The Role of Dermal Irritation in the Skin Tumor Promoting Activity of Petroleum Middle Distillates

49, 48 –55 (1999) Copyright © 1999 by the Society of Toxicology TOXICOLOGICAL SCIENCES The Role of Dermal Irritation in the Skin Tumor Promoting Act...
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49, 48 –55 (1999) Copyright © 1999 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

The Role of Dermal Irritation in the Skin Tumor Promoting Activity of Petroleum Middle Distillates Craig S. Nessel, James J. Freeman, Richard C. Forgash, and Richard H. McKee 1 Exxon Biomedical Sciences, Inc., East Millstone, New Jersey 08875–2350 Received July 16, 1998; accepted November 20, 1998

primarily or entirely produced from the distillation of crude oil at atmospheric pressure. These products are called “straightrun” middle distillates. Alternatively, it is possible to produce hydrocarbons of similar boiling range by processes that break down larger and more complex molecules by catalytic or thermal methods, generally referred to as “cracking”. Products such as diesel fuel or residential heating oils may contain cracked stocks, and may have higher levels of aromatic components than straight-run materials of a similar boiling range. The carcinogenic activity of some petroleum-derived materials is associated with the presence of biologically active 4 – 6 ring polycyclic aromatic compounds (PACs) (Bingham et al., 1980; Roy et al., 1988). While these compounds may be present in cracked stocks, 4 – 6 ring PACs are typically found at very low concentrations in straight-run PMDs (McKee et al., 1989). However, both straight-run PMDs and products blended with cracked stocks may produce tumors following chronic application to mouse skin (Biles et al., 1988). Unlike PACmediated carcinogenesis, the tumorigenic responses of straight-run PMDs are characterized by low tumor yield and long latency. These factors raise questions as to the tumorigenic mechanism of these materials. Additional studies indicate that straight-run PMDs generate skin tumors through a nongenotoxic mechanism. These materials have low or no activity in the Salmonella mutagenesis test (Deininger et al., 1991; McKee, 1994; McKee et al., 1989), lack tumor-initiating activity (Jungen et al., 1995; McKee et al., 1989), and their response is likely elicited through a promotional effect (McKee et al., 1989). With repeated application to the skin, middle distillates cause chronic irritation and inflammation (Freeman et al., 1990; Grasso et al., 1988). The irritation is characterized in part by epidermal hyperplasia. It has been hypothesized that mouse skin tumors arise as a secondary response to chronic skin irritation produced by PMDs (Ingram et al., 1993; McKee et al., 1989). Przygoda et al. (1994) showed that the skin tumors associated with PMDs might be due to the promotion of preexisting, spontaneously initiated cells. It has been suggested that the tumor incidence from some chemicals in animals is in fact a secondary response to cell proliferation. Cell proliferation is often a result of the cytotox-

Petroleum middle distillates (PMDs), a class of hydrocarbons which boil between 350 –700°F, are tumor promoters in mouse skin. The promotional activity is produced under conditions that also result in local changes, including chronic irritation and epidermal hyperplasia. The present study was conducted by comparing equal weekly doses of irritating and minimally or nonirritating test materials, to assess whether tumor promotion was a secondary response to these effects. Four PMDs, C10 –C14 normal paraffins (NP), lightly refined paraffinic oil (LRPO), Jet Fuel A (JF), and steam-cracked gas oil (SCGO), were evaluated. Test materials were applied undiluted (23/week) or as 28.6% (73/week) or 50% (43/week) concentrations in mineral oil for 52 weeks following initiation with dimethylbenzanthracene (DMBA). When applied undiluted, all materials produced moderate irritation and significant increases in tumor incidence. When NP, LRPO, or JF were applied in mineral oil diluent, skin irritation was generally ameliorated and few, if any, tumors were produced. SCGO was irritating and produced a significant increase in tumor frequency when administered in mineral-oil diluent. These data indicate that the promotional activity of straight-run PMDs is likely related to chronic irritation at the application site and not to dose. Thus, when used appropriately in the absence of prolonged irritation, these materials should not present a tumorigenic hazard to humans. Key Words: petroleum middle distillates; C10 –C14 normal paraffins; lightly refined paraffinic oil; jet fuel; steam cracked gas oil; skin irritation; tumor promotion.

