Petroleum hydrocarbon toxicity studies

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

Petroleum XII.

Animal

and

36,451-412

Hydrocarbon Human

Response

(1976)

Toxicity to Vapors

Studies of “40 Thinner”‘p2

C. P. CARPENTER, D. L. GEARY, JR., R. C. MYERS, D. J. NACHREINER, L. J. SULLIVAN,AND J. M. KINGS The Chemical Hygiene Fellowship, Carnegie-Mellon Institute of Research, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213 Received June 2,1975; accepted December 22,1975

Petroleum Hydrocarbon Toxicity Studies. XII. Animal and Human Responseto Vapors of “40 Thinner”. CARPENTER, C. P., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., AND KING, J. M. Toxicol. Appl. Pharmacol. 36, 457-412. The suggestedhygienic standard for “40 Thinner”, basedupon the results of inhalation studieswith rats and dogsand the sensoryresponseof human subjects,is 0.15 mg/liter (25 ppm). Ratstolerated 0.20mg/liter (33ppm) anddogs0.25mg/liter (41ppm) for 7 and 8 hr, respectively,without visible discomfort. Inhalation by rats for 7 hr of a
457

458

CARPENTER ETAL.

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 “40 Thinner” was furnished by one of the major producers in the United States and was assigned Chemical Hygiene Fellowship No. 35-173. Pertinent physicochemical properties are presented in Table 1 and composition in Table 2. The minor differences between mass spectral and glc data presented in Table 2 probably result from differences in data acquisition and their calculation. TABLE 1 PHYSICOCHEMICALPROPERTIESOF “~~THINNER”

Boiling Range’ Percent

Temperature (“F)

I.B. pt 5 10 20 30 40 50

368 376 378 379 381 384 387

FlashPointb Refractive Index’ SpecificGravityd Mean Mol Wt’ At 25°C and 760 mm:

Percent

Temperature C’F)

60 70 80 90 95 End pt.

390 394 401 414 428 447

120°F(tag closedcup) 1.4583at 20°C 0.8198at 60/6O”F 148 1 mg/liter = 165.2ppm 1 ppm = 0.00605mg/liter

aDeterminedby ASTM methodD-86. bDeterminedby ASTM methodD-56. cAbbe-typeinstrument. 1Determinedby ASTM methodD-1298. eCalculatedfrom massspectrography data. Analytical methodfor monitoring “40 Thinner”. 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 response 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 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.

VAPOR

INHALATION

OF

“40

459

THINNER”

TABLE 2 COMPOSITION OF “40 TH!NNER”

MassSpectralAnalysis (Weight percentage) Carbon number

Paraffins

c7 CL3 G GO Cl1 Cl2 Cl3 Subtotals

0.2 0.2 0.5 2.0 18.7 9.8 4.0 35.4

Monocycloparaffins Dicycloparaffins Alkyl benzene

23.3

0.2 1.0 1.1 11.8 7.7 1.4 0.4 23.6

9.6

Indansand Tetralins Naphthalenes Cycle-oleflnsand Diolofins Benzene

5.0 2.9 0.2 0.0

glc Compositionalanalysis(Weight percentage) Carbon number Paraffins Cycloparathns Alkyl benzenes C9 C10 Cl1 G2 C13

0.76 2.28 14.69 7.13 1.01

0.35 13.38 2.59

5.14 21.46 12.63 1.98

Unknowns (mostly Cl1 and Cl* aromatics) Naphthalene 1,2,3,4-Tetrahydronaphthalene(tetralin) Indane

14.38 1.44 0.45 0.33

TABLE 3 GAS CHROMATOGRAPHIC PROGRAM FOR “40 THINNER”’

Column Coating Support Conditions Solvent for calibration Carrier gas Burner Vapor samplesize Lower limit of detection

Coppertubing, +-in. o.d. x 13 ft 10% Poly I-l 10(Applied ScienceLabs, State College,Pa.) Gas ChromeQ, 80/100mesh Temperaturelimits = 125to 175”C, programmedat 2.5”C/ min with a IO-min upper limit delay Methylene chloride ‘ Helium Beckman: Hydrogen at 45 ml/min, air 450 ml/min 2 to 10ml 0.1 Pg

aA temperature-programmed gaschromatograph equippedwith a flameionizationdetectorwas usedfor all analyses.

