Toxicology, 59 (1989) 297--309 Elsevier Scientific Publishers Ireland Ltd.

Thirteen-week toxicity studies of 3,3'-dimethoxybenzidene and C.I. Direct

15 in the Fischer 344 rat Blue

Daniel L. Morgan a, C .W. Jameson a, John H. Mennear b, Borge M. Ulland ¢ and Joan K. Lemen ¢

*National Institute o f Environmental Health Sciences, Research Triangle Park, NC, bSchool of Pharmacy, Campbell University, Buies Creek, NC and ¢Hazleton Laboratories America, Inc., Vienna,

VA (U.S.A.)

(Received February 13th, 1989; accepted July 12th, 1989)

Summary

The benzidine congener 3,Y-dimethoxybenzidine (DMOB), and C.I. Direct Blue 15 (Blue 15), a prototypical compound of the DMOB-derived class of dyes, were evaluated in 13-week studies to characterize the toxicity and establish dose levels for subsequent chronic studies. Groups of 10 Fischer 344 rats of each sex were administered either DMOB, or Blue 15, at 1 of 5 concentrations in drinking water for 13 weeks. DMOB concentrations were 0, 0.017, 0.033, 0.063, 0.125, and 0.25% for males and females. For Blue 15, the concentrations were 0.063, 0.125, 0.25, 0.50, and 1.0% for females and 0, 0.125, 0.25, 0.50, 1.0, and 3.0% for male rats. Rats showed dose-related decreases in water con- sumption and weight gains. All DMOB-treated rats and their controls survived the 13-week treatment. There were 7 deaths in the 3% level of male rats treated with Blue 15. Liver and kidney weights were increased in rats treated with both compounds. Target organs for DMOB-treated rats were the kidney and thyroid. These lesions were characterized by chronic nephropathy, and increased pigment in the follicular cells of the thyroid. The kidney and liver were identified as target organs for Blue 15-treated rats. In the high-dose rats that died before termination of the study, renal effects were characterized by degeneration and focal necrosis of proximal tubular epithelial cells. Liver lesions in this group con- sisted of degeneration and necrosis of hepatocytes, fatty metamorphosis, and minimal megalocytosis. Mild chronic nephropathy was the principal histological effect in Blue 15-treated rats surviving to study termination.

Key words: 3,3'-Dimethoxybenzidine; o-Dianisidine; C.I. Direct Blue 15; Nephropathy; Toxicity

Introduction

Benzidine is a widely recognized human carcinogen, causing cancer of the uri- nary bladder [1--3]. Several benzidine-based dyes have been shown to be carcino-

Address all correspondence to: Dr. Daniel L. Morgan, National Institute of Environmental Health Sciences, MD AO-01, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.).

0300-483X/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

297

genic in rats [4], and were found to be metabolized to benzidine in animals [4,5] and humans [6].

Dyes based on the benzidine congener 3,3'-dimethoxybenzidine (DMOB, also called o-dianisidine) are also of concern because of their structural similarities to benzidine, and their potential for reductive metabolism to the parent aromatic amines [5]. The azo dyes have been shown to be metabolized by rat, hamster, and human tissue to free aromatic amines [7]. Although azo reduction can occur by liver enzymes [8,9], enzymes in the intestinal bacterial flora appear to be the primary mechanism for azo reduction of benzidine-based and benzidine congener- based dyes after oral administration [8,10--12]. Although the benzidine conge- ners and derived dyes have not been shown to be human carcinogens, they have been demonstrated to be mutagenic in the Salmonella assay after reductive metabolism [13] and are classified as animal carcinogens [14].

Dimethoxybenzidine is an intermediate in the synthesis of a number of bisazobiphenyl dyes used for coloring textiles, paper, plastic, rubber and leather. A small quantity of DMOB is reportedly used to manufacture an ingredient in isocyanate adhesives and a component of polyurethane elastomers [15]. Recent production volumes of DMOB are not available. Approximately 106 000 pounds of DMOB were imported in 1983 [16]. Production and importation of dyes made from DMOB was reported to be 1 329 000 pounds in 1978 [17].

C.I. Direct Blue 15 (Blue 15) is a widely used bisazobiphenyl dye synthesized from DMOB (Fig. 1). In addition to being a potential contaminant of the finished dye, DMOB has also been shown to be a metabolite of Blue 15 [5]. Blue 15 is a representative compound of the DMOB class of benzidine congener-based dyes. The most recent production volume reported for Blue 15 was about 270 000 pounds in 1982 [18] with imports of 7716 pounds in 1980 [19].

