petroleum hydrocarbon toxicity studies: i. methodology

17
TOXICOLOGY AND APPLIED PHARMACOLOGY 32, 246-262 (1975) Petroleum Hydrocarbon Toxicity Studies I. Methodology’ C. P. CARPENTER, E. R. KINKEAD,~ D. L. GEARY, JR., L. J. SULLIVANAND J. M. KINGS The Chemical Hygiene Fellowship, Carnegie-Mellon Institute of Research, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213 Received June 29,1974; accepted January 7,1975 PetroleumHydrocarbon Toxicity Studies.I. Methodology. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J. AND KING, J. M. (1975). Toxicol. Appl. Pharmacol. 32,246-262.Theobjectives, protocols, and procedures followed in the study of the inhalation toxicity of a series of petroleum hydrocarbons are presented herein. To conserve journal space reference will be made to this report in order to avoid repetition in succeed- ing reports on selected commercially available petroleum hydrocarbons. The compoundsare arranged in a matrix basedon boiling range and aromaticity. It is hoped that the information gatheredin this series of 12 or more studies will provide a basis for predicting the toxicity of closely related hydrocarbon mixtures that are articles of commerce. This investigation deals with the response to the inhalation of representative com- mercially available hydrocarbons. The primary objective of these studieswas to obtain experimental data which couid be usedas a guide for recommending hygienic standards for hydrocarbons, as well as the confirmation or modification of those standards which have already been adopted. The hydrocarbons under consideration fall under the broad category of solvents with an approximate benzene content ordinarily not exceeding 2.0 %. In all of these studies, a major objective was to make certain that the vapor phase generated be as truly representative of the liquid phaseas possible. The candidate materials are those of commercial importance, produced in quantity, and well characterized by chemical class. Early emphasis was on those hydrocarbons and mixtures under review by the Threshold Limits Committee of the American Conference of Governmental Industrial Hygienists (ACGIH). Subsequent selections were based upon two major parameters: namely, boiling range and percentage of aromatics. It is hoped that the proposed matrix will provide sufficient breadth of coverage so that the toxicity of any mixture within these parameters can be predicted with reasonable confidence. METHODS To preserve the absolute anonymity of the supplier, a Committee appointee from the American Petroleum Institute (API), familiar with the products and their suppliers, 1 Supported by TheAmerican Petroleum Institute. 2 Present address: University of California, Irvine, Wright Patterson Air Force Base, Dayton, Ohio 45431. 3Present address: NewYork State Veterinary College, Cornell University,Ithaca,N.Y. 14850. Copyright 0 1975 by Academic Press, Inc. 246 All rights of reproduction in any form reserved. Printed in Great Britain

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Page 1: Petroleum hydrocarbon toxicity studies: I. Methodology

TOXICOLOGY AND APPLIED PHARMACOLOGY 32, 246-262 (1975)

Petroleum Hydrocarbon Toxicity Studies

I. Methodology’

C. P. CARPENTER, E. R. KINKEAD,~ D. L. GEARY, JR., L. J. SULLIVAN AND J. M. KINGS

The Chemical Hygiene Fellowship, Carnegie-Mellon Institute of Research, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213

Received June 29,1974; accepted January 7,1975

Petroleum Hydrocarbon Toxicity Studies. I. Methodology. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J. AND KING, J. M. (1975). Toxicol. Appl. Pharmacol. 32,246-262.The objectives, protocols, and procedures followed in the study of the inhalation toxicity of a series of petroleum hydrocarbons are presented herein. To conserve journal space reference will be made to this report in order to avoid repetition in succeed- ing reports on selected commercially available petroleum hydrocarbons. The compounds are arranged in a matrix based on boiling range and aromaticity. It is hoped that the information gathered in this series of 12 or more studies will provide a basis for predicting the toxicity of closely related hydrocarbon mixtures that are articles of commerce.

This investigation deals with the response to the inhalation of representative com- mercially available hydrocarbons. The primary objective of these studies was to obtain experimental data which couid be used as a guide for recommending hygienic standards for hydrocarbons, as well as the confirmation or modification of those standards which have already been adopted. The hydrocarbons under consideration fall under the broad category of solvents with an approximate benzene content ordinarily not exceeding 2.0 %. In all of these studies, a major objective was to make certain that the vapor phase generated be as truly representative of the liquid phase as possible.

The candidate materials are those of commercial importance, produced in quantity, and well characterized by chemical class. Early emphasis was on those hydrocarbons and mixtures under review by the Threshold Limits Committee of the American Conference of Governmental Industrial Hygienists (ACGIH). Subsequent selections were based upon two major parameters: namely, boiling range and percentage of aromatics. It is hoped that the proposed matrix will provide sufficient breadth of coverage so that the toxicity of any mixture within these parameters can be predicted with reasonable confidence.

METHODS

To preserve the absolute anonymity of the supplier, a Committee appointee from the American Petroleum Institute (API), familiar with the products and their suppliers,

1 Supported by The American Petroleum Institute. 2 Present address: University of California, Irvine, Wright Patterson Air Force Base, Dayton, Ohio

45431. 3 Present address: New York State Veterinary College, Cornell University, Ithaca, N.Y. 14850.

Copyright 0 1975 by Academic Press, Inc. 246 All rights of reproduction in any form reserved. Printed in Great Britain

Page 2: Petroleum hydrocarbon toxicity studies: I. Methodology

HYDROCARBON TOXICITY METHODS 247

selected three or four representative commercially available solvents in each of the several categories. From this list, we made the final single selection.

