45.95
TABLE 3 GASCHROMATOGRAPHICPROGRAM:TOLUENECONCENTRATE'
Column Coating Support Conditions Solvent for calibration Carrier gas Burner Vapor samplesize Lower limit of detection
Coppertubing + in. o.d. x 25 ft. 15% Apiezon M Gas Chrom Q 80/100mesh 90 to 140°Cat 2.5”C/min Hexadecane Helium Beckman Hydrogen : 45 ml/min Air: 450ml/min l-10 ml 0.05 ,ug
’ A temperature-programmed gaschromatographequippedwith a flameionizationdetectorwasusedfor all analyses.
of days elapsed in the 13-week study, it refers to the actual number of days the animal inhaled the vapor and not calendar days. Any deviation from this practice will be noted. PrPcis of experimental design. A stepwise approach was undertaken to determine a no ill effect level by inhalation in rats and dogs. First, rats were subjected to a series of single 4-hr inhalation experiments to arrive at an LC50 and to detect cellular injury. Cats then were subjected to this LC50 concentration to detect any overt evidence of central nervous system effects that might occur. Based on these data, sublethal concentrations were set up to determine the earliest persistent signs of toxic stress as an aid in setting levels for the 6-hr-per-day, Sday-per-week, 66-day inhalation study. At the termination of the 66-day repeated study, some of the surviving rats were subjected
VAPOR
INHALATION
OF TOLUENE
CONCENTRATE
477
to a massive concentration of 1.3 times the 4-hr LC50 for 1.5 hr to demonstrate whether they had become more resistant, more susceptible, or neither as a result of their repeated inhalation of Toluene Concentrate. Respiratory irritation was studied in mice by a technique described by Alarie (1966). To complete the investigation, odor thresholds and sensory responses were determined through inhalation of Toluene Concentrate by human volunteers. RESULTS Acute Inhalation Toxicity Prior to determining the 4-hr acute response to the vapor it was determined empirically that fog formation (saturation) did not occur until a concentration in the neighborhood of 500 mg/liter (130,000 ppm) was generated. At a concentration of about 173 mg/liter (44,000 ppm), calculated on the dilution air-flow and weight of compound utilized, four of six rats succumbed in 30 min. Progressive signs of distress included: eye irritation in 2 min, loss of coordination in 5 to 6min, anesthesia in 8.5 min, one dead in 14 min, tonic convulsions in another rat in 16 min and a total of 3 deaths in 17 min, with a fourth dying in 30 min. Two of the four victims had slightly congested lungs but no other notable gross pathological changes. Acute 4-hr LC.50 rats. Groups of 10 young male albino Harlan-Wistar rats (130200 g) were subjected to measured concentrations of Toluene Concentrate for single 4-hr periods. At a measured 49 mgjliter (12,000 ppm) all succumbed following tremors (5 min) and prostration (15 min). The first death occurred within 1 hr and the remainder during the next 3.5 hr. Three of an additional group of six animals in each concentration were to be sacrificed immediately after the exposure and the other three, 2 days later. However, only one of these six survived the exposure and was sacrificed 2 days thereafter. At roughly one-half the above concentration, 25 mg/liter (6300 ppm), head tremors were seen in 1 hr and prostration occurred in 2 hr, but at 3-hr postexposure all appeared to be normal. Fourteen-day weight gains were normal for rats of this age. At 13 mg/liter (3300 ppm) the only sign was slight loss of coordination and at 6.8 mgjliter (1700 ppm) the rats were normal throughout the 4-hr exposure. The calculated LC50 is 35 (31 to 38) mg/liter or 8800 (7800 to 9600) ppm. The only micropathology noted other than the commonly found sporadic lesions was lung atelectasis in 4 of 8 rats examined that died in the 4-hr exposure to 49 mg/liter (12,000 ppm) of Toluene Concentrate. The sole survivor killed 2 days after the exposure had chronic tracheitis, slight bile duct proliferation, and slight tubular regeneration. Bone marrow smears on these rats were not remarkable. At the lower levels there were no micropathological changes related to the inhalation of this vapor. Dogs. Two female beagles that inhaled 6.0 mg/liter (1500 ppm) 6 hr per day successively for 3 days responded with slight lacrimation and head tremors. Thereupon the concentration was reduced to 4.1 mg/liter (1000 ppm) but the intermittent head tremors still persisted. Finally, another pair of female beagles were exposed 6 hr/day for 2 days at 3.0 mg/liter (760 ppm), rested for 4 days and then exposed for 3 successive days, One had a weight loss of 1.l kg after the fifth exposure and the other recovered unchanged. Both appeared normal throughout the ensuing 1Zday observation period. Cats. Four male cats of mixed breed inhaled a mean measured concentration of 31 mg/liter (7800 ppm) for 6 hr. These cats were carefully observed for signs of central.
