Exposure of laboratory animals to polychlorinated dibenzodioxins and polychlorinated dibenzofurans from commercial rodent chow

Pergamon

0045-6535(95)00328-2

Chemosphere,Vol. 32, No. 3, pp. 501-508, 1996 Copyright © 1996 Published by Elsevier Science Ltd Printed in Great Britain. All fights reserved 0045-6535/96 $15.00+0.00

EXPOSURE OF LABORATORY ANIMALS TO POLYCHLORINATED DIBENZODIOXINS AND POLYCHLORINATED DIBENZOFURANS FROM COMMERCIAL RODENT CHOW

Schecter, A.J. A, Olson, j.B, P~tpke, O. c A Department of Preventive Medicine, Clinical Campus, State University of New York, Health Science Center-Syracuse, 88 Aldrich Ave, Binghamton, NY 13903, USA. 8 University at Buffalo, SUNY Medical Center, Department of Pharmacology and Toxicology, 102 Farber Hall, Buffalo, NY 14214, USA. c ERGO Forschungsgesellschaft mbH, Albert-Einstein Ring 7, D-22761 Hamburg, Germany.

ABSTRACT

Polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/PCDFs) are ubiquitous low level environmental contaminants. Present finding of PCDDs and PCDFs in the human food supply suggested that laboratory animals maintained under controlled conditions may also receive unwanted exposure to these compounds from food. In this study, a commercial rodent chow, Prolab-RMH 1000, was analyzed for the toxicologically active 2,3,7,8-substituted PCDDs and PCDFs. The results show that the feed had a total PCDD/F dioxin toxic equivalence (I-TEq) of 0.13 pg/kg (ppt) wet weight. OCDD was the most abundant congener present, followed by 1,2,3,4,6,7,8-HpCDD, with these congeners representing the 5th and 6th greatest contributors to the calculated TEq. Daily ingestion of I-TEqs for the rats is calculated to be 3.9-6.5 pg/kg/day, and 13.0-32.5 pg/kg/day for mice. It is therefore important to consider the potential for background exposure to PCDDs and PCDFs from the diet when conducting studies with laboratory animals. This is particularly important when sensitive responses, such as enzyme induction, immunotoxicity, and developmental/reproductive toxicity are being investigated at very low exposures to this class of compounds.

501

502 INTRODUCTION

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are a concern to human health and the environment due to their persistence and profound toxicological activity.<~'2~ PCDDs and PCDFs are produced as byproducts from various combustion and chemical processes which have resulted in their widespread global contamination. The environmental and biological stability and lipophilicity of PCDDs and PCDFs has also led to their effective transport in the food chain, with bioaccumulation at higher trophic levels, including humans.(3,4) Although specific point sources are important in highly exposed individuals, dietary intake represents the primary source of exposure to PCDDs and PCDFs in the general background human population. Greater than 90% of background human exposure has been reported to occur through the diet, with foods of animal origins being the predominant sources.The potency of individual PCDDs and PCDFs have been investigated in laboratory animals and in vitro models using endpoints such as affinity of congeners for the Ah receptor, enzyme induction and overt toxicity, including carcinogenicity, developmental toxicity, immunotoxicity, thymic atrophy, and/or decrease in body weight gain. The potency of individual congeners has been expressed relative to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), the most potent and extensively studied congener which has a Toxic Equivalency Factor (TI::F) of 1.0. With the assumption of additive toxicological activity, the combined potency of complex environmental mixtures of PCDDs and PCDFs has been expressed as TCDD or dioxin toxic equivalents (TEq). (~5"~8) Therefore, analysis of PCDDs and PCDFs is generally expressed on a congener specific basis and TEqs. Studies in laboratory animals assume that under controlled laboratory conditions, animals are not exposed to PCDDs or PCDFs. However, the ubiquitous nature of these environmental contaminants and their presence in the human food supply suggest that laboratory animals maintained under controlled conditions may receive unwanted exposure to these compounds. The present study was conducted to assess whether commercial laboratory animal feed contains PCDDs and PCDFs. The daily intake of PCDDs and PCDFs in lab animals through consumption of commercial chow will also be estimated and the toxicological significance discussed.

