Methylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1. Mutagenicity Test Data of Existing Chemical Substances, 1997; Ames Test, 0, 0.05, 0.1, 0.5, 1, 5, 10, 50 %.
2.Caspary, W. J. and Myhr, B. Mutagenicity of methylisocyanate and its reaction products to cultured mammalian cells. 1986, Mouse Lymphoma Assay; 0, 50, 100, 150, 200, 300 and 400 nl/mL (100 nl/mL is equivalent to 1.3 mM).

3. In vitro Mammalian Gene Mutation Test using the Hprt and xprt genes, 2020, HPRT Test using Chinese hamster ovary cell line CHO; 250, 500, 1000 and 2000 µg/mL.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: 1. According to the Guidelines for the Ministry of Labour, Japan 2. Some study data are in appendices, which were not available for review 3. No GLP certificate

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Ministry of Labour, Japan (1979, 1985, 1988)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Performed by the following methods: Ames et al. (1975), Maron and Ames (1983), and Matsushima et al. (1980)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: The Salmonella strains were histidine deficient.

Species / strain / cell type:

S. typhimurium, other: TA104

Additional strain / cell type characteristics:

other: The Salmonella strain was histidine deficient

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: The Salmonella strain was histidine deficient.

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: The E. coli strain was L-tryptophan deficient.

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: The E coli. strain was L. tryptophan deficient.

Metabolic activation:

with and without

Metabolic activation system:

S9 from rat liver induced with sodium phenobarbital and 5,6-benzofravone

Test concentrations with justification for top dose:

0, 0.05, 0.1, 0.5, 1, 5, 10, 50%

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other:

Remarks:

Further substances: BLM: bleomycin, PA: pyruvic aldehyde

Details on test system and experimental conditions:

PREPARATION OF S9 FRACTION
- Male Sprague-Dawley rats (7 weeks old, 200g) were used for the preparation of liver S9 fractions
- Sodium phenobarbital and 5,6-benzofravone were used to induce the rat metabolic activation system by i.p. injection
- S9 was prepared from rat liver samples, it was frozen and stored at -80 deg C

PREINCUBATION METHOD
- The test substance was dissolved in 0.05 or 0.1 mL of the solvent and supplemented with 0.5 mL of S9 mix (with metabolic activation) or 0.1M phosphate buffer pH 7.4 (without metabolic activation) and 0.1 mL of tester strains which had been cultured in nutrient broth
- The mixture was incubated for 20 min. at 37 deg C, then rapidly mixed with agar containing 0.05 umol/mL of L-histidine and biotin for the Salmonella test
- In the E.coli test, 0.05 umol/mL of L-tryptophan was used instead of L-histidine and biotin
- All plates were incubated for 48 hours at 37 deg C and the number of revertant colonies were scored

Evaluation criteria:

DATA EVALUATION
- Two-hold rule criteria was used for data evaluation.
- The chemicals are considered to be mutagenic when a dose-related increase in revertant colonies is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when a reproducibility of test result is observed
- Mutagenic potency was calculated by the following equation and maximum potency was expressed as a specific activity on the data sheet: mutagenic potency (induced revertants / mg test substance) = (number of induced revertants on the dose X - number of revertant on the solvent control) / mg of test chemical on the dose X

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium, other: 104

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks on result:

other: all strains/cell types tested

Table 2

Tester Strain	Solvent Control		Positive Control
	-S9 Mix	+S9 Mix	-S9 Mix	+S9 Mix
TA100	138 +/- 27	139 +/- 29	728 +/- 196	1011 +/- 210
TA1535	14 +/- 5	13 +/- 4	300 +/- 81	255 +/- 70
TA98	20 +/- 8	26 +/- 7	413 +/- 82	404 +/- 118
TA1538	16 +/- 3	23 +/- 5	376 +/- 79 (2NF)	556 +/- 216
			343 +/- 34 (4NQO)
TA1537	8 +/- 2	11 +/- 4	497 +/- 254	196 +/- 70
TA102	260 +/- 49	317 +/- 52	758 +/- 175	1676 +/- 562
TA104	269 +/- 40	332 +/- 48	1973 +/- 755	1196 +/- 252
WP2uvrA	29 +/- 11	34 +/- 11	273 +/- 126	879 +/- 177
WP2uvrA/pKM101	141 +/- 43	198 +/- 49	2080 +/- 884	928 +/- 250

Control values (mean +/- standard deviation) for solvent controls and positive controls

Conclusions:

Interpretation of results:
- negative with metabolic activation
- negative without metabolic activation

Methylamine is negative in the Ames Test.