478
CARPENTER
ET AL.
nervous system effect. Prior to inhalation, they had been examined and found normal in placing reactions, righting reflexes, aversion to foot pad pain, extensor thrust reflex, and pupillary contraction. Responses of cats included slight loss of coordination, mydriasis, and slight hypersensitivity to light within 20 min. These effects were progressive and were accompanied by ear twitching, sneezing, body jerks, and prostration all of which occurred within 80 min. Increasing tremors were noted in two of the cats. Within 2 hr all appeared to be under light anesthesia. This condition persisted throughout the remainder of the 6-hr exposure. All survived the exposure and only one succumbed during the ICday observation period. Gross autopsy revealed pneumonia to be the cause of death. One surviving cat had lost 0.59 kg the day following exposure, but body weights of the three survivors appeared normal prior to sacrifice at the conclusion of the 1Cday holding period. Sporadic lesions, common to cats, were reported but aside from the purulent bronchopneumonia and the attendant round cell foci in two of four animals, undoubtedly present before exposure, the other lesions were single occurrences or of no importance. None of these pathological changes were related to treatment. Short-term massive concentration. Groups of five male rats were subjected to a mean measured concentration of 180 mg/liter (45,000 ppm) for time-periods varied by a factor of 2 to determine the Lt50. This concentration is five times greater than the 4-hr LC50 for rats of 35 mg/liter (8800 ppm) and approximates substantial saturation of air with Toluene Concentrate vapor at room temperature. Signs of toxicity included lacrimation and loss of coordination within 2 min, and prostration and anesthesia within 5 min. The Lt50 was 11 (7.2 to 16) min. Microscopic examination of the entire lung, including the bronchi, trachea, bifurcation of the trachea, thyroid, parathyroids, and esophagus revealed no lesions related to treatment. Osmotic erythrocytefragility. No increase in erythrocyte fragility was observed among six rats that inhaled a metered concentration of 80 mg/liter (20,000 ppm) of Toluene Concentrate vapor for 45 min. The mean initial hemolysis was in 0.51% NaCl solution and final in 0.31%. These values were not statistically significantly different from those of five air-exposed controls, namely, 0.47 % initial and 0.31% final. Mouse respiratory tract irritation. The mouse respiratory irritation study was patterned after the method described by Alarie (1966). Two Charles River mice, 45 to 50 g, were tested simultaneously. A 50% or greater depression in respiratory rate was registered by four of six and three of six mice, which had inhaled measured concentrations of 60 mg/liter (15,000 ppm) and 34 mg/liter (8600 ppm), respectively. A similar decrease in rate did not occur when another group of six inhaled 20 mg/liter (5000 ppm). Subacute Inhalation Toxicity To determine the response of rats and dogs to repeated daily inhalation, in order to provide a basis to establish acceptable inhalation standards, groups of 25 male Harlan-Wistar rats and 4 male beagles were assigned randomly to each of three graded’ levels of Toluene Concentrate and to a solvent-free air-control. Three rats from each level were sacrificed for histopathological examination after 14- and 38-day intervals. After repeated inhalation for 6 hr/day, 5 days/week for 71 days, 10 randomly preselected
479
VAPORINHALATIONOFTOLUENECONCENTRATE
