DRUG AND CHEMICAL TOXICOLOGY, 1(3), 219-230 (1978)

ACUTE TOXICITY OF : , ISODURENE AND

Dennis ;J. Lynch Vernon R. Perone Ronald L. Schuler William B. Ushry Trent rl. Lewis

3epartment of Bealth, Education, and Welfare Public Health Service Center for Disease Control National Institute for Occupational Safety and Health 4676 Columbia Parkway Cincinnati, Ohio 45226

ABSTRACT

Oral LD50 (rat), primary skin irritation (rabbit), cutaneous

sensitization (guinea pig) and eye irritation (rabbit) studies

For personal use only. were conducted on the three tetramethylbenzene isomers: durene,

isodurene and prehnitene. The order of oral toxicity was

isodurene > prehnitene > durene. Durene was not a skin irritant,

while isodurene and prehnitene each produced a mild positive skin

response (erythema). None of the tetramethylbenzenes were skin

sensitizers or eye irritants. qurene, isodurene and prehnitene

are only slightly toxic on an acute toxicologic basis and only

pose an acute health hazard when injested in excessive quantities. Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12

219

Copyright 0 1978 hy Marcel Dekker, InL All Rights Reserved Neither this work nor dny pdrt may be reproduced or transmitted in dny form or by any means, electronic or mechanicdl including photocopying, microfilming, and recording, or by any information storage and retrieval system without permission in writing from the publisher 220 LYNCH ET AL.

INTRODUCTION

Durene (1,2,4,5-tetramethylbenzene), isodurene (1,2,3,5-

tetramethylbenzene) and prehnitene (1,2,3,4-tetramethylbenzene)

are used as starting materials in the organic synthesis of

plastics and resins. At room temperature durene is a solid,

while the other isomers are liquids. Physical constants for the

tetramethylbenzenes are presented in Table 1.

TABLE 1

Physical Constants for Tetramethylbenzene Isomers 9

Durene Isodurene Prehnitene (1,2,4,5-tetra- (1,2,3,5-tetra- (1,2,3,4-tetra- methylbenzene) methylbenzene) methylbenzene)

Formula '10H14 '10H14 '10H14 Mol Wt 134.22 134.22 134.22

Physical State Solid Liquid Liquid For personal use only. (21OC)

Specific Gravity 0.838 0.890 0.905

Melting Point 79 -24 -6 ("C)

Boiling Point 196.8 198 205 ("C)

Flash Point 74 68 73 ("C)

Solubility Insolub-3 Insolu Insolu Le

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12 (H20) Solubility - Organic Compounds Alcohol Very Soluble Very soluble Very soluble Ether Very soluble Very soluble Very soluble ACUTE TOXICITY OF TETRAMETHYLBENZENES 221

The tetramethylbenzenes are also important industrially

because they possess extremely high reactivity relative to

and , and they can form only one mono- and one di-

nuclear derivative. This enables high yields of high purity

products under relatively mild treatment conditions. 1 Durene is

important for its oxidation product, pyromellitic anhydride, used

in high molecular weight polymers, particularly in some

engineering plastics. Durene has also been employed experi-

mentally as a fungi~ide.~*~

In the course of production and utilization of tetramethyl-

in industry, the working environment and employees may

become contaminated. Data on the acute toxicity of the

tetramethylbenzenes are sparse.

Gerarde6 orally administered a homogenized suspension of

durene (in olive oil warmed to 4OoC) to fasted 250g rats and had

For personal use only. no deaths among the animals dosed. He determined that the oral

LD was greater than 5000 mg/kg body weight. The oral LD for 50 50 durene has been estimated at 6800 mg/kg.?

It is essential, therefore, to obtain additional data on the

acute toxicity of industrial compounds in order to ascertain what

health hazards, if any, are associated with their industrial

uses.

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12 The present study describes the acute toxicity of durene,

isodurene, and prehnitene for three routes of industrial

exposure, i.e., oral, cutaneous and ocular. 222 LYNCH ET AL.

