Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
other: Expert statement
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
No study covering all the relevant information was available, hence, an extensive assessment of the toxicokinetic behaviour of manganese (II) acetate was performed, taking into account the chemical structure, the available physico-chemical and toxicological data.

Data source

Reference
Reference Type:
other: Expert Statement
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
toxicokinetics
Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
An extensive assessment of the toxicokinetic behaviour of Manganese (II) acetate was performed, taking into account the chemical structure, the available physico-chemical and toxicological data.
GLP compliance:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Manganese di(acetate)
EC Number:
211-334-3
EC Name:
Manganese di(acetate)
Cas Number:
638-38-0
Molecular formula:
C2H4O2.1/2Mn
IUPAC Name:
manganese(2+) diacetate
Test material form:
other: not applicable
Details on test material:
not applicable
Radiolabelling:
other: not applicable

Test animals

Species:
other: not applicable
Strain:
other: not applicable
Details on test animals or test system and environmental conditions:
not applicable

Administration / exposure

Route of administration:
other: all relevant routes of administration are discussed in the expert statement
Vehicle:
other: not applicable
Details on exposure:
not applicable
Duration and frequency of treatment / exposure:
not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
not applicable
No. of animals per sex per dose / concentration:
not applicable
Control animals:
other: not applicable
Positive control reference chemical:
not applicable
Details on study design:
not applicable
Details on dosing and sampling:
not applicable
Statistics:
not applicable

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Animal data

The absorption efficiency of manganese from soluble salts after oral uptake is very low and comparable in animals and healthy human individuals: 1-13%. Animal experiments have shown that absorption is mediated by active transport in the upper gastrointestinal tract, whilst no active transport is present in the colon. The rate of manganese absorption is rapid and reaches maximal blood concentrations after a very short time. The human absorption of manganese from the soluble sulphate or acetate is likely to be lower than the one from the soluble chloride salt (Bales et al, 1987; Cirkt and Vostal 1969).

Under normal conditions, the level of absorption from the intestinal tract is low; in man it is said to be about 3 %. However the individual differences can be as great as a factor of five.
A homeostatic mechanism regulates the absorption from and excretion into the gastrointestinal tract (GI). When excess iron is present the absorption takes place by diffusion, when iron is deficient by active transport in the intestine. Iron deficiency increases the absorption of manganese. It is conceivable that a common mechanism is involved in the absorption of the two metals and that it is stimulated by iron deficiency.

Calculation of the daily uptake of manganese through the gastrointestinal tract and the lungs of workers revealed that the amount taken up by inhalation is much larger than that absorbed after oral intake (Oberdoerster and Cherian 1988). Assuming absorption of 3 % of the manganese taken in with the diet (on average 4 mg/day), the amount of manganese absorbed daily is about 120 µg. In contrast, about 4000µg of manganese would be absorbed daily after 8-hour inhalation exposure concentration of 1mg/m3 (assuming steady-state conditions for manganese retention, 10 m3 inhaled in 8 hours, about 40 % deposited in the lungs and thus absorbed).

The following factors influence the oral uptake of manganese (in humans and animals):
• Iron status (heme and non heme iron, anaemia, ferritin. Transport of manganese is obviously in both cases via transferring and, as such, antagonistic relationship is likely),
• Dietary matrix (especially the presence of transitional metal ions can influence uptake in the upper GI-tract) and the ionic-form and source of manganese e.g. as a MnCl2 solution (higher) or as in food (lower),
• Bioavailability from manganese-rich matrices is decreased, while manganese poor matrices and fasting lead to increased biovailability,
• A high existing body burden of manganese will decrease further absorption, whilst a low body-burden will increase absorption.

In animal experiments it could be shown that oral and inhalation bioavailability of insoluble manganese compounds is less efficient compared with soluble compounds.

The monitoring of manganese in urine as a marker of recent manganese exposure seems to be possible as shown in humans, but it is not fully validated; blood levels seem to be less predictive of recent exposure and appear to better reflect the overall body-burden of manganese (cumulative exposure and retention). Both urine- and blood-manganese monitoring as markers of manganese exposure show a good correlation on a group basis, but there is limited correlation on an individual basis.

The three routes of entry for manganese by inhalation are:
• Transport across the pulmonary epithelial lining and subsequent distribution in lymph and blood
• Clearance from the lung by mucocilliary elevator and subsequent ingestion from the GI tract
• Through the nasal mucosa

But the relative proportion absorbed by each process is not accurately known. The absorption via the lungs depends from the particle size, the solubility and from the geometry of the respiratory tract (Schlesinger 1996). It is known, that the absorption of the ultrafine particles (UFPs) of an insoluble form of manganese in rats is of the same magnitude as the one from a soluble form of manganese (Elder et al. 2006). In case of the intratracheal instillation to rats of either soluble manganese chloride or insoluble manganese tetroxide ( 90% of Particles < 1µm), the manganese uptake, distribution and excretion was far more rapid from the soluble form (Drown et al.1986). Maximal brain manganese concentration were of the similar magnitude, although the absorption from the lungs of the two forms of manganese proceeded at different rates : hours for the soluble manganese chloride and days for the insoluble manganese tetroxide; the overall exposure was very similar. The variation in manganese levels in the intestinal contents with time showed a similar profile as many other tissues, with the soluble group having a higher and earlier maximum of manganese concentration. The mechanism involved in the clearance of insoluble manganese is not clear and it is not investigated hitherto (Drown et al. 1986 ) no study could be found in the further literature.

The absorption of manganese from inhaled soluble salts in rats is partly mediated by direct transport along the olfactory neurons into the brain. This route of entry is postulated for
humans as well, but no study in humans exists to prove this hypothesis. It has been proposed that the percentage of air flow that reaches the rats´ olfactory mucosa is comparable to humans, suggesting that potential absorption of airborne metals by rats and humans would be similar (Thompson et al 2007). But this is in contrast to the results of the studies of other scientists (Kimbell 2006). It cold be shown, that a direct comparison between rats and humans is complicated by interspecies differences in nasal and brain anatomy and physiology.

