Registration Dossier

Administrative data

Description of key information

Oral (gavage) administration of the read-across substance SiMn slag to Sprague-Dawley rats at doses up to 1000 mg/kg/day for 13 weeks did not cause any evidence of systemic toxicity, including neurotoxicity. The no-observed-adverse-effect level (NOAEL) for systemic toxicity in this study was considered to be 1000 mg/kg/day.

The NOAEL for male rats exposed to MnSO4 was 1700 mg/kg/day. The NOAEL for female rats was 2000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
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:
guideline study
Reason / purpose:
other: Target substance
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
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 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
Details on route of administration:
Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Vehicle:
corn oil
Details on oral 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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.
Duration of treatment / exposure:
13 weeks (90 days)
Frequency of treatment:
Once daily
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:
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.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A viability check was performed near the start and end of each working day. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
- Cage side observations included: Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the treatment period, detailed observations were recorded three times daily in week 1 and twice daily in the remainder of the study period to note any signs associated with dosing.
- Observations included: Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal. After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the treatment period and on the day of necropsy. More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored.
Group mean weight changes were calculated from the weight changes of individual animals surviving the specified period.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: 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 treatment period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No effect was observed and, consequently, quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and week 12.
- Dose groups that were examined: All animals (including spares) were examined pre-treatement and Groups 1 and 4 in week 12. The eyes of the animals were examined by means of a binocular indirect ophthalmoscope and a slit-lamp biomicroscope may also have been used.
Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein and collected into tubes containing EDTA anticoagulant or citrate (for PT and APTT).
- Anaesthetic used for blood collection: Yes; light anaesthesia under isoflurane.
- Animals fasted: Yes; withdrawal of food overnight.
- How many animals: All animals
- Parameters examined: Haematocrit (Hct), haemoglobin concentration (Hb), erythrocyte count (RBC), absolute reticulocyte count (Retic), mean cell haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), mean cell volume (MCV), red cell distribution width (RDW), total leucocyte count (WBC), differential leucocyte count (neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M) and large unstained cells (LUC)), platelet count (Plt), morphology (anisocytosis, microcytosis, macrocytosis, hypochromasia and hyperchromasia), prothrombin time (PT) and activated partial thromboplastin time (APTT).
Blood film was prepared for all samples (Romanowsky stain) and examined for abnormalities by light microscopy, in the case of flags relating to the analyses above. Confirmation or a written description from the blood film was made where appropriate. In the presence of platelet clumping a manual count of the differential white blood cell parameters was performed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant.
- Anaesthetic used for blood collection: Yes; light anaesthesia under isoflurane.
- Animals fasted: Yes; withdrawal of food overnight.
- How many animals: All animals.
- Parameters examined: Alanine amino-transferase (ALT), aspartate amino-transferase (AST), blood urea nitrogen (BUN), creatinine (Creat), glucose (Gluc), total cholesterol (Chol), sodium (Na), potassium (K), total protein (Total Prot), albumin (Alb) and albumin/globulin ratio (A/G Ratio).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Sensory reactivity, grip strength and motor activity assessments were performed (before dosing) on all animals during Week 12 of treatment.
- Dose groups that were examined: All
- Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1: No reaction or ignores probe/walks past probe
2: Normal awareness and reaction e.g. approaches and/or sniffs probe
3: Active avoidance, abnormally fearful or aggressive reaction

- Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1: No response
2: Normal response e.g. ear twitches/flattens or animal shakes its head
3: Abnormally fearful or aggressive response

- Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1: No response
2: Weak response e.g. ear twitch only
3: Normal response e.g. obvious flinch or startle
4: Exaggerated response e.g. all feet off floor

- Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1: No response
2: Weak response e.g. turns around slowly or weak vocalisation without moving away
3: Normal response e.g. jumps forward or turns around sharply, usually with vocalisation
4: Exaggerated response e.g. excessive vocalisation, body movement or aggression

- Grip strength
Forelimb and hind limb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

- Motor activity
The motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson Technical Services and software developed and maintained by the testing facility.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity, respectively.

IMMUNOLOGY: No

OTHER: 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).
Sacrifice and pathology:
SACRIFICE
Animals were killed following 13 weeks of treatment by carbon dioxide asphyxiation with subsequent exsanguination.

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen testes, thymus and uterus were weighed.

