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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
19988-08-16 to 1988-11-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reliable guideline study. Also by ATSDR (2003) expert judgement, no significant deficiencies in study design and evaluation were reported and the study is said to provide reliable, quantitative estimates of a NOAEL and LOAEL.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
, no post-treatment period observation group; no data on ophthalmological examination; no data on sensory reactivity test; several parameters in serum, urine and pathology were not stated to be analysed
GLP compliance:
yes
Remarks:
USFDA Good Laboratory Practices regulations (21 CFR 58)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): sodium selenite
- Molecular formula (if other than submission substance): Na2SeO3
- Molecular weight (if other than submission substance): 172.95
- Substance type: technical product
- Physical state: solid
- Analytical purity: ca. 98 %
- Impurities (identity and concentrations): e.g. potassium, sodium, sulfur, water, sodium selenate
- Lot/batch No.: 43489
- Stability under test conditions: because literature references indicate that sodium selenite is stable under normal laboratory conditions (NTP, 1986b), no accelerated stability studies were performed on the bulk chemical.
- Storage condition of test material: sodium selenite was stored in the dark at 4° ± 3° C; periodic reanalyses performed by the study laboratory using TLC and infrared or visible spectroscopy indicated no decomposition of the bulk chemical.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: 30 to 32 days old at receipt; 6 weeks old when the studies began
- Weight at study initiation: 140-142 g (mean, males); 122-124 g (mean, females)
- Housing: housed five per cage by sex
- Diet: NIH-07 Open Formula Diet (Zeigler Brothers, Inc., Gardners, PA) was available ad libitum
- Water: filtered, deionised water (with or with added test substance), ad libitum
- Acclimation period: 12 to 14 days quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9 °C (69° to 75° F)
- Humidity (%): 35% to 65% relative humidity
- Air changes (per hr): at least 10 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours of fluorescent light per day

IN-LIFE DATES: From: 16./18. August 1988 (first dose) To: 15-18. November 1988 (last dose and necropsy)

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Drinking water solutions were prepared by mixing sodium selenite with filtered, deionised water and stirring the mixtures for 1.5 minutes.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability studies of the drinking water solutions were performed at MRI using ion chromatography. The results indicated that aqueous solutions of 3.9 g/mL (3.9 ppm) sodium selenite were stable for 3 weeks when stored in the dark at room temperature and for 4 days when stored under animal room conditions.
- Periodic analyzes of the drinking water formulations and animal room samples by visible light (421 nm) spectroscopy showed that all dose formulations administered to rats and mice were within 10% of the theoretical concentrations.
- 14 of 15 animal room samples for rats in the sodium selenite study were within 10% of the theoretical concentrations. In each study, the same dose formulations were administered to rats and mice; therefore no animal room samples were analyzed for mice.
- Results of referee analyses performed by MRI on the drinking water solutions were within 10% of study laboratory results; discrepancies between the results of MRI and the study laboratory occurred for one sample from the sodium selenite studies. For the 32 ppm sodium selenite formulation prepared on 26 September 1988, the study laboratory determined an actual concentration of 32.3 ppm; MRI found concentrations of 38.2 and 38.9 ppm in repeated analyses. No reason for these discrepancies was discovered.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
7 days a week (continuously in drinking water)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2 ppm sodium selenite (0.9 ppm Se)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
4 ppm sodium selenite (1.8 ppm Se)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
8 ppm sodium selenite (3.7 ppm Se)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
16 ppm sodium selenite (7.3 ppm Se)
Basis:
nominal in water
Remarks:
Doses / Concentrations:
32 ppm sodium selenite (14.6 ppm Se)
Basis:
nominal in water
No. of animals per sex per dose:
Main study: groups of 10 rats per sex
Clinical pathology study: 10 male rats
Control animals:
yes
Details on study design:
- Dose selection rationale: exposure levels selected for the 13-week studies were based on increased mortality and decreased body weights and water consumption observed at higher concentrations in previous 2-week studies (EG&G Mason Research Institute, 1988a,b,c,d).
- Rationale for animal assignment (if not random): randomised
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: recorded weekly

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the study, weekly thereafter, and at necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: twice weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule: from supplemental male clinical pathology study rats on day 3, 14, 42, 70, and 90; from main-study rats at the end of the study.
- Anaesthetic used for blood collection: Yes, anesthetized with CO2.
- Animals fasted: No data.
- Blood samples were drawn from the retroorbital sinus.
- Parameters checked: haematocrit (Hct), haemoglobin (Hgb) concentration, erythrocyte (RBC) count, reticulocyte count, nucleated erythrocyte count, mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC) and leukocyte (WBC) count and differential.

CLINICAL CHEMISTRY: Yes
- Time schedule: from supplemental male clinical pathology study rats on day 3, 14, 42, 70, and 90; from main-study rats at the end of the study.
- Anaesthetic used for blood collection: Yes, anesthetized with CO2.
- Animals fasted: No data.
- Blood samples were drawn from the retroorbital sinus.
- Parameters checked: urea nitrogen, creatinine, alanine aminotransferase (ALT), alkaline phosphatase, sorbitol dehydrogenase (SDH), 5N-nucleotidase and total bile acids.