rats from each level were subjected to a challenge exposure to determine whether they had become more or less sensitive to the vapor. The remaining rats and all beagles were killed after 65 days and 66 days, respectively. The criteria of toxic response monitored for the subacute study included body weight change and analyses of blood and urine. Evaluation on blood included packed cell volume (hematocrit), total erythrocyte count, reticulocyte count, total and differential leukocyte counts, serum alkaline phosphatase activity (SAP), serum glutamic pyruvic transaminase activity (SGPT), serum glutamic oxalacetic transaminase activity @GOT), and blood urea nitrogen (BUN) on both species, with the addition of bilirubin and blood glucose on dogs. Preliminary or baseline values were established on blood drawn from the jugular vein of all dogs allocated to this subacute study prior to the first day of vapor inhalation. The dog blood values were determined again, prior to the sacrifice, after 61 days. Preliminary values were not established for the rats because not enough blood could be taken to accomplish the tests without debilitating the animals. The values for the treated rats were compared to the values of the air-controls concomitantly sacrificed at the 14-, 38-, and 65-day intervals. Rat blood was taken from the tip of the tail prior to sacrifice for the hematological determinations and from the severed cervical vessels after section of the cord, for clinical biochemistry. Procedures for blood determinations are outlined in Carpenter et al. (1975a). Food consumption was followed 1 day of each week, electrocardiograms were taken and urine analyses were performed on the dogs prior to and at termination of the study. At sacrifice, tissues taken from rats for microscopic examination included adrenal, brain, pituitary, trachea, thyroid, parathyroid, lung, heart, liver, kidney, spleen, stomach, duodenum, pancreas, ileum, jejunum, colon, skeletal muscle, sciatic nerve, and bone marrow impression smear. Similar tissues were collected from the dogs at sacrifice, with the addition of the pharynx, tonsil, bifurcation of the trachea, nasal mucosa, and bone marrow section. Gas chromatographic analyses of the nominal 6, 3, and 1.5 mg/liter vapor: air mixtures yielded mean measured concentrations of 3.9, 1.9, and 0.95 mg/liter corresponding to 980,480, and 240 ppm, as shown in Table 4. Rats. Two deaths occurred among rats during the repeated subacute inhalation. One moribund rat was killed after 27 and another died after 42 exposures to the 0.95-mg/ liter (240 ppm) concentration. Both rats had endemic pneumonia judging from the TABLE 4 GAS CHROMAT~GRAPHIC
ANALYSES OF TOLUENE CONCENTRATE VAPOR CONCENTRATIONS INHALEDBYRATSANDDOGSFOR 13 WEEKS
Number of analyses 85 Metered concentration (mg/liter) 6.0 Measured concentration (mg/liter) 3.9 95% Fiducial limits for measured concentrations (r&liter) 1J-5.94 Measured concentration (ppm) 980 Measured as percentages of metered concentration 65.0 Coefficient of variation 26.37
92 3.0 1.9
81 1.5 0.95
0.98-2.87 0.25-l .65 480 64.0 24.75
240 63.3 36.84
35 0.0 0.0
0.0 -
Hmtmmxx4t
6.99 7.45 7.94
6.83 7.83 7.36
3 8 13
3 8 13
0.95
a 0.01 > p > 0.001.
(24’3
13
7.53 7.47 7.70
83
18.2 15.5 12.7
16.4 12.7 15.4
15.3 12.7 11.4
15.4 16.1 12.2
6.76 7.82 7.70
(i;890)
WBC (thousands/ mm3)
FOR GROUPS
RBC (millions/ mm3)
83 13
Period Wk)
FttmtNGs
$0)
Concn mg/liter (m-W
MEAN
41 .o 43.7 42.1
42.1 41.5 43.2
45.0 41.6 43.4
42.2 42.1 42.4
Ht (%)
OF THREE,
5
15.2 16.3 15.4
15.4 15.3 15.5
16.6 15.4 15.7
15.2 15.5 15.2
THAT
2.93 2.00 3.10
0.0 0.0 0.2
0.0 0.0 0.0
0.0 0.0 0.0
2.67 1.80 3.20 2.80 1.87 2.75
0.0 0.0 0.0
Baso
-
INHALED
2.53 1.73 3.10
Reticulocytes (%I
THREE, AND FOUR Rnrs 13 WEEKS,FCESPECTIVELY
TABLE
2.0 2.0 4.0
4.3 1.7 4.2
4.3 5.0 4.5
5.3 3.0 3.5
Eosino
-
CONCENTRATE
VAPOR
FOR
5.0 5.7 5.8
5.3 5.0 5.5
3.7 4.3 7.0
6.7 5.0 3.5
Immature neutro
56.3 53.7 49.5
50.0 54.0 51.5
54.3 55.0 50.2
51.0 51.7 49.5
Neutro
126.7 131.7 134.5
134.7 136.0 132.5
131.3 130.3 131.5
128.3 134.3 137.0
Lymph0
Based on a 200-cell count. To convert to percentage, divide by 2.
