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Administrative data

Link to relevant study record(s)

Reference
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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
Reason / purpose for cross-reference:
reference to same study
Objective of study:
absorption
Qualifier:
no guideline available
Principles of method if other than guideline:
In view of the nature of this study its design did not meet any specific regulatory guideline.
The purpose of this study was to assess the toxicokinetics (proof of absorption) of the test material in a seven week oral study in Sprague-Dawley rats. Three groups, each comprising four males and four females, received the test material at doses of 10, 100 or 1 000 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 (proof of absorption), clinical condition, body weight and food consumption investigations were undertaken.
GLP compliance:
yes (incl. QA statement)
Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
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 Crl:CD(SD) strain was used because of the historical control data available at the Test Facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 37 to 43 days.
- Weight at study initiation: Males: 166 to 208 g; females: 152 to 189 g.
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations. Number of animals per cage: Four of the same sex.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Eight days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20 - 24 °C.
- Humidity (%): Monitored and maintained within the range of 40 - 70 %.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.
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.
- Frequency of preparation: Weekly.
- Storage of formulation: Refrigerated (nominally 2 - 8 °C).
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure. A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
- Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
- Treated at: Constant doses in mg/kg.
- Volume dose:
Group 1, 3 and 4: 5 mL/kg body weight.
Group 2: 1 mL/kg body weight.
- Individual dose volume: Calculated from the most recently recorded scheduled body weight.

Duration and frequency of treatment / exposure:
Once daily at approximately the same time each day for seven weeks.
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:
Three males and three females in each group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study (0, 10, 100 and 1 000 mg/kg/day) were selected in conjunction with the Sponsor.
In a preliminary embryo-foetal study (Envigo Study Number: RV86PS) 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 a study performed on silico-manganese slag (considered to be an analogue substance), the same doses (0, 10, 100 and 1000 mg/kg/day) were selected.

- 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.
- Animal Replacement: On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ± 20 % of the mean for the appropriate sex. No individuals were rejected on this criterion.
- Three males and three females in each group were sampled for toxicokinetic evaluation. The remaining male and female were allocated to each group in the event of any premature deaths.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY: Absorption
- Tissues and body fluids sampled: Blood, plasma
- Time and frequency of sampling: 1 h, 4 h and 24 h after dosing.
- Blood sample site: Tail vein.
- Anaesthetic: None.
- Anticoagulant: Lithium heparin.
- Blood volume: 0.5 mL
- Treatment of samples: Mixed on an automatic mixer until centrifugation within 30 minutes of blood withdrawal.
- Centrifugation conditions: At 2300 g for 10 minutes at approximately 4 °C.
- Number of aliquots per sample: One.
- Plasma tubes: Clear polypropylene tubes.
- Plasma volume per aliquot: All available plasma, at least 0.2 mL.
- Temporary storage conditions: Plasma was placed on dry ice following collection.
- Final storage conditions: Protected from light and deep frozen (approximately -20 °C).
- Fate of plasma samples: Dispatched to the Principal Investigator on dry ice.

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.
- During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

SIGNS ASSOCIATED WITH DOSING
- Daily during the first week of treatment and twice weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Pre-dose
One to two hours after dosing
As late as possible in the working day (Week 1 only)

CLINICAL SIGNS
- 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. These data are retained in the study data but are not reported.

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.

NECROPSY
- Following completion of sampling the animals were humanely killed and discarded without necropsy.
Statistics:
See below.
Type:
absorption
Results:
With the exception of one intermediate dose male there were no quantifiable levels of the elements Mn, Si, Al and Ba (i.e. the values were < 0.5 mg/L for Mn, < 2.5 mg/L for Ba, < 5 mg/L for Al and < 1 000 mg/L for Si) in any of the samples analysed.
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 < 1 000 mg/L for silicon) in any of the samples analysed.
Metabolites identified:
not measured

Formulation Analysis

The mean achieved concentrations of the test material in test formulations analysed during in Week 1 and 7 were in the range -8.9 to +5.5 % of the nominal concentrations. For Week 1 the results were from residual formulation samples since the original results had to be discounted due to tare weights of the centrifuge tubes not being available. For Week 7 the first sample analysed for Group 3 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 and was therefore excluded from all calculations.

Overall, these 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).

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 1 000 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 1 000 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 similarity of change between the sexes, these variations were considered to result from 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 1 000 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 1 000 mg/kg/day but, in the absence of a similar finding at 100 mg/kg/day, was considered to represent normal biological variation.

Conclusions:
Under the conditions of the study 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 quantification.
Executive summary:

The toxicokinetics of the test material, specifically absorption, was assessed. In view of the nature of this study its design did not meet any specific regulatory guideline but was conducted in compliance with GLP.

The toxicokinetics (proof of absorption) of the test material was assessed in a seven week oral study in Sprague-Dawley rats. Three groups, each comprising four males and four females, received the test material at doses of 10, 100 or 1 000 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 (proof of absorption), clinical condition, body weight and food consumption investigations were undertaken.

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

Under the conditions of the study 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 quantification.