503 METHODS

Prolab RMH 1000 Standard diet for laboratory rats, mice and hamsters was obtained from Agway Inc., Syracuse, NY. Approximately 200 grams of the rodent diet, in the form of dried pellets, was randomly sampled from the AALAC accredited laboratory animal care facility at SUNY Buffalo. The analysis of the rodent chow was conducted in a laboratory certified by the World Health Organization for dioxin analysis of human tissues. The laboratory also passed interlaboratory dioxin food validation testing. The rodent diet was analyzed for PCDDs and PCDFs by high resolution gas chromatography - high resolution mass spectroscopy (HRGC/HRMS) following extraction and clean-up by previously described methods: Sample amounts of 20 grams - spiked with 13 13C 2,3,7,8 substituted PCDD/F standards - were extracted. The duplicate was extracted with a mixture of hexane/ acetone (1:1) within 24 hours. The same procedure was performed with a so-called "method blank". The following clean-up procedure was undertaken by the use of a multicolumn system, including carbon on glass fiber. (19) After consecutive measurement by HRGC/HRMS, using a SP 2330 fused silica column, the quantification was done by the isotope dilution method. Recoveries of the internal standards ranged from 65 to 105%. The Prolab RMH 1000 diet contains 14.5% protein, 6.5% fat, 1.1% linoleic acid, 4% fiber, 7.5% ash, and 57.5% nitrogen free extract. Ingredients of RMH 1000 include ground wheat, ground corn, wheat middlings, dried whey product, meat and bone meal, animal fat preserved with BHA, soybean meal, dehydrated alfalfa meal, brewers dried yeast, dicalcium phosphate, salt, calcium carbonate, choline chloride, magnesium oxide, iron sulfate, iron carbonate, manganous oxide, zinc oxide, copper oxide, calcium iodate, cobalt carbonate, vitamin A acetate, D-activated animal sterol (source of vitamin D3), vitamin E supplement, niacin, riboflavin, menadione sodium bisulfite complex (source of vitamin K activity), pyridoxine hydrochloride, thiamine mononitrate, calcium pantothenate, folic acid, biotin, vitamin B12 supplement.

RESULTS AND DISCUSSION

Table 1 summarizes the PCDD and PCDF analysis of the Prolab RMH 1000 rodent chow. Most 2,3,7,8-substituted congeners were detected in the rodent diet, with OCDD being the most abundant congener followed by 1,2,3,4,6,7,8-HpCDD and 1,2,3,4,6,7,9-HpCDD. On a whole or wet weight basis, the rodent diet had a I-TEq content for total PCDDs and PCDFs of 0.13 ng/kg (ppt). The I-TEq content of the

504

rodent diet was similar to that reported for bologna and crunchy haddock from a U.S. supermarket and about 10-fold less than that in ground beef. ¢5~ Animal fat is generally considered a major source of PCDDs and PCDFs in the human diet. <12) Animal fat is also one of the ingredients in the Prolab RMH 1000 rodent chow and thus is probably the main source of PCDDs and PCDFs in the rodent diet. In a related study, Vanden Heuvel et al ¢2s~reported that 2,3,7,8-TCDD, 1,2,3,6,7,8-HxCDD, 1,2,3,4,6,7,8-HpCDD, OCDD, 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF, 2,3,4,6,7,8-HxCDF, and 1,2,3,4,6,7,8-HpCDF accumulated in the liver of control laboratory rats. Standard laboratory rat feed was identified as one potential source of the background exposure to these persistent compounds, however the feed was only consistently found to contain OCDD and 1,2,3,4,6,7,8-HpCDD c25). Our results suggest that the sensitivity of the analysis was not adequate to detect other congeners since the limits of detection in the Vanden Heuvel study 12slwere greater than the levels of each congener reported in rat feed in Table 1. Therefore, our results suggest that the commercial rat feed could be the source of the PCDDs and PCDFs retained in the liver of control laboratory rats. The daily intake of PCDDs and PCDFs (I-TEq) in laboratory animals from the consumption of commercial rodent chow is estimated in Table 2. Daily ingestion of I-TEqs range from 3.9-6.5 pg/kg/day for the rat to 13.0-32.5 pg/kg/day for the mouse. This is slightly higher than the estimated background adult human exposure from the U.S. diet of between 0.3 and 3.0 pg TEQ/kg/day and lower than that of a nursing infant which may consume from 35 to 53 pg TEQ/kg/day in its first year of life36~ Thus, as with humans, laboratory animals are exposed to PCDDs and PCDFs through consumption of food in the form of commercial diets. It is therefore important to consider the potential for background exposure to PCDDs and PCDFs when conducting studies with laboratory animals. In addition, it is important to consider that other Ah-agonists (eg. heterocyclic amines, PAHs, PCBs) may be present in laboratory animal diets and that some of these compounds, including PCDDs and PCDFs, have a potential to accumulate in animals with chronic dietary exposure. This is particularly important when sensitive responses, such as enzyme induction, immunotoxicity, and developmental/reproductive toxicity, are being investigated at very low exposures to this class of compounds (Table 3). Although only one commercial rodent diet was investigated, it is likely that other laboratory animal diets with animal derived ingredients will contain low levels of PCDDs