TOLUENE
8.7 6.7 5.2
5.7 2.7” 5.8
5.7 4.3 6.2
7.0 5.3 5.5
Mono
3, 8, AND
VAPOR
INHALATION
OF TOLUENE
481
CONCENTRATE
precipitate weight loss of 96 g in 7 days in the first case and 54-g loss by the second in the same time period. The only hematological finding that was determined to be statistically significant was not dosage-related, as it occurred only in the lowest level (0.95 mg/liter, 240 ppm) after approximately 8 weeks or 37 exposures. This was a reduction in the monocytes based upon evaluation of the differential white blood cell counts (Table 5). Although the serum glutamic oxalacetic transaminase (SGOT) values were high when compared to the controls at 13 weeks, they are all within our range of normal values (Carpenter et al., 1975a) and it is obvious that this was forced by the low control value. To determine whether the statistically significant values noted were primarily the result of unusual control values (Weil et al., 1969), the individual SGOT and SGPT values were correlated with the measured concentration the rats received. The correlations were run with and without the values of the control group. Coefficient of Correlation
Without control values With control values
SGOT
SGPT
-0.036 +O. 508
-0.519 -t-o.144
(r)
For SGOT, the “r” without the control value was practically zero; indicating no correlation between the amount of Toluene Concentrate received and SGOT. When the control values were added, the correlation became positive, indicating that the difference found when the means of each group were compared to the control mean was the result of an unusual control group. A slightly different situation was present with the SGPT values. In this case, without the control values, the correlation approached significance, but in a negative direction. Thus, the trend was that the higher the exposure concentration, the lower the SGPT values. The addition of control values, again, changed this to a positive, but not significant, correlation. Therefore, in summary, there is no deleterious, dosage-related, effect between the amount of Toluene Concentrate received and elevation of SGOT or SGPT values (Table 6). All other criteria of effect, including mean body weight and body weight change (Table 7) that were monitored showed no significant differences when compared to the controls. After 3 weeks of 6-hr per day inhalation of Toluene Concentrate vapor, there were no lesions in any of the treated or control groups among the three rats per group killed for examination. The incidence of slight kidney tubular regeneration was 0, 1, 1, 1 for the three concentrations and controls in descending order of concentration. At 8 weeks, after a similar sacrifice, the incidence of this condition was 3,1,3,0 while after 13 weeks this incidence was 6,6,4,6 in groups of nine rats killed. However, there were only two cases of moderate tubular regeneration at 13 weeks and both rats were from the middle level, 1.9 mg/liter (480 ppm). We have demonstrated that this rat stock normally has a better than 20 % incidence of slight or scattered tubular regeneration and for this reason the finding will be ignored (Carpenter et al., 1975b). The condition termed moderate regeneration is considered an effect only if dosage-related, and obviously it is not. Numerous sporadic lesions common to the rat were found in various tissues but none is considered by the pathologist as being related to the inhalation regimen. Furthermore, none differ in a statistically significant manner from the controls.
482
CARPENTER
TABLE MEAN
BLOOD INHALED
Concn mg/liter (ppm)
CHEMISTRY TOLUENE
Period VW
ET AL.
6
FINDINGS FOR GROUPS OF THREE, THREE, AND FOUR RATS CONCENTRATE VAPORS FOR 3,8, AND 13 WEEKS, RESPECTIVELY
BUN (mg/lOO ml)
SGOT (Sigma units/ml)
SGPT (Sigma units/ml)
Alkaline phosphatase (Sigma units/ml)
3 8 13
20.2 21.5 20.2
225.0 148.3 142.5”
41.7 33.7 27.5
6.35 6.51 4.66
3 8 13
20.7 22.3 19.4
305.0 228.3 I 50.0b
49.0 40.3 32.0”
7.55 5.25 4.18
0.95 (240)
3 8 13
20.5 20.8 18.9
240.0 251.7 142.5b
40.0 37.7 33.0b
8.17 6.02 6.08
(9;po)
3 8 13
21.5 21.7 19.9
230.0 303.3 96.2
36.3 44.0 23.0
9.00 5.76 4.18
$0)
THAT
a 0.05 >p > 0.01. D 0.01 >
p > 0.001.
