Slags, silicomanganese-manufg.

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-02 to 2016-04-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

The systemic toxic potential of the test material when administered for 90 days was investigated in a study conducted in accordance with the standardised guideline OECD 408 under GLP conditions.
Additional blood samples were taken in weeks 7 and 13 to investigate the toxicokinetic of the test material and determine if any absorption took place.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague-Dawley strain was used because of the historical control data available at the testing laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD(SD)
- Age at study initiation: 41 to 47 days
- Weight at study initiation: Males: 209 to 259 g; females: 143 to 198 g
- Fasting period before study: No (except overnight for blood sampling for haematology and blood chemistry).
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Five animals of the same sex were housed per cage, unless reduced by mortality or isolation. Bedding was softwood based bark-free fibre, sterilised by autoclaving. Aspen Chew Blocks (soft white untreated wood blocks) and a plastic shelter were provided as environmental enrichment.
- Diet: Pelleted diet non-restricted.
- Water: Potable water ad libitum from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 12 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: The manganese content was determined to be 92.88 and 91.76 mg/kg in the two batches used (acceptable range 60 to 150 mg/kg).
In the most recently available analysis, the manganese content in the water supply was determined to be <0.187 μg/L.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23 °C
- Humidity: 40 to 70 % (relative)
- Air changes (per hr): The air supply was filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 hours of light:12 hours of darkness.

IN-LIFE DATES
From: 02 September 2015
To: 14 and 15 December 2015

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test material was ground in a mortar using a pestle and mixed with the vehicle to form a smooth paste. The suspension was poured into a measuring cylinder, which was wetted with vehicle and the mortar was thoroughly rinsed. The required volume was made up with vehicle and mixed for a minimum of 5 minutes with a magnetic stirrer and transferred to final containers, via syringe whilst magnetic stirring. The remaining concentrations were formulated in ascending order. Formulations were prepared weekly, prepared in advance of the first day of dosing. Formulations were stored refrigerated (nominally 2 to 8 °C). Individual dose volume was calculated from the most recently recorded scheduled body weight.
Doses were administered once daily at approximately the same time each day.

VEHICLE
- Concentration in vehicle: 0, 10, 20 and 200 mg/mL for the 0, 10, 100 and 1000 mg/kg/day dose groups, respectively.
- Amount of vehicle (if gavage): The dose volumes were 5 (vehicle), 1, 5 and 5 mL/kg for the 0, 10, 100 and 1000 mg/kg/day dose groups, respectively.

Duration and frequency of treatment / exposure:

Once daily for 13 weeks (90 days)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose / concentration:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses were selected in conjunction with the Sponsor and were based on findings from a preliminary toxicity study performed at the testing facility for 7 days at 500 mg/kg and 14 days at 1000 mg/kg, as well as the scientific understanding of the bioavailability potential of the test material and the GHS/CLP categories for specific target organ toxicity.
In the preliminary toxicity study, doses up to 1000 mg/kg/day were administered for 14 days. In this study there were no deaths and no effect of treatment on body weight, food consumption and organ weights or any macroscopic findings. Based on this, the highest dose level for this main study was 1000 mg/kg/day. The low and intermediate doses were 10 and 100 mg/kg/day, to meet the GHS classification requirements.
- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Details on dosing and sampling:

TOXICOKINETICS (PROOF OF ABSORPTION)
- Time schedule for collection of blood: 1, 4 and 24 hours after completion of dosing in weeks 7 and 13. Blood samples (0.5 mL) were obtained via the tail vein using lithium heparin as the anticoagulant. Samples were mixed on an automatic mixer until centrifugation (2300 g for 10 minutes at 4 °C) within 30 minutes of blood withdrawal. Plasma was placed on dry ice following collection and final storage conditions were protected from light and deep frozen (nominally <-20 °C).
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 3 animals per sex per group
- Parameters examined: The determination of manganese, silicon, aluminium and barium in the plasma samples was performed using ICP-MS (Agilent 7500cx; Mass Hunter software, version: B.01.01).

ANALYSIS OF PLASMA SAMPLES
Analysis of the plasma samples for manganese, silicon, aluminium and barium concentrations was carried out using inductively coupled plasma with mass spectrometric (ICP-MS) detection.
The ICP-MS method for the measurement of manganese, aluminium, barium and silicon in plasma samples was successfully validated and successful calibration of the analytical system was performed prior to sample analyses.

- Control Matrices
Samples of independent Sprague-Dawley rat plasma were taken (off-white solid liquid; stored at approximately -20 ˚C in the dark). The plasma was used in the preparation of standards and basic diluent.

- Preparation of Matrix Calibration Standards
Stock solutions of aluminium, barium and manganese ICP analytical standards (1000, 10 000 and 1000 ppm, respectively) were prepared in ultra-gradient water to give final concentrations of 100 mg/L aluminium, 50 mg/L barium and 10 mg/L manganese (termed stock 100). This stock solution was further diluted with ultra-gradient water to give final concentrations of 10 mg/L aluminium, 5 mg/L barium and 1 mg/L manganese (termed stock 10).
The stock solutions, control matrix rat plasma (100 μL), silicon ICP analytical standard (1000 ppm) and basic diluent (5 mL) and an amount of water were mixed so that the final volume was 20 mL. These calibration standards were used to determine the aluminium, barium, silicon and manganese sample concentrations.
The accuracy of the preparation of the test material solution was verified against a dilution of a second stock solution, prepared similarly, over the same concentration range.
The working standard blank was prepared by diluting 250 μL plasma, 12.5 mL basic diluent with 37.25 mL water. The calibration standards and QC sample were prepared and then stored on the instrument for use in the study. Standard 6 was also used as a QC sample and analysed throughout the analytical sequence to confirm continued system suitability.