A stepwise approach to the 13-wk inhalation study, to determine a no-ill-effect level in rats and dogs, was followed. First, rats were subjected to inhalation of a series of concentrations, for single 4-hr periods to arrive at an LC50 (most probable concentra- tion lethal to 50 % of the animals within a postexposure period of 14 days) and to detect major sites of cellular injury. Cats, the animal model of choice for central nervous system (CNS) effects, then inhaled a concentration equivalent to the rat LC50 to allow detection of any overt signs of CNS effect that might occur. Following this, the highest concentration which caused no overt signs of toxic stress in rats and dogs was deter- mined, These data served as a guide to setting concentration levels for the 6 hr/day, 5 day/wk, 13-wk inhalation study.

At the termination of the 65 exposure days, representative rats were subjected to a massive concentration (1.25-2 times the 4-hr LC50) for 6 hr to demonstrate whether their resistance had increased or decreased or had not been affected. A method described by Alarie (1966) was used to determine the irritant potential of the hydrocarbons to the respiratory tract of mice. To round out the investigation, human subjects inhaled the vapor to determine odor threshold and to delineate sensory response during 1 5-min inhalation periods.

Theprotocol,4jointlyestablished bytheAPIToxicology Committeeand thechemical Hygiene Fellowship, is outlined below:

A. ACUTE INHALATION TOXICITY

I. Acute LC.50 and No-Ill-EfSect Concentrations Species and number. Groups contained 15 or 16 male rats (100-200 g) per exposure

level. Concentrations. Inhalation levels were varied by a factor of 2, until a level which

produced no adverse signs in rats was found. Duration. Exposures were of 4-hr duration followed by a l/l-day observation period.

When mortality could not be obtained in 4-hr periods, 8-hr periods were used. For the determination of acute toxicity it is more convenient for the investigator to utilize a 4-hr instead of an 8 hr period. Response, generally speaking, is related to con- centration x time. In maintaining a 4-hr period for acute exposure, the acute toxic effects of different materials in relationship to concentration can be compared at a constant time base.

Observations. Animals were observed continuously for the first 5 min of exposure, at 15 and 30 min, and then at 30-min intervals until 1 hr postexposure, again at 2 hr, and daily thereafter.

Procedure. Ten of the animals on each level were preselected for the LC50 calculation. The remainder were sacrificed immediately after the exposure or several days’ post- exposure for tissue examination.

Autopsy andpathology schemata. Gross autopsy performed on all animals at death or sacrifice included visual examination of all organs and tissues. Histopathological

4 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.

Page 3: Petroleum hydrocarbon toxicity studies: I. Methodology

248 CARPENTER ET AL.

evaluation was made of the respiratory tract (trachea, bronchi, and lung parenchyma not distended with fixative) and liver from three animals per level (or less depending on the number of survivors) sacrificed at the conclusion of the 4-hr exposure and similarly from 2 days’ postexposure. Histopathological evaluation of respiratory tract, liver, kidney, brain, and bone marrow was made from three animals (or less) per level sacrificed following the 1Cday postexposure observation.

Statistical analysis. Based on mortality during a 1Cday observation period, the most probable LC50 with its fiducial range was calculated by the Thompson (1947) method of moving averages using tables by Weil(1952) and other unpublished tables.

The results of the quantitative continuous variables, such as body weight changes, were intercompared for the dosage groups and the controls by the use of the following tests: Bartlett’s homogeneity of variance, analysis of variance, rank sum (Snedecor and Cochran, 1967), and Duncan’s multiple range (Duncan, 1955,1957; Harter, 1960). The latter was used, if F for analysis of variance was significantly high, to delineate which groups differed from the controls. If Bartlett’s test indicated heterogeneous variances, the F test was used for each group versus the control. If these individual F tests were not significant, Student’s t test was used; if significant, the means were compared by the Cochran t test (Snedecor and Cochran, 1967) or the rank sum test.

Frequency data, such as incidences of mortality or of micropathological conditions, were intercompared by the normal deviate of chi square calculated with Yates’ correc- tion for continuity (Fisher, 1950). In all cases the fiducial limit of 0.05 was employed to delineate the critical level of significance.

In general, only criteria that differed statistically significantly from the control group are discussed. Omission of comment is indicative that no valid differences were found. An attempt has been made to round off machine calculated data but if it is obvious that the data portend fictitious accuracy the implication should be ignored.

Erythrocyte osmotic fragility. Five additional rats were subjected to the highest acute vapor exposure level used to determine the rat LC50 and sacrificed immediately after exposure for erythrocyte fragility determinations.

2a. Acute Inhalation-Cats

Species, number, and concentration. Four mature male cats were exposed to the previously determined LC50 for rats.

Duration. A 4-hr inhalation period was followed by a 1Cday postexposure observa- tion period.

Observations. Continuous observations were made during the first 5 min, at 15 min, and then at 30-min intervals until 1 hr postexposure, again at 2 hr and daily thereafter.

Autopsy andpathology. Gross autopsy was performed on all cats which died or were sacrificed and histopathological evaluation of the respiratory tract, liver, kidney, brain, bone marrow, skeletal muscle, and peripheral nerves from each animal was made.

Analytical verljication. Concentrations were quantified by gas chromatography.

2b. Acute Inhalation-Dogs

The preliminary data necessary to set levels for the 13-wk subacute toxicity study were determined by finding the concentration rats tolerated in a 4-hr acute exposure

Page 4: Petroleum hydrocarbon toxicity studies: I. Methodology

HYDROCARBON TOXICITY METHODS 249

without adverse signs. Then the highest concentration which did not elicit signs of outward distress in dogs during a 4-hr inhalation period was sought. This maximum concentration was used as the highest level for the repeated inhalation studies, if it was lower than the concentration found not to cause outward signs of distress in rats.