Dogs. There were no signs of distress noted among the beagles at any time during the 6-hr per day inhalation for 13 weeks and no statistically significant differences were found between the treated groups and the controls in any of the parameters monitored (Tables 7-10). Challenge
At the conclusion of the 70-day rat repeated inhalation study, 10 rats from each level, the air controls, and an additional 20 “naive controls” randomized from the same stock of Harlan-Wistar rats as those on study but not subjected to daily handling, were exposed to a challenge concentration of 47 mg/liter (12,000 ppm) until 25% had died. This concentration is 1.3 times the 4-hr LC50 of 35 mg/liter (8800 ppm). It was anticipated that this procedure might demonstrate whether the animals had been rendered more susceptible or more resistant by virtue of their prior treatment. The rat responses presented in Table 11 indicate that there were more deaths among the “naive controls” than any of the other groups including the air-exposed controls. It is our opinion that these rats, which had not become acclimated to daily handling and exposure, were much more inquisitive and active than the others that had been handled and consequently they breathed more rapidly and probably acquired higher blood concentration even though deaths occurred in relatively the same time interval. This conclusion was reached because no significant difference in time to recovery from prostration (regaining ability to move about) was noted when treated animals were compared to the exposed controls but the difference became significant for all treated
209.6 587.9
Body weight (g) 0 64
10.8 12.2
3.9
Body weight changes (kg) 2 -0.1 22 0.4 36 0.6 51 1.2 64 1.4
-3 64
Body weight (kg)
Body weight changes (g) 3 17.8 22 193.7 36 268.0 51 337.3 64 373.3
Mean
Days of exposure
0.3 0.7 0.4 0.7 1.0
1.3 2.1
6.6 27.5 36.9 43.1 45.7
23.3 62.3
SD
-0.2 0.3 0.4 0.7 0.8
10.7 11.5
20.2 187.2 253.3 303.9 331.6
223.4 548.0
Mean
1.9
Concentration
0.3 0.6 0.5 0.6 0.7
1.2 1.9
4.0 32.9 41.8 46.1 45.3
22.4 48.8
SD
(mg/liter) 0.95
-0.5 -0.2 0.0 0.4 0.6
10.1 10.6
Dogs
18.0 184.5 257.1 319.7 351.5
0.2 0.6 0.5 0.5 0.6
2.4 2.8
5.5 32.3 43.5 50.0 58.4
26.8 70.9
SD
Concentrate
Rats 202.9 551.8
Mean
Toluene
-0.4 0.0 0.4 0.6 0.8
10.1 10.9
16.2 190.8 263.5 323.2 354.5
197.9 553.7
Mean
0.0
0.3 0.6 0.8 1.2 1.1
1.9 2.6
6.0 28.9 36.4 47.6 45.3
26.0 56.5
..__ SD
TABLE 7 MEAN BODY WEIGHTS AND BODY WEIGHT CHANGES FOR RATS AND DOGS
0.28 0.08 0.95 2.54 1.35
1.68 0.55
5.95 1.46 1.21 -0.02 1.43
1.08 2.96
Homogeneity of variance (23
1.64 1.00 0.72 0.67 0.67
0.20 0.34
2.11 0.38 0.59 1.64 2.34
2.39 1.81
Analysis of variance
$ w
2 ;;I
z 8 3 ki?
% i: 2
8.02 7.32
7.63 8.00
7.50 7.30
7.61 6.66
Preexposure Plus 61 days
(&
0.95 Preexposure (240) Plus 61 days
0.0 Preexposure (0.0) PIUS61 days
RBC (millions/ m3)
HEMATOLOGICAL
3.9 Preexposure (980) Plus 61 days
Concn mg/liter bpm)
MEAN
9.6 9.9
9.6 9.2
12.9 10.7
12.3 11.2
51.6 44.4
51.6 50.2
52.6 54.7
56.2 49.8
Ht (%I
FOR GROUPS
WBC (thousands/ mm”)
FINDINGS
18.8 16.8
18.7 18.8
19.6 19.5
20.0 18.4
Hb (g/100 ml)
1.45 1.10
0.80 0.95
1.25 1.20
1.50
0.90
0
0
Baso
THAT INHALED
Reticulocytes (%>
DOGS
TABLE 8 OF FOUR
CONCENTRATE
VAPORS
7.2 10.8
5.2 7.5
7.2 8.2
9.2 8.5
Eosino
9.5 8.8
7.5 8.0
9.5
10.2 9.0
Immature neutro
100.2 102.5
108.5 109.8
106.2 112.5
111.8 114.2
Neutro
76.0 68.2
71.0 67.5
60.0 68.8
62.0 59.5
Lymph0
Based on a 200-cell count. To convert to percentage, divide by 2.