Several studies have documented the tumorigenic potential of petroleum-derived middle distillate fuels (Biles et al., 1988; Clark et al., 1988; Lewis et al., 1984; McKee et al., 1989), a class of hydrocarbons which boil between approximately 350 and 700°F (176 –371°C). Kerosene, jet fuel, diesel fuel, and residential heating oils are representative products of this class. The compositions of different petroleum middle distillates (PMDs) vary, but include linear and branched chain aliphatics, cycloparaffins, and aromatics in approximately the C10 –C20 range. Some of the fuels, including kerosene and jet fuel, are 1

To whom correspondence should be addressed. Fax: (732) 873-6009. E-mail: [email protected]. 48

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ROLE OF IRRITATION IN PMD TUMOR PROMOTION

TABLE 1 Physico-Chemical Characteristics of Test Samples

Sample

Boiling range (°C)

Aromatic content (vol%)

Sulfur content (PPM)

Nitrogen content (PPM)

C10–C14 normal paraffins (NP) Steam cracked gas oil (SCGO) Lightly refined paraffinic oil (LRPO) Jet fuel A (JF)

193–220 238–267 259–309 163–273

0.03 92.0 22.8 21.8

NM a 445 414 425

NM a 139 ,1 23

a

NM, not measured.

icity that occurs following repeated administration of high doses of these materials (Ames and Gold, 1990; Cohen and Ellwein, 1990; Iversen, 1992). Many such chemicals are nongenotoxic and appear to be tumor promoters. The likelihood that a promotional mechanism associated with chronic irritation and skin injury is the cause of the tumorigenic response of PMDs in experimental animals was examined in the present study. In this study, the role of skin irritation and injury on the tumor-promoting activity of PMDs was evaluated by comparing the effect of equal weekly doses of irritating and minimally or nonirritating test materials. The effect of irritation was compared by administering test compounds in pure and diluted forms, and adjusting the number of administrations per week to maintain a constant weekly dose. A study based on a similar experimental design, but assessing complete carcinogenesis in mouse skin, was also conducted recently (CONCAWE, 1997; Nessel et al., 1998). The tumor promotion study was conducted for a full year, rather than 26 weeks, due to the weak tumorpromoting activity of some of the test materials. The study was preceded by a range-finding study to evaluate the irritation potential of PMDs in mice and to determine the appropriate doses and dilutions for the tumor-promotion study. MATERIALS AND METHODS Test materials. Four petroleum-derived middle distillates, C10 –C14 normal paraffins (NP), lightly refined paraffinic oil (LRPO), Jet Fuel A (JF), and steam-cracked gas oil (SCGO) were assessed for tumor-promotion activity in mouse skin. NP was also evaluated for mutagenic activity. NP, LRPO, and JF are representative of straight-run middle distillates. As the name implies, SCGO was cracked in the presence of steam, and contained a much higher level of aromatic hydrocarbons than the other test materials. Descriptions of the test materials are presented in Table 1. Mutagenicity studies—Salmonella assay. NP was tested in the Salmonella typhimurium mutagenicity assay in accordance with the method of Ames et al. (1975). Bacterial tester strains (TA98, TA100, TA1535, TA1537, and TA1538) were originally supplied by Dr. Bruce Ames (Berkeley, CA). Bacteria were plated on minimal agar with limited histidine and exposed to NP (100 –10,000 mg/plate) both in the presence and absence of an exogenous metabolic activation system from the livers of Aroclor 1254-pretreated Sprague Dawley rats. Two negative-control groups were used, one which was nontreated and a second which was exposed to DMSO, the vehicle for NP dilution. Positive-control groups were exposed to 2-aminoanthracene, 9-aminoacridine, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), or 2-nitroflu-

