Chromium tin calcium silicon sphene

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics, other

Remarks:

mass balance

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-11-19 to 2019-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

mass balance

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010-07-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 2017-05-08

Specific details on test material used for the study:

not applicable

Radiolabelling:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at dosing: males: 62 - 75 days; females: 55 - 68 days
- Weight at dosing: males: 396.9 – 459.8 g; females: 240.9 – 284.1 g
- Housing (exception: sampling period): kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 x 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (ad libitum): drinking water
- Acclimation period: males: 20 days; females: 13 days
- Health status: an initial health check was performed upon delivery of the animals. Only animals free of signs of illness were selected for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Humidity: 55 % ± 15 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: test item: 0.5 % aqueous hydroxyl propyl methylcellulose gel; reference item (mixture of chromium(III) acetate hydroxide and sodium stannate trihydrate): tap water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1) Chromium tin calcium silicon sphene
The formulations were freshly prepared on the administration day by dissolving the test item in the respective vehicle to the appropriate concentrations. During and after preparation the formulation was continuously stirred and the homogeneity of the suspension was checked by visual appraisal.

Administration volume: 10 mL/kg bw

The amount of the test item was adjusted to each animal's current body weight on the administration day.

2) Reference item (mixture of chromium(III) acetate hydroxide (Purity: Cr: 23.5 %; dose level:15.5 mg/kg bw) and sodium stannate trihydrate (Purity: Sn: 43.8 %: dose level: 80.5 mg/kg bw))
The formulations were freshly prepared on the administration day by dissolving the reference item in the respective vehicle to the appropriate concentrations. During and after preparation the formulation was continuously stirred and the homogeneity of the suspension was checked by visual appraisal.

Administration volume: 10 mL/kg bw

The amount of the reference item was adjusted to each animal's current body weight on the administration day.

Duration and frequency of treatment / exposure:

single administration

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose / concentration:

5 males / 5 females

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: the dose levels for this study had been selected based on available toxicity data:

The oral LD50 values for chromium acetate, basic and sodium stannate trihydrate are stated as being > 5000 and approximately 2400 mg/kg bw (recalculated from SnCl2 and SnSO4), respectively; oral bioavailabilities of soluble Sn and Cr(III) substances are given in the public domain with approximately 2 % (Sn) with as much as 98% being excreted directly in the faeces at intakes around 10 mg/day or higher, and 0.1 – 2 % (Cr), respectively.

The test item oral dose of 1000 mg/kg bw corresponds to the limit dose used in a separate 28-day oral toxicity study, which is considered the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg/kg bw of chrome tin pink sphene equates to a dose of 4 mg Cr/kg bw and 358.4 mg Sn/kg bw, corresponding to a dose of 15.5 mg chromium acetate, basic/kg bw and 80.5 mg sodium stannate trihydrate/kg bw, respectively.

The dosage for the reference item group was set equal to the dose of test item group on a stoichiometric basis from the test item for Cr(III) and was reduced to 10% for Sn. This equates to a dose of 4 mg Cr/kg bw and 35.8 mg Sn/kg bw, corresponding to a dose of 15.5 mg chromium acetate, basic/kg bw and 80.5 mg sodium stannate trihydrate/kg bw, respectively.

The dose level for the reference item have been confirmed in a preliminary experiment (non-GLP) employing two animals.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine and faeces
- Time and frequency of sampling: all animals of the test item, vehicle and reference item groups were scheduled for urine and faeces sampling. After the single administration, the animals were kept in metabolism cages. Urine and faeces were collected in 3 fractions/animal (sampling periods: 0 - 24 hours, 24 - 48 hours, and 48 - 72 hours).
The urine and faeces weight per collected fraction and animal were determined upon removal of the sample fraction. All samples were frozen at - 20 °C or colder until analysis.

The different biological matrices were handled differently for the analytical measurements. Urine samples were directly analysed for chromium and tin content by ICP-MS without any pre-treatment except for an acidification with HNO3 and dilution of samples (according to concentration of chromium and tin) and filtration. Faeces samples were initially freeze-dried (lyophilisation), followed by homogenization and microwave digestion and were analysed for chromium and tin content using ICP-MS.

The ICP-MS measurements were performed with an Agilent 7700 ICP-MS (Agilent Technologies, Waldbronn, Germany).

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn Germany
Nebulizer: Concentric nebulizer, from Agilent
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.93 L/min
Make-up/Dilution Gas gas flow: 0.11 L/min
RF power: 1550 W
Isotopes: 52Cr, 53Cr, 54Cr, 116Sn, 118Sn, 120Sn and 103Rh (internal standard)
Gas mode: [NoGas] = no gas mode; [He] = Helium (flow rate 4.3 mL/min) gas mode; [HEHe]
= high Helium mode (flow rate 10 mL/min)

At least three internal measurements for each sample were performed and the mean was calculated and printed by the instrument software.

LOD (urine samples): Cr: 0.005 - 0.122; Sn: 0.013 - 0.028
LOQ (urine samples): Cr: 0.016 - 0.366; Sn: 0.040 - 0.085
LOD (faeces samples): Cr: 0.002 - 0.033; Sn: 0.006 - 0.153
LOQ (faeces samples): Cr: 0.005 - 0.098; Sn: 0.017 - 0.460

The mass balance was calculated based on analytical information on urine and faeces that were measured, as described above, and using the raw data on urine and faeces weight. The calculation procedure was as follows:

For each animal, the mass of chromium and tin excreted via urine in each 24h time period (in mg/24h) was calculated by multiplying the concentration measured in urine with the volume of urine that was sampled for each individual animal. The volume of urine was obtained by correcting the recorded mass of urine with the density of urine (1.036 g/mL; reference: Ferrets, Rabbits and Rodents, 2nd Edition, Quesenberry and Carpenter,ISBN: 978-0-7216-9377-4).

Animals not treated with either chrome tin pink sphene or a mixture of
Cr3(OH)2(CH3COO)7 and Na2SnO3 · 3H2O served as the control group. The mean mass of chromium and tin excreted via urine by the control animals over 24h was <0.07 μg/<0.04 μg /24h (Cr, male and female), and <0.04 μg/<0.02 μg/24h (Sn, male and female).

The measured mean background masses in urine were subtracted from the mass of the respective elements Cr and Sn excreted by the treated animals (for m/f respectively). If this background correction resulted in a negative figure, the corrected excretion was set to zero for that animal for further calculations.

The mean mass of Cr, and Sn excreted via faeces by the control animals over 24h, calculated by multiplying the concentrations measured for a specific sample with the total weight of the faeces (wet weight) was 27.04 μg/24h and 10.22 μg/24h (Cr, male and female), and 0.97 μg/24h and 0.57 μg/24h (Sn, male and female). These background excretions were utilised to correct the masses of Cr, and Sn excreted by the treated animals (for m/f respectively). If this background correction resulted in a negative figure, the corrected excretion was set to zero for that animal.

The received actual dose of chromium, and tin was calculated, by multiplying the target dose with the actual body weight of each animal. For the respective doses for each dosing group (Group 2: Chrome tin pink sphene; Group 3: mixture Cr3(OH)2(CH3COO)7 and Na2SnO3 · 3H2O) please refer to section "Overall remarks, attachments" below.

For each treated animal the fractions of received chromium and tin that was excreted via urine or faeces was calculated for each 24h time period (0-24h, 24-48h, 48-72h).