Executive summary:

Methylamine was assayed for mutation (according to OECD 471, Klimisch 2 - reliable with restrictions) in seven histidine requiring strains (TA98, TA100, TA102, TA104, TA1535, TA1537 and TA1538) of Salmonella typhimurium and in two strains of Escherichia Coli (WP2uvrA and WP2uvrA/pKM101) both in the absence and presence of metabolic activation by an sodium phenobarbital and 5,6-benzofravone induced rat liver post-mitochondrial fraction (S-9).

An initial toxicity range-finder experiment was carried out. No toxicity was observed at 2000 µg/plate, therefore that concentration was chosen as the top dose for the mutation experiments.

In the mutation experiment each strain (0.1 mL) was treated with the test substance (test substance was dissolved in 0.05 or 0.1 mL of the solvent and supplemented with 0.5 mL of S9 mix (with metabolic activation) or 0.1M phosphate buffer pH 7.4 (without metabolic activation).

The mixture was incubated for 20 min. at 37 deg C, then rapidly mixed with agar containing 0.05 µmol/mL of L-histidine and biotin for the Salmonella test. In the E.coli test, 0.05 µmol/mL of L-tryptophan was used instead of L-histidine and biotin. Incubations for 48 h were carried out and the number of colonies were scored. Negative (solvent) and positive control treatments were included for all strains. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. So the study met the acceptance criteria and is considered to be valid.

Treatment of the strains in the absence of SS-9 and in the presence of S-9 did not rise the numbers of revertant colonies significantly. So methylamine did not induce revertants in all bacterial strains tested both with and without metabolic activation.

It is concluded that the results fully satisfy the requirements for a non-mutagenic response, the compound being considered non-mutagenic in this assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported, published 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented publication which meets basic scientific principles.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Methylamine was tested only without metabolic activation

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

tk-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cells deficient in thymidine kinase (TK) due to the mutation TK+/- -→ TK-/- are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell division. Thus mutant cells are able to proliferate in the presence of TFT, whereas normal cells, which contain thymidine kinase, are not.

Metabolic activation:

without

Vehicle / solvent:

water

Negative solvent / vehicle controls:

yes

Remarks:

dist. water

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF CELLS EVALUATED: The cells were then plated for both cloning efficiency (600 cells) and mutant selection (3000,000 cells, 3 µg/mL TFT).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Mutant colony count (MC)
- Group average mutation frequency (MF)

The mutant count was divided by the product of the cloning efficiency and the number of cells at risk (3000,000 cells) to yield the mutant fraction. The highest test dose was limited by solubility or toxicity but did not exceed 5 mg/mL.

Evaluation criteria:

All data were evaluated statistically for both trend and peak response. An experiment was considered positive if the p value was less than 0.05 for at least 1 of the 3 highest dose sets (peak response) and if there was a significant trend (p<0.05). If there was only a trend response or a single dose increase without a trend, the evaluation was 'questionable'; a 'negative' evaluation was made when there was no trend or peak response. A chemical was considered 'positive' only if the positive response was confirmed in a repeat test. Quality control criteria and response categories have been published elsewhere (Myhr et al., 1985).

Statistics:

The statistical analysis consisted of a trend test and a pairwise comparison of the variance of the mutation frequency of the treated samples against the solvent control. The variance of the mutation frequency was determined by using a series of statistical assumptions associated with a mathematical model of the assay that was described previously (Lee and Caspary, 1983).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Methylamine induced mutagenic responses in two experiments in the absence of S9 at concentrations around 200-300 nL/mL (3-4 mM) and became lethal at approximately 400 nL/mL (5 mM).l (Table 1). Increasing the dose increased both the toxicity and the mutagenic response in a dose-related manner.

