Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Trimethylamine (read-across)_OECD 471_ S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A_negative

Trimethylamine (read-across)_OECD 471_TA 97, TA98, TA100, TA1535 and TA1537_negative

Trimethylamine (read-across)_OECD 477_L57178 tk +/- (3.7.2C) mouse lymphoma cells_negative

Trimethylamine (read-across)_OECD 473_human lymphocytes_negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). More precisely, TMA-HCl is a tertiary ammonium salt.
The class of tertiary amines includes amines in which three alkyl groups are attached to the nitrogen atom and can be described by the structure R-N(R1)-R2. 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.
The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. The genetic toxicity potential of Trimethylamine / Trimethylammonium HCL is believed to be negligible, as the organic part - the Trimethylammonium cation does not interact with biomolecules. Trimethylamine has been shown not to bind to DNA, producing an adverse genotoxicity effects. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical: Trimethylamine (TMA, C3H9N, SMILES CN(C)C, CAS 75-50-3, EC 200-875-0)
Target chemical: Trimethylammonium chloride (TMA-HCl, C3H9N.ClH, SMILES CN(C)C.Cl, CAS 593-81-7, EC 209-810-0)

3. ANALOGUE APPROACH JUSTIFICATION
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines. The fundamental properties of different amine classes (primary, secondary and tertiary) – basicity and nucleophilicity – are very much the same (Morrison and Boyd, 1987). The nitrogen in an amine bears an unshared pair of electrons, and this non-binding electron pair is localized in one of the nearly perfectly sp³-hybridized molecule orbitals. The ammonium cation of the TMA-HCl is also sp³-hybrized, whereas the electron pair of the nitrogen is shared with the proton originating from HCl. Formally, the resulting positive charge is attributed to the nitrogen, whereas in reality the electrons are equally distributed in all orbitals. In consequence, there are generally relevant differences when regarding both TMA and TMA-HCl on their own. Nevertheless, the sp³-hybridized nitrogen with three methyl groups can be regarded as a common structure / functional group of both TMA and TMA-HCl. Additionally, taking however in account that, in order to exhibit any toxicological properties, the interactions of both molecules with the body, i.e. body fluids, enzymes, cellular structures etc., are relevant, these differences are indeed minor. Upon solvation, both substances dissociate into identical Trimethylammonium cations and exhibit very similar physico-chemical and toxicological properties, as outlined below.
The dissociation constants of TMA allow the conclusion that virtually all molecules of Trimethylamine - when dissolved in an excess of water are present as the Trimethylammonium cation. Moreover, the available pka-data and the titration curve of TMA with hydrochloric acid shows clearly, that there will be no relevant amounts of the amine available once in contact with the bodies’ fluids and only the ionic form is the relevant species present. This applies to all relevant exposure routes, i.e. inhalation, dermal, and oral. So, in consequence, the solvation of both Trimethylamine and Trimethylammonium chloride in water would result in solutions of the Trimethylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. Trimethylamine solutions are accompanied by the hydroxyl anion OH-, resulting in alkaline solutions, whereas the chloride anion of the Trimethylammonium chloride solutions is not expected to trigger significant changes in the pH and exhibit any significant (eco)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. Some differences may need to be taken into account when assessing the possible irritating properties of the substances which need to be considered during read-across. However, with regard to irritating properties, both OH- and H3O+ are known to exhibit irritating / corrosive properties. However, TMA-HCl solutions do not bear alkaline properties. Here, false positive effects are expected due to the strong irritating effects of these high-alkaline solutions. Taking into account this fact, the corrosive effects of TMA can certainly be explained by the high concentration of hydroxyl anions in TMA solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. TMA is legally classified as Skin Irrit. 2 and Eye Dam. 1, TMA-HCl has no legal classification. The available data suggest additionally a corrosive potential for TMA, dependent on its concentration, whereas TMA-HCl is only minor irritating / corrosive compared to TMA, only classifiable as irritating to the skin and Eye Irrit. 2 (60% aqueous solution). Consequently, the enhanced absorption of TMA compared to TMA-HCl due to damage of the skin barrier should be regarded when the substances are applied in corrosive concentrations or without pH neutralization.
So if irritating properties are observed it can be expected they do not result from the amine but rather the evolving ions.
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 Trimethylammonium cation.
The similar findings (refer to 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 Trimethylammonium cation, is responsible for the observed effects. In consequence, the Trimethylammonium cation is what is left to be considered in both cases and similar effects can be reasonably expected when testing TMA-HCl for the lacking endpoints, compared to the data obtained with TMA. Hence, TMA may perfectly serve as read-across substance for TMA-HCl and vice versa. So, the available data on TMA can be used to cover all systemic endpoints currently lacking from TMA-HCl, making further testing obsolete.

