Reaction mass of disodium [2,2'-(imino-kappaN)dibutanedioato-kappa2O1,O4(4-)]manganese(2-) and sodium sulphate

Endpoint summary

Description of key information

- IDHA chelating agent: OECD 407; 28-day oral(gavage) study, rats, NOAEL: 200 and 1000 mg/kg bw in males and females, respectively;
- Mn(2Na)EDTA: OECD 422; rats, NOAEL: 500 mg/kg bw (males/females);
- Manganese and its inorganic compounds (oral exposure): ATSDR, 2012: interim guidance value of 0.16 mg/kg bw ;
- Manganese and its inorganic compounds (inhalation exposure): SCOEL, 2011; ATSDR, 2012: IOELV of 0.2 mg/m³ (inhalable fraction);
- Estimated dose levels for Mn(2Na)IDHA: NOAEL of 2.016 mg/kg bw for humans (oral); NOAEC of 2.52 mg/m³ for humans (inhalation).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2.016 mg/kg bw/day

Study duration:

chronic

Species:

other: human

Quality of whole database:

High quality (there is sufficient data for hazard assessment).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2.52 mg/m³

Study duration:

chronic

Species:

other: human

Quality of whole database:

High quality (there is sufficient data for hazard assessment).

Additional information

No repeated dose toxicity studies are available for the target substance Mn(2Na)IDHA. Therefore, the data on free IDHA chelating agent and Mn(2Na)EDTA have been used to assess toxicity potential of Mn (2Na) IDHA at repeated exposures (please refer to read-across statement). Due to the fact that Mn(2Na)IDHA can dissociate at low pH level of stomach, the toxicity results of studies conducted with inorganic manganese compounds were also taken into account.

28 -Day oral (gavage) study with the chelating agent IDHA in rats

Iminodisuccinic acid, sodium salt was administered orally to Wistar rats (5 males and 5 females per dose) once a day, by gavage in target doses of 0 (vehicle control), 40, 200, and 1000 mg/kg body weight over a period of 4 weeks (Stropp and Popp, 1997; Report No. PH 26446). In addition, 5 male and 5 female rats per group were treated with the vehicle or 1000 mg/kg body weight and observed for reversibility, continuance or delayed occurence of toxic effects during a recovery period of 14 days. Mortality was unaffected by treatment with the test substance. Appearance, clinical findings and general behaviour were not altered by treatment with the test substance up to and including 1000 mg/kg body weight. Growth and food consumption were not affected by the treatment.

Hematological investigations gave no indication of toxicologically relevant damage to blood, hematopoetic organs or coagulability up to and including 1000 mg/kg body weight. Clinical laboratory tests produced no evidence of treatment-related metabolic or organ damage.

Gross and histopathological investigations of various organs and tissues gave no indication of test-compound-related functional or morphological changes in both sexes up to and including 1000 mg/kg body weight. The organ weights were unaffected with the exception of a decrease of relative thymus weight in the recovery group (1000 mg/kg body weight). Taking into account the discrepancy of observations in the main groups and in the recovery groups and the fact that there was no evidence of treatment related effect in haematology examinations as well as in the histopathological examinations of thymus in the main group and the recovery group and of adrenals, spleen, draining and distant lymph nodes and bones in the main groups the decrease of relative thymus weight is considered to be of no toxicological relevance.

The assessment of motor activity (horizontal activity) showed a wide variety of individual values. There was no effect on the motor activity up to 200 mg/kg body weight in both sexes. With 1000 mg/kg body weight the motor activity in the males in the main groups and in the recovery groups was below that of the respective control group at the end of the sampling period. Functional observations as another marker for neurotoxicity gave no evidence of a neurotoxic potential. In addition, the histopathological examination of brain, spinal cord and sciatic nerve gave no evidence of a neurotoxic action of the test material. In conclusion, taking into account all available data with regard to neurotoxicity, the toxicological relevance of the findings with regard to motor activity is questionable.

Under the conditions described the administration of Iminodisuccinic acid, sodium salt, to male and female rats was tolerated without treatment-related lesions up to and including 200 mg/kg body weight in the males and up to and including 1000 mg/kg body weight in the females. Therefore the no observed adverse effect level (NOEL) for the daily administration of Iminodisuccinic acid, sodium salt, is considered to be 200 mg/kg body weight in males and 1000 mg/kg body weight in females. The toxicological relevance of the isolated effect on motor activity in the males with 1000 mg/kg body weight is questionable.