Remarks on result:

other: all strains/cell types tested

TABLE 1. RESULTS OF THE MOUSE LYMPHOMA CELL FORWARD MUTATION ASSAY
METHYLAMINE
Nonactivation trial 1 ( + )						Nonactivation trial 2 ( + )
Conc. (nl/ml)	CE	RTG	MC	MF	Ave. MF	Conc. (nl/ml)	CE	RTG	MC	MF	Ave. MF
Dist.	106	98	122	38		Dist.	105	123	64	20
0.	103	123	111	36		0.	96	85	127	44
	86	117	103	40			64	92	115	60
	101	62	112	37	38		43R	44R	113	89R	41
50.	55	64	51	31		25.	70	68	138	66
	77	76	70	30			100#	169	107	36
	83	82	114	46	36		63	101	102	54	52
100.	69	74	102	50		50.	54	80	117	72
	67	95	107	53			59	112	87	49
	62	39	93	50	51		51	85	91	60	61
150.	86	93	99	39		100.	114	57	178	52
	67	87	71	35			112	159	169	50
	87	96	68	26	33		51	99	106#	69	57
200.	85	60	68	27		200.	99	147	116	39
	93	81	148	53			52	76	122	78
	105	83	145	46	42		47	79	119	84	67
300.	103	56	128 #	41		300.	62	38	196	106
	71	42	119	56			61	45	175	96	101
	82	54	181	73	57		Lethal	_	_	_
400.	104	22	333	107		400.	Lethal	_	_	_
	109	25	253	78	92		Lethal	_	_	_
	Lethal	_	_	_			Lethal	_	_	_
MMS						MMS
5.	51	29	462	301		5.	40	54	413	349
	36	24	607	565			43	23	611	477
	41	37	624	509	458		35	24	253	243	356
100 nL/mL is equivalent to 1.3 mM.

CE = cloning efficiency %,

RTG = relative total growth;

MC = mutant colony count;

MF = mutation frequency (per 1 mio. cells),

Ave.MF = group average mutation frequency;

underline = P<0.05%,

# = Data from two acceptable plates normalized to three.

MMS methyl methanesulfonate

Conclusions:

Interpretation of results: positive without metabolic activation

Methylamine caused the mutagenic response in a dose-related manner.

Executive summary:

The test substance methylamine (MMA) was investigated similar to OECD TG476 for its potential to cause mutagenic responses at the tk locus in the mouse lymphoma cell forward mutation assay (Caspary and Myhr, 1986). However, the test included only experiments without metabolic activation. Under the conditions of this experiment MMA was found to cause mutagenic responses in the absence of any exogenous metabolic activation. From 3 to 5 mM, the increase of the mutagenic responses was obtained in a dose-dependent manner. In conclusion, it can be stated that, the test material caused mutations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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 as in this case 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, Toxicokinetics, oral Repeated dose toxicity, Genetic toxicity in vitro and 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. 67.52 (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 some extent to aquatic algae and cyanobacteria (shown in Desmodesmus subspicatus, 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 (Acute tox 4 - H302 for MMA aqueous solution and for MMA HCl) and are also classified as Acute tox 3 - H331 (MMA - gaseous form, MMA - aqueous solution) and Acute tox 4 - H332 (MMA HCl) via the inhalation route of exposure: MMA (gaseous form and aqueous solution) are also classified 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). Concerning genetic toxicity in vitro, both substances have been shown to be not mutagenic in Gene mutation in bacteria (Ames) tests. Furthermore, MMA and MMA HCl have been tested for their potential to cause gene mutation in mammalian cells. For MMA an older result gives a positive result when tested only without metabolic activation. However, the very recent new investigation conducted with MMA-HCl showed it not to be mutagenic Chinese hamster ovary cells. 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 for cross-reference:

read-across source

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH values of test item solutions did not show any significant alterations compared to the concurrent control groups
- Data on osmolality: In the Main Mutation Assay the osmolality values of treatment solutions were similar compared to the concurrent control.
- Water solubility: 100 mg/mL as stated in the CoA from the supplier
- Precipitation and time of the determination: There was no precipitation of the test item at any dose level tested.

RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected on the basis of the result of a pre-test on toxicity. In the pre-test on toxicity, precipitations were not observed at the applied concentrations. In addition, pH and osmolality was considered for dose level selection. Based on the result of the preliminary cytotoxicity assay, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended for lower- cytotoxic substances in OECD Guideline 476 (updated 2016). Four concentrations were selected for the treatment in the main mutation assay with and without metabolic activation.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
without S9-mix [MUTANT FREQ. X 10^-6]:
Solvent control: 7.00, 7.00, 7.07, 7.00
Pos. control (EMS 1.0 µL/mL): 1519.05**, 1449.23**, 1476.56**, 1475.76**
** = p < 0.01 to the concurrent negative (solvent) control and to the historical control

with S9-mix [MUTANT FREQ. X 10^-6]:
Solvent control: 6.93, 8.00, 7.07, 8.08
Solvent control of DMBA: 7.00, 7.00, 8.00, 8.00
Pos. control (DMBA 20 µg/mL): 716.22**, 701.32**, 718.92**, 708.00**
** = p < 0.01 to the concurrent negative (solvent) control and to the historical control

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: not observed
- Statistical analysis:
positive controls showed statistically significant increase in mutant freqencies (p < 0.01), test item showed no statistically significant increase in mutant frequencies


Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency : no significant decrease in relative population growth, relative survival or cloning efficiency observed for test item treated cultures


- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: Approximately 5x10^6 cells/concentration were used and incubated for 24 hours before treatment and approximately 20x10^6 cells / concentration were treated.
o Number of cells plated in selective medium: 2x10^5 cells /dish for mutant selection (4x 5 dishes, yielding 4x10^6 cells per treatment)
o Number of resistant colonies in selective medium, and related mutant frequency:
MEAN COLONY NUMBER +/- S.D. and MUTANT FREQ. X 10^-6 of solvent control (4x 5 replicates without and with S9): [-S9] 202.7 ± 2.52 and 7.00, 201.7 ± 0.58 and 7.00, 203.0 ± 1.00 and 7.07, 201.7 ± 0.58 and 7.00, [+S9] 199.7 ± 0.58 and 6.93, 200.0 ± 1.00 and 8.00, 198.0 ± 1.00 and 7.07, 198.7 ± 1.15 and 8.08


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95 % control limits for the distribution as well as the number of data)
- Positive historical control data:
Without S9 mix EMS:
Mean: 1487.41
SD: 27.12
Range: 1357.81 – 1671.46
Lower confidence interval: 1432.61
Upper confidence interval: 1542.21
n: 40

With S9 mix DMBA:
Mean: 735.07
SD: 17.11
Range: 667.11 – 810.29
Lower confidence interval: 700.49
Upper confidence interval: 769.66
n: 40

- Negative (solvent/vehicle) historical control data:
Without S9 mix (Ham's F12 medium)
Mean: 6.58
SD: 0.59
Range: 4.90 – 8.91
Lower confidence interval: 5.36
Upper confidence interval: 7.81
n: 20

With S9 mix (Ham's F12 medium)
Mean: 6.95
SD: 0.84
Range: 4.90 – 11.76
Lower confidence interval: 5.19
Upper confidence interval: 8.71
n: 20

Without S9 mix (DMSO)
Mean: 6.66
SD: 0.58
Range: 4.95 – 10.89
Lower confidence interval: 5.44
Upper confidence interval: 7.89
n: 20

With S9 mix (DMSO)
Mean: 6.85
SD: 0.69
Range: 4.12 – 11.88
Lower confidence interval: 5.41
Upper confidence interval: 8.29
n: 20

Conclusions:

In conclusion, Methylamine hydrochloride >= 98 % tested up to the maximum recommended concentration (2000 µg/mL) without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control. Thus, it is concluded that the test item, Methylamine hydrochloride >= 98 %, was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

Executive summary:

The test item, Methylamine hydrochloride >= 98 % was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells in accordance with OECD Guideline 476 and under GLP compliance. The purpose of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells. The test item was dissolved in Ham's F12 medium and the concentrations of the main test were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver.

Based on the result of the preliminary cytotoxicity assay, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended for lower- cytotoxic substances in OECD Guideline 476 (updated 2016). The Mutation Assay was performed at the concentrations and treatment intervals given below:

Mutation Assay 5-hour treatment period without S9-mix: 250, 500, 1000 and 2000 µg/mL

Mutation Assay 5-hour treatment period with S9-mix: 250, 500, 1000 and 2000 µg/mL

Following treatment and 19 h incubation period, phenotypic expression was allowed for 8 days followed by a mutant selection period for another 8 days.

