Lime (chemical), hydraulic

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Annex VIII, point 8.8.1 of Regulation No 1907/2006 specifies that "assessment of the toxicokinetic behaviour … to the extent that can be derived from the relevant available information" is required. For this specific information requirement, however, column 2 of Annex VIII does not provide any rules for deviating from standard testing requirements. The toxicokinetic behaviour of calcium and magnesium released from hydraulic lime is therefore assessed in this dossier based on sources gathered from the public domain where the fate of the chemical species in question was investigated or evaluated, independent of the counter-ion (which is, in the respective toxicokinetic studies, typically different from the hydroxyl ion characteristic of the substance discussed in the technical dossier). Toxicokinetic behaviour of calcium has already been assessed in the form of official evaluation documents by EU bodies (e.g. EFSA) and also by the WHO and FAO. Much of this information consists of human data. This approach therefore follows Annex XI, points 1.1.3 and 1.2, specifying general rules for adaptation of the standard testing regime. The information on toxicokinetics of calcium available from EFSA and FAO/WHO is summarised as follows: Biological function: Calcium is an essential mineral nutrient for humans, with daily requirements ranging between 400 mg for infants, up to 1200 mg for pregnant women, as assessed by the Scientific Committee on Food. Calcium serves as a structural element in bone and tooth formation, mainly as hydroxyapatite, and is furthermore involved in a broad range of physiological processes: It plays a central role in blood coagulation, is involved in cell adhesion, hormone and neurotransmitter release, muscle contraction, cellular differentiation, several intracellular signalling pathways, and many others. Absorption: From dietary sources and when ingested as a salt, calcium is absorbed in the intestine by, on average, 32 %, varying between 10 and 40 %, independent of the solubility of the salt. Calcium absorption is partly regulated (active transport), or may take place by passive diffusion over an electrochemical gradient. Absorption rates vary by age, reflecting the dietary needs of subjects in the various age classes, and are under genetic control. Distribution: More than 99 % of the calcium stores in the body are located in the bones and teeth. The soft tissues accordingly contain less than 1 % of total body calcium. In extracellular fluids calcium is tightly regulated at a concentration of approximately 2.5 mmol/L (10 mg/dL). In blood, calcium is available as free Ca2+ by approximately 45 %, the remainder being complexed to citrate, phosphate, sulphate, and carbonate (ca. 10 %). Regulation of Ca levels is effected via three hormones, parathyroid hormone, 1,25-dihydroxycholecalciferol, and calcitonin. The extracellular calcium is involved in blood coagulatoin and cell andhesion, and serves as a source for bone metabolism. Intracellular calcium is primarily bound to to membrane structures of the nucleus, mitochondria, endoplasmatic reticulum, or stored in special vesicles. The intracellular concentration of free Ca2+ is only 0.1 µmol/L, i.e. approximately 25000 times below the extracellular level. Excretion: Absorbed calcium is predominantly excreted via urine, but also via faeces and sweat. Renal calcium excretion is the result of glomerular filtration (about 8 to 10 g calcium per day in adults) and tubular reabsorption (normal more than 98 % of the filtered load), which is predominantly passive, taking place in the proximal tubules, and by 20 % active in the distal part of the convoluted tubules and connecting tubules. Active transport is under the control of parathyroid hormone, calcitonin and 1,25(OH)2D. Average 24-hour excretion of calcium amounts to 40 mg in young children, 80 mg in prepubertal children and reaches about 150-200 mg in adults, largely independent of dietary calcium intake in healthy persons. Metabolism: As a mineral, calcium is not metabolised. In conclusion, it is considered that a wealth of information on the toxicokinetics and biological function of calcium is publicly available, the most significant ones being officially approved and peer-reviewed opinions of intergovernmental bodies. Since calcium cannot be considered as a xenobiotic, but instead is an essential mineral nutrient, the existing information is considered as sufficient for assessment of its physiological behaviour and for hazard assessment of lime (chemical) hydraulic containing calcium. The conduct of further studies is therefore not considered to be required.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

distribution

excretion

other: retention

Qualifier:

no guideline followed

Principles of method if other than guideline:

The distribution, retention and excretion of different radionuclides administered in a single i.v. dose per test animal were determined.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

45Ca radioisotopes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8 months old
- Diet: For several weeks before and throughout the experiment, the rats were fed a diet containing 0.5 % Ca and 0.5 % P.
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
No further details are given.

Route of administration:

intravenous

Vehicle:

other: acetate buffer solution of pH 4

Details on exposure:

no data

Duration and frequency of treatment / exposure:

single administration

Remarks:

Doses / Concentrations:
Group I: 16 µCi of 45Ca and 5 µCi of 85Sr;
Group II: 0.8 µCi 85Sr and 1.7 µCi 133Ba;
Group III: 0.75 µCi 226Ra.

No. of animals per sex per dose / concentration:

120 albino rats were used in total.

Control animals:

not specified

Positive control reference chemical:

No positive control substance was tested.

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood, plasma and other tissues, bones
- Time and frequency of sampling: The animals were sacrificed serially in ether narcosis at 1, 2, 4, 6, 12, 18, 24, 48, 96 and 408 or 432 hours after administration of the isotopes. Blood was withdrawn at death, and the plasma was obtained by centrifugation in the presence of heparin.
From each animal at least one femur and one tibia were sampled for calcium assay and activity measurements.
Urine and faeces were collected quantitatively in metabolic cages in selected 20 hour periods from the first through the sixteenth day after injection of the radionuclides.
- Other: Plasma, bones, urine, faeces and in some cases whole carcasses were wet ashed with nitric acid and hydrogen peroxide, diluted to a constant volume, and appropriate aliquots analysed for stable calcium by the method of Bett and Fraser. The total and specific activity of 45Ca in the whole body was determined from beta-counting of the wet-ashed bones and the residual carcasses, corrected for self-absorption and radioactive decay.

METABOLITE CHARACTERISATION STUDIES
not examined

Statistics:

no data

Preliminary studies:

not performed

Details on absorption:

not examined

Details on distribution in tissues:

The calcium concentration in plasma was close to 100 µg/mL (mean value of 100.4 µg/mL for all groups). It appears that 24 hours after injection, the radioactivity per g Ca in the plasma fell off exponentially.
One day after injection, the retention of the 3 elements Ca, Ba, and Sr was almost identical; later, however, the strontium tracer disappeared from the body faster than did those of calcium and barium.
After 96 hours the highest retention in the bones was that for 133Ba with values for the other isotopes in the decreasing order 45Ca > 226Ra > 85Sr. At that time, levels in the tibiae were consistently lower than in the femora, by 10 to 35 % for all the elements studied. The relative retention for the different radionuclides in single bones is similar to that for whole-body retention.

