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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied, based on the chemical structure of the potential analogues, their toxicokinetic behaviour and other available (eco-)toxicological data. Please refer also to the detailed read-across justification attached in section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar toxicological properties due to their structural similarity, resemblance to their chemical reactivity, and biotransformation products in toxicological compartments. In other words, there is a clear chemical analogy (“biotransformation to common compound", scenario 1 of the Read Across Assessment Framework (ECHA 2017)).
The solvation of both methylamine and methylammonium chloride in water results in solutions of the methylammonium cation (“common breakdown product", scenario 1 of the Read Across Assessment Framework (ECHA 2017)). The toxicity of the respective counterion (“non-common compound” – Cl-) is in this case negligible; as Cl does not drive toxicity effects in mammalian species because it is one of the main electrolytes and is required in large amounts in living organisms. So any toxicity of the amounts originated from the methylammonium chloride at maximum dose levels that would be used in toxicity studies is not expected.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

source substance:
methylamine hydrochloride (methylammonium chloride)
structural formula: CH5N.ClH
SMILES: [Cl-].C[NH3+]
CAS 593-51-1
purity: not specified

target substance:
methylamine
structural formula: CH5N
SMILES: CN
CAS 74-89-5
purity: ≥ 80 – 100 %

No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
Read across from the structural analogue to the target substance is based on the high structural similarity of the analogue with the substance of interest and the similarity of their toxicological characteristics.
The target substance MMA (CAS 74-59-5), as well as the source substance MMA-HCl (CAS 593-51-1) belong to the category of the “Aliphatic amines” according to the profiler “US EPA New Chemical Categories” in the OECD QSAR Toolbox v4.1.
The basicity of amines increases with the length of the aliphatic rest due to electron releasing properties of alkyl groups: the higher the pKa value, the weaker the acid, so the stronger the base. Monomethylamine (the primary amine with a central nitrogen atom and 1 methyl group and 2 hydrogen-atoms) is the least basic of the aliphatic (primary) amines. Monomethylammonium chloride (composed also of one methyl group attached to the nitrogen atom, which is regarded as the common structure / functional group) is no longer basic, but neutralised (please also refer to the following paragraphs). Therefore, it has to be kept in mind that MMA-HCL is an example of a worst case read-across according to RAAF, as MMA tested as salt may achieve very high doses because it is not corrosive.
Furthermore, the chemicals are characterized by a common Mode Of Action (MOA) in detail as “narcotic amines” according to Acute aquatic toxicity MOA by OASIS in the OECD QSAR Toolbox v4.1.
Methylamine and methylammonium chloride - as primary aliphatic amines - undergo similar reactions and resemble each other in their physico-chemical properties. The fundamental properties of different amine classes (primary, secondary and tertiary) – basicity and nucleophilicity – are very much the same (Morrison and Boyd, 1987).
Typical reactions of amines are salt formation, alkylation, and conversion into amides and Hofmann elimination from quaternary ammonium salts (Morrison and Boyd, 1987).
As already mentioned above, they are linked by the common functionality of one central nitrogen atom which bears an unshared pair of electrons and tends to share these electrons determining a similar chemical behaviour. This unshared electron pair can accept a proton - forming a substituted ammonium ion. The tendency to share this electron pair underlies the entire chemical behaviour of amines as a group and this was considered as main / basic parameter, which is suitable for read-across in an analogue approach within an/a analogue/category approach.
The dissociation constant of MMA allows the conclusion that virtually all molecules of methylamine - when dissolved in an excess of water - are present as the methylammonium cation. Moreover, the available data of MMA with hydrochloric acid shows clearly that there will be no relevant amounts of the amine available once in contact with the bodies’ fluids. Only the ionic form is the relevant species present. This applies to all relevant exposure routes, i.e. inhalation, dermal, and oral. So, in consequence, the solvation of both methylamine and methylammonium chloride in water would result in solutions of the methylammonium cation (“common breakdown product"). Therefore, one must only regard the physico-chemical properties of the respective counterion. Methylamine solutions are accompanied by the hydroxyl anion OH-, resulting in alkaline solutions, whereas the chloride anion of the methylammonium chloride solutions is not expected to trigger significant changes in the pH and exhibit any significant toxicological effects. Both anions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions.
The corrosive effects of MMA can certainly be explained by the high concentration of hydroxyl anions in MMA solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. MMA (gas) is legally classified as Skin Irrit. 2 and Eye Dam. 1, MMA (aqueous solution) is legally classified as Skin Corr. 1B; MMA-HCl has no legal classification for Skin or Eye irritation/corrosion. There is no data available on skin irritation of MMA. Experimental data showed MMA-HCl to be only transiently mildly irritating to the eyes of rabbits (no C & L is triggered). MMA-HCl is expected to be minor irritating when compared to MMA, because of the pH neutralisation caused by HCl. Consequently, the enhanced absorption of MMA compared to MMA-HCl due to damage of the skin barrier should be regarded when the substances are applied in corrosive concentrations or without pH neutralization.
Besides the influence of HCl on the pH value of an aqueous solution, it does not bear a relevant intrinsic property, allowing one in general to focus on the methylammonium cation. Generally, it should be denoted that very commonly in literature there is no differentiation made between MMA and MMA-HCl.
Primary amines are considered to be hydrolytically stable and both substances have been shown to be readily biodegradable. The log Koc values are both negative and relatively close and support the read-across approach. Furthermore, the results show that the substances do not have a significant potential for persistence (not P not vP) or bioaccumulation in organisms (not B not vB). Moreover, they are similar in their toxicity endpoints (despite the above mentioned missing corrosivity in the case of MMA-HCl).
The similar findings (refer also to the data matrix outlined below) for both substances support the conclusion that the identical molecule will be formed from both substances when applied systemically, and this molecule, i.e. the methylammonium cation, is responsible for their behaviour and the observed effects. In consequence, the methylammonium cation is indeed what is left to be considered in both cases and similar effects can reasonably be expected when using data from MMA-HCl for the lacking endpoints, compared to the data obtained with MMA.
Hence, MMA-HCl may perfectly serve as a worst case read-across substance for MMA. So, the available data on MMA-HCl can be used to cover all systemic endpoints currently lacking from MMA, making further testing obsolete.