3. Short-Term Inhalation of a Massive Concentration

Species, number, and concentration. Groups of five male rats were subjected to five times the rat LC50 or to substantially saturated vapor, if the former was unobtainable.

Duration. The length of time necessary to establish an LT50 (the time period lethal to 50 % of the rats) was determined.

Observations. Animals were observed constantly during exposure; and at 0.5, 1, 2, and 4 hr postexposure; and once daily thereafter for 7 days.

Autopsy andpat1zoZog.v. A complete gross autopsy was performed on all animals that died or were sacrificed. Histopathological evaluation of the trachea, bronchi, and lung parenchyma from animals which died during the exposure or the first 24-hr post- exposure were made. If gross lesions were present after 7 days, tissues were taken from the animals surviving 14 days.

B. SUBACUTE INHALATION TOXICITY

Species and number. Twenty-five male rats and four dogs per level were used. Another 20 rats, from the same week of production, were maintained for use as challenge- exposure controls (naive rats). The challenge exposures were run to determine whether the 6-hr daily inhalation of a nonlethal level of hydrocarbon, whether by acclimatiza- tion or induction of enzymes, would result in the rat becoming more or less resistant than nonexposed or naive controls from the same lot of animals.

Concentrations. A control group (exposed to dilution air) plus groups exposed to three graded levels of test material were utilized for each compound.

Duration. Exposures were 6 hr/day, 5 days/wk for 13 wk. Observations. Once each week, body weights of both species and food consumption

for dogs were determined. Overall appearance and behavior were checked daily. Procedure. At 3, 8, and 13 wk of exposure three, three, and four rats, respectively,

from each group, including controls, were sacrificed. After 13 wk of exposure 10 surviving rats from each group and 20 unexposed rats of the same age were used to detect any significant differences in time to death or in occurrence of signs of distress among the groups. The groups were “challenged” simultaneously by exposure to a vapor-air concentration 1.25-2 times the 4-hr rat LC50 until 25 % of the group succumbed or for no more than a 6-hr period. The remaining rats, or those surviving the subacute exposure, exclusive of the ten per level reserved for the challenge exposure were sacrificed after 13 wk and tissues were taken for histopathological interpretation following gross autopsy. The assignment of rats to each of the above groups was made by random selection before the start of the study.

Clinical and hematological schemata. These tests were performed on all dogs initially and prior to sacrifice; and on rats, prior to sacrifice at 3, 8, and 13 wk of exposure. Minimum hematological evaluation for each animal included hematocrit, total erythrocyte count, reticulocyte count, and total and differential leucocyte counts.

Page 5: Petroleum hydrocarbon toxicity studies: I. Methodology

250 CARPENTER ET AL.

The biochemical survey included serum alkaline phosphatase, serum glutamic oxal- acetic transaminase, serum glutamic pyruvic transaminase, and blood-urea-nitrogen on the rats, and these tests plus bilirubin and blood glucose on the dogs. Initial and preterminal electrocardiograms were performed on all dogs. Urinalyses were conducted with both species.

Autopsy andpathology schemata. Gross examinations were made of all organ systems. Histopathological examination included brain (three sections), respiratory tract (three sections minimum, based on acute exposure data results), heart, thyroid, liver, kidney, adrenal, spleen, pancreas, stomach and intestines, skeletal muscle, bone marrow, and peripheral nerves. The reproductive organs and eyes were not scheduled for histopathological study unless abnormalities were discovered upon gross examina- tion. Tissues were taken from all dogs and from rats sacrificed after 3, 8, and 13 wk of inhalation of the vapor.

Analytical verijicution. Exposure chamber concentrations were verified at least two times daily by gas chromatography, unless forestalled by uncontrollable events.

C. MOUSE UPPER RESPIRATORY TRACT IRRITATION

The mouse respiratory irritation study was performed using a slight modification of the method described by Alarie (1966). Two Swiss-Webster male mice (25-30 g) were tested simultaneously. Each animal was placed in a Plexiglas cylinder 9.5 cm in length and 3.2 cm in diameter. The head of the animal protruded through a 1.3-cm hole in a rubber dam which covered one end while the mouse was prevented from retreating by an adjustable stainless steel plate. The rubber dam was reinforced with vinyl plastic tape to prevent the animal from pushing through. When the cylinder was sealed properly, it then became a body plethysmograph (Fig. 1).

The vapor-air chamber consisted of a Plexiglas cylindrical tube, 14.4 cm long and 9.5 cm in diameter which could be separated longitudinally to form two semicylindrical halves. When joined, these halves had an approximate volume of 1 liter. Both circular ends had gum rubber gaskets which sealed the cylinder to form an airtight exposure chamber. Two and four-tenths centimeter x 5.1 cm tubes, chemically welded into the center of both ends, conducted the flow of vapor through the chamber. The tubes in which the mice were confined were chemically welded at right angles to the sides of the main cylindrical chamber in such a way that only the head of the mouse protruded into the vapor passing through the chamber.

Vapor-air mixtures were generated as previously described. During the preexposure period, the mice inhaled room air to provide a baseline respiration rate and to acclimate the animals to the confined space. By use of a series of stopcocks, the vapor-air mixture, delivered at a rate of 30 liters/min, was directed into the chamber for the I-min exposure (Fig. 2). The respirations were monitored and divided into four 15-set periods. After the I-min exposure, room air was again drawn through the chamber and the mouse respirations were monitored for an additional 15 min.