Details on excretion:

The average urinary excretion of calcium over a 16 day period was 0.044, 0.062 and 0.058 mg/hour in group I, II and III, respectively.
The corresponding values for the total faecal calcium were 1.42, 1.02 and 1.47 mg/hour. The endogenous faecal calcium in group I was 0.05 mg/hour.

Metabolites identified:

not measured

Details on metabolites:

not examined

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

distribution

Qualifier:

no guideline followed

Principles of method if other than guideline:

Behaviour of 241Am and 45Ca in the skeleton and growth and senescence of the skeleton of female rats.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

45Ca

Species:

rat

Strain:

other: CRCD

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: rats free of disease raised by Charles River Breeding Laboratories, North Wilmington, Massachusetts
- Age at study initiation: 11 days
- Individual metabolism cages: yes
No further details are given.

Route of administration:

intramuscular

Vehicle:

other: isotonic sodium citrate

Details on exposure:

Isotopes were diluted with isotonic sodium citrate and administered to rats.

Duration and frequency of treatment / exposure:

single exposure; effect observations: 180 days after injection

Remarks:

Doses / Concentrations:
241Am: 0.1 µCi/rat;
45Ca: 10 µCi/rat

No. of animals per sex per dose / concentration:

45Ca: 8 female rats housed in individual cages
241Am: 4 groups of rats (three rats per cage)

Control animals:

not specified

Positive control reference chemical:

No positive control substance was tested.

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces and other tissues
- Time and frequency of sampling: excreta were collected from Ca-injected rats.
Soluble soft tissue ash was removed from insoluble bone ash by gentle rinsing with alkaline water. Rinse from each rat carcass was analysed as a separate sample designated "soft tissue balance".
- Other: Three radiochemical procedures were used: a) 45Ca and 241Am in ashed excreta were precipitated as oxalates according to the method of Comar. b) Bone and tissue ash were evaporated on glass plates as Ca3(PO4)2, according to the method of Barr. c) When Am levels in excreta were very low, Am was first concentrated by the oxalate method, then redissolved and coprecipitated with LaF3 as described by Scott et al.
Bones were fixed in 80% alcohol, sawed, dehydrated in alcohol and acetone, and embedded in Bioplastic. Autoradiographs were prepared by exposure to X-ray film, NTA plates and spectrographic plates.

METABOLITE CHARACTERISATION STUDIES
not examined

Statistics:

no data

Preliminary studies:

not performed

Details on absorption:

no data

Details on distribution in tissues:

45Ca kinetics: After 45Ca injection 4 general processes were identified - their rates were sufficiently different to be distinguishable:
- Component A1 (T1/2 = 2 to 4 days) was attributed to early exchange loss of diffuse label;
- Component A2 (T1/2 = 20 to 30 days) was associated with resorption during growth remodeling;
- Component A3 (t1/2 = 150 to 300 days) was related to slow growth remodeling as in rip and mandible and maintenance remodeling in long bones and vertebrae;
- Component A4 (T1/2 = 2600 days) was associated with cortical bone remodeling and structural loss in very old age.
The recirculation of 45Ca is demonstrated by continued deposition in incisors and in metaphysal trabeculae formed during the first 180 days PI. When 45Ca enters bone, it partitions between diffuse deposition in pre-existing bone and concentrated deposition in regions of new bone formations.

Details on excretion:

no data

Metabolites identified:

not measured

Details on metabolites:

not examined

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Early exchange loss of diffuse label, resorption during growth remodeling,
Redistribution trend of Ca was mainly towards new bone formation in temporary bony structures

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Well documented and scientifically acceptable. Under physiological conditions, the hydroxyl-ions released from hydraulic lime following oral adminstration are neutralised in the GI tract and are therefore not relevant for consideration of toxicokinetics. Therefore for assessment of the metabolic fate of the systemically relevant species of hydraulic lime following administration via the oral route, the calcium ion Ca2+ is the chemical species of interest. In the current study, calcium was administered in the form of calcium gluconate (plus glycine). Gluconate is an integral component of mammalian energy metabolism and therefore toxicologically not relevant. The objective of the study was the evaluation of the absorption of calcium, including its retention and fate in the human body. In view of the limited relevance of the anionic counter-ions discussed here, calcium released both from hydraulic lime and calcium gluconate can be considered as structurally equivalent (analogue), and the results of the study can be used by read-across.

Objective of study:

other: bioavailablility

Qualifier:

no guideline followed

Principles of method if other than guideline:

Determination of the bioavailability, biodistribution and toxicity of a calcium source.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

Compounds were intrinsically labeled with 45Ca (specific activity 370 GBq/g).

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 400-450 g for the male and 300-350 g for the female rats
- Fasting period before study: Before the administration of the compounds, the animals were deprived of any solid food for 10 hours. Food was not provided until 1 hour after the compounds intake.
- Housing: Animals were housed in stainless steel cages of 315 mm by 445 mm by 240 mm high with grated floor and a collection tray of the same material.
- Diet: ad libitum; normalised diet (Nutrimentos Diet No.3)
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 hours dark/light cycle
No further details are given.

Route of administration:

oral: gavage

Vehicle:

not specified

Remarks:

The brief description of the procedure for preparation and adminstration of the test substance suggests that the vehicle may have been water. However, this is in fact not further specified in the publication.

Details on exposure:

Animals were administered by means of a syringe coupled to a gastric catheter, which allowed the intake volume to be standardised to one millilitre.

Duration and frequency of treatment / exposure:

12 days

Remarks:

Doses / Concentrations:
30 mg Ca per kg body weight

No. of animals per sex per dose / concentration:

74 male and 60 female rats

Control animals:

not specified

Positive control reference chemical:

No positive control substance was tested.

Details on study design:

- Bioavailability studies: Carried out with two groups of 7 male rats each. One of them received calcium gluconate as reference standard and the other one calcium gluconate stabilised with glycine.
- Biodistribution studies: Carried out with six groups of 10 rats/sex.
- Toxicity study: In addtion, toxicity of Biocal was investigated using six groups of ten rats/sex, receiving doses of 10, 11, 12, 13, 14 and 15 g Biocal/kg bw.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
Bioavailability study
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: Each rat was placed in a stainless steel metabolic cage, allowing the separation of urine from faeces. The urine was collected for a period of 12 days and its volume was measured daily. Samples of 1 mL were taken in triplicate and were kept for subsequent radioactivity determination.
- Other: Bioavaiability was determined by measurement of 45Ca urine excretion as a function of time and given as the percentage of the total amount of 45Ca activity in urine.