4. DATA MATRIX
There is mainly data available on the toxicological properties of TMA, Data on TMA-HCl covers merely the toxicokinetic endpoint. Hence, the identification and discussion of common properties of TMA and TMA-HCl will be mainly based on this and physicochemical data.
The different physical state of the two substances (TMA is as a pure substance, gaseous at room temperature, TMA-HCL is a solid tertiary 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 TMA 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 relevant for absorption into living organisms, are very similar. Both substances are small molecules with a molecular weight of 59.1103 (TMA) resp. 95.5712 (TMA-HCl), they are both very soluble in water (410 g/L at 19°C (TMA) and 758 g/L at 20°C (TMA-HCl)), have a small to negative logPow (- 0.245 (aqueous solution, 25°C, pH 10) / <-3.5 (neutralisation with hydrochloric acid, 25°C, pH 7) / ca. -2.9 (neutralisation with acetic acid, 25°C, pH 7) (TMA) and < -2.25 (Trimethylamine part) / < -3.4 (Chloride part) (TMA-HCl)), and at least TMA is readily biodegradable, making it very like for TMA-HCl to bear the same property. Although being expected to be hydrolytically stable in the natural environment, as a result, they both have a very low potential for bioaccumulation in aquatic and terrestrial organisms. Most importantly, TMA has a pKa of 9.78 - 9.8 at 20°C, which indicates that Trimethylamine exists almost entirely in the cationic form at pH values of 5 to 9.
For the following toxicological endpoints there is data derived from both substances available: Acute toxicity oral, Skin irritation / corrosion, Eye irritation /corrosion, Repeated dose toxicity oral and Toxicity to reproduction (fertility / developmental toxicity).
For the acute oral toxicity, the values for TMA are generally lower than the one of TMA-HCl, which trigger a classification as acute toxic cat. 4, i.e. LD50 values ranging from 397-820 mg/kg. Taking into account however the fact that TMA-HCl was applied as 58% solution, resulting in a LD50 of ca. 2 g/kg, and the generally higher molecular weight of TMA-HCl due to the additional HCl, the values for both substances can be considered as similar within normal biological variations.
Regarding Skin/Eye Irritation, TMA must be considered as (potentially) corrosive, whereas TMA-HCl is only irritating. These differences are likely attributable to differences in the pH value. The NOAELs derived for the endpoints Repeated dose toxicity oral and Toxicity to reproduction (fertility / developmental toxicity) also only differ marginally, which can be easily explained by the study design and the differences in molecular weight.
Reason / purpose:
read-across source
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: all strains/cell types tested
Remarks:
Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2 uvrA; Pre-incubation Method

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the Ames Test.

Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

Conclusions:
Interpretation of results:negative
Trimethylamine showed no gene mutation effects to S.typhimurium strains TA98, TA100, TA1535, and TA1537 and E.coli strain WP2 uvr A.

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the Ames Test.
Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).
Executive summary:

Gene mutation properties of trimethylamine were investigated in a bacterial reverse mutation assay (Ames test) (Nakajima et al., 2001). The test was performed according to OECD guideline 471 with Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 as well as with Escherichia coli strain WP2 uvr A. The concentrations of the test substance ranged from 0 to 5000 µg and the test result was negative in all strains, with and without metablic activation system.

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the Ames Test.

Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting 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.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). More precisely, TMA-HCl is a tertiary ammonium salt.
The class of tertiary amines includes amines in which three alkyl groups are attached to the nitrogen atom and can be described by the structure R-N(R1)-R2. 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.
The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. The genetic toxicity potential of Trimethylamine / Trimethylammonium HCL is believed to be negligible, as the organic part - the Trimethylammonium cation does not interact with biomolecules. Trimethylamine has been shown not to bind to DNA, producing an adverse genotoxicity effects. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical: Trimethylamine (TMA, C3H9N, SMILES CN(C)C, CAS 75-50-3, EC 200-875-0)
Target chemical: Trimethylammonium chloride (TMA-HCl, C3H9N.ClH, SMILES CN(C)C.Cl, CAS 593-81-7, EC 209-810-0)