The measurement of effect was based upon the decrease in the frequency of individual respirations caused by an increase in the duration of the expiratory phase of the breath- ing cycle. Four 15set segments of the preexposure period were chosen as near to the actual exposure period as possible and the mean respiratory rate was determined for

Page 6: Petroleum hydrocarbon toxicity studies: I. Methodology

HYDROCARBON TOXICITY METHODS 251

the baseline value. The exposure period of 1 min was divided into four 15-set segments and the number of respirations per segment was determined. The number of respira- tions was determined for the 15-set interval following 0.5, I, 3, 5, 10, and 15 min postexposure. The exposure and postexposure effects were determined by dividing the number of respirations in each 15-set interval by the preexposure average. This value was then determined to be either a 50 % or greater inhibition (a positive effect) or less than 50 % inhibition of respiratory rate, a negative effect.

d

0 Ja -__- .- - -_-- _ _ --, I.--- - - - -- -- _ - . - - - ---

Jb

..+.,, t--2%‘. : 1 jb “1.“

-_---_------_------_---- - _-_- -- -.rf*‘-

SIDE VIEW, EXPWDED

S.S. Hose Clamp

END VIEW, ASSEMBLED

FIG. 1. Modified Alarie chamber. a---Gum rubber gasket (covers perimeter of circular collar); &port for plethysmograph chamber; c-circular stainless steel baffle; &-circular collar; e- Plethysmograph chamber, lf- in. o.d.; 14 in. i.d.; f-no. 7 rubber stopper; g--& in. stainless steel rod; /r---l& in. stainless steel disc; i---j in. calibration port (to be sealed); j-tube to transducer; k-gum rubber gaskets (separate halves of chamber lengthwise); I-diaphragm to fit neck of mouse; ~fi- 4) in. o.d.; 33 in. i.d.; n-exhaust or inlet tube.

D. HUMAN SENSORY RESPONSE

These studies were conducted in a glass-lined room with a terrazzo floor which was entered via an airlock. The room had a volume of 12.8 m3 and the vapor-air mixture was exhausted at 2.3-3.0 m3/min. Human volunteers, not selected for olfactory acuity, entered through the airlock, and then proceeded into the chamber.

Page 7: Petroleum hydrocarbon toxicity studies: I. Methodology

252 CARPENTER ETAL.

For odor threshold detection, the volunteers inhaled (in random order) either room air or metered concentrations spaced at ten-fold intervals, for periods of lo-set duration. No analytical verification of the nominal concentrations was supplied for evaluation of odor thresholds.

To select a concentration that would be accepted as comfortable for a working environment, a 15-min inhalation period was used. Such trials were limited to one per day to prevent any build-up of positive responses. Four measured concentrations were presented in random order and a confidential written record of response was kept by

- = -+ To Exhaust -

To Exhaust f- = - (Slight Vacuum) ZZ

Rotameter A - -

u u

Rotameter B

1 =

All Vapor Conduits 10 mm i.d.

Liquid De" ..^_.. 5

To Transducer and Polygraph

A

Sampling Port Septum

Room ”

Air 5 P

I I 1

Stopcock A Stopcock B 6 mu bore 6 mm bore II ' Plethysmograph *.*-.

: -.-.._.. '; :..J..:

:*- .*. L."" ,.....; II Chamber (one on

'Q.' either side)

tLWing pm- and postexposure periods, the positions of Alarie Chamber

L4 plugs in stop&cks A and B One Liter -

are delineated by solid q

tines; during exposure periods, by dotted tines. 1 k!!l

Rvaporator

FIG. 2. Alarie chamber modified for vapor exposures.

each volunteer at I-min intervals throughout the 15-min inhalation period. If olfactory fatigue was encountered, the volunteers returned within lo-15 min to sniff the con- centrations again in order to judge whether it seemed as strong as it did at the beginning of the period. Gas chromatographic analysis of each concentration was made. Con- centrations inhaled never exceeded the 13-wk no-ill-effect level for rats and dogs.

Sample

Sufficient sample was procured from a single production run to carry out the entire study and was stored in the original containers until needed. Sample numbers were assigned and a code name devised to conceal the supplier’s identity. Referee samples taken from each drum in the shipment were pooled and shipped back to the

Page 8: Petroleum hydrocarbon toxicity studies: I. Methodology

HYDROCARBON TOXICITY METHODS 353

manufacturer, who furnished complete physical properties data. Two-gallon samples of each solvent were retained by the CHF Laboratory as referee samples. The samples tested during this study are indicated in the matrix (Fig. 3).

100

B 20 we-,-,-,-

A -,-,-a-

10 C J -o-,-1- R P G - , , , , ,~~d

100 150 200 250 300 350 400 450 500 550

Boiling Point in OF

FIG. 3. Petroleum hydrocarbon toxicity matrix. Materials: (1969 --.-)-A. VM&P naphtha. B. Stoddard solvent. C. Rubber solvent. (1970 =3-)--D. Mixed xylenes. E. 60 Solvent. F. 70 Solvent. G. 140” Flash aliphatic solvent. (1971 -2)-H. 80 Thinner. I. 50 Thinner. J. Deodorized kerosene. (1972 IIIIIIIIIIIIIIII&-L. 40 Thinner. M. Toluene concentrate. 0. High aromatic solvent. (1973 0)-P. High naphthenic solvent Q. Naphthenic aromatic solvent. R. Nonane.

Production of Vapor-Air Mixtures

The hydrocarbons were vaporized by passage through an electrically heated, vertical, 59 x 8 cm Pyrex tube (Fig. 4). The middle 35 cm of the tube had a spiral groove to increase the surface area and evaporating efficiency. Bare 16-gauge coiled nichrome wire was wound around the tube and nested in the groove between the ridges that created the spiral. An air insulating jacket, made from Pyrex tubing, 40 x 12 cm, was held in place around the wired portion of the inner tube by means of a wrap of 1 in. asbestos cloth tape. A small hole blown 2.5 cm from each end of the jacket provided for passage of the nichrome heater wires, which then led to a rheostat (Carpenter et al., 1949).