Because dyes based on benzidine and benzidine congeners such as DMOB are generally found in the same industry, it is difficult to determine accurately the numbers of exposed workers and the extent of exposure to DMOB or Blue 15 alone [20]. Non-occupational exposure to these dyes may occur through contact with paper, fabrics, and leather to which these dyes have been applied, and through the use of dyes in arts and crafts. Exposure may occur by inhalation, ingestion and skin absorption [21--23].

The National Toxicology Program's Benzidine Dye Initiative is a collaborative effort of the National Institute of Environmental Health Sciences, the National Center for Toxicological Research, the National Institute of Occupational Safety and Health, the Environmental Protection Agency, the Consumer Product Safety Commission and the Occupational Safety and Health Administration, under the aegis of National Toxicology Program (NTP), to evaluate toxicologically prototypical benzidine- and benzidine congener-dyes. Because of the large number of dyes, this approach was initiated to better utilize resources and to reduce the number of long term studies on these dyes. The integrated research and testing of representative dyes will provide data applicable to a generic assess- ment of the carcinogenicity or other toxicity associated with benzidine, benzidine congeners, and derived dyes.

Dimethoxybenzidine, an important benzidine congener, and Blue 15, a

298

3,3'-DIMETHOXYBENZIDINE • 2HCI CAS No. 20325-40-0

DYE SYNTHESIS (diazo coupling)

f

METABOLISM (azo reductase)

OH NH 2

1-HYDROXY, 2,8-OIAMINO, 3,6-NAPHTHALENE DISULFONIC ACID

NH z OH H3CO OCH3

: N ~ N H 2

SO No S03Na

C.I. DIRECT BLUE 15 CAS No. 2429-74-5

Fig 1.

representative DMOB-derived dye, were selected for evaluation to establish the toxicity and carcinogenicity of these compounds. Thirteen-week studies of DMOB and Blue 15 were performed to characterize the toxicity, identify target organs and establish dose levels for use in designing the 2-year chronic studies. The results of these prechronic studies are presented in this report.

Materials and methods

Chemicals C.I. Direct Blue 15 was provided by the Dye Environmental and Toxicology

Organization (DETO), Scarsdale, NY. The study chemical was desalted by dialysis and found to be approximately 40070 pure by high performance liquid chromatography (HPLC) analysis [24]. Although the material was found to contain only about 4007o of Direct Blue 15, it was representative of the

299

commercial product and therefore used in the toxicity studies. Benzidine and DMOB concentrations were determined in the desalted Direct Blue 15 dye sample by HPLC analysis following solvent extraction of the dye. The desalted direct Blue 15 contained < 1 ppm benzidine and 780 ppm DMOB. This amount of DMOB contaminant would result in a maximum concentration of 0.002% in the highest dose of Direct Blue 15 in dosed water (3%).

3,3'-Dimethoxybenzidine dihydrochloride was obtained from Sigma Chemical Co., St. Louis, MO and was found to be 98% pure by perchloric acid titration and HPLC analysis. 3,3'-Dimethoxybenzidine dihydrochloride was unstable in rodent feed (200/ag/g) under all storage conditions at or above 5 °C. Dosed feed stored open to air and light in a rat cage lost 12.4% and 18.2% of chemical after 3 and 7 days, respectively. Solutions of DMOB and Blue 15 stored at room tem- perature in rat cage water bottles under simulated dosing conditions, were stable for at least 72 h. Dosing solutions stored in the dark at 5 °C were stable for up to 3 weeks.

Animals Fischer 344 rats (35--39 days old) of each sex were acclimated to laboratory

conditions for at least 2 weeks prior to initiation of the study. Animals were housed by sex and by dose level with 5 animals per cage. Rats were fed powdered NIH-07 open formula diet ad libitum, and fresh water (control and test) was supplied twice weekly.

DMOB and Blue 15 treatment Because DMOB was unstable in feed, the drinking water route of administra-

tion was selected for both the dye and parent amine. Concentrations for the 13- week studies were selected based on mortality, body weight gain, and histological data obtained from 14-day repeated-dose toxicity studies. Groups of 10 rats of each sex received either DMOB or Blue 15 in drinking water for up to 13 weeks. DMOB concentrations were 0, 0.017, 0.033, 0.063, 0.125, and 0.25% in drinking water for both male and female rats. Blue 15 concentrations were 0, 0.063, 0.125, 0.25, 0.50, and 1.0%0 for female rats and 0, 0.125, 0.250, 0.50, 1.0, and 3.0% for male rats.