METHODS

The following compounds were studied: durene (1,2,4,5-

tetramethylbenzene), isodurene (1,2,3,5-tetramethylbenzene) and

prehnitene (1,2,3,4-tetramethylbenzene). The chemicals were

analyzed for purity by gas chromatography using a flame

ionization detector. The durene sample was better than 99+%

pure; isodurene had a purity of 98% (the major impurity appeared

to be prehnitene): and prehnitene was 96% pure (the major

impurity appeared to be isodurene). All the test materials were

administered undiluted, except for durene which was suspended in

corn oil for the oral LD studies. 50

Acute Oral LD50. In rat oral LD50 studies, each test material

was administered by gastric intubation as a single dose. The

experimental subjects were male, cesarean-derived, Sprague-Dawley

albino rats weighing 175-250g that were fasted overnight prior to For personal use only. dosing. Dose levels were seven to eight in number (durene 5200-

9800 mgfkg, prehnitene 4000-9000 mg/kg and isodurene 2000-8200

mg/kg). The number of rats employed per dose level was eight.

The 14-day oral LD50 values and their 95% confidence limits were

calculated by the exact probit analysis of Finney.8 When the

data were nonlinear on log-probit graph paper, the moving average

method of Thompson was employed .g Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12

Primary Skin Irritation. In the primary skin irritation studies,

the three test materials and a negative control (distilled ACUTE TOXICITY OF TETRAMETHYLBENZENES 223

water), were tested on six rabbits, each rabbit receiving the

three tetramethylbenzenes plus the distilled water control. For

each test and control material, two skin sites were used, one

abraded and one intact. A 0.1 ml volume or 100 mg (durene) of

each undiluted test compound, and 0.1 ml of the control compound

(distilled water) was applied to each 20 mm2 test site. The

durene test sites were moistened with 1-2 ml of distilled water

immediately prior to application of the test compound. The test

sites were covered with a gauze patch. Each animal was provided

with a leather harness to prevent the animal from disturbing the

test site." Following a 24-hr exposure the harness and patches

were removed and the skin reactions evaluated. A second

evaluation was performed 48 hr after administration of the

compounds. The method employed for determining the degree of

primary skin irritation was a modification of the Draize

For personal use only. technique." The reactions were evaluated on the basis of the

designated values in the following table:

React ion Intact Skin Abraded Skin

No irritation 0 (Nonirritant) 0 (Nontoxic)

Erythema (regard- 1 (Mild irritant) 1 (Xild cellular less of degree) toxicant)

Erythema and edema 2 (Irritant) 2 (Cellular toxi- confined to test cant) area Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12

Erythema and edema 3 (Strong irritant) 3 (Strong cellular extending beyond toxicant) test area

Eschar 4 (Corrosive) 4 (Corrosive) 224 LYNCH ET AL.

Skin Sensitization. Fifteen male albiqo guinea pigs, weighing

300-400g, were employed for each test material in the repeated

insult test for cutaneous sensitization. Five animals were

assigned to the control group. The testing method was similar to

that described by Landsteiner and Jacobs.13 A 0.1 ml volume or

100 mg (durene) of undiluted test compound was applied topically

to separate abraded skin sites on the animals three times weekly

for a total of nine treatments. Each durene test site was

moistened with distilled water. Following a two week rest period

for an immune response to develop, during which time no further

treatments were made, a challenge dose of a test compound was

administered to both test and control animals in the same dosage

and by the same method used during the initial treatment period.