URINALYSIS: Yes
- Time schedule for collection of urine: from supplemental male study rats on days 7, 14, 42, 70, and 90; over a 16-hour period.
- Metabolism cages used for collection of urine: Yes.
- Animals fasted: No data.
- Parameters checked: alkaline phosphatase, N-acetyl-beta-D-glucosaminidase (NAG), volume, specific gravity and pH.
- Due to contamination by spilled feed, the urine samples collected on day 7 were not analyzed.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
- The median lobes of the livers of all surviving male rats in the main study were analyzed for selenium at the end of the study (LOD: 0.1728 ppm, %RSD: 1.4 to 4.0 %).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Complete necropsies and pathology were performed on all main study animals and on male rats designated for clinical pathology testing.
- Organs and tissues were examined for gross lesions and fixed in 10% neutral buffered formalin. Tissues to be examined microscopically were trimmed, embedded in paraffin, sectioned, and stained with haematoxylin and eosin.
- The following organs were weighed: brain, heart, right kidney, liver, lungs, right testis and thymus.

HISTOPATHOLOGY: Yes.
- Complete histopathologic examinations were performed on all animals in the control and highest exposure groups. Gross lesions and selected organs of rats in lower exposure groups were examined until a no-observed-effect level was determined.
- The following tissues were examined: adrenal glands, brain (three sections), clitoral glands, oesophagus, eyes (if grossly abnormal), femur and marrow, gallbladder (mice only), gross lesions and tissue masses, heart, kidneys, large intestine (cecum, colon, rectum), liver, lungs, lymph nodes (mandibular and mesenteric), mammary gland, nasal cavity and turbinates (three sections), ovaries, pancreas, parathyroid glands, pharynx (if grossly abnormal), pituitary gland, preputial glands, prostate gland, salivary gland, seminal vesicle, small intestine (duodenum, jejunum, ileum), spinal cord/sciatic nerve (if neurological signs were present), spleen, stomach (forestomach and glandular stomach), testes (with epididymis), thigh muscle, thymus, thyroid gland, trachea, urinary bladder, uterus, and vagina (females in vaginal cytology studies only).
- Gross lesions of rats in all lower exposure groups were examined.
- Organs examined in lower exposure groups included: kidneys, mandibular lymph node, and thymus of male and female rats and clitoral glands, femur and marrow, liver, mammary gland, mesenteric lymph node, pancreas, salivary gland, and uterus in female rats.
Other examinations:
Examinations on reproductive parameters were performed in addition.
Statistics:
Two approaches were employed to assess the significance of pairwise comparisons between dosed and control groups in the analysis of continuous variables:
- Organ and body weight data, which are approximately normally distributed, were analyzed with the parametric multiple comparisons procedures of Williams (1971, 1972) or Dunnett (1955).
- Clinical chemistry, haematology, spermatid, and spermatozoal data, which typically have skewed distributions, were analyzed with the nonparametric multiple comparisons methods of Shirley (1977) or Dunn (1964). Jonckheere's test (Jonckheere, 1954) was used to assess the significance of dose-response trends and to determine whether a trend-sensitive test (Williams, Shirley) was more appropriate for pairwise comparisons than a test capable of detecting departures from monotonic dose response (Dunnett, Dunn). If the P-value from Jonckheere's test was greater than or equal to 0.10, Dunn's or Dunnett's test was used rather than Shirley's or Williams' test.
- The outlier test of Dixon and Massey (1951) was employed to detect extreme values. No value selected by the outlier test was eliminated unless it was at least twice the next largest value or at most half of the next smallest value. The extreme values chosen by the statistical test were subject to approval by NTP personnel.
- In addition, values indicated by the laboratory report as being inadequate due to technical problems were eliminated from the analysis.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
majority of the changes were attributed to reduced water intake and resulting dehydration
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
majority of the changes were attributed to reduced water intake and resulting dehydration
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
only in the highest dose group
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
only in the highest dose group
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Two females exposed to 32 ppm sodium selenite died or were killed moribund before the end of the studies.
- Male and female rats in the 32 ppm groups had abnormal posture.
- Females exposed to 32 ppm were emaciated and had ruffled fur and urine stain.

BODY WEIGHT AND WEIGHT GAIN
- Final mean body weights and mean body weight gains of males and females in the 16 and 32 ppm groups were lower than those of the control.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
- Water consumption by male and female rats decreased with increasing exposure concentration.
- Water consumption by male rats in the 32 ppm group and female rats in the 16 and 32 ppm groups was notably lower than that by the controls.
- Water concentrations of 2, 4, 8, 16, and 32 ppm sodium selenite were estimated to deliver 0.08, 0.13, 0.2, 0.4, 0.8 (males), and 0.9 (females) mg Se/kg per day.

HAEMATOLOGY
- Haematology changes observed in rats were sporadic and minimal.