The liquid was introduced at the top of the vaporizing tube and allowed to flow downward over the inner wall heated to temperature required to effect complete vaporization. Resultant vapors were carried into the chamber by a countercurrent air stream entering the bottom of the tube. The desired concentration was produced by controlling the amount of liquid vaporized into the metered air stream (Fig. 5). All

Page 9: Petroleum hydrocarbon toxicity studies: I. Methodology

254 CARPENTER ET AL.

I

588

1

‘t----- 18/7 Ball and Socket Joint - See Figure B

L---

i" : - O.D. 80 mm.

mm. 408

Asbestos Cloth Tape 2.8 mm. wide

Glass Jacket O.D. 124 mm. "Pyrex"

The spiral grooves in the vaporizer are made with a graphite mandrel, 10 spirals per 350 mm.

I.D. of Hole 4.0 mm. Glass Bollard

I/ L---- Air Inlet

FIG. 4. Vaporizer, 3000 liter/min capacity.

1

i-

ic ai hc mds r

FIG. 5. Simplified schematic of vapor-air delivery and exhaust system for an inhalation chamber. .ai-air inlet of electrically heated vaporizer; ef-exhaust fan; gv-gate valve; hc-heat control for vaporizer; ic-inhalation chamber; If-liquid flow; m-manometer, to indicate vacuum (1 cm H,O); mds-motor-driven syringes ; am-orifice meter ; r-reservoir ; sp-sampling port ; t-thermometer.

Page 10: Petroleum hydrocarbon toxicity studies: I. Methodology

HYDROCARBON TOXICITY METHODS 255

analyzed vapor concentrations were reported as “measured” and all nominal con- centrations as “metered” to avoid any possibility of confusion.

With dilution airflows of 1000 liters/min for the 4000-liter chamber solvent deliveries up to 20 ml/min were utilized. The amount of solvent required was an inverse function of the boiling point. The maximum vapor concentration attainable for any given mixture was dependent upon the highest boiling component. Because the vapor phase generated matched liquid composition as determined by gas chromatographic profile, no significant pyrolysis could have occurred. Aerosol generation was eliminated by observation for a Tyndall effect and consistency of chromatographic analysis.

Inhalation Chambers: Data and Operation

Pertinent data concerning chambers and air turnover are presented in Table 1. The t99 (the length of time necessary for the chamber to reach 99% concentration) was calculated for the chambers using an average airflow.

TABLE 1 EXPOSURE CHAMBER DATA

Protocol outline

reference

B

C

D

E

Inhalation study Species”

Approx chamber capacity Chamber (liters) constructionb

Acute, 4-hr LCSO

Acute, 4-hr (at rat LC50)

Acute, 4-hr

Massive 5 x 4-hr rat LC50 and/or saturated vapor

Subacute, 6 hr x 65 day

Respiratory irritation

Sensory response

Odor threshold

R

C

D

R

200 TM, P

550 TM, G 550 TM, G

200 TM, P

R, D 4000 TM, G 18.0

M 1 P 0.15

H 13,000 G, TF 20.0

H 13,000 G, TF 20.0

h9 {min)

---.. 7.0

10.0

10.0

7.0

LI C = cat, male. D = dog, male or female, beagle. H = human, male and female. M = mouse, male, Swiss-Webster. R = rat, male, albino, Harlan-Wistar.

b G = glass. P = Plexiglas. TF = terrazzo floor. TM = tempered Masonite.

All chambers were operated under negative pressure of no more than 1 mmHg. The vapor concentrations in the acute single inhalation and the human sensory response 15-min exposure were analyzed at least once per exposure and, in the rat and dog repeated inhalation study, twice each day. Chamber air samples, taken with gas-tight syringes manufactured by Precision Scientific, were injected within 30 set after capture into the gas chromatograph. Prior to use, all areas of the chambers were sampled to verify an even distribution throughout. All sampling was then done from a single port at the center of the chamber.

The dogs were placed in the rear of the chambers on Mondays, Wednesdays, and

Page 11: Petroleum hydrocarbon toxicity studies: I. Methodology

256 CARPENTER ET AL.

Fridays and the rats in the front. This procedure was reversed on Tuesdays and Thurs- days to compensate for any possible but undetected variation in vapor distribution.

Animal: Sources, Feed, Husbandry

Rats. Male, albino rats of the Harlan-Wistar strain, from our own breeding colony, were used in all rat inhalation experiments. Food and water were available ad Zibitum when the animals were not in the inhalation chambers. The rats were housed in an air- conditioned room maintained at 72 f 2°F and 50 + 10 % relative humidity. For acute studies, rats were approximately 5 wk of age and for the 13-wk subacute, 6 wk of age. Each rat was handled and eyes, nose, muscle tone, condition of fur, etc., were observed twice daily during the subacute inhalation study. Each rat was weighed twice a week prior to the first subacute inhalation period, daily during the first week, and once per week thereafter. The animals and cages were coded so that the animals were always in the same home cage and inhalation cage to minimize the risk of spreading any infections. The control group was handled in the same manner as the test groups. All rats were maintained on Wayne Lab Blox5 prior to and during the inhalation study. This diet contained a minimum of 24.0% crude protein, 6.0% crude fat, and a maximum of 4.5 % crude fiber according to the manufacturer.