Skin reactions were examined and recorded at 24, 48 and 72

hr following the challenge dose. For personal use only. Eye Irritation. Yale albino rabbits, weighing 2 to 3 kg, were

used in the eye irritation studies. A 0.1 ml volume or 100 mg

(durene) of each undiluted test material was placed in one eye of

each animal by pulling the lower lid of the eye to form a cup

into which the compound under investigation was instilled. The

eyelids were held together for approximately one second and the

animal was then released. The rabbit eye was exposed to the test Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12 compound for either 5 min or 24 hr before washing the eye with

approximately 300 ml of distilled water over a 2 min period. The

eyes were examined at 1, 24, 48 and 72 hr, and again at 7 days; ACUTE TOXICITY OF TETRAMETHYLBENZENES 225

if any injury persisted, the eyes were re-examined at 14 and 21

days. The nontreated eye served as a control. Grading of the

severity of the eye irritation was performed according to the

United States Food and Drug Administration method13 based upon

the presence and degree of ulceration or opacity of the cornea

and iris and erythema, chemosis and ulceration or necrosis of the

conjuctival mucosa.

RESULTS

Acute Oral LD The oral LD values and their 95% confidence 50' 50 limits for the three test compounds are presented in Table 2.

Isodurene was more toxic than prehnitene which was more

toxic than durene. Lethal doses of the tetramethylbenzenes

(durene > 6000 mgfkg, prehnitene and isodurene > 4400 mgfkg)

induced stomach and intestinal hemorrhage, labored breathing,

lacrimation, bladder distention and adhesions of abdominal organs

For personal use only. (stomach, pancreas, spleen, liver and small intestines). Death

was related to the degree of blockage of the gastrointestind

tract from the resultant adhesions and/or leakage of bloody

TABLE 2

Acute Oral LD50 Values in Rats

Number of Animals Number Estimated LD50 95% Confidence

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12 Compound per Dose of Doses (mglkg) Limits

Isodur ene a 7 5157 4 53 2- 570 1

Prehnitene a a 6408 5488-7688

Durene 8 7 6989 5948-7892 226 LYNCH ET AL.

exudate into the peritoneal cavity. Behavioral effects noted for

survivors of the LDjO study were initial hyperexcitability,

followed by apathy and weakness, which persisted for several

hours to several days, depending upon the dosage administered.

The signs and symptoms observed were essentially identical for

the three compounds tested. All deaths occurred within three

days, except for one rat which received isodurene and died on day

six.

Primary Skin Irritation. Results of the skin irritation studies

with rabbits revealed that isodurene and prehnitene produced a

mild positive response (erythema) which did not persist beyond 48

hr. Durene did not produce a positive response and was

considered to be a nonirritant to skin. Primary irritation

scores for each compound (average rating, intact vs abraded

skin) and the interpretation of the scores are presented in Table For personal use only. 3. The tetramethylbenzenes were not irritating to intact or

abraded skin.

TABLE 3

Primary Skin Irritation Scores in Rabbits

Average Ratinga Interpretation Intact Abraded Intact Abraded Compound Skin Skin Skin Skin

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12 Durene 0 0 Nonirritant Nontoxic

Isodurene 0.7 0.9 Nonirritant Nontoxic

Prehnitene 0.4 0.9 Nonirritant Nontoxic

a Average score of six rabbits ACUTE TOXICITY OF TETRAMETHYLBENZENES 227

Skin Sensitization. There was minor irritation of the test sites

after each of the nine sensitizing cutaneous applications, with

the challenge dose eliciting an identical response. These

results indicate that the tetramethylbenzenes tested have little

or no potential for inducing cutaneous sensitization in man.

Eye Irritation. Isodurene, prehnitene and durene are nonirritants,

since no ocular reactions were induced by the substances beyond the

one-hour observation period.

DISCUSSION

The results of these studies provide a basis for evaluating

the absolute and relative acute toxicity of durene, isodurene and

prehnitene. There are no prior studies in the literature in

which these compounds were assessed simultaneously for their oral For personal use only. toxicity, primary skin irritation, cutaneous sensitization and

eye irritation responses. The oral LD50 results obtained in this

study for durene are supported quantitatively by the studies of

Gerarde6 and Uzhdavini, et al.? Gerarde estimated the LD50 to be

in excess of 5000 mglkg, and Uzhdavini reported the LD50 to be

6800 mg/kg, which is in agreement with the 6989 mglkg value

obtained in this study. The relatively low oral toxicity of the Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12 tetramethylbenzenes can be explained in terms of their biotrans-

formation products, carboxylic acids and alcohols, which have

a very low order of t0~icity.l~The necropsy findings are similar

to those reported in rats dosed orally with alkyl derivatives

of benzene. 6 228 LYNCH ET AL.