4. DATA MATRIX
There is data available on the environmental fate and behaviour, ecotoxicological and toxicological properties of MMA. Data on MMA-HCl covers data on Biodegradation, Toxicity to aquatic algae and cyanobacteria, Toxicokinetics, oral Repeated dose toxicity, Genetic toxicity in vivo and Toxicity to reproduction/Developmental toxicity. Hence, the identification and discussion of common properties of MMA and MMA-HCl will be mainly based on this and physicochemical data.
The different physical state of the two substances (MMA is - as a pure substance - gaseous at room temperature, MMA-HCl is a solid primary ammonium salt) triggers some differences in the physico-chemical properties like Melting point, Boiling point, Decomposition temperature and Vapour pressure. Nevertheless, regarding the application of both substances, i.e. their distributed form, the gaseous character of MMA becomes less relevant as the substances are usually not applied in their pure forms but rather as aqueous solutions.
The available data for the following physico-chemical properties, which are among others relevant for absorption, are very similar. Both substances are small molecules with a low molecular weight of 31.042 (MMA) resp. 67.019 (MMA-HCl), they are both very soluble in water (completely miscible in water (MMA) and at least 1080 mg/L at 20°C (MMA-HCl)), have a negative logPow (-0.713 (aqueous solution, 25°C, pH 11.1 - 11.4; MMA) and -3.82 (MMA-HCl)), and both are readily biodegradable. Although being expected to be hydrolytically stable in the natural environment, they both have a very low potential for bioaccumulation in aquatic and terrestrial organisms. Most importantly, MMA has a pKa of 10.79 at 20°C (≙ pKb = 3.21), which indicates that methylamine exists almost entirely in the cationic form as methylammonium cation at pH values of 5 to 9.
MMA (not neutralised) has a toxicity potential towards fish and aquatic invertebrates, but does not have to be classified as hazardous. MMA (neutralised) and MMA-HCl have a lower toxicity potential to aquatic organisms and do not have to be considered to be acutely harmful to fish or aquatic invertebrates, nor to microorganisms (no C & L). MMA and MMA-HCL have been shown to be toxic to aquatic algae and cyanobacteria (shown in Pseudokirchnerella subcapitata, Scenedesmus obliquus and Microcystis aeruginosa). Their aquatic toxicity potential under real environmental conditions has to be judged carefully, since, methylamine and methylamine hydrochloride are readily biodegradable in nature. As such, both can be considered as non-toxic to aquatic organisms and thus do not have to be classified as hazardous as per the CLP classification criteria.
Regarding their toxicity towards mammals, both substances exert their acute toxicity oral toxicity in the same range and both are classified as Acute tox 4 and MMA also as STOT SE 3 (C≥5 %). Moreover, MMA is corrosive / irritative to the skin and the eyes(MMA (gas) = Skin Irrit. 2 and Eye Dam 1; MMA (aqueous solution) = Skin Irrit 1B); in the case of MMA –HCl no classification is warranted.
Regarding their repeated dose toxicity, the No-observed-adverse-effect-levels of both substances to differ to a certain extent (NOAEL ≥ 10 versus NOAEL = 500 mg/kg bw/day). For MMA HCL there is also data for toxicity to reproduction and developmental toxicity available (NOEL (reproduction/systemic tox) = 500 mg/kg bw/day and NOAEL (maternal/developmental tox.) = 155 mg/kg bw/day).
No data on long-term toxicity to fish; on long-term toxicity to aquatic invertebrates or carcinogenicity were available.
Reason / purpose:
read-across source
Preliminary studies:
listed in the original paper
Type:
metabolism
Results:
MA is a substrate for the semicarbazide-sensitive amine oxidase (SSAO)
Metabolites identified:
yes
Details on metabolites:
Vt was determined. Vt=product formation from a mixture of the two substrates (A and B). Specific inhibition of MMA-metabolism by semicarbazide. These results suggest that MA is predominantly, if not exclusively, a substrate for the soluble amine oxidase in human plasma.