orene. Revertants were scored 72 h following exposure to test chemicals, using a Biotran-III colony counter. Mutagenicity studies—Bone marrow micronucleus test. The clastogenic effect of NP was examined in the in-vivo mammalian bone marrow micronucleus test, as described by McKee et al. (1994). Briefly, groups of male and female CD-1 mice were given 1.0, 2.5, or 5.0 mg/kg NP in corn oil. Corn oil and cyclophosphamide (40 mg/kg) served as the negative and positive controls, respectively. Mice treated with NP were sacrificed 24, 48, or 72 h posttreatment, and bone marrow was collected. Slides for microscopic examination were prepared, and 1000 erythrocytes were examined to determine the percentage of polychromatic (PCE) and normochromatic (NCE) erythrocytes. PCE (n, 1000) from each animal were examined to determine the frequency of micronuclei (MNE) per 1000 PCE. Animals (rangefinding and tumor promotion studies). Male CD-1 mice (25–35 g) were obtained from Charles River Laboratories (Quebec, Canada) and acclimated for a minimum of 7 days prior to dosing. They were housed in stainless steel cages with wire bottoms, and lighting was maintained on a 12 h light-dark cycle. Animals were allowed access to water and commercial rodent chow (PMI Feeds Inc., Richmond, IN) ad libitum. Room temperature and relative humidity were controlled to 68 –76°F and 40 –70%, respectively. Dose rangefinding study. In order to determine a non-irritating dose of test materials for the tumor promotion study, a 6-week rangefinding study was conducted with NP. This study supplemented a rangefinding study previously conducted with kerosene and a kerosene blend (Nessel et al., 1998). NP was applied to the dorsal skin of groups of male CD-1 mice (10/test group) neat (37.5 ml, 23/week; 25 ml, 33/week) and as 50% (v/v; 37.5 ml, 43/week and 25 ml, 63/week) and 28.6% (v/v; 37.5 ml, 73/week) concentrations in highly refined mineral oil. Mineral oil served as the control and was applied in 37.5 ml aliquots, 73/week. Irritation was evaluated by clinical observation during the study, by gross changes at postmortem, and by histopathologic assessment of treated skin at necropsy. Skin irritation was semi-quantified; briefly, an irritation index (II) of 0 – 4 was calculated for each treatment group based on gross signs of skin irritation as shown in Table 2. Tumor promotion study. During the initiation phase of the study, mice in groups of 30, were each given a single dermal application of 25 ml mineral oil (negative control) or 50 mg DMBA (25 ml of a 0.2% solution w/v in acetone) on day 0. During the promotional phase of the study, animals were treated 2–7 times weekly for 52 weeks with mineral oil, 12-O-tetradecanoyl-phorbol-13acetate (TPA; 25 ml of a 0.01% solution w/v in acetone/dose), a known tumor promoter, or PMD test materials starting on day 14. All PMD-treated animals received 75 ml of test material weekly during the promotional phase as indicated in the study design (Table 3). Mice were examined for viability twice daily. Tumors were counted and grossly diagnosed on a weekly basis. Animals were examined for skin irritation weekly, beginning prior to dose initiation on day 0. Skin irritation was semi-quantified according to the evaluation system presented in Table 2. Following termination of the study, all tumors and skin sections from treatment areas were examined microscopically. Tumor incidence was reported after both 26 (the normal length of a tumor promotion study) and 54 weeks. Interim

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TABLE 2 Dermal Irritation Scoring System

Statistical analysis. The tumor incidence data are presented as the number of mice that developed tumors after the 26- and 52-week promotion phases, the number of each tumor type, and the percentage of mice that developed tumors. Calculations were based on the number of mice initially on test. Tumor incidences were compared by chi square and Fisher’s exact tests (Bradley, 1968).

Degree of irritation (score) a Slight (1)

Moderate (2)

Marked (4)

Slight erythema Slight edema Desquamation

Moderate erythema Moderate/marked edema Atonia Cracking Leathery Pinpoint scabs Thickening

Extreme erythema Eschar Exfoliation Fissuring Necrosis Open sores Blanching

Note: Irritation index for each treatment group 5 (sum of the single highest score for dermal irritation for each animal)/(number of animals in group). a Erythema was graded as slight, moderate, or marked. Edema was graded as slight or moderate/marked. All other signs of irritation were recorded as present or not present. Animals could exhibit several signs of irritation at one time, but the irritation score for each animal was based on the single most severe sign of irritation (i.e., maximum score, 4).

(26 week) tumor counts were based on gross visual examination, and final tumor counts were based on microscopically identified tumors. No treatmentrelated findings, other than epidermal findings, were noted, and therefore visceral organs were not examined. A review of dermal carcinogenicity studies on petroleum hydrocarbons has shown that papillomas and squamous cell carcinomas are the only tumor types clearly associated with dermal application of these materials (Freeman and McKee, 1993). Therefore, although all tumor data are shown in Table 7, only tumors of these types are considered treatment-related and are included in tumor incidence tabulations and statistical evaluations. A number of keratoacanthomas were found in the study and all were in groups that also contained squamous cell carcinomas and papillomas. Thus, they may have been treatment-related. However, since the keratoacanthomas did not comprise more than 5% of the total tumors in any experimental group, the inclusion of these tumors in the statistical analysis was unlikely to affect the conclusions.