Combined oral repeated dose toxicity study with reproduction /developmental toxicity screening test with Mn(2Na)EDTA in rats

In a study, the possible effects of Mn(2Na)EDTA on reproductive performance and development, and its sub-chronic toxicity were examined in groups of 12 male and 12 female Wistar rats (OECD 408, OECD 422, Wolterbeek, 2010; Report No. V8650). Mn(2Na)EDTA was administered daily by gavage during a premating period of 10 weeks and during mating, gestation and lactation until postnatal day 4. The dose levels were 0 (tap water only), 150, 500 and 1500 mg/kg bw/day. Additionally, an extra group was included in the study. The animals of this group were treated with the highest concentration of the test item by gavage and received a surplus dietary level of Zn. This group with additional dietary zinc was added to the study to compensate for possible (repro-) toxic effects, if any, due to the zinc-chelating properties of EDTA.

Data with regard to fertility/reproduction/developmental toxicity are presented under “toxicity to reproduction”, and “developmental toxicity”.

Daily clinical observations during the premating, mating, gestation and lactation period did not reveal any treatment-related changes in the animals’ appearance, general condition or behaviour. Detailed clinical observations, functional observational battery observations and motor activity assessment did not indicate treatment related effects on neurobehaviour. No treatment-related effects on body weights and body weight changes of male and female animals were observed except for females in the highest dose groups that showed a decreased mean body weight during the last week of the gestation period which was most probably related to an increased foetal mortality. No statistically significant adverse effects were observed on food consumption of male and females animals during the entire study. Water consumption was measured during 2 consecutive days of two weeks during the premating period. During all these 4 days, water consumption of particularly male and also of female animals treated with the highest concentration of the test item was increased. Most probably, this effect was due to the high sodium exposure of these animals via the test item. No treatment-related adverse effects were observed on haematology and clinical chemistry parameters between the control and treatment groups.

The volume of urine was increased in the male animals of the mid- and highest dose groups and in the female animals of the high dose group which resulted in an increased concentration of creatinine. The absolute amount of creatinine excreted was not affected. The sodium concentration and the sodium/creatinine ratio were statistically significantly increased in both male and female animals of the two groups treated with the highest concentration of the test item (irrespectively of dietary zinc supplementation).

Both the absolute and relative weights of the kidneys of the male and females of the two groups treated with the highest concentration of the test item (irrespectively of dietary zinc supplementation) were statistically significantly increased. Furthermore, the absolute and relative weights of the spleen of the female animals of the highest dose group with supplementary zinc were statistically significantly decreased. At necropsy no treatment related gross changes were observed in male and female animals.

In the two groups treated with the highest concentration of the test item (irrespectively of dietary zinc supplementation) an increase in the incidence of rats showing very slight diffuse subcortical tubular dilatation was observed in the kidneys, reaching the level of statistically significance in the female animals only.

Based on the results of this repeated study (specifically water consumption, urinary sodium concentration, weight of and histopathological effects in kidneys as observed in the animals treated with the highest concentration of the test item), the No Observed Adverse Effect Level (NOAEL) is 500 mg/kg body weight/day. The NOAEL is comparable to that found by free EDTA's (EDTA-Na4 and EDTA-H4; RAR, 2004).

Repeated oral toxicity study with rats using manganese acetate.

The purpose of this study was to investigate the effect of oral administration of manganese acetate on the kidneys and urinary bladder of Sprague-Dawley (SD) rats (Ponnapakkam et al., 2003). Male and female SD rats (150 to 175 g), 6 weeks old, were administered 0, 306, 612, 1225 and 1838 mg/kg bw of manganese acetate for 63 days by oral gavage. At the end of 63 days, 50% of the animals were sacrificed and kidney tissue was isolated and fixed for histopathological studies (study A). The remaining 50% were cross-mated and dosing ceased. Animals were sacrificed after 2 weeks (study B). Male treated animals were noted to have viscous, gritty urine in the urinary bladder, and the high-dose groups had urinary bladder stones (uroliths). Histopathologically, the most striking lesions were observed in the kidneys and prostate glands of male animals. Mild-to-moderate tubulointerstitial nephritis with tubular proteineous and glomerulosclerosis was observed in animals of all treatment groups. Urolithiasis in the urinary bladder was confirmed in 33% to 66% of treated animals. Female animals did not show a significant difference above controls in renal tissues. Results of this study suggest that male rats are more sensitive to the effects of high levels of manganese given orally than female rats and that the genitourinary structures represent target organs of toxicity. The lowest dose level of 306 mg/kg bw (1.25 mmol Mn per kg bw.) was considered to be LOAEL because of histopathological changes in kidneys and urinary bladder. For comparison, Mn(II)Na2 -EDTA produced only very slight renal changes at the highest dose of 1500 mg/kg bw (3.86 mmol Mn per kg bw), indicating a much lower renal toxicity of Mn(II)Na2 -EDTA because of (a) higher NOAEL, (b) longer duration of treatment, and (c) only very slight renal toxicity.