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
Biodistribution study
- Tissues and body fluids sampled: blood, other tissues
- Time and frequency of sampling: Biological distribution was carried out 12 days after administration of the compounds, in order to determine the calcium biodistribution. The rats received i.v. 1500 I.U. of heparin per kg body weight, then they were anaesthetised with diethyl ether and finally bled by sinus puncture, collecting about 15 mL blood from each rat. Afterwards, liver, spleen, bone, the gut with its content, muscle, lungs, heart, brain and kidneys were removed, washed with isotonic saline solution and weighed.
- Other: The organs were kept for subsequent radioactivity determination. The results were given as percentage of radioactivity concentration, C %.

METABOLITE CHARACTERISATION STUDIES
not examined

Statistics:

The data are presented as mean ± SD. The results were evaluated by one-way analysis of variance (ANOVA). Differences among means were tested using the Student-Newman-Keuls test. Probability levels < 0.05 were considered to be statistically significant.
Toxicity study: LD50 including confidence limits, according to the method by Litchfield and Wilcoxon.

Preliminary studies:

not performed

Details on absorption:

not examined

Details on distribution in tissues:

The highest values of radioactivity concentration were found in bone, giving a value of 97.1 ± 1.3 % for Biocal and 98.7 % for calcium gluconate.

Observation:

not determined

Details on excretion:

Bioavailability was expressed as the accumulated percentage of 45Ca excreted in urine as a function of time, giving a value of 2.436 ± 1.337 % for Biocal and 1.241 ± 0.473 % for calcium gluconate. This difference was statistically significant (p<0.05).
Other excretion pathways (e.g. via faeces) were not investigated in this study.

Metabolites identified:

not measured

Details on metabolites:

Not examined.

Toxicity study with 60 female and 60 male rats:

- "Biocal": LD50 = 13.5 g/kg (12.8 -14.3 g/kg)

- Calcium gluconate: LD50 = 10 g/kg

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results Estimation of bioaccumulation was not an objective of this study. However, any bioaccumulation potential of calcium can be ruled out beforehand, due to its physiological role as an essential mineral nutrient. Calcium is regulated homoestatically.
The study authors concluded that "calcium from 'Biocal' has a higher bioavailability with the same metabolic behaviour than calcium from calcium gluconate with lower toxicity". However, the assessment of bioavailability only by determination of urinary excrection is not meaningful. Nevertheless, considering the retention of Ca from both sources in bone being 97.1 % (Biocal) and 98.7 % (calcium gluconate) bioavailability values of approximately 99.54 and 99.94 %, respectively, may be derived from this study. It may therefore be concluded that calcium, when available in water-soluble form (the test materials are well soluble in water), is absorbable in the intestine by almost 100 %.

Reference 5

Endpoint:

dermal absorption

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In the absence of measured data on dermal absorption of calcium from hydraulic lime, current guidance suggests the assignment of either 10 % or 100 % default dermal absorption rates. In contrast, the currently available scientific evidence on dermal absorption of metals (predominantly based on the experience from previous EU risk assessments) yields substantially lower figures, which can be summarised briefly as follows:   Measured dermal absorption values for metals or metal compounds in studies corresponding to the most recent OECD test guidelines are typically 1 % or even less. Therefore, the use of a 10 % default absorption factor is scientifically not supported for metal salts. This is corroborated by conclusions from previous EU risk assessments (Ni, Cd, Zn), which have derived dermal absorption rates of 2 % or far less from liquid media (but with considerable methodical deviations from existing OECD methods).   However, considering that under industrial circumstances many applications involve handling of dry powders, substances and materials, and since dissolution is a key prerequisite for any percutaneous absorption, a factor 10 lower default absorption factor may be assigned to such “dry” scenarios where handling of the product does not entail use of aqueous or other liquid media. This approach was adopted in the EU RA on zinc. A reasoning for this is described in detail elsewhere (Cherrie and Robertson, 1995), based on the argument that dermal uptake is dependent on the concentration of the material on the skin surface rather than its mass.   The following default dermal absorption factors for metal cations are therefore proposed (reflective of full-shift exposure, i.e. 8 hours): From exposure to liquid/wet media: 1.0 % From dry (dust) exposure: 0.1 % This approach is consistent with the methodology proposed in the HERAG guidance for metals (HERAG fact sheet - assessment of occupational dermal exposure and dermal absorption for metals and inorganic metal compounds; EBRC Consulting GmbH, Hannover, Germany; August 2007). This approach shall be applicable to calcium contained in lime (chemical) hydraulic.

Reference 6

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Estimation whether magnesium ions (Mg2+) and calcium ions (Ca2+) are able to pass through isolated patches of human skin and investigation of the dynamics of ion diffusion.

GLP compliance:

no

Radiolabelling:

no

Species:

human

Strain:

other: not applicable, human tissue

Sex:

not specified

Details on test animals or test system and environmental conditions:

not applicable, since no test animals were used

Type of coverage:

other: in solution

Vehicle:

physiological saline

Duration of exposure:

20 hours at 37 °C for each experiment.

Doses:

Concentrations of 2.0 and 7.6 mg Ca2+/L or 0.3 and 2.1 mg Mg2+/L were tested

No. of animals per group:

not applicable

Control animals:

no

Remarks:

not applicable

Details on study design:

DOSE PREPARATION
no data

APPLICATION OF DOSE:
in solution

VEHICLE
no further details as described in the field "Vehicle"

TEST SITE
not applicable

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: no (not applicable)

REMOVAL OF TEST SUBSTANCE
not applicable

SAMPLE COLLECTION
not applicable

SAMPLE PREPARATION
- Storage procedure: Samples were frozen in -20°C and stored for discriminations.
- Preparation details: no data

ANALYSIS
Ion chromatography method was used to determine cation and anion levels. Ion chromatograph Metrohm operated with Metrodata 714 IC version 2.06 software equipped with Super SEP anionic and cationic columns was applied.

OTHER:
no further details

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Human skin samples from mammectomised breasts of 27 breast cancer patients, aged 34 to 55 years.
- Ethical approval if human skin: The study was approved by University Ethic Committee.
- Preparative technique: A dermatome was used to sample the skin in all cases.
- Thickness of skin (in mm): 0.5
- Storage conditions: Skin samples were transported in ice-cold saline and frozen (-20 °C) until used in the experiments. Storage did not exceed 3 weeks.