Hematology and clinical chemistry Blood samples were collected from the retroorbital sinus of all animals at the

termination of the study for hematologic measurements. Samples were analyzed for: hemoglobin, hematocrit, red blood cell and white blood cell (WBC) counts, and differential WBC counts. Blood samples were collected prior to necropsy from the abdominal aorta of all animals and analyzed for" blood urea nitrogen, serum creatine, lactate dehydrogenase, sorbitol dehydrogenase, and serum glutamic pyruvic transaminase. Animals were not fasted prior to obtaining blood samples.

Clinical and pathologic examinations The animals were observed twice dally for mortality and moribundity. Body

300

weights were recorded at the initiation of the study, and weekly thereafter. Food consumption was measured weekly, and water consumption was measured twice weekly; no adjustments were made for spillage. A complete necropsy was performed on rats that died early and on rats sacrificed at the end of the experi- ment. Tissues were fixed in 1007o formalin and processed for histological evalua- tion. Analyses of selected organ weights as well as organ to body weight ratios were performed. A complete histopathological examination was performed on tis- sues f rom control and the highest dose-level group animals. Target organs were examined in lower doseqevel groups down to a no-effect level.

Statistical analyses The data f rom groups of the same sex were evaluated by Bartlett 's test for

homogeneity of variance. This analysis was followed by one-way classification analysis of variance (ANOVA) [25] if the variance proved to be homogeneous. If ANOVA of the non-transformed data was significant, Scheffe's [26] multiple pair-wise comparison procedure was used to compare the group means. If the ANOVA of heterogeneous data was significant, Game's and Howell 's [27] multiple pair-wise comparison was used to compare the group means. The null hypothesis was rejected only if P < 0.05. One-tailed tests were used in all cases.

Results

Water consumption Water consumption and estimated mean daily intake of DMOB and Blue 15

are summarized in Table I. Animals dosed with DMOB showed a dose-related trend towards reduced water consumption. Estimated mean daily doses of DMOB were higher in females than males.

The trend for reduced water consumption in rats dosed with Blue 15 was not as marked as it was for rats dosed with DMOB. In males, only the highest Blue 15 concentration (3.0070) was associated with a decrease in consumption, while in females the 1.0070 concentration had reduced water intake. Estimated mean daily doses for females were higher than for males.

Mortality All DMOB-treated rats and their controls survived the 90-day test period.

There were 7 deaths in the 3070 level of male rats treated with Blue 15. The first death occurred in week 3 and the last in week 13.

Body weight gains Male and female rats exposed to 0.125°70 DMOB exhibited 14070 decreases in

body weight gain (Table II). Exposure to 0.25070 DMOB resulted in body weight gain decrements of 30% and 24070 in male and female rats, respectively. Exposure to 1.0070 Blue 15 produced a 17070 decrement in body weight gain of male and female rats. In male rats treated with 307o Blue 15, a 4307o decrease in body weight gain was observed.

301

TABLE I

CONSUMPTION OF DRINKING WATER CONTAINING 3,3"-DIMETHOXYBENZIDINE OR C.I. DIRECT BLUE 15 BY F344 RATS

% in 3, Y-Dimethoxybenzidine" drinking

water Male Female

Water Estimated dose Water Estimated dose consumption (mg/kg/day) consumption (mg/kg/day) (mean nil/day) (mean ml/day)

0 22 0 25 0 0.017 21 13 23 24 0.033 18 22 24 49 0.063 17 39 15 60 0.125 14 70 13 103 0.250 11 120 11 187

C.I. Direct Blue 15"

Male Female

% in Water Estimated DMOB b Water Estimated DMOB drinking consumption dose equivs, consumption dose equivs. water (mean nil/ (mg/kg/day) (mg/kg/day) (mean mi/ (mg/kg/day) (mg/kg/day)

day) day)

0 22 0 0 22 0 0 0.063 c c c 23 83 26 0.125 25 115 38 24 174 54 0.250 25 232 73 20 290 93 0.50 26 473 150 21 592 188 1.00 23 845 268 19 1083 332 3.00 16 2396 767 c c c

*13-week exposure. Values represent means of 10 rats per group. bDMOB equivalents represent the amount of DMOB the animals would he exposed to if 100% of Blue 15 was reductively metabolized. cNot treated at this concentration.