- Preparation of Test Samples
Test samples and control samples were allowed to thaw at room temperature and then analysed. A volume (50 μL) of each sample was transferred by micropipette to a centrifuge tube (15 mL), basic diluent (2.5 mL) and water (7.45 mL) was then added and the sample mixed. This solution was then analysed.

- Instrumental Parameters
Computerized system: ICP-MS 7500cx -Chemstation/Masshunter version B.01.01
Instrument: Agilent 7500cx series
Acquisition mode: multi-tune
Element and mass: Aluminium 27, silicon 29, manganese 55, barium 137
Peak pattern: full quant. (3)
Repetitions: 5
Interference equation: none
Detector setting: auto
Integration time: 0.3 seconds
Internal standard: scandium (45) for the analysis of aluminium, silicon and manganese; indium (115) for the analysis of barium

- Calculations
A calibration plot was prepared using a linear plot of the form y = mx + a
The concentration “x” of element in an injected sample was calculated by the following equation:
x = [(y - a) / m].F
Where:
x = concentration of element in the injected sample [mg/L element]
y = mean response of element in the injected sample [counts]
m = slope of the calibration plot [counts per mg/L element]
a = y-axis intercept [counts]
F = Sample preparation factor

Element refers to each of the analysed elements aluminium, barium, silicon and manganese. A calibration curve was produced for each element and the samples quantified accordingly.

- Validation of the Analytical Method
Full validation was carried out and found to be acceptable.
The LOQ of the method was found to be 0.0025 mg/L manganese, 0.0125 mg/L barium, 0.025 mg/L aluminium and 5 mg/L silicon in rat plasma.

- Test Linearity
The aluminium data was found to have a linear correlation within the calibration range of 0 to 2.5 mg/L. The R² fit of the calibration curve to the data was 0.9999, and was considered to be acceptable.
The silicon data was found to have a linear correlation within the calibration range of 0 to 50 mg/L. The R² fit of the calibration curve to the data was 0.9997, and was considered to be acceptable.
The manganese data was found to have a linear correlation within the calibration range of 0 to 0.25 mg/L. The R² fit of the calibration curve to the data was 0.9999, and was considered to be acceptable.
The barium data was found to have a linear correlation within the calibration range of 0 to 1.25 mg/L. The R² fit of the calibration curve to the data was 0.9998, and was considered to be acceptable.

FORMULATION ANALYSIS
Chromatographic analytical techniques were not suitable for this study due to the nature of the test material. It was considered appropriate to perform gravimetric analysis of the test material as a method of determining the achieved content, in terms of percentage dry matter content only, of the test material as supplied.
Samples of each formulation prepared for administration in Weeks 1, 12 and 13 (residual doses) of treatment were analysed for achieved concentration of the test material.

- Preparation of Test Samples
A representative sample (1 mL, accurately weighed) of test formulation was mixed with acetone (8 mL) to dissolve the corn oil. The sample was then centrifuged (4000 rpm, 10 minutes) to separate the test material sediment from the corn oil/acetone supernatant. The supernatant was then decanted off and the process of centrifuging and decanting repeated a second and third time with acetone (8 mL); finally acetone (8 mL) was mixed with the test material sediment and the centrifuge tube vortex mixed to ensure all corn oil had been removed, the process of centrifuging and decanting was repeated again. The samples were then dried off by nitrogen blow-down in a dry block (30 °C) for 15 minutes and weighed for information only. The samples were re-weighed to determine the mass of the sample after leaving overnight at ambient temperature

- Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (corn oil) with known amounts of test material. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

Calculations
The concentration was determined using the following equation:
Analysed concentration (mg/mL) = ((W2 - W1) / W3) × 1000 × D
Procedural recovery analysed concentration, mg/mL = ((W2 - W1) / V) × 100

Procedural recovery values were determined using the following equation:
Procedural recovery = (Analysed concentration (mg/mL) / Fortified concentration (mg/mL)) × 100

Where:
W1 = Original mass of centrifuge tube (mg)
W2 = Final mass of centrifuge tube and solid residue (mg)
W3 = Weight of sample taken (mg)
V = Total amount of recovery sample (1.0 g)
D = Density of sample (g/mL)

- Validation of The Analytical Procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the analysis;
The method accuracy and precision, by determining six procedural recoveries at nominal concentrations of 10, 20 and 200 mg/mL during the method validation.

The analytical procedure was successfully validated for the test material in corn oil with respect to the specificity of analysis, method accuracy and precision.
The specificity assessed the analytical process with vehicle only and was acceptable as there was no significant change in the weight of the glass tubes during sample analysis.
Method accuracy and precision were confirmed; a mean procedural recovery value of 100.8 % (CV = 0.80 %, n = 6) was obtained for 10 mg/mL; 101.9 % (CV = 1.80 %, n = 6) was obtained for 20 mg/mL; and 100.6 % (CV = 0.05 %, n = 6) was obtained for 200 mg/mL.


