Registration Dossier

Administrative data

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study, conducted to good scientific principles

Data source

Reference
Reference Type:
publication
Title:
High Levels of Inorganic Sulfate Cause Diarrhea in Neonatal Piglets
Author:
Gomez G, Sandler RS & Seal E
Year:
1995
Bibliographic source:
J Nutr 125: 2325–2332

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Artificially reared neonatal piglets were used to study the effect of inorganic sulfate on bowel function in human infants. Two experiments were conducted to evaluate the effect of high levels of inorganic sulfate on the growth, feed intake and feces consistency of artificially reared piglets, and to determine the dose at which at least 50 % of piglets develop nonpathogenic diarrhea. The effect of sulfate level on kidney weight and concentration of inorganic sulfate in urine was also assessed. In each experiment 40 pigs with an average initial age of 5 days were individually caged and reared with an automatic feeding device. Ten pigs per dietary treatment were fed one of four diets containing the following levels of inorganic sulfate (mg/L of diet), as anhydrous sodium sulfate (USP): 0, 1200, 1600 and 2000 for Experiment 1 (18 day study), and 0, 1800, 2000, and 2200 for Experiment 2 (16-day study).
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
repeated dose toxicity: oral

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material : Anhydrous sodium sulfate

Test animals

Species:
pig
Strain:
other: cross-bred pigs
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: North Carolina State University Swine Farm
- Age at study initiation: 5 days
- Weight at study initiation: Piglets in experiment 2 had lower body weights than those in experiment 1 as they were from first parrity litters, not third.
- Housing: Piglets were kept in an Autoso, a maching containing individual cages which regulates food release.
- Diet : Prior to study initiation, the piglets were fed a diet with no additional sulfate until moved onto the experimental diets. The experimental diets were liquid. Feeding intervals in each experiment was 1.5 hours. Pigs were weighed daily and the volume of diet for each pig was adjusted according to its bodyweight.
- Water : Piglets had no access to drinking water
- Acclimation period: 4 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 °C for the first week then lowered to 27-29 °C
- Humidity (%): 55-75%
- Photoperiod (hrs dark / hrs light): Lights were on at all times

Administration / exposure

Route of administration:
oral: feed
Vehicle:
water
Remarks:
distilled
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with: A liquid diet containing per L of diet 840 ml deionized distilled water, 80 g 8/50 SPL (supplying crude protein and 50 % ether extract), 143 g nonfat dry milk, 5 mL Trace mineral premix (containing mg/L of diet: CuSO4.5H2O, 5.1; FeSO4.7H2O, 78; ZnO, 12). and 1.6 g vitamin premix.
- Storage temperature of food: Food was refrigerated in the Autosow
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
18 days in experiment 1 and 16 days in experiment 2
Frequency of treatment:
In experiment 1 the daily volume of diet was divided into 16 equal portions during the first 8 days of the trial and 13 portions for the remainder. In experiment 2 the piglets were fed 13 times per day throughout the entire trial.
Doses / concentrationsopen allclose all
Dose / conc.:
1 200 other: mg/L of diet
Remarks:
Experiment 1 (18 day study)
Dose / conc.:
1 600 other: mg/L of diet
Remarks:
Experiment 1 (18 day study)
Dose / conc.:
2 000 other: mg/L of diet
Remarks:
Experiment 1 (18 day study)
Dose / conc.:
1 800 other: mg/L of diet
Remarks:
Experiment 2 (16-day study)
Dose / conc.:
2 000 other: mg/L of diet
Remarks:
Experiment 2 (16-day study)
Dose / conc.:
2 200 other: mg/L of diet
Remarks:
Experiment 2 (16-day study)
No. of animals per sex per dose:
10 pigs in each group (40 in each experiment).
Control animals:
yes, plain diet
Details on study design:
Data were analyzed as a randomized complete block design, using individual piglets as the experimental unit, and following the general linear model procedures of SAS. Values are reported as means for each diet group with either SEM or pooled SD. Following a significant F test (P < 0.05), the Duncan's multiple range test was used to identify differences among individual groups.