3. ANALOGUE APPROACH JUSTIFICATION
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines. The fundamental properties of different amine classes (primary, secondary and tertiary) – basicity and nucleophilicity – are very much the same (Morrison and Boyd, 1987). The nitrogen in an amine bears an unshared pair of electrons, and this non-binding electron pair is localized in one of the nearly perfectly sp³-hybridized molecule orbitals. The ammonium cation of the TMA-HCl is also sp³-hybrized, whereas the electron pair of the nitrogen is shared with the proton originating from HCl. Formally, the resulting positive charge is attributed to the nitrogen, whereas in reality the electrons are equally distributed in all orbitals. In consequence, there are generally relevant differences when regarding both TMA and TMA-HCl on their own. Nevertheless, the sp³-hybridized nitrogen with three methyl groups can be regarded as a common structure / functional group of both TMA and TMA-HCl. Additionally, taking however in account that, in order to exhibit any toxicological properties, the interactions of both molecules with the body, i.e. body fluids, enzymes, cellular structures etc., are relevant, these differences are indeed minor. Upon solvation, both substances dissociate into identical Trimethylammonium cations and exhibit very similar physico-chemical and toxicological properties, as outlined below.
The dissociation constants of TMA allow the conclusion that virtually all molecules of Trimethylamine - when dissolved in an excess of water are present as the Trimethylammonium cation. Moreover, the available pka-data and the titration curve of TMA with hydrochloric acid shows clearly, that there will be no relevant amounts of the amine available once in contact with the bodies’ fluids and only the ionic form is the relevant species present. This applies to all relevant exposure routes, i.e. inhalation, dermal, and oral. So, in consequence, the solvation of both Trimethylamine and Trimethylammonium chloride in water would result in solutions of the Trimethylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. Trimethylamine solutions are accompanied by the hydroxyl anion OH-, resulting in alkaline solutions, whereas the chloride anion of the Trimethylammonium chloride solutions is not expected to trigger significant changes in the pH and exhibit any significant (eco)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. Some differences may need to be taken into account when assessing the possible irritating properties of the substances which need to be considered during read-across. However, with regard to irritating properties, both OH- and H3O+ are known to exhibit irritating / corrosive properties. However, TMA-HCl solutions do not bear alkaline properties. Here, false positive effects are expected due to the strong irritating effects of these high-alkaline solutions. Taking into account this fact, the corrosive effects of TMA can certainly be explained by the high concentration of hydroxyl anions in TMA solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. TMA is legally classified as Skin Irrit. 2 and Eye Dam. 1, TMA-HCl has no legal classification. The available data suggest additionally a corrosive potential for TMA, dependent on its concentration, whereas TMA-HCl is only minor irritating / corrosive compared to TMA, only classifiable as irritating to the skin and Eye Irrit. 2 (60% aqueous solution). Consequently, the enhanced absorption of TMA compared to TMA-HCl due to damage of the skin barrier should be regarded when the substances are applied in corrosive concentrations or without pH neutralization.
So if irritating properties are observed it can be expected they do not result from the amine but rather the evolving ions.
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 Trimethylammonium cation.
The similar findings (refer to 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 Trimethylammonium cation, is responsible for the observed effects. In consequence, the Trimethylammonium cation is what is left to be considered in both cases and similar effects can be reasonably expected when testing TMA-HCl for the lacking endpoints, compared to the data obtained with TMA. Hence, TMA may perfectly serve as read-across substance for TMA-HCl and vice versa. So, the available data on TMA can be used to cover all systemic endpoints currently lacking from TMA-HCl, making further testing obsolete.