Toxicity results of studies conducted with manganese inorganic compounds

Manganese is a naturally occurring element found in rock, soil, water, and food (ATSDR, 2012). In humans and animals, manganese is an essential nutrient that plays an important role in bone mineralization, protein and energy metabolism, metabolic regulation, cellular protection from damaging free radical species, and formation of glycosaminoglycans. Manganese acts as both a constituent of metalloenzymes and an enzyme activator. Manganese, in its activating capacity, can bind either to a substrate, or to a protein directly. Manganese has been shown to activate numerous enzymes involved with either a catalytic or regulatory function (e.g., transferases, decarboxylases, hydrolases). Although manganese is an essential nutrient, exposure to high levels via inhalation or ingestion may cause some adverse health effects.

The most common health problems in workers exposed to high levels of manganese involve the nervous system, although adverse respiratory and cardiovascular effects were also reported. The neurological health effects include behavioral changes and other nervous system effects, which may include psychiatric manifestations. Among neurotoxic signs and symptoms movements that may become slow and clumsy is frequently observed. These combination of symptoms when sufficiently severe is referred to as “manganism” (Parkinson’s like effects) (ATSDR, 2012).

Oral exposure:

Manganese has been shown to cross the blood-brain barrier and a limited amount of manganese is also able to cross the placenta during pregnancy, enabling it to reach a developing fetus (ATSDR, 2012). Nervous system disturbances have been observed in animals after very high oral doses of manganese, including changes in behavior. Sperm damage and adverse changes in male reproductive performance were observed in laboratory animals fed high levels of manganese (see section "Toxicity to reproduction"). Impairments in fertility were observed in female rodents provided with oral manganese before they became pregnant. Illnesses involving the kidneys and urinary tract have been observed in laboratory rats fed very high levels of manganese. These illnesses included inflammation of the kidneys and kidney stone formation.

Effects in children:

Studies in children have suggested that extremely high levels of manganese exposure may produce undesirable effects on brain development, including changes in behavior and decreases in the ability to learn and remember. In some cases, these same manganese exposure levels have been suspected of causing severe symptoms of manganism disease (including difficulty with speech and walking). In case of Mn(2Na)IDHA, the direct exposure to children is unlikely since the substance is an ingredient of professional fertilizers used only by farmers not by consumers. An extremely high level of manganese exposure mentioned in ASTDR 2012 is rather not common and concerns children and adults who live on contaminated area e.g. nearmetallurgicalplants, foundries, mines etc. It was also observed on area where food from sources irrigated with sewage, so the children may have been exposed to increased levels of other metals, such as lead or mercury.

Adequate intake (AI) value for manganese for men and women of 2.3 and 1.8 mg manganese/day, respectively (for 70-kg individuals, this would result in exposures of 0.033 and 0.026 mg manganese/kg/day, respectively) was established. An interim guidance value of 0.16 mg manganese/kg/day for adults of 19-year and older is lower than AI and can serve as systemic DNEL . The interim guidance value is necessary because of the prevalence of manganese at hazardous waste sites and the fact that manganese is an essential nutrient.

Inhalation exposure:

The inhalation of a large quantity of dust or fumes containing manganese may cause irritation of the lungs which could lead to pneumonia (ATSDR, 2012). Loss of sex drive and sperm damage has also been observed in men exposed to high levels of manganese in workplace air (see section “Toxicity to reproduction”). The manganese concentrations that cause effects such as slowed hand movements in some workers are approximately twenty thousand times higher than the concentrations normally found in the environment. Manganism has been found in some workers exposed to manganese concentrations about a million times higher than normal air concentrations of manganese. Other less severe nervous system effects such as slowed hand movements have been observed in some workers exposed to lower concentrations in the work place.