OBSERVATIONS
- clinical signs: before and after dosing as well as regularly throughout the working day (7.30 a.m. to 4.30 p.m.) and on Saturdays and Sundays (8.00 a.m. to 12.00 noon; final check at approx. 4.00 p.m).
- mortality: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays (final check at approx. 4.00 p.m).
- body weight: at the time of group allocation and on the day of administration

GROSS PATHLOLOGY / HISTOPATHOLOGY
On test day 4 (approx. 72 hours after the administration) the animals were dissected. The animals were sacrificed, weighed, dissected, and inspected macroscopically.

All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland, and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.

The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole. The stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes, and accessory reproductive organs were recorded.

The weights of the following organs of all animals were determined: adrenal gland (2), brain, heart, kidney (2), liver, lungs, lymph nodes (cervical (1), mesenteric (1)), ovary (2), pituitary, prostate, spleen, testicle (2), thymus, and thyroid (1).
Paired organs were weighed individually and identified as left or right.
The residual carcasses were weighed.

Statistics:

The test and reference item treated groups were compared statistically to the control group.

The following statistical method was used for body weight/relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01): Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics, 482-491 (September 1964)

The statistical evaluation of the parametrical values captured by Provantis was done using the following settings:

Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Preliminary studies:

Please refer to the field "Details on study design" above.

Details on absorption:

The calculation of the mass balance show that the elements Cr3+ and Sn4+ of chromium tin calcium silicon sphene are not absorbed in the gastrointestinal tract to any significant extent but pass the animal effectively unchanged. Urinary excretion for all two elements was negligible and below 0.001 % for Cr and 0.002 % for Sn.

Please also refer to the section "Overall remarks, attachments".

Details on distribution in tissues:

not examined

Details on excretion:

1) Test item (Chromium tin calcium silicon sphene)
Animals that received 1000 mg test item /kg bw excreted 91.6 % Cr, and 79.4% Sn of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 84 % of Cr, and 71.6 % of Sn were excreted via faeces as largest fraction. Further 7.43 % and 0.13 % (Cr), and 7.63 % and 0.17 % (Sn) were excreted via faeces on the second and third day. Urinary excretion for all two elements was negligible and below 0.001 % for Cr, and 0.002 % for Sn.

2) Reference item (mixture of chromium(III) acetate hydroxide and sodium stannate trihydrate)
Animals that received a mixture of 4 mg Cr /kg bw (administered as Cr3(OH)2(CH3COO)7), and 35.8 mg Sn/Kg bw (administered as Na2SnO3 · 3H2O) excreted 86 % (Cr) and 95.6 % (Sn) of the administered dose (as mean, male and female animals) via urine and faeces during the first three days after exposure. The largest fraction (72.5 % for Cr, and 79.39 % for Sn) was excreted via faeces and urine (0.13 % for Cr and 0.01 % for Sn) already within the first 24h.

Please also refer to the section "Overall remarks, attachments".

Metabolites identified:

not measured

Details on metabolites:

not measured

Enzymatic activity measured:

not measured

Bioaccessibility (or Bioavailability) testing results:

not measured

CLINICAL SIGNS / MORTALITY /BODY WEIGHTS / GROSS PATHOLOGY

1) Control group

- none of the rats died prematurely.

- no signs of systemic intolerance.

- faeces of test item-treated animals were normally formed.

- body weight of the male and female animals were within the normal range on test day 1.

- no pathological findings were recorded.

 

2) Test item group (chromium tin calcium silicon sphene)

- none of the rats died prematurely.

- none of the rats showed any changes in behaviour or external appearance during the study.

- faeces of test item-treated animals were normally formed. However, male and female animals treated with the test item showed a reversible purple discolouring of the faeces on TD 2 caused by excreted amount of the test item.

- body weight of the male and female animals were within the normal range on test day 1.

- no pathological findings were recorded.

- no test item-related changes in relative and absolute organ weights were
noted for the male and female rats. However, statistically significant differences in relative and absolute organ weights compared to the
control which are not considered to be test item-related were noted, as follows:

Relative liver weight of male animals was statistically significant (p ≤ 0.05) increased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

Absolute liver weight of male animals was statistically significant (p ≤ 0.05) increased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

 

Please also refer to the section "Overall remarks, attachments".

 

3) Reference item group (mixture of chromium(III) acetate hydroxide and sodium stannate trihydrate)

- none of the rats died prematurely.

- none of the rats showed any changes in behaviour or external appearance during the study.

- faeces of test item-treated animals were normally formed.

- body weight of the male and female animals were within the normal range on test day 1.

- no pathological findings were recorded, except for a slight reduced size of the left adrenal gland of one male animal (no reference item-related finding).

- no reference item-related changes in relative and absolute organ weights were
noted for the male and female rats. However, statistically significant differences in relative and absolute organ weights compared to the
control which are not considered to be reference item-related were noted, as follows:

Relative liver weight of male animals was statistically significant (p ≤ 0.01) increased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

Relative liver weight of female animals was statistically significant (p ≤ 0.05) decreased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

Relative (right) kidney weight of female animals was statistically significant (p ≤ 0.05) decreased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

Absolute liver weight of male animals was statistically significant (p ≤ 0.01) increased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

 

Please also refer to the section "Overall remarks, attachments".

 

Conclusions:

Animals that received 1000 mg chromium tin calcium silicon sphene /kg bw excreted 91.6 % Cr, and 79.4% Sn of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 84 % of Cr, and 71.6 % of Sn were excreted via faeces as largest fraction. Further 7.43 % and 0.13 % (Cr), and 7.63 % and 0.17 % (Sn) were excreted via faeces on the second and third day. Urinary excretion for all two elements was negligible and below 0.001 % for Cr, and 0.002 % for Sn. In total, the calculation of the mass balance show that the elements Cr3+ and Sn4+ of chromium tin calcium silicon sphene are not absorbed in the gastrointestinal tract to any significant extent but pass the animal effectively unchanged.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-05-25 to 2020-05-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010-07-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

not applicable

Radiolabelling:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for toxicokinetic studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at dosing: 61 days
- Weight at dosing: males: 299.4 to 430.3 g; females: 168.7 to 294.3 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of
approx. 39 cm × 23 cm and a height of approx. 18 cm
- Diet (ad libitum): certified commercial pellet diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany); bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 5 days
- Health status: an initial health check was performed upon delivery of the animals. Only animals free of signs of illness were selected for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Humidity: 55 % ± 15 % (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: chrome tin pink sphene: oral (gavage); reference item: oral (gavage) & intravenously injected

Vehicle:

other: chrome tin pink sphene: 0.5% aqueous hydroxylpropyl methylcellulose gel; reference item: tap water (oral adminsitration) or 0.9% NaCl solution (intravenous administration)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1) Chromium tin calcium silicon sphene
The test item was freshly suspended in the vehicle to achieve the appropriate concentration on the administration day. The administration formulation was continuously agitated by stirring throughout the entire administration procedure.

Administration volume: 10 mL/kg bw

The amount of test item was adjusted to each animal's current body weight on the administration day.