1.Comparison of MA metabolism determined by colorimetric and radiochemical assay:

Mean (± S.E.) specific enzyme activities for the group were 281 ± 32 nmol H2O2 (mg protein)/1 h for the colorimetric assay and 366 ± 31 nmol MA metabolized (mg protein)/1 h for the radiochemical assay.

2.Effects of clorgyline and semicarbazide upon MA metabolism in rat aorta, human umbilical artery and plasma:

The deamination of both MA and BZ was largely resistant to inhibition by clorgyline concentrations up to 1 mM, whereas similar concentrations of semicarbazide produced a progressive degree of inhibition which resulted in virtually complete inhibition of MA metabolism, and around 80% inhibition of BZ metabolism at 1 mM semicarbazide. These results suggest that MA is predominantly, if not exclusively, a substrate for the soluble amine oxidase in human plasma.

3. Kinetic constants for MA metabolism in rat aorta, human umbilical artery and plasma

Mean values (± S.E.) from four different samples were: Km(µM) of 516 ± 74 (MA) and 225 ± 36 (BZ): Vmax (nmol (mL serum)/1 h) of 48 ± 5 (MA) and 28 ± 3(BZ).

4. Inhibition of [14C]MA metabolism in human umbilical artery and plasma by unlabelled BZ

BZ was found to be a competitive inhibitor of MA metabolism. Mean values (± S.E.) for Ki (µM) from experiments with different samples of each enzyme source were 220 ±10 (umbilical artery, n=3) and 172 ± 27 (plasma, n=4).

5. Kinetic constants for (14C]BZ metabolism in human umbilical artery and product formation from mixtures of[14C]BZ and [14C]MA

200 µM [14C]BZ and 800 µM [14C]MA were cocultivated with homogenates of human umbilical artery. The substrate concentrations were chosen to approximate closely to their Km values-found in the earlier work reported in the original paper. If a single enzyme metabolizes two different substrates at the same catalytic site, the product formation calculated (for detailed calculations see original paper) should be comparable with that experimentally determined. Table 1 shows the results obtained in this experiment:

Table 1. Comparison of predicted and actual metabolite formation in mixtures of [14C]BZ and [14C]MA.

Expt. no.

vA

vB

Predicted vT

Actual vT

Ratio (Actual vT/ Predicted vT)

d min-1

d min-1

d min-1

d min-1

 

1

623

3526

2832

2743

0.97

2

577

3460

2757

2822

1.02

3

858

5401

4274

4759

1.11

4

721

4481

3552

3681

1.04

 

 

 

 

Mean ratio= 1.04 ± 0.03

vAand vBrepresent [14C]metabolite formation (in d min-1) from 200 µMBZ and 800 µMMA, respectively, in assays containing the appropriate amine individually. The predicted metabolite formation (vT) from a mixture of the two amines if metabolized by the same enzyme was determined as described in the text, and compared with actual experimentally determined values. Experiments were carried out on 4 different arteries, assays on each artery involving concurrent determinations (in triplicate) of BZ and MA metabolism alone, and in a mixture.

 

6. Effects of β-aminopropionitrile (BAPN) upon MA metabolism in human umbilical artery

BAPN was found to be a competitive inhibitor of MA metabolism

Conclusions:
SSAO metabolizes MA.
The vasculature and plasma contain semicarbazide-sensitive amine oxidase (SSAO) which metabolizes MA
Executive summary:

