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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and guideline. For justification of read-across see CSR chapter 1.3.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Calcium sulfate dihydrate
- Analytical purity: 99.9%
- Lot/batch No.: Sigma Aldrich Corporation, Lot No. - 109H0166

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 weeks
- Weight at study initiation: 254.2 - 297.8 g (males) and 182.7 - 208.2 g (females)

Administration / exposure

Route of administration:
oral: gavage
Duration of treatment / exposure:
35 days for male animals and 41-45 days for female animals
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day
Basis:

No. of animals per sex per dose:
60
Control animals:
yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:
- Clinical observations performed and frequency: Clinical symptoms were observed once a day but were observed once a week in detail; a death rate was observed twice a day; and body weight was observed once a week and just before the necropsy, but in case of pregnant females, it was measured on the day 0, 7, 14, 20 of gestation period, date of delivery, and 4 days after the delivery; consumption rate of fodder was observed once a week except mating period.

Tests for sensory organ and reflex action: 5 animals were randomly selected from each test group. Both preyer reflex test and corneal reflex test were performed before necropsy and during lactation for males and females, respectively.

Behaviour test: 5 animals were randomly selected from each test group to do grip strength test in terms of behaviour test. This test was performed before necropsy and during lactation for males and females, respectively.

- Haematological and biochemical test of blood: randomly selected 5 male and female animals from each test group were fasted a day before necropsy for both tests. Animals were anesthetized using ether and cut the abdomen open to collect blood. In case of the haematological test, blood coagulation preventative chemicals for the test of blood coagulation and the calculation of blood-corpuscles were 3.2 % sodium citrate and EDTA- 2K, respectively. On the other hand, blood coagulation preventative chemical was not used for the biochemical test, but gathered blood was left itself in the room temperature then the sera were separated using a centrifuge. For haematological test, 6 following items were measured; Haematocrit, hemoglobin concentration, erythrocyte count, total and different leucocyte count, platelet count, prothrombin time, and active partial thromboplastin time. For biochemical test of blood, eleven following items were measured; sodium, potassium, glucose, total cholesterol, blood urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, aspatate aminotransferase, and total bilirubin.

Organs examined at necropsy: Organ weight: testes, epididymider (all males) liver, kidney, adrenals, thymus, spleen, brain and heart (5 male and female animals from each test group).

Fixation: 22 kinds of tissues were fixed to do histopathologic tests such as testes, epididymides, ovaries, accessory sex organs for all animals, brain (including cerebrum, cerebellum and pons), spinal cord, stomach, small and large intestines (including peyer¿s patches), liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea, lungs, uterus, urinary bladder, lymph nodes (cervical mesenteric), peripheral nerve (sciatic or tibial), and bone marrow.
Statistics:
Statistical decision tree, but in case of recovery group, either two-side Students t-test or two-side Apsin Welch t-test was used. In case of categorical data, two-sided Fishers exact test was used.

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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality: There was one death at day 8 for male, and each death on the day 7 and 14 for female in the treatment group of 300 mg/kg/day. These were occurred during the administration process, so it did not have relationship with test substance.

Body weight: In male groups, temporarily, a case of diminishment in the amount of body weight change was observed at week 2 within the
control group in which some clinical signs were observed such as damage to esophagus. Body weight loss for female animals was observed several times during lactation period in every treatment group including the control group. However, these were occurred temporarily because of the lactation.

- Clinical signs: In male control group, a case of salivation and bloodlike secretion was observed on the day 11 and 12. In the 1,000 mg/kg/day treatment group, a case of depilation, dcab and pus was observed on the left cheek between the day 25 and the closing day. However, the frequency of occurrence was low and no dose-response correlation was observed. Thus these symptoms were not influenced by test substance. In female control group, a case of genitalia blood-like secretion was observed at day 29. In the 100 mg/kg/day treatment group, each case of hypoactivity and depilation was observed on the day 8 and 9, and between day 44 and the closing day, respectively. However, these symptoms were disappeared in short, thus these did not have relationship with test substance.

Amount of fodder consumption: No crucial difference between the reatment group and the control group was observed for both male and female animals during test period. For recovery group, no significant change was observed within themselves.

Test of reflex action: Five male and female animals were randomly selected from each test group, in which no specific reaction was observed.

Grip strength test: For male animals, 6 animals were left out from the treatment group. For female animals, 5 animals were left out from the control group, and the treatment group. All things being considered, there was no dose-response correlation and was no illness at the related organs such as the cerebellum and muscle.

Organ weight: Both absolute weight of the liver and the left kidney were increased at the recovery group with administration of 1,000 mg/kg/day as compared with that of the control group within the recovery group. There was no histopathological illness at the organs, so increased organ weight did not have relationship with test substance.

