Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
4, 8, or 12 weeks
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Well-documented and corresponded to the criteria set for assessing animal studies in (Klimisch, et. al. 1996)- A Systematic Approach for Evaluating the Quality of Experimental Toxicological and Ecotoxicological Data, giving due consideration to the published data quality criteria in place at the time the study was conducted.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1987

Materials and methods

Principles of method if other than guideline:
Followed guidelines of an EPA chronic feeding study.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River CD
- Age at study initiation: 30-day old rats
- Weight at study initiation: 85-100g BW
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum):Teklad AIN-76A purified diet for rats and mice (Cat. No. 170481)
- Water (e.g. ad libitum): Allowed access to drinking water
- Acclimation period: Three days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Remarks:
demineralized water
Details on oral exposure:
Seventy-two male 30-day-old rats, 85-100g body-wt (Charles River CD, Sprague Dawley Crl:CD (SD) BR, Kingston, NY) were placed on Teklad AIN76A purified diet for rats and mice (Cat. No. 170481) and allowed access to tap drinking water. This diet is intended to supply 5.1551 g Ca/kg and 0.0314 Zn/kg, comparable to the recommended dietary allowance of 5.0 g Ca/kg and 0.012 g Zn/kg. Three days later, the animals were dived into four gorups of 18 rats that received, respectively, 0, 200, 500, or 1000 ppm Pb as acetate in demineralized water; all groups received water containing equimolar amounts of acetate ( as sodium) to that of 1000 ppm Pb water.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In a previous study, 200 ppm of lead was found to be the minimally toxic lead dose using a diet with similar trace metal content (Six and Goyer, 1970).
Duration of treatment / exposure:
4, 8, and 12 weeks
Frequency of treatment:
ad libitum
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm
Remarks:
Pb as acetate in demineralized water
Dose / conc.:
200 ppm
Remarks:
Pb as acetate in demineralized water
Dose / conc.:
500 ppm
Remarks:
Pb as acetate in demineralized water
Dose / conc.:
1 000 ppm
Remarks:
Pb as acetate in demineralized water
No. of animals per sex per dose:
18 male rats per dose level
Control animals:
yes
Details on study design:
Two experimental groups of animals were used. The primary experiment was conducted with Group A animals. Group B animals were treated similarly; however, additional tissues were obtained for zinc analysis.
Group A: Seventy-two male 30-day-old rats, 85-100g body-wt (Charles River CD, Sprague Dawley Crl:CD (SD) BR, Kingston, NY) were placed on Teklad AIN76A purified diet for rats and mice (Cat. No. 170481) and allowed access to tap drinking water. This diet is intended to supply 5.1551 g Ca/kg and 0.0314 Zn/kg, comparable to the recommended dietary allowance of 5.0 g Ca/kg and 0.012 g Zn/kg. Three days later, the animals were divided into four groups of 18 rats that received, respectively, 0, 200, 500, or 1000 ppm Pb as acetate in demineralized water; all groups received water containing equimolar amounts of acetate ( as sodium) to that of 1000 ppm Pb water. One-third of tha animals in each group were placed in plastic metabolism cages at weeks 4, 8, or 12 (for 4 days while continuing on treatment water). Water consumption was measured daily and urine was collected daily for each of 4 consecutive days. The animals were then killed to obtain blood and tissue specimens for metal analysis.
Broup B: A second group of twenty-five rats was given 0.200. or 500 ppm Pb in drinking water for 12 weeks and then killed to obtain blood and other tissues as described for Group A. Since there were no differences between these groups, results were combined for these two groups.