Organ weights Dose-related depressions in organ weights and organ weight to body weight

ratios were observed in DMOB-treated rats (Table III). A significant increase was observed in absolute liver weights of males at the 0.0630/0 and 0.125070 dose lev- els. Liver to body weight ratios were significantly greater for all treated male groups and for the 2 highest dose groups of females as compared to controls. Absolute kidney weights were significantly increased for females at the 3 highest dose levels. Kidney weight to body weight ratios were significantly increased for males receiving doses of 0.063% DMOB or more, and for females receiving solu- tions of 0.033°70 or more.

Dose-related increases in the absolute kidney weights and kidney weight ratios were observed in male and female rats treated with Blue 15 (Table IV). Kidney

302

TABLE II

BODY W E I G H T GAIN IN RATS T R E A T E D W I T H 3 ,Y-DIMETHOXYBENZIDINE OR C.I. DIRECT BLUE 15 FOR 13 WEEKS

% in Body weight gain (g) (% control) drinking water 3,Y-Dimethoxybenzidine % in C.I. Direct Blue 15

drinking Male Female water Male Female

0 211 (100)* 87 (100) 0 190 (100) 75 (IO0) 0.017 206 (98) 83 (95) 0.063 b 68 (91) 0.033 208 (99) 85 (98) 0.125 165 (87) 70 (93) 0.063 200 (95) 80 (92) 0.250 176 (93) 70 (93) 0.125 181 (86)* 75 (86) 0.500 176 (93) 69 (92) 0.250 147 (70)* 66 (76)* 1.0 157 (83) 62 (83)

3.0 109 (57)** b

• Values represent means o f 10 rats per group. Numbers in parentheses bNot dosed at this concentrat ion. • Significantly different f rom controls, P < 0.05. • *Significantly different f rom controls, P < 0.01.

= % body weight o f controls.

TABLE III

ORGAN W E I G H T S IN F344 RATS T R E A T E D W I T H 3 ,Y-DIMETHOXYBENZIDINE FOR 13 WEEKS

% in Liver weights drinking water Male Female

Weight ratio %" Absolute weight b Weight ratio %" Absolute weight b

0 2.51 -4- 0.06 c 8.17 4- 0.48 2.59 4. 0.13 4.64 4. 0.29 0.017 2.77 4. 0.06** 8.84 4. 0.53 2.62 4. 0.11 4,62 4. 0.32 0.033 2.79 4. 0.07** 9.07 4. 0.40 2.70 4. 0.12 4.81 4- 0.27 0.063 2.93 4. 0.10"* 9.33 4. 0,62* 2.84 4. 0.31 4,97 4. 0.52 0.125 3.13 4. 0.11"* 9.26 4. 0.80* 2.83 4. 0.08** 4.92 4. 0.30 0.25 3.28 -4- 0.18"* 8.71 -4- 0.95 3.02 4. 0.15"* 4.94 4. 0.31

Kidney weights

0 0.296 + 0.013 0.963 + 0.060 0.318 + 0.014 0.570 4. 0.036 0.017 0.310 4. 0.013 0.991 4. 0.090 0.327 4. 0.016 0.576 4. 0.038 0.033 0.318 -4- 0.011 1.033 4. 0.048 0.352 4. 0.017" 0.627 4. 0.030 0.063 0.336 4. 0.012"* 1.070 -4- 0.075 0.394 4. 0.019"* 0.691 4. 0.043** 0.125 0.354 4. 0.018"* 1.046 4. 0.077 0.400 4. 0.027** 0.695 4. 0.056** 0.25 0.402 4. 0.018"* 1.065 4. 0.067 0.419 4. 0.015"* 0.687 4. 0.028**

• Weight ratio % in organ weight /body weight x 100. bOrgan weight in grams. cValues represent means + S.D., n = 10. • Significantly different f rom controls at the one-tailed 5% level. • *Significantly different f rom controls at the one-tailed 1% level.