CLINICAL CHEMISTRY
- Increases in ALT activity occurred in male rats exposed to 32 ppm and in females exposed to 16 or 32 ppm.
- SDH activity was also elevated in clinical pathology study males at day 70.
- 5N-Nucleotidase activity and bile acid concentration were increased in clinical pathology study male rats in the 32 ppm group on days 42 and 70 and at week 13. These changes also occurred at week 13 in main study male and female rats exposed to 32 ppm.
- Alkaline phosphatase activity was increased sporadically in various groups in the clinical pathology and base studies.

URINALYSIS
- Decreases in urine volume and increases in urine specific gravity occurred in multiple exposure groups at various time points in the clinical pathology study males.

ORGAN WEIGHTS
- Differences in organ weights occurred primarily in the rats in the 32 ppm groups.
- Absolute and relative thymus weights of rats exposed to 32 ppm were significantly decreased.
- Relative right kidney weights were increased in rats in the 32 ppm groups.
- Other statistically significant differences in organ weights were considered related to decreases in body weight gain in exposed rats.

GROSS PATHOLOGY
- Treatment-related gross lesions observed at necropsy consisted of decreased thymus, seminal vesicle, and uterus sizes in male and female rats in the 32 ppm groups.

HISTOPATHOLOGY:
- Most microscopic diagnoses of atrophy or cellular depletion in the thymus, lymph nodes, bone marrow, spleen, salivary gland, pancreas, liver, mammary gland, uterus, clitoral gland, and metaphyseal plate of the femur were considered secondary to the marked decrease in body weight gain in males and the body weight loss in females exposed to 32 ppm.
- Lymphoid organ changes diagnosed as cellular depletion consisted of decreased numbers of lymphocytes in the thymic cortex and decreased size of the lymphoid follicles and periarteriolar sheaths in the lymph node and spleen.
- Cellular depletion in the bone marrow consisted of a decrease in the density of erythroid and myeloid cellular components.
- Atrophy of the bone metaphyseal plate ranged from decreased size and number of metaphyseal trabeculae to the complete absence of the trabeculae, which are normally present in growing, young adult rats.
- Atrophy in other glandular organs was based on decreased organ and cell size or absence of normal cytoplasmic granules or secretory product in ductular lumen.
- Degeneration of the renal papilla was a concentration- and treatment-related histopathologic effect that occurred in the kidneys of male and female rats. This papillary lesion varied from minimal to marked in severity. Minimal degeneration consisted of a focal area of oedema of the interstitium at the most distal tip of the renal papilla. This was characterized by a slight loss of cytoplasmic and nuclear detail of the interstitial cells, with increased eosinophilia and granularity of the cytoplasm. Epithelial cell lining of the distal portion of collecting ducts were swollen, and the cytoplasm often contained large, clear vacuolar spaces. When the degeneration involved a large area of the distal portion of the papilla, the lesion was diagnosed as mild. In rats with degeneration of moderate or marked severity, the morphologic features were more prominent and included necrosis of the interstitial cells and renal epithelium covering the distal end of the papilla. The incidence and average severity of this lesion were greater in females than in males. In male rats, papillary lesions were of minimal severity except at the highest exposure concentration (32 ppm), where the degeneration was mild to moderate. Papillary degeneration was not present in male rats in the 2 ppm group, and the single occurrence in a male in the 4 ppm group was similar in morphology and severity to the minimal focal area of papillary degeneration in the kidney of one control male. Papillary degeneration, which varied in severity from minimal to moderate, was present in most female rats exposed to 8 ppm or greater. Minimal degeneration occurred in 1 of 10 females in the 2 ppm group and 3 of 10 females in the 4 ppm group.

OTHER FINDINGS
- Livers of all base-study male rats were analyzed for selenium concentration.
- The following average selenium concentrations (per dry weight of tissue) were detected:
2 ppm group, 3.4 ± 0.2 ppm
4 ppm group, 4.1 ± 0.5 ppm
8 ppm group, 5.8 ± 0.4 ppm
16 ppm group, 7.9 ± 0.8 ppm and
32 ppm group, 12 ± 1 ppm.
- Selenium concentration in control males was less than the minimum quantifiable level.

Effect levels

Dose descriptor:
NOAEL
Remarks:
based on selenium
Effect level:
0.4 mg/kg bw/day (actual dose received)
Based on:
element
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Calculation of NOAEL for ZnSeO3:

molar weight ZnSeO3: 192.3672 g/mol

molar weight Se: 78.96 g/mol

--> 0.4 mg Se = 0.97 mg ZnSeO3 --> NOAEL (rat, oral) ZnSeO3 = 0.97 mg/kg bw/d

Applicant's summary and conclusion

Conclusions:
Based on mortality, body weight depression, decreased water consumption and renal papillary lesions, the estimated no-observed-adverse-effect level (NOAEL) in rats was 0.4 mg selenium/kg body weight per day (16 ppm sodium selenite). This corresponds to 0.97 mg/kg bw/d ZnSeO3 based on molecular weight. Sodium selenite was more toxic to rats than to mice.