460

CARPENTER

ET AL.

PrPcis of experimental design. A stepwise approach was undertaken to determine a no-ill effect level by vapor inhalation in rats and dogs. Rats were subjected first to a I-hr vapor inhalation experiment and this was followed by a IO-day, Sday/week, 6-hr/day vapor inhalation study with rats and dogs. No deviation from prior practice was instituted in the 65day repeated vapor inhalation study of rats and dogs nor in the human sensory studies. In these endeavors, the object was to evaluate a vapor phase as truly representative of the liquid phase as possible. However, because of the low volatility and the lack of acute toxicity in the vapor phase, aerosol concentrations were substituted for vapor in some of the acute studies. Both vapor and aerosol phases were studied by the mouse respiratory irritation technique of Alarie (1966). For the aerosol studies, droplets of05 to l-pm size were verified as described in Carpenter et al. (1976k). RESULTS Acute Inhalation Toxicity Acute “no ill effect level”. Rats. A group of 16 male Harlan-Wistar 90- to 120-g rats was subjected for 7 hr to a metered 0.5 mg/liter which produced a measured concentration of 0.20 mg/liter (33 ppm) of “40 Thinner”. This concentration represents substantial saturation of air with this vapor at room temperature. The rats appeared normal throughout the inhalation period. Only a few sporadic lesions were found in those sacrificed either immediately after the exposure, after 2 days, or after 14 days. Nothing remarkable was found in bone marrow impression smears. Dogs. In separate trials, two female beagles, each weighing about 13 kg, were subjected to measured vapor concentrations of 0.25 (41 ppm) and 0.26 mg/liter (43 ppm) for 8-hr periods. Both animals appeared normal throughout the exposures. One lost 0.2 kg and the other was unchanged 24 hr after the exposure. Exposure chamber temperatures were roughly 20°C. Ten-day repeated exposure to rats and dogs. Two beagles plus 10 female HarlanWistar rats were exposed 6 hr/day, 5 days/week, for 2 weeks to a mean measured concentration of 0.19 mg/liter (31 ppm). All animals appeared normal throughout the trials and during the subsequent observation period. Chamber temperatures averaged between 23 and 24°C. Mean weights of the 10 exposed rats were 109 g initial and 186.3 g final, and for the controls 110 vs 181.6 g. One beagle gained 0.4 kg and the other lost 0.9 kg in weight during this 2-week period. Cats. Three cats were subjected to a 7.0 mg/liter aerosol of cl-,nm droplets. During the 6-hr exposure, throughout which a visible fog was present, the three cats had wet noses and wet fur. One of the three died the next day but immediate postexposure placing reactions, righting reflex, response to foot pad pain, extensor thrust reaction, and pupillary contraction were normal for this animal as well as the other two. One of the surviving cats had a slight diarrhea 24 hr after exposure and the other a discharge from the left eye. These findings are not related to the exposure because the diarrhea reoccurred on postexposure Day 5 after disappearing on Day 2 and only one eye of the other cat had the discharge. Short-term massive concentration. After finding no signs of distress among rats inhaling saturated vapor for 7 hr, another group of rats was subjected to an 8-hr inhalation period in an aerosol of 8.3 mg/liter. The liquid “40 Thinner” was metered to

VAPOR

INHALATION

OF

“40

THINNER”