Examinations

Observations and examinations performed and frequency:
Water consumption was measured daily and urine was collected daily for each of four consecutive days. Body weights and kidney weights and kidney weights as a percentage of body weight after different periods of lead exposure. Blood and kidney lead concentrations, average daily lead excretion rates, and urinary lead clearance obtained over 12 weeks of the study at each drinking water lead concentration.
Sacrifice and pathology:
The animals were anesthetized with metofane (Pitman-Moore, Washington Crossing, NJ) by inhalation via nose cone until anesthesia was achieved and then were exsanguinated via the abdominal aorta. Approximately 5 ml heparinized blood was obtained by syringe and ionized calcium was determined either on whole blood or on the subsequently separated plasma using an automated CO2 equilibration turntable (Radiometer, Copenhagen). Samples for whole PbB analysis and hematocrit were pipetted prior to plasma separation. Plasma and tissues (kidney, liver, and brain) were frozen for metal analysis at a later date. Plasma creatinine and blood urea nitrogen were measured using a Baker Encore Centrifugal Chemistry System (Allentown, PA). One kidney was fixed in a mixture of glutaraldehyde (2.6%), paraformaldehyde (2%), and sucrose (3%) in 0.1 M phosphate buffer, pH 7.4 which was suitable for both light and electron microscopy.
Statistics:
All grouped data are expressed aas means +/- standard error of mean. For some distributions, a mean +/- two standard deveiations from the mean is also shown. Statisitcal comparisons are by analysis of variance. Duncan's test for multiple comparisons to one control, Dunnett's test for differences between lead-exposed groups, and Student's t test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Comparisons were made between lead-exposed and the respective control animals at each point.Table 1 presents body weights and kidney weights as a percentage of body weight after different periods of lead exposure. Beginning at 4 weeks, all animals on lead treatment (with the exception of 200 ppm animals at 12 weeks) had mean body weights that were significantly lower than controls at the same time points. There were no differences between absolute kidney weights between groups.; however, kidney weight as a percentage of body weight was elevated at all doses from Week 4 to the conclusion of the experiment. There appeared to be no change in hematocrit values.with these doses of lead. Blood and kidney lead concentrations, average daily lead excretion rates, and urinary lead clearance obtained over 12 weeks of the study were analyzed in two ways: (1) level of lead exposure-comparisons were made between doses at the same length of exposure (horizontal in table 2); and (2) length of exposure: comparisons were made between different lengths of exposure at each dose (vertical in table 2). At each time point, comparison between different dose levels of lead in the drinking water revealed some dose-dependent differences in blood and kidney lead concentrations and urinary lead excretion rates. There were no differences in lead clearance rates between the doses at each month's exposure period. At the 200 ppm dose, blood lead peaked at 8 weeks, while with 500 and 1000 ppm, blood lead concentration peaked at 4 weeks. At 200 ppm, kidney lead values reached a plateau at 16-20 ug/g wet wt and did not change appreciably with further exposure. At 500 ppm, kidney levels were highest at 4 weeks but did not change significantly with longer exposure. At 1000 ppm, kidney values were similar at 4 and 12 weeks. These results indicate, therefore, that lead dose is more important than duration of exposure for determining kidney lead levels. Average daily lead intake was nearly constant over time for each dose level (mean daily intake of 3.1, 7.2, or 16.2 mg/day at 200, 500, and 1000 ppm, respectively). Excretion rate, however, was time dependent, declining from 0.22% of ingested lead at 4 weeks to 0.01% at 8 weeks and 0.06% at 12 weeks (the slopes for the regression lines are significantly different at p<0.01). Excretion rate of lead was also dose dependent:absolute excretion rates in ug/day are given in table 2. Lead clearance from whole blood was calculated as the mass of pb excreted per day in ug/day divided by blood concentration in ug/ml. This gives the theoretical blood volume in milliliters cleared of lead per day (see last section of table 1). Lead clearance as determined differs from true renal clearance of lead in that measurements were done on whole blood rather than the ultrafilterable fraction of plasma lead. At each lead dose, the volume cleared becomes smaller with increasing length of exposure (in spite of a tendency for blood lead concentration to fall over time at any dose level). The clearance volumes are similar between the threee doses at each time point, approximately 20 ml/day at 4 weeks, 10 ml/day at 8 weeks, and 7-8 ml/day at 12 weeks, indicating that excretion rate is falling even more rapidly than blood lead.
At each time point in lead exposure, urinary zinc excretion was elevated in a dose-dependent pattern when compared to controls. Statistical significance was observed in each exposure group at 4 weeks, only in the two highest doses at 8 weeks, while at 12 weeks with the highest dose only. The rate of zinc excretion in control rats at the different collection periods was not significantly different by ANOVA and, therefore, an overall mean was calculated to be 9.95 +/- 0.87 ug/kg/day. At 4-week lead exposure, all rats had zinc excretion greater than the control value + 2 SD (17.35 ug/kg/day); at 8 weeks, 12 of 18 animals were outside this range, while after 12 weeks exposure, 10 of 18 fell outside this range. There was a trend toward increased calcium excretion in all exposure groups, although the increase in calcium excretion is statistically different from controls only at the highest dose. Calcium excretion rates from control animals at the different collection periods were not significantly different by ANOVA (p=0.057) and, therefore a mean value for all control animals was calculated to be 1.31 mg/kg/day. The mean calcium excretion for all control animals is 1313 ug/kg/day; the mean value + SD are shown as lines on the graph. most of the values above the latter line were obtained from animals at 4 weeks of lead exposure. Correlation between urine lead an calcium excretion is statistically significant. (r squared = 0.88).
Urinary sodium, potassium, and magnesium were also measured for all samples. in summary, sodium and potassium excretion in lead-exposed animals were not significantly different from controls at any level or length of lead exposure.
Tissue and plasma zinc concentrations in all animals exposed to lead for 12 weeks are given in Table 3. Plasma zinc tended to be lower in lead-exposed rats, although there was no statistical difference. Kidney, liver, and erythrocyte zinc were not affected by lead exposure but brain, testes, and bone zinc were significantly depressed. Pancreatic zinc was significantly elevated in 500 ppm rats. At kidney lead concentrations of 60 ug/g wet wt or above, there was a marked increase in the kidney calcium concentration. While the group means of renal calcium did not differ, some individual animals had very high values. Plasma concentrations of calcium and ionized calcium were not different at any time point between control and exposed rats nor were there differences in liver calcium concentration. There were no differences in plasma creatinine or urea nitrogen at any exposure or dose levels. Examination of kidneys by light microscopy did not reveal any nuclear inclusion bodies in proximal tubular epithelial cells at 4 weeks, but all kidneys at each dose had nuclear inclusion bodies at 8 and 12 weeks of lead exposure.