- Homogeneity in Corn Oil Formulations
The homogeneity in corn oil formulations was assessed at nominal concentrations of 10, 20 and 200 mg/mL during ambient temperature and refrigerated storage. Freshly prepared specimen formulations (400 mL) were equally sub-divided (4 × 100 mL) into four amber glass screw top bottles and submitted for analysis.

- Ambient Temperature Storage (nominally +21 °C)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for 20 minutes (representing 0 hour), 1 hour and 2 hours, single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the continuously stirred formulation.
The remainder of the bottle was stored at ambient temperature and after 1 day and 8 days of storage the contents were remixed and sampled as detailed above.

- Refrigerated Storage (nominally +4 °C)
The remaining bottles were refrigerated on receipt and on Day 1, Day 8 and Day 15 the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for 20 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.

- Results
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 8 days and refrigeration for up to 8 days. At each time-point, the mean analysed concentration for the three samples remained within 7 % of the initial time zero value and the coefficient of variation was less than 5 %.
With the exception of two 10 mg/mL procedural recoveries prepared during Day 15, recovery results during the trial remained within ±7.5 % of the mean recovery found during validation showing the continued accuracy of the method. The Day 15 10 mg/mL procedural recoveries are considered to both be out of validated limits due to analytical issues, the recovery of 112.7 % is considered to be due to the vehicle not being washed away fully, and the recovery of 38.4 % is considered to be a result of washing the test material away during the sample process. The homogeneity of the three formulation samples analysed on this occasion is acceptable, confirming that these samples were analysed effectively.

- Concentration of Dose Formulations
For Week 1 and 12 of treatment, freshly prepared test formulations were sampled (2 × 10 mL, accurately weighed) and submitted for analysis. Duplicate samples were taken from the first sample and analysed in accordance with the analytical procedure, the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved.
In Week 13, residual formulations were submitted for analysis. The bottles were mixed by 20-fold inversion followed by magnetic stirring for 20 minutes and duplicate samples (nominally 1 mL) were removed for analysis from the middle of the formulation. These duplicate samples were analysed in accordance with the analytical procedure.
The mean concentrations were within applied limits ± 10 % for Week 1, confirming the accuracy of formulation.
Results of the Week 12 formulations for both Groups 3 and 4 did not meet the defined acceptance criteria. The achieved concentration of the Group 3 formulated dose was -21.5 % from the nominal concentration. When the Group 4 dose formulation was analysed there was wide precision which was outside the acceptance limit of <5 %.
These results were investigated and it was agreed that the contingency samples for these two groups should be analysed; however, the results from the analyses of the Week 12 contingency sample results did not confirm the original results nor meet the defined acceptance criteria.
As the Week 12 results did not meet acceptance criteria, the Week 13 residue formulations were analysed. The results from these residual formulations were inconclusive. Procedural recoveries prepared concurrently with the Week 12 contingency samples and the Week 13 residue analysis were unacceptable, meaning that neither of these occasions can be reported.
The results obtained from the Group 2 and 4 formulations in Week 12 were within acceptance criteria for mean achieved concentration, and the analysed concentration for Group 3 confirms that the Group 3 dose formulation contained more test material than Group 2 and less than Group 4. The reason for these poor results is considered to be the analytical procedure rather than issues that occurred during the preparation of the formulations. The Week 12 contingency and the Week 13 residue results were not reported due to this and a record of the results kept in the raw data.

- Conclusion
The analytical procedure was successfully validated with respect to specificity of analysis and method accuracy and precision.
The homogeneity and stability was confirmed for the test material in corn oil formulations at nominal concentrations of 10, 20 and 200 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 8 days and refrigerated storage for up to 15 days.
The mean concentrations in test formulations analysed for the study were within ± 10 % of nominal concentrations, confirming accurate formulation, with the exception of Week 12 Group 3.

Statistics:

All statistical analyses were carried out separately for males and females. The following sequence of statistical tests was used for clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1 % level. The F1 approximate test was applied. If the F1 approximate test for monotonicity of dose-response was not significant at the 1 % level, Williams' test for a monotonic trend was applied; if it was significant, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1 % level following both logarithmic and square-root transformations. The H1 approximate test, the non-parametric equivalent of the F1 test described above, was applied. If the H1 approximate test for monotonicity of dose-response was not significant at the 1 % level, Shirley's test for a monotonic trend was applied; if it was significant, Steel's test was performed instead.

For clinical pathology data, if 75 % of the data (across all groups) were the same value, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

Significant differences between Control and treated groups were expressed at the 5 % (p<0.05) or 1 % (p<0.01) level.

Details on absorption:

PROOF OF ABSORPTION
Plasma manganese, aluminium, barium and silicon concentrations were found to be less than the limit of quantification (LOQ) in all groups, indicating that no quantifiable absorption had occurred. The LOQ of the method was found to be 0.0025 mg/L manganese, 0.0125 mg/L barium, 0.025 mg/L aluminium and 5 mg/L silicon in rat plasma, from the validation study.