461

the nebulizer by means of a Monodrum driven syringe at a rate of 0.213 ml/min. The aerosol was delivered to a 60-liter glass cube containing 10 male albino Harlan-Wistar rats, having a mean weight of 118 g. Five were sacrificed after 7 days and five after 14 days. The mean weight gain of the 1Cday survivors was 99 g vs 89 g for nontreated controls. Early nasal irritation (evidenced by the animals rubbing their noses) was followed within 5 hr by wet fur, irritated extremities, slight loss of coordination, and hyperactivity. After 3 days all rats appeared completely normal. Lung weights of treated rats, recorded at sacrifice, were not statistically significantly different from their controls. Osmotic erythrocyte fragility. There was no statistically significant difference in red blood cell fragility between five rats that inhaled 8.3 mg/liter of “40 Thinner” for 7 hr and five nonexposed control rats. Mean initial and complete hemolysis of red cells of the treated rats occurred in 0.47 and 0.30% saline solution, respectively, and in 0.45 and 0.30 % solutions for controls. Mouse upper respiratory tract irritation. Preliminary tests indicated “40 Thinner” vapor to be nonirritating to the upper respiratory tract of mice at a metered concentration of 0.5 mg/liter (83 ppm) approximating the highest attainable vapor-phase concentration. For that reason, aerosols of “40 Thinner” were generated by means of a Dautrebande generator. Concentrations were varied by adjusting the chamber dilution air flow. Five of six mice registered a 50 % or more depression in respiratory rate when they inhaled a metered aerosol concentration of 8.7 mg/liter. A similar response was noted in six mice inhaling 4.4 mg/liter. In this exposure, one mouse died within a minute after exposure, probably because of strangulation by the restraining device. During inhalation of 2.7 mg/liter of “40 Thinner”, two of six mice exhibited a 50 % or greater depression of respiratory rate. Further exposures were deemed impractical, since very large volumes of dilution air would be necessary to produce significant concentration decrease. Subacute Inhalation Toxicity

To determine the response of rats and dogs to repeated daily inhalation in order to provide a basis for establishing acceptable inhalation standards, groups of 25 male rats and 4 male beagles were assigned randomly to each of three graded levels of “40 Thinner” and to a solvent-free air-control. Three rats from each level were sacrificed for histological examination after 14- and 44-day intervals, and four after 65 days. The challenge exposure, described in Carpenter et al. (1975a), was obviated since rats and dogs inhaled the highest attainable concentration of 0.22 mg/liter (36 ppm) for 13 weeks without gross signs of toxic effects. Therefore, the remaining rats and all beagles were killed after 65 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 oxalacetictransaminase activity(SGOT), 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

462

CARPENTER

ET AL.

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 sacrifice at 66 days. Preliminary blood values were not established for the rats because not enough blood could be taken to accomplish the tests without debilitating the animal. The values for the treated rats were compared to the values of the air-controls concomitantly sacrificed at the 14-, 44-, and 65day 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 the 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 the 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, duodenum, pancreas, ileum, jejunum, colon, skeletal muscle, sciatic nerve, and bone marrow. Similar tissues were collected from the dogs at sacrifice, with the addition of the bifurcation of the trachea, pharynx, tonsil, nasal mucosa, and stomach. Analyses of the metered 0.5, 0.25, and 0.125 mg/liter vapor: air mixtures yielded mean measured concentrations of 0.22, 0.10, and 0.05 mg/liter, respectively, by gas chromatography as shown in Table 4. These concentrations correspond to 36, 17, and 8 ppm based on mean molecular weight as determined by mass spectrography. TABLE 4 GAS

CHROMAT~GRAPHIC

ANALYSIS OF “40 THINNER” RATS AND Does FOR

Number of analyses Metered concentration (mg/liter) Measured concentration (mg/liter) 95% Fiducial limits for measured concentrations (mg/liter) Measured concentration (ppm) Measured as percentage of metered concentration Coefficient of variation

VAPOR

CONCENTRATIONS

INHALED

BY

13 WEEKS 104 0.5 0.22

105 0.25 0.10

97 0.125 0.05

48 0.0 0.0

0.10-0.33 36

0.05-0.14 17

0.03-0.08 8

0

44.0 26.0

40.0 22.6

40.0 24.5

-

Ruts. Hematocrits from rat blood taken at 8 weeks were on the borderline of statistical significance for the two upper levels; both were slightly higher than the controls. After 13 weeks at 0.22 mg/liter (36 ppm), hemoglobin was decreased by 0.4 g/l00 ml, as compared to the control mean. This is presumed to be a statistical artifact caused by the almost identical values for rats on this, the highest level. Certainly it is not an adverse finding as the controls are numerically higher than the other two levels as well, but they were not statistically significantly different (Table 5). Blood urea nitrogen values for the 0.22- and 0.05-mg/liter levels were slightly lower than the tightly bound control values after 13 weeks. Again we can see no real deviation from essentially normal values throughout and, therefore, consider the findings to be valid statistical deviations but not meaningful effects (Table 6).