RESULTS

Mutagenicity Studies In the Salmonella mutagenesis assay, NP did not induce a significant dose-related increase in the mutation frequencies in any of the tester strains (Table 4). The positive and negative controls responded in a manner consistent with results of previous assays. Under the conditions of the assay, NP was not mutagenic at doses up to and including 10,000 mg/plate. In the bone marrow micronucleus test, NP did not produce a decrease in the percentage of PCE, indicating that there was no evidence of bone marrow toxicity. Furthermore, the test material did not produce an increase in micronucleus frequency at 24, 48, or 72 h post-treatment in either male or female mice (Table 5), indicating that NP was not clastogenic. Dose Rangefinding Study The skin irritant effects of equal weekly doses of neat (100%), 50%, and 28.6% concentrations of NP were compared, following dermal application for 6 weeks. Weekly dermal irritation scores over the 6-week range-finding study are shown in Table 6. Application of neat NP produced moderate skin irritation, with the irritation index ranging up to 2.0 during the course of the study. NP diluted to 50% in mineral oil produced no more than minimal skin irritation (irritation index up to 0.30). No irritation was observed at any time interval in mice

TABLE 3 Experimental Dose Groups

Group

Initiator a

Promotional test material b

1 2 3 4 5 6 7 8 9 10 11 12

Mineral oil DMBA (0.2%) DMBA (0.2%) DMBA (0.2%) DMBA (0.2%) DMBA (0.2%) DMBA (0.2%) DMBA (0.2%) DMBA (0.2%) DMBA (0.2%) DMBA (0.2%) DMBA (0.2%)

Mineral oil TPA Mineral oil NP NP NP SCGO SCGO LRPO LRPO JF JF

Concentration of test material in mineral oil [% (V/V)]

Test material dose volume (ml)

Doses per week

Total weekly test material dose (ml)

— 0.01 c — 100.0 50.0 28.6 100.0 28.6 100.0 28.6 100.0 28.6

37.5 25.0 37.5 37.5 37.5 37.5 37.5 37.5 37.5 37.5 37.5 37.5

7 3 7 2 4 7 2 7 2 7 2 7

— 0.0075 — 75 75 75 75 75 75 75 75 75

25 ml application on day 0. Applications began on day 14 and continued until day 379 (52-week promotional phase). c 0.01% (W/V) in acetone. a b

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ROLE OF IRRITATION IN PMD TUMOR PROMOTION

TABLE 4 Results of Salmonella Mutagenesis Assays on C10 –14 Normal Paraffins Revertants/plate c TA98

TA100

TA1535

TA1537

TA1538

Dose (mg/plate)

2S9

1S9

2S9

1S9

2S9

1S9

2S9

1S9

2S9

1S9

10000 3200 1000 320 100 2AA a Positive control b DMSO Untreated

9.7 10.7 11.7 9.3 13.0 — 554.7* 14.0 10.7

13.0 13.0 13.0 16.7 15.0 1910.0* — 15.3 19.7

73.0 72.0 75.7 73.7 72.3 — 1572.7* 80.0 81.3

70.0 78.7 73.0 74.0 73.0 1920.3* — 76.3 73.3

7.0 8.0 6.7 8.0 9.0 — 1718.7* 9.7 8.7

9.3 10.0 10.0 9.0 9.3 230.7* — 11.7 11.3

2.7 3.0 3.7 4.3 3.7 — 1180.7* 5.3 4.7

3.3 5.0 4.0 3.0 5.3 303.3* — 3.7 5.3

7.3 9.7 6.7 8.3 8.0 — 799.7* 12.0 8.7

8.7 8.7 9.7 9.0 10.3 697.0* — 10.7 10.3

a

2AA, 2-aminoanthracene (positive control). Positive control is 2-nitrofluorene for strains TA98 and TA1538, N-methyl-N-nitro-N-nitrosoguandine (MNNG) for strains TA100 and TA1535, and 9aminoacridine for strain TA1537. c Values are the means of three plates. * Signifies a positive point $ 3 times the vehicle control. b

treated with 28.6% NP. NP caused slightly more irritation when administered in 37.5 ml aliquots than it did when applied as 25 ml aliquots. These results indicated that 37.5 ml doses of PMDs at 100, 50, and 28.6% concentrations could be applied in the tumor promotion study to compare the promotional activity of irritating, slightly irritating, and nonirritating doses of PMDs.