2) Reference item (mixture of chromium (III) acetate hydroxide (Purity: Cr: 23.5 %) and sodium stannate trihydrate (Purity: Sn: 43.8 %)
The reference item compounds - chromium(III) acetate hydroxide and sodium stannate trihydrate - were combined according to the following dosage regimen:
- Reference item (oral adminsitration): 15.5 mg/kg bw of chromium(III) acetate hydroxide and 80.5 mg/kg bw of sodium stannate trihydrate
- Reference item (intravenous adminsitration): 0.78 mg/kg bw of chromium(III) acetate hydroxide and 0.81 mg/kg bw of sodium stannate trihydrate

The mixture was dissolved or suspended in the respective vehicle for oral/intravenous administration.The administration formulations were continuously agitated by stirring throughout the entire administration procedure.

Administration volume: 10 mL/kg bw
Injection speed (intravenous administration): approx. 15 seconds
The amount of reference item was adjusted to each animal's current body weight on the administration day.

Duration and frequency of treatment / exposure:

single administration

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose / concentration:

15 males / 15 femlaes

Control animals:

no

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: the dose levels for this preliminary study had been selected based on available toxicity data:

The oral LD50 values for chromium acetate, basic and sodium stannate trihydrate are stated as being > 5000 and approximately 2400 mg/kg bw (recalculated from SnCl2 and SnSO4), respectively. Oral bioavailabilities of soluble Sn and Cr(III) substances are given in the public domain with approximately 2% (Sn) with as much as 98% being excreted directly in the faeces at intakes around 10 mg/day or higher, and 0.1% to 2% (Cr), respectively.

The test item oral dose of 1000 mg/kg bw corresponded to the limit dose used in a separate 28-day oral toxicity study, which is considered the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg/kg bw of chrome tin pink sphene equates to a dose of 4 mg Cr/kg bw and 358.4 mg Sn/kg bw, corresponding to a dose of 15.5 mg chromium acetate, basic/kg bw and 805 mg sodium stannate trihydrate/kg bw, respectively.

The dosage for oral administration (gavage) of the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) was set equal to the dose of the test item on a stoichiometric basis from the test item for Cr(III) and was reduced to 10% for Sn. This equates to a dose of 4 mg Cr/kg bw and 35.8 mg Sn/kg bw, corresponding to a dose of 15.5 mg chromium acetate, basic/kg bw and 80.5 mg sodium stannate trihydrate/kg bw, respectively.

The dosage for intravenous injection of the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) was set to 5 % of the dose of the reference item administered via gavage on a stoichiometric basis for Cr(III) and to 1% for Sn, thereby lowering the dose for reasons of tolerability of the test animals. This equates to doses of 0.36 mg Sn/kg bw and 0.2 mg Cr/kg bw, respectively, corresponding to doses of 0.81 mg sodium stannate trihydrate/kg bw, and 0.78 mg chromium acetate, basic/kg bw, respectively.

The dose levels for the reference item (gavage and intravenous administration) were confirmed in a preliminary experiment (non-GLP) employing two animals per group.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling: in order to obtain approx. 100 μL LiHeparin plasma per animals and sampling time, a sufficient volume of blood was collected from the retrobulbar vvenous plexus of all animals under isoflurane anaesthesia. The blood was withdrawn at the following sampling times: 0 (predose), 1, 2, 4, 8, 12, 24, 48 and 72 hours after administration. At each sampling time point 5 animals/sex/group were used and three sampling points used the same animals.

The blood samples were cooled using an IsoTherm-Rack system until centrifugation. Immediately after centrifugation the isolated plasma was frozen at -20 °C ± 2 °C and stored at this temperature until analysis. The total time between blood withdrawal and freezing the plasma did not exceed 30 minutes.

For plasma samples, a pre-treatment by a microwave digestion with HNO3 was necessary to digest the proteins in plasma. Afterwards chromium and tin concentrations in digested samples were determined by ICP-MS.

The ICP-MS measurement were performed with an Agilent 7700 ICP-MS (Agilent
Technologies, Waldbronn, Germany).

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn Germany
Nebulizer: Conical nebulizer, from Glass Expansion
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.93 L/min
Dilution Gas flow: 0.11 L/min
RF power: 1500 W
Isotopes: 52Cr; 53Cr; 116Sn; 118Sn; 120Sn and 103Rh (internal standard)

LOD: Cr: 0.003 - 0.011; Sn: 0.005 - 0.077
LOQ: Cr: 0.009 - 0.033; Sn: 0.014 - 0.232

Toxicokinetic evaluation of the data from the plasma analysis was performed and a non-compartment model was employed. The following parameters were determined:
AUC0-inf = extrapolated area from zero to infinity
AUC0-t last = extrapolated area from zero to the last quantifiable plasma concentration > LLOQ
Kel = elimination rate constant
t1/2 = elimination half-life
Cmax values are the highest measured plasma concentrations and tmax values are the time points of highest plasma concentrations.

Cmax values are the highest measured plasma concentrations and tmax values are the time points of highest plasma concentrations.

Elimination rate constants (Kel) and plasma elimination half-lives (t½) were calculated by linear regression analysis of the log/linear portion of the individual plasma concentration-time curves (c = concentration, t = time).

Half-life:
t0.5 = ln2/Kel [h]
(dc/dt) = Kel * c [h]

Area under the curve (AUC) values were calculated using the linear trapezoidal method and extrapolated to infinite time by dividing the last measurable plasma concentration by the elimination rate constant. Plasma concentrations at time zero were taken to be those at the first blood sampling time.

A dose correction to the analyte content for AUC values was performed for chromium and tin.

As far as possible based on the available data, the bioavailability was calculated for the oral route in comparison to the intravenous route based on AUC0-t last/dose and AUC0-inf/dose values as follows:

- reference item (oral adminsitration) vs. reference item (intravenous administration)
Bioavailability (abs.) = (AUC/dose (reference item; oral administration) / AUC/dose (reference item; intravenous administration)) x 100
- test item (oral adminsitration) vs. reference item (intravenous administration)
Bioavailability (rel..) = (AUC/dose (test item; oral administration) / AUC/dose (reference item; intravenous administration)) x 100

OBSERVATIONS:
- clinical signs: before and after dosing as well as regularly throughout the working day (7:00 a.m. to 3:45 p.m.) and on Saturdays and Sundays (7:00 a.m. to 11:00 a.m.; final check at approx. 3:30 p.m).
- mortality: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays (final check at approx. 3:00 p.m).
- body weight: at the time of group allocation and on the administration day.

Statistics:

Reference item (oral administration) and reference item (intravenous administration were compared to the test item.

The following statistical method was used for body weight (p ≤ 0.05 and p ≤ 0.01): Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics, 482-491 (September 1964)

The statistical evaluation of the parametrical values captured by Provantis was done using the following settings:

Homogeneity of variances and normality of distribution were tested using the
BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Preliminary studies:

Please refer to the field "Details on study design" above.

Details on absorption:

BIOANALYSIS
1) Chromium
Following oral administration of 1000 mg Chromium tin calcium silicon sphene/kg bw (test item, oral administration), peak mean chromium concentrations were noted at 72 hours p.a. for the male animals and at 1 hour p.a. for the female animals. The mean peak values were 0.0060 μg chromium/g sample for the males and 0.0195 μg chromium/g sample for the females.

The peak mean chromium plasma concentrations observed for the animals orally treated with the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate; oral administration) were slightly higher compared to the test item-treated group with Cmax values of 0.0132 and 0.0258 μg chromium/g sample for males and females, respectively.