Metabolites identified:

no

Conclusions:

Under the conditions of this study, there was no evidence of any absorption of the test material.

Executive summary:

The systemic toxic potential of the test material when administered for 90 days was investigated in a study conducted in accordance with the standardised guideline OECD 408 under GLP conditions.

The ground, granular test material was administered by gavage to Sprague-Dawley (Crl:CD (SD) rats over a period of 13 weeks. Groups of 10 male and 10 female rats received the test material suspended in corn oil at doses of 10, 100 and 1000 mg/kg/day, with a similarly constituted control group receiving the vehicle (corn oil) only.

Blood samples were taken in weeks 7 and 13 to investigate the toxicokinetics of the test material and determine if any absorption took place. Animals were sacrificed at the end of the study by carbon dioxide asphyxiation followed by exsanguination and submitted for necropsy.

Administration of the test material was well tolerated. The plasma samples taken in Week 7 for proof of absorption analysis were analysed (using inductively coupled plasma with ICP-MS detection) and manganese, aluminium, barium and silicon concentrations were found to less than the limit of quantification (0.0025, 0.025, 0.0125 and 5 mg/L, respectively) in all groups indicating that no quantifiable absorption had occurred.

It was concluded that oral (gavage) administration of the granulated test material to Sprague-Dawley rats at doses up to 1000 mg/kg/day for 13 weeks did not cause any evidence of systemic toxicity. The NOAEL for systemic toxicity in this study was considered to be 1000 mg/kg/day.

Under the conditions of this study, there was no evidence of any absorption of the test material.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Initiated 29 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

This study is being used as read across to address the data requirement on the similar substance being registered. Please see attached RAAF document.

In this study the absorption behaviour of ferromanganese slag (FeMn slag), a substance considered to be analogous to silico-manganese slag (SiMn slag), was investigated at oral doses of 10, 100 or 1000 mg/kg/day for 43 days. The study was designed to mimic the conditions of a 13-week study using SiMn slag to assess the proof of absorption in Week 7 (Day 43).

Reason / purpose for cross-reference:

read-across source

Objective of study:

absorption

Qualifier:

no guideline followed

Principles of method if other than guideline:

In view of the nature of this study, its design did not meet any specific regulatory guideline.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD(SD)
- Age at study initiation: 37 to 43 days old
- Weight at study initiation: Males: 166 to 208 g; females: 152 to 189 g
- Fasting period before study: No
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Animals were housed in groups of 4 per sex. An aspen soft white untreated wood block was provided to each cage throughout the study and replaced when necessary. A plastic shelter was provided to each cage throughout the study and replaced when necessary.
- Diet (e.g. ad libitum): Non-restricted
- Water (e.g. ad libitum): Potable water from the public supply was provided as libitum via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20 - 24 °C. There were no deviations.
- Humidity (%): Monitored and maintained within the range of 40 - 70 % (relative). There were no deviations.
- Air changes (per hr): Not specified; filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting; 12 hours of light:12 hours of darkness.

IN-LIFE DATES
- From: 29 June 2016
- To: 19 August 2016

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test material was weighed, transferred to a mortar and ground to a fine powder using a pestle. Small amounts of the pre-weighed vehicle were added and mixed to form a smooth paste. The suspension was poured into a measuring cylinder which had been wetted with the vehicle and the mortar was rinsed thoroughly with the vehicle and the rinsed residue was added to the measuring cylinder. The required volume was achieved by addition of the remaining vehicle and the suspension was transferred to a beaker for mixing. The final suspension was transferred to the issue container using a syringe. The formulations were prepared in ascending group order. The formulation was prepared weekly and refrigerated (nominally 2 to 8 °C).

VEHICLE
- Concentration in vehicle: 0, 10, 20 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg for the control, mid and high dose groups; 1 mL/kg for the low dose group

Duration and frequency of treatment / exposure:

Once daily for seven weeks

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Formulated concentration: 10 mg/mL

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Formulated concentration: 20 mg/mL

Dose / conc.:

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

Remarks:

Formulated concentration: 200 mg/mL

No. of animals per sex per dose / concentration:

4 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study were selected in conjunction with the Sponsor. In a preliminary embryo-foetal study, females receiving 100, 500 or 1000 mg/kg/day from gestation Day 6 to 19 had slightly low body weight gains but there was no mortality or signs of toxicity. Based on this information, and the requirement for this study to match the study performed on the substance to be registered, the same doses were selected.
- Rationale for animal assignment: Random

Details on dosing and sampling:

SERIAL OBSERVATIONS
- Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
Daily during the first week of treatment and twice weekly thereafter, detailed observations were recorded pre-dose, one to two hours after dosing and as late as possible in the working day (Week 1 only).
A detailed weekly physical examination was performed on each animal to monitor general health.

- Body Weight
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and on the day the animals were culled.
More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored.

- Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

- Toxicokinetics
Blood samples were obtained on Day 43 in week 7. Samples were taken from 3 animals per group at 1, 4 and 24 hours after dosing.
Following completion of sampling the animals were humanely killed by carbon dioxide asphyxiation with subsequent exsanguination. All animals were discarded without macroscopic examination or any retention of tissues.
Blood samples were taken from the tail vein without anaesthetic. 0.5 mL of blood was drawn into clear polypropylene plasma tubes containing lithium heparin. Samples were mixed on an automatic mixer until centrifugation within 30 minutes of blood withdrawal at 2300 g for 10 minutes at approximately 4 °C. There was one aliquot per sample with all available plasma, at least 0.2 mL. Plasma was placed on dry ice following collection then stored protected from light and deep frozen (approximately -20 °C).

