Registration Dossier

Administrative data

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).

Link to relevant study records

Referenceopen allclose all

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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Ministry of Labour, Japan (1979, 1985, 1988)
Qualifier:
equivalent or similar to
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:
other: S.typhimurium strains TA98, TA100, TA102, TA104, TA1535, TA1537, TA1538 and E.coli WP2uvrA and WP2uvrA/pKM101
Additional strain / cell type characteristics:
other: Salmonella were histidine deficient; E. coli were 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:
9-aminoacridine
Remarks:
2AA: aminoantracene was used without metabolic activation

Migrated to IUCLID6: Further substances: sodium azide, 2-nitrofluorene, 4NQO: 4-nitroquinoline-N-oxide, 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
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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 (migrated information):
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 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. 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key 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:
according to
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:
no 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 lethaat 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
Remarks:
Migrated from field 'Test system'.

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 (migrated information):
positive without metabolic activation

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

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.

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, 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
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

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). 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.

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.