Tumor Promotion Study The results of the skin tumor promotion study are shown in Table 7. Tumor yields after 26 and 52 weeks of promotion are reported. The dermal irritation index is a quantitative estimate of chronic skin irritation over the course of the study (weeks 4 –54). The tumor promoting activity of the

TABLE 5 Results of Bone Marrow Micronucleus Tests on C10 –14 Normal Paraffins Mean PCE 6 SD (%) b

Mean MNE 6 SD/1000 PCE c

Time

Treatment (dose)

Na

Female

Male

Female

Male

24 hours

Corn oil NP (1.0 g/kg) NP (2.5 g/kg) NP (5.0 g/kg) Cylcophosphamide (40 mg/kg) Corn oil NP (1.0 g/kg) NP (2.5 g/kg) NP (5.0 g/kg) Corn oil NP (1.0 g/kg) NP (2.5 g/kg) NP (5.0 g/kg)

5 5 5 5 5 5 5 5 5 5 5 5 5

51.4 6 5.0 54.6 6 2.6 52.5 6 3.2 56.8 6 3.7 53.6 6 3.4 53.6 6 2.7 53.6 6 5.1 54.6 6 2.7 56.0 6 2.2 55.1 6 2.8 58.3 6 3.5 56.3 6 3.4 57.5 6 3.2

52.4 6 4.2 51.6 6 4.4 52.9 6 1.6 49.1 6 4.2 52.5 6 1.9 52.9 6 2.9 53.9 6 2.0 53.1 6 2.4 53.2 6 1.7 49.0 6 10.7 56.1 6 2.2 54.6 6 1.4 56.1 6 1.8

1.6 6 1.3 2.2 6 1.1 1.8 6 1.3 2.8 6 1.8 24.0 6 2.9* 2.6 6 0.5 2.0 6 0.7 2.8 6 1.5 2.0 6 2.3 1.0 6 1.4 1.0 6 1.2 2.2 6 0.8 2.2 6 2.2

1.6 6 0.9 2.6 6 1.3 2.6 6 2.5 1.8 6 2.0 20.4 6 6.0* 2.2 6 0.8 1.2 6 1.3 2.4 6 0.9 2.2 6 1.6 2.0 6 1.6 1.8 6 1.3 3.2 6 0.8 2.4 6 1.9

48 hours

72 hours

a

Number of animals in dose group. Mean PCE, Mean % polychromatic erythrocytes. c Mean MNE/1000 PCE, Mean micronuclei per 1,000 polychromatic erythrocytes. * Significantly different from corn oil control at p , 0.01. b

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TABLE 6 Irritant Effect of Normal Paraffins in Rangefinding Study Irritation index a Treatment

Dose volume (ml)

Doses/week

Day 0

Day 7

Day 14

Day 21

Day 28

Day 35

Day 42

Mineral oil 28.6% NP 50.0% NP 50.0% NP 100.0% NP 100.0% NP

37.5 37.5 25.0 37.5 25.0 37.5

7 7 6 4 3 2

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0.30 0.30

0 0 0 0 1.20 1.50

0 0 0 0.30 1.50 1.70

0 0 0 0.30 1.25 2.00

0 0 0 0.10 1.63 2.00

a

Mean irritation index in group as determined by method in Table 2.

test materials generally correlated with the level of dermal irritation (Fig. 1). Dermal irritation was greatest in mice that received the positive control, TPA, and in groups that were dosed with undiluted PMDs twice per week. In general, there was an increased incidence of animals with moderate to marked irritation in these groups. Irritant effects included exfoliation, pinpoint scabbing, eschar, sores, and leathery or thickened skin. Treatment-related non-neoplastic changes were also observed in the skin sections of animals in all groups. The predominant changes were thickening of the epidermis due to acanthosis and/or hyperkeratosis, dermal inflammation, and areas of superficial epidermal necrosis. The incidence and

severity of these lesions were much greater in groups treated with neat PMDs than those administered diluted test materials. One mouse in the initiated negative control group (DMBA/ mineral oil) developed a squamous cell carcinoma. Nearly all mice in the positive control group (DMBA/TPA) developed tumors. Undiluted PMDs produced marked skin irritation and significant increases in tumor incidence. Dilution of straightrun materials (NP, LRPO, JF) in mineral oil resulted in minimal skin irritation and injury. There was no significant tumor response following the administration of these diluted test materials. Unlike the straight-run PMDs, dilution of SCGO in mineral oil did not result in a reduction in the level of skin irritation when compared to the undiluted material. SCGO