In the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 2000 µg/mL.

In this In Vitro Mammalian Cell Gene Mutation Test, the frequency of the cells with mutations did not show biologically and statistically significant increases compared to the concurrent and historical controls when Methylamine hydrochloride >= 98 % was examined in the absence and in the presence of metabolic activation. All values were within the range of the laboratory historical control data and no dose-response relationships were noted.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was well within the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µL/mL) and 7, 12-Dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increase of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, Methylamine hydrochloride >= 98 % tested up to the maximum recommended concentration (2000 µg/mL) without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control.

Thus, it is concluded that the test item, Methylamine hydrochloride >= 98 %, was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

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 conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo:

1. BASF (2007). Mammalian Erythrocytes Micronucleus test with Methylamine hydrochloride. OECD 474; 500, 1000, 2000 mg/kg bw.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

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 as in this case 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, Toxicokinetics, oral Repeated dose toxicity, Genetic toxicity in vitro and 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. 67.52 (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 some extent to aquatic algae and cyanobacteria (shown in Desmodesmus subspicatus, 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 (Acute tox 4 - H302 for MMA aqueous solution and for MMA HCl) and are also classified as Acute tox 3 - H331 (MMA - gaseous form, MMA - aqueous solution) and Acute tox 4 - H332 (MMA HCl) via the inhalation route of exposure: MMA (gaseous form and aqueous solution) are also classified 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). Concerning genetic toxicity in vitro, both substances have been shown to be not mutagenic in Gene mutation in bacteria (Ames) tests. Furthermore, MMA and MMA HCl have been tested for their potential to cause gene mutation in mammalian cells. For MMA an older result gives a positive result when tested only without metabolic activation. However, the very recent new investigation conducted with MMA-HCl showed it not to be mutagenic Chinese hamster ovary cells. 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 for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Remarks:

The rate of micronuclei was close to the same range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data.

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

other: vehicle control served as negative control

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: single dose of 2000 mg/kg bw
- Solubility: well soluble in water
- Clinical signs of toxicity in test animals: piloerection, squatting posture, irregular respiration, eyelid closure and the general state was poor.
- Rationale for exposure: determination of the acute oral toxicity. The recommended highest dose of 2 000 mg/kg body weight was tolerated with no deaths. On account of the test results, 2 000 mg/kg body weight was administered as the highest dose. 1 000 mg/kg and 500 mg/kg body weight were selected as further doses.

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei and ratio of PCE/NCE (for Micronucleus assay):

The single oral administration of purified water in a volume of 10 mL/kg body weight led to 1.4 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 0.8 ‰ after the 48-hour sacrifice interval (Table 1).
After the single administration of the highest dose of 2 000 mg/kg body weight, 1.6 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.2 ‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of about 1.7 ‰ (1 000 mg/kg group) and 2.0 ‰ (500 mg/kg group) were detected after the 24-hour sacrifice interval in each case.
The positive control substance for clastogenicity, cyclophosphamide, led to a clear increase (15%) in the number of polychromatic erythrocytes containing mainly small micronuclei, as expected. Vincristine, a spindle poison agent, produced a 30.6‰ increase in micronuclei in polychromatic erythrocytes. A significant portion of this increase (8.0‰) was attributable to large micronuclei.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.
No inhibition of erythropoiesis induced by the treatment of mice with Methylamine hydrochloride was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

- Appropriateness of dose levels and route:

The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms.
The administration of the test substance led to evident clinical signs of toxicity. Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Table1: Results of all groups for polychromatic and normochromatic erythrocytes