Dogs. Male beagle dogs, less than 2 yr in age, were randomly distributed into four groups of four dogs each. During the course of the study, appetite, body functions, and general behavior of each dog were observed daily. Their food consumption and body weights were measured once each week. Water was available ad libitum when the dogs were not in the inhalation chambers. The dogs were fed 4 cups of wet Friskie@ Mix daily at the conclusion of the 6-hr inhalation period. Each dog was moved from home cage to exposure cage and back each day. Dog and cage were numbered so that the same dog was always in the same inhalation cage and the same home cage. The control dogs were handled and exposed to room air each day in parallel with the test dogs.

The Friskies Mix dog diet contained a minimum of 23.0 % crude protein, 7.0 % crude fat, and a maximum of 5.0% crude fiber, 10.0% ash and 12.0% moisture according to the manufacturer’s analysis.

Analytical Methodfor Monitoring the Material

Gas chromatograms of the hydrocarbons were obtained on a temperature pro- grammed gas chromatograph using various liquid phases and column lengths. An F&M 770 model gas chromatograph, converted to direct injection for two columns within one oven was used. Each column was equipped with a Beckman GC2A flame ionization detector, an electrometer, and a recorder which provided two independent analyses with a single program run.

The analytical procedure developed depended upon measurement of peak height of a minimum of two principal peaks with reasonable resolution and good symmetry characteristics. Of the peaks chosen for measurement, one appeared early and another late in the chromatogram. Further selection of measurement peaks depended on symmetry and resolution from other components of the mixture. Attainment of the same mg/liter for both peaks demonstrated the composition of the material in air to

5 Allied Mills, Inc., Chicago, Ill. 60606. 6 Carnation Co., Los Angeles, Calif. 90036.

Page 12: Petroleum hydrocarbon toxicity studies: I. Methodology

HYDROCARBON TOXICITY METHODS 257

be the same as the original starting material. Calibration curves were constructed from solutions of known weight per unit of volume of the material dissolved in a suitable solvent. Microliter samples were injected into the chromatograph at three concentrations covering the range of analysis. Vapor-air samples, taken volumetrically from the chambers, were injected directly into the chromatograph by means of randomly selected gas-tight syringes. Standards were run each day to verify the analytical repro- ducibility of the calibration curves. Reference chromatograms were placed on file in the Chemical Hygiene Laboratory of Carnegie-Mellon University for future reference if required.

Calibration Technique

A IO-ml volumetric flask was filled to approximately 80% of its capacity with a predetermined solvent and weighed. Using a microliter syringe, an appropriate amount of API sample was added to the flask and immediately reweighed to determine the weight of the sample added. Next, enough solvent was added to bring the volume up to the mark and the solution was thoroughly mixed, using wrist action. One microliter of this standard solution (weight/volume) was then injected into the gas chromatograph and peak heights from the resulting chromatogram were used to plot the standard curve.

Efect of sample size on peak height. The effect of variation in sample size of hydro- carbon calibration standard solutions was investigated because it was deemed necessary to verify the use of liquid calibration standards in the determination of a wide range of air concentrations. Several weight/volume standards were prepared in a suitable solvent with the concentrations differing by factors of 2, 5, and 10. The samples were injected directly into the gas chromatograph and peak heights measured.

It was found that a l-p1 sample of the mixture produced the same peak height as 10 ~1 of a 1: 10 dilution of the sample. The same effect was shown for several other concentrations as long as the same total mass per total volume was injected into the chromatograph. Solvent volumes in excess of 10 ~1 were found to cause distortions.

Estimation qf the Mean Molecular Weight of Hydrocarbon Mixtures

An estimation of the mean molecular weight of the hydrocarbon mixtures was made by inspection of mass spectral data and American Society for Testing and Materials distillation data (unless supplied by the producer) as outlined below (Table 2):

1. A partial listing of possible isomers for each mass spectral group was made by correlating boiling point and carbon number from analysis data.

2. The mean molecular weight for these selected isomers was then obtained and used as the molecular weight for those compounds boiling in the specified range.

3. These data were converted to a weight percent basis. 4. The weight percent of each boiling range was multiplied by the corresponding

molecular weight of each boiling range to arrive at a percent weighted mean. This information (Table 2) was also used to provide a rough estimate of the con-

centration in parts per million (ppm) for those not facile or familiar with the more accurate method of reporting concentrations of these mixtures in mg/iiter or /cubic m of air. The values in ppm are probably dependable only to two places, and to forestall fictitious accuracy, should be rounded to reflect this fact.

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258 CARPENTER ET AL.

TABLE 2

SAMPLE CALCULATION FOR COMPARISON WITH DATA PUBLISHED BY NAU et al. (1966)

Mol wt Mol % wt % wt % Component (assumed) (given) G (alkyl benzenes) (chemical groups)

C9-Clo Fraction, boiling range = 31 l-392°F

Paraffins 13.5 20 27.00 20.86

Cycloparaffins 145 6 8.70 6.72

Alkyl benzenes 72.41 C9 120 42 50.40 38.95 Cl0 134 29 38.86 30.03 Cl1 148 3 4.44 3.43

- Total 100 129.40 72.41 99.99

% Aromatics = 72.41 wt % Estimated mean mol wt = 136 (vs Nau’s 125 value)

Cl,-C,, Fraction, boiling range = 392480°F

Paraffins 148 6 8.88 Alkyl benzenes

c9 120 7 8.40 5.63 C 10 134 10 13.40 8.99 Cl1 148 24 35.52 23.83 C 12 160 5 8.00 5.37 C 13 172 1 1.72 1.15

Indans 150 27 40.50

Naphthalenes 142 23 32.66 -

Total 103” 149.08 44.97

% Aromatics = 44.97 + 27.17 + 21.91 = 94.05 wt % Estimated mean mol wt = 147 (vs Nau’s 150 value)

5.96

44.97

27.17

21.91

100.01

a This is not an addition error; it is based on Nau’s figures.

