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THE REPRODUCTIVE AND DEVELOPMENTAL TOXICITY OF HIGH FLASH AROMATIC NAPHTHA RICHARD H. McKEEl, ZACHARY A. WONG:!, SUSAN SCHMITI3, PATRICK BEAITy4, MARK SWANSONs, CEINWEN A. SCHREINER' AND JAMES L. SCHARDEIN Catalytic reforming is a refining process that converts naphthenes to aromatics by dehydrogenation to make higher octane gasoline blending components. A portion of this wide boiling range hydrocarbon stream can be separated by distillation and used for other purposes. One such application is a mixture of predominantly 9-carbon aromatic molecules (C9 aromatics, primarily isomers of ethyltoluene and trimethylbenzene], which is removed and used as a solvent-High Flash Aromatic Naphtha. A program was initiated to assess the toxicological properties of High Flash Aromatic Naphtha since there may be human exposure through inhalation or external body contact. The current study was conducted to assess the potential for developmental toxicity in the mouse and for reproductive toxicity in the rat. In the developmental toxicity study in CD-] mice, exposure of dams by inhalation to near lethal levels (1500 ppm) resulted in 1. Exxon Biomedical Sciences, Inc., Mettlers Road, CN 2350, East Millstone, NJ 08875·2350. 2. Chevron Environmental Health Center, 15229San Pablo Avenue, P.O. Box 4054, Richmond, CA 94804. 3. Amoco Corporation, 200 East Randolph Drive, Chicago, IL 60601. 4. Shell Oil Company, P.O. Box 4320, Houston, TX 77210. Currently Chevron Environmental Health Center, 15229 San Pablo Avenue, P.O. Box 4054, Richmond, CA 94804. 5. American Petroleum Institute, 1220 L Street NW, Washington D.C. 20005. Currently, Atlantic Richfield Company, 515 South Flower Street, Los Angeles, CA 90051. 6. Mobil Oil Corporation, Toxicology Division, P.O. Box 1029, Princeton, NJ 08540. 7. International Research and Development Corporation, 500 North Main St., Mattawan, MI 49071. Abbreviations: ASTM (American Society for Testing and Materials), CAS (Chemical Abstract Service), g (gram), G (gestation), HVAC (heating, ventilation, air conditioning), L (lactation), LC50 (a concentration lethal to 50% of the exposed animals) Key Words: C9 Aromatic Hydrocarbons, Developmental Toxicity, Ethyltoluene, High Flash Aromatic Naphtha, Reproductive Toxicity, Trimethylbenzene Toxicology and Industrial Health, 6:3/4, pp. 441-460 Copyright 1989 Princeton Scientific Publishing Co., Inc. ISSN: 0748-2337

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fetal mortality, reduced weight, delayed ossification, and an increased incidence of cleft palate. At 500 ppm, a level at which maternal weight gain was slightly reduced, fetal weight gain was also reduced, but there was no other evidence of developmental effects. The lowest exposure level (l00 ppm) did not cause any maternal or developmental toxicity. There was no consistent evidence of reproductive toxicity in rats, even at exposure levels which resulted in significantly reduced parental weight gain. In addition, when parental exposure was stopped on GD (gestation day) 20, birth weights as well as postnatal survival were generally similar to control values, even in the 1500 ppm exposure group. Postnatal weight gain was also similar to controls early in weaning, but, if maternal exposure was reinitiated, weight gain was reduced in the high exposure group. However, when exposure was continued until delivery, pups in the high exposure group exhibited reduced litter size, birth weight and poor survival. Thus it was likely that the reduction in fetal weight, seen in the developmental toxicity study in mice, was transient and had no postnatal consequences if maternal exposure was terminated at any time prior to delivery.

INTRODUCTION Catalytic reforming is a refining process which dehydrogenates naphthenes (i.e., cycloparaffins) to produce hydrocarbon streams enriched in aromatics. These hydrocarbon streams are predominantly used as gasoline blending stocks; however, one product mixture which can be removed and used as a solvent is High Flash Aromatic Naphtha (CAS No. 64742-95-6). This material contains 70-80% C9 aromatic hydrocarbons, primarily isomers of ethyltoluene and trimethylbenzene, and conforms to the specifications for High-Flash Aromatic Naphtha, Type I, as defined by ASTM test method D-3734 (1987). In previous studies it was found that inhalation exposure to High Flash Aromatic Naphtha and similar materials produced minimal acute (Carpenter et al., 1977) or subchronic (Carpenter et al., 1977; Clark et al., 1989; Nau et al., 1966) toxicity in rats. Additionally, High Flash Aromatic Naphtha and similar materials were inactive in a number of in vitro and in vivo assays of genetic toxicity (American Petroleum Institute, 1973; 1978; Schreiner et aI., 1989). Thus such products are unlikely to cause systemic toxicity or mutation. In a study in rats of the developmental and reproductive toxicity of Aromatol, a branded product conforming to the specifications of High Flash Aromatic Naphtha (Ungvary, et aI., 1983), continuous inhalation exposure (i.e., 24 hours/