PRINCIPLES OF ASSAY
- Diffusion cell: Each of 3 systems consisted of dual (transmission and reception chamber of 20 mL volume, respectively) diffusion chambers. A fraction collector and a peristaltic pump were attached to each system.
- Receptor fluid: Isotonic saline solution.
- Test temperature: 37 °C
- Other: For each of the ions, 5 to 8 experiments were performed resulting in a total of 27 experiments; samples for analysis were taken at 60-minute intervals.

Signs and symptoms of toxicity:

not specified

Dermal irritation:

not specified

Absorption in different matrices:

not applicable

Total recovery:

no data

Remarks on result:

other: not determined

Conversion factor human vs. animal skin:

not examined

The aim of the study was to exemplify the changes in ion concentrations in the first 20 hours of perfusion.
In this study it was shown that human skin, under in vitro conditions, was permeable to calcium ions.

Continuous sampling of solution performed in 1-hour intervals and consecutive refilling the chamber with equal volume of perfusion solution would lead to gradual steady decrease in concentration in the reception chamber.

Conclusions:

Human skin is permeable to magnesium and calcium ions at concentrations comparable to those found in rainwater from Polish regions with different levels of air pollution. However, specific dermal dermal absorption rates cannot be derived from the parameters reported.

Reference 7

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

distribution

Qualifier:

no guideline followed

Principles of method if other than guideline:

In this study the C14 tracer isotope has been administered by implantation of a pellet of CaC14O3 in the peritoneal cavity and by application of powdered CaC14O3 over the peritoneal viscera of the rat.

GLP compliance:

no

Radiolabelling:

yes

Species:

rat

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 473 g (expt 1) and 422 g (expt 2)

Route of administration:

intraperitoneal

Vehicle:

other: Pellets of CaCO3 were prepared using gelatin (expt 1)

Details on exposure:

Experiment 1: The rat was given about 0.40 millicuries of C14 by implanting, through an abdominal incision, a pellet of the test material in each of the lower quadrants of the peritoneal cavity.

Experiment 2: About 145 mg of powdered CaC14O3 (0.065 millicurie or 1.3x10^6 counts per minute) was distributed over the peritoneal viscera of the rat.

Duration and frequency of treatment / exposure:

Experiment 1: 6 days

Experiment 2: 40 days

Remarks:

Doses / Concentrations:
Experiment 1: The rat was given about 0.40 millicuries of C14 (8.3 x 10^6 counts per minute).

Experiment 2: The rat was given about 0.065 millicuries of C14 (1.3 x 10^6 counts per minute).

No. of animals per sex per dose / concentration:

One animal was used

Control animals:

not specified

Details on dosing and sampling:

Experiment 1: Collections of excreta and expired air were made, beginning 2 hours after the operation, over the time intervals indicated in Table 1. Six days after the implantation of the pellets the animal was sacrificed by slitting its throat.

The tissues and body fluids sampled were blood, liver (for total C14 determination), muscle glycogen, body and mesenteric fat and proteins from visceral organs, the testes and the central nervous system. After skinning, the carcass was boiled in slightly alkaline water, permitting the bones and teeth and skeletal muscle to be isolated. The pelt was also analysed.

Experiment 2: Collections of expired air were made at intervals over a period of 40 days following the implantation of the CaC14O3. After sacrifice the carcass and pelt were analysed.

Details on absorption:

While dissecting the rat several small particles which apparently consisted of unabsorbed CaC14O3 were found. Their C14 content, present as inorganic carbon was found to be 12.9% of the total administered dose. The fat-free residue from the mesenteric fat depot contained an amount of C14, as inorganic carbon, which accounted for about 20% of the total dose of C14. Therefore, approximately 30% of the administered CaC14O3 remained unabsorbed.

Details on distribution in tissues:

See Table 2

Experiment 1: About 0.75% of the administered dose or about 1.0% of the absorbed dose was retained in the tissues. The C14 recovered in the tissues, excreta and expired air was 108% of the administered amount.

It is apparent that the greatest incorporation of C14 in bone and teeth took place in the inorganic carbonate fraction.
The radioautograph of the kidney shows that the C14 was concentrated in the cortical region.
The greater specific activity of C14 in the enamel of the incisor teeth than in the dentin is probably due to the presence in enamel of a greater amount of inorganic carbon, which has a high uptake of C14, than is present in dentin. No results as to the C14 content of the enamel of molar teeth were obtained because of the poor yields of enamel from these teeth.
In the fat the greater part of the activity resides in the glycerol fraction but a definite incorporation of C14 in the fatty acids took place.

Details on excretion:

See Table 1

Experiment 1: The administration of C14 as pellets of CaC14O3 prolonged the period of elimination of the labelled carbon and resulted in the body fluids having a high and constant C14 specific activity for the 48 hour period preceding the sacrifice.

Experiment 2: The largest amount of the administered C14 was present in the expired air of the rat on the 7th-8th day and no detectable C14 was present in the expired air on the 22nd day following implantation. The C14 retained 40 days after administration was 0.38 ± 0.05% of the total administered dos, 0.21 ± 0.03% being present in the pelt. These results indicate that a large fraction of C14 is excreted even when the isotope is present in the body as an insoluble inorganic compound.

Metabolites identified:

no

The weight of the animal just before being sacrificed was 474 g.

Table 1: Excretion of C14 by mature rat after implantation of CaC¹⁴O₃in the peritoneal cavity

Time elapsed after administration	% injected dose excreted			Specific activity* x 10^6		Relative specific activity (long bones = 100)
	Expired air	Urine	Faeces	Expired air	Urine	Expired air	Urine
2-21 h	1.09	0.0052	0.00	255	39.0	480	73
21-45 h	3.77	0.0156	0.0047	580	88.5	1100	170
45-69 h	9.42	0.0414	0.0052	1380	212	2600	400
69-93 h	15.4	0.0595	0.0150	2190	283	4100	530
93-117 h	20.8	0.0802	0.0253	2830	374	5300	700
117-141 h	20.9	0.0666	0.0165	2820	388	5300	730
141-142 h	0.80	-	-	2650	-	5000	-

* % of injected dose per mg carbon

 

 

Table 2: Distribution of C14 in tissues and their components after implantation of CaC¹⁴O₃in the peritoneal cavity