Effect levels

Dose descriptor:
LOAEL
Effect level:
ca. 200 ppm
Based on:
other: dose
Sex:
male
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: See 'Remarks':
Remarks:
Blood lead values in 200 ppm animals averaged 40 to 60Ug/dl and corresponded, according to the authors, to values observed in children with low-level or subclinical toxicity. At the two higher exposure levels, the rats had blood lead values between 100 and 150ug/dl, and thus would be comparable to those found in children and adults with severe overt toxicity.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1 Body and Kidney Weights of Rats Exposed to One of Several Doses of lead For indicated Time periods (mean +/-SE)

Exposure Weeks  Lead Dose ppm   Body wt, g   Kidney wt, g   Kidney wt, % body weight   n  
358.9 +/- 7.9  2.638 +/- 0.100  0.733 +/- 0.019 
4 200  327.9 +/- 9.9*  2.799 +/- 0.107  0.853 +/- 0.021** 
500  287.2 +/- 13.2**  2.992 +/- 0.234  1.033 +/- 0.036 ** 
1000  284.7 +/- 6.9**  2.765 +/-0.114  0.973 +/- 0.040** 
441.9 +/- 8.3  2.850 +/- 0.042  0.646 +/- 0.007 
200  389.6 +/- 6.6**  2.992 +/- 0.103  0.770 +/- 0.032** 
500  381.1 +/- 12.7**  3.459 +/- 0.295   0.902 +/- 0.050**
1000  365.1 +/- 19.7 **  3.153 +/- 0.167  0.864 +/- 0.018** 
12  492.3 +/- 24.8  2.945 +/- 0.162  0.599 +/- 0.020 
12  200  451.6 +/- 18.8  3.216 +/- 0.094  0.716 +/- 0.068** 
12  500  422.8 +/- 13.5*  3.110 +/- 0.140  0.734 +/- ).015** 
12  1000  379.3 +/- 11.48**  3.367 +/- 0.166  0.888 +/- 0.036 ** 

Table 2 Dose and Time Related Changes in Blood and Kidney Lead Concentrations, Urinary lead Excretion and Ratio of Excretion to Blood Values in Rats (mean +/- SE; n = 6 for all values)

Exposure Level   4 weeks   8 weeks   12 weeks     Statistical Comparison (weeks)
Blood Lead ug/dl - 200 ppm       p<0.05: 4 vs. 8; 8 vs 12
500 ppm         No differences
1000 ppm         No differences
Statistical Comparison   p<0.05*,**    p<0.05**   p<0.01**,***   
Kidney lead ug/g -200 ppm         No differences  
500 ppm         No differences
1000 ppm         No differences
Statistical Comparison  p<0.05*,**   p<0.05**  p<0.05**  
Urine pb - ug/day 200 ppm          <0.05:4 vs 8;  p<0.01: 4 vs 12
500 ppm          p<0.01: 4vs 8; 4 vs 12
1000 ppm          p<0.01: 4 vs 8; 4 vs 12
Statistical Comparison   p<0.05*,p<0.01**  p<0.05*, p<0.01**,***  p<0.01**,***
 Lead Clearance urine Pb/blood Pb (ug/day)/(ug/ml) - 200 ppm       p<0.01: 4 vs 8; 4 vs 12
 500 ppm        No differences
 1000 ppm        No differences
Statistical Comparison  No differences  No differences  No differences  