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day from day 7 to 15 of gestation) to approximately 120 ppm was without effect, but exposure to 200 or 400 ppm resulted in developmental delays. Fetal mortality and/or malformations were not observed at any concentration. Since maternal weight gain during gestation was slightly but significantly reduced at all exposure levels, the developmental delays may have been direct fetal effects or were secondary to maternal toxicity. In a one generation reproduction study in rats (Lehotzky et al., 1985) it was found that if the dams were continuously exposed from days 7-15 of gestation to 120,200, or 400 ppm and then maintained without exposure until parturition, there were no significant differences in birth weight, postnatal weight gain, or postnatal survival. Additionally, there were no apparent effects on nervous system development. Thus the delays seen in the developmental study (Ungvary et al., 1983)appeared to have been transient and without postnatal consequences. The objectives of the current study were to examine the developmental toxicity of High Flash Aromatic Naphtha in the mouse, a species not previously tested and to also extend the evaluation of reproductive toxicity by conducting a three generation study in rats. These studies were part of a testing program mandated by the U.S. Environmental Protection Agency under section 4(a) of the Toxic Substances Control Act (Fed. Reg. 50: 20662-20667, May 17, 1985). MATERIALS AND METHODS Materials The test sample conformed to the specifications for High-Flash Aromatic Naphtha, Type 1 (CAS No. 64742-95-6) as defined by the ASTM method D-3734. Additionally, the sample was analyzed by gas chromatography to measure the concentrations of each of the components of the mixture (Table 1). Although there are commercially available materials which conform to these ASTM specifications, the material used for these and all other mandated tests was specially prepared to meet the compositional requirements (i.e., at least 22% ethyltoluene isomers, at least 15% trimethylbenzene isomers, and at least 75% total ethyltoluenes and trimethylbenzenes) specified in the test rule (Fed. Reg. 50: 2066220667, May 17, 1985.). Methods Inhalation Exposures All animals were exposed in 16 cubic meter glass and stainless steel chambers. Chamber ventilation was provided by an HVAC system separate from the general laboratory air handling system. This air was particulate-filtered and controlled for temperature and humidity. Chamber air flow rate, temperature, and relative humidity were monitored every half-hour during exposure periods.

To generate test atmospheres, nitrogen was heated to 200°C by passage through

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TABLE 1 Composition of High Flash Aromatic Naphtha Weight Percent

Compound

3.20 2.74 3.97 7.05 15.1 5.44 8.37 40.5 6.18 6.19

a-xylene cumene n-propylbenzene 4-ethyltoluene 3-ethyltoluene 2-ethyltoluene 1,3,5-trimethylbenzene 1,2,4-trimethylbenzene 1,2,3-trimethylbenzene ~ClO's

Total 98.74

Mean Chamber Concentrations (PPM) Developmental Toxidty Study Nominal Concentrations

Target Concentrations

Actual Concentrations

Mean

SD

Mean

SD

102 463 1249

3.5 5.3 16.5

102 500 1514

2.6 3.7 22.9

0 100 500 1500 Reproductive Toxicity Study Nominal Concentrations

Actual Concentrations

Target Concentrations

Mean

SD

Mean

SD

0 100 500 1500

107 513 1483

2.4 12.8 33.0

103 495 1480

8.0

2.1 20.5

a one-liter stainless steel cylinder fitted with a 1500-watt band heater and was then introduced at the bottom of a glass column 7.6 em. in diameter and 30 em long, packed with glass beads. The liquid test material was delivered by a fluid metering pump from a stainless steel safety can, through Teflon tubing, to the bottom quarter of the column. The test sample was vaporized as it flowed up the column with the nitrogen. The vapors were passed to the chamber inlet where dilution with chamber ventilation air reduced the concentration to the desired exposure levels. The exposure levels were monitored with a gas-phase infrared spectrometer.

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Measurements were made on an hourly basis throughout the study. The accuracy of this method was confirmed by the use of vapor standards. Additionally, the composition (on a weight percentage basis) of the test material within each exposure chamber was determined by gas chromatography during the first exposure week. Animals Virgin female CO-l mice, 8 weeks old at the time of receipt, as well as 6 week old male and female rats (Charles River COBS CD) were obtained from Charles River Laboratories, Portage, MI. These animals were individually housed in wire mesh cages and maintained for either 2 or 3 weeks of acclimation. These housing conditions were maintained throughout the study except during the reproduction study when male and female rats cohabitated (1: 1) for mating and also between gestational day (GO) 20 and lactational day (LO) 4 when pregnant dams were individually housed in plastic cages with wood chips supplied for nesting material. Water was provided ad libitum, and Purina Certified Rodent Chow No. 5002 was available except during exposures. Other details of animal husbandry were in accordance with standards promulgated by the U.S. Department of Health, Education, and Welfare (DHEW, 1985). Developmental Toxicity Study in Mice Female mice, confirmed to have mated by the presence of vaginal plug, were randomly assigned to one of four treatment groups, each containing 30 animals. The animals were exposed 6 hr/day from GD6 through day GD15 to target vapor concentrations of 100, 500 or 1500 ppm. A control group was exposed to room air only.