Lyles et al. investigated in 1990 the metabolism of methylamine hydrochloride in vitro. An ion exchange radiochemical assay has been developed to study the deamination of [14C]methylamine (MA) in homogenates of rat aorta and human umbilical artery, as well as in samples of human plasma. MA metabolism was found to be inhibited almost completely by 1 mM semicarbazide, but virtually unaffected by 0-1 mM clorgyline, suggesting that MA is a substrate for the semicarbazide-sensitive amine oxidase (SSAO) activities which also metabolize benzylamine (BZ) in these sources. Mean Km values for MA metabolism by aorta, umbilical artery and plasma were 182, 832 and 516 µM, respectively, with corresponding Vmax values in aorta and umbilical artery of 100 and 590 nmol /(mg prot.) h, and in plasma of 48 nmol /(mL serum) h. Kinetic constants determined for [14C]BZ metabolism in plasma (by an organic solvent extraction assay) and in umbilical artery (by the ion exchange assay) yielded mean Km values of 225 µM (plasma), 222 µM (umbilical artery), and Vmax values of 28 nmol (mL serum)/ 1 h (plasma) and 377 nmol /(mg prot.) h (umbilical artery). The deamination of [14C]MA was inhibited competitively by unlabelled BZ, with Ki values in umbilical artery and plasma of 220 and 172 µM, respectively. Also, metabolite formation from mixtures of [14C]BZ (200 µM) and [14C]MA (800 µM) was extremely close to that predicted for a single enzyme capable of metabolizing two alternative substrates in a competitive fashion. β-Aminopropionitrile was found to be a reversible, competitive inhibitor (Ki of 165 µM) of [14C]MA metabolism in umbilical artery, inhibitory properties characteristic of those found previously for the effects of β-aminopropionitrile upon BZ-metaboIizing SSAO activities in other tissues.

The target substance methylamine and the source substance methylammonium chloride used in this study belong to the group of primary aliphatic amines. The solvation of both, methylamine and methylammonium chloride in water results in solutions of the methylammonium cation (common "breakdown product"). Both respective counterions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions. Besides the influence on the pH value of an aqueous solution (OH-), they do not bear a relevant intrinsic property, allowing one in general to focus on the methylammonium cation. The methylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms.

Therefore both substances are expected to follow the same toxicokinetic pattern.

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied, based on the chemical structure of the potential analogues, their toxicokinetic behaviour and other available (eco-)toxicological data. Please refer also to the detailed read-across justification attached in section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar toxicological properties due to their structural similarity, resemblance to their chemical reactivity, and biotransformation products in toxicological compartments. In other words, there is a clear chemical analogy (“biotransformation to common compound", scenario 1 of the Read Across Assessment Framework (ECHA 2017)).
The solvation of both methylamine and methylammonium chloride in water results in solutions of the methylammonium cation (“common breakdown product", scenario 1 of the Read Across Assessment Framework (ECHA 2017)). The toxicity of the respective counterion (“non-common compound” – Cl-) is in this case negligible; as Cl does not drive toxicity effects in mammalian species because it is one of the main electrolytes and is required in large amounts in living organisms. So any toxicity of the amounts originated from the methylammonium chloride at maximum dose levels that would be used in toxicity studies is not expected.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

source substance:
methylamine hydrochloride (methylammonium chloride)
structural formula: CH5N.ClH
SMILES: [Cl-].C[NH3+]
CAS 593-51-1
purity: not specified

target substance:
methylamine
structural formula: CH5N
SMILES: CN
CAS 74-89-5
purity: ≥ 80 – 100 %