Clinical Test Procedures Blood-Urea-Nitrogen. Urograph, folder 3006250, urea nitrogen assay system,

General Diagnostics Division, Warner-Chilcott Lab., Morris Plains, N.J. Alkalinephosphatase. The calorimetric determination of serum alkaline phosphatase,

Sigma Technical Bulletin no. 104 (revised March 1963), Sigma Chemical Co., St. Louis, MO.

SGOT and SGPT. The calorimetric determination of glutamic oxalacetic and glutamic pyruvic transaminases at 490-520 nm in serum or other fluids, Sigma Technical Bulletin no. 505, Sigma Chemical Co., St. Louis, MO.

Blood glucose. Dextrostix Reagent Strips, Ames Co., Division of Miles Labs, Inc., Elkhart, Ind.

Bilirubin. Determined by the method of Allen (1963).

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HYDROCARBON TOXICITY METHODS 259

Hematology Procedures Hematocrit. Heparinized capillary tube was filled with blood and sealed with

Critoseal.’ Sample was centrifuged for 5 min at 11,500 rpm and percent blood cell volume determined by means of a hematocrit reader.

White blood count-Coulter counter. One hundred milliliters of 0.9 % saline was pipetted into suitable vessel; then 1 .O ml of 0.5 % saponin solution plus 100 ,ul of blood was added and the mixture agitated periodically during next 10 min. A Coulter counter was loaded and readings were made. Background count on 0.9% NaCl-saponin solution was subtracted to correct the final reading.

Differential smears. Blood smears were stained with Wright’s stain (Reich, 1954) and counted by means of the battlement system. A total of 200 cells were identified to determine the percentage distribution of cell types.

Hemoglobin-Fisher hemophotometer (Flo-thru model). Five milliliters of Drabkins’ solution was pipetted into a suitable vessel and 20 ~1 of blood added, thoroughly mixed and allowed to stand for 3-10 min. Sample was placed in a cuvet, read, and hemoglobin recorded as g Hb/lOO ml.

Red blood cell count-Coulter counter. One hundred milliliters of 0.9 % NaCl solution was pipetted into a suitable vessel and 2 ~1 blood delivered by repeatedly rinsing the pipette with vessel contents before mixing by inversion. Readings were made in a Coulter counter subtracting the background count on the 0.9 % NaCl solution. Reduced counts were corrected for coincidence using chart provided. Chart corrected red cell counts were multiplied by 100 to obtain final red cell count.

Reticulocyte count-new methylene blue staining method. Four drops of blood and an equal quantity of stain were left in contact for 15-20 min in a small test tube. A thin film was prepared from the mixture in the same manner as for a differential smear and oil immersion magnification was employed for counting reticulocytes. Fields were selected at random and the total number of erythrocytes and of reticulocytes were counted in each field. When 500 erythrocytes were counted, those reticulated were expressed as a percentage.

Bone marrow impression smear. The femur was removed from the animal and split using a bone-cutting tool. The marrow was gently removed with a surgical knife and placed on a piece of filter paper and an impression smear made directly on the slide. The slide was stained with a modified Wright’s stain (Reich, 1954), examined and interpreted by the pathologist.

Erythrocyte osmotic fragility tests. Five rats inhaled the maximum concentration used in determining the 4-hr acute LC50. Five matching rats, breathing air alone, served as controls. Immediately following exposure, the animals were sacrificed by severing the cervical cord and neck vessels. The blood was quickly collected and one drop added to each of several glass tubes, using a 22-gauge needle. The series of tubes contained saline concentrations that ranged from 0.28 to 0.56% with a 0.02% change from one concentration to the next. One series of concentrations was used for each rat.

The tubes of saline that contained the drop of blood were centrifuged at 2100 rpm for I min. They were then examined visually to determine the highest concentration of saline causing initial and complete hemolysis.

’ Biological Research, St. Louis, MO. or Seal-Ease, Clay Adams, Inc., New York, N.Y. 10010.

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260 CARPENTER ET AL.

The normal ranges for hematological and blood chemistry values for rats and dogs are given in Table 3.

TABLE 3

NORMALRANGESFORHEMATOLOGICALANDBLOODCHEMISTRYVALUES FOR RATS AND DOGS’**

Dog Rat

RBCs millions/mm3 WBCs thousands Hematocrit, % Hemoglobin, g/100 ml blood Reticulocytes, % Basophils, % Eosinophils, % Stabs or Staff cells (band cells), % Neutrophils, % Lymphocytes, % Monocytes, % BUN, mg/lOO ml Blood glucose, mg/lOO ml Bilirubin, mg/lOO ml SGOT, Sigma units/ml SGPT, Sigma units/ml Alkaline phosphatase, Sigma units/ml

0 Coffin (1953). * Brunk (1969). c Mean values only are available.

6.4-8.0 5-10 6-20 8-15

M-55 5oc 12-17.8 15.6” o-1.4 2-5 O-2 O-2 2-10 o-4 O-4.2* e 60-75 8-40 IO-30 50-80 2-12 2-7 10-28.8 15-26* 50-78 e O-O.56 e 15-34s 67-356* 14-4Of 25-60* 0.2-2.6* 1.4-l 1.4*

* Data obtained on 52 beagles and 66 control rats at the Chemical Hygiene Fellowship Laboratory, Carnegie-Mellon University.

e Data not available. f Cramer et al. (1969).