Throughout the study the mice were examined twice daily for viability and overt changes in appearance and behavior. The presence and duration of clinical signs were recorded daily from G06 to G015 and also on GDI8. Maternal body weight was measured on GDO and daily from G06 to GD18. Females which died prior to scheduled sacrifice were necropsied, and fetuses, if present, were examined externally and discarded. Blood samples were taken on GD15 from dams in the 0, 500, and 1500 ppm groups. Hematological parameters evaluated included leukocyte and erythrocyte counts, and measurements of hemoglobin and hematocrit. On day GD18 surviving females were sacrificed, and the uterine contents were examined. The number and location of viable and nonviable fetuses, early and late resorptions, and the number of total implantations and corpora lutea were recorded. The uteri were excised and weighed, and then the fetuses were removed. Lung, liver, and kidneys were also removed from the dams and weighed. Fetuses were individually weighed, sexed, tagged, and examined for external malformations and variations. Approximately one-half of the fetuses were dissected, internally sexed and examined for visceral malformations and variations.

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The hearts were dissected by a modification of the method described by Staples (1974). The heads were placed in Bouin's solution for soft tissue examination by the razor blade sectioning technique (Wilson, 1965). The remaining fetuses were fixed in alcohol, macerated with potassium hydroxide, stained with Alizarin Red S and cleared with glycerin by a method similar to that described by Dawson (1926). All of these features were then subjected to skeletal examination.

Reproductive Toxicity Study in Rats FO Generation Male and female rats were randomly assigned to one of four treatment groups, each of which contained 30 animals of each sex. The rats were exposed for ten weeks (6 hours/day, 5 days/week) to 100, 500, or 1500 ppm High Flash Aromatic Naphtha. A control group was exposed to room air only. Males and females were then cohoused (1: 1) for a two week mating period. The exposure schedule was maintained during the mating period. Once mating was confirmed, males were removed, and the females were exposed 6 hours/day, 7 days/week from GOO (the day on which mating was confirmed) to G020. At this point exposure was discontinued, and the females were placed in nesting boxes and allowed to deliver. Exposure of the dams was reinitiated on postnatal day 5 (LOS) and continued until weaning (LD21). Parental males were sacrificed and necropsied at the end of the mating period; parental females were sacrificed and necropsied following weaning. F1 Generation The Fl litters were culled to 8 on L04, but kept with their dams until the end of the weaning period (LD2l). On day LD2l all pups were counted, sexed, and weighed individually, and the weights were recorded by sex. Exposure of randomly selected individuals (30/sex/exposure group) to produce the F2 generation was initiated approximately a week after all had completed the weaning period. Thus, the F1 rats were between 5 and 7 weeks of age at the time exposure was initiated. These animals were subsequently exposed for 10 weeks, and then mated to produce the F2 generation. F2 Generation The experiment was initially planned as a 2-generation study. However, in order to assess the effects of exposure immediately following weaning, the study was extended for a third generation. The F2 litters were treated as described above, but exposure of each animal used to produce the F3 generation was initiated immediately after it completed the lactation period (i.e., on postnatal day 22). Because of concern for toxicity, 40 animals/sex/dose group were initially randomly selected with the intention of reducing this number to 30 by random selection at the time of mating. This was done in the control, 100, and 500 ppm groups. However, the majority of the pups in the high dose group died during the first week of exposure, so all survivors in that group were mated.