HISTOPATHOLOGY: Yes
The following tissues were sampled and fixed for histopathology: Abnormalities, adrenals, aorta, brain (cerebellum, cerebrum, midbrain), caecum, colon, duodenum, epididymides, eyes, femur (femorotibial joint), head, heart (including auricular and ventricular regions), ileum, jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), lymph nodes (mesenteric and left axillary), oesophagus, ovaries, pancreas, pituitary, prostate, salivary glands (submandibular, sublingual and parotid), sciatic nerves, seminal vesicles, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), spleen, sternum (with bone marrow), stomach, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus with cervix and vagina.
Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of the testes (modified Davidson’s fluid) and the eyes (Davidson’s fluid).

- Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
- Tissues examined: The full list (except eyes and head) was examined for the animal that died prematurely (Group 3 male No. 3) and all animals of Groups 1 and 4. Any abnormalities in animals of Groups 2 and 3 were also examined.
- Routine staining: Sections were stained with haematoxylin and eosin.
- Light microscopy: The full list (except eyes and head) was examined for the animal that died prematurely (Group 3 male No. 3) and all animals of Groups 1 and 4. Any abnormalities in animals of Groups 2 and 3 were also examined.
Statistics:
All statistical analyses were carried out separately for males and females. The following were analysed at each time-point separately: Grip strength and motor activity, body weight, haematology, blood chemistry and organ weights.
The following sequence of statistical tests was used for grip strength, motor activity, body weight, food consumption, organ weight and 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 grip strength, motor activity and 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.
For organ weight data, analysis of covariance was performed using terminal body weight as covariate, unless non-parametric methods were applied.
The treatment comparisons were made on adjusted group means in order to allow for differences in body weight which might influence the organ weights.
Significant differences between Control and treated groups were expressed at the 5 % (p<0.05) or 1 % (p<0.01) level.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The appearance and behaviour of the animals were unaffected by treatment.
There was transient piloerection after dose administration on Day 1 in a few males given 1000 mg/kg/day, with a further male given 100 mg/kg/day being affected on Day 1 and a female given 1000 mg/kg/day showing this sign on Day 2. In the absence of any other treatment-related signs, this transient finding was considered of no toxicological significance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no treatment-related deaths.
Male No. 3 given 100 mg/kg/day died in Week 8 after experiencing a convulsive episode. As this was an intermediate dose animal, and there were no similar signs at the highest dose, the death of this animal was not attributable to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect on body weight.
The overall weight gains of males given 10 mg/kg were approximately 8 % lower than that of the controls but, in contrast, females given this dose gained approximately 13 % more weight than the control females. There were no similar trends at the higher doses and, consequently, these inter-group differences from controls in the low dose animals were considered fortuitous.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was considered unaffected by treatment.
The amount of food consumed by males and females receiving 10 mg/kg/day was consistently higher than that of the controls but this was not attributed to treatment as there was no similar finding at 100 and 1000 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related ophthalmic findings.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The haematology examination performed in Week 13 did not reveal any findings that were attributable to treatment.
All inter-group differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included a small reduction of reticulocyte count in males receiving 100 or 1000 mg/kg/day and high mean cell haemoglobin concentration in males receiving 1000 mg/kg/day and at all doses in females. In each case the difference from controls was minimal and there were no other erythrocytic findings. They also included the statistically significant increase of prothrombin time, predominantly in males, as there was no dose response and the magnitude of the increase, compared with controls, was minimal and of no toxicological importance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma performed in Week 13 did not reveal any findings that were attributable to treatment.
All inter-group differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included a slight increase of blood urea nitrogen concentration in females receiving 1000 mg/kg/day since the results for the other treated females, though slightly higher than controls, showed that there was clearly no dose-response. They also included the statistically significant low sodium and high potassium concentrations in males receiving 1000 mg/kg/day since the magnitude of the differences from controls was minimal and there was no similar finding in the females.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Sensory reactivity and grip strength
There was no effect of treatment at the sensory reactivity and grip strength investigations performed in Week 12.
There was a statistically significant decrease of the group mean fore- and hind-limb grip strength values in females receiving 1000 mg/kg/day and, in addition, the mean forelimb grip strength values at all doses in males were also low, but there was no dose-response as the mean values at 10 and 100 mg/kg/day were lower than at 1000 mg/kg/day. A comparison against the historical control data (HCD) indicated that all values, including the controls (except for hind limb grip strength in females), were below the background range in both sexes. As the magnitude of the differences from controls was small, and lacked dose-relationship in males, they were not clearly attributable to treatment.