Necropsy opinions: For male animals, in the control group within the recovery group, a case of left and right caput epididymis cyst was observed and the 1,000 mg/kg/day recovery group had symptom of right caput epididymis cyst. However, its frequency of occurrence was low and it was even observed at the control group within the recovery group, so it did not have relationship with test substance. For female animals, in the 300 mg/kg/day treatment group, each animal was dead on the day 7 and 14 and; each case of lung dark-red discolouration was observed, but white particle in a lobe of the lung was observed just from one of carcasses. A case of spleen white nodule was observed for an animal in the 300 mg/kg/day treatment group. There was a case of right adrenal gland white spots at the 1,000 mg/kg/day treatment group. In the control group within the recovery group, each case of right adrenal gland hemorrhagia and atrophy and liver adhesion with diaphragm was observed.

Analysis of haematological test of blood: In the 1,000 mg/kg/day male recovery group, segments were increased (p < 0.05) in contrast with that of the control group within the recovery group. Prothrombin time (PT) was decreased in the 1,000 mg/kg/day female recovery group in comparison with that of the control group within the recovery group. However, these symptoms were not observed in the definitive test
groups, so these symptoms were not influenced by test substance. In addition, level of WBC (white blood cell) was increased (p < 0.001) in the 100 mg/kg/day female treatment group in contrast with that of the control group. However, its increased value was in the normal range and no
dose-response correlation, so it did not have relationship with test substance.

- Analysis of biochemical test of blood: In the 100 mg/kg/day treatment group for male animals, BUN (Blood urea nitrogen) was decreased as compared with that of the control group. The male treatment groups with both administration of 300 mg/kg/day and the 1,000 mg/kg/day decreased in TP (Total protein), ALB (Albumin), AST (Aspatate aminotransferase), ALT (Alanine aminotransferase), BUN (Blood urea nitrogen), CREA (Creatinine), Na (Sodium), TCHO (Total cholesterol), and Cl (Chloride) as compared with those of the control group. In case of the 1,000 mg/kg/day male recovery group, the value of AST was decreased significantly in contrast with that of the control group within the recovery group.

No significant difference was found at every test item between female control and treatment group. The female recovery group with administration of 1,000 mg/kg/day decreased in AST in contrast with that of the control group within the recovery group, but TCHO and GLU (Glucose) were increased. In fact, decreased values of AST and ALT could be no toxicological effects. In addition, the changed values of AST, TCHO, and GLU in this
test were in the normal range and no histopathological opinion in terms of related organs, so these changed values were not influenced by test substance. However, decreased values of TP, ALB, BUN, and CREA were possibly influenced by excretion process or metabolism of test substance in relation to the kidney, and these symptoms were possibly recovered in 2 weeks from reversible effects.

- Histopathology: For male animals, in the control group, each case of heart focal inflammatory cell infiltration, submadibular lymph node blood absorption, liver mononuclear cell foci, and adrenal gland cortical vacuolation was observed. In the 300 mg/kg/day treatment group, two cases of pancreas vacuolation, and a case of liver mononuclear cell foci were observed. In the treatment group with administration of 1,000 mg/kg/day, there were three cases of liver mononuclear cell foci; and a case of heart focal inflammatory cell infiltration was found.

For female animals, in the control group, two cases of liver mononuclear cell foci, a case of kidney cortical scaring, and a case of pancreas vacuolation were observed. In the treatment group with administration of 100 mg/kg/day, one case of esophagus submucosal gland proliferation was observed. In the 300mg/kg/day treatment group, a case of trachea submucosal gland proliferation was observed. In the 1,000 mg/kg/day treatment group, each case of pancreas vacuolation and liver mononuclear cell foci was observed. However, these symptoms for both sexes were just subtle level and were occurred spontaneously, so there was no significant difference between the treatment group and the control group.

Effect levels

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Dose descriptor:
NOAEL
Effect level:
79 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
237 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: The LOAEL for calcium sulfate dihydrate was determined to be 300 mg/kg/day for males. The value has been corrected for calcium sulfate anhydrous

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The study was performed with calcium sulfate dihydrate and the results calculated for calcium sulfate anhydrous.
The NOAEL for calcium sulfate anhydrous was 79 mg/kg/day for males based on a significant decrease in TP (Total Protein), ALB (Albumin), BUN (Blood Urea Nitrogen), and CREA (Creatinine) at the 300 mg/kg b.w./day and 1,000 mg/kg b.w./day groups. No effects were observed in females.
Executive summary:

Calcium sulfate dihydrate was tested in a combined repeated dose/reproductive toxicity screening test according to OECD Guideline 422. The test material was administered to male and female Sprague Dawley rats by gavage at daily doses of 0, 100, 300 and 1000 mg/kg bw/day. The NOAEL for calcium sulfate anhydrous was 79 mg/kg/day for males based on a significant decrease in TP (Total Protein), ALB (Albumin), BUN (Blood Urea Nitrogen), and CREA (Creatinine) at the 300 mg/kg b.w./day and 1,000 mg/kg b.w./day groups. No effects were observed in females.