3 8 13

83 13

3 8 13

0.10 (17)

0.05 03)

0.0 (0)

“0.05 >p> 0.01. bO.O1 >p>O.ool.

3 8 13

0.22 (36)

Period (Weeks)

HEMATOLOGICAL

Concn mg/liter (ppm)

MEAN

6.73 7.25 7.82

6.66 8.04 7.72

6.73 8.14 7.78

6.81 8.14 7.74

RBC (millions/ mm3)

FINDINGS

13.1 13.7 12.7

13.3 13.6 13.7

13.1 12.4 12.6

11.4 12.3 13.5

WBC (thousands/ mm3)

FOR GROUPS

40.8 40.1 44.6

38.6 43.0 42.2

40.1 44.9” 42.4

42.3 44.6 43.6

OF THREE,

15.3 16.1 15.8*

15.0 14.5 16.2

14.3 15.5 15.7

14.6 16.0 15.4

TABLE

5

2.53 2.73 2.10

2.13 3.00 2.05

2.07 3.40 2.20

2.87 2.53 2.15

Reticulocytes (%I

AND FOUR RATS THAT RESPECTIVELY

(g/lFFrn*)

THREE,

0 0 0

00 0

0 0 0

0 0 0

Baso

VAPOR

FOR

3.0 6.7 4.5

3.3 5.0 3.5

5.7 5.7 4.8

3.7 5.3 3.8

5.3 3.7 6.5

4.3 6.0 5.2

5.7 6.7 7.2

5.7 5.3 6.5

neutro

Immature

56.7 47.7 55.5

52.7 55.7 57.2

60.3 51.7 57.2

52.3 45.0 61.8

Neutro

129.7 134.7 126.2

134.0 128.0 129.0

122.3 131.0 124.2

131.3 135.3 121.5

Lymph0

4.7 7.0 6.2

5.3 5.0 4.8

5.7 4.0 6.0

6.0 8.3 6.0

Mono

3,8, AND 13 WEEKS,

Based on a 200-cell count. To arrive at a percentage, divide by 2.

“40 THINNER”

Eosino

INHALED

3 gz

: % 2

2 g

F2 5

8

5

464

CARPENTER

ET AL.

TABLE MEAN

6

BLOOD CHEMISTRY FINDINGS FOR GROUPS OF THREE, THREE, AND FOUR RATS INHALED “40 THINNER” VAPORS FOR 3, 8, AND 13 WEEKS, RESPECTIVELY

SGOT

Alkaline phosphatase (Sigma

mg/liter (ppm)

Period (Weeks)

BUN (mg/lOO ml)

units/ml)

SGPT (Sigma units/ml)

0.22 (36)

3 8 13

20.2 22.0 19.8“

271.7 270.0 263.8

36.7 42.0 46.5

9.42 4.50 6.11

0.10 (17)

3 8 13

18.2 22.2 18.2

211.7 188.3 176.2

34.0 34.7 37.5

6.70 7.23 4.28

0.05

3 8 13

21.5 20.0 19.9”

226.7 291.7 251.2

35.7 42.0 48.2

6.94 5.87 4.84

3 8 13

20.2 19.5 20.8

206.7 175.0 290.0

36.0 35.7 51.5

8.16 6.15 6.49

Concn

63)

0.0 (0)

(Sigma

THAT

units/ml)

“0.05 >p > 0.01.