4. DATA MATRIX
There is mainly data available on the toxicological properties of TMA, Data on TMA-HCl covers merely the toxicokinetic endpoint. Hence, the identification and discussion of common properties of TMA and TMA-HCl will be mainly based on this and physicochemical data.
The different physical state of the two substances (TMA is as a pure substance, gaseous at room temperature, TMA-HCL is a solid tertiary 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 TMA 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 relevant for absorption into living organisms, are very similar. Both substances are small molecules with a molecular weight of 59.1103 (TMA) resp. 95.5712 (TMA-HCl), they are both very soluble in water (410 g/L at 19°C (TMA) and 758 g/L at 20°C (TMA-HCl)), have a small to negative logPow (- 0.245 (aqueous solution, 25°C, pH 10) / <-3.5 (neutralisation with hydrochloric acid, 25°C, pH 7) / ca. -2.9 (neutralisation with acetic acid, 25°C, pH 7) (TMA) and < -2.25 (Trimethylamine part) / < -3.4 (Chloride part) (TMA-HCl)), and at least TMA is readily biodegradable, making it very like for TMA-HCl to bear the same property. Although being expected to be hydrolytically stable in the natural environment, as a result, they both have a very low potential for bioaccumulation in aquatic and terrestrial organisms. Most importantly, TMA has a pKa of 9.78 - 9.8 at 20°C, which indicates that Trimethylamine exists almost entirely in the cationic form at pH values of 5 to 9.
For the following toxicological endpoints there is data derived from both substances available: Acute toxicity oral, Skin irritation / corrosion, Eye irritation /corrosion, Repeated dose toxicity oral and Toxicity to reproduction (fertility / developmental toxicity).
For the acute oral toxicity, the values for TMA are generally lower than the one of TMA-HCl, which trigger a classification as acute toxic cat. 4, i.e. LD50 values ranging from 397-820 mg/kg. Taking into account however the fact that TMA-HCl was applied as 58% solution, resulting in a LD50 of ca. 2 g/kg, and the generally higher molecular weight of TMA-HCl due to the additional HCl, the values for both substances can be considered as similar within normal biological variations.
Regarding Skin/Eye Irritation, TMA must be considered as (potentially) corrosive, whereas TMA-HCl is only irritating. These differences are likely attributable to differences in the pH value. The NOAELs derived for the endpoints Repeated dose toxicity oral and Toxicity to reproduction (fertility / developmental toxicity) also only differ marginally, which can be easily explained by the study design and the differences in molecular weight.
Reason / purpose:
read-across source
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
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 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks on result:
other: all strains/cell types tested
Remarks:
TA 97, TA98, TA100, TA1535 and TA1537

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the Ames Test.

Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

Conclusions:
Interpretation of results: negative
Trimethylamine was negative in Bacterial Reverse Mutation Test with and without metabolic activation.
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the Ames Test.
Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).
Executive summary:

A bacterial reverse mutation assay (Ames test) was done to investigate trimethylamine for gene mutation (Mortelmanns, 1986). The experiment was performed in accordance with OECD guideline 471 without GLP. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 were investigated with and without metabolic activation, whereas Salmonella typhimurium strain TA97 was tested only with metabolic activation system, which was rat / hamster S9 - mix (Aroclor 1254 included). A preincubation assay was done and the test concentration were 10, 33, 100, 333, and 1000 µg/plate, as vehicle water was used. The genotoxicity test showed for all strains under all circumstances a negative result.

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the Ames Test.

Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). More precisely, TMA-HCl is a tertiary ammonium salt.
The class of tertiary amines includes amines in which three alkyl groups are attached to the nitrogen atom and can be described by the structure R-N(R1)-R2. 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.
The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. The genetic toxicity potential of Trimethylamine / Trimethylammonium HCL is believed to be negligible, as the organic part - the Trimethylammonium cation does not interact with biomolecules. Trimethylamine has been shown not to bind to DNA, producing an adverse genotoxicity effects. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical: Trimethylamine (TMA, C3H9N, SMILES CN(C)C,CAS 75-50-3, EC 200-875-0)
Target chemical: Trimethylammonium chloride (TMA-HCl, C3H9N.ClH, SMILES CN(C)C.Cl, CAS 593-81-7, EC 209-810-0)