The male and female animals treated intravenously with the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) revealed peak mean plasma concentrations of 0.0884 and 0.0752 μg chromium/g sample for males and females, respectively, corresponding approximately to the 15-fold (males) and 4-fold (females) of the peak mean concentrations observed for the test item-treated animals.

Please also refer to the section "Overall remarks, attachments".

2) Tin
Following oral administration of 1000 mg Chromium tin caclium silicon sphene/kg bw (test item, oral administration), peak mean tin concentrations of 0.0635 μg tin/g sample for the males and 0.0290 μg tin/g sample for the females were observed at 1 hour p.a.

The peak mean tin plasma concentrations observed for the animals orally treated with the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) were slightly lower compared to the test item-treated group for the male animals (0.0458 vs. 0.0635 μg tin/g sample) but slightly higher when the values of the females are compared (0.0645 vs. 0.0290 μg tin/g sample).

The male and female animals treated intravenously with the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) revealed peak plasma concentrations of 0.0180 and 0.0354 μg tin/g sample for males and females, respectively, being lower with regard to the males and slightly higher with regard to the females compared to the peak concentrations observed for the test item-treated animals.

Please also refer to the section "Overall remarks, attachments".

TOXICOKINETIC EVALUATION
1) Chromium
The highest Cmax value for chromium in the plasma samples was noted following the intravenous administration of the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate), followed by the oral administration of the reference item and the oral administration of the test item (chrome tin pink sphene).

The plasma concentrations of chromium declined post dosing with an elimination half-life (t½) ranging approximately from 20.1 to 43.5 hours across sexes and treatments.

The AUC0-t last values ranged from 0.1469 to 1.6272 h μg/g across sexes and treatments. The animals intravenously treated with the reference item revealed the highest exposure towards chromium, whereas the lowest exposure was observed following treatment with the test item chromium tin caclium silicon sphene.

Although the female animals orally treated with the reference item appeared to show a slightly higher exposure to chromium than the male animals, no general differences with regard to exposure were noted between the sexes at all treatments.

The calculation of t½, Kel, and AUC0-∞ was not possible for the male animals of test item group (oral administration) and the female animals of the reference item group (oral administration) because the chromium concentrations measured increased towards the end of the kinetics period. Further, the AUC%extrapolated for the male animals of the reference item group (oral administration) was larger than 20%, rendering the AUC0-inf value unreliable.

Please also refer to the section "Overall remarks, attachments".

2) Tin
The highest Cmax value for tin in the plasma samples was noted following the oral administration of the test item chromium tin calcium silicon sphene, followed by the oral administration of the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate), and the intravenous administration of the reference item.

The elimination half-life (t½) could be calculated only for the test item-treatment of the male animals and the intravenous treatment with the reference item of both sexes, and ranged from 4.3 to 23.5 hours.

The AUC0-t last values ranged from 0.0872 to 0.3522 h μg/g across sexes and treatments. The male animals revealed the highest exposure towards tin following intravenous administration of the reference item, the lowest exposure was noted following oral treatment with the reference item. For the female animals, the highest exposure was observed following oral administration of the test item chromium tin calcium silicon sphene, whereas the oral treatment with the reference item resulted in the lowest exposure towards tin.

The calculation of t½, Kel, and AUC0-∞ was not possible for the female animals of test item group (oral adminsitration) and the male and female animals of reference item group (oral administration) because there were insufficient measurement data and/or because the tin concentrations measured increased towards the end of the kinetics period. Further, for both the males and the females of the reference item group (intravenous administration), the AUC%extrapolated was larger than 20%, rendering the AUC0-inf values unreliable.

Please also refer to the section "Overall remarks, attachments".

Details on distribution in tissues:

not applicable

Details on excretion:

not applicable

Toxicokinetic parameters:

other: "see Remarks"

Remarks:

Abs. bioavailability of 0.63/2.27% (m/f) was calculated for Cr(III) acetate hydroxide following the oral administration of the reference item compared to the i.v. administration, & a rel. bioavailability of approx. 0.43/1.17% (m/f) for the test item.

Toxicokinetic parameters:

other: see "Remarks"

Remarks:

Rel. bioavailability of tin was calculated to be approx. 0.05%/0.25% (m/f) following treatment with the test item. For the oral treatment with the reference item (Na stannate trihydrate), an abs. bioavailability of 0.56%/0.63% (m/f) was calculated.

Metabolites identified:

not measured

Details on metabolites:

not measured

Enzymatic activity measured:

not measured

Bioaccessibility (or Bioavailability) testing results:

1) Chromium
For chromium, an absolute bioavailability (based on AUC0-t last/dose) of 0.63/2.27% (males/females) was calculated from soluble chromium(III) acetate hydroxide following the oral administration of the reference item compared to the intravenous administration, and a relative bioavailability of approximately 0.43/1.17% (males/females) for the test item (chromium tin calcium silicon sphene).

2) Tin
The relative bioavailability (based on AUC0-t last/dose) of tin was calculated to be approx. 0.05% and 0.25% for male and female animals, respectively, following treatment with the test item chromium tin calcium silicon sphene. For the oral treatment with the reference item, an absolute bioavailability (based on AUC0-t last/dose) of 0.56% was calculated for the males and of 0.63% for the females.

CLINICAL SIGNS AND MORTALITY (males and females)

1) Chromium tin calcium silicon sphene (oral administration)

- no deaths were noted during the study course.

- no abnormal findings with regard to behaviour, external appearance, or consistency of faeces.

2) Reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate; oral administration)

- no deaths were noted during the study course.

- no abnormal findings with regard to behaviour, external appearance, or consistency of faeces.

3) Reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate; intravenous administration)

- no deaths were noted during the study course.

- no abnormal findings with regard to behaviour, external appearance, or consistency of faeces.

- none of the animals treated intravenously with the reference item revealed any signs of local intolerance reactions at the injection site.

 

BODY WEIGHT (males and females)

The body weight of all animals employed in the study was in the normal range. There were no noteworthy differences between the three test groups.

Conclusions:

For chromium, a relative bioavailability of approximately 0.43/1.17% (males/females) was calculated for chromium tin calcium silicon sphene. Furthermore, the relative bioavailability of tin was calculated to be approx. 0.05% and 0.25% for male and female animals, respectively, following treatment with chromium tin calcium silicon sphene.

Reference 3

Endpoint:

basic toxicokinetics in vitro / ex vivo

Remarks:

Bioaccessibility - transformation/dissolution in artificial physiological media

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-17 to 2012-09-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Objective of study:

bioaccessibility (or bioavailability)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media

Deviations:

yes

Remarks:

Bioaccessibility testing: loading of 100 mg/L; five artificial physiological media agitated at 100 rpm, at 37 °C ± 2 °C; sampling after 2h and 24h; determination of Cr and Sn concentrations after filtration by ICP-MS (samples) and ICP-OES (mass balance).

Principles of method if other than guideline:

An internationally agreed guideline does not exist for this test (e.g. OECD). However, similar tests have been conducted with several metal compounds, including steels, in previous risk assessments (completed under Regulation (EEC) No 793/93) and in recent preparation for REACH regulation (EC) No 1907/2006.
The test was performed on the basis of the guidance for OECD-Series on testing and assessment Number 29 and according to the bioaccessibility test protocol provided by the study monitor.