- Motor activity
There was no effect of treatment at the motor activity investigation performed in Week 12.
When compared with the controls, there were a few isolated statistical significances seen in the treated groups but the magnitude of these differences from controls was small and were not dose-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Organ weights were not affected by treatment.
All inter-group differences from controls were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the trend towards low liver weight in males since the difference from controls was small and there was no similar trend in the females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination revealed mucosal depressions in the stomach of one male given 1000 mg/kg/day (No. 36) and one female given 100 mg/kg/day (No. 75).
The incidence and distribution of all the other findings were consistent with the common background of macroscopic lesions seen in Sprague-Dawley rats at the testing laboratory.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with the test material were seen in the stomach.
- Nonglandular Stomach
Ulceration of the nonglandular stomach (forestomach) was observed in one female (No. 75) and one male (No. 36) given 100 and 1000 mg/kg/day, respectively. These focal changes were associated with slight to moderate inflammation and slight oedema and correlated with the macroscopic findings of mucosal depression recorded during the post mortem examination in female No. 75.
Hyperplasia/hyperkeratosis was observed in two males (Nos. 31 and 33) and spongiosis of the nonglandular stomach was also present in one of these animals (No. 31).

- Glandular stomach
In the glandular stomach, inflammatory cell infiltrate (composed of a variable number of neutrophils and eosinophils) was seen in two males (Nos. 31 and 32) given 1000 mg/kg/day.
These inflammatory changes were associated with infiltration of globule leukocytes (No. 31) and foveolar hyperplasia (Nos. 31 and 32). Increased globule leukocytic infiltrate in the glandular stomach is an unusual finding that has been occasionally reported in literature with different classes of chemicals (Ueda et al., 2011).
A summary of treatment related findings in the stomach for animals killed after 13 weeks of treatment can be seen in Table 1.
The incidence and distribution of all the other findings were consistent with the common background of lesions seen in Sprague-Dawley rats.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
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.
Details on results:
Gastric irritation occurred in a few animals (one female given 100 mg/kg/day and four males given 1000 mg/kg/day) and was attributed to the physical properties of the test material since it was a hard, granular material which was considered to have caused physical irritation leading to localised damage to the gastric mucosa in these animals.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no

Table 1: Summary of treatment related findings in the stomach for animals killed after 13 weeks of treatment

 

Males

Females

Dose (mg/kg/day)

0

10

100

1000

0

10

100

1000

Ulceration, Non-glandular Region

Slight

Moderate

Total

0

0

0

-

-

-

0

0

0

0

1

1

0

0

0

-

-

-

1

0

1

0

0

0

Inflammation, Non-glandular Region

Slight

Moderate

Total

0

0

0

-

-

-

0

0

0

0

1

1

0

0

0

-

-

-

1

0

1

0

0

0

Oedema

Slight

Total

0

0

-

-

0

0

1

1

0

0

-

-

1

1

0

0

Hyperplasia/Hyperkeratosis, Non-glandular Region

Minimal

Slight

Total

0

0

0

-

-

-

0

0

0

1

1

2

0

0

0

-

-

-

0

0

0

0

0

0

Spongiosis, Non-glandular Region

Slight

Total

0

0

-

-

0

0

1

1

0

0

-

-

0

0

0

0

Foveolar Hyperplasia

Minimal

Total

0

0

-

-

0

0

2

2

0

0

-

-

0

0

0

0

Inflammatory Cell Infiltrate, mucosal/submucosal, Glandular Region

Minimal

Slight

Total

0

0

0

-

-

-

0

0

0

1

1

2

0

0

0

-

-

-

0

0

0

0

0

0

Infiltration, Globule Leukocytes, Glandular Region

Slight

Total

0

0

-

-

0

0

1

1

0

0

-

-

0

0

0

0

No. tissues examined

10

0

1

10

10

0

2

10

Conclusions:
The no-observed-adverse-effect level (NOAEL) for systemic toxicity in this study was considered to be 1000 mg/kg/day.
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.