Slight tubular regeneration of the rat kidneys was reported by the pathologist at each sacrifice. As there were no cases of moderate or marked response, the finding is considered to be representative of the endemic conditions present in the Harlan-Wistar rats used for these studies and not related to the inhalation of the “40 Thinner” vapor. Bile duct proliferation was noted in rat liver at each sacrifice. At no time did the incidence become statistically significant nor did the pathologist find any reason for the apparent dosage-relationship seen after 8 and 13 weeks. It is noteworthy that we had an incidence of this condition in 8 of 10 of the control rats used for the acute 7-hr exposure at a measured 0.2 mg/liter while among those exposed at this level the incidence was 5 of 9. It is evident that this condition is endemic in the Harlan-Wistar rats used in this series of studies. Bone marrow impression smears were interpreted as normal by the pathologist. No statistically significant differences in body weight or body weight change between treated and control groups were found (Table 7). Dogs. The change from preexposure values of the number of immature neutrophils in dog blood at the 0.22 mg/liter level was statistically significant. However, the original preexposure count of immature forms was statistically significantly lower than control values and, therefore, we believe the final increase was forced by this fact. No toxicological significance is attached to this finding because the values in question are all within the normal range for beagles (Table 8). Bilirubin values were statistically significantly lower than control values after 13 weeks at the 0.22 and 0.10 mg/liter levels (Table 9). In addition, the change from preexposure value indicated a statistically significant decrease in serum bilirubin after

24.1 60.9

5.4 24.6 34.8 44.0 49.5

2.1 2.4

0.2 0.6 0.4 0.2 0.4

257.8 556.3

Body weight changes (g) 4 23.3 19 133.5 34 205.5 47 257.4 62 299.8

8.8 9.1

Body weight (g) 0 62

Body weight (kg) 0 62

Body weight changes (kg) 4 0.0 19 0.0 34 0.4 47 0.5 62 0.2

SD

Mean

Days of expbsure

0.22

MEAN

WEIGHTS

0.2 -0.2 0.2 0.3 0.3

9.4 9.7

24.6 142.5 214.2 266.7 312.1

261 .O 574.0

Mean

0.10 SD

0.1 0.4 0.3 0.4 0.7

1.7 2.3

5.9 21.1 29.5 36.6 42.7

7 CHANGES

0.1 0.2 0.7 0.6 0.5

10.1 10.6

Dogs

25.2 147.4 204.1 264.1 297.1

259.3 564.0

Rats

Mean

0.05

0.1 0.2 0.1 0.2 0.2

1.3 1.5

6.1 31.5 35.4 39.1 54.8

22.7 46.6

SD

“40 Thinner”

WEIGHT

TABLE

(mg/liter)

AND BODY

24.8 56.1

Concentration

BODY

0.0 0.3 0.4 0.2 0.6

9.8 10.4

23.4 139.3 199.0 255.2 297.6

248.2 547.5

Mean

0.0

0.1 0.5 0.6 0.7 0.9

1.2 1.8

5.3 22.4 33.6 40.9 38.0

21.6 37.4

SD

FOR RATS AND DOGS

2.72 2.21 7.66 5.51 5.58

1.14 0.74

0.80 4.33 0.84 1.13 2.20

0.50 4.64

Homogeneity of variance (x2)

0.74 1.25 0.91 1.02 0.31

0.44 0.43

0.63 1.18 0.78 0.34 0.44

1.49 0.92

Analysis of variance

“0.05 >p > 0.01. bO.O1 >p > 0.001.

11.6 11.8

Preexposure Plus 60 days Change from preexposure

0.0 (0)

7.40 8.04

Preexposure Plus 60 days Change from preexposure

0.05 (8)

9.3 9.0

11.5 10.4

7.80 7.70

Preexposure Plus 60 days Change from preexposure

0.10 (17)

8.07 7.44

11.4 11.5

7.46 7.12

Preexposure Plus 60 days Change from preexposure

0.22 (36)

.-

49.0 55.1

55.0 51.5

53.0 52.4

50.9 52.5

FOR GROUPS

WBC (thousands/ mm3)

FINDINGS

RBC (millions/ m3>

HEMATOLOGICAL

Concn mg/liter (pm)

MEAN

-.