No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
Read across from the structural analogue to the target substance is based on the high structural similarity of the analogue with the substance of interest and the similarity of their toxicological characteristics.
The target substance MMA (CAS 74-59-5), as well as the source substance MMA-HCl (CAS 593-51-1) belong to the category of the “Aliphatic amines” according to the profiler “US EPA New Chemical Categories” in the OECD QSAR Toolbox v4.1.
The basicity of amines increases with the length of the aliphatic rest due to electron releasing properties of alkyl groups: the higher the pKa value, the weaker the acid, so the stronger the base. Monomethylamine (the primary amine with a central nitrogen atom and 1 methyl group and 2 hydrogen-atoms) is the least basic of the aliphatic (primary) amines. Monomethylammonium chloride (composed also of one methyl group attached to the nitrogen atom, which is regarded as the common structure / functional group) is no longer basic, but neutralised (please also refer to the following paragraphs). Therefore, it has to be kept in mind that MMA-HCL is an example of a worst case read-across according to RAAF, as MMA tested as salt may achieve very high doses because it is not corrosive.
Furthermore, the chemicals are characterized by a common Mode Of Action (MOA) in detail as “narcotic amines” according to Acute aquatic toxicity MOA by OASIS in the OECD QSAR Toolbox v4.1.
Methylamine and methylammonium chloride - as primary aliphatic amines - undergo similar reactions and resemble each other in their physico-chemical properties. The fundamental properties of different amine classes (primary, secondary and tertiary) – basicity and nucleophilicity – are very much the same (Morrison and Boyd, 1987).
Typical reactions of amines are salt formation, alkylation, and conversion into amides and Hofmann elimination from quaternary ammonium salts (Morrison and Boyd, 1987).
As already mentioned above, they are linked by the common functionality of one central nitrogen atom which bears an unshared pair of electrons and tends to share these electrons determining a similar chemical behaviour. This unshared electron pair can accept a proton - forming a substituted ammonium ion. The tendency to share this electron pair underlies the entire chemical behaviour of amines as a group and this was considered as main / basic parameter, which is suitable for read-across in an analogue approach within an/a analogue/category approach.
The dissociation constant of MMA allows the conclusion that virtually all molecules of methylamine - when dissolved in an excess of water - are present as the methylammonium cation. Moreover, the available data of MMA with hydrochloric acid shows clearly that there will be no relevant amounts of the amine available once in contact with the bodies’ fluids. Only the ionic form is the relevant species present. This applies to all relevant exposure routes, i.e. inhalation, dermal, and oral. So, in consequence, the solvation of both methylamine and methylammonium chloride in water would result in solutions of the methylammonium cation (“common breakdown product"). Therefore, one must only regard the physico-chemical properties of the respective counterion. Methylamine solutions are accompanied by the hydroxyl anion OH-, resulting in alkaline solutions, whereas the chloride anion of the methylammonium chloride solutions is not expected to trigger significant changes in the pH and exhibit any significant toxicological effects. Both anions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions.
The corrosive effects of MMA can certainly be explained by the high concentration of hydroxyl anions in MMA solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. MMA (gas) is legally classified as Skin Irrit. 2 and Eye Dam. 1, MMA (aqueous solution) is legally classified as Skin Corr. 1B; MMA-HCl has no legal classification for Skin or Eye irritation/corrosion. There is no data available on skin irritation of MMA. Experimental data showed MMA-HCl to be only transiently mildly irritating to the eyes of rabbits (no C & L is triggered). MMA-HCl is expected to be minor irritating when compared to MMA, because of the pH neutralisation caused by HCl. Consequently, the enhanced absorption of MMA compared to MMA-HCl due to damage of the skin barrier should be regarded when the substances are applied in corrosive concentrations or without pH neutralization.
Besides the influence of HCl on the pH value of an aqueous solution, it does not bear a relevant intrinsic property, allowing one in general to focus on the methylammonium cation. Generally, it should be denoted that very commonly in literature there is no differentiation made between MMA and MMA-HCl.
Primary amines are considered to be hydrolytically stable and both substances have been shown to be readily biodegradable. The log Koc values are both negative and relatively close and support the read-across approach. Furthermore, the results show that the substances do not have a significant potential for persistence (not P not vP) or bioaccumulation in organisms (not B not vB). Moreover, they are similar in their toxicity endpoints (despite the above mentioned missing corrosivity in the case of MMA-HCl).
The similar findings (refer also to the data matrix outlined below) for both substances support the conclusion that the identical molecule will be formed from both substances when applied systemically, and this molecule, i.e. the methylammonium cation, is responsible for their behaviour and the observed effects. In consequence, the methylammonium cation is indeed what is left to be considered in both cases and similar effects can reasonably be expected when using data from MMA-HCl for the lacking endpoints, compared to the data obtained with MMA.
Hence, MMA-HCl may perfectly serve as a worst case read-across substance for MMA. So, the available data on MMA-HCl can be used to cover all systemic endpoints currently lacking from MMA, making further testing obsolete.