Animals were observed daily for mortality and clinical signs. Detailed physical examinations took place weekly, as did measurement of body weight and food consumption. In week 12, animals were subjected to sensory reactivity, grip strength and motor activity investigations as well as ophthalmoscopic examination. Haematology and clinical chemistry parameters were investigated in week 13. 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. 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, causing no adverse clinical signs or any changes in the sensory reactivity, grip strength and motor activity assessments and no effect on body weight, food consumption, ophthalmology, haematology, blood chemistry and organ weights.

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.

The only findings in this study occurred in the stomach. The macroscopic examination revealed the presence of mucosal depressions in one female given 100 mg/kg/day and in one male given 1000 mg/kg/day. At the histopathological examination there were findings in the glandular and non-glandular stomach of one female given 100 mg/kg/day and four males given 1000 mg/kg/day. The findings comprised ulceration with inflammation and oedema in the non-glandular region and foveolar hyperplasia associated with eosinophilic globules and inflammatory cell infiltrate in the glandular region. In one of the males the inflammatory changes were associated with infiltration of globule leukocytes.

In this study these changes were attributed to the physical properties of the test material, rather than a consequence of a systemic toxic response. The test material was a hard, granular material which was considered to have caused physical irritation leading to localised damage to the gastric mucosa in a few animals.

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 no-observed-adverse-effect level (NOAEL) for systemic toxicity in this study was considered to be 1000 mg/kg/day.

Endpoint:
repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to GLP and according to peer reviewed methods.
Reason / purpose:
other: Target
Qualifier:
according to
Guideline:
other: NTP peer reviewed methods
Principles of method if other than guideline:
Groups of 10 male and 10 female rats were fed diets containing 0, 1600, 3130, 6250, 12500 or 25000 ppm manganese sulphate. Clinical findings were recorded weekly, feed consumption was recorded weekly by cage. Rats were weighed at the beginning of the studies and weekly thereafter. At the end of the exposure period, blood was collected for haematology analyses. A necropsy was performed on all animals and organs were weighed. A complete histopathological analysis was performed on all control and high-dose animals.
GLP compliance:
yes
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories (Stone Ridge, NY)
- Age at study initiation: 50 days



Route of administration:
oral: feed
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 1600, 3130, 6250, 12500 or 25000 which was equivalent to doses from 110 to 1700 mg/kg in males and 115 to 2000 mg/kg in females
Basis:
nominal in diet
No. of animals per sex per dose:
10 male and 10 female per dose group
Control animals:
yes
Observations and examinations performed and frequency:
Clinical findings were recorded weekly, feed consumption was recorded weekly by cage. Rats were weighed at the beginning of the studies and weekly thereafter. At the end of the exposure period, blood was collected for haematology analyses.
Sacrifice and pathology:
A necropsy was performed on all animals and organs were weighed. A complete histopathological analysis was performed on all control and high-dose animals.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
-BODY WEIGHT AND WEIGHT GAIN: The mean bodyweight gain in males receiving 3130 ppm was marginally lower than that of the controls and was significantly lower in the three highest female dose groups than the controls.
-FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed consumption by exposed rats was similar to that of the controls. Females ingested an average of 20% more manganese sulphate than males in the corresponding exposure groups.
-HAEMATOLOGY: Neutrophil counts were significantly higher in all exposed male groups. Lymphocyte counts were significantly lower in the three highest dose groups.
In females: leukocyte counts were significantly lower in the three highest dose groups.
A significant increase in the percent haematocrit and erythrocyte counts occurred in males exposed to the three highest dose levels.
-ORGAN WEIGHTS: Absolute and relative liver weights of all exposed males and of the female 25000 ppm group were significantly lower than the controls.
Dose descriptor:
NOAEL
Effect level:
1 700 mg/kg bw/day (nominal)
Based on:
other: food intake and concentration in food.
Sex:
male
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day (nominal)
Based on:
other:
Sex:
female
Critical effects observed:
not specified

No clear relationship between observed differences and the ingestion of manganese sulphate has been defined.