TOLUENE
6.8 8.5
7.2 6.8
7.5 8.5
6.2 7.8
Mono
14.4 14.9 15.1 15.4 14.8 14.1
1.9 Preexposure (480) Plus61 days
0.95 Preexposure (240) Plus61 days
0.0 Preexposure (0.0) Plus61 days
132.5 131.2
128.8 128.8
130.0 123.8
128.8 126.2
0.24 0.30
0.28 0.27
0.28 0.31
0.23 0.32
Bilirubin (mg/lOOml)
TABLE 10
Blood glucose (mg/lOOml)
24.0 21.8
19.2 18.2
22.0 18.2
27.2 20.0
29.5 21.2
26.2 20.0
27.5 19.8
33.8 23.5
Liver weights(g) Liver weights,percentageof body weight Kidney weights(g) Kidney weights,percentageof body weight
319.8 2.58 45.0 0.36
Mean
3.9
112.5 0.55 11.3 0.05
SD
344.8 3.11 43.8 0.38
Mean
1.9
37.4 0.89 5.7 0.04
SD
272.8 2.61 39.2 0.38
Mean
0.95
47.9 0.29 7.3 0.07
SD
Concentration (mg/liter) Toiuene Concentrate
264.8 2.38 40.5 0.38
Mean
0.0
91.0 0.29 5.9 0.04
SD
1.74 1.47
1.33 1.07
2.27 1.64
1.51 1.58
Alkaline SGOT SGPT phosphatase (Sigmaunits/ml) (Sigmaunits/ml) (Sigmaunits/ml)
MEAN ORGAN WEIGHTS OF GROINS OF FOUR BEAGLES AFTER THEY INHALED TOLUENE CONCENTRATE VAPOR FOR 66 DAYS
15.5 14.4
BUN (mg/lOOml)
3.9 Preexposure (980) Plus 61 days
Concn mg/liter (mm)
TABLE 9
MEAN BLEND CHEMISTRY FINDINGS OF GROUTS OF FOUR DOGS THAT INHALED TOLUENE CONCENTRATE VAPORS
2110 3/10 l/10 2110 1o/20
During exposureb 2/10 3110 l/10 2110 11120
Total within 7 days
Number of rats that died Number exposed
Time to death
70 83 59 76 78
Median time to death (min)
HAD SURVIVED GRADED CONCENTRATIONS 90-MIN CHALLENGE EXPOSURE TO 47
None None None None
vs Control
0.05 > p > 0.01 None p = 0.05 -
vs Naives
Statistical significance”
Time to prostration
18.5 18.5 20.0 22.0 35.0
Median time (min)
of naive rats is not statistically significantly different.
14.0 13.5 13.0 13.8 12.0
Median time (min)
-.
mg/liter
None None None None
0.05 >p > 0.01 0.05 > p > 0.01 0.05 > p > 0.01 None
Statistical significance’ ~ ~~ ~~ vs vs Control Naives
Time to recovery”
-
FOR 6 HR PER DAY, 5 DAYS PER WEEK FOR 70 DAYS TO A (12,000 ppm) OF TOLUENE CONCENTRATE
11
OF THIS SOLVENT
TABLE
a Regaining ability to move about. b Exposure was terminated after 25% of the animals had died. Mortality e Rank sum test of significance used.
3.9 mg/liter 1.9 mg/liter 0.95 n&liter 0.0 mg/liter
70 Days 70 Days 70 Days 70 Days Naives
x x x x
OF RATS THAT
RESPONSE
2 F
h
2 E
F z
VAPOR
INHALATION
OF TOLUENE
CONCENTRATE
487
groups versus the naive controls. The latter group recovered in 35 min as opposed to 22 for the air-exposed controls and 18.5 to 20 min for the treated groups. Human Sensory Response. Odor threshold. Vapor-air
concentrations for the odor threshold study were varied by a factor of 10. These concentrations were not analyzed by gas chromatography. Therefore, metered concentrations were reduced 36% to correspond with the mean percentage recovery in the repeated inhalation study. A group of six volunteers, between 23 and 50 years of age, inhaled a series of concentrations (each for approximately 10 set) in the following sequence: 0.64,0.00,0.0064,0.064,0.064,0.0064,0.64, and 0.00 mg/ liter. The responses are summarized in Table 12. A corrected concentration of 0.064 mg/liter (16 ppm) was readily perceived, as indicated by the 100% incidence of detection. Volunteers were able to detect 0.0064 mg/liter (1.6 ppm) at an incidence of 33 %. By the method of moving averages (Carpenter et al. 1975a) the most probable corrected concentration for the threshold lies between these limits or at 0.01 mg/liter (2.5 ppm). Sensory threshold. All concentrations were determined by gas chromatography. In order to determine the sensory threshold, six volunteers between 20 and 63 years of age inhaled measured vapor-air concentrations for 15-min periods in the following order: 1.9 mg/liter and 3.7 mg/liter, corresponding to 480 and 930 ppm, respectively. Exposures were limited to 1 per day to prevent any buildup of symptoms (Table 12). TABLE 12 ODOR DETECTION AND SENSORY THRESHOLD FOR TOLUENE CONCENTRATE(HUMAN
SUBJECTS)