TABLE 7 Results of One-Year Tumor Promotion Study Initiator/promoter and concentration (%)

Number of mice: Examined With tumors (week 26) a With tumors (week 54) b Mean irritation index (weeks 4–54) Tumor types: Basal cell Carcinoma, squamous cell Fibrosarcoma Keratoacanthoma Lymphosarcoma Papilloma a

MO/MO

DMBA/TPA

DMBA/MO

DMBA/NP







100

50

28.6

100

28.6

100

28.6

100

28.6

30 0 0

30 30** 29**

30 1 1

30 14** 15**

30 1 1

30 1 3

30 4 17**

30 2 9**

30 3 7*

30 1 0

30 12** 11**

30 0 0

0.02

2.12

0.03

1.98

0.21

0.09

1.40

1.48

2.08

0.31

2.12

0.18

0 0 0 0 0 0

0 11/16 c 1 10/12 c 0 25/72 c

0 1 0 0 0 0

0 7/8 c 0 1 0 14/32 c

0 1 0 0 0 0

0 0 0 0 0 3

1 3 0 1 0 15/19 c

0 4/5 c 0 1 1 7/16 c

0 4/5 c 0 0 0 5/9 c

0 0 0 0 0 0

0 2 0 1/2 c 0 10/24 c

0 0 0 0 1 0

Number of mice with tumors based on visual inspection. Number of mice with histologically confirmed papillomas or squamous cell carcinomas. c Number of animals with specified tumor/total number of tumors per group. * Statistically different from negative control at p , 0.05. ** Statistically different from negative control at p , 0.01. b

DMBA/SCGO

DMBA/LRPO

DMBA/JF

ROLE OF IRRITATION IN PMD TUMOR PROMOTION

FIG. 1. Comparison of dermal irritation and skin tumor incidence caused by petroleum middle distillates in a one-year tumor promotion study. Petroleum middle distillates tested as promoters were C10-C14 normal paraffins, steam cracked gas oil (SCGO), lightly refined paraffinic oil (LRPO), and Jet Fuel A. Mineral oil and TPA were used as the negative and positive controls, respectively. Tumor incidence was based on the number of mice (n, 30) with histologically confirmed squamous cell carcinomas or dermal papillomas. Dermal irritation is expressed as the mean irritation index from weeks 4 –54 of the study.

caused a significant increase in tumor incidence when applied both neat and in the diluted form. DISCUSSION

Previous studies have shown that prolonged and repeated application of PMDs to mouse skin results in the development of skin tumors (Biles et al., 1988; Clark et al., 1988; Lewis et al., 1984). Straight-run PMDs have low or no mutagenic activity, lack tumor initiating activity, and are active skin tumor promoters (McKee et al., 1989). The tumor development may be secondary to the repeated dermal irritation caused by PMDs (Ingram et al., 1993; McKee et al., 1989), and it may involve the promotion of spontaneously initiated cells (Przygoda et al., 1994). There are two previous studies on PMDs that are consistent with the hypothesis that the promotional activity and tumor development in mice is secondary to prolonged dermal irritation. Skisak (1991) showed that tumors were not produced if irritation and inflammation were reduced by treatment with dexamethasone. However, it is unknown whether dexamethasone influenced the process in ways other than reducing skin irritation. Freeman et al. (1993), using dilution and intermittent