Sample description	% of total administered dose x 10^3	Specific activity* (SA), x 10^6	Relative (SA) (long bones = 100)
Long bones (less marrow)	22.0	53.1 ± 0.72	100
Short bones	68.0	49.9 ± 0.54	94
Inorganic CO2(long bones)	-	428 ± 2.5	810
Inorganic CO2(short bones)	-	408 ± 2.4	770
Long bone protein	-	10.6 ± 0.64	20
Short bone protein	-	12.1 ± 0.21	23
Long bone marrow	-	52.0 ± 1.3	98
Molar teeth	0.37 ± 0.10	-	-
Incisor teeth	1.30 ± 0.032	-	-
Molar dentine	-	28.7 ± 0.81	54
Incisor dentine	-	28.1 ± 0.53	53
Inorganic CO2of incisor dentine	-	474 ± 12	890
Incisor enamel	-	54.2 ± 4.0	102
Whole blood**	11.2	16.8 ± 1.1	32
Haemin	-	3.1 ± 0.15	5.8
Red blood cell protein	-	4.06 ± 0.27	7.6
Plasma albumin	-	74.1 ± 0.48	140
Plasma globulin	-	60.6 ± 0.65	110
Liver (intact)	365	48.7 ± 0.54	92
Liver glycogen	-	24.9 ± 0.28	47
Liver fatty acids	-	9.54 ± 0.15	18
Muscle protein	62.9	6.93 ± 0.16	13
Muscle glycogen	-	6.8 ± 0.9	13
Myosin from muscle	-	5.47 ± 0.17	10
Body fat	30.5	1.73 ± 0.11	3.3
Fatty acids from body fat	-	1.27 ± 0.09	2.4
Glycerol from body fat	-	10.1 ± 0.2	19
Fat from viscera	-	2.13 ± 0.068	4.0
Fat from bones & teeth	-	1.70 ± 0.055	3.2
Protein – GI tract	25.3	37.1 ± 0.41	70
Protein – spleen	2.64	34.1 ± 0.34	64
Protein – testicles	3.97	27.5 ± 0.26	52
Protein - kidney	4.79	30.8 ±0.33	58
Protein – lungs	3.28	21.7 ± 0.17	41
Protein – heart	1.72	12.1 ± 0.39	23
Protein – brain & spinal cord	-	6.39 ± 0.17	12
Brain & spinal cord (intact)	1.50	5.24 ± 0.15	9.8
Pelt	130	2.20 ± 0.22	4.1
Debris & pelt remains	34600	-	-

* % of injected dose per mg carbon

** the haemoglobin concentration was 14.9%

Conclusions:

A significant incorporation of C14 was found in the inorganic carbonate fraction of bone and in bone protein, the dentin and enamel of teeth, fatty acids, glycerol, haemin, red cell protein, plasma proteins, central nervous system protein, liver and muscle glycogen, muscle protein and in the proteins of the testes and of the thoracic and abdominal viscera.

Reference 8

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

other: Bioavailability

Principles of method if other than guideline:

The study examined whether the bioavailability of calcium carbonate and calcium citrate could be improved by reducing the particle size. The study characterised the size distribution and morphology of nano calcium carbonate and nano calcium citrate. Because nanoscale supplements are novel formulas in health foods, the acute toxicity (see separate IUCLID entry), sub-chronic toxicity (see separate IUCLID entry) and bioavailability needs to be determined in both sexes of mice in advance. The anti-osteoporosis activity was demonstrated by an ovariectomised (OVX) mice model. A bilateral OVX ICR mouse model was utilised to mimic the condition in postmenopausal women. Bone mineral density (BMD) was examined after administering nano calcium carbonate and nano calcium citrate, respectively.

GLP compliance:

no

Radiolabelling:

no

Species:

mouse

Strain:

ICR

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Taiwan University Hospital, Taipei, Taiwan
- Age at study initiation: 8-10 weeks
- Diet: Pelleted mouse feed available ad libitum
- Water: Reverse osmosis water available ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 h/12 h day/night cycle


Sham surgery (n = 6, SHAM) or bilateral ovariectomy (n = 30, OVX) was performed from a dorsal approach at 6 to 8 week old mice. Surgical removal of the ovaries is a well-represented approach to mimic the postmenopausal condition in mice. In the sham operation, ovaries were exteriorised and then replaced.

Route of administration:

oral: gavage

Duration and frequency of treatment / exposure:

Daily for 28 days

Remarks:

Doses / Concentrations:
Micro calcium carbonate: 1.3 g/kg bw
Nano calcium carbonate: 1.3 g/kg bw

Vitamin D3 was also administered by gavage along with the test materials at a concentration of 261 U/kg bw.

No. of animals per sex per dose / concentration:

At 2 months post ovariectomy, the OVX group of mice were randomly divided into groups of 6 animals.

Control animals:

yes, sham-exposed

Details on study design:

Biofunctionality of nano calcium carbonate on OVX ICR mice:
The test materials plus vitamin D3 were administered every day by gavage to the groups of mice for 28 days.

In vivo evaluation of serum calcium:
Female mice were randomly divided into groups of 24 animals (n = 6 in each group). Vitamin D3 (261 U/kg bw) plus micro calcium carbonate or nano calcium carbonate was administered by gavage to groups of mice at a dose of 1.3 g/kg bw.

Details on dosing and sampling:

Biofunctionality of nano calcium carbonate on OVX ICR mice:
The BMD of the whole body was analysed by dual-energy x-ray absorptiometry (pDEXA).

In vivo evaluation of serum calcium:
After 2, 6, 12 and 24 hours, blood sample were collected from the inferior vena cava of each mouse before autopsy. Serum calcium ion concentration was estimated using analysis kits.

Statistics:

Data were analysed statistically using ANOVA followed by the least-significant difference multiple comparison test to establish the significance of any differences. The level of statistical significance was set at p<0.05.

Serum calcium concentration:

Serum calcium concentrations were significantly higher in the nano calcium carbonate group than in the micro calcium carbonate group at 6 h post-administration and tended to be higher at 2 h post-administration. The nano calcium carbonate thus exhibited a higher efficacy than the microsized equivalent form.

The primary site of calcium absorption is the small intestine, where some 90% of calcium is absorbed. Serum calcium concentration is up-regulated following administration of nano calcium carbonate, especially at 6 and 12 h post-administration.

BMD of the whole body:

The BMD of the whole body in OVX mice was significantly lower than in SHAM mice.

The BMD value of the whole body in the micro and nano calcium carbonate treated OVX mice was greater than in the placebo treated OVX mice.

The BMD of the nano calcium carbonate treated OVX mice was significantly higher than in the micro calcium carbonate treated OVX mice.

Oestrogen enhances active calcium absorption; therefore, the BMD of OVX mice was significantly attenuated compared to the SHAM mice.