18.3 20.0

20.0 19.0

19.3 19.0

18.8 19.1

Hb (gtly

OF FOUR

THAT

0.95 1.75

1.oo 1.75

1.15 2.00

0.95 1.80

“40

0 0

0 0

0 0

0 0

Baso

INHALED

Reticulocytes (%I

DOGS

TABLE 8 VAPORS

FOR

13 WEEKS

7.0 4.0

7.2 5.2

4.2 6.5

Eosino 4.5 5.8

-1.0

8.5 7.5

-1 .o

8.2 7.2

-0.5

8.2 7.8

4.2b

4.8“ 9.0

Immature neutro

106.5 114.0

112.2 116.5

112.5 115.8

111.8 115.2

Neutro

71.0 68.0

64.5 63.2

67.8 62.0

70.8 61.5

Lymph0

Based on a 200-cell count. To arrive at a percentage, divide by 2.

THINNER”

6.5 6.2

6.8 6.8

Mono

OF GROUPS

c 0.01 > p > 0.001.

used.

15.1 17.8

Preexposure Plus 60 days Change from preexposure

0.0 (0)

E Rank sum test of significance * 0.05 > p > 0.01.

14.1 17.8

Preexposure Plus 60 days Change from preexposure

0.05 (8)

14.0 14.6

Preexposure Plus 60 days Change from preexposure

BUN (mg/lOO ml) ~__ 14.2 17.9

FINDINGS

0.10 (17)

CHEMISTRY

Preexposure Plus 60 days Change from preexposure

BLEND

0.22 (36)

Concn mgjiter (ppm>

MEAN

TABLE 9 FOUR Does

127.5 128.8

125.0 131.2

126.2 128.8

126.2 130.0

Blood glucose” (mg/l~ ml>

OF

INHALED

0.33 0.36 0.04

0.27 0.35 0.08

0.32 0.19’ -0.14b

0.21 0.23* 0.02

Bilirubin (mg/lOO ml)

THAT

THINNER”

17.5 22.2

16.5 18.0

14.8 18.2

19.0 18.8

SGOT (Sigma units/ml)

“40 FOR

15.8 22.2

19.2 18.2

19.0 18.8

17.8 18.5

SGPT (Sigma units/ml)

13 WEEKS

1.20

1.23

0.86 0.98

1.06

0.93

0.90 0.93

Alkaline phosphatase (Sigma units/ml)

ORGAN

0.42

Kidney weight (% body weight)

260.8 2.89

9.1

Mean

OF GROUPS

38.8

WEIGHTS

Kidney weight (g)

Liver weight (g) Liver weight (% body weight)

Body weight (kg)

MEAN

0.04

11.5

62.0 0.14

2.4

“40 THINNER”

0.43

40.5

297.2 3.14

9.7

Mean

0.10

0.09

7.9

52.2 0.64

2.3

SD

0.40

41.2

294.2 2.80

10.6

Mean

0.05

0.08

8.0

0.40

41.5

280.5 2.74

10.4

__. Mean

FOR 13 WEEKS

48.2 0.42

1.5

SD

VAPOR

Concentration (mg/liter) “40 Thinner”

AFTER THEY INHALED

TABLE 10 BEAGLES

SD

FOUR

0.22 ____

OF

0.0

0.06

2.4

21.5 0.46

1.8

SD

$

0 % IT+ 5 B yh

VAPOR

INHALATION

OF

“40

469

THINNER”

13 weeks at the O.lO-mg/liter level. However, all values are within the normal range for dogs and as metabolic and liver dysfunction are associated with hyperbilirubinemia (an increase rather than a decrease in serum bilirubin), the statistically significant bilirubin values are considered an expected chance occurrence. Such findings are expected to occur occasionally when p = 0.05 is employed as the fiducial level of significance. No statistically significant differences in body weight, body weight change, or organ weights between treated and control groups were found (Tables 7 and 10). No treatment-related lesions were observed during gross autopsy after 13 weeks nor during micropathological evaluation of tissues. 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 57 % 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 approx. 10 set) in the following sequence: 0.02, 0.00,0.002,0.0002,0.0002,0.02, 0.00, and 0.002 mg/liter. The responses are summarized in Table 11. A corrected concentration of 0.002 mg/liter TABLE 11 ODORDETECTIONAND

SENSORY THRESHOLD TO “40 THINNER"(HUMAN

SUBJECTS)

Odor threshold Metered concentration (mg/liter) Corrected concentration (mg/liter) Correctedconcentration (ppm) Number detecting odor/number of subjects (two trials)