Description of key information

Cooper (2019)

Under the conditions of the study 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 quantification.

Key value for chemical safety assessment

Additional information

TEST MATERIAL: Slags, Ferromanganese manufacturing (EC Number 273-728-1; CAS Number 69012-28-8; common name: FeMn Slag

 

Physical and chemical description

The test material is an industrial by-product from the manufacture of ferromanganese alloy.

It is a solid grey/green material composed of a mixture of mainly metallic oxides present at varying concentrations. The test material exists in a solid physical state of varying sizes ranging from dust; 10 - < 1 cm (Butler and O’Connor 2009) to rocks (5.5 x 3 x 3 cm) (Anderson 2009). The major and minor crystalline phases of the test material consist of a variety of calcium silicates (LSM 2010). The X-ray diffraction (XRD) phase detection analysis identified several crystalline components including; diopside, aluminian, wollastonite, and gehlenite. Chemical analysis of a typical FeMn slag has shown that it consisted of a mixture of the following oxides; silicon (16-44%), manganese (10-44%), calcium (11-45%), aluminium (0-24%), magnesium (1-12%), barium (< 5%), potassium (< 3%), iron (< 2%) and titanium (<2%) (LSM 2010).

 

Absorption 

The test material, FeMn slag, is of low solubility in water (4.3 x 10^-4g/L) (Butler & O’Connor 2009). For the FeMn slag test sample, in order to produce a worst-case scenario potential inhalation risk, the larger particles were removed and only the smaller particles and dust particles were sampled for particle size determination. From the sub-sample, the percentage of test material with a particle size of < 100 µm was determined to be 3.3% by sieve analysis. This indicates that the test material was of a particle size that was essentially non-inhalable.This indicates an extremely low potential for absorption via the respiratory route. Once any of the test material is absorbed, the potential for FeMn slag to become bioavailable is unlikely and the possibility of breakdown into its various components is minimal. This is confirmed in the very low percentage (0.024%) of manganese leaching out of the substance when exposed to artificial lung fluid in a bioaccessability study (Anderson 2009).

 

FeMn slag is not readily available for absorption via the gut due to its low solubility and due to the homeostatic control of manganese in the body. Although the level of Mn release was determined to be 24% following exposure to artificial gastric fluid in a bioaccessibility test (Anderson 2009), the uptake of manganese via the gastric route is very low in humans (<5%). It is not considered that the potential uptake via this route of any released manganese will make a substantial contribution over and above the normal daily nutritional requirement IOM (2002) that is homeostatically-controlled by the liver (IEH, 2004). The level of release determined in the bioaccessibility study may be artificially, and unrealistically high since the test material was ground to a fine powder prior to exposure; this is likely to have altered the physico-chemical nature of the substance, making it more susceptible to dissolution.

In a seven week oral gavage study in Sprague-Dawley rats conducted to assess the absorption, three groups, each comprising four males and four females, received FeMn Slag at doses of 10, 100 or 1000 mg/kg/day (Cooper, 2019). 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 quantification.This is supported by a lack of systemic effects in in vivo studies in rats with their NOAEL being the limit dose of 1000 mg/kg bw/day for the registered substance and its analogue substance (Cooper, 2016; 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 the registered substance. In the rabbit study on the registered substance (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.

As the test material is of very low solubility in water, coupled with its physical inorganic nature, means that it is very unlikely to be absorbed through the skin. As such, the test material has an exceedingly low potential for any absorption by inhalation or dermal absorption. Any potential absorption via the oral route is likely to be extremely low, particularly, as noted above, that the uptake of manganese from the gut in humans is less than 5% (IEH, 2004). Consideration has been given to any inhaled material reaching the gut via the mucociliary escalator but, as described above, the test material is essentially non-inhalable, thus this potential contribution is thought to be negligible.

 

Metabolism, Distribution and Excretion

Since the test material has a low potential for absorption by the inhalation and dermal route, it means that the test material will not be readily bioavailable by these two principle routes of potential human exposure. Most test material that is ingested orally is likely to pass through the gastrointestinal tract unchanged and be excreted in the faeces. Some small amount of manganese from the test material that is absorbed by the gut will enter the essential manganese pool along with that is absorbed from the daily nutritional requirement of manganese and the circulating amount will be controlled by the normal homeostatic mechanism provided by the liver that controls the manganese balance. As noted above, although the test material is essentially non-inhalable, is likely that any inhaled (non-respiratory) particles will be cleared from the lungs by the mucociliary escalator into the gastrointestinal tract.

 

Further information

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

 

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 O'Connor, J. B. (2009). FeMn slag (Ferroatlantica): Determination of water solubility and particle size determination, 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.

IEH (2004) Occupational Exposure Limits: Criteria Document for Manganese and Inorganic Manganese Compounds (Web Report W17), Leicester, UK, MRC Institute for Environment and Health

IOM (2002) Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc, Washington DC, USA, National Academy Press, available [December 2002] athttp://books. nap.edu/books/0309072794/html/394.html

LSM (2010) Certificate of Analysis for FeMn Slag

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.