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-02-07

Species:

other: in vitro (simulated human body fluids)

Details on exposure:

Test principle in brief:
- five different artificial physiological media,
- single loading of test substance of 100 mg/L,
- samples taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C,
- two method blanks per artificial media were tested; measurement (ICP-MS) of dissolved chromium and tin concentrations after filtration (0.2 µm, Supor membrane)
- measurement of mass balance samples by ICP-OES
- the study was performed in duplicates

The aim of this test was to assess the dissolution of IPC-2013-004 (Chrome tin pink sphene) in five artificial physiological media: Artificial lysosomal fluid (ALF, pH = 4.5), Artificial sweat solution (ASW, pH = 6.5), Gamble´s solution (GMB, pH = 7.4), Artificial gastric fluid (GST, pH = 1.5), Phosphate buffered saline (PBS, pH = 7.4). The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. a substance entering the human body by ingestion into the gastrointestinal tract and by inhalation.

Duration and frequency of treatment / exposure:

Samples were taken after 2 h and 24h.

Dose / conc.:

100 other: mg of the test item /L artificial media

Details on study design:

Reagents
The water (resistivity >18 MΩ·cm.) used for this test was purified with a Pure Lab Ultra water purification system from ELGA LabWater, Celle, Germany.
- Nitric acid - “Supra” quality (ROTIPURAN® supplied by Roth, Karlsruhe, Germany).
- Hydrochloric acid – “instra-analyzed plus” quality (J.T. Baker, Griesheim, Germany).

Metal analysis
- Standards: chromium and tin standards containing 1000 mg/L Cr and Sn (lot no. HC114885 and HC077712, respectively) in 2-3 % nitric acid were applied. - Certified reference materials. TMDA-70 (lot no. 0310) and TM-DWS.2 (lot no. 1010) obtained from Environment Canada and a multielement standard (19 elements, CPI, Amsterdam, The Netherlands, lot no. 09K023 and 12A089)

Instrumental and analytical set-up for the ICP-OES instrument:
Thermo IRIS Intrepid II from Thermo Electron Corporation, Germany
Nebulizer: Concentric glass nebulizer, from Thermo
Spray chamber: Glass cyclonic spray chamber, from Thermo
Nebulizer gas flow: 0.68 L/min
Make-up gas flow: 0.5 L/min
RF power: 1150 W
Wavelengths:
Cr: 267.716 nm, 283.563 nm, 284.325 nm and 357.869 nm;
Sn 175.790 nm (axial and radial), 181.120 nm (axial and radial), 189.989 nm (axial and radial), 224.605 nm (axial and radial), 226.891 nm (axial and radial) and 283.999 nm (axial and radial)
Calibration: Cr: blank, 500 µg/L, 2500 µg/L, 5000 µg/L, 7500 µg/L and 10000 µg/L.
Correlation coefficients (r): at least 0.9998.

One measurement series was performed for the determination of chromium and tin concentrations of the mass balance samples.
The applied LOD/LOQ calculations are:
LOD: 3 * method standard deviation from calibration line;
LOQ: 10 * method standard deviation from calibration line.
These data were read directly from the Thermo IRIS Intrepid II ICP-OES instrument output (data calculated by internal algorithms of the instrument software).

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn, Germany
Nebulizer: Concentric glass nebulizer, from GlassExpansion
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.91 L/min
Dilution/Make-up gas flow: 0.13 L/min
RF power: 1500 W
Isotopes: 52Cr, 53Cr, 103Rh (internal standard), 118Sn, 119Sn, 120Sn
Calibrations: blank, 0.1 µg/L, 0.25 µg/L, 0.5 µg/L, 0.75 µg/L, 1.0 µg/L, 2.5 µg/L, 5.0 µg/L, 7.5 µg/L, 10.0 µg/L, 25.0 µg/L, 50.0 µg/L, 75.0 µg/L, 100 µg/L, 250 µg/L and 500 µg/L
Correlation factors (r): at least 0.9977
The certified reference materials TMDA-70 (certified with 389 µg/L chromium and 19.5 µg/L tin) and TM-DWS.2 (certified with 44.4 µg/L chromium and 12.2 µg/L tin) were analyzed as quality assurance samples along with the test samples. To meet quality assurance requirements, Cr and Sn recovery needs to be in the range of ± 15 % of the respective certified value. Mean accuracy and precision for the analysis of TMDA-70 were 104 ± 8.2% (n = 18) for Cr and 103 ± 11.3% (n = 21) for tin. Mean accuracy and precision for TM-DWS.2 by ICP-MS were 105 ± 8.6 % (n = 25) for Cr and 109 ± 8.3% (n = 20) for Sn.

In sum, five series of measurements were performed for the determination of total dissolved chromium and tin concentrations in samples from test vessels, blanks to determine background levels of elements, fortified samples and samples of filters and syringes for calculating the mass balance.

Details on dosing and sampling:

Loading:
The nominal loading in this test was 100 mg/L. However, due to weighing uncertainties the actual loadings range from 100.000 mg/L to 100.032 mg/L in the test vessels.

Type:

other: Bioaccessibility

Results:

Highest dissolution (loading: 0.1g/L, after 24h) in ALF: Cr: 3.81 ± 0.71 µg/L, Sn: 161 ± 2.51 µg/L, in GST: Cr: 5.22 ± 0.15 µg/L, Sn: 144 ± 2.34 µg/L

Method validation summary (ICP-OES)

validation parameter	results	Comment
Selectivity	similar data with two different Cr and Sn wavelengths for ICP-OES method	no interferences observed
Linearity	applied calibration functions were linear	correlation coefficient at least 0.9998
Limit of detection	Cr: 1.65 µg/L / Sn: 212 µg/L
Limit of quantification	Cr: 5.51 µg/L / Sn: 708 µg/L
Accuracy & reproducibility	mean recovery for CRM TMDA-70: Cr: 97.1 ± 1.5 % (n =3) Sn: concentration <LOD	high concentration range (389 µg Cr/L; 19.5 µg Sn/L)
Trueness chromium	mean recovery for recalibration standard: Cr: 97.7 ± 0.7 % (n = 3)	mid concentration range (100 µg/L)
Trueness tin	mean recovery for recalibration standard: Sn: 99.3 ± 0.9 % (n = 3)	high concentration range (5000 µg/L)