4. DATA MATRIX
There is data available on the environmental fate and behaviour, ecotoxicological and toxicological properties of MMA. Data on MMA-HCl covers data on Biodegradation, Toxicity to aquatic algae and cyanobacteria, Toxicokinetics, oral Repeated dose toxicity, Genetic toxicity in vivo and Toxicity to reproduction/Developmental toxicity. Hence, the identification and discussion of common properties of MMA and MMA-HCl will be mainly based on this and physicochemical data.
The different physical state of the two substances (MMA is - as a pure substance - gaseous at room temperature, MMA-HCl is a solid primary ammonium salt) triggers some differences in the physico-chemical properties like Melting point, Boiling point, Decomposition temperature and Vapour pressure. Nevertheless, regarding the application of both substances, i.e. their distributed form, the gaseous character of MMA becomes less relevant as the substances are usually not applied in their pure forms but rather as aqueous solutions.
The available data for the following physico-chemical properties, which are among others relevant for absorption, are very similar. Both substances are small molecules with a low molecular weight of 31.042 (MMA) resp. 67.019 (MMA-HCl), they are both very soluble in water (completely miscible in water (MMA) and at least 1080 mg/L at 20°C (MMA-HCl)), have a negative logPow (-0.713 (aqueous solution, 25°C, pH 11.1 - 11.4; MMA) and -3.82 (MMA-HCl)), and both are readily biodegradable. Although being expected to be hydrolytically stable in the natural environment, they both have a very low potential for bioaccumulation in aquatic and terrestrial organisms. Most importantly, MMA has a pKa of 10.79 at 20°C (≙ pKb = 3.21), which indicates that methylamine exists almost entirely in the cationic form as methylammonium cation at pH values of 5 to 9.
MMA (not neutralised) has a toxicity potential towards fish and aquatic invertebrates, but does not have to be classified as hazardous. MMA (neutralised) and MMA-HCl have a lower toxicity potential to aquatic organisms and do not have to be considered to be acutely harmful to fish or aquatic invertebrates, nor to microorganisms (no C & L). MMA and MMA-HCL have been shown to be toxic to aquatic algae and cyanobacteria (shown in Pseudokirchnerella subcapitata, Scenedesmus obliquus and Microcystis aeruginosa). Their aquatic toxicity potential under real environmental conditions has to be judged carefully, since, methylamine and methylamine hydrochloride are readily biodegradable in nature. As such, both can be considered as non-toxic to aquatic organisms and thus do not have to be classified as hazardous as per the CLP classification criteria.
Regarding their toxicity towards mammals, both substances exert their acute toxicity oral toxicity in the same range and both are classified as Acute tox 4 and MMA also as STOT SE 3 (C≥5 %). Moreover, MMA is corrosive / irritative to the skin and the eyes(MMA (gas) = Skin Irrit. 2 and Eye Dam 1; MMA (aqueous solution) = Skin Irrit 1B); in the case of MMA –HCl no classification is warranted.
Regarding their repeated dose toxicity, the No-observed-adverse-effect-levels of both substances to differ to a certain extent (NOAEL ≥ 10 versus NOAEL = 500 mg/kg bw/day). For MMA HCL there is also data for toxicity to reproduction and developmental toxicity available (NOEL (reproduction/systemic tox) = 500 mg/kg bw/day and NOAEL (maternal/developmental tox.) = 155 mg/kg bw/day).
No data on long-term toxicity to fish; on long-term toxicity to aquatic invertebrates or carcinogenicity were available.
Reason / purpose:
read-across source
Details on excretion:
Administration of Choline 100 mg to rats: mean excretion of dimethylamine increasing from 37.5 +/- 5.4 µg to 73.5 +/- 4.8 µg per mg creatinine (t = 9.3; P < 0.01).
The formation of dimethylamine from ingested choline was confirmed by giving [Me-14C]choline chloride to two rats. The results show the incorporation of a small but significant amount of radioactivity into dimethylamine. In contrast to the findings in the rats given choline orally, there was no significant increase in urinary dimethylamine, when the same dose of choline was administered by intraperitoneal injection. It seemed, therefore, that the conversion of choline to dimethylamine depended upon the activities of the intestinal flora. To test this, two rats were treated with neomycin (100 mg each, twice daily for 4 days) and were then fed the same dose of radioactive choline chloride as used previously. There was no incorporation of radioactivity into dimethylamine, isolated as the DNP-derivative from urine. Since ingested lecithin can be converted to choline by enzymes in the gastrointestinal tract, the excretion of dimethylamine was studied in three rats before and after administration of 1 g egg lecithin. Dimethylamine excretion increased considerably during 48 h, indicating that the phospholipid is another source of dimethylamine.
Metabolites identified:
yes
Details on metabolites:
Dimethylamine is build when choline, lecithin methylamine hydrochloride or trimethylamine hydrochloride are administered to rats
Conclusions:
No bioaccumulation potential based on study results
In conclusion, urinary dimethylamine is traced to two sources: (a) from dietary choline; (b) from methylamine, by transmethylation, and methionine serves as the methyl donor.
Executive summary:

The study performed by Asatoor et al, in 1965, already gives important data concerning DMA and the fate of MMA in the rats. They investigated whether DMA is generated in the mammalian body by feeding rats (2 -4 rats; in cases also administered via injection) with either choline chloride, lecithin, [14C]methylamine, or [Me14C]choline chloride (orally) or egg lecithin or trimethylamine hydrochloride (orally) or trimethylamine hydrochloride (intraperitoneal) and then analytical verification of DMA in Urine, to test whether these substances can be transformed in DMA and excreted that way. They could prove that MMA is partly methylated to form DMA.

The target substance methylamine and the source substance methylammonium chloride used in this study belong to the group of primary aliphatic amines. The solvation of both, methylamine and methylammonium chloride in water results in solutions of the methylammonium cation (common "breakdown product"). Both respective counterions are naturally and ubiquitous occurring ions and are also to a certain extent required for the maintenance of various body functions. Besides the influence on the pH value of an aqueous solution (OH-), they do not bear a relevant intrinsic property, allowing one in general to focus on the methylammonium cation. The methylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms.

Therefore both substances are expected to follow the same toxicokinetic pattern.