* 200 ppm vs 500 ppm; ** 300 ppm vs 1000 ppm; ***500 ppm vs 1000 ppm Statisitcal Comparisons by ANOVA and Duncan's Multiple range test

Table 3 Tissue Zinc Concentrations in Rats Exposed to lead for 12 weeks (Mean +/-; Number of Animals in Parentheses)

*p<0.05

p<0.01, from controls

***N.D., no observations


  Control   200 ppm   500 ppm   1000 ppm  
Plasma (ug/ml)  1.18 +/- 0.05 (16)  1.04 +/-0.04 (11)  1.04 +/-0.05 (15)  1.04 +/- 0.03 (6) 
Kidney (ug/g wet wt)  21.68 +/-n0.35 (16)  21.26 +/-0.34 (11)  21.92 +/-0.78 (15)  20.68 +/-0.69 (6) 
Liver (ug/g wet wt)  28.20 +/- 0.79 (16)  30.91 +/-1.06 (12)  31.50 +/-1.09 (14)  30.17 +/-1.30 (6) 
Brain (ug/g wet wt)  9.47 +/- 0.17 (60  9.36 +/- 0.12 (6)  8.88* 0.18 (6)  8.90* +/- 0.15 (6) 
erythrocytes (ug/g dry wt)  30.96 +/- 0.78 (10)  35.18 +/-2.68 (6)  33.38 +/-0.75 (9)  N.D.*** 
Testes (ug/g wet wt)  25.71 +/- 1.05 (10)  21.47** +/-0.21 (6)  22.34* +/-0.43 (9)  N.D. 
Pancreas (ug/g wet wt)  23.19 +/- 0.19 (9)  25.12 +/-1.53 (6)  30.21* +/-1.40 (9)  N.D. 
Bone (ug/g dry wt)  207.28 +/- 4.96 (9)  191.62* +/-4.55 (6)  181.13** +/- 3.58 (9)  N.D. 

Applicant's summary and conclusion

Conclusions:
In this experiment, toxic effects of lead observed as early as 4 weeks of exposure and at all dose levels, were decreased body weight and increased kidney weight as a percentage of body weight. Rats continuously exposed to lead via drinking water exhibit dose- and time-dependent changes in urinary excretion rates of three metals, lead, zinc, and calcium. Lead excretion decreased with continued lead exposure and the maximum lead effect on the other two metals was seen at 4 weeks of lead exposure. The relative magnitude of the hyperzincuria along with evidence of decreased tissue levels in testis, bone, and brain suggest that lead exposure can reduce body stores of zinc. Effects of the decrease in zinc on zinc-dependent function were not investigated. Hypercalciuria is most pronounced with the highest lead dose. This finding is thus additional evidence of lead-induced alterations in calcium metabolism.
Executive summary:

Influence of lead on tissue content and urinary excretion of lead, zinc, and calcium in rats was studied following various exposure periods. Weanling male rats were fed a trace mineral-sufficient diet with either 0, 200, 500, or 1000 ppm lead (as acetate) in drinking water for 4, 8, or 12 weeks. Blood lead ranged from 40 to over 100 ug/dl; kidney lead was highest at 4 weeks. Urinary lead excretion was highest at 4 weeks and declined with longer exposure. Urinary zinc excretion correlated positively with lead excretion at the lower excretion rates but plateaued at higher lead excretion rates. After 12 weeks exposure at each lead dose employed, decreased zinc concentration was observed in testis, bone, and brain. Plasma, erythrocyte, and kidney zinc were not affected, while pancreas and liver zinc were slightly elevated. Urine calcium was increased significantly only in rats exposed to 1000 ppm possibly reflecting renal cell damage as determined by elevated renal calcium levels. These results indicate that lead dose is more important than exposure period for determining kidney lead levels, while urinary lead excretion rate is both dose and time dependent. Blood lead clearance values are relatively independent of dose and fall as exposure continues. Essential trace metal balance for zinc, especially, and to a lesser extent for calcium, is affected by the dose and length of chronic lead exposure.