121
Twenty-six week toxicity study with KASAL| (basic sodium aluminum phosphate) in beagle dogs John C. Pettersen*, Dennis S. Hackett*, Gary M. Zwicker 1 and Gary L. Sprague 2 Stauffer Chemical Co., Environmental Health Center, 400 Farmington Ave., Farmington, Connecticut 06032, USA; 1Merrell Dow Pharmaceutical, Inc., Toxicology Department, P.O. Box 68470, 9550 Zionsville Rd, Indianapolis, IN 46268, USA; 2Smith Kline-Beckman Co., One Franklin Plaza, P.O. Box 7929, Philadelphia, PA 19101, USA
Introduction
Sodium aluminum phosphate in its basic form (KASAL) is a white odorless powder comprised of an autogenous mixture of alkaline sodium aluminum phosphate and dibasic sodium phosphate. KASAL is used primarily as an emulsifier in the production of processed cheese. This food additive constitutes a significant source of dietary aluminum in the USA (Pennington, 1987). Recent studies implicated parenteral administration of aluminum to a variety of maladies including osteomalacia and dialysis dementia (Ellis et al., 1979; Goodman, 1986; Will and Savory, 1985). However, relatively few long-term studies in laboratory animals have been reported. Because sodium aluminum phosphate formulations are potentially important sources of aluminum exposure it was important to study their toxicity and contribution to aluminum deposition in potentially important biological tissues (i.e., bone and brain). In the present study, Beagle dogs were fed diets containing up to 30,000 p.p.m. KASAL for 26 weeks. Mild toxicity was observed in high-dose males. No toxicologically significant increase of tissue aluminum concentrations were observed in KASAL treated dogs.
determined monthly by analysis of aluminum content using atomic absorption spectrophotometry.
Animals Male and female Beagle dogs (20 to 22 weeks of age) were obtained from Marshall Research Animals (North Rose, NY). They were housed individually in stainless steel cages and were exercised weekly. The animals were quarantined for approximately five weeks.
Study design Groups of four male and four female dogs were fed constant dietary concentrations of 0, 3,000, 10,000 or 30,000 p.p.m. KASAL for 26 weeks. Four-hundred grams of blended diet containing KASAL was provided to each dog in a 3-hour feeding period. Blood, urine, and faecal samples were collected for clinical laboratory analyses from all animals prior to study initiation and at termination. Blood specimens only were collected near the midpoint of the study for the haematological and blood chemistry tests listed below. The dogs were fasted for at least 16 hours prior to sample collection. Urine and faecal samples were collected overnight using metabolism cages. Blood samples were collected from the jugular vein the following morning. Hematocrit, haemoglobin concentration, erythrocyte count, leukocyte count (total and differential), and platelet count were determined. Serum chemistry measurements included blood urea nitrogen, creatinine, sodium, potassium, chloride, phosphorus, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transferase, sorbitol dehydrogenase, total bilirubin, total protein, albumin, globulin, glucose, calcium, cholesterol, and triglyceride. Organs weighed at necropsy included heart, liver, kidneys, ovaries, testes, thyroid, adrenals, and brain. Specimens from all major organs were fixed by immersion in 10% neutral buffered formalin or 2.5% buffered glutaraldehyde and processed for light microscopic examination.
Test diets A sample of KASAL, representative of commercial production, was obtained from Stauffer Chemical Co. (Chicago Heights, IL). The test diets were prepared by blending the basal diet (Purina Certified Canine Diet #5007 ground meal, Ralston Purina, St Louis, MO) with KASAL and a small amount of Mazola corn oil (0.5% w/w) to suppress dust. The test diets were blended biweekly and stored at room temperature. The concentration and homogeneity of KASAL in each of the blended diets were
Bone aluminum analysis The aluminum concentration of trabecular bone and brain specimens were determined. Trabecular bone specimens were obtained from the fight and left femurs and brain specimens from the frontal cortex. Tissue samples were prepared for aluminum analysis in a Class 10-100 clean room. The samples were digested with NBS (National Bureau of Standards Purified Reagent) nitric acid. Aluminum in the resulting solution was determined with a Perkin-Elmer 5000 (Norwalk, CT)
Materials and Methods
122
Twenty-six week toxicity study with KASAL
Table 1 Group mean KASAL and elemental aluminum consumption.
Nominal dietary KASAL concentration (p.p.m.).
0 3,000 10,000 30,000
Mean KASAL consumption (mg/kg/day)
Mean a elemental aluminum dose (mg/kg/day)
Male Female
Male Female
0 0 112 106 390 323 1,143 1,251
4 10 27 75
3 10 22 80
a Aluminum levels contributed by the basal diet were included in these calculations.