Conclusions:

Administration of nano calcium carbonate to OVX mice was more effective at inducing calcium uptake (as shown by increased serum calcium concentrations) and maintained their BMD. These data suggest that nano calcium carbonate is more bioavailable than the micro form.
Thus, nano calcium carbonate is a potential and convenient calcium supplement.

Reference 9

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was conducted to evaluate the ability of the large intestine to absorb calcium and magnesium from their sparingly water-soluble salts and also to determine whether fructooligosaccharides (FOS) stimulate the absorption of these minerals in rat large intestine in vivo. Rats were fed Ca- and Mg- free diets with and without 5% FOS. An aqueous suspension of CaCO3 and MgO was infused into the stomach via a gastric tube or into the caecum via an implanted catheter.

GLP compliance:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Clea Japan, Tokyo, Japan
- Age at study initiation: 4 weeks
- Mean weight at study initiation: 221 - 227 g
- Housing: Individually housed in stainless steel wire mesh cages
- Diet: Stock diet (MF, Oriental Yeast, Tokyo, Japan) available ad libitum
- Water: Deionised water available ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 °C
- Humidity (%): 55 %

Route of administration:

other: Direct implantation into the caecum and the stomach

Details on exposure:

A polyethylene tube was implanted directly into the caecum of each rat.
All rats were fed the Ca and Mg free diet. Two groups were subsequently fed a fructooligosaccharide (FOS) free diet (control diet) and the other two groups were fed a FOS-containing diet (50 g/kg diet). In half of the rats from each diet group, 0.3 mL of the suspension of CaCO3 and MgO, which contained 930 µmol of Ca and 136 µmol of Mg, was infused into the caecum via the implanted tube. In the remaining rats from each group, the same suspension was infused into the stomach by intubation. The suspension was infused twice per day (9:00 and 18:00) for 10 days. For the last 7 days, the rats were subjected to a Ca and Mg balance study and all faeces and urine were collected.

No. of animals per sex per dose / concentration:

7 animals/group

Control animals:

yes

Details on dosing and sampling:

The amounts of Ca, Mg and P in the diet, infusion, faeces, urine and femur of each rat were determined.

Details on absorption:

Ca BALANCE:
The apparent absorption of Ca infused into the stomach was higher than that absorbed into the caecum. FOS feeding increased the apparent absorption and retention of Ca irrespective of the infusion route. The apparent absorption and retention of Ca increased in the case of infusion into the stomach in the FOS fed groups.

Mg BALANCE:
The apparent absorption of Mg into the caecum in rats was equivalent to the Ca and Mg infusion into the stomach for both the control and FOS-feeding groups. FOS feeding increased the apparent absorption of Mg irrespective of the infusion route. However, the retention of Mg did not significantly differ among all of the groups.

P BALANCE:
The apparent absorption of P was higher in rats with Ca and Mg infusion into the caecum than in rats with Ca and Mg infusion into the stomach. On the other hand, the retention of P was lower in rats with Ca and Mg infusion into the caecum than in rats with Ca and Mg infusion into the stomach. The apparent absorption of P was decreased by FOS feeding.

APPARENT ABSORPTION EFFICIENCY OF Ca, Mg AND P:
The apparent absorption ratio of Ca in FOS fed rats with Ca and Mg infusion into the stomach was higher than in the other 3 groups. The apparent absorption ratio of Ca did not differ among these 3 groups. Regardless of FOS feeding, the apparent absorption efficiency of Mg infused into the caecum was similar to that of Mg infused into the stomach. The apparent absorption efficiency of P was higher in the case of infusion in the caecum than in infusion in the stomach.

RETENTION EFFICIENCY OF Ca, Mg AND P:
The retention ratio of Ca in FOS fed rats with Ca and Mg infusion into the stomach was higher than in the other 3 groups. The retention efficiency of Ca did not differ among these 3 groups. The retention efficiency of Mg was similar among all of the 3 groups. The retention efficiency of P was decreased by FOS feeding, being lower in rats with Ca and Mg infusion into the caecum than in rats with Ca and Mg infusion into the stomach.

Ca, Mg and P CONTENTS IN SERUM AND FEMUR:
The serum concentration of Ca in the FOS fed groups was higher when Ca was infused into the stomach than when Ca was infused into the caecum. The dry and ash weights of femur samples and the serum concentrations of Mg and P did not differ among the groups. The P content of the femur samples in the FOS fed groups with Ca and Mg infusion into the caecum was lower than that in the other 3 groups.

pH AND WEIGHT OF CAECAL CONTENTS:
The pH of the caecal contents was decreased by FOS feeding while the weight of the caecal contents was increased by FOS feeding.

The initial and final body weights did not significantly differ between the groups. FOS feeding decreased food intake.

Conclusions:

The large intestine has the ability to absorb large amounts of Ca and Mg from CaCO3 and MgO, which are respectively very sparingly soluble in water and suggest that the stimulatory effect of FOS on the absorption of Mg takes place mainly in the large intestine.

Description of key information

Short description of key information on bioaccumulation potential result:

Calcium is an essential mineral nutrient. In the human body it serves as a structural element in bone (99 % of the body's Ca stores are located in bone and teeth). It is the fifth most abundant element by mass (1.5 %). Calcium is a common cellular ionic messenger with many functions. Further, calcium plays an  important role in neurotransmitter release, muscle contraction, and many other physiological processes. Intestinal absorption and excretion (mostly via urine), intra- and extracellular levels of dissolved Ca2+ are tightly regulated, constituting homoeostasis, depending on the body's needs and dietary supply.

Short description of key information on absorption rate:

Following the HERAG guidance for metals and inorganic metal compounds, the following conservative default values for dermal absorption are proposed:

0.1 % for dry (dust) exposure

1.0 % for exposure to liquid/wet media

These absorption rates are applicable to calcium present in lime (chemical) hydraulic. However, since lime (chemical) hydraulic is irritating to skin, dermal exposure has to be minimised as far as technically feasible. Thus, an absorption rate for dermal exposure is not considered for the chemical safety assessment.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Due to its ubiquitous occurrence in the environment and its function as an essential mineral for human nutrition (see also technical dossier section 7.10), calcium is among the most extensively investigated elements with respect to physiological behaviour.

In the human body calcium serves as a structural element in bone. It is the fifth most abundant element by mass in the human body (1.5 %). Calcium is a common cellular ionic messenger with a broad range of functions. Further functions of calcium include, e.g., involvement in neurotransmitter release, and in muscle contraction.