Method validation summary (ICP-MS)

validation parameter	results	Comment
Selectivity	chromium: similar in Helium and HiHelium mode; Tin: similar in all three gas modes (NoGas, Helium, HiHelium)	appropriate Isotope and gas mode were selected for interference free measurements
Linearity	applied calibration functions were linear	correlation coefficients ≥0.9977
Limit of detection	Cr: 0.014 – 0.067 µg/L Sn: 0.004 – 0.015 µg/L
Limit of quantification	Cr: 0.048 – 0.224 µg/L Sn: 0.013 – 0.050 µg/L
Cr and Sn concentrations in method blanks	< LOD/LOQ (< 0.067 / < 0.224 µg/L) of Cr in GST < LOD/LOQ (< 0.010 / < 0.034 µg/L) of Sn in GST < LOQ (< 0.050 µg/L) of Sn in PBS; in all other media above LOD/LOQ ; well below concentrations in test samples Cr: at least 15 % lower; Sn: at least thirtyfold lower	Elevated concentrations of Cr and Sn in method blanks origin from chemicals applied to prepare different media
Accuracy & reproducibility	mean recovery for CRM TMDA-70: Cr: 104 ± 8.2 % (n = 18) Sn: 103 ± 11.3 % (n = 21)	higher concentration range (389 µg Cr/L; 19.5 µg Sn/L)
Accuracy & reproducibility	mean recovery for CRM TM-DWS.2: Cr: 105 ± 8.6 % (n = 25) Sn: 109 ± 8.3 % (n = 20)	lower concentration range (44.4 µg Cr/L; 12.2 µg Sn/L)
Trueness	mean recovery for recalibration standard: Cr: 103 ± 3.4 % (n = 3) Sn: 97.2 ± 3.8 % (n = 3)	higher concentration range (200 µg/L)
Trueness	mean recovery for recalibration standard: Cr: 99.4 ± 2.9 % (n = 3) Sn: 106 ± 9.9 % (n = 7)	high concentration range (50 µg/L)
Trueness	mean recovery for recalibration standard: Cr: 98.3 ± 4.5 % (n = 3) Sn: 95.2 ± 1.4 % (n = 3)	mid-high concentration range (20 µg/L)
Trueness	mean recovery for recalibration standard: Cr: 101 ± 6.1 % (n = 7) Sn: 102 ± 14.7 % (n = 11)	mid-low concentration range (10 µg/L)
Trueness	mean recovery for recalibration standard: Cr: 96.8 ± 1.1 % (n = 3) Sn: 95.5 ± 0.8 % (n = 3)	low concentration range (5 µg/L)
Trueness	mean recovery for recalibration standard: Cr: 92.9 ± 4.4 % (n = 6) Sn: 98.2 ± 2.0 % (n = 6)	low concentration range (2.5 µg/L)
Trueness	mean recovery for recalibration standard: Cr: 98.2 ± 7.7 % (n = 10) Sn: 95.5 ± 9.0 % (n = 10)	low concentration range (1 µg/L)
Trueness	Fortification of samples: Cr: 96.9 – 114 % Sn: 96.9 – 110 %

Solution pH value

The pH of ALF, GST and PBS solutions remained stable. The pH of the ASW solutions containing the test item, increased in the first 2 h. After 24h, the pHs had decreased to 6.3 in both vessels. The pH of ASW method blanks decreased from 6.5 to 6.1. In GMB medium, the pH in all vessels (including method blanks) increased during the time of the test from 7.4 to 8.9. Therefore, an effect of the test substance can be excluded. In fact, the pH of the GMB media does not seem to be stable under the conditions of the test.

pHs of the physiological test media prior to the test

media	target pH	measured pH	temp. [°C]
ALF	4.5	4.5	36.0
ASW	6.5	6.5	35.8
GMB	7.4	7.4	35.2
GST	1.5 – 1.6	1.6	35.3
PBS	7.2 - 7.4	7.2	35.7

 

Analytical measurements

The results of the ICP-MS measurements of total dissolved Cr and Sn in test samples are presented below. Some samples had to be diluted so that dissolved Cr and Sn concentrations were in the optimal working range of calibration.

Concentration of chromium in artificial media, calculated nominal chromium concentration and dissolved amount of chromium

media and sample	total Cr ± SD in method blanks [µg/L]	total Cr ±SD in sample vessels [µg/L]	Cr ± SD in sample vessels with blank subtraction [µg/L]	dissolved amount of Cr in artificial media [%] normalised for measured background in method blank #
ALF 2h	6.69 ± 0.19	8.15 ± 0.07	1.46 ± 0.07	0.39 ± 0.02
ALF 24h	6.80 ± 1.71	10.6 ± 0.71	3.81 ± 0.71	1.00 ± 0.19
ASW 2h	0.19 ± 0.02	0.54 ± 0.09	0.36 ± 0.09	0.09 ± 0.02
ASW 24h	0.23 ± <0.01	1.98 ± 0.06	1.75 ± 0.06	0.46 ± 0.02
GMB 2h	0.19 ± <0.01	0.39 ± 0.1	0.20 ± 0.10	0.05 ± 0.03
GMB 24h	0.17 ± 0.01	1.06 ± 0.15	0.90 ± 0.15	0.24 ± 0.04
GST 2h	<LOD	1.08 ± 0.08	1.08 ± 0.08	0.28 ± 0.02
GST 24h	<LOD	5.22 ± 0.15	5.22 ± 0.15	1.37 ± 0.04
PBS 2h	0.74 ± 0.06	1.07 ± 0.08	0.33 ± 0.08	0.09 ± 0.02
PBS 24h	0.72 ± 0.08	2.90 ± 0.36	2.18 ± 0.36	0.57 ± 0.09

# initial nominal loading of 100 mg/L * 0.38 (according to CoA, Cr content of test item is 0.38 %) = nominal Cr concentration [0.38 mg/L] * 1000 = nominal Cr concentration [380 µg/L]

In five different artificial physiological media, dissolved Cr levels ranged from 0.05 to 1.37 % of nominal chromium contained in the respective loading of 100 mg/L chrome tin pink sphene (IPC-2013-004) depending on solution parameters and test duration.

Concentration of tin in artificial media, calculated nominal tin concentration and dissolved amount of tin

media and sample	total Sn ± SD in method blanks [µg/L]	total Sn ±SD in sample vessels [µg/L]	Sn ± SD in sample vessels with blank subtraction [µg/L]	dissolved amount of Sn in artificial media [%] normalised for measured background in method blank #
ALF 2h	0.04 ± <0.01	35.3 ± 1.28	35.3 ± 1.28	0.10 ± <0.01
ALF 24h	0.10 ± 0.01	161 ± 2.51	161 ± 2.51	0.45 ± 0.01
ASW 2h	0.07 ± 0.01	11.8 ± 1.27	11.8 ± 1.27	0.03 ± <0.01
ASW 24h	0.07 ± 0.02	154 ± 16.6	154 ± 16.6	0.43 ± 0.05
GMB 2h	0.02	0.62 ± <0.01	0.60 ± <0.01	0.002 ± <0.001
GMB 24h	0.06	2.58 ± 0.08	2.52 ± 0.08	0.01 ±<0.01
GST 2h	<LOQ (0.01)	28.0 ± 2.86	28.0 ± 2.86	0.08 ± 0.01
GST 24h	<LOQ (0.01)	144 ± 2.34	144 ± 2.34	0.40 ± 0.01
PBS 2h	<LOQ (0.03)	5.41 ± 1.90	5.41 ± 1.90	0.02 ± 0.01
PBS 24h	<LOQ (0.04)	38.4 ± 8.10	38.4 ± 8.10	0.11 ± 0.02

# initial nominal loading of 100 mg/L * 35.9 (according to CoA, Sn content of test item is 35.9 %) = nominal Sn concentration [35.9 mg/L] * 1000 = nominal Sn concentration [35900 µg/L]

In five different artificial physiological media, dissolved Sn levels ranged from 0.002 to 0.45 % of nominal tin contained in the respective loading of 100 mg/L chrome tin pink sphene (IPC-2013-004) depending on solution parameters and test duration.

Mass balance calculation

Total dissolved chromium and tin concentrations in test samples measured by ICP-OES and dissolved residues on filters and syringes measured by ICP-MS indicate an incomplete dissolution of the test item in all physiological media after addition of aqua regia to the sample vessels.

