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Description of key information

The submission substance consists of naturally occuring components of generally low toxicity. Evaluation of oral exposure to phosphorous compounds by various international bodies (JECFA, EFSA) yielded a maximum tolerable daily intake) of 70 mg P/kg bw/day. Ca sulfate has been evaluated (EFSA, JECFA) as food additive (no ADI allocated). 
A combined repeated dose - reproductive / developmental toxicity screening test according to OECD 422 with the related substance triple superphosphate (TSP) resulted in a NOAEL of 250 mg/kg bw/day. A siimilar study with Ca sulfate dihydrate revealed a NOAEL of 79 mg/kg bw/day.
Contributions of of the ligand humate to the overall toxicity of the submission substance are considered to be small due to the small contribution (<1% on w/w basis).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
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)
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
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
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.
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.
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
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
79 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
good

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Phosphate:

In a combined repeated dose - reproductive / developmental toxicity screening test according to OECD 422 (Dent 2002) (GLP, RL1) triple superphosphate (TSP) was administered to Sprague-Dawley rats orally (gavage). Animals were either exposed for 6 weeks (males and females of toxicity subgroup) or 2 weeks before mating, during mating and gestation until postnatal day 3 at doses of 0, 250, 750 or 1500 mg/kg bw/day (doses are calculated as TSP added to feed, natural phosphate in feed not counted). Several haematological and clinical chemistry parameters were changed at the two highest dose levels and neurobehavioural changes were observed at the highest dose. Minimal kidney and stomach effects observed at the lowest dose administered were not considered adverse or caused by a locally irritating effect, respectively. No

effects on reproductive parameters or developmental toxicity were observed other than slight reductions in pup body weights at the highest dose level. Therefore, a NOAEL of 250 mg/kg bw/day for repeated dose toxicity was derived from this study performed with TSP.

In an older, but reliable (RL2) published study (Dymsza et al., 1959) normal and high levels of phosphorous (0.43 and 1.3% P) inthe form of ortho- (dibasic potassium phosphate) or metaphosphate (Na hexamethphosphate)were fed to male rats over 150 days. Neither the types nor the levels of phosphates impaired growth conclusively as measured by weight geined and femur length. No adverse physiological effects were observed from autopsy, histological and clinical studies. There appeared to be adequate absorption and utilization of calcium, phosphorous and iron with any of the tested diets. The study is not as detailed and comprise fewer investigations compared to current guideline studies, but focuses on potential deleterious effects of phosphate, and includes histopathological examination of the target organ kidney (examination of nephrocalcinosis). Therefore it is considered reliable with respect to the evaluation of phosphates. The NOAEL from this subchronic feeding study is 1.3% P in food, which translates to (ECHA Guidance on IR and CSA, R.8) to 520 mg/kg day for male rats.

Several reviews are available which summarise the results of various older (years 1950 to 1975) feeding studies with various phosphate compounds (JECFA 1982, Weiner et al. 2001; Lang 1959). Very high levels of phosphate in feed may cause an increase of plasma phosphorus and a decrease in serum calcium. In consequence, hypocalcaemia, bone resorption and loss and calcification of soft tissues may occur, especially in the kidney. In rats nephrocalcinosis was observed at high phosphate concentrations in the diet. A LOAEL of 10000 mg P/kg diet was derived by JECFA from studies with rats, which are - according to JECFA - especially susceptible to tissue calcification. A MTDI (maximum tolerable daily intake) of 70 mg P/kg bw/day was established for the intake by humans of phosphates from food.

Calcium sulfate:

Calcium and sulfate are natural constituents of food and essential food components. It is of limited water solubility.

Calcium sulfate as an food addtive has been evaluated by the EU Commission's Scientific Committee on Food (SCF) and the FAO/WHO Joint Expert Committee on Food Additives (JECFA). These committees allocated an Acceptable Daily Intake (ADI) not specified for the respective calcium and sulfate ions, due to its non-toxicity after oral exposure. It is generally permitted in the EU and the USA as an approved food additive (EU: E516). The European Food Safety Agency EFSA (2008) evaluated the use of Calcium sulfate in food supplements as a source of calcium based on the tolerable upper intake level of 2500 mg calcium/day as established by SCF. EFSA concluded that calcium sulfate as a source of calcium in food supplements is of no safety concern. Similarly, in 2004 EFSA concluded that its use as a mineral substance in food is of no safety concern. Any contribution to the overall exposure from handling the submission substance is considered to be low compared to exposure from food.

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.

Humic acid potassium salt:

The substance was tested for subacute toxicity using the EU method B.7.Repeated Dose (28-days) Toxicity (Oral), study with Wistar rats. The test substance was administered as solution in water by stomach tube; oral application of rats was made daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 5 males and 5 females; each satellite group consisted of 5 males and 5 females.Main groups contained 3 treated groups (doses 250, 500, 1000 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (1000 mg/kg/day). The administration period lasted 28 days.  

Based on slightly reduced body weights and changes in values of haematological and biochemical parameters detected at the hightest dose level a NOAEL of 500 mg/kg bw/day was derived.

Several published studies on the repeated dose toxicity of humic acids or humate salts are available. Two subchronic oral studies did not observe advese effects up to levels of 1000 mg/L drinking water (Daniel 1991; Condie 1985). One study of low reliability reported slight effects on the thyroid gland (Seffner 1985).

Justification for classification or non-classification

The main components of the submission substance are naturally occuring substances of generally low toxicity. LOAELs from repeated dose toxicity studies are well above the classification criterion of 10 mg/kg bw/day for oral studies. No classification is required.