Examinations

Examinations:
Feeding:
Feed scores were recorded according to the following: 1 = eating normally; 2 = off feed; 3 = not eating. Scores were recorded 3 times a day (morning, evening and night).
Feces:
Feces consistency or diarrhea scores were based on the following scale: 1 = normal, solid feces; 2 = soft, looser than normal stools; and 3 = liquid diarrheal feces. Scores were recorded 3 times a day (morning, evening and night). Every morning, feces with scores of 2 or 3 were further confirmed at the time of taking rectal swabs. Rectal swabs were assayed for hemolytic E. coli and rotavirus assays.

At the begining of experiment 1, urine samples were obtained from 5 to 6 pigs of each group using a bladder puncture technique. Urine samples were analyzed for inorganic sulfate by a turbidimetric analysis.

At the end of each experiemtn, piglets were sedated with an intramuscular injection of a mixture of 0.8 mL of Ketamine hydrochloride and killed with an intracardiac lethal dose of an euthanasia solution. In each experiment, (first, third, fifth, seventh and ninth) of pigs from the heaviest to the lightest were used to obtain kidney weights. Kidneys were removed from the abdominal cavity, connective tissue was trimmed and organs blotted on paper towels and weighed. Samples of each kidney were dessicated to determine the dry matter content.

Results and discussion

Details on results:
Food intake and weight gain: At the beginning of each trial the piglets were found to consume similar amounts of feed, of all four experimental diets throughout either of the experiments, indicating that added sulfate did not affect food intake. The calculated overall food intakes per pig were 3.76 ± 0.19 (n = 40) and 3.10 ± 0.10 (n = 40) kg of dry matter for Experiments 1 and 2 respectively. Average initial body weights of piglets at 5 days of ager were 1.94 ± 0.08 kg (n = 40) and 1.85 ± 0.07 kg (n = 40) for experiments 1 and 2 respectively. Final body weights were similar among the four experimental groups in each trial (6.47 ± 0.34 and 6.01 ± 0.41 kg n = 40 each for experiments 1 and 2 respectively). In each experiment, weight gains were similar for piglets fed the basal diet and the sulfate-supplemented diets. The overall average gains were 267 ± 18 and 278 ± 24 g, n = 40 for experiments 1 and 2 respectively.

Feces consistency: At the beginning of the experiments, between 80 and 100 % of the piglets had solid, normal stools. In experiment 1, the proportion of piglets showing liquid feces increased with the levels of sulfate but diarrhea response to the highest sulfate level varied between 40 and 80 %. In experiment 2 practically all (90-100 %) of piglets with 2000 or 2200 mg/L had liquid feces from day 2 onwards. In both experiments, rectal swabs of piglets having softer stools were negative for either of the pathogens.

Relative kidney weigh and sulfate concentration in urine: There were no differences in fresh kidney weights or in their dry matter content. Sulfate concentration in urine of piglets at the beginning of Experiment 1 at 5 days of age, was 2.4 ± 0.2 mol of inorganic sulfate per L (n = 22). By the end of the experimental periods the sulfate concentration in the urine of piglets fed the basal diet rose to 8.7 ± 1.1 (n = 10) and 8.8 ± 1.1 (n = 10) mol/L for Experiments 1 and 2 respectively. Concentration of inorganic sulfate in urine was affected by dietary levels of inorganic sulfate. The highest sulfate concentrations were found in the urine of piglets fed diets with added sulfate levels of 1600 and 1800 mg/L for Experiments 1 and 2 respectively.

Applicant's summary and conclusion

Conclusions:
The study has been considered to be relevant to calcium sulfate as the main effects are caused by the sulfate ion rather than the metal counterion. This study demonstrated that added inorganic sulfate levels as high as 2200 mg/L of diet did not affect growth of artificially reared neonatal piglets. Although 1200 mg added sulfate/L of diet had essentially no effect of feces consistency, levels > 1800 mg/L of diet resulted in persistent, non-pathogenic diarrhea in neonatal piglets.