When groups of monkeys were exposed to either air or MnSO4 ( up to 1.5 mg Mn/m3, MMAD 1.72-2.12 µm) for 65 exposure days before tissue analysis, monkeys at the lowest dose level developed increased manganese concentration in the olfactory epithelium, olfactory bulb, olfactory cortex, globus pallidus, putamen, and cerebellum.
A greater than 3-5-fold increase in mean tissue concentration was observed in the globus pallidus, putamen and caudate of monkeys exposed at the highest dose level. Results from this work were combined with MRI, pallidal index (PI), and T(I) relaxation rate (RI) to establish a direct association between MRI changes and pallidal manganese concentrations (Dorman et al. 2006c). The authors stated that their results indicated that the RI can be used to estimate regional brain manganese concentration and may be a reliable biomarker of occupational manganese exposure. Furthermore, they suggested that this study was the first to provide indirect evidence of direct olfactory transport of an inhaled metal in a nonhuman primate. However, they also stated, that to their knowledge significant neuronal connection between the monkey olfactory bulb and the globus pallidus do not exist. As such a transport of manganese was not likely to go beyond the olfactory bulb into deeper brain tissue like the globus pallidus, a situation that was also likely to be operable in humans. Instead, they concluded that the pallidal delivery of manganese was likely to have arisen primarily from systemic delivery and not directly from olfactory transport.

Furthermore, the absorption of the acetate, i.e. the absorption of the salt of acetic acid, has to be regarded for risk assessment, too:

Administration of 0.01-0.25% (e. g. 8-210 mg/kg bw/d) in the drinking water for 9-15 weeks, did not affect food and water consumption and body weight grain.
Doses of 0.5% ( e. g. 410 mg /kg bw / d), for 9 weeks, caused decreases in food consumption and body weight gain, but not in water consumption (Henschler 1973)

Rats that have received 0,5% acetic acid in drinking water for up to 15 weeks gained weight more slowly and ate less food than controls (Lundberg 1988).

Guinea pigs exposed for one hour to concentrations of 5, 39, 119, 568 ppm of acetic acid showed in increase in pulmonary flow resistance, a decrease in pulmonary compliance and an increase in the time constant of lungs. These changes suggest bronchial constriction as the first action of acetic acid. At 5 ppm there was a 20% increase in airway resistance (p=0,001), accompanied by 15 % reduction in increase in compliance. In case of exposure to 100ppm, recovery was complete within one hour while the recovery was not complete after exposure to 500 ppm (Amdur 1961).

Instillation of 0.5 ml of 1 % acetic acid in the eyes of rabbits caused a severe burn (Smyth et al 1951). Solutions of 5% induced injury in eyes of rabbits which were healed by 14 days while a 10% solution resulted in severe permanent damage (Henschler 1973).

No skin corrosion was observed when 0.5 ml undiluted glacial acetic acid was applied to the shave backs and flanks of rabbits (patch testing for 4 hour) (Vernon et al, 1977). Based on the average of mean scores for intact and abraded skin (reading at 4, 24 and 48 h) a 10% solution was concluded to be slightly and negligibly irritating to rabbits and guinea pigs, respectively (Nixon et al , 1975).

In rats 4.500 mg/kg body weight /day for 30 days induced gastric lesions. (Leung and Paustenbach, 1990))

The non dissociated acetic acid can be taken up by the intact skin: therefore lethal dermal doses as LD50-values could be investigated:
Guinea pig (LD50) dermal: 3.360 mg/kg for 28 % acetic acid
Guinea pig (LD50) dermal > 21 mg/kg bw for 5% acetic acid
Rabbit (LD50) dermal: 1060 mg/kg (Katz and Guest, 1994)

The LC50 (oral) for mice was found to be 5.620 ppm for one hour exposures. Symptoms were mainly irritations of the upper respiratory tract and of the conjunctiva. Most of the surviving animals recovered quickly and showed no abnormal conditions.


Human data

Uptake, administration, manganese cation

Oral uptake

In a Long-term drinking-water study in a rural northern area of Germany (Vieregge et al.1995) found no neurological effects following ingestion of increased manganese. No significant differences in neurological tests were found in older people (41 subjects older than 40 years with a mean age of 57,7) consuming well-water containing at least 0.3 mg/L of manganese (0.3 to 2.16 mg/L of manganese for 10-40 years. The control group (74 subjects, mean age 56.9 years ) was exposed to water containing less than 0.05mg/L of manganese. Subjects of both groups were randomly selected and matched with respect to age, sex, nutritional habits and drug intake. However, like the Kondakis et al.(1989) study, this study lacks exposure data from other routes and sources, and the manganese concentration range in the water was very broad.

Two other studies involving ingestion exposure to manganese reported no increase in adverse health effects. In one area of Japan, a manganese concentration of 0.75 mg/L in the drinking-water supply had no apparent adverse effects on the health of consumers (Suzuki, 1970). No signs of toxicity were observed in patients given 30 mg of manganese citrate (9 mg of manganese) per day for many month (Schroeder et al., 1966).

Another case study (Woolf, 2002) reported increased manganese levels in the hair and blood of a 10-year-old child exposed to increased manganese in drinking-water. The child had been ingesting drinking-water supplied by a well for 5 years prior to a clinic visit for evaluation of over-exposure to manganese. In addition, the family lived in a house near a toxic waste dump. An evaluation of the well water performed four months prior to the child ´s health assessment indicated that manganese and iron levels in the water were both elevated, with concentrations of 1.21 (reference level, 0.05 mg/L) and 15.7 mg/L, respectively.
The child ´s whole blood and serum manganese levels were 3.82xg/100ml (reference normal, < 1.4xg/100 ml) and 0.90x g/100ml (reference normal <0.265 x g/100 ml) respectively.
The child ´s hair manganese level was 3,091 ppb of washed, digested hair any clinical effects (reference normal <260ppb hair). Although the child ´s 16-year-old brother did not exhibited elevated blood manganese, he did have increased manganese in his hair.

Inhalation

The most investigations included workers which worked as miners, welders, smelters or in the ceramic industry and they were exposed to the manganese oxides, most MnO2 . No information on Manganese (II) acetate is available.
Only the investigations of Roels (1987, 1992) and Lauwerys (1992) included dust (aerosols of manganese salts .