Gerarde4 reported that his unpublished data indicated that

guinea pigs did not develop a sensitivity to durene injected

intradermally. This finding is in agreement with the current

study in which topically applied durene failed to elicit a

sensitization response. Uzhdavini, et a1.15 reported that

durene had a lower local irritating effect than pseudocumene

(1,2,4-trimethylbenzene), 2-isopropylnaphthalene and

dodecylbenzene.

Brattsten and Wilkinsonl6 working with the southern armyworm

and Fabacher and Hodgsonl7 working with mice, have both demon-

strated that durene is an enzyme inducer. The other tetra-

methylbenzenes have not been tested for enzyme induction

activity. These facts should be considered in terms of

xenobiotic metabolism in workers exposed to tetramethylbenzene

isomers in the workplace.

For personal use only. The data from this study and other laboratories suggest that

durene, isodurene and prehnitene should not present acute

toxicological hazards to workers as long as standard good hygiene

practices are employed when handling these tetramethylbenzene

compounds.

ACKNOWLEDGMENTS

The authors wish to express their gratitude to Dr. Jeanne Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12 Burg for her help with statistics; to Ms. Ardith Grote for the

gas chromatographic analyses of purity; to Mr. Paul Wright and

Mr. Clinton Gray for their care of the animals; and to Els. Evelyn

Munro for typing this manuscript. ACUTE TOXICITY OF TETRAMETHYLBENZENES 229

REFERENCES

1. H. W. Earhart, "Polymethylbenzenes," Noyes Development Corp.,

Park Ridge, N.J., 1969.

2. "Handbook of Chemistry and Physics," R. C. Weast, ed., CRC

Press, Cleveland, 1973, pp C-164-165.

3. A. L. Waddams, "Chemicals from Petroleum,'' Wiley,

New York, 1973.

4. H. W. Gerarde, "Toxicology and Biochemistry of Aromatic Hydro-

carbons," Elsevier, New York, 1960.

5. R. E. 3cDonald and W. R. Buford, Plant Dis. Rep., 58, 1143 (1974). 6. H. W. Gerarde, Arch. Ind. Health, 19, 403 (1959).

7. E. R. Uzhdavini, E. Z. Liswanskii and B. 14. Talalaev, Tr. Nauch.-Issled. Inst. Neftekhim. Proizvod., L, 179 (1969).

8. D. J. Finney, "Statistical Methods in Biological Assay," Hafner,

New York, 1971.

For personal use only. 9. W. R. Thompson, Bacteriol. Rev., 2, 115 (1947).

10. E. A. Newmann, Lab. Anim. Care, 13, 207 (1963).

11. J. H. Draize, G. Woodard and H. D. Clavery, J. Pharmacol. Exp.

Ther., g,377 (1944).

12. K. Landsteiner and J. Jacobs, J. Exp. Med., 61, 643 (1935). 13. Food and Drug Administration, Fed. Reg., 37, 8534 (1972). 14. H. W. Gerarde, Arch. Ind. Health, 12, 468 (1956).

Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12 15. E. R. Uzhdavini, E. 2. Lisnyanskii, N. K. Karimulina, Z. Kh.

Filippova and V. M. Talalaev, Toksikol. Gig. Prod. PJeftekhim.- Neftekhim. Proizvod., 1, 126 (1968.) 2 30 LYNCH ET AL.

16. L. B. Brattsten and C. F. Wilkinson, Pestic. Biochem.

Physiol., 2, 393 (1973).

17. D. L. Fabacher and E. Hodgson, J. Toxicol. Environ.

Health, -2, 1143 (1977). For personal use only. Drug and Chemical Toxicology Downloaded from informahealthcare.com by Hacettepe Univ. on 11/24/12