The focus of toxicokinetics, metabolism and distribution for hydraulic lime is on calcium sincein aqueous media hydraulic limedissociates, resulting in the formation of calcium cations and hydroxyl anions. Dissociation in water is accompanied by generation of heat.

Neither the alkaline reaction nor the generation of heat is of concern regarding systemic effects. The other main constituents dicalcium-silicate and limestone (calcium carbonate) as well as the impurities tricalcium-silicate and "calcined clay and/or silica" are not classified according to Directive67/548/EEC; they are therefore regarded to be of no toxicological significance.Thus, only calcium (Ca²⁺) is considered in the current section. The key conclusion for this section is that calcium, as an essential mineral nutrient, underlies homoeostatic regulation and therefore cannot be considered as a xenobiotic.

Absorption

Oral

From dietary supplements such as calcium carbonate, calcium acetate, calcium lactate, calcium citrate, or calcium gluconate, net absorption amounts to 30 %. The oral absorption rate is independent of the solubility of the calcium salt and is therefore applicable to calcium contained in hydraulic lime.

Dermal

Following an approach consistent with the methodology proposed in the HERAG guidance for metals(Anonymous, 2007: HERAG fact sheet - assessment of occupational dermal exposure and dermal absorption for metals and inorganic metal compounds; EBRC Consulting GmbH, Hannover, Germany; August 2007), the following default dermal absorption factors for metal cations are proposed (reflective of full-shift exposure, i.e. 8 hours) for calcium present in lime (chemical) hydraulic:

For exposure to liquid/wet media: 1.0 %

For dry (dust) exposure: 0.1 %

In view of (1) the physiological role of calcium as an essential mineral and (2) the fact that any effects of lime (chemical) hydraulic upon dermal exposure are characterised as local irritation (pH effect), dermal absorption of calcium from lime (chemical) hydraulic is proposed to be insignificant.

Dermal absorption of calcium from lime (chemical) hydraulic is therefore considered to be negligible compared to its physiological importance.

Inhalation

Lime (chemical) hydraulic technical material is supplied as a powder, from which airborne particles may be generated during handling.

The estimates for inhalation absorption are composed of 100 % absorption for material deposited in the pulmonary region, plus material deposited in the tracheobronchial and the head region (transported to the pharynx and swallowed), corrected for intestinal absorption (30 %). As a result, total inhalation absorption of calcium is 21.74 %.

Excretion

Absorbed calcium is predominantly excreted via urine, and to a minor degree via faeces and sweat. Renal calcium excretion is the result of glomerular filtration (about 8 to 10 g calcium per day in adults) and tubular re-absorption (passive and active). Average 24 -hour excretion of calcium amounts to 40 mg in young children, 80 mg in prepubertal children and reaches about 150-200 mg in adults, largely independent of dietary calcium intake in healthy persons.

Distribution

The physiological importance of calcium has been extensively evaluated by the Scientific Committee on Food (SCF) as follows:

Over 99 % of the total calcium of the body is located in the bones, where it accounts for 39 % of the total body bone mineral content, and in the teeth, mostly as hydroxyapatite. Bone mineral provides structure and strength to the body and, very important, a reservoir of calcium that helps to maintain a constant concentration of blood calcium. Less than 1 % of total body calcium is found in soft tissues (~7 g) and body fluids (~1 g). Calcium in the extracellular fluid and the blood are kept constant at 2.5 mmol/L (10 mg/dL) (between 2.25 and 2.75 mmol/L) via cell surface calcium-sensing receptors in parathyroid, kidney, intestine, lung, brain, skin, bone marrow, osteoblasts and other organs. Calcium is present in blood in three different forms: as free Ca2+ ions, bound to protein (about 45 %), and complexed to citrate, phosphate, sulphate and carbonate (about 10 %). Ionised calcium is kept within narrow limits by the action of three hormones, parathyroid hormone, 1,25-dihydroxycholecalciferol, and calcitonin. Extracellular calcium serves as a source for the skeleton and participates in blood clotting and intercellular adhesion. Intracellular calcium varies widely between tissues and is predominantly bound to intracellular membrane structures of the nucleus, mitochondria, endoplasmatic reticulum or contained in special storage vesicles. Free Ca2+is only 0.1μmol/L in the cytosol, which is 25,000 times lower than in the extracellular fluid (2.5 mmol/L). Intracellular calcium rises in response to stimuli interacting with the cell surface receptor. The increase of intracellular calcium comes from influx of extracellular calcium or from release of intracellular calcium stores. This activates specific responses like hormone or neurotransmitter release, muscle contraction, cellular differentiation and many others.

Therefore, due to its function as an essential element, distribution of calcium is actively regulated according to the body's requirements. Calcium levels in the body are subject to homoeostasis.

Discussion on bioaccumulation potential result:

Toxicokinetic behaviour of calcium is assessed utilising mostly publications focusing on the nutritional and physiological role of calcium. First and foremost, however, secondary literature in the form of official evaluation documents by EU bodies (e.g. EFSA) is extensively referred to. Much of this information consists of human data. This approach therefore follows the provisions of Annex XI, points 1.1.3 and 1.2 of Commission Regulation No 1907/2006, specifying general rules for adaptation of the standard testing regime.

Biological function and essentiality of calcium:

In humans, calcium is an essential mineral nutrient, with daily requirements ranging between 400 mg for infants, up to 1200 mg for pregnant women, as assessed by the Scientific Committee on Food. Calcium serves as a structural element in bone and tooth formation (mainly as hydroxyapatite) and is furthermore involved in a broad range of physiological processes: It plays a central role in blood coagulation, is involved in cell adhesion, hormone and neurotransmitter release, muscle contraction, cellular differentiation, several intracellular signalling pathways, and many others.

Biological significance of the hydroxyl ion:

The anionic counter ion released from hydraulic lime, irrespective of being present as oxide or hydroxide, is the hydroxyl ion. As an element of the acid-base system hydroxyl ions are not relevant in terms of toxicokinetics: If hydraulic lime is ingested, hydroxyl ions will be neutralised by gastric juice. Upon either inhalation exposure or deposition on the skin, hydroxyl ions released from hydraulic lime may lead to irritation of the skin or the respiratory tract, depending on the amount of substance dissolved, due to a pH effect. This is a local effect and therefore need not be considered further in the assessment of toxicokinetics and metabolism.

Biological significance of other constituents and major impurities:

The other main constituents dicalcium-silicate and limestone (calcium carbonate) as well as the impurities tricalcium-silicate and "calcined clay and/or silica" are not classified according to Directive67/548/EEC; they are therefore regarded to be of no toxicological significance. The toxicological properties of lime (chemical) hydraulic are considered to be governed by calcium hydroxide.