The following abstracts are taken from the Publications :

Roels et al. (1992)
Same collective as studied by Lauwerys et al.(1992), the study group comprised 102 employees of a factory producing cell batteries, duration of exposure 0.2 to 17.7 years (average 5.3), manganese concentration (as total dust) 0.046 to 10.84 mg/m3 (arithmetic mean 0.301 t, geometric mean 0.215). The control group comprised 102 employees who were not exposed to manganese.
Neurological tests and numerous biochemical tests were carried out. Blood parameters and short-term memory were unaffected.

Wennberg et al. (1991, 1992)
Data were collected from 30 employees of two foundries, duration of exposure 1 to 45 years, manganese dust concentration between 0.03 mg/m3 and 1.62 mg/m3, average values 0.19 and 1.39 mg/m3, depending on the kind of work. The average concentration value for all workplaces in the first foundry was 0.18 mg/m3 and in the second 0.41 mg/m3, conditions unchanged for 17 and 18 years, respectively.
Electroencephalographic and psychiatric studies revealed no apparent effects.


Uptake and metabolism, acetate anion

Acetic acid is absorbed from the gastrointestinal tract and through the lungs. The acetate ion is a normally-occuring metabolite in catabolism or anabolic synthesis e.g. in the formation of glycogen or cholesterol, and in the degradation of fatty acids or in the acetylation of amine and alcohols.

It is estimated that the level of acetate ion in humans is about 50-60 µmol/l (3,0-3,6 mg/l) in plasma and 116 µmol/l (7 mg/l) in cerebrospinal fluid. Daily turnover of the acetate ion in humans is estimated to be about 7.5 µmol/kg/min representing some 450 g/day
(Simeneau et al. 1994).

Acceptable daily intakes (ADI´s) for acetic acid have not been proposed as sensory properties will limit intakes. Estimations of the daily intake of acetic acid vary from about 1 gram (Elias 1987) to 2.1 g/day for subjects older than 2 years (Katz and Guest, 1994). No adverse health effects are reported at these intakes.

The main adverse health effects are the corrosion und the irritation caused by higher concentrated acetic acid. The target organs are the skin and the mucosa of the eyes, the nose, the upper airways and the lung or the gastrointestinal tract.
Acetic acid at very high concentrations of 24.000 ppm and above causes irritation of the eyes and upper respiratory tract in humans (von Oettingen, 1960).

Humans unaccustomed to acetic acid vapours experience extreme irritation of the eyes and nose at concentrations of 25 ppm or more, and 50 ppm is considered unendurable. Acclimatised persons can tolerate 30 ppm without difficulty: exposures over several years to concentrations higher than 10 ppm of acetic acid at workplaces producing acetic acid from wine caused no symptoms of poisoning leading to the view that concentrations of 20 to 30 ppm are harmful (Vigliani and Zurlo 1955).

In a well conducted human volunteer study 11 individuals (5 men and 6 women) with a mean age of 27y (range 27-41) were exposed on 3 separate occasions to air (the control exposure) and to acetic acid vapour at 5 and 10 ppm in an exposure chamber with 18-20 air changes per hour. One additional male subject was only exposed to air and the 10 ppm vapour concentration. Subjects were exposed in pairs for 2 hours under resting conditions while seated. Results: no exposure related effects were observed on pulmonary function, nasal swelling, nasal airways resistance, or plasma inflammatory markers measured before and 3 h post exposure. Subjects were also asked to complete a questionnaire to rate their acute symptoms on a 0-100mm visual scale. Subjective ratings of nasal irritation and increased smell increased with exposure but were only significant at the 10 ppm concentrations; the reported irritation was constant throughout the 2 hours suggesting a real irritant effect rather than a response to smell only. Apart from the smell the rating for nasal effects at the effects ranged from the lower end of the scale with median values at 6 mm (rated `hardly noticeable at all`) and the highest at 26 mm in a category of `somewhat noticeable`.´

Eye blinking frequency increased during and after exposure to 10 ppm but was not significantly different from the control exposure. There were no observed effects on any of the measured parameters at 5 ppm acetic acid vapour and this value can be regarded as a NOAEL in naive human subjects (Ernstgard et al. 2006).

In patch tests with human volunteers over 4, 24 and 48 hours a 10% aqueous solution of acetic acid caused slight irritation (Nixon et al 1975) that did no lead to EC classification as “irritant to the skin” (Griffiths et al. 1997)

On the basis of industrial experience, it has been stated that exposure at 10 ppm is relatively non-irritating (Henschler 1973).

The odour threshold for non-acclimatised individuals is 1-5 ppm (Greim, 2000)

SCOEL ( 2010) The recommendation on a OEL is : 8hr TWA: 2ppm (5mg/m3 ) and
STEL: 5 ppm (12,5 mg/m3 )
Details on distribution in tissues:
Animal data

Three methods were used for the investigation of distribution of Manganese in the tissues. In animal experiments using radioactive Mn the tissue and organs can separated at necropsy and sub-samples can be prepared for measurement of the radioactivity.
The use of radiolabelled 54 Mn is useful for the animal experiments but not for humans. For humans it is impossible to pinpoint the exact location of radioactivity.

The concentration of (non-radiolabelled) manganese in samples of blood, serum, plasma, hair and excreta in humans and animals can be readily measured after sample processing using atomic absorption spectroscopy or neutron activation. The manganese concentration of animal tissue taken at necropsy can be analysed. Interpretation of these results rely on the assessment of background levels of manganese. The background data can be obtained from historical data, control groups or assessment of manganese levels in the same subject pre-administration or re-assessment at time periods after the cessation of treatment.

The third method for assessing manganese distribution is a qualitative method that exploits the paramagnetism of manganese using magnetic resonance imaging (MRI).This technique
can clearly distinguish between several separate and specific regions of the brain since the spin-lattice relaxation time,T1, is strongly influenced by tissue characteristics such as water, and fat content. This technique has recently been expanded to use MRI, pallidal index (PI) , and T(1) relaxation rate(R1) in concert with chemical analysis to establish a direct association between MRI changes and pallidal manganese concentration in rhesus monkeys following subchronic inhalation of manganese sulfate (Dorman et al 2006c).