3. ANALOGUE APPROACH JUSTIFICATION
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines. The fundamental properties of different amine classes (primary, secondary and tertiary) – basicity and nucleophilicity – are very much the same (Morrison and Boyd, 1987). The nitrogen in an amine bears an unshared pair of electrons, and this non-binding electron pair is localized in one of the nearly perfectly sp³-hybridized molecule orbitals. The ammonium cation of the TMA-HCl is also sp³-hybrized, whereas the electron pair of the nitrogen is shared with the proton originating from HCl. Formally, the resulting positive charge is attributed to the nitrogen, whereas in reality the electrons are equally distributed in all orbitals. In consequence, there are generally relevant differences when regarding both TMA and TMA-HCl on their own. Nevertheless, the sp³-hybridized nitrogen with three methyl groups can be regarded as a common structure / functional group of both TMA and TMA-HCl. Additionally, taking however in account that, in order to exhibit any toxicological properties, the interactions of both molecules with the body, i.e. body fluids, enzymes, cellular structures etc., are relevant, these differences are indeed minor. Upon solvation, both substances dissociate into identical Trimethylammonium cations and exhibit very similar physico-chemical and toxicological properties, as outlined below.
The dissociation constants of TMA allow the conclusion that virtually all molecules of Trimethylamine - when dissolved in an excess of water are present as the Trimethylammonium cation. Moreover, the available pka-data and the titration curve of TMA with hydrochloric acid shows clearly, that there will be no relevant amounts of the amine available once in contact with the bodies’ fluids and only the ionic form is the relevant species present. This applies to all relevant exposure routes, i.e. inhalation, dermal, and oral. So, in consequence, the solvation of both Trimethylamine and Trimethylammonium chloride in water would result in solutions of the Trimethylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. Trimethylamine solutions are accompanied by the hydroxyl anion OH-, resulting in alkaline solutions, whereas the chloride anion of the Trimethylammonium chloride solutions is not expected to trigger significant changes in the pH and exhibit any significant (eco)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. Some differences may need to be taken into account when assessing the possible irritating properties of the substances which need to be considered during read-across. However, with regard to irritating properties, both OH- and H3O+ are known to exhibit irritating / corrosive properties. However, TMA-HCl solutions do not bear alkaline properties. Here, false positive effects are expected due to the strong irritating effects of these high-alkaline solutions. Taking into account this fact, the corrosive effects of TMA can certainly be explained by the high concentration of hydroxyl anions in TMA solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. TMA is legally classified as Skin Irrit. 2 and Eye Dam. 1, TMA-HCl has no legal classification. The available data suggest additionally a corrosive potential for TMA, dependent on its concentration, whereas TMA-HCl is only minor irritating / corrosive compared to TMA, only classifiable as irritating to the skin and Eye Irrit. 2 (60% aqueous solution). Consequently, the enhanced absorption of TMA compared to TMA-HCl due to damage of the skin barrier should be regarded when the substances are applied in corrosive concentrations or without pH neutralization.
So if irritating properties are observed it can be expected they do not result from the amine but rather the evolving ions.
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 Trimethylammonium cation.
The similar findings (refer to 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 Trimethylammonium cation, is responsible for the observed effects. In consequence, the Trimethylammonium cation is what is left to be considered in both cases and similar effects can be reasonably expected when testing TMA-HCl for the lacking endpoints, compared to the data obtained with TMA. Hence, TMA may perfectly serve as read-across substance for TMA-HCl and vice versa. So, the available data on TMA can be used to cover all systemic endpoints currently lacking from TMA-HCl, making further testing obsolete.

4. DATA MATRIX
There is mainly data available on the toxicological properties of TMA, Data on TMA-HCl covers merely the toxicokinetic endpoint. Hence, the identification and discussion of common properties of TMA and TMA-HCl will be mainly based on this and physicochemical data.
The different physical state of the two substances (TMA is as a pure substance, gaseous at room temperature, TMA-HCL is a solid tertiary 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 TMA 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 relevant for absorption into living organisms, are very similar. Both substances are small molecules with a molecular weight of 59.1103 (TMA) resp. 95.5712 (TMA-HCl), they are both very soluble in water (410 g/L at 19°C (TMA) and 758 g/L at 20°C (TMA-HCl)), have a small to negative logPow (- 0.245 (aqueous solution, 25°C, pH 10) / <-3.5 (neutralisation with hydrochloric acid, 25°C, pH 7) / ca. -2.9 (neutralisation with acetic acid, 25°C, pH 7) (TMA) and < -2.25 (Trimethylamine part) / < -3.4 (Chloride part) (TMA-HCl)), and at least TMA is readily biodegradable, making it very like for TMA-HCl to bear the same property. Although being expected to be hydrolytically stable in the natural environment, as a result, they both have a very low potential for bioaccumulation in aquatic and terrestrial organisms. Most importantly, TMA has a pKa of 9.78 - 9.8 at 20°C, which indicates that Trimethylamine exists almost entirely in the cationic form at pH values of 5 to 9.
For the following toxicological endpoints there is data derived from both substances available: Acute toxicity oral, Skin irritation / corrosion, Eye irritation /corrosion, Repeated dose toxicity oral and Toxicity to reproduction (fertility / developmental toxicity).
For the acute oral toxicity, the values for TMA are generally lower than the one of TMA-HCl, which trigger a classification as acute toxic cat. 4, i.e. LD50 values ranging from 397-820 mg/kg. Taking into account however the fact that TMA-HCl was applied as 58% solution, resulting in a LD50 of ca. 2 g/kg, and the generally higher molecular weight of TMA-HCl due to the additional HCl, the values for both substances can be considered as similar within normal biological variations.
Regarding Skin/Eye Irritation, TMA must be considered as (potentially) corrosive, whereas TMA-HCl is only irritating. These differences are likely attributable to differences in the pH value. The NOAELs derived for the endpoints Repeated dose toxicity oral and Toxicity to reproduction (fertility / developmental toxicity) also only differ marginally, which can be easily explained by the study design and the differences in molecular weight.
Reason / purpose:
read-across source
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: human lymphocyte cultures prepared from the pooled blood of three male volunteers.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: all strains/cell types tested