Description of key information

Short description of key information on bioaccumulation potential result:
Lyles et al, Further studies on the metabolism of methylamine by semicarbazide-sensitive amine oxidase activities in human plasma, umbilical artery and rat aorta, J.Phar. Pharmacol. 1990, 42: 322-338;
Asatoor et al., The urinary origin of dimethylamine, Biochimica et biophysica acta, 111 (1965) 384-392;
Dar et al., The Enzymatic Systems Involved In The Mammalian Metabolism Of Methylamine, Gen. Pharmac. Vol. 16, No. 6, pp. 557 560, 1985;
Gubisne-Haberle et al, Protein Cross-Linkage Induced by Formaldehyde Derived from Semicarbazide-Sensitive Amine Oxidase-Mediated Deamination of Methylamine, The Journal Of Pharmacology And Experimental Therapeutics, Vol. 310, No. 3, S.1125-1132;
Zhang et al, Dimethylamine Formation in the Rat from Various Related Amine Precursors, Food and Chemical Toxicology, 36, 923-27, 1998


Short description of key information on absorption rate:
Jeevaratnam et al., 1993, Do the Hydrolysis Products, Methylamine and N,N’-Dimethylurea, Play any Role in the Methyl Isocyanate- Induced Haematological and Biochemical Changes in Rabbits?, Human &Experimental Toxicology, 12, 135-139

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
100
Absorption rate - inhalation (%):
100

Additional information

The metabolism, disposition and toxicokinetics of the biogenic amine monomethylamine have been characterized in different in vitro experiments, additionally in rats and mice in vivo. First of all, it is important to take into account, that methylamine has three different sources: the diet (for example in fish), only negligible bacterial synthesis in the intestinal tract and mostly endogenous synthesis. Endogenously formed methylamine was found in human urine with the range of values spreading from 1.68 to 62.30 mg (Mitchell and Zhang, 2001). The methylamine content was also investigated in a variety of foods. Urinary excretion of MA increased after eating fish products as well as after creatinine ingestion (Mitchell and Zhang, 2001). The major source of MA is endogenous with the contributions from the diet. In total the knowledge of the endogenous occurrence influences mainly the categorization and classification of methylamine.

Methylamine and methylamine hydrochloride were rapidly absorbed from the diet or after gut bacterial degradation of dietary precursors, uniformly distributed and eliminated mainly as carbon dioxide (about 52 % in 24 hours after methylamine hydrochloride administration) via exhalation in both sexes of rats (only negligible amounts are excreted via urine) (Dar et al., 1985).

The in vitro metabolism is mediated via semicarbazide-sensitive amine oxidase (oxidative deamination) (Lyles et al, 1990). This was reinvestigated in 2004 by Gubisne-Haberle et al, and they found that methylamine is catalysed by SSAO to formaldehyde and that this membrane-bound enzyme is mainly present in the endothels of the vascular system. This metabolite is classified as carcinogenic, but at higher concentrations than 6 ppm, since those are not likely to be reached during the metabolism of MA, the carcinogenic potential of methylamine can not be deduced from the available data. Additionally methylamine can also be methylated to form dimethylamine (after administration of 9.4 µmole 14C-methylamine to rats there occurred a radioactivity in dimethylamine of 4.8 x 10³ counts per min and µmole, Asatoor et al., 1965). But this is not a main pathway, since Zhang et al, 1998 could not prove a significant rise of urinary dimethylamine output after the administration of monomethylamine. It is not possible to exclude the possibility of nitrosamine formation after metabolism to dimethylamine, but it seems rather unlikely that a significant amount of nitrosamine can be built, since this reaction occurs mainly at low pH, like in the stomach and requires nitrite.

Based on the results obtained in these studies, methylamine may be considered to be rapidly absorbed, and mainly via exhalation well eliminated substance in rats, mice and humans. It bears the slight possibility to be metabolized to a carcinogenic substance (N-Nitrosodimethylamine or formaldehyde).

Discussion on bioaccumulation potential result:

Lyles et al. investigated in 1990 the metabolism of methylamine hydrochloride in vitro. An ion exchange radiochemical assay has been developed to study the deamination of [14C]methylamine (MA) in homogenates of rat aorta and human umbilical artery, as well as in samples of human plasma. MA metabolism was found to be inhibited almost completely by 1 mM semicarbazide, but virtually unaffected by 0-1 mM clorgyline, suggesting that MA is a substrate for the semicarbazide-sensitive amine oxidase (SSAO) activities which also metabolize benzylamine (BZ) in these sources. Mean Km values for MA metabolism by aorta, umbilical artery and plasma were 182, 832 and 516 µM, respectively, with corresponding Vmax values in aorta and umbilical artery of 100 and 590 nmol /(mg prot.) h, and in plasma of 48 nmol /(mL serum) h. Kinetic constants determined for [14C]BZ metabolism in plasma (by an organic solvent extraction assay) and in umbilical artery (by the ion exchange assay) yielded mean Km values of 225 µM (plasma), 222 µM (umbilical artery), and Vmax values of 28 nmol/ (mL serum) h (plasma) and 377 nmol /(mg prot.) h (umbilical artery). The deamination of [14C]MA was inhibited competitively by unlabelled BZ, with Ki values in umbilical artery and plasma of 220 and 172 µM, respectively. Also, metabolite formation from mixtures of [14C]BZ (200 µM) and [14C]MA (800 µM) was extremely close to that predicted for a single enzyme capable of metabolizing two alternative substrates in a competitive fashion. β-Aminopropionitrile was found to be a reversible, competitive inhibitor (Ki of 165 µM) of [14C]MA metabolism in umbilical artery, inhibitory properties characteristic of those found previously for the effects of β-aminopropionitrile upon BZ-metabolizing SSAO activities in other tissues.