ANALYSIS OF PLASMA SAMPLES
Analysis of the plasma samples for manganese, silicon, aluminium and barium concentrations was carried out using inductively coupled plasma with mass spectrometric (ICP-MS) detection.
The ICP-MS method for the measurement of manganese, aluminium, barium and silicon in plasma samples was successfully validated and successful calibration of the analytical system was performed prior to sample analyses.

- Control Matrices
Samples of independent Sprague-Dawley rat plasma were taken (off-white solid block; frozen and stored at approximately -20 ˚C in the dark). The plasma was used in the preparation of standards and basic diluent.

- Preparation of Matrix Calibration Standards
Stock solutions of aluminium, barium and manganese ICP analytical standards (1000, 10 000 and 1000 ppm, respectively) were prepared in ultra-gradient water to give final concentrations of 100 mg/L aluminium, 50 mg/L barium and 10 mg/L manganese (termed stock 100). This stock solution was further diluted with ultra-gradient water to give final concentrations of 10 mg/L aluminium, 5 mg/L barium and 1 mg/L manganese (termed stock 10).
The stock solutions, control matrix rat plasma (100 μL), silicon ICP analytical standard (1000 ppm) and basic diluent (5 mL) and an amount of water were mixed so that the final volume was 20 mL. These calibration standards were used to determine the aluminium, barium, silicon and manganese sample concentrations.
The accuracy of the preparation of the test material solution was verified against a dilution of a second stock solution, prepared similarly, over the same concentration range.
The working standard blank was prepared by diluting 250 μL plasma, 12.5 mL basic diluent with 37.25 mL water. The calibration standards and QC sample were prepared and then stored on the instrument for use in the study. Standard 6 was also used as a QC sample and analysed throughout the analytical sequence to confirm continued system suitability.

- Preparation of Test Samples
Test samples and control samples were allowed to thaw at room temperature and then analysed. A volume (50 μL) of each sample was transferred by micropipette to a centrifuge tube (15 mL), basic diluent (2.5 mL) and water (7.45 mL) was then added and the sample mixed. This solution was then analysed.

- Instrumental Parameters
Computerized system: ICP-MS 7500cx -Chemstation/Masshunter version B.01.01
Instrument: Agilent 7500cx series
Acquisition mode: multi-tune
Element and mass: Aluminium 27, silicon 29, manganese 55, barium 137
Peak pattern: full quant. (3)
Repetitions: 5
Interference equation: none
Detector setting: auto
Integration time: 0.3 seconds
Internal standard: scandium (45) for the analysis of aluminium, silicon and manganese; indium (115) for the analysis of barium

- Calculations
A calibration plot was prepared using a linear plot of the form y = mx + a
The concentration “x” of element in an injected sample was calculated by the following equation:
x = [(y - a) / m].F
Where:
x = concentration of element in the injected sample [mg/L element]
y = mean response of element in the injected sample [counts]
m = slope of the calibration plot [counts per mg/L element]
a = y-axis intercept [counts]
F = Sample preparation factor

Element refers to each of the analysed elements aluminium, barium, silicon and manganese. A calibration curve was produced for each element and the samples quantified accordingly.

- Validation of the Analytical Method
Full validation was carried out and found to be acceptable.
The LOQ of the method was found to be 0.5 mg/L manganese, 2.5 mg/L barium, 5 mg/L aluminium and 1000 mg/L silicon in rat plasma.

- Test Linearity
The aluminium data was found to have a linear correlation within the calibration range of 0 to 2.5 mg/L. The R² fit of the calibration curve to the data was 0.9997, and was considered to be acceptable.
The silicon data was found to have a linear correlation within the calibration range of 0 to 50 mg/L. The R² fit of the calibration curve to the data was 0.9995, and was considered to be acceptable.
The manganese data was found to have a linear correlation within the calibration range of 0 to 0.25 mg/L. The R² fit of the calibration curve to the data was 0.9998, and was considered to be acceptable.
The barium data was found to have a linear correlation within the calibration range of 0 to 1.25 mg/L. The R² fit of the calibration curve to the data was 0.9998, and was considered to be acceptable.

FORMULATION ANALYSIS
Homogeneity was confirmed at concentrations of 10 and 200 mg/kg/day for 15 days when stored at refrigerated temperature and 1 day when stored at ambient temperature.
Samples of each formulation prepared for administration in weeks 1, 6 and 7 (Day 43) of treatment were analysed for achieved concentration of the test material.

- Preparation of Test Samples
A representative sample (1 mL, accurately weighed) of test formulation was mixed by vortex with acetone (8 mL) to disperse the sample. The sample was centrifuged (4000 rpm, 10 minutes) to separate the test material sediment from the corn oil/acetone supernatant. The supernatant was decanted off and the process of centrifuging and decanting (using gentle mixing rather than vortexing, with the exception of the final wash where vortex mixing was again used) repeated a further three times with acetone (8 mL). The samples were dried by nitrogen blow-down in a dry block (30 °C) for 15 minutes and weighed for information only (with the exception of the first analysis of the Week 1 samples). The samples were reweighed to determine the mass of the sample after leaving overnight at ambient temperature.

- Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (Corn oil) with known amounts of test material. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

- Unforeseen events
The week 1 samples were received and analysed by Dose Formulation Analysis (DFA). On review of the results both Groups 2 and 3 were variable; the procedural recoveries prepared alongside these groups were also very low. The Group 4 results were within specification.
On review of the data it was noted that the tubes into which samples were placed had not had their weights recorded as required in the method of analysis and study plan. This would not adversely affect Group 4 due to the high nominal concentration. However it could affect Groups 2 and 3 due to the lower concentrations. Although this was not the only problem with the analysis (the low recoveries would not be affected by this) the fact that this weight was omitted meant that results from Groups 2 and 3 could not be calculated with confidence, therefore these results were discounted.
Contingency samples were not analysed as the tube weights had also not been recorded for these.
The residues were sent for analysis. These were received and sampled and analysed in duplicate by DFA (Groups 2 and 3 only). The Group 3 results were acceptable. The Group 2 results showed one result within acceptable limits and one high result. The high result was investigated and confirmed but was found at a later date to be due to a calculation error, meaning the Group 2 result was also acceptable.
The Group 2 residue was resampled in triplicate to give more confidence in the results obtained. The three results were all within specification giving added confidence that the dose was prepared correctly.

- Results and Conclusion
The mean concentrations were within 16 % of the nominal concentration (for non-discounted results), confirming the accuracy of formulation.
For Week 7, Group 3 the first sample analysed was lower than the remaining samples. A Dixon’s Q Test was performed and this value was found to be an outlier with 90 % confidence. Therefore this value was excluded from all calculations.
The mean achieved concentrations in test formulations analysed during in Week 1 and 7 were in the range -8.9 to +5.5 % of the nominal concentrations.
Overall, the results were within ± 10 % of the target concentrations and demonstrated acceptable formulation.
The residual samples for Day 43 (i.e. the day of toxicokinetic sampling) were also analysed and whilst the results for Group 2 and 4 were within ± 10 % of the target concentrations, the results for Group 3 were slightly high (+ 16 % of the nominal concentration).

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analysed at each time point separately: Body weight, using gains over appropriate study periods.
The following comparisons were performed: Group 1 vs Groups 2, 3 and 4

Significant differences between the groups compared were expressed at the 5 % (p<0.05) or 1% (p<0.01) level.

Details on absorption:

With the exception of one intermediate dose male which had quantifiable concentrations of aluminium (male No. 10 had an aluminium concentration of 7.64 and 8.90 mg/L at one and 24 hours post-dose but no aluminium was detected at 4 hours post-dose in this animal), there were no quantifiable levels of the elements manganese, silicon, aluminium and barium (i.e. the values were <0.5 mg/L for manganese, <2.5 mg/L for barium, <5 mg/L for aluminium and <1000 mg/L for silicon).

Metabolites identified:

no

Clinical Observations

The appearance and the behaviour of the animals were not affected by treatment, there were no signs after dose administration and there were no deaths.

 

Body Weight

There was considered to have been no effect of treatment on body weight and bodyweight gain.

When compared to controls the overall bodyweight gain by the end of treatment was low for males receiving the test material at 10 or 100 mg/kg/day (69 % of control for both groups) but was similar to controls for males receiving 1000 mg/kg/day. In contrast, body weight gains by the end of treatment was slightly high for females receiving the test material at 10 or 1000 mg/kg/day (114 and 121 % of control, respectively) but was similar to control for females receiving 100 mg/kg/day. In view of the absence of any dose-response or similarly of change between the sexes, these variations were considered due to normal biological variation.

 

Food Consumption

Food consumption was considered unaffected by treatment.

When compared to controls, the food consumption for males receiving 10 or 100 mg/kg/day was slightly low but was similar to controls for males receiving 1000 mg/kg/day. This reflected a trend that was present before treatment commenced and was therefore not attributable to treatment. Food consumption was slightly high for females receiving 10 or 1000 mg/kg/day but, in the absence of a similar finding at 100 mg/kg/day, was considered to represent normal biological variation.

 

Discussion

Administration of the test material was well tolerated and there were no treatment-related signs or any effect on body weight and food consumption.

There was no evidence of any significant absorption of the test material. One intermediate dose male had quantifiable concentrations of aluminium at one and 24 hours post-dose but, otherwise, the plasma levels of the elements measured (manganese, silicon, aluminium and barium) were all below the limit of detection.