It is likely that manganese is metabolised by the body in the form of converting manganese from the Mn 2+ valence state to the Mn 3+ valence state (Gibbons et al., 1976). The authors proposed the following hypothesis to help to explain the homeostasis of manganese:

“ingested manganese is absorbed as Mn 2+ possible bound to alpha2-macroglobulin or albumin. In transversing the liver it is removed nearly quantitatively. However a small proportion is oxidised to the Mn 3+ valence state, bound to transferrin and enters the systemic circulation to be transported to tissues”.
Gibbons (et al. 1976) investigation in the binding of manganese ( 54MnCl2) to bovine and caprine blood constituents had the following key findings:

• Mn 2+ bound to transferring after incubation with fresh serum, but would not bind to purified transferring in vitro without the presence of an oxidizing agent,
• A Mn3+ -transferrin complex was removed much more slowly (half-life of about 3 hours) from cow´s blood (in vivo) than either free Mn 2+ or a Mn 2+ alpha2macroglobulin complex.

More recently Reany et al. 2006. utilised older female rats (8 month old) and found that the tissue accumulation of manganese was significant higher (>25%) following ip injections of (Mn3+) than following equimolar ip injections of( Mn2+). Both the manganese chloride solution (Mn2+ ) and the manganese pyrophosphate solution (Mn3+ ) were administered at nominal doses of 0.2 or 6.0 mg Mn/kg via ip dose 3 times a week for 5 weeks. The Mn 3+ exposures produced significantly higher blood and brain concentrations than Mn2+ exposures at both treatment levels. There was no differences in manganese concentration between the region of the brain examined within a dose level or oxidation state.
The authors concluded that these data substantiate the heightened susceptibility of the globus pallidus to manganese, and they indicate that the oxidation state of manganese exposure may be an important determinant of tissue toxicodynamics.


In a combination of fast protein liquid chromatography (FPLC) with a combination of anion exchange and gel filtration columns, transferring as identified as the major manganese-binding protein in rat plasma (Davidsson et al. 1989c). The authors concluded that this result was independent of the route of administration (oral or iv) or the length of time after administration of 54 Mn. When 54Mn3+ was added to plasma in vitro , more 54 Mn was identified as bound to transferring than when 54 Mn 2+ was added, if the 54 Mn was added immediately before injection into the FPLC system. However no difference was observed between 54 Mn3+ and 54 Mn 2+ binding if allowed to incubate first (5 min.).

The addition of ceruplasmin, which is associated with the oxidation of Fe2+ to Fe3+ , to the incubation of 54 MnCl2 in rat plasma significant increased the transferring-bound fraction of
54 Mn (Aschner and Aschner, 1990). Further the authors described that the 54 Mn rat brain uptake levels were significantly reduced, when the rats were pre-treated for 6 hours with a continuous infusion of ferric hydroxide dextran complex. The authors concluded that the manganese uptake across the blood-brain barrier (BBB) might also be modulated by plasma iron homeostasis.
The rapid uptake of 54 Mn 2+ into brain and choroid plexus from the circulation was studied using the in situ rat brain perfusion technique (Rabin et al., 1993). The authors concluded that the results demonstrated that 54 Mn 2+ is readily taken up into the CNS, most likely as the free ion, and that transport is critically affected by plasma protein binding. Further the results supported the hypothesis that Mn 2+ transport across the blood-brain barrier is facilitated by either an active or a passive mechanism. More recently the carrier-mediated influx of manganese citrate, as well as Mn2+ and manganese transferring, using an in situ rat brain perfusion technique through the BBB has been reported (Crossgrove et al., 2003). In a companion paper, same workers also demonstrated that the rate of manganese efflux from the brain was consistent with diffusion (Yokel et al., 2003)

The relationship between manganese and iron transport is attributed to the fact that both metals can be transported via the same molecular mechanisms. Whether brain manganese distribution patterns due to increased manganese exposure compared to iron deficiency are the same , or whether iron supplementation would reverse or inhibit manganese deposition were investigated in a specific series of experiments in rats. Three treated groups of rats all received weekly injections of manganese chloride (3 mg Mn/kg) for 14 weeks with one group on an iron diet (FeD), another on an iron supplement diet (FeS) and one on a standard control diet. The distribution of manganese in the brains was determined by both MRI and atomic absorption spectroscopy (AAS). An increase in manganese accumulation and a difference in distribution was seen, as expected, in the rats on the FeD diet compared to the treated rats on the control diet. However the same accumulation and regionally specific pattern of manganese distribution was also seen in the brains of the FeS rats compared to the FeD rats. The authors were quite surprised to find, that FeS and FeD diets did not have the opposite effects in brain manganese deposition since manganese and iron compete for the same transporter system. Further work must be done to explain these findings (Fitsanakis et al., 2008).

Iron deficiency or supplementation can considerable affect both the oral absorption of manganese as well as the uptake of manganese following inhalation (Thomson et al.,1971; Davis et al. 1992b; Brain et al. 2006; Heilig et al. 2006; Thompson et al. 2006 ).

Following a single ip injection of MnCl2 at either 2.5, 10 or 40 mg Mn /kg to adult male rats, the manganese content of selected tissues was measured for up to 24 hours post-dose (Keen et al., 1984). A dose response increase in manganese concentration was found in the plasma, liver, kidney, and brain with the liver concentration of manganese returning to basal level by 24 hours post-dose. Gel filtration chromatography of rat livers from the 10 mg Mn/kg dose level showed that manganese was initially (up to 1 hour post-dose) distributed between a protein fraction of MW 80000 that co-eluted with transferring and lower molecular weight substance.

When adult male rats were exposed to 0.5 % manganese as MnCl2 in their drinking water for 1, 4 or 6 weeks, the manganese concentration in the blood, brain, liver and kidney, were the highest after one week of exposure (Hietanen et al., 1981). The authors concluded that the results suggested an adaptation during continuous exposure.