1.     The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures.

2.     The proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the normal range.

3.     At least 160 cells out of an intended 200 were suitable for analysis at each test article concentration and vehicle controls. In Experiment 2, <10 cells showing structural aberrations other than gaps were observed on one slide from the ‘B’ replicate of the CPA (12.5 µg/mL) positive control (from a total of 26 metaphases analysed). However, a clear positive response was observed, therefore this did not affect the interpretation of the data.

4.     The positive control chemicals induced statistically significant increases in the proportion of cells with structural aberrations.

 

Table 2 :Mitotic Index Determinations - Experiment 1

Treatment

(µg/mL)

Mitotic index (%)

3+17 hours, -S-9

3+17 hours, +S-9

 

A/C

B/D

MIH*

A/C

B/D

MIH*

 

 

 

 

 

 

 

Vehicle

8.0/8.5

7.4/9.4

-

7.3/6.9

9.9/8.3

-

37.50

NS

NS

-

NS

NS

-

75.00

NS

NS

-

NS

NS

-

150.0

NS

NS

-

8.5

9.7

0

250.0

NS

NS

-

8.5

11.1

0

350.0

10.8

10.0

0#

9.5

10.2

0#

450.0

8.5

7.8

2#

8.0

7.1

7#

591.1

9.3

12.7

0#

6.7

9.0

3#

 

NS = Not scored

NT = Not tested

*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

# Highlighted concentrations selected for analysis

 

Table 3: Mitotic Index Determinations – Experiment 2

Treatment

(µg/mL)

Mitotic index (%)

20+0 hours, -S-9

3+17 hours, +S-9

 

A/C

B/D

MIH*

A/C

B/D

MIH*

 

 

 

 

 

 

 

Vehicle

6.4/6.6

6.7/6.0

-

9.2/10.8

10.2/8.4

-

50.00

7.6

4.9

3

NS

NS

-

100.0

4.9

5.4

20

11.3

8.5

0

150.0

5.5

4.8

20

NT

NT

-

200.0

5.6

6.0

10#

10.4

8.0

5

230.0

3.4

6.1

26

NT

NT

-

260.0

4.3

5.2

26#

NT

NT

-

290.0

5.2

5.3

18

NT

NT

-

300.0

NT

NT

-

8.7

6.9

19

320.0

3.3

3.7

46#

NT

NT

-

350.0

2.4

4.5

46

NT

NT

-

400.0

3.3

1.9

60#

7.9

5.8

29#

500.0

1.0

1.3

82

10.0

8.9

2#

591.1

1.5

0.8

82

7.6

9.3

12#

 

NS = Not scored

NT = Not tested

*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

 # Highlighted concentrations selected for analysis

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the in vitro mammalian chromosome aberration test. Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

Conclusions:
Treatment of cultures with Trimethylamine (TMA-opl in water) in the absence and presence of S-9 in Experiments 1 and 2 resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within normal ranges.
No increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were generally observed in cultures treated with Trimethylamine (TMA-opl in water) in the absence and presence of S-9 in Experiments 1 and 2. The only exception to this was observed in Experiment 2 in the absence of S-9 at one intermediate concentration (320.0 µg/mL), where the frequency of cells with numerical aberrations marginally exceeded the 95th percentile of the normal range (but fell within the observed normal range in both cultures. The increases were almost entirely attributable to hyperdiploid cells but they were small and there was no evidence of a concentration-related response, therefore they were not considered biologically relevant.
It is concluded that Trimethylamine (TMA-opl in water) did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of cytotoxicity for 20+0 hours in the absence of S-9 and up to a maximum of 10 mM for 3+17 hours in the absence and presence of S-9.
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the in vitro mammalian chromosome aberration test. Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).
Executive summary:

Trimethylamine (TMA-opl in water) was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments(Key study, 2010).