Odor threshold Metered concentration (mg/liter) 1.0 0.1 0.01 0.0 Corrected concentration (mg/liter) 0.64 0.064 0.0064 0.0 Correctedconcentration (ppm) 160 16 1.6 0.0 Number detectingodor/number of subjects (two trials) 12/12 12/12 4112 o/12 Conclusion: The odor thresholdlies between0.0064and 0.064 mg/liter, the most probable concentration being 0.01mg/liter or 2.5 ppm. Sensorythresholds Measuredconcentration(mg/liter) 1.9 3.7 Measuredconcentration(ppm) 480 930 First Exposureorder for six subjects Number detectingodor 6 Number olfactory fatigue 0 Number throat irritation 0 Number eyeirritation 1 Number noseirritation 0 Number reporting “dizziness” or “lightheadedness” 0 Number tasting “something” 1 Number with effects1 hr after exposure 0 Conclusion: Number of subjectsconsideringconcentrationstolerable for 8-hr working day wassix of six at 1.9r&liter andthree of six at 3.7mg/liter.
488
CARPENTER
ET AL.
One volunteer reported a transitory mild drying sensation of the eyes after inhaling 1.9 mg/liter for 15 min. In the 3.7-mg/liter concentration one individual experienced transient nasal irritation and another transient throat irritation. The nasal irritation was described as a mild sensation of dryness experienced only during the final minute of inhalation. The throat irritation, reported as a mild burning sensation, persisted only from the third through the sixth min of the period. The same volunteer reported a cooling sensation in the throat for about 1 min immediately following the inhalation TABLE 13 RECAPITULATION
Acute 4-hr, no ill effect concn, rats Rat LC50 Acute 6-hr, no ill effect concn, dogs Acute 6-hr inhalation, cats Osmotic erythrocyte fragility, rats Short-term massive concn, rats Mouse respiratory tract irritation
13-Week subacute inhalation, and dogs
rats
Statistically significant finding Monocyte count, 8 weeks, rats SGOT, 13 weeks, rats SGPT, 13 weeks, rats
Challenge exposure to rats, 6 hr
Odor threshold, human subjects
Sensory threshold, human subjects
Suggested hygienic standard
6.8 mg/liter (1700 ppm) 35 (31 to 38) mg/liter or 8800 (7800 to 9600) pm 3.0 mg/liter (760 ppm) 31 mg/liter (7800 ppm); signs observed were suggestive of CNS depression Metered 80 mg/liter (20,000) ppm), 45 tin inhalation caused no statistically significant changes in hemolysis 180 mg/liter (45,000 ppm); lethal to five of five, in 20 min; Lt 50 = 11 (7.2 to 16) min Lowest concentration at which 50 % depression of respiratory rate occurred: 34 mg/liter (8600 ppm); no effect concentration: 20 mg/liter (5000 ppm) 3.9 mg/liter or 980 ppm; no ill effects
Concn (mg/liter) 0.95 3.9;1.9;0.95 1.9;0.95
Assessment Not dosage related, ignored Low control value; ignored Low control value and not dosage related; ignored
47 mg/liter (12,000 ppm); statistically significantly increased mortalities among “naive” controls. Assessment: due to increased activity and respiration of unacclimated animals 0.01 mg/liter or 2.5 ppm after application of the correction factor of 64% obtained from the measured to metered relationship in the subacute study 1.9 mg/liter or 480 ppm was considered as a tolerable concentration for an 8-hr working day. This concentration would contain 0.87 mgtliter or 220 ppm of toluene per se. 1.9 mg/liter (480 ppm) as a ceiling or limiting value based on response of human volunteers
VAPOR
INHALATION
OF TOLUENE
CONCENTRATE
489
period. Two volunteers reported feeling “slightly light-headed” after 15 min in 3.7 mg/liter and another reported a “stuffy, drowsy feeling.” All volunteers detected the odor of the Toluene Concentrate at both concentrations and none reported complete olfactory fatigue. Following each inhalation period, the volunteers were asked if they thought they could work for 8 hr in that particular concentration. Six gave an affirmative response after inhaling 1.9 mg/liter, while only three thought they could work in the 3.7-mg/liter concentration. Based on the foregoing data, 1.9 mg/liter (480 ppm) of Toluene Concentrate vapor would seem to be an acceptable level for an 8-hr daily inhalation by normal healthy individuals of working age. This concentration corresponds to 0.87 mg/liter (220 ppm) of toluene based on the 46 % aromatic content of the Toluene Concentrate. Table 13 summarizes the findings. DISCUSSION