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Parental Observations All animals were examined twice daily for viability and overt signs of toxicity. A detailed clinical examination was conducted weekly. Body weights were recorded weekly until confirmation of mating. In addition, females were weighed on GD 0, 7,14, and 21 and LD 0,7, 14, and 21. Food consumption was measured weekly except during mating, gestation, and lactation. At sacrifice the following organs and tissues were collected for microscopic examination: epididymis, lung, ovary, pituitary, prostate, seminal vesicle, testis, uterus, vagina, and regional lymph nodes. These organs were examined in the control and high dose groups only. In addition, all masses and gross lesions from all groups were examined. Finally, epididymis, lung, ovary, testis, prostate/seminal vesicle, and uterus/ vagina were weighed. Examination of Of/spring The litters were examined as soon as possible after delivery for litter size, stillborns, live births, and any gross anomalies. The litters were culled by random selection to 8 pups on day LD4. The culled pups as well as any which died spontaneously during the lactation period were necropsied and examined for anomalies. Pups were weighed individually on LD 0,4,7, and 14. On day LD21, all pups were counted, sexed, and weighed individually, with the weights recorded by sex. Statistical Analysis Fertility indices and male to female fetal sex ratios were compared by the Chisquare test criterion with Yates' correction for 2 x 2 contingency tables, and the proportions of litters with malformations were compared by the Fisher's exact probability test as described by Siegel (1956) to determine the significance of differences. The proportions of resorbed and dead fetuses, pre- and postimplantation losses, and pup survival indices were compared by the Mann-Whitney V-Test as described by Siegel (1956) and Weil (1970) to determine the significance of the differences. Numbers of corpora lutea, total implantations and live fetuses, mean fetal body weights, parental body weights, maternal body weight changes, organ weights and hematological parameters were compared by analysis of variance (one-way classification), Bartlett's test for homogeneity of variance and the appropriate t-test (for equal or unequal variance) as described by Steel and Torrie (1960) using Dunnett's (1964) multiple comparison tables to determine the significance of differences. Mean numbers of liveborn pups per litter and mean pup weight were also compared by analysis of variance and the appropriate t-test as described above. All statistical analyses compared the treatment groups to control with the levels

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of significance at p < 0.05 and p < 0.01. All means were accompanied by standard deviations. RESULTS

Developmental Toxicity Study Mean chamber concentrations are given in Table 1. Maternal Toxicity The highest concentration (1500 ppm) produced severe maternal toxicity; 12 pregnant mice died between days GO 8-16, and 2 other animals died on OD6 and were replaced. Thus, mortality in the high dose group was 44% (14/32) (Table 3). Additionally, mean maternal body weight gain in the 1500 ppm group was significantly reduced during treatment (006-15) and overall (000-18) (Table 2). Finally, clinical observation revealed abnormal gait (18), labored breathing (9), hunched posture (9), weakness (7), inadequate grooming (7), circling (8) and ataxia (8). (The number of affected individuals is given in parentheses.) Two mice in the 500 ppm group died during the exposure period. In one case the death was due to injury; the cause of death for the other mouse was undetermined. All mice survived to terminal sacrifice in the control and 100 ppm exposure groups. Maternal body weight gain between GD6 and 15 was significantly reduced in the 500 ppm group; a similar reduction was also seen in the 100 ppm group, but it was not statistically significant (Table 2). Additionally, there were no clinical signs of toxicity at either the 100 or 500 ppm levels. There were no significant differences in maternal organ weights in any of the exposure groups (Table 3). Hematological evaluations revealed significant decreases in the percent hematocrit (p < 0.01) and mean corpuscular volume (p < 0.05) values of the high exposure group as compared to controls. The leukocyte count was decreased in the 500 ppm group (p < 0.01) but was assumed to have been unrelated to treatment since the 1500 ppm group was not significantly different from control (data not shown). To summarize the maternal effects, exposure to 1500 ppm produced severe maternal toxicity. At the 500 ppm level there was slight maternal toxicity (i.e., significantly reduced weight gain, one unexplained death). At the 100 ppm level weight gain was reduced but was not significantly different from control. Thus, if there was maternal toxicity at the 100 ppm level, it was minimal.

Developmental Toxicity There was also evidence of developmental toxicity at the 1500 ppm level. The number of live fetuses per litter and the mean fetal body weight were significantly reduced (Table 2); post implantation loss was significantly elevated (Table 2);

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TABLE 2 Reproductive Parameters Among Pregnant Mice Exposed to High Flash Aromatic Naphtha High Flash Aromatic Naphtha Concentration (ppm)

Number of deaths/number females Number pregnant/number mated Number of litters with viable fetuses Corpora luteal dam Implantations/dam Live fetuses/litter Postimplantation loss/dam Fetal body weight (grams) Fetal sex ratio, males: females

0

100

500

1500

0/30

0/30

2/30

14/32(')

26/30

26/30

27/30

22/30(b)

24 12.9 ± 1.8(d) 11.6 ± 1.5(d) 10.7 ± 1.8(d) 0.9 ± 0.9(d) 1.25 ± 0.14(d) 57:41

21 12.6 ± 1.8 11.0 ± 1.9 8.7 ± 4.6* 2.3 ± 4.1 1.24 ± 0.08 51:49

23 12.7 ± 2.3 11.3 ± 1.6 9.3 ± 3.1 2.0 ± 3.1 1.16 ± 0.11* 54:46

13.8 12.3 7.9 4.3 0.82

131

The reproductive and developmental toxicity of High Flash Aromatic Naphtha.

Catalytic reforming is a refining process that converts naphthenes to aromatics by dehydrogenation to make higher octane gasoline blending components...
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