Calculation of chromium mass balance

media	dissolved Cr after addition of aqua regia [mg]	nominal amount [mg] #	recovery [%]
ALF 24h A	0.02	0.19	13.0
ALF 24h B	0.03	0.19	13.9
ASW 24h A	0.03	0.19	14.8
ASW 24h B	0.03	0.19	14.3
GMB 24h A	0.02	0.19	11.8
GMB 24h B	0.03	0.19	16.1
GST 24 A	0.03	0.19	15.0
GST 24h B	0.03	0.19	17.7
PBS 24h A	0.03	0.19	14.5
PBS 24h B	0.03	0.19	14.5

# nominal concentration Cr = 0.55 % as Cr₂O₃ in test item = 68.42 % Cr in Cr₂O₃ => 0.1882 mg Cr in 50 mg test item==> 0.1882 * initial weight / 50 mg

Calculation of tin mass balance

media	value for dissolved Sn after addition of aqua regia [mg]	nominal concentration [mg] #	recovery [%]
ALF 24h A	2.72	17.9	15.2
ALF 24h B	2.84	17.9	15.8
ASW 24h A	3.29	17.9	18.4
ASW 24h B	3.30	17.9	18.4
GMB 24h A	2.77	17.9	15.5
GMB 24h B	3.64	17.9	20.3
GST 24 A	3.12	17.9	17.4
GST 24h B	3.77	17.9	21.0
PBS 24h A	1.85	17.9	10.3
PBS 24h B	2.54	17.9	14.2

# nominal concentration Sn = 45.50 % as SnO₂in test item = 78.77 % Sn in SnO₂ => 17.920 mg Sn in 50 mg test item => 17.920 * initial weight / 50 mg  

Conclusions:

The bioaccessibility of chromium tin caclium silicon sphene has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation). Dissolved Cr and Sn concentrations were below 5.22 µg/L and 161 µg/L, respectively, even at the highest loading of 0.1 g/L, referring to a solubility of 0.005 % and 0.161 %, respectively. Therefore, chromium tin caclium silicon sphene may reasonably be considered not bioaccessible.

Executive summary:

The bioaccessibility of chromium and tin has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation), as follows:

 

- Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions,

- Phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum,

- Artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating,

- Artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and

- Artificial gastric fluid (GST, pH 1.5), which mimics the very harsh digestion milieu of high acidity in the stomach.

 

Under the conditions of this test (flasks with different artificial physiological media; loadings of 0.1 g/L, 37 °C, sampling after 2h and 24h), the concentrations of dissolved chromium and tin were between 0.002 and 1.37 % based on total Cr and Sn content. Therefore, chromium tin caclium silicon sphene may reasonably be considered not bioaccessible.

Description of key information

The in-vitro and in-vivo experiments described above are in very good agreement with regards to the negligible level of bioavailability of the elements Cr and Sn contained in the pigment.

1) In in-vitro dissolution experiments in five different artificial physiological media, dissolved Cr and Sn concentrations were below 5.22 µg/L and 161 µg/L, respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.005 % and 0.161 %, respectively.

2) In a 28-day oral toxicity study with 1,000 mg/kg pigment the uptake of Sn and Cr during 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.05 % of the dose was excreted via urine for both metals, mirrored by either minimal or no increase in blood plasma concentrations.

3) In a mass balance study with a single oral dose of 1,000 mg/kg of the pigment, 91.6 % Cr, and 79.4 % Sn of the dose were excreted via urine and faeces during the first three days after exposure, with only <0.002 % of the dose being excreted via urine.

4)  In a relative bioavailability study, a relative bioavailability of approx. 0.43/1.17 % (males/females) and 0.05/0.25 % (males/females) was calculated for Cr and Sn, respectively, following treatment with the pigment.

In conclusion, the oral relative bioavailability of the pigment "Chromium tin calcium silicon sphene" can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by an in-vitro dissolution experiment in five different artificial physiological media.

A rounded value of <<0.05 % for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg pigment/kg bw/d (<<0.05 % for both metals) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.001 % for Cr and 0.002 % for Sn).

 

Absorption rate - oral: 0.05 %

 

 

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

0.05

Additional information

The toxicity data in this registration dossier refer explicitly to the pigment Chromium tin calcium silicon sphene and document its negligible bioavailability and the complete lack of any human health hazard. Experiments on the bioavailability of the pigment are summarised and discussed in this section.

Summary of in-vitro bioaccessibility experiments (Knopf, 2013):

The chemical and physiological properties of the pigment Chromium tin calcium silicon sphene are dominated by inertness due to the characteristics of the production process (calcination at high temperatures, approximately 1000 °C), rendering the substance to be of a unique, stable crystalline structure. This fundamental process leads to a very low bioaccessibility for the elements contained in the pigment. This has been investigated experimentally in vitro by simulating dissolution under physiological conditions of the presumed exposure routes (oral, dermal and inhalation), as follows:

1.) Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions,

2.) Phosphate‐buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum,

3.) Artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating,

4.) Artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and

5.) Artificial gastric fluid (GST, pH 1.5), which mimics the very harsh digestion milieu of high acidity in the stomach.

An oral, dermal or inhalative uptake of the substance for humans exposed to the pigment is negligible.

The dissolution of Cr of the test item is between 0.20 µg/L /0.91 µg/L (GMB) and 1.08 µg/L/ 5.22 µg/L (GST) at a loading of 0.1g/L after 2 and 24 hours.

The dissolution of Sn of the test item is in a range of 0.60 µg/L/ 2.52 µg/L, (GMB) and 35.3 µg/L/ 161 µg/L (ALF) at a loading of 0.1g/L after 2 and 24 hours.

A pH dependent dissolution can be observed. In conclusion, since the dissolved Cr and Sn concentrations were below 161 µg/L even at the highest loading of 0.1g/L, referring to a solubility of 0.161 %, this pigment may reasonably be considered biologically inert.

 

Toxicokinetic screening data from a 28-day repeated dose oral toxicity study (Leuschner, 2021)

In a 28 day repeated dose toxicity study, male and female rats were given a daily dose of the pigment "Chromium tin calcium silicon sphene" of 1,000 mg/kg bw/day via gavage. Individual urine samples were collected from all animals prior to sacrifice in one cumulated 24-h fraction/animal after the last oral application, and blood samples were collected from each animal upon sacrifice. The plasma and urine samples were analysed for total chrome and tin content.

The uptake of tin and chromium during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.05 % of the dose was excreted via urine for both metals, mirrored by either minimal or no increases in blood plasma concentrations.

The tin and chromium concentrations of the 24h-urine samples, collected during the day before final sacrifice, ranged from: 0.673 - 1.08 μg/L urine (mean: 0.85 µg/L ± 0.173) and 0.561 - 2.14 μg/L urine (mean: 1.18 µg/L ± 0.58) for Sn and from 5.22 - 6.46 μg/L urine (mean: 5.76 µg/L ± 0.59) and 2.63 - 67.9 μg/L urine (mean: 27.8 µg/L ± 28.1) for Cr for the male and female animals of the control group, respectively.

For the dosed group, the concentrations were 0.885 - 1.42 μg/L urine (mean: 1.07 µg/L ± 0.23) and 0.545 - 2.57 μg/L urine (mean: 1.30 µg/L ± 0.81) for Sn and 6.63 - 31.6μg/L urine (mean: 20.1 ± 11.9) and 5.69 - 85.8 μg/L urine (mean: 35.4 µg/L ± 38.9) for Cr for the male and female animals, respectively.