303

TABLE IV

ORGAN WEIGHTS IN RATS TREATED WITH C.I. DIRECT BLUE 15 FOR 13 WEEKS

070 in Liver weights drinking water Male Female

Weight ratio %" Absolute weight b Weight ratio %" Absolute weight b

0 2.79 .4- 0.37 c 9.74 .4- 1.66 2.50 .4- 0.11 4.86 .4- 0.28 0.063 d d 2.53 .4- 0.06 4.74 4- 0.18

0.125 3.01 .4- 0.41 9.91 .4- 1.68 2.53 .4- 0.08 4.80 .4- 0.24 0.25 3.12 .4- 0.40 10.42 .4- 2.05 2.51 .4- 0.17 4.80 .4- 0.29 0.50 3.11 .4- 0.41 10.20 ± 1.64 2.50 ± 0.13 4.75 ± 0.34 1.0 3.17 .4- 0.49 10.21 .4- 1.95 2.70 .4- 0.16" 5.04 .4- 0.43 3.0 3.74 .4- 0.60 8.75 .4- 3.07 d d

Kidney weights

0 0.279 .4- 0.020 0.968 _ 0.081 0.311 .4- 0.022 0.604 .4- 0.053

0.063 d d 0.321 .4- 0.016 0.602 ± 0.052

0.125 0.298 .4- 0.010 0.979 .4- 0.069 0.311 .4- 0.014 0.591 .4- 0.031

0.25 0.308 .4- 0.019" 1.018 _ 0,084 0.332 4- 0.019 0.634 .4- 0.034

0.50 0.342 .4- 0,018"* I.I17 __. 0.074 0.351 __. 0,014" 0.666 .4- 0.040

1.0 0.357 .4- 0.013"* 1.145 _ 0.091"* 0.402 .4- 0.030** 0.750 .4- 0.075**

3.0 0.631 .4- 0.176 1.403 .4- 0.343"* d d

• Weight ratio % = organ weight/body weight x 100. bOrgan weight in grams. cValues represent means .4- S.D., n = l0 (n = 3 for 3% males). dNot dosed at this concentration. • Significantly different from controls at the one-tailed 5°7o level. • *Significantly different from controls at the one-tailed 1 070 level.

weights of male and female rats at the 1.0°70 dose level and males at the 3.0070 level were significantly greater than controls. Kidney ratios were significantly increased for males at the 0.25070 level, for males and females at the 0.5°70 and the 1.0070 level, and although not statistically significant, the kidney ratios for the 3.0070 level males were markedly increased (45070 increase over control ratios). Due to the small number of survivors in the 3070 dose group, and the variability of weights among these animals, the statistical analysis does not reinforce this biologically significant finding.

In female rats, the only statistically significant change in liver weights occurred in the liver to body weight ratios in the 1 070 Blue 15 dose group.

Hematology and clinical chemistry Treatment with DMOB, and Blue 15 produced no consistent or dose-related

changes in hematology or clinical chemistry of biological significance (data not shown).

Histopathology A very slight increase in the incidence and severity of chronic nephropathy

304

TABLE V

HISTOPATHOLOGICAL EFFECS OF 3,3 '-DIMETHOXYBENZIDINE" AND C.I. DIRECT BLUE 15 IN F344 RATS

Tissue 3,3'-Dimethoxybenzidine (070 in water)

Control 0.125 0.25

Male Female Male Female Male Female

Thyroid pigment in follicular cells

Kidney multifocal chronic nephropathy

0 0 l0 (1.6) 10 (1.5) 10 (3.1) 10 (3.0)

10 (1.0) 0 10 (1.0) 0 10 (1.8) 8 (1.0)

C.I. Direct Blue 15 (070 in water)

Control 0.5°70 1.0070

Male Female Male Female Male Female

Kidney multifocal chronic nephropathy

10 (1.0) 0 10 (1.0) l (1.0) 10 (1.4) 9 (1.1)

• 13-week treatment. Number of rats with lesion out of 10 rats per group (severity of lesion). Severity of lesion: 1.0 = minimal; 2.0 = mild; 3.0 = moderate; 4.0 = marked.

characterized by foci of regenerative tubular epithelium was detected in female rats treated with 0.25°70 DMOB (Table V). Although all males (treated and controls) were affected, a slight increase in severity was observed in the 0.25070 group. Both males and females dosed with 0.25070 had moderately severe amounts of yellowish-brown pigment in the cytoplasm of thyroid follicular cells, and this effect decreased in intensity as dose decreased. The pigment stained weakly posi- tive for lipofuscin.