Autopsy and Histological Techniques

Animals were anesthetized using methoxyflurane* for rats and aqueous solution of pentobarbital sodium9 for dogs. The rat cervical cord and neck vessels were severed and the carcass suspended head down until exsanguination was complete. In the anesthetized dog, the aorta and vena cava were severed after laparotomy. The abdominal cavity was opened and the diaphragm incised. The ribs were bisected with cartilage scissors to expose the heart and lungs. All abdominal and thoracic organs were examined grossly for lesions or abnormalities. Representative samples of the following tissues as well as any tissues in which lesions or abnormalities were observed were taken and fixed in 10% formalin solution: lung (12 sections), liver, kidneys, heart, spleen, adrenal, thyroid, parathyroid, trachea, bifurcation of trachea, stomach, duodenum, pancreas, ileum, jejunum, colon, brain, pituitary, skeletal muscle, sciatic nerve, nasal mucosa, pharynx, and tonsil. Both femurs were removed and a bone marrow impression was made. A bone marrow section was embedded and stained for micropathological studies.

8 Pitman-Moore, Inc., Zionsville, Ind. 46077. 9 Diamond Laboratories, Inc., Des Moines, Iowa.

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HYDROCARBON TOXICITY METHODS 261

Five to 6-mm slices of tissue were fixed for 3 days or longer, with daily shaking, in 10% formalin solution (4500 ml 3740% formaldehyde + 13,500 ml distilled water). Tissues were placed in the Autotechnicon and sequentially passed through alcohols, toluenes, and into paraffin. Tissuemat (mp 565°C) was used for embedding via metal molds and a rotary microtome was used for cutting 5- to 6-,um slices of the blocked tissue after hydration in ice water. The tissues were floated on a 43°C water bath to remove wrinkles, mounted on alcohol-cleansed slides, and placed on a warming table at 43°C for no longer than 4 hr. Slides were stained with hematoxylin-eosin stain

(Table 4). After staining was completed, the slides were mounted in Canada balsam, using a cover slip cleaned with alcohol.

TABLE 4 COMPOSITION OF TISSUE STAINS

Aqueous alum hematoxylin ~- ..__ Hematoxylin 2g Distilled water Distilled water 200 ml Thymol Aluminum ammonium sulfate 40 g KMnOJ

Alcoholic eosin

600 ml 2g

0.354 g

Eosin Y 2g Distilled water 160 ml

95 % Alcohol Acetic acid, coned

640 ml 8 drops

Micropathological Interpretation

Finished slides were shipped to our nonresident pathologist who interpreted the slides, without knowledge of concentration of vapor inhaled, and recorded his findings by dictaphone. The records and slides were then returned to the Chemical Hygiene Laboratory for compilation of synoptic tables. After the results had been transposed and tabulated according to concentration, they were returned to the pathologist for final evaluation and comment.

Electrocardiogram Monitoring

Dogs were restrained manually by their handler, needle electrodes were inserted subcutaneously and the electrocardiogram taken while the dog was held on his left side. Extraneous electrical interference was kept to a minimum by placing the dog on a rubber insulating pad.

Electrocardiographic leads I, II, and III were taken both prior to and following the 13-wk inhalation regimen. The lead positions for monitoring the electrical potentials across the heart were as follows: Lead I, left foreleg to right foreleg; Lead II, right foreleg to left hind leg; Lead III, left hind leg to left foreleg. The animals were grounded during the process by a lead placed on the right hind leg.

Electrocardiograms were taken using a model P-5 Grass Polygraph with a suitable amplifier and recorded at a chart speed of 25 mm/set. The unit was calibrated so that a 1-mV current caused a l-cm deflection. The tracings were examined critically for any abnormalities.

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262 CARPENTER ET AL.

Inhalation of Aerosols

Aerosols were produced by a glass Dautrebande D18 aerosol generator operated at a pressure of 30 psi. The resultant droplets, less than 5 pm in size, were conducted directly into the animal chamber. All chamber concentrations were based upon weight loss of the sample and airflow. Liquid hydrocarbon samples were taken from the generator for analysis prior to, during, and following animal inhalation of the aerosol to determine whether components of the sample were being preferentially lost during aerosol generation. A motor-driven syringe was used to meter the hydrocarbon sample into the generator at a rate approximately equal to the rate of delivery.

ACKNOWLEDGMENTS

PARTICIPATING STAFF U. C. Pozzani, Project Manager to July 1970; R. C. Myers, Inhalation Operations;

D. J. Nachreiner, Inhalation Operations; C. R. Humes, Equipment Construction and Main- tenance; P. A. Baker, Gross Pathology, Histology, and Hematology; P. A. Crawford, Histology and Clinical Chemistry; M. A. McGee, Histology; G. J. Haines, Technical Assistance; K. L. Wells, Technical Assistance; J. M. Eldridge, Analytical Procedures; L. W. Keller, Analyst to September 1971; S. J. Kozbelt, Analyst; C. S. We& Statistical Design; R. C. Myers, Statistical Analyses; M. D. Woodside, Statistical Analyses, Animal Husbandry, and Technical Assistance; N. S. Bellich, Animal Care; and M. J. Cardella, Animal Care.

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ALLEN, T. E. (1963). The direct reading newborn bilirubin method as used in our routine clinical laboratory. Amer. J. Med. Tech. 29, 395-396.

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CARPENTER, C. P., SMY~H, H. F., JR. AND POZZANI, U. C. (1949). The assay of acute vapor toxicity, and the grading and interpretation of results on 96 chemical compounds. J. Znd. Hyg. Toxicol. 31, 343-346.

COFFIN, D. L. (1953). Manual of Veterinary Clinical Pathology, 3rd Ed. Comstock Publishing Co., Inc., Cornell University Press, Ithaca, N.Y.

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