Table 1.Survival, Body Weights, and Feed Consumption of Rats in the Rats in the 13-Week Feed Study of Manganese (II) Sulphate Monohydrate

 

Concentration (ppm)

Survivala

Mean body weight and weight changesbrelative

Final Weight Feed to controls (%)

Consumptionc

Initial

Final

Change

Week 1

Week 13

 

Male

 

 

 

 

 

 

 

0

10/10

136±5

291±4

155±4

 

14.9

13.1

1, 600

10/10

142±4

294±5

152±4

101

14.5

13.5

3,130

10/10

149±3

291±4

141±4

100

14.8

13.6

6, 250

10/10

148±2

294±3

146±3

101

15.0

9.6

12, 500

10/10

150±11

290±6

140±11

99

14.9

14.9

25, 000

10/10

140±4

284±6

144±4

97

14.1

14.4

Female

 

 

 

 

 

 

 

0

10/10

99±1

184±2

84±2

 

10.7

9.2

1, 600

10/10

103±1

181±2

79±2

99

10.8

9.3

3,130

10/10

96±1

175±2*

80±3

95

10.9

9.2

6, 250

10/10

101±1

176±2*

75±1**

96

10.7

14.3

12, 500

10/10

106±1**

178±1*

73±2**

97

10.7

10.5

25, 000

10/10

104±1**

174±3**

70±2**

95

12.1

10.3

* Significantly different (P≤0.05) from the control group by Williams’ or Dunnett’s test

** P≤0.01

a Number of animals surviving at 13 weeks/ number initially in group

b Weight given as mean ± standard error

c Feed consumption is expressed as grams per animal per day

 

Conclusions:
The high level of MnSO4 consumed on a daily basis for 13 weeks in this study, without mortality, supports the lack of acute toxicity. Although some changes in lung weight and certain haematological parameters were significant compared to controls, the lack of clinical and histopathological findings despite the very high daily oral dose over a sub-chronic period, is an indication of the relatively low toxicity of MnSO4, at least for the parameters studied in this report.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The subchronic study was conducted in accordance with a standardised guideline under GLP conditions and was awarded a reliability score of 2 in accordance with the criteria of Klimisch et al (1997) as the data is read-across. The quality of the database is therefore considered to be high.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

The systemic toxic potential of the read-across substance SiMn slag 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.

Animals were observed daily for mortality and clinical signs. Detailed physical examinations took place weekly, as did measurement of body weight and food consumption. In week 12, animals were subjected to sensory reactivity, grip strength and motor activity investigations as well as ophthalmoscopic examination. Haematology and clinical chemistry parameters were investigated in week 13. 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. 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, causing no adverse clinical signs or any changes in the sensory reactivity, grip strength and motor activity assessments and no effect on body weight, food consumption, ophthalmology, haematology, blood chemistry and organ weights.

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.

The only findings in this study occurred in the stomach. The macroscopic examination revealed the presence of mucosal depressions in one female given 100 mg/kg/day and in one male given 1000 mg/kg/day. At the histopathological examination there were findings in the glandular and non-glandular stomach of one female given 100 mg/kg/day and four males given 1000 mg/kg/day. The findings comprised ulceration with inflammation and oedema in the non-glandular region and foveolar hyperplasia associated with eosinophilic globules and inflammatory cell infiltrate in the glandular region. In one of the males the inflammatory changes were associated with infiltration of globule leukocytes.

In this study these changes were attributed to the physical properties of the test material, rather than a consequence of a systemic toxic response. The test material was a hard, granular material which was considered to have caused physical irritation leading to localised damage to the gastric mucosa in a few animals.

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 no-observed-adverse-effect level (NOAEL) for systemic toxicity in this study was considered to be 1000 mg/kg/day.

The systemic toxic potential of the read-across substance MnSO4 when administered for 90 days was investigated in a study conducted using NTP peer reviewed methods. Groups of 10 male and 10 female rats were fed diets containing 0, 1600, 3130, 6250, 12500 or 25000 ppm manganese sulphate. The high level of MnSO4 consumed on a daily basis for 13 weeks in this study, without mortality, supports the low toxicity of this substance. Although some changes in lung weight and certain haematological parameters were significant compared to controls, the lack of clinical and histopathological findings despite the very high daily oral dose over a sub-chronic period, is an indication of the relatively low toxicity of MnSO4 for the parameters studied in this report. The NOAEL for male rats exposed to MnSO4 was 1700 mg/kg/day. The NOAEL for female rats was 2000 mg/kg/day.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to repeated dose toxicty.