Tissue accumulation of manganese (from MnCl2 ) was greater following ip administration
(6 mg Mn/kg/day) than by oral administration (75 mg Mn/kg/day) for 4 weeks to groups of rats (Missy et al., 2000). After a two week rest period, increases in manganese concentration were observed in most of the tissues, particularly significant in the nervous system (brain and spinal cord), the stomach, spleen and femur. Whole blood concentrations of manganese were at 191± 39µg/L, an 18.5-fold increase compared to controls following ip administration.; however plasma levels were not significantly different to controls. The authors believes that the increase of manganese in whole blood but not in plasma was due to manganese being associated with certain blood elements, such as haemoglobin, instead of iron. Thus the progressive disappearance of the high levels of manganese from the blood would have been closely related to the length of life and renewal of the erythrocytes. This hypothesis correlated with the increase in manganese concentration seen in the spleen, where old erythrocytes are destroyed.

The changes in the concentration of manganese in regions of the brain of a non-human primate (the common marmoset, Callithrix jacchus) following four systemic injections of 30 mg/kg MnCl2 x 4H2O in the tail vein using MRI and ICP-MS were compared these changes in the rat following the same exposure route and dose. The doses were spaced 48 hours apart and the animals were imaged 48 hours after the final dose. The brain structures that had a significantly greater increase in enhancement in the marmoset versus the rat were the visual cortex, the striatum, the globus pallidus, the ventral pallidum and the substantia nigra. Two of these structures are proximal to a large volume of CSF in the marmoset but not in the rat. In the marmoset, the visual cortex is adjacent to the posterior horn of the lateral ventricle. In the rat, the posterior horn does not extend to this structure. As well, the caudate of the striatum in the marmoset forms the lateral wall of the anterior horn and body of the lateral ventricle. While the same is true in the rat, the CSF space is much smaller than in the marmoset. The authors suggested that these two species differences suggest that the CSF-brain route of uptake is important in the marmoset and that the stronger manganese uptake in the marmoset brain is because the geometry of the lateral ventricles. There was also a significantly higher accumulation of manganese in marmoset brains even though the dose was normalised by body weight. When gross pathology was performed post-mortem it was found, that two of the four marmosets had significant liver damage, which suggested that marmosets were more susceptible to liver damage than rats. As such, the higher accumulation of manganese into the marmosets brains could have been due to a longer lifetime of manganese in the blood due to poor hepatobiliary clearance and thus compromising the study. Table 1 (see attachment) shows the comparison of brain manganese concentration (µg/g wet tissue) after repeated manganese iv administration (30 mg/kg MnCl2 x H2 O ) in the rat and marmoset (Bock et al. 2008).

The brain manganese concentration of Cynomolgus macaques following 45 weekly injections of manganese sulphate (10-15 mg/kg, equivalent to 3.26-4.89 mg Mn/kg) showed a greater increase in the globus pallidus compared to the other brain regions (Guilarte et al., 2006). In addition to measuring tissue concentration post-mortem, manganese distribution using T1 weighted MRI was performed and analysed using a pallidal index (PI) equivalent approach at 18 and 41 weeks. Only small increases (not statistically significant) in PI equivalents were seen in several brain tissues which was a lot less than the increases seen in blood manganese at the time-points and also in the increases seen in the tissues post mortem. The authors explained that these differences were due to concurrent increases in manganese levels in the frontal white matter, which is used as the denominator for the PI equivalent ratio calculation. They also suggested that better correlation was not seen as the tissue concentration of manganese were measured later (4 weeks) than the MRI investigations. It is also interesting to note that although the increase in blood manganese was significant at 18 weeks into the study, the level had dropped at 41 weeks and further still 45 weeks becoming no longer statistically significant (at p<0.05)).

Table 2 (see attachment) shows the distribution of brain manganese concentration (µg/g wet tissue) and blood manganese levels (µ/L) after 45 weekly manganese iv administrations (10-15 mg/MnSO 4 ) in the monkey (Guilarte et al., 2006).


Olfactory transport:

Following 54MnCl2 dosing to the olfactory chambers of pikes 54Mn2+ was taken up in the olfactory receptor cells and was transported at a constant rate along the primary olfactory neurones into the brain (Tjalve et al.,1995). The 54Mn2+ accumulated in the entire olfactory bulbs, although most marked in central and caudal parts. The metal was also seen to migrate into large areas of the telencephalon, apparently mainly via the secondary olfactory axons present in the medial olfactory tract. The results also showed that there was a pathway connecting the two olfactory bulbs of the pike and that this can carry the metal. The authors concluded that it appeared that manganese has the ability to pass the synaptic junctions between the primary and the secondary olfactory neurones in the olfactory bulbs and to migrate along secondary olfactory pathways into the telencephalon and the diencephalons in pike. Therefore it was concluded that the olfactory route might be a crucial pathway by which manganese gains access to the brain.

The dose-dependence of the uptake and subcellular distribution of the manganese in the olfactory epithelium and the brain of rats was examined after a single intranasal instillation of (54MnCl2, (Henrikson et al, 1999). The results indicate that manganese transport was a saturable process, both regarding the uptake into the olfactory epithelium and the transfer to the olfactory bulb. The data also indicated that manganese moves relatively freely from the olfactory bulb to the olfactory cortex at an amount dependent of the level of influx into the bulb. The transport to the rest of the brain was related to the amounts in the olfactory bulb and the olfactory cortex, but the relative proportion reaching this area increased with increasing doses. The authors concluded that their results showed that the olfactory neurons provided a pathway with a considerable capacity to uptake via this route.
This statement failed to take into account the difference in the relative size of the olfactory bulb in rats and humans and presumably assumed that the same pathway with the same capacity was present in humans, although unfortunately this was discussed. Similarly it fails to address the suitability of the rat as a model for manganese toxicity in humans. Additionally the study failed to provide evidence to support the proposal that the neurotoxicity of inhaled manganese is related to an uptake via this route, and thus it can only be speculation.