Histopathological findings in Blue 15-treated rats consisted primarily of renal and hepatic toxicity in high-dose males that died before termination (data not shown). The liver lesion was characterized by minimal megalocytosis, degeneration and necrosis of hepatocytes, fatty metamorphosis, and blue pigment (likely Blue 15) in Kupffer cells of the hepatic sinusoids. Kidney lesions in the high-dose male rats were characterized by degeneration and focal necrosis of proximal tubular epithelial cells.

In the 1.0070 dose level animals which were sacrificed at termination (Table V) mild chronic nephropathy was the principal histopathology finding among males and females. No differences existed between controls and lower doses.

Discussion

Dimethoxybenzidine and Blue 15 doses (mg/kg per day) were limited by

305

decreases in water consumption with increasing dose level (Table I). Because the measurement of water intake is not precise, these data were presented to show trends rather than to express precise dose levels. A problem with palatibility of the drinking water was anticipated; however, it was considered that the advantages of a more constant intake of the chemicals outweighed any disadvan- tages of the administration of single doses via gavage, and the risk of animal loss due to gavage trauma or accident. Doses in females were higher than in male rats for both chemicals. This difference was due to the fact that although females weighed less than males, they consumed nearly the same amount of water.

Microscopic examination of the tissues revealed that the kidney is a target organ for both DMOB and Blue 15. Mild renal lesions, characterized by foci of regenerative tubular epithelium, which appeared to be an exacerbation of the chronic progressive nephropathy common to the Fischer 344 rat, were detected in male rats at all dose levels. A slight increase in severity of this lesion was observed in males exposed to 0.25°?0 DMOB and 1% Blue 15. An increased incidence of nephropathy was detected in female rats exposed to 0.50?o and 1.0% Blue 15 and to 0.250?o DMOB. These early renal changes, when compounded by naturally occurring progressive nephropathy, may result in shortened life spans of rats given these doses in a longer term or 2-year study.

Treatment-related histopathological alterations were observed in the thyroid of animals treated with 0.125 and 0.250?o DMOB. Pigment deposition indicated an increase in thyroid activity or function. This effect was not observed in Blue 15- treated rats.

The liver was also found to be a target organ for Blue 15-treated rats based upon mild histological lesions in the high dose (30?o) group male rats which died before termination of the study. Hepatic lesions were not detected in female rats exposed to a maximum dose of only 1070 Blue 15. In addition, these hepatic lesions were not observed in rats treated with DMOB. The occurrence of hepatotoxicity in Blue 15-exposed rats and not in rats exposed to DMOB may have been caused by differences in the administered doses of the 2 compounds, metabolites of Blue 15, or impurities in the Blue 15 dye.

In the high-dose groups, exposure concentrations of Blue 15 were slightly higher than the administered doses of DMOB, suggesting that hepatotoxicity may have been caused directly by the higher concentrations of unmetabolized Blue 15. Dimethoxybenzidine produced by metabolism of Blue 15 is not likely to be the cause of hepatotoxicity since similar lesions would be expected to have occurred in DMOB-treated rats. In studies by Lynn et al. [5], only 1--3% of the administered Blue 15 was excreted as DMOB in the urine of treated rats.

The aromatic amine metabolite resulting from azo reduction of Blue 15 (1- hydroxy-2,8-diamino-naphthalene sulfonic acid) may have contributed to the hepatotoxicity observed with Blue 15 and not DMOB. This same compound is a potential metabolite of the carcinogenic benzidine-based dyes C.I. Direct Blue 6, C.I. Direct Blue 14, and C.I. Acid Red 10B. Positional isomers of this metabolite may also be produced by reduction of a number of azo dyes (e.g. Acid Red dyes 18, 26, 27, and 33, Mordant Red 9, Acid Orange 10), many of which are hepatotoxic [28]. These small sulfonated molecules are generally well absorbed

306

and may undergo further metabolic change before excretion by the liver (via the bile) or kidney. There is a general lack of informat ion on the toxicological poten- tial o f such probable metabolites of the azo dyes.

No information on target organs for short- term exposure to DMOB or Blue 15 was found. After long-term (1 year) exposures of Fischer rats to DMOB [29] target organs for the carcinogenic effects o f DMOB were the lower intestinal tract, skin, ear duct, bladder and forestomach. Renal toxicity was not reported in this earlier study, and hepatotoxicity was observed in only 1 male rat given 1 mg DMOB/day . Apparent differences in target organs in these 2 studies may have been due, in part , to differences in dose levels, durat ion of exposure, and routes of administration (gavage vs. dosed water).

