Chromium tin calcium silicon sphene

Basic toxicokinetics

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Objective of study:

mass balance

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Chromium tin calcium silicon sphene

EC Number:

701-417-7

Cas Number:

68187-12-2

Molecular formula:

Ca(x)Cr(y)Sn(1-y)SiO5 0,7≤x≤1,0 0

IUPAC Name:

Chromium tin calcium silicon sphene

Test material form:

solid: particulate/powder

Details on test material:

- OLD EC Name: Chrome tin pink sphene
- C.I. name: PIGMENT RED 233
- NEW EC Name: Chromium tin calcium silicon sphene
- Substance type: inorganic pigment
- Physical state: solid, odourless powder with a pink colour
- Stability under test conditions: Stable under normal conditions.
- Storage condition of test material: Keep the container tightly closed. Keep container in an adequately ventilated storage.

Specific details on test material used for the study:

not applicable

Radiolabelling:

no

Test animals

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

Administration / exposure

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

Doses / concentrations

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).

Results and discussion

Preliminary studies:

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

Toxicokinetic / pharmacokinetic studies

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".

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

not measured

Enzymatic activity

Enzymatic activity measured:

not measured

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

not measured

Any other information on results incl. tables

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".

 

Applicant's summary and conclusion

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.