“Experiments examing the dosimetry of inhaled manganese generally focus on pulmonary deposition and subsequent delivery of manganese in arterial blood to the brain. Growing evidence suggests that nasal deposition and transport along olfactory neurons represent another route by which inhaled manganese is delivered to certain regions of the rat brain. The purpose of this study was to evaluate the olfactory uptake and direct brain delivery of inhaled manganese phosphate (54MnHPO4). Male 8-wk-old CD rats with either both nostrils patent or the right nostril occluded underwent a single, 90-min, nose-only exposure to a (54MnHPO4 aerosol (0.39 mg 54Mn /m³;MMAD 1.68 micron sigma (g) 1.42) The left and right sides of the nose, olfactory pathway, striatum cerebellum, and rest of the brain were evaluated immediately after the end of the ((54)Mn HPO(4) exposure and at 1,2,4, 8 and 21 d post exposure with gamma spectrometry and autoradiography. Rats with two patent nostrils had equivalent (54) Mn concentrations on both sides of the nose, olfactory bulb and striatum, while a symmetrical (54)Mn delivery occurred in rats with one occluded nostril.
High levels of (54)Mn activity were observed in the olfactory bulb and tubercle on the same side (i.e., ipsilateral) to the open nostril within 1-2 d following (54)MnHPO(4) exposure, while brain and nose samples on the side ipsilateral to the nostril occlusion had negligible levels of (54)Mn activity. Our results demonstrate that the olfactory route contributes to (54) Mn delivery to the rat olfactory bulb and tubercle. However, this pathway does not significantly contribute to striatal(54) Mn concentrations following a single, short-term inhalation exposure to (Mn) HPO(4)”. (Dorman et al. 2002; Brenneman et al. , 2000).

Distribution following inhalation or pulmonary instillation

Adult male CD rats were exposed (inhalation) for 6h/day/week (14 exposures) to either manganese sulphate (MnSO4), manganese tetraoxide (Mn3O4) or manganese phosphate in the mineral form hureaulite (Mn5(PO4)2(PO3)((OH)2 x 4H2O) at three different dose levels (Vitarella et al., 2000b, Dorman et al., 2001a) significant increases in the manganese concentration in some tissues were found following this repeated short-term exposure at 0.3 mg Mn/m3 exposure levels and in most tissues at the 3 mg Mn/m3 exposure levels but not at the 0.03 mgMn/m3 exposure level compared to controls. A summary of the manganese tissue concentrations, which were taken immediately after the last exposure following the 3 mg Mn/m3 exposure level is presented in the Table. Although the lungs showed the largest increases in manganese concentration which were taken, the increase in manganese sulphate group as significantly les than the other two forms of manganese salts. Since the aerosols were aerodynamic similar with equivalent exposure concentrations of manganese, the results suggested that more soluble manganese sulphate were cleared more rapid than the less soluble particles from the manganese tetra oxide and phosphate forms following inhalation. Conversely, the olfactory bulb and striatal levels of manganese were significantly higher following the soluble manganese sulphate exposure than the less soluble particles from the manganese tetra oxide and phosphate forms. This suggests that the more soluble forms of manganese are more readily delivered to the olfactory bulb and striatum, adding credence to the direct olfactory transport theory (Brenneman et al. 2000 ; Dorman et al. 2002). Overall the authors concluded that dissolution rat could influence the pulmonary clearance of a metal and thus affect its delivery to the brain and other organs. In the earlier inhalation study (Brenneman et al., 2000) with the soluble manganese chloride (MnCl2) significant increases in the level of manganese in the striatum were generally not seen. However, the manganese exposure was at much lower dose (0.54 Mn/m3 ) and also this study was a single 90-minute exposure as opposed to repeated exposure.

In a further study in rats by the same workers, the influence of the old age and gender on the toxicokinetics of inhaled manganese sulphate (MMAD 1.85-2.03 µm) and manganese phosphate (hureaulite, MMAD 1.47µm) was investigated (Dorman et al., 2004a.). Gender and age did not affect manganese delivery to the striatum, a known target site for neurotoxicity in humans, but did not influence manganese concentrations in other tissues. at the end of the exposure olfactory bulb, lung, and blood manganese concentrations were higher in young rats than in female or aged rats and the authors concluded that this may reflect a portal-of-entry effect.

Adult male Sprague-Dawley rats were exposed (inhalation) for 6 h/day for 5 days/ week for 13 consecutive weeks to manganese sulphate at 3 different dose levels in order to sasses the effect of the subchronic exposure to manganese on locomotor activity, neuropathology and blood serum biochemical parameters (Tapin et al., 2006). The tissue collections were performed approximately 42 hours after last manganese exposure to allow for locomotor assessments to be made. The results are shown in Table 3 (see attachment)

Dissolution rate can influence the pulmonary clearance of metal and thus affect its delivery to the brain and other organs.The goal of the study was to determine the exposure-response relations for the relatively soluble sulfate (MnSO4 ) and the insoluble manganese tetroxide Mn3O4 forms of inhaled manganese in adult male CD rats. Rats were exposed 6h/day for 7days/week (14 exposures) to either manganese sulfate or manganese tetroxide at : 0, 0.03, 0.3 or 3 mg Mn/m3 . End-of-exposure olfactory bulb, striatum, cerebellum, bile, lung, liver, femur serum, and testes (n= 6 rats/Concentration/chemical) manganese concentrations and whole-body (54)Mn elimination were then determined. Increased whole-body clearance rates (54)Mn clearance rates were observed an animals from the high dose 3 mg Mn/m3 MnSO4 and Mn 3 O 4 exposure groups. Elevated manganese concentration in the lung were observed following MnSO4 and Mn3O4 exposure to < or = 0.3 mg Mn/m3.
Increased olfactory bulb and femur concentrations were also observed following MnSO4 exposure at >0.3 mg Mn/m3 . Elevated striatal, testes, liver and bile manganese concentrations were observed following exposure to Mn3O4 at 3 mg Mn/m3. Animals exposed to MnSO4 (3mg Mn/m3 ) had lower lung and higher olfactory bulb and striatal manganese concentrations compared with the levels achieved following similar Mn3O4 exposures. The results suggested that inhalation exposure to soluble forms of manganese results in higher brain manganese concentrations than those achieved following exposure to an insoluble form of manganese (Dorman 2001b).