Three benzidine-based dyes, Direct Blue 6, Direct Black 38 and Direct Brown 95 caused hepatocelluar carcinomas and neoplastic nodules of the liver after exposure of rats to 190--3000 p p m in feed for only 13 weeks [30]. The presence of benzidine in the urine and of liver lesions identical to those caused by benzidine alone suggested that benzidine released by metabolism of the dyes was responsible for the liver lesions. In these studies no neoplastic effects and only mild toxicity were detected after exposure of rats to DMOB or Blue 15 for 13 weeks. Differences in the doses, in metabolism of the different dyes, and in the carcinogenicity of benzidine relative to DMOB may account for the different results of these 2 studies.

The kidney appeared to be a target organ for both DMOB and Blue 15 based on increased kidney weights and an increased incidence of nephropathy in female rats, and slight increases in severity of nephropathy in male rats. The no-effect level for nephropathy was 0.125°70 for DMOB-exposed rats, and 0.5°70 for Blue 15-exposed rats. The liver was also found to be a target organ for Blue 15-treated rats based upon mild histological lesions in the high-dose (3°70) group of male rats which died before termination of the study. Reductions in food and water consumption, as well as decreases in weight gain were also observed with high doses of both DMOB and Blue 15. These results were used to select doses for chronic studies evaluating the carcinogenicity and long-term toxicity of DMOB and Blue 15. Chronic studies of DMOB and Blue 15 will provide data that can be used in evaluating the potential toxicity and carcinogenicity of other benzidine congener dyes.

Acknowledgements

The authors sincerely thank Dr. M. Elwell and Dr. K. Abdo for their valuable suggestions in the preparat ion of this manuscript .

References

1 T. Haley, Benzidine revisited: A review of the literature and problems associated with the use of benzidine and its congeners. Clin. Toxicol., 8 (1975) 13.

2 M.R. Zavon, U. Hoegg and E. Bingham, Benzidine exposure as a cause of bladder tumors. Arch. Environ. Health, 27 (1973) 1.

307

3 IARC, Benzidine. In IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Vol. 1. World Health Organization, International Agency for Research on Cancer, Lyon, 1972, p. 80.

4 C.R. Nony, M.C. Bowman, T. Cairns, L.K. Lowry and W.P. Tolos, Metabolism studies of an azo dye and pigment in the hamster based on analysis of the urine for potentially carcinogenic aromatic amine metabolites. J. Anal. Toxicol., 4 (1980) 132.

5 R.K. Lynn, D.W. Donielson, A.M. Ilias, J.M. Kennish, K. Wong and H.B. Matthews, Metabolism of bisazobiphenyl dyes derived from benzidine, 3,3'-dimethylbenzidine, or 3,3'- dimethoxybenzidine to carcinogenic aromatic amines in the dog and rat. Toxicol. Appl. Pharma- col., 56 (1980) 248.

6 L.K. Lowry, WoP. Tolos, M.F. Boeniger, C.R. Nony and M.C. Bowman, Chemical monitoring of urine from workers potentially exposed to benzidine-derived azo dyes. Toxicol. Lett., 7 (1980) 20.

7 C.R. Nony, J.R. Althaus and M.C. Bowman, Chromatographic assays for traces of potentially carcinogenic metabolites of two azo dyes, direct red 2 and direct blue 15 in rat, hamster and human urine. J. Anal. Toxicol., 7 (1983) 40.

8 R.P. Bos, M.A.M. Groenen, J.L.G. Theuws, CH.-M. Leijdekkers and P.Th. Henderson, Metabolism of benzidine-based dyes and the appearance of mutagenic metaholites in urine of rats after oral or intraperitoneal administration. Toxicology, 31 (1984) 271.

9 C.N. Martin and J.C. Kennelly, Rat liver microsomal azoreductase activity on four azo dyes derived from benzidine, 3,3'-dimethylbenzidine, or 3,3'-dimethoxybenzidine. Carcinogenesis, 2 (1981) 307.

10 J.P. Brown and P.S. Dietrich, Mutagenicity of selected sulfonated azo dyes in the Salmonella/ microsome assay: Use of aerobic and anaerobic activation procedures. Mutat. Res., 116 (1983) 305.