The study performed by Asatoor et al, in 1965, already gives important data concerning DMA and the fate of MMA in the rats. They investigated whether DMA is generated in the mammalian body by feeding rats (in cases also administered via injection) with either choline chloride, lecithin, [14C]methylamine hydrochloride, or [Me14C]choline chloride (orally) or egg lecithin or trimethylamine hydrochloride (orally) or trimethylamine hydrochloride (intraperitoneal) and then analytical verification of DMA in Urine, to test whether these substances can be transformed in DMA and excreted that way. They could prove that MMA is partly methylated to form DMA.

In 1985, Dar et al. studied the excretion of radioactivity in the expired air and the urine of rats, to indirectly assess the role of the monoaminooxidase in the metabolism of methylamine hydrochloride. It was postulated, that the metabolism of methylamine hydrochloride falls in the category separate from the amines metabolized by monoaminooxidase. Methylamine hydrochloride undergoes rapid oxidative degradation and an average of 52% of the injected [14C]-activity appears in the expired air as [14C]O2 in rats in 24 hr; over 30% of [14C]-activity as respiratory [14C]O2 excreted during the initial 2- 6hr of methyl- [14C]amine hydrochloride administration.

Gubisne-Haberle et al. investigated in 2004 the metabolism of methylamine in rats and mice. They found that the semicarbazide-sensitive amine oxidase (SSAO) catalyzes the conversion of methylamine to formaldehyde. This enzyme is located on the surface of the cytoplasmic membrane and in the cytosol of vascular endothelial cells, smooth muscle cells, and adipocytes. Increased SSAO activity has been found in patients with diabetes mellitus, chronic heart failure, and multiple types of cerebral infarcts and is associated with obesity. Increased SSAO-mediated deamination may contribute to protein deposition, the formation of plaques, and inflammation, and thus may be involved in the pathophysiology of chronic vascular and neurological disorders, such as diabetic complications, atherosclerosis, and Alzheimer’s disease. They demonstrate the induction of cross-linkage of formaldehyde with the lysine moiety of peptides and proteins. Formaldehyde-protein adducts were reduced with sodium cyanoborohydride, hydrolyzed in hydrochloric acid, and the amino acids in the hydrolysates were derivatized with fluorenylmethyl chloroformate and then identified with high-performance liquid chromatography. Additionally they demonstrate that incubation of methylamine in the presence of SSAO-rich tissues, e.g., human brain meninges, results in formaldehyde-protein cross-linkage of particulate bound proteins as well as of soluble proteins. This cross-linkage can be completely blocked by a selective inhibitor of SSAO. The data support the hypothesis that the SSAO induced production of formaldehyde may be involved in the alteration of protein structure, which may subsequently cause protein deposition associated with chronic pathological disorders.

In 1998, Zhang et al. investigated the excretion of DMA after administration of various related amine precursors. They found trimethylamine N-oxide (at a dose rate of 1 mmol/kg body weight) to be 20 times more efficient at providing dimethylamine than an equivalent dose of choline. Additionally less than 1 % of the choline dose was converted to dimethylamine. Slightly more of an equivalent trimethylamine dose (1.6%) was converted to dimethylamine. So trimethylamine N-oxide is undoubtedly a significant dietary source of dimethylamine and it carries the potential of being converted to carcinogenic nitrosodimethylamine. However, other dietary sources of dimethylamine probably lie undetected amidst the myriad of anutrient chemicals within foodstuffs, and the continued excretion of small amounts of dimethylamine from antibiotic treated and germfree rats (Asatoor et al., 1967; Zeisel et al., 1985) implies that dietary components, although probably the major source, are not the only originator of dimethylamine. Whereas those compounds containing the monomethylamino function (methylamine, N-methylglycine) did not give rise to any significant increases in urinary dimethylamine output, suggesting that methylation of monomethylamines is not a major source of dimethylamine.

Discussion on absorption rate:

Jeevaratnam et al. administered methylamine in 1993 subcutaneous to the animals to investigate whether the observed haematological and biochemical changes observed after MIC administration to the rabbits were due only to MIC itself or if its hydrolysis metabolites play any role in these effects. Both MA and DMU administered subcutaneously in an equimolar dose to that of 1.0 LD50 MIC, 2.2 mmol/kg had no influence on these parameters, although there was a marginal increase in the plasma urea level shortly after the administration of DMU. This study establishes that the observed haematological and biochemical changes induced by MIC intoxication in rabbits are mostly due to MIC.