Monkey´s brains were imaged before and after manganese administration (as manganese chloride) in coronal and horizontal planes that included the basal ganglia and substantia nigra (Newland et al: 1989).One monkey was exposed to manganese chloride aerosol (20-40 mg Mn/m3 ; mean particle size 1.2 µm) for 2 hr/day, 4 days /week) and had its brain imaged after 3 to 5 month´ exposure. Other monkeys received either 5, 10 or 20 mg Mn/kg by iv injection and had their brains imaged starting at 2 days after administration. The kinetics of manganese accumulation was important in determining the imaged intensity of these regions, but the route of administration (inhalation or iv injection) was not. Spin-lattice relaxation times showed that T1 was shortened at lower doses of manganese and remained shortened longer in the globus pallidus and pituitary gland while little effect appeared in grey and white matter. T1 effects in caudate and putamen effects were intermediate. These data suggested selective affinity for manganese in the gluobus pallidus and pituitary regions of the brain.

These findings were consistent with the tissue manganese concentration observed in young male rhesus monkeys following subchronic manganese sulphate inhalation for 6 h/day, 5 days/week (Dorman et al., 2006b). Groups of monkeys were exposed to either air or MnSO4 (0.06, 0.3 or 1.5 mg Mn/m3 , MMAD 1.72-2.12 µm) for 65 exposure days before tissue analysis. Monkeys at the lowest dose level developed increased manganese concentrations in the olfactory epithelium, olfactory bulb, olfactory cortex, globus pallidus, putamen and cerebellum. A greater than 3- to 5-foldincrease in mean tissue concentration was observed in the globus pallidus, putamen and caudate of monkeys exposed at the highest dose level.
Results from this work were combined with MRI, pallidal index (PI) and T(1) relaxation rate (R1) to establish a direct association between MRI, changes and pallidal concentration (Dorman et al., 2006c). The authors stated that their results indicate that the R1 can be used to estimate regional brain manganese concentrations and may be a reliable biomarker of occupational manganese exposure. Further, they suggested that this study was the first to provide indirect evidence of direct olfactory transport of inhaled metal in a nonhuman primate. They concluded that the pallidal delivery of manganese, however, likely arises primarily from systemic delivery and not directly from olfactory transport.


Human data

The amount of manganese in the body of an adult weighing 70 kg is estimated to be about 10-20 mg and because of uptake regulation, is relatively constant in the blood and tissues of non-occupationally exposed persons.
Most human tissue contain manganese in concentration s between 0.1 and 1,0 µg/g wet weight.
The levels in liver, pancreas, kidneys and brain are higher than in other organs (WHO, 1981). In general, tissue rich in mitochondria contain higher levels of manganese. The storage of manganese in the mitochondria can affected the enzymes responsible for oxidation reactions. In animal studies, inhibition of succinic dehydrogenase, cytochrome oxidase and lactic dehydrogenase by manganese has been demonstrated. Manganese also accumulates in the hair and more in darker hair .The levels in hair are below 4µg/g. The manganese levels are higher during the day than during the night (Hiroshi and Shunichi, 1988). The manganese concentration in erythrocytes are higher than in plasma; in the erythrocytes manganese occurs as a porphyrin complex.

In serum, Mn3+ ions are bound to transferrin and Mn2+ ions to macroglobulin, independent of the administration route (Davidsson et al.1989, Murphy et al., 1991). Since in plasma Mn 2+ is oxidised to Mn 3+ , the substance is mostly in the form of the manganese-transferrin complex in this body fluid.
In the most enzymes which require manganese for activity, the manganese is present in the divalent form (Keen and Leach, 1988). Mn3+ can be reduced in the tissues. Manganese can cross the placenta and the blood-brain barrier. This is essential for the brain function, because manganese is a cofactor for glutamine synthetase.

The exposure route determines the rate of changes in manganese concentration in the brain. After iv or sc injection, the levels change much more rapidly than after inhalation. For example, the half- life in man after iv injection of manganese is 54 days (Cotzias et al. 1968) and in macaque monkeys after sc. injection 53days. After inhalation by 2 macaque monkeys, the half-life of manganese in the brain was found to be 223 and 267 days (Newland et al. 1987) The longer half-life after inhalation of the substances can be put down to the fact that manganese is absorbed slowly from the lungs and so is present in the circulation and can go on entering the brain over a longer period. It is conceivable that the accumulation of manganese in the brain results from the transfer of the substances from the lungs and possible also from other organs as well as from the fact that manganese concentration of the brain decreases only slowly. With Sprague-Dawley rats given MnCl2 or Mn3O4 by intratracheal instillation, it could be shown that on days 3 and 7 , that is , in the initial phase, the manganese clearance from the lungs was higher for the soluble manganese salts than for poorly soluble oxide. However two weeks after application of the substances the manganese concentration in the organs liver, kidneys, spleen, brain and testes were similar in the two groups of animals (Drown et al. 1986).
The half-times for retention of manganese in the lungs depend on the amounts inhaled: lung clearance rates are higher after inhalation of high concentrations than after lower concentrations (Wiezorek et al. 1987). In rats, orally administered 54 Mn had a slightly shorter half-life (21.4 days) than administered intramuscularly (26.5 days) or intraperitoneally (25.7 days) ( Lee and Johnson 1988).
Details on excretion:
Animal data

The elimination of absorbed manganese is primarily through the bile (>95%) and, as such the main excretion route for manganese is ultimately in the faeces, however this route can also include unabsorbed dose following oral dosing, which is one of the complicating factors when trying to measure proportion of dose absorbed.
Very little manganese is excreted in the urine or other potential routes such as milk or sweat.
The biological half-life of manganese in human urine is estimated to be less than 30 hours following cessation of exposure to manganese (Roels et al. (1987b).
Chelation therapy with EDTA leads to forced excretion of stored manganese via urine. An increased uptake of manganese results in faster elimination through the body ´s natural homeostatic regulation.