 

Table 1: Body Weight - Group Mean Values (g)

Week		Dose (mg/kg)
		Males				Females
		0	10	100	1000	0	10	100	1000
P1	Mean SD N	137 6.1 4	133 4.8 4	135 5.4 4	137 10.1 4	124 7.1 4	123 8.5 4	126 8.4 4	118 7.5 4
0	Mean SD N	204 5.1 4	184 13.8 4	190 9.2 4	197 9.1 4	160 6.4 4	167 6.8 4	166 17.2 4	160 4.9 4
1	Mean SD N	254 10.3 4	221 20.6 4	228 12.8 4	245 10.3 4	178 9.6 4	188 9.4 4	185 23.9 4	184 7.6 4
2	Mean SD N	301 14.8 4	258 29.4 4	259 14.1 4	286 18.9 4	205 5.2 4	208 10.4 4	208 30.8 4	210 5.9 4
3	Mean SD N	337 22.9 4	292 55.4 4	282 15.5 4	320 30.7 4	220 3.6 4	231 18.1 4	231 33.5 4	230 3.6 4
4	Mean SD N	368 27.2 4	306 44.9 4	303 21.3 4	353 39.8 4	229 4.3 4	245 17.1 4	238 36.6 4	245 10.1 4
5	Mean SD N	398 30.1 4	321 50.4 4	324 25.7 4	380 47.5 4	238 8.5 4	262 20.8 4	250 38.7 4	260 10.2 4
6	Mean SD N	427 35.9 4	339 52.3 4	344 31.4 4	401 48.6 4	250 8.3 4	270 28.9 4	262 35.7 4	269 12.7 4
Change 0 - 6	Mean SD N %*	223 33.2 4 -	155 38.9 4 69	155 25.5 4 69	204 45.2 4 91	91 14.3 4 -	103 25.0 4 114	97 19.5 4 107	110 16.2 4 121

*% of Control

Conclusions:

Under the conditions of this study, there was no evidence of any significant absorption of the test material.

Executive summary:

The purpose of this study was to assess the toxicokinetics of the test material, in a seven week oral study in Sprague-Dawley rats conducted under GLP conditions.

Three groups, each comprising four males and four females, received the test material at doses of 10, 100 or 1000 mg/kg/day. A similarly constituted control group received the vehicle, corn oil, at a volume dose of 5 mL/kg/day.

During the study, toxicokinetics, clinical condition, body weight and food consumption investigations were undertaken.

One intermediate dose male had quantifiable concentrations of aluminium at one and 24 hours post-dose but, otherwise, the plasma levels of the elements measured (manganese, silicon, aluminium and barium) were all below the limit of detection.

There were no treatment-related clinical signs, no unscheduled deaths and no effect on bodyweight and food consumption.

Under the conditions of this study, there was no evidence of any significant absorption of the test material.

Description of key information

The substance is poorly soluble in artificial bodily fluids (gastric juices and alveolar fluid) and is a solid of largely non-inhalable size. The substance is therefore not readily bioavailable. If ingested, the substance is likely to pass through the gastrointestinal (GI) tract unchanged and be excreted in the faeces.If inhaled, the substance is likely to be cleared from the lungs by the mucocilliary elevator into the GI tract and again excreted unchanged in the faeces.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

0

Absorption rate - dermal (%):

0

Absorption rate - inhalation (%):

0

Additional information

TEST MATERIAL: Slags, Silicomanganese-Manufg (EC Number 273-733-9; CAS Number 69012-33-5; common name: SiMn slag)

 

The substance is an industrial by-product from the manufacture of silico-manganese alloy. The major crystalline phase of the substance is silicon-manganese with a minor crystalline phase of calcium oxide and a trace crystalline phase of silicon oxide (LSM 2010). The substance exists in a crystalline physical state of varying size rocks, pebbles and sand (Anderson 2009). The substance is of a crystalline structure and X-ray diffraction (XRD) analysis of SiMn slag identified several crystalline components: akermanite (calcium magnesium silicate) was the main compound with calcium aluminium silicide, potassium magnesium silicate, manganese oxides, larnite, quartz and sulphides (mainly manganese) also identified (Frias, Sanchez et al. 2006). Chemical analysis of a typical SiMn slag has shown that it consisted of a mixture of oxides, primarily of silicon (36-43%), calcium (25-28%), aluminium (10-12%), manganese (10-11%), magnesium (4%), barium (3%) and potassium (1-2%). Smaller proportions (<2%) of oxides of titanium, iron, sodium and sulphur were also present (Frias, Sanchez et al. 2006; LSM 2010). However, since the substance is an industrial by-product, the individual proportions of the chemical components will vary depending on its source, for example, manganese content can be as high as 21% (Frias, Sanchez et al. 2006).

 

Absorption

 

The substance, SiMn slag, has a very low solubility in artificial gastric juice (<1%) and even lower solubility (<0.01%) in artificial alveolar fluid (Anderson, 2009). A detailed analysis using particle-size distribution of the substance indicates that it is not considered an inhalation hazard since the particle size of over 96% of the substance was greater than 100 µm (Butler and O'Connor 2010; O'Connor and Woolley 2010). As the substance is very poorly soluble in physiological media, coupled with its physical inorganic nature (crystalline solid), means that it is very unlikely to be absorbed through the skin. Toxicokinetic work in the 90 day study with the registered substance (Cooper, 2016) showed no detectable material in plasma up to the limit dose of 1000 mg/kg bw/day after 7 weeks of dosing.

Poor oral absorption of the registered substance is also supported by a lack of systemic effects in the prenatal developmental toxicity study in the rat up to the limit dose of 1000 mg/kg bw/day conducted on an analogue substance, FeMn Slag (CAS Number 69012-28-8) (Thacker, 2016).  The available prenatal developmental toxicity studies may indicate a potential inter-species difference in the sensitivity of foetal development following oral administration of FeMn Slag. In the rabbit study conducted on FeMn Slag (Stannard, 2020), concerns over fertility were observed at much lower doses, however, the toxicological pathway remains unclear as there were some vehicle related confounding factors in this study as well as species specific nuances which cast doubt on the relevance of this data to other species.