	Interval: 24 hours				Interval: 48 hours
	Total No. of		MN  ( ‰ ) in		Total No. of		MN  (‰ ) in
	PCE's	NCE's	PCE's	NCE's	PCE's	NCE's	PCE's	NCE's
Vehicle pur. water	10000	3227	1.4	1. 9	10000	3447	0.8	0.9
500 mg/kg	10000	3465	2.0	0.6
1000 mg/kg	10000	3770	1. 7	0.5
2000 mg/kg	10000	3949	1. 6	0.8	10000	3870	1.2	0.5
CPP 20 mg/kg	10000	4708	15.0**	1. 9
VCR 0.15 mg/kg	10000	3813	30.6**	0.3
WILCOXON TEST (ONE-SIDED) : *: p <= 0.05, **: p <= 0.01
A pairwise comparison of each dose group with the vehicle control group

PCE's: polychromatic erythrocytes;

NCE's: normochromatic erythrocytes;

MN: micronuclei

Table 2: Differentiation between small and large micronuclei

	Interval: 24 hours			Interval: 48 hours
	Total No. of PCE's	Cells  (‰) with		Total No. of PCE's	Cells  (‰ ) with
		MN.d<D/4	MN.d  ≥   D/4		MN.d<D/4	MN.d  ≥   D/4
Vehicle pur. water	10000	1.4	0.0	10000	0.8	0.0
500 mg/kg	10000	2.0	0.0
1000 mg/kg	10000	1. 7	0.0
2000 mg/kg	10000	1. 6	0.0	10000	1.2	0.0
CPP 20 mg/kg	10000	14.9**	0.1
VCR 0.15 mg/kg	10000	22.6**	8.0**
WILCOXON TEST (ONE-SIDED): *: p ≤ 0.05, **: p ≤ 0.01
A pairwise comparison of each dose group with the vehicle control group

PCE's: polychromatic erythrocytes;

MN: micronuclei;

CPP: Cyclophosphamide;

VCR: Vincristine Sulphate;

d = diameter of micronucleus, D = cell diameter.

Conclusions:

Under the experimental conditions chosen of the test, the test substance Methylamine hydrochloride has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Executive summary:

The substance Methylamine hydrochloride was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method according to the OECD guideline 474. The study was conducted without deviations from the protocol of the guideline and therefore is considered of the highest quality (Klimisch 1). For the purpose of the test, the test substance, dissolved in purified water, was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The dose levels were determined in a range-finding study.

As a negative control, male mice were administered merely the vehicle, purified water, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive controls chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Hence, the vehicle and the positive control substances fulfilled validity criteria of the test system.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2 000 mg/kg body weight and in the vehicle controls. In the test groups of 1 000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

According to the results of the present study, the single oral administration of Methylamine hydrochloride did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the same range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen in the test, the test substance Methylamine hydrochloride does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis(aneugenic activity) in bone marrow cells in vivo.

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 conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

 

Methylamine was assayed for mutation (according to OECD 471, Klimisch 2 - reliable with restrictions) in seven histidine requiring strains (TA98, TA100, TA102, TA104, TA1535, TA1537 and TA1538) of Salmonella typhimurium and in two strains of Escherichia Coli (WP2uvrA and WP2uvrA/pKM101) both in the absence and presence of metabolic activation by an sodium phenobarbital and 5,6-benzofravone induced rat liver post-mitochondrial fraction (S-9).

An initial toxicity range-finder experiment was carried out. No toxicity was observed at 2000 µg/plate, therefore that concentration was chosen as the top dose for the mutation experiments.

In the mutation experiment each strain (0.1 mL) was treated with the test substance (test substance was dissolved in 0.05 or 0.1 mL of the solvent and supplemented with 0.5 mL of S9 mix (with metabolic activation) or 0.1M phosphate buffer pH 7.4 (without metabolic activation).

The mixture was incubated for 20 min at 37 deg C, then rapidly mixed with agar containing 0.05 µmol/mL of L-histidine and biotin for the Salmonella test. In the E.coli test, 0.05 µmol/mL of L-tryptophan was used instead of L-histidine and biotin. Incubations for 48 h were carried out and the number of colonies were scored. Negative (solvent) and positive control treatments were included for all strains. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments. So the study met the acceptance criteria and is considered to be valid.

Treatment of the strains in the absence of S-9 and in the presence of S-9 did not rise the numbers of revertant colonies significantly. So methylamine did not induce revertants in all bacterial strains tested both with and without metabolic activation.

It is concluded that the results fully satisfy the requirements for a non-mutagenic response, the compound being considered non-mutagenic in this assay.