0.05 0.02 3.3

0.005 0.002 0.33

0.0005 0.0002 0.033

0.00 0.00 0.0

12/12

9/12

4112

l/l2

Conclusion: The odor threshold lies between0.0002and 0.002 mg/liter, the most probable concentration being0.001mg/liter or 0.17 ppm. Sensorythreshold Metered concentration (mg/liter) 0.5 0.5 0.25 Measuredconcentration(mg/liter) 0.12” 0.21 0.10 Measuredconcentration(ppm) 20 35 17 First Exposureorder for six subjects Third Second Number detectingodor 6 6 6 0 Number with slight olfactory fatigue 0 3 Number tasting “something” 3 2 3 Number reporting dizziness 1 0 0 2 Number willing to work in concentration for 8 hr 4 6 Conclusion: A concentration of 0.21 mg/liter would not be expected to causeirritation, although somehumansmay find the odor disagreeable in the working environment. uLow valuedueto analyticaldifficulties 17

470

CARPENTER ETAL.

(0.33 ppm) was readily perceived, as indicated by the 75% incidence of detection. Volunteers were able to detect 0.0002 mg/liter (0.033 ppm) with an incidence of 33 % (Table 11). The odor threshold lies between these two values with the most probable concentration being 0.001 mg/liter or 0.17 ppm. Sensory thresholds. All concentrations were determined by gas chromatography. In order to determine the sensory response, six volunteers between 23 and 63 years of age inhaled measured vapor: air concentrations, for 15min periods, in the following order: 0.12,0.10, and 0.21 mg/liter (20, 17, and 35 ppm, respectively). Exposures were limited to one per day to prevent any build-up of symptoms. Responses are summarized in Table 11. There were analytical difficulties (low results) during the first 15-min inhalation period at a metered 0.5 mg/liter, so the 0.5 mg/liter trial was repeated. The responses from the first trial are recorded, nevertheless, because the final judgment as to acceptance was the same in both cases. In the first trial of a metered 0.5 mg/liter (0.12 mg/liter measured) all subjects reported a strong odor but little or no olfactory fatigue. Two reported a dry feeling of the eyes and one claimed a slight dizzy feeling after 11 min which another subject reported as a feeling of pressure behind his left eye. All symptoms were transitory and no nausea resulted from “tasting” the material. Only two of the six subjects thought they could work in this concentration. During the second trial metered at one-half the concentration of the first (0.10 mg/ liter measured), three persons thought they had some olfactory fatigue, but all could detect the odor at the end of the 15-min exposure. Three reported they could taste the chemical but not in an attitude of complaint. All agreed they could work in this concentration. Upon repeating the metered 0.5 mg/liter (0.21 mgfliter measured) there were fewer symptoms reported and four of six said they could work in this concentration while another said he would if it were demanded of him. A summary of the results appears in Table 12.

DISCUSSION The amount of “40 Thinner” in air saturated at an ambient room temperature of 25°C is on the order of 0.25 mg/liter (41 ppm). No deaths among rats or dogs resulted at this limiting concentration in single exposures of 7- to 8-hr duration. Indeed, no illeffects were observed in either rats or dogs during 10 repeated 6-hr/day exposures within 2 weeks at 0.19 mg/liter (31 ppm). It is not surprising that no dosage-related ill effects were found in either species during or at the conclusion of 65 exposures, 6 hr/day, 5 days/week, at concentrations of 0.22, 0.10, 0.05, and 0.0 mg/liter corresponding to 36,17,8, and 0 ppm. To investigate the effect of an aerosol of about l-pm or less droplet-size, rats were subjected to 8.3 mg/liter for 8 hr with mild loss of coordination but no deaths during the 14day observation period. Another group of rats had no evidence of osmotic erythrocyte fragility following 7 hr in a similar concentration of aerosol. Cats had no visible signs suggestive of central nervous system depression during 4 hr in a similar aerosol exposure at 7.0 mg/liter.