In conclusion, the substance has an extremely low potential for any absorption by oral ingestion, inhalation or dermal absorption.

 

Metabolism, Distribution and Excretion

 

Since the substance has an exceedingly low potential for absorption by any route it means that the substance will not be readily bioavailable. Any substance that is ingested orally is likely to pass through the gastrointestinal (GI) tract unchanged and be excreted in the faeces. Any substance that is inhaled is likely to be cleared from the lungs by the mucocilliary elevator into the GI tract and again excreted unchanged in the faeces.

 

Further information

A toxicokinetic assessment has been made on manganese and its inorganic compounds, the full report is attached to this endpoint summary (Bounds 2009).

Two studies (Cooper, 2016, 2019)h ave been conducted to investigate the in vivo toxicokinetics of silicomanganese slags and ferromanganese slags with particular focus on the absorption. These studies are summarised below.

The systemic toxic potential of silicomanganese slags when administered for 90 days was investigated in a study conducted in accordance with the standardised guideline OECD 408 under GLP conditions (Cooper, 2016).

The ground, granular test material was administered by gavage to Sprague-Dawley (Crl:CD (SD) rats over a period of 13 weeks. Groups of 10 male and 10 female rats received the test material suspended in corn oil at doses of 10, 100 and 1000 mg/kg/day, with a similarly constituted control group receiving the vehicle (corn oil) only.

Blood samples were taken in weeks 7 and 13 to investigate the toxicokinetics of the test material and determine if any absorption took place. Animals were sacrificed at the end of the study by carbon dioxide asphyxiation followed by exsanguination and submitted for necropsy.

Administration of the test material was well tolerated. The plasma samples taken in Week 7 for proof of absorption analysis were analysed (using inductively coupled plasma with ICP-MS detection) and manganese, aluminium, barium and silicon concentrations were found to less than the limit of quantification (0.0025, 0.025, 0.0125 and 5 mg/L, respectively) in all groups indicating that no quantifiable absorption had occurred.

It was concluded that oral (gavage) administration of the granulated test material to Sprague-Dawley rats at doses up to 1000 mg/kg/day for 13 weeks did not cause any evidence of systemic toxicity. The NOAEL for systemic toxicity in this study was considered to be 1000 mg/kg/day.

Under the conditions of this study, there was no evidence of any absorption of the test material.

An additional study was carried out to assess the toxicokinetics of the similar material ferromanganese slags in a seven week oral study in Sprague-Dawley rats conducted under GLP conditions (Cooper, 2019).

Three groups, each comprising four males and four females, received the test material at doses of 10, 100 or 1000 mg/kg/day. A similarly constituted control group received the vehicle, corn oil, at a volume dose of 5 mL/kg/day.

During the study, toxicokinetics, clinical condition, body weight and food consumption investigations were undertaken.

One intermediate dose male had quantifiable concentrations of aluminium at one and 24 hours post-dose but, otherwise, the plasma levels of the elements measured (manganese, silicon, aluminium and barium) were all below the limit of detection.

There were no treatment-related clinical signs, no unscheduled deaths and no effect on bodyweight and food consumption.

Under the conditions of this study, there was no evidence of any significant absorption of the test material.

References

 

Anderson, K. A. (2009). Bioaccessibility of manganese from manganese Materials in Gastric and Lung (Alveolar) Biofluids, Oregon State University.

Bounds, S.V.J (2009). A toxicokinetic assessment for the Registration, Evaluation and Authorisation of Chemicals, Regulation (EC) No. 1907/2006 (REACH), Manganese and it inorganic compounds, Bounds Consulting Ltd.

Butler, R. E. and J. B. O'Connor (2010). SiMn slag (Erachem): Determination of particle size distribution, Harlan Laboratories Ltd.

Cooper, S. (2016) Silico-Manganese Slag (SiMn slag): toxicity study by oral administration to Sprague-Dawley rats for 13 weeks including proof of absorption analysis. Report No. PIQ0003. Unpublished report.

Cooper, S. (2019). Ferromanganese slag (FeMn slag): Toxicokinetic study by oral gavage administration to Sprague-Dawley rats for 7 weeks. Envigo CRS Ltd., FL12FP, 2019-03-26.

Frias, M., d. R. Sanchez, M.I., et al. (2006). "Recycling of silicomanganese slag as pozzolanic material in Portland cements: Basic and engineering properties."Cement and Concrete Research36: 487-491.

LSM (2010). XRD analysis of SiMn slag.

O'Connor, J. B. and S. M. Woolley (2010). SiMn Slag (Ferroatlantica): Determination of particle size distribution, Harlan Laboratories Ltd.

Stannard, D. (2020) Ferromanganese Slag: study for effects on embryo-foetal development in the rabbit by oral gavage administration. Report No. VK72FT. Unpublished report.

Thacker, K. (2016) Ferromanganese Slag: study for effects on embryo-fetal development in the rat by oral gavage administration. Report No. PR23XK. Unpublished report.