There is a substantial literature on the effects of manganese on the human nervous system (SCOEL, 2011). Of the 28 studies considered, three key studies were identified. These were the cross-sectional studies of Roels et al. (1992), Gibbs et al. (1999) and Myers et al. (2002) (cited in SCOEL, 2011). There were used in the development of the IOELV proposals of this SCOEL Recommendation. Roels et al. (1992) identified adverse effects on reaction time, tremor and hand–eye coordination in 92 workers exposed to manganese dioxide dust (with current average total and respirable fractions, 0.95 and 0.22 mg/m³ (GM), respectively, measured in each worker by personal sampling). LOAEL for total and respirable manganese dust were established 3.575 and 0.730 mg/m³, respectively. According to Gibbs et al. (1999), no neurofunctional effects were observed among manganese metal (electrolytic) production workers at average total and respirable fractions 0.11 and 0.04 mg/m3 (GM), respectively. Myers et al. (2003) observed "no exposure-response relationship" in workers of manganese smelters at a cumulative exposure of 1.3 mg/m³ x years (inhalable). This was considered as LOAEL. The results of further human studies are: a LOAEL for non-clinical hand tremor was around the mean manganese exposure to 0.301 inhalable and 0.036 mg/m³ respirable ( Bast-Pettersen et al., 2004, cited in SCOEL, 2011); LOAEL of 0.423 mg/m³ for total dust and 0.338 mg/m³ for respirable dust was established by Ellingsen et al. (2008), this corresponds to NOAEL of 0.137 mg/m³ for total dust or 0.110 mg/m³ for respirable dust. A reference concentration (RfC) of 0.05 μg/m3 for manganese (environmental exposure) was established by EPA (cited in ATSDR, 2012). This value is derived from a benchmark dose calculation of 0.02 mg/m³ (5% increased risk based on 5 last years average exposure levels).

Based on this information, a reasonable respirable IOELV of 0.05 mg/m³ and a reasonable inhalable IOELV of 0.2 mg/m³ were recommended.

 

An MRL of 0.0003 mg manganese/m3 (manganese in respirable dust; 0.3 μg manganese/m³) has been derived for chronic inhalation exposure (365 days or more) to manganese (ATSDR, 2012). This value includes uncertainties factors covering neonates, children, pregnant women, age and stage of health. For workers, OSHA set a legal limit of 5 mg/m3 for manganese compounds and dust in air averaged over an 8-hour work day. A concentration limit of 0.2 mg/m³ as 8 -hour TWA was set by ACGIH for manganese (element).

Estimation of an equivalent realistic NOAEL(C) for Mn(2Na)IDHA

Mn(2Na)EDTA and Mn(2Na)IDHA are stable compounds but they can de-complex under acidic conditions of stomach, releasing manganese ions which can become systemically available. Moreover, intestinal absorption of free IDHA chelating agent (37 %) is higher than oral absorption determined in experimental studies with free EDTA (5 %) or EDTA metal complexes (less than 1 -2 %). Therefore, the read-across from NOAEL established for systemic effects for Mn(2Na)EDTA (500 mg/kg bw) or IDHA chelating agent (NOEL of 200 and 1000 mg/kg bw for males and females, respectively) may underestimate the risk of systemic toxicity of Mn(2Na)IDHA. Especially the risk of systemic toxicity of manganese being systemic available after exposure to Mn(2Na)IDHA could be underestimated because of higher absorption percentage in case of IDHA. Moreover, the toxicity of manganese is expected to be more considerable comparing with the toxicity of the equivalent quantities of free chelating agent IDHA.