atomic absorption spectrophotometer equipped with a graphite furnace and Zeeman background correction system. Limits of detection and quantitation of tissue aluminum concentrations were calculated as described by Long and Winefordner (1983) and the American Chemical Society's Committee on Environmental Improvement (1980). The limit of detection for a sample was the lowest concentration measured that was statistically different from the blank sample concentration. In a similar manner, the limit of quantitation was also calculated from the blank analysis data that assured that the measured value was a precise number. For ,these studies, concentration values for aluminum below the detection limit were reported as not detected, values between the limit of detection and the limit of quantitation could not be precisely measured and were therefore reported as a range, and values that exceeded the limit of quantitation were assigned numerical values. Statistical analyses Tissue aluminum concenl~ations were analyzed statistically by a one-way analysis of variance followed by Neuman-Keuls tests. Statistical significance was accepted at p
Twenty-six week toxicity study with KASAL| (basic sodium aluminum phosphate) in beagle dogs John C. Pettersen*, Dennis S. Hackett*, Gary M. Zwicker 1 and Gary L. Sprague 2 Stauffer Chemical Co., Environmental Health Center, 400 Farmington Ave., Farmington, Connecticut 06032, USA; 1Merrell Dow Pharmaceutical, Inc., Toxicology Department, P.O. Box 68470, 9550 Zionsville Rd, Indianapolis, IN 46268, USA; 2Smith Kline-Beckman Co., One Franklin Plaza, P.O. Box 7929, Philadelphia, PA 19101, USA
Introduction
Sodium aluminum phosphate in its basic form (KASAL) is a white odorless powder comprised of an autogenous mixture of alkaline sodium aluminum phosphate and dibasic sodium phosphate. KASAL is used primarily as an emulsifier in the production of processed cheese. This food additive constitutes a significant source of dietary aluminum in the USA (Pennington, 1987). Recent studies implicated parenteral administration of aluminum to a variety of maladies including osteomalacia and dialysis dementia (Ellis et al., 1979; Goodman, 1986; Will and Savory, 1985). However, relatively few long-term studies in laboratory animals have been reported. Because sodium aluminum phosphate formulations are potentially important sources of aluminum exposure it was important to study their toxicity and contribution to aluminum deposition in potentially important biological tissues (i.e., bone and brain). In the present study, Beagle dogs were fed diets containing up to 30,000 p.p.m. KASAL for 26 weeks. Mild toxicity was observed in high-dose males. No toxicologically significant increase of tissue aluminum concentrations were observed in KASAL treated dogs.
determined monthly by analysis of aluminum content using atomic absorption spectrophotometry.
Animals Male and female Beagle dogs (20 to 22 weeks of age) were obtained from Marshall Research Animals (North Rose, NY). They were housed individually in stainless steel cages and were exercised weekly. The animals were quarantined for approximately five weeks.
Study design Groups of four male and four female dogs were fed constant dietary concentrations of 0, 3,000, 10,000 or 30,000 p.p.m. KASAL for 26 weeks. Four-hundred grams of blended diet containing KASAL was provided to each dog in a 3-hour feeding period. Blood, urine, and faecal samples were collected for clinical laboratory analyses from all animals prior to study initiation and at termination. Blood specimens only were collected near the midpoint of the study for the haematological and blood chemistry tests listed below. The dogs were fasted for at least 16 hours prior to sample collection. Urine and faecal samples were collected overnight using metabolism cages. Blood samples were collected from the jugular vein the following morning. Hematocrit, haemoglobin concentration, erythrocyte count, leukocyte count (total and differential), and platelet count were determined. Serum chemistry measurements included blood urea nitrogen, creatinine, sodium, potassium, chloride, phosphorus, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transferase, sorbitol dehydrogenase, total bilirubin, total protein, albumin, globulin, glucose, calcium, cholesterol, and triglyceride. Organs weighed at necropsy included heart, liver, kidneys, ovaries, testes, thyroid, adrenals, and brain. Specimens from all major organs were fixed by immersion in 10% neutral buffered formalin or 2.5% buffered glutaraldehyde and processed for light microscopic examination.
Test diets A sample of KASAL, representative of commercial production, was obtained from Stauffer Chemical Co. (Chicago Heights, IL). The test diets were prepared by blending the basal diet (Purina Certified Canine Diet #5007 ground meal, Ralston Purina, St Louis, MO) with KASAL and a small amount of Mazola corn oil (0.5% w/w) to suppress dust. The test diets were blended biweekly and stored at room temperature. The concentration and homogeneity of KASAL in each of the blended diets were
Bone aluminum analysis The aluminum concentration of trabecular bone and brain specimens were determined. Trabecular bone specimens were obtained from the fight and left femurs and brain specimens from the frontal cortex. Tissue samples were prepared for aluminum analysis in a Class 10-100 clean room. The samples were digested with NBS (National Bureau of Standards Purified Reagent) nitric acid. Aluminum in the resulting solution was determined with a Perkin-Elmer 5000 (Norwalk, CT)
Materials and Methods
122
Twenty-six week toxicity study with KASAL
Table 1 Group mean KASAL and elemental aluminum consumption.
Nominal dietary KASAL concentration (p.p.m.).
0 3,000 10,000 30,000
Mean KASAL consumption (mg/kg/day)
Mean a elemental aluminum dose (mg/kg/day)
Male Female
Male Female
0 0 112 106 390 323 1,143 1,251
4 10 27 75
3 10 22 80
a Aluminum levels contributed by the basal diet were included in these calculations.
atomic absorption spectrophotometer equipped with a graphite furnace and Zeeman background correction system. Limits of detection and quantitation of tissue aluminum concentrations were calculated as described by Long and Winefordner (1983) and the American Chemical Society's Committee on Environmental Improvement (1980). The limit of detection for a sample was the lowest concentration measured that was statistically different from the blank sample concentration. In a similar manner, the limit of quantitation was also calculated from the blank analysis data that assured that the measured value was a precise number. For ,these studies, concentration values for aluminum below the detection limit were reported as not detected, values between the limit of detection and the limit of quantitation could not be precisely measured and were therefore reported as a range, and values that exceeded the limit of quantitation were assigned numerical values. Statistical analyses Tissue aluminum concenl~ations were analyzed statistically by a one-way analysis of variance followed by Neuman-Keuls tests. Statistical significance was accepted at p