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dosing, showed that a high level of skin irritation was necessary for tumor development in mouse skin, and that diluted test materials did not produce skin tumors. However, the dilution and intermittent dosing resulted in a reduction in the overall dose that the mice received in this study. The present study investigated the role of dermal irritation on the skin tumor-promoting activity of middle distillates by separating the effect of irritation from dose. Test materials were administered both neat and diluted in a nonirritating mineral oil. The weekly doses of diluted test materials were kept equal to that of undiluted materials by administering a greater number of doses of diluted PMDs to mice each week. All four PMDs, NP, LRPO, JF, and SCGO, caused dermal irritation and were active skin tumor promoters when applied undiluted to the skin. Tumor incidence in mice ranged from 23–57% following application of undiluted PMDs. The promoting activity of these materials was very weak compared to that of TPA, which caused tumors in nearly all mice at a dose four orders of magnitude below that of the PMDs (0.0075 ml/week vs. 75 ml/week). Dilution of NP, LRPO, and JF to concentrations of 28.6 – 50% in mineral oil substantially reduced the level of dermal irritation in mouse skin. Skin irritation was generally ameliorated, and few, if any, tumors were produced when straight-run PMDs were applied in mineral oil. However, 28.6% SCGO caused an equivalent level of skin irritation as undiluted SCGO, and produced a significant increase in tumor incidence (30%). Generally, the results of this experiment were consistent with those of a conventional lifetime skin painting assay of similar design (CONCAWE, 1997; Nessel et al., 1998). In that study, two straight-run PMDs, a kerosene and a gas oil, were applied neat, and at 50% and 28.5% concentrations to mouse skin. As in the present study, these PMDs produced moderate to marked skin irritation and increased tumor frequency when applied undiluted. However, they did not produce tumors in the absence of prolonged skin irritation. The tumor development was likely a result of a nongenotoxic process, possibly resulting from frequent cell damage and repair. NP was included in this study because of previous data from Sice (1966) and Baxter and Miller (1987) which showed that normal paraffins of approximately 12 carbons (n-dodecane) were active skin tumor promoters. Sice (1966) showed that the tumor promoting activity of n-alkanes was related to their chain length, with maximal activity at C12–14. Brown and Box (1970), in a comparison of the irritancy of n-alkanes from C6 –C24, showed that irritation peaked at approximately C14 (n-dodecane was not included in the study). Thus the previous data indicated that the tumor promoting activity of n-alkanes could be related to skin irritancy. The data from the present study have confirmed that the skin tumor promoting activity of C10 –C14 normal paraffins was secondary to skin irritation. In addition to the tumor promotion activity, several studies have suggested that n-dodecane may have “accelerating” or

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cocarcinogenic activity (Horton et al., 1957; Bingham and Falk, 1969). In these studies, application of a known carcinogen (e.g., benzo(a)pyrene [BaP], DMBA) in n-dodecane resulted in an increased tumor yield and/or decreased latency period as compared to treatment with the carcinogen alone. N-dodecane is not mutagenic (Lankas et al., 1978; Tummey et al., 1992) and was shown to be noncarcinogenic in a lifetime mouse skin painting study (Horton et al., 1957). Likewise, NP was not mutagenic in the Salmonella mutagenesis and in vivo bone marrow micronucleus tests conducted in the present study. The suggestion that the tumor response from BaP and DMBA in n-dodecane was indicative of cocarcinogenic activity is most likely only a result of the difference in experimental design from a promotion study, and not a difference in effect. The data on NP in the present study suggests that the “accelerating” activity of n-dodecane is more likely a result of simultaneous initiation (from BaP or DMBA) and promotion (from n-dodecane) in the earlier studies. Furthermore, the present study suggests that the promotional activity is likely a consequence of the irritating effect of n-dodecane. SCGO was the one cracked PMD included in the study. SCGO differs from straight-run PMDs in that it is produced by cracking in the presence of steam and contains a high content of aromatic compounds, including approximately 1% 3 or 4 ring PAHs. Unlike straight-run PMDs, dilution in mineral oil did not remove the prolonged irritation caused by SCGO, and the diluted SCGO was an active skin tumor promoter. These results are consistent with the hypothesis that the tumor promoting activity of PMDs is a secondary response to prolonged skin irritation. However, it is more likely that the tumor response of SCGO, and possibly other cracked gas oils, is more complex. In addition to its high aromatic content, SCGO has been shown to cause a different pattern of irritation than straight-run PMDs (Freeman et al., 1990). In this 90-day dermal study, SCGO caused a transient necrotic response during the first week of exposure, while LRPO and JF caused a slower, more progressive response characterized by moderately severe inflammation and proliferative changes. The SCGO results in the present study are inconsistent with those of a dermal carcinogenicity bioassay which found SCGO to be noncarcinogenic in C3H mice (Freeman et al., 1993). In that study, SCGO caused chronic skin irritation, but did not produce any tumors when applied in 37.5 ml aliquots, 23/week for 2 years. There were several differences in the two studies, including use of different mouse strains and application of the tumor initiator, DMBA, in the tumor promotion study. However, whether these factors were responsible for the inconsistent results is unknown. CD-1 mice, used in the present study, are more responsive to hyperplasia than C3H mice, and could be more susceptible to tumor development. It is less likely that a single dose of DMBA would have led to the inconsistent results. The initial severe inflammatory response caused by SCGO may also contribute to the complexity of the results.