Following a subtraction of the background urinary element excretion (control group), and taking into account the excreted urine volume (mean 13 mL (m) and 19 mL (f)) and the body weight of the animals at the end of the study (mean 429.92 g (m) and 267.0 g (f)), the following conclusion can be made:

From a final dose of 1,000 mg/kg pigment that the animals received on the last day of the study, the uptake of Sn and Cr during 24 hour urine sampling period was demonstrated to be negligible considering that <<0.05 % of the dose was excreted via urine for both metals.

Summary of comparative Mass-Balance Study (Leuschner, 2021)

In a comparative mass balance study involving oral dosing of (i) the inorganic pigment "Chromium tin calcium silicon sphene" and (ii) soluble salts of the elements contained therein (Cr3⁺and Sn4+), the gastrointestinal absorption as well as urinary and faecal excretion were compared. For details, please refer to the corresponding robust study summary.

In brief, 10 (5m/5f) animals per group received a single oral dose of 1000 mg/kg of the pigment or 15.5 mg/kg bw Cr3(OH)2(CH3COO)7, and 80.5 mg/kg bw Na2SnO3 * 3H2O/kg bw(corresponding to 4 mg/kg Cr, and 35.8 mg/kg Sn). A third group served as vehicle treated control. Animals were individually housed in metabolic cages and daily samples of urine and faeces were collected for three days. All samples were analysed for chromium and tin. The averaged “background” excretion via urine and faeces of the control animals was subtracted from the amounts excreted by the dosed animals, and a mass balance was calculated.

Animals that received a mixture of 4 mg Cr/Kg bw (administered as Cr3(OH)2(CH3COO)7) and 35.8 mg Sn/Kg bw (administered as Na2SnO3 * 3H2O) excreted 86 % (Cr), and 95.6% (Sn) of the administered dose (as mean, male and female animals) via urine and faeces during the first three days after exposure. The largest fraction (72.5 % for Cr, and 79.39 % for Sn) was excreted via faeces and urine (0.13 % for Cr and 0.01 % for Sn) already within the first 24h.

Animals that received 1000 mg pigment/kg bw excreted 91.6 % Cr and 79.4 % Sn of the administered dose via urine and faeces during the first three days after exposure. The largest fraction (approx. 84 % for Cr, and 71.6 % for Sn) was excreted via faeces and urine already within the first 24h. Further 7.43 % and 0.13 % (Cr), and 7.63 % and 0.17 % (Sn) were excreted via faeces on the second and third day. Urinary excretion for all two elements was negligible and below 0.001 % for Cr, and 0.002 % for Sn.

Urinary excretion for all elements was negligible and below 0.001 % for Cr, and 0.002 % for Sn. In total, the calculation of the mass balance show that the elements Cr3+ and Sn4+ of chromium tin calcium silicon sphene are not absorbed in the gastrointestinal tract to any significant extent but pass the animal effectively unchanged.

Summary of relative bioavailability study (Haferkorn, 2021)

A relative bioavailability study involving serum kinetics over a period of 72 hours p. a. involving an i. v. dosing of a soluble Cr and Sn reference substances (chromium(III) acetate hydroxide, sodium stannate trihydrate) compared to single oral doses of the same substances and the pigment was performed. For details, please refer to the corresponding robust study summary. In brief, 30 animals (15m/15f) per group received single doses of (1) mixture of 0.78 mg/kg bw chromium(III) acetate hydroxide and 0.81 mg/kg bw sodium stannate trihydrate intravenously, (2) a mixture of 15.5 mg/kg chromium(III) acetate hydroxide, and 80.5 mg/kg bw sodium stannate trihydrate via oral gavage, and (3) 1000 mg/kg of the pigment via oral gavage.

Blood samples were taken at 0, 1, 2, 4, 8, 12, 24, 48 and 72 hours post exposure and blood plasma samples were prepared and analysed for the elements Cr and Sn.

The highest Cmax value for chromium in the plasma samples was noted following the intravenous administration of the mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate, followed by the oral administration of the mixture and the oral administration of the pigment. The plasma concentrations of chromium declined post dosing with an elimination half-life (t½) ranging approximately from 20.1 to 43.5 hours across sexes and treatments. The AUC0-t last values ranged from 0.1469 to 1.6272 h μg/g across sexes and treatments. The animals intravenously treated with the mixture revealed the highest exposure towards chromium, whereas the lowest exposure was observed following treatment with the pigment.

The highest Cmax value for tin in the plasma samples was noted following the oral administration of the pigment, followed by the oral administration of the mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate, and the intravenous administration of the mixture. The elimination half-life (t½) could be calculated only for the pigment-treatment of the male animals and the intravenous treatment with the mixture of both sexes, and ranged from 4.3 to 23.5 hours. The AUC0-t last values ranged from 0.0872 to 0.3522 h μg/g across sexes and treatments. The male animals revealed the highest exposure towards tin following intravenous administration of the mixture, the lowest exposure was noted following oral treatment with the mixture. For the female animals, the highest exposure was observed following oral administration of the pigment, whereas the oral treatment with the mixture resulted in the lowest exposure towards tin.

For chromium, an absolute bioavailability (based on AUC0-t last/dose) of 0.63/2.27% (males/females) was calculated from soluble chromium(III) acetate hydroxide following the oral administration of the mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate compared to the intravenous administration, and a relative bioavailability of approximately 0.43/1.17% (males/females) for the pigment.

The relative bioavailability (based on AUC0-t last/dose) of tin was calculated to be approx. 0.05% and 0.25% for male and female animals, respectively, following treatment with the pigment. For the oral treatment with the mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate, an absolute bioavailability (based on AUC0-t last/dose) of 0.56% was calculated for the males and of 0.63% for the females.

Overall conclusion:

The in-vitro and in-vivo experiments described above are in very good agreement with regards to the negligible level of bioavailability of the elements Cr and Sn contained in the pigment.

1) In in-vitro dissolution experiments in five different artificial physiological media, dissolved Cr and Sn concentrations were below 5.22 µg/L and 161 µg/L, respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.005 % and 0.161 %, respectively.

2) In a 28-day oral toxicity study with 1,000 mg/kg pigment the uptake of Sn and Cr during 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.05 % of the dose was excreted via urine for both metals, mirrored by either minimal or no increase in blood plasma concentrations.

3) In a mass balance study with a single oral dose of 1,000 mg/kg of the pigment, 91.6 % Cr, and 79.4 % Sn of the dose were excreted via urine and faeces during the first three days after exposure, with only <0.002 % of the dose being excreted via urine.

4)  In a relative bioavailability study, a relative bioavailability of approx. 0.43/1.17 % (males/females) and 0.05/0.25 % (males/females) was calculated for Cr and Sn, respectively, following treatment with the pigment.

In conclusion, the oral relative bioavailability of the pigment "Chromium tin calcium silicon sphene" can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by an in-vitro dissolution experiment in five different artificial physiological media.

A rounded value of <<0.05 % for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg pigment/kg bw/d (<<0.05 % for both metals) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.001 % for Cr and 0.002 % for Sn).

 

Absorption rate - oral: 0.05 %