Registration Dossier

Administrative data

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
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
29 June 2016 to 14 October 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In view of the nature of this study its design to address toxicookinetics, in particular absorption, 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)
Limit test:
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 this laboratory.
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 ºC.
- 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 oral 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Homogeneity:
The homogeneity of formulations during storage have been determined as part of another study, Envigo Study Number: PR23XK.
Formulations at concentrations of 10 and 200 mg/mL of test material in corn oil were shown to be homogenous following re-suspension after storage for 15 days when stored refrigerated and for one day at ambient temperature conditions.
In view of the nature of the test material (by-product of the production of manganese alloys), assessment of stability within the vehicle was not possible/relevant for this study.

Achieved concentration:
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. The samples required from the Day 43 formulations were taken from the residual doses returned to Pharmacy from the animal facility on completion of dosing.
 
Analytical Procedure:
The samples were analysed in accordance with the validated Envigo Analytical Procedure DFA/M049/16. The analytical method involved removing the corn oil using acetone, and drying the residue. Sample concentrations were determined by weighing the final residue remaining in the sample.
 
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 (4 000 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 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 the test material. The prepared procedural recoveries were analysed in accordance with the analytical procedure.
 
Unforeseen Events:
The week 1 samples were 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. However, these were not reported due to the poor procedural recoveries results.
 
Deviation from SOP:
SOP PFI-MET-006 states that for 3 inclusion levels, only 1 out of 3 procedural recovery samples may be excluded as an outlier. For the Week 1 analysis, the procedural recovery samples prepared at both 10 mg/mL and 20 mg/mL were excluded and only the 200 mg/mL procedural recovery sample was acceptable.
The only results reported from this sample set were for the Group 4 samples (200 mg/mL) and Group 1 (0 mg/mL) samples. The other two groups were reported from a subsequent analysis.
It is considered that since the samples were not corrected for procedural recovery the Group 4 samples could be reported from this sample set. The analysis and recovery for this concentration level performed consistently well during the study.
 
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.
Duration of treatment / exposure:
Seven weeks
Frequency of treatment:
Once daily at approximately the same time each day.
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:
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 1 000 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 1 000 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.
Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
NECROPSY
- Following completion of sampling the animals were humanely killed and discarded without necropsy.
Other examinations:
Toxicokinetics - Proof of Absorption
Following completion of sampling the animals were humanely killed and discarded without necropsy.
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 2300g 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.
Statistics:
See below.
Clinical signs:
no effects observed
Description (incidence and severity):
The appearance and the behaviour of the animals were not affected by treatment, there were no signs after dose administration.
Mortality:
no mortality observed
Description (incidence):
There were no deaths.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
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 and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Toxicokinetics: 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.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
no

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).

Conclusions:
Under the conditions of the study there were no treatment-related clinical signs, no unscheduled deaths and no effect on bodyweight and food consumption up to 1 000 mg/kg bw/day.
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 was no evidence of any significant absorption of the test material. Furthermore, there were no treatment-related clinical signs, no unscheduled deaths and no effect on bodyweight and food consumption up to 1 000 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Objective of study:
absorption
Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
Grey/green
Radiolabelling:
no

Test animals

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.

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Results and discussion

Main ADME results
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.

Toxicokinetic / pharmacokinetic studies

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.

Metabolite characterisation studies

Metabolites identified:
not measured

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.