VAPOR

INHALATION

OF

“40

471

THINNER”

TABLE 12 RECAPITULATION

OF “40

THINNER”

0.2 mg/liter (33 ppm) Not attainable since saturated vapor did not cause death 0.25 mg/liter (41 ppm)

Acute 7-hr no ill effect concn, rats Rat LC50 Acute 8-hr no ill effect concn, dogs lo-Day repeated inhalation, no ill effect concn, rats and dogs

0.19 mg/liter (31 ppm)

13-Week subacute inhalation, rats and dogs Significant findings

Concn (mgfliter)

Blood hematocrit, 8 weeks, rats Blood hemoglobin, 13 weeks, rats Blood urea nitrogen, 13 weeks, rats

0.22; 0.10 0.22 0.22; 0.05

Bilirubin values, 13 weeks, dogs Immature neutrophils, 13 weeks, dogs

0.22; 0.22

Challenge exposure, rats

Odor threshold, human volunteers Sensory thresholds, human volunteers Acute 4-hr inhalation of aerosol, cats Erythrocyte

fragility, rats

Short-term massive concn., rats Mouse respiratory tract irritation

Suggested hygienic standard

0.10

Assessment by CHF Not seen at 13 weeks, ignored Statistical nuance, ignored Statistical significance forced by control values, ignored Chance occurrence Forced by low preexposure count

Eliminated because 0.22 mg/liter was the highest attainable concentration and rats had inhaled that concentration for 13 weeks without adverse effect Between 0.0002 and 0.002 mg/liter, the most probable concentration is 0.001 mg/liter (0.17 mm) 0.21 mg/liter (35 ppm) did not cause adverse effects but was considered disagreeable by two of six 7.0 mg/liter; signs of CNS depression were not observed; no deaths 8.3 mg/liter aerosol; 7-hr inhalation caused no statistically significant differences in hemolysis 8.3 mg/liter aerosol; 8-hr inhalation caused mild coordination loss, but no deaths Fifty percent respiratory rate depression in five of six mice inhaling a 4.4 mg/liter aerosol; similar effect in two of six inhaling 2.7 mg/liter; vapor at metered 0.5 mg/liter was nonirritating 0.15 mg/liter (25 ppm) based on human and animal data

Human volunteers considered 0.21 mg/liter (35 ppm) of vapor disagreeable in a 15min inhalation period but had no upper respiratory tract or eye irritation at 0.10 mg/liter (17 ppm). This response leads to the suggested hygienic standard of 0.15 mg/liter (25 mm>.

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ET AL.

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, 433-449.

C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING J. M., (1975a).Petroleumhydrocarbontoxicity studies.I. Methodology. Toxicol. Appl. Pharmacol. 32,246-262. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING, J. M. (1975b).Petroleumhydrocarbon toxicity studies.II. Animal and humanresponseto 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).Petroleumhydrocarbon toxicity studies.III. Animal and humanresponseto 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. 33,526-542. 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. (19750.Petroleumhydrocarbon toxicity studies.VI. Animal andhumanresponseto vapors of “60 Solvent”. Toxicol. Appl. Pharmacol. 34, 374394. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., SULLIVAN, L. J., AND KING. J. M. (19758).Petroleumhydrocarbontoxicity studies.VII. Animal andhumanresponseto 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). Petroleum hydrocarbon toxicity studies.VIII. Animal and human responseto vapors of “140” Flash Aliphatic Solvent”. Toxicol. Appl. Pharmacol. 34,413-429. CARPENTER, C. P., KINKEAD, E. R., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., AND KING, J. M. (19761).Petroleum hydrocarbon toxicity studies.IX. Animal and human responseto vapors of “80 Thinner”. Toxicol. Appl. Pharmacol. 36, CARPENTER,

409-425.

C. P., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., J. M. (1976j). Petroleumhydrocarbon toxicity studies.X. Animal and human responseto vapors of “50 Thinner”. Toxicol. Appl. Pharmacol. 36,427-442. CARPENTER, C. P., GEARY, D. L., JR., MYERS, R. C., NACHREINER, D. J., SULLIVAN, L. J., AND KING, J. M. (1975k).Petroleumhydrocarbon toxicity studies.XI. Animal and human responseto vapors of Deodorized Kerosene.Toxicol. Appl. Pharmacol. 36,443-456. CARPENTER, AND KING,