Oral absorption of calcium:

Calcium must be present in a soluble form, generally ionised, at least in the upper small intestine or bound to or complexed by a soluble organic molecule before it can cross the wall of the intestine. Absorption in the intestine is the result of two processes: (1) Active transport across membranes in the duodenum and the upper jejunum, which is regulated depending on dietary intake and the needs of the body. Active transport involves three stages, namely entry across the brush border of the enterocyte via calcium channels and membrane-bound transport proteins, diffusion across the cytoplasma attached to the calcium binding protein calbindin-D9K, and secretion across the basolateral membrane into the extracellular fluid against an electrochemical gradient either in exchange for sodium or via a calcium pump, a Ca-ATPase activated by calbindin, calcium and calmodulin. Active transport is negatively correlated with dietary calcium intake. This control is mediated via parathyroid hormone and 1,25(OH)2D. The renal production of 1,25(OH)2D is stimulated by increased parathyroid hormone secretion in response to a decrease of Ca²⁺in blood. Furthermore, it stimulates the expression of the gene encoding calbindin, thereby enhancing calcium absorption in the intestine. Both parathyroid hormone and 1,25(OH)2D also increase renal re-absorption of calcium and bone resorption. (2) Passive diffusion occurs throughout the small intestine, but mainly in the ileum and very little in the large intestine. On average, calcium is abosorbed in the intestine by approximately 30 %.

Most retained calcium is stored in the skeleton (99 % of the body’s calcium), depending on its needs. The main factors affecting the efficiency of calcium storage in bone are not dietary; they are physiological, related to growth, pregnancy and lactation, for example. Deposition and resorption of bone are regulated by several hormones.

Inhalation absorption:

Systemic availability of calcium from lime (chemical) hydraulic is a function of regional deposition in the respiratory tract, depending on the particle size of airborne dust. Dust may be released to air under practically relevant workplace conditions, for example during manual operations such as filling and emptying of bags, or during mechanical agitation as in mixing and weighing operations.

The particle size distribution of the airborne fraction during mechanical agitation in a rotating drum was determined according to the modified Heubach method (see section 4.5 of the technical dossier). The relative density of lime (chemical) hydraulic powder was measured according to OECD 109 (gas pycnometer method; section 4.4 of the technical dossier). From these data, the mass median aerodynamic diameter (MMAD) as well as the relevant parameter for predicting airway deposition of particulate matter was estimated as follows (also see section 4.5 of the technical dossier):

MMAD (lime (chemical) hydraulic) = 6.68 µm

Therefore, airborne particles may partly be deposited in the respiratory tract. From the particle size distribution, the proportions of airborne dust deposited in the extra-thoracic (head), tracheo-bronchial (TB) and alveolar (PU) region, respectively, have been estimated by the MPPD model (see section 4.5 of the technical dossier) as:

	Head	TB	PU	Total
Lime (chemical) Hydraulic	63.9 %	0.9 %	2.3 %	67.0 %

Only particles deposited on mucous membranes are available for absorption. For material deposited in the alveolar (PU) region a default absorption factor of 100 % is assumed in absence of specific data. Particles deposited in the head and the TB region are transported to the pharynx by mucociliary excitation, and subsequently swallowed. In the GI tract, calcium underlies intestinal absorption kinetics, hence contributes to systemic availability according to its oral absorption factor of 30 % (see above). Total inhalation absorption is therefore obtained by summing up the fraction deposited in the pulmonary region (100 % absorption) and the TB and head fractions corrected for intestinal absorption. As a result, total inhalation absorption of calcium from natural hydraulic lime is 21.74 %.

Distribution and elimination:

The main focus of toxicokinetics, metabolism and distribution for the substances of interest is on calcium sincein aqueous media lime (chemical) hydraulic dissociates under formation of calcium cations and hydroxyl ions. Carbonate is either released as CO2 if ingested (acidity of gastric juice) or remains undissolved and is therefore not bioavailable via the dermal or inhalation route. Calcium silicate is very poorly soluble (see section 4.8 of the technical dossier) and is therefore also not bioavailable to a relevant degree. Dissociation of calcium hydroxide in water is accompanied by generation of heat. Neither the alkaline reaction nor the generation of heat is of concern regarding systemic effects. Thus, further evaluation of hydroxyl ions is not considered to be necessary.

Calcium:

More than 99 % of the calcium stores in the body are located in the bones and teeth. The soft tissues accordingly contain less than 1 % of total body calcium. In extracellular fluids calcium is tightly regulated at a concentration of approximately 2.5 mmol/L (10 mg/dL). In blood, calcium is available as free Ca^{2 +}by approximately 45 %, the rest being complexed to citrate, phosphate, sulphate, and carbonate (ca. 10 %). Regulation of Ca levels is effected via three hormones, parathyroid hormone, 1,25-dihydroxycholecalciferol, and calcitonin. The extracellular calcium is involved in blood coagulation and cell adhesion, and serves as a source for bone metabolism. Intracellular calcium is primarily bound to membrane structures of the nucleus, mitochondria, endoplasmatic reticulum, or stored in special vesicles. The intracellular concentration of free Ca²⁺is only 0.1 µmol/L, i.e. approximately 25,000 times below the extracellular level.

Calcium is an essential mineral nutrient for humans and therefore cannot be considered as a xenobiotic substance. Calcium is homoeostatically regulated, with the skeleton serving as a reservoir to compensate for short-term fluctuations in dietary supply. In view of its essentiality for human nutrition, the existing information on calcium is considered as sufficient for assessment of its physiological behaviour, and for hazard assessment of lime (chemical) hydraulic.

Discussion on absorption rate:

A published in vitro study on dermal absorption of calcium constituting academic research is available. However, this study does not provide endpoint information suitable for derivation of an absorption rate that could be of use in a regulatory context. The study by Laudanska et al. (2002) is therefore considered as supportive data but will not be used for derivation of dermal absorption rates.

Nevertheless, following an approach consistent with the methodology proposed in the HERAG guidance for metals (HERAG fact sheet - assessment of occupational dermal exposure and dermal absorption for metals and inorganic metal compounds; EBRC Consulting GmbH, Hannover, Germany; August 2007), the following default dermal absorption factors for metal cations are proposed (reflective of full-shift exposure, i.e. 8 hours) for calcium present in lime (chemical) hydraulic:

For exposure to liquid/wet media: 1.0 %

For dry (dust) exposure: 0.1 %