11 C.E. Cerniglia, J.P. Freeman, W. Franklin and L.D. Pack, Metabolism of azo dyes derived from benzidine, 3,3'-dimethylbenzidine and 3,3'-dimethoxybenzidine to potentially carcinogenic aromatic amines by intestinal bacteria. Carcinogenesis, 3 (1982) 1255.

12 R.P. Bos, W. Van Der Krieken, L. Smeijsters, J.P. Koopman, H.R. De Jonge, J.L.G. Theuws and P.Th. Henderson, Internal exposure of rats to benzidine derived from orally administered benzidine-based dyes after intestinal azo reduction. Toxicology, 40 (1985) 207.

13 M.J. Prival, S.J. Bell, V.D. Mitchell, M.D. Peiperl and V.L. Vaughn, Mutagenicity of benzidine and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay. Mutat. Res., 136 (1984) 33.

14 IARC, In Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man, Vol. 4, World Health Organization, International Agency for Research on Cancer, Lyon, 1974.

15 P.F. Woolrich and W.A. Rye, Urethanes. J. Occup. Med., 11 (1969) 184. 16 USITC, U.S. International Trade Commission. Imports of Benzenoid Chemicals and Products

1983. Washington, DC: U.S. Government Printing Office. Publication No. 1548, 1984, p. 17. 17 NIOSH, National Institute of Occupational Health, Health hazard alert - - benzidine, o-tolidine-,

and o-dianisidine-based dyes. Am. Ind. Hyg. Assoc. J., 42 (1980) A36. 18 USITC, U.S. International Trade Commission. Synthetic Organic Chemicals, U.S. Production

and Sales, 1982. Washington, DC: U.S. Government Printing Office. Publication No. 1422, 1983, p. 60.

19 USITC, U.S. International Trade Commission. Imports of Benzenoid Chemicals and Products 1980. Washington, DC: U.S. Government Printing Office. Publication No. 1163, 1981, p. 57.

20 USEPA, TSCA Chemical Assessment Series, Preliminary Risk Assessment Phase I, Benzidine, its Congeners, and their Derivative Dyes and Pigments. U.S. Environmental Protection Agency- 560/11-80-019, 1980.

21 J.W. Meigs, L.J. Sciarini and W.A. Van Sandt, Skin penetration by diamines of the henzidine group. A.M.A. Arch. Ind. Hyg., 9 (1954) 122.

22 J.W. Meigs, R.M. Brown and L.J. Sciarini, A study of exposure to benzidine and substituted benzidines in a chemical plant. A.M.A. Arch. Ind. Hyg., 4 (1951) 533.

23 A. El-hawari, M. Sawyer, and M. Hainje, Absorption and elimination of ~4C-labeled Direct

308

Black 14 and Direct Black 38 following dermal application to male New Zealand rabbits.

Midwest Research Institute Report prepared for I.B.M. Corporation, M.R.I. Project No. 4612- B(2). Kansas City, MO (1979).

24 A. Shah, D. Harbin and C.W. Jameson, Analyses of two azo dyes by high-performance liquid chromatography. J. Chromatog., 26 (1988) 439.

25 G.W. Snedecor and W.G. Cochran, Statistical Methods, 6th Edn., Iowa State University Press, Ames, Iowa, 1967.

26 H. Scheffe, A method for judging all contrasts on the analysis of variance. Biometrika, 40 (1953) 87.

27 P.A. Games and J.F. Howell, Pairwise multiple comparison procedures with unequal N's and/ or variances: A Monte Carlo Study. J. Ed. Statist., 1 (1976) 113.

28 DETO, Comments on the Testing Recommendations of the TSCA Interagency Testing Committee for Benzidine based, o-Tolidine based and o-Dianisidine Based Dyes. Dyes Environ- mental and Toxicology Organization, Inc., Scarsdale, NY, 1980.

29 Z. Hadidian, T.N. Frederickson, E.K. Weisburger, J.H. Weisburger, R.M. Glass and N. Man- tel, Tests for chemical carcinogenesis: Report on the activity of derivatives of aromatic amines, nitrosoquinolines, nitroaldanes, amides, epoxides, aziridines, and purine antimetabolites. J. Natl. Cancer Inst., 41 (1968) 985.

30 NCI, National Cancer Institute, 13-Week subchronic studies of direct blue 6, direct black 38, and direct brown 95 dyes. NCI Technical Report No. 108. U.S. Department of Health, Educa- tion and Welfare, Public Health Service, Nati,onal Institutes of Health, Bethesda, MD., 1978.

309