Therefore, an estimated dose level based on the toxicity data available for manganese is considered more appropriate. For example, the lowest intermediate-duration daily dose associated with the absence neurobehavioral effects in adult rats is 5.6 mg manganese/kg/day for severely impaired cognitive performance in a maze test following a 30-day exposure (Shukakidze et al. 2003, cited in ATSDR, 2012) while NOAEL of 200 and 1000 mg/kg bw were established in a 28-day study for IDHA in males and females, respectively. According to equation:

4H+ + MnIDHANa2 + H2O⇔H4IDHA + 2Na+ + Mn2+

5.6 mg Mn would correspond to 35.3 mg of Mn(2Na)IDHA ((MW of Mn(2Na)IDHA is 346.08 g/mol/ MW of manganese is 54.94 g/mol) x 5.6). Taking into account 50 % oral absorption established for Mn(2Na)IDHA (please refer to toxicokinetic section), the estimated NOAEL would result in 70.6 mg/kg bw Mn(2Na)IDHA: 35.3 mg/kg bw x (100 %/ 50 %). 70.6 mg/kg bw is lower than NOAEL of 500 mg/kg bw established for Mn(2Na)EDTA. Therefore, an estimated dose level for Mn(2Na)IDHA is considered more appropriate approach than the read-across from NOAEL established for the read-across substances. The interim guidance value of 0.16 mg Mn/kg bw (ATSDR, 2012) is considered to be a sufficiently safe level to estimate an equivalent dose for Mn(2Na)IDHA for systemic effects for humans. It corresponds to 1.008 mg/kg bw for Mn(2Na)IDHA: ((MW of Mn(2Na)IDHA is 346.08 g/mol/ MW of Mn is 54.94 g/mol) x 0.16 mg/kg bw). Taking into account 50% oral absorption established for Mn(2Na)IDHA, the estimated DNEL would be 2.016 mg/kg bw: 1.008 x (100 %/ 50%).This dose level however may be overprotective but it can serve as an internal DNEL for systemic effects (no assessment factors are needed; see DNEL calculation section).

Similarly as for oral exposure route, an estimated NOAEC for humans for inhalation exposure route make sense to be derived.An IOELV value of 0.2 mg/m³ exists for inhalable fraction for manganese (SCOEL, 2011; ATSDR, 2012). Since no respirable fraction is expected for Mn(2Na)IDHA (particles of Mn(2Na)IDHA are > 100 µm), this IOELV represents an appropriate reference level to calculate an equivalent amount for Mn(2Na)IDHA. This limit would correspond to 1.26 mg/m³ of Mn(2Na)IDHA: ((MW of Mn(2Na)IDHA 346.08 g/mol/ MW of manganese 54.94 g/mol) x 0.2 mg/m³). Taking into account 50 % absorption by inhalation (equal to oral absorption), the estimated NOAEC (=DNEL for inhalation, systemic effects) would result in 2.52 mg/m³: 1.26 x (100 %/50 %). The estimated concentration of 2.52 mg/m³ is of the same order of magnitude as the limit of 5 mg/m³ set by OSHA for manganese compounds and dusts.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
No study is selected since NOAEL is estimated for Mn(2Na)IDHA based on the interim guidance value available for manganese. The reason is that toxicity is believed to be mediated by manganese rather than by chelate moiety. Additionally, IDHA (and its chelates) are expected to be absorbed more extensively due to the higher oral absorption of IDHA comparing to EDTA and its salts.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No study is selected since NOAEC is estimated for Mn(2Na)IDHA based on the IOELV available for inhalable fraction of manganese. The reason is that toxicity of Mn(2Na)IDHA is believed to be mediated by manganese rather than by chelate moiety. Mn(2Na)IDHA is expected to be swallowed after its deposition in the airways. Therefore, inhalation absorption, if occur, is confined to oral absorption. Additionally, IDHA (and its chelates) are expected to be absorbed more extensively due to the higher oral absorption of IDHA comparing to EDTA and its salts.

Justification for classification or non-classification

NOAEL of 200 and 1000 mg/kg bw were established for the chelating agent IDHA in the 28-day oral (gavage) study in male and female rats, respectively. No target organ toxicity was observed in this study. NOAEL of 500 mg/kg bw was established in the combined study in rats with Mn(2Na)EDTA. Target organs were kidneys. For Mn(2Na)IDHA, an estimated NOAEL of 2.016 mg/kg bw was derived based on interim guidance value of 0.16 mg/kg bw (ATSDR, 2012) for manganese. No target organ toxicity is expected at this dose level. Similarly, no systemic or target organ toxicity can be expected at the estimated concentration of 2.52 mg/m³ based on the existed IOELV of 0.2 mg/m³ for manganese. Therefore, Mn(2Na)IDHA does not meet the criteria for classification and labelling for systemic target organ toxicity after repeated exposures (STOT-RE) in accordance with European regulation (EC) No. 1272/2008.

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