The test substance methylamine (MMA) was investigated according to OECD TG476 for its potential to cause mutagenic responses at the tk locus in the mouse lymphoma cell forward mutation assay (Caspary and Myhr, 1986). Under the conditions of this experiment MMA was found to cause mutagenic responses in the absence of any exogenous metabolic activation. From 3 to 5 mM, the increase of the mutagenic responses was obtained in a dose-dependent manner. In conclusion, it can be stated that, the test material caused mutations.

 

To confirm the conclusion that MMA is non-mutagenic, the analogue methylamine hydrochloride was tested in an In Vitro Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells according to OECD 476 was conducted.

The test item was dissolved in Ham’s F12 medium and the concentrations of the main test were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver.

In the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration of 2000 µg/mL. The frequency of the cells with mutations did not show biologically and statistically significant increases compared to the concurrent and historical controls when Methylamine hydrochloride >= 98 % was examined in the absence and in the presence of metabolic activation. All values were within the range of the laboratory historical control data and no dose-response relationships were noted. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was well within the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µL/mL) and 7, 12-Dimethyl benzanthracene (20 µg/mL) caused the expected biologically relevant increase of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

 

According to the results of the present study, Methylamine hydrochloride tested up to the maximum recommended concentration (2000 µg/mL) without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control and thus MMA-HCl is not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

 

In vivo studies

 

There are no in vivo genetic toxicity studies on methylamine (CAS No. 74 -89 -5) available. For methylamine hydrochloride (CAS No. 593 -51 -1), however, there is a GLP OECD guideline 474 study, in which it was tested for chromosomal damage (clastogenicity) or its ability to induce spindle poison effects (aneugenic activity). Methylamine hydrochloride is a related substance to methylamine and represents salt of methylamine, in which unshared electron pair on nitrogen forms a coordinate bond with the proton hydrogen, accompanied by chlorine bond ionically. Under alkalic conditions the salt dissociates liberating free methylamine. Hence, under specific conditions the toxicological and ecotoxicological properties of the gaseous methylamine and its salt (also in aqueous solution) are equal and as a result, read-across is justified (please also refer to the justification for type of information in the respective endpoint study record).

 

For the purpose of the study, the test substance - methylamine hydrochloride - dissolved in purified water, was administered once orally to male NMRI mice at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The dose levels were determined in a range-finding study.

As a negative control, mice were administered merely the vehicle, purified water, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive controls chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Hence, the vehicle and the positive control substances fulfilled validity criteria of the test system.

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2 000 mg/kg body weight and in the vehicle controls. In the test groups of 1 000 mg/kg and 500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2 000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

According to the results of the study, the single oral administration of methylamine hydrochloride did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the same range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen in the test, the test substance methylamine hydrochloride does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis(aneugenic activity) in bone marrow cells in vivo.

 

Conclusion

Methylamine was negative in in vitro bacterial reverse mutation assay (Ames Test) both with and without metabolic activation. In the mouse lymphoma cell forward mutation assay (OECD 476) methylamine was tested only without metabolic activation where it is turned to be positive. Nevertheless, in in vivo OECD 474 chromosome aberration assay methylamine hydrochloride, an analogue substance to methyalamine, did not induce chromosome-damaging effect (clastogenicity) or spindle poison effects (aneugenicity).

The absence of a mutagenic potential was confirmed in an in vitro mammalian gene mutation test (HPRT Test) with the analogue substance methylamine hydrochloride, following OECD 476 in chinese hamster cells, both in the absence and presence of metabolic activation.

Based on the results of these studies, methylamine is considered to be non-mutagenic.

Justification for classification or non-classification

According to the column 2 in the Annex VIII of REACh Regulation, if the in vitro study results are ambiguous, an in vivo study is needed to conclude about genetic toxicity potential of a substance. Since methylamine in an in vitro study is negative (Ames Test) and in another in vitro study is positive (Mouse Lymphoma Assay), the outcomes of in vivo study are decisive about the genetic toxicity potential. Moreover, the absence of a genetic toxicity potential was confirmed by another negative in vitro study (HPRT).

Due to the negative results of in vivo chromosome damage assay with methylamine hydrochloride, classification for methylamine is not warranted according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.