505

TABLE 1 PCDDs and PCDFs in Prolab RMH 1000 Laboratory Rodent Diet Congener

TEF

wet weight ppt.

I-TEq (NATOCCMSIEPA)

2378-TCDD

t

<0.04 (m)

0.02

Total TCDD 12378-PnCDD Total PnCDD 1234578-HxCDD 123678-HxCDD 123789-HxCDD Total HxCDD 1234678-HpCDD Total HpCDD OCDD 2378.TCDF Total TCDF 12378-PnCDFI12348-PnCDF 23478-PnCDF Total PnCDF 123478-HxCDFI123479-HxCDF 123678.HxCDF 123789.HxCDF 234678.HxCDF Total HxCDF 1234678-HpCDF 1234789-HpCDF Total HpCDF OCDF

0.5 0.1 0.1 0.1

OlOl 0.001 0.1 0.06 0.5 0.1 0.1 0.1

0.18 (m) 0.64 0,02

0.03 (m) 0.26 0.07 (m) 0.03 n.d. (0.01)

0.016 0.008 0.009 0.004 0.0146 0.0111 0.018

0.001 0.015 0.007 0.003 0.0006

0.02 0.24 0.26 0.03 0.43 0.33

0.002

Total PCDD

14.12

0.08

Total PCDF

1.80

0.05

15.92

0.13

Total PCDDIF

0.1

0.06 0.03 0.16 0.08 0.09 0.04 (m) 0.38 1.46 2.45 11.08

0.01 0.01 0.001

n.d. = not detected, detection limit in ( ) (m) = maximum value with possible contribution of artifacts

0.0026 0.0003 0.0003

506

TABLE 2 Estimated Daily Intake of PCDDs PCDFs in Laboratory Animals from the Consumption of Commercial Rodent Chow Rat

Mouse

Hamster

Adult Male Body weight(g)

300-400

20-40

85-110

Daily food consumption(g)"

12-15

4-5

8-15

Daily I-TEQ from chow (pglday) b

1.56-1.95

0.52-0.65

1.04-1.95

3.9-6.5

13.0-32.5

9.5-22.9

(pglkglday)

• from Agway Inc., Syracuse, NY b Based on rodent chow in Table 1 with 0.13 I-TEq (ppt, nglkg).

Table 3 Sensitive Responses to TCDD Exposure Observed in Laboratory Animals Effect

Animals

TCDD Exposure

References

Induction of Hepatic cytochrome P-

Rat

Single dose of 1 nglkg body weight

(20)

Immunosuppression (enhanced viral susceptibility)

Mouse

Single dose of 10 nglkg body weight

(21)

Decreased glucose uptake into adipocytes

Guinea Pig

Single dose of 30 nglkg body weight

(22)

Developmental, behavioral, endocrine, decreased sperm count

Rat

Single Maternal dose of 64 nglkg body weight on gestation day 15

(23)

Liver and lung cancer

Rat

100 nglkg per day for 2 year

(24)

4501A1

507

and PCDFs. Elimination of animal fat and other animal derived materials from commercial lab animal diets is a possible means of reducing the low level exposure of laboratory animals to these compounds. ACKNOWLEDGEMENTS: Ms. Heather Kessler, research assistant, assisted with the

preparation of this manuscript. REFERENCES:

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