In summary, data from the both the present tumor promotion study and recent skin painting studies on straight-run PMDs suggest that the development of initiated cells into tumors are a consequence of repeated dermal irritation. When skin irritation is prevented, tumor induction does not occur. These data suggest that PMDs, when used appropriately in the absence of prolonged irritation, should not present a tumorigenic hazard to humans. REFERENCES Ames, B. N., and Gold, L. S. (1990). Chemical carcinogenesis: Too many rodent carcinogens. Proc. Natl. Acad. Sci. U S A 87, 7772–7776. Ames, B. N., McCann, J., and Yamasaki, E. (1975). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res. 31, 347–364. Baxter, C. S., and Miller, M. L. (1987). Mechanism of mouse skin tumor promotion by n-dodecane. Carcinogenesis 8, 1787–1790. Biles, R. W., McKee, R. H., Lewis, S. C., Scala, R. A., and DePass, L. R. (1988). Dermal carcinogenic activity of petroleum-derived middle distillate fuels. Toxicology 53, 301–314. Bingham, E., and Falk, H. L. (1969). Environmental carcinogens: The modifying effect of cocarcinogens on the threshold response. Arch. Environ. Health 19, 779 –783. Bingham, E., Trosset, R. P., and Warshawsky, D. (1979). Carcinogenic potential of petroleum hydrocarbons: A critical review of the literature. J. Environ. Pathol. Toxicol. 3, 483–563. Bradley, J. V. (1968). In Distribution Free Statistical Tests, pp. 195–203. Prentice Hall, Englewood Cliffs, NJ. Brown, V. K. H., and Box, V. L. (1970). Skin arginase activity as a measure of skin change under the influence of some alkanes and alkenes. Br. J. Dermatol. 82, 606 – 612. Clark, C. R., Walter, M. K., Ferguson, P. W., and Katchen, M. (1988). Comparative dermal carcinogenesis of shale and petroleum-derived distillates. Toxicol. Ind. Health 4, 11–22. Cohen, S. M., and Ellwein, L. B. (1990). Cell proliferation in carcinogenesis. Science 249, 1007–1011. CONCAWE (1997). Overview of the CONCAWE middle distillate programme. Report No. 96/62. Deininger, G., Jungen, H., and Wenzel-Hartung, R. P. (1991). Middle distillates: Analytical investigations of mutagenicity studies. Research Reports No. 412–1. DGMK, Hamburg, Germany. Freeman, J. J., Federici, T. M., and McKee, R. H. (1993). Evaluation of the contribution of chronic skin irritation and selected compositional parameters to the tumorigenicity of petroleum middle distillates in mouse skin. Toxicology 81, 103–112. Freeman, J. J., and McKee, R. H. (1993). The objectives and goals of dermal carcinogenicity testing of petroleum liquids. In Health Risk Assessment: Dermal and Inhalation Exposure and Absorption of Toxicants (R. G. M. Wang, J. B. Knaak, and H. I. Maibach, Eds.), pp. 283–289. CRC Press, Boca Raton, FL. Freeman, J. J., McKee, R. H., Phillips, R. D., Plutnick, R. T., Scala, R. A., and Ackerman, L. J. (1990). A 90-day toxicity study of the effects of petroleum middle distillates on the skin of C3H mice. Toxicol. Ind. Health 6, 475– 491. Grasso, P., Sharratt, M., and Ingram, A. J. (1988). Early changes produced in mouse skin by the application of three middle distillates. Cancer Lett. 42, 147–155. Horton, A. W., Denman, D. T., and Trosset, R. P. (1957). Carcinogenesis of

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