When interpreting data on the absorption of manganese or manganese elimination, the body ´s natural homeostatic control of manganese must be taken into account, particularly so if is following oral absorption. The typical whole-body terminal half-lives for manganese in healthy volunteers are around 30-40 days (Mena et al. 1969, Johnson et al. 1991, Finley et al. 1994 and 2003). As with the absorption of manganese), factors such as pre-body burden of manganese and dietary levels of manganese, considerable affect its rate of elimination.

Following the iv dosing of a Mn 54 tracer it was established that the elimination of manganese is at least biphasic with an initial fast elimination phase, typically around 3-4 days in healthy human subjects (Mahony and Small, 1968) as with the slower terminal elimination phase , the initials fast phase can be modulated by manganese body burden.
The elimination of absorbed manganese is dependent on bile production and flow into the small intestine. The efficiency of this process is delayed in the rat neonate following birth and may lead to manganese retention following high doses of manganese.
However in human neonate, efficient bile flow occurs within the first days after birth, therefore the accumulation of manganese in human neonates may not be as much as of a potential problem with neonates (Inoue et al., 1997). The manganese eliminated in the bile usually then undergoes enterohepatic recirculation . There is evidence from a study in rats that auxiliary GI routes of elimination to that of the bile are also in operation (Wieczorek and Oberdorster, 1989), namely through the intestinal wall and with the pancreatic juice. However this was from an inhalation study and so this could have been due to cklearance of manganese from the lung by mucocilliary elevator and subsequent direct excretion into the GI-tract.
As already discussed earlier, it has been shown that manganese can enter the brain via carrier-mediated transport and can leave the brain via the slower processes of diffusion only, and thus the elimination of manganese from the brain has been observed to be slower than for other tissues.

In rats given radio- labelled acetate in the diet, 50% of the radiolabel was excreted as CO2; (see human data for daily turnover)


Human data

The most important excretion route for manganese is via the bile into the faeces. Manganese has been shown to bind to bile acids for biliary excretion. It has been demonstrated that 40% to 70% of a dose of 54 MnCl2 or 54 Mn2O3 inhaled by man is excreted with the faeces within four days. The elimination of absorbed manganese is dependent on bile production and flow into the small intestine. The efficiency of this process is delayed in the rat neonate following birth and may lead to delayed elimination following high doses of manganese. However in human neonate, efficient bile flow normally occurs within the first three days after birth, therefore the accumulation of manganese in human neonates may not be as much of a potential problem as with rat neonate. The manganese elimination in the bile usually undergoes enterohepatic recirculation. The enterohepatic circulation plays an important role in the maintenance of the steady- state manganese concentrations in body. The pancreas and the whole of the small intestine have a capacity to excrete low levels of manganese.
Renal elimination accounts for only about 0.1% to 0.3 % of administered manganese. Only traces are eliminated with perspiration, tears, hair and finger- and toe nails.

The half-life of manganese in urine is estimated to be less than 30 hours following cessation of exposure of manganese, see attachment for details.

The exceedingly fast initial clearance of manganese from blood means that the measurement of blood manganese levels is not suitable for estimation of very recent exposure but is more a reflection of overall body burden.

The level of manganese in scalp hair has shown to be useful in determining a population ´s overall exposure to manganese (Stauber at al., 1987). However the analytical procedure and the interpretation of the data are very difficult, because of the need to distinguish between endogenous manganese ( from the body) and exogenous manganese from the environment.

Interpreting data on oral absorption of manganese or manganese elimination is difficult, because the body ´s natural homeostatic control of manganese must be taken into account, particularly so if this is following oral absorption. The typical whole-body terminal half-lives for manganese in healthy volunteers are around 30-40 days (Mena et al, 1969, Finley et al 2003). The elimination of manganese depends from factors such as prebody-burden of manganese and dietary levels.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
It is likely that manganese is metabolised by the body in the form of converting manganese from the Mn2+ valence state to the Mn3+ valence state, and that ingested manganese is absorbed as Mn2+ possible bound to alpha2-macroglobulin or albumin. In transversing the liver it is removed nearly quantitatively, but a small proportion is oxidised to the Mn3+ valence state, bound to transferrin and enters the systemic circulation to be transported to the tissues. It had been proposed that the oxidation state of manganese exposure may be an important of the toxicokinetics of manganese and tissue toxicodynamics and subsequently neurotoxicity (Reaney et al. 2006).
The kinetic processes for the uptake and elimination of orally absorbed manganese are likely to be different following iv or inhalation administration whereby the manganese enters the systemic circulation without passing through the liver. The influence of the absorption process followed by direct delivery to the liver (first pass effect) can considerably reduce the bioavailability of a material compared to iv administration. Manganese from inorganic compounds is almost entirely excreted in the bile where it undergoes enterohepatic recirculation.
Manganese as a natural element can only change its valence state by oxidation or reduction. Manganese can exist under natural conditions in the valence state 0 to 7 (synthetic -1). In the body of humans and animals it exist in most cases in the valence state +2 or +3.

Any other information on results incl. tables

see attached expert statement

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study results
The present expert statement covers all relevant toxicokinetic parameters to assess the behaviour of manganese (II) acetate (Mn(acO)2) in the body, the available information is sufficient to enable one to perform a proper risk assessment. Hence, no further information needs to be gathered and further studies can be omitted due to animal welfare. In conclusion, Mn(acO)2 has only a low potential for bioaccumulation, and its absorption during normal environmental or slightly elevated exposure does not bear any potential for adverse effects but is also required to maintain the normal functionality of the body.
Executive summary:

In order to assess the toxicological behaviour of Manganese (II) acetate, the available physico-chemical and toxicological data have been evaluated. The substance is expected to be well absorbed after oral exposure. Concerning the absorption after exposure via inhalation, as the chemical has really low vapour pressure, a decomposition temperature and is furthermore distributed as aqueous solution; it is clear, that the substance has a low availability for inhalation. However, in case inhalable dusts are formed by the less present water-free salt, inhalative absorption has to be considered. The substance has only a low bioaccumulative potential, which is only due to the manganese cation. Its absorption during normal environmental or slightly elevated exposure does not bear any potential for adverse effects, because Manganese is also a required trace element to maintain the normal functionality of the body and acetate is a common metabolite in many metabolic pathways.