There is no evidence that either rats or beagles were harmed as a result of inhaling 3.9,1.9, or 0.95 mg/liter of Toluene Concentrate for 13 weeks, 6 hr/day. A concentration of 1.9 mg/liter (480 ppm) was considered as acceptable for 8-hr continuous inhalation by a panel of six human subjects but 3.7 mg/liter (930 ppm) was not. Based upon these findings, the hygienic standard should be set at 1.9 mg/liter (480 ppm) which we realize is slightly over twice the current (1975) TLV recommended by the American Conference of Governmental Industrial Hygienists and the National Institute for Occupational Safety and Health for toluene alone. The fact that Toluene Concentrate contains 46 % aromatics (only 0.06 % benzene) accompanied by some 15 % naphthenics and 39 % paraffinics raises the legitimate question as to whether the net effect ofall three is additive, less than additive, or greater than additive as regards both toxicity and irritation (Pozzani et al. 1959). The evidence at hand denies the latter and we suggest that whether the final effects are simply additive or less than additive (antagonistic) will rest on future monitoring of airborne concentrations coincident with medical surveillance of those exposed. Therefore, our interpretations of the findings presented may or may not be justified by further observations. In any case, it is evident that any claim of human discomfort will result in the prompt reduction of the TLV. ACKNOWLEDGMENTS
Practically the entire staff of the Chemical Hygiene Fellowship has been involved in this project and the authors wish to extend sincere appreciation and thanks for their faithful attention and cooperation. Analytical Procedures: J. M. Eldridge and S. J. Kozbelt; Hematology and Histopathology: P. A. Baker, P. A. Crawford, and M. A. McGee; Experimental Assistance: C. S. Weil, and M. D. Woodside. REFERENCES ALARIE, Y. (1966). Irritating properties of airborne materials to the upper respiratory tract. Arch. Environ. Health 13,433+9. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975a). Petroleum hydrocarbon toxicity studies. I. Methodology. Toxicol. Appl. Pharmacol. 32,246-262.
490 CARPENTER,
CARPENTER C. P., KINKEAD,
E. R.,
GEARY,
ET AL. D.
L.,
JR., SULLIVAN,
L. J., AND
KING,
J. M.
(1975b). Petroleum hydrocarbon toxicity studies. II. Animal and human response to vapors of Varnish Makers’ and Painters’ Naphtha. Toxicol. Appl. Pharmacol. 32, 263-281. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975c). Petroleum hydrocarbon toxicity studies.III. Animal and human responseto vapors of Stoddard Solvent. Toxicol. Appl. Pharmacol. 32,282-297. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975d). Petroleum hydrocarbon toxicity studies.IV. Animal and human responseto vapors of Rubber Solvent. Toxicol. Appl. Pharmacol. 33,526542. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975e).Petroleumhydrocarbon toxicity studies.V. Animal and humanresponseto vapors. of Mixed Xylenes. Toxicol. Appl. Pharmacol. 33,543-558. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975f).Petroleumhydrocarbon toxicity studies.VI. Animal and humanresponseto vapors of “60 Solvent”. Toxicol. Appl. Pharmacol. 34, 374-394. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975g).Petroleum hydrocarbon toxicity studies.VII. Animal and human responseto vapors of “70 Solvent”. Toxicol. Appl. Pharmacol. 34, 395-412. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975h).Petroleumhydrocarbon toxicity studies, VIII. Animal and human responseto vapors of “140” Flash Aliphatic Solvent”. Toxicol. Appl. Pharmacol. 34,413429. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., AND KING, J. M. (1976i). Petroleum hydrocarbon toxicity studies.IX. Animal and human responseto vapors of “80 Thinner.” Toxicol. Appl. Pharmacol. 36, 409425. CARPENTER, C. P., GEARY, D. L., JR., MYERS, R. C., NACHREINER, KING, J. M. (1976j).Petroleumhydrocarbontoxicity studies.X.
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