Treatments covering a broad range of concentrations were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The highest concentration used in the Main Experiments, 591.1 mg/mL (equivalent to 10 mM) was determined following a preliminary cytotoxicity Range-Finder Experiment. The test article concentrations for chromosome analysis were selected by evaluating the effect of Trimethylamine on mitotic index. Treatment of cultures with trimethylamine in the absence and presence of S-9 resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within normal ranges. No increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were generally observed in cultures treated with Trimethylamine in the absence and presence of S-9. The only exception to this was observed in Experiment 2 in the absence of S-9 at one intermediate concentration (320.0 µg/mL), where the frequency of cells with numerical aberrations marginally exceeded the 95th percentile of the normal range (but fell within the observed normal range) in both cultures. The increases were almost entirely attributable to hyperdiploid cells but they were small and there was no evidence of a concentration-related response, therefore they were not considered biologically relevant.

It is concluded that Trimethylamine (TMA-opl in water) did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of cytotoxicity for 20+0 hours in the absence of S-9 and up to a maximum of 10 mM for 3+17 hours in the absence and presence of S-9.

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the in vitro mammalian chromosome aberration test. Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). More precisely, TMA-HCl is a tertiary ammonium salt.
The class of tertiary amines includes amines in which three alkyl groups are attached to the nitrogen atom and can be described by the structure R-N(R1)-R2. 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.
The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. The genetic toxicity potential of Trimethylamine / Trimethylammonium HCL is believed to be negligible, as the organic part - the Trimethylammonium cation does not interact with biomolecules. Trimethylamine has been shown not to bind to DNA, producing an adverse genotoxicity effects. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemical: Trimethylamine (TMA, C3H9N, SMILES CN(C)C, CAS 75-50-3, EC 200-875-0)
Target chemical: Trimethylammonium chloride (TMA-HCl, C3H9N.ClH, SMILES CN(C)C.Cl, CAS 593-81-7, EC 209-810-0)

3. ANALOGUE APPROACH JUSTIFICATION
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines. The fundamental properties of different amine classes (primary, secondary and tertiary) – basicity and nucleophilicity – are very much the same (Morrison and Boyd, 1987). The nitrogen in an amine bears an unshared pair of electrons, and this non-binding electron pair is localized in one of the nearly perfectly sp³-hybridized molecule orbitals. The ammonium cation of the TMA-HCl is also sp³-hybrized, whereas the electron pair of the nitrogen is shared with the proton originating from HCl. Formally, the resulting positive charge is attributed to the nitrogen, whereas in reality the electrons are equally distributed in all orbitals. In consequence, there are generally relevant differences when regarding both TMA and TMA-HCl on their own. Nevertheless, the sp³-hybridized nitrogen with three methyl groups can be regarded as a common structure / functional group of both TMA and TMA-HCl. Additionally, taking however in account that, in order to exhibit any toxicological properties, the interactions of both molecules with the body, i.e. body fluids, enzymes, cellular structures etc., are relevant, these differences are indeed minor. Upon solvation, both substances dissociate into identical Trimethylammonium cations and exhibit very similar physico-chemical and toxicological properties, as outlined below.
The dissociation constants of TMA allow the conclusion that virtually all molecules of Trimethylamine - when dissolved in an excess of water are present as the Trimethylammonium cation. Moreover, the available pka-data and the titration curve of TMA with hydrochloric acid shows clearly, that there will be no relevant amounts of the amine available once in contact with the bodies’ fluids and only the ionic form is the relevant species present. This applies to all relevant exposure routes, i.e. inhalation, dermal, and oral. So, in consequence, the solvation of both Trimethylamine and Trimethylammonium chloride in water would result in solutions of the Trimethylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. Trimethylamine solutions are accompanied by the hydroxyl anion OH-, resulting in alkaline solutions, whereas the chloride anion of the Trimethylammonium chloride solutions is not expected to trigger significant changes in the pH and exhibit any significant (eco)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. Some differences may need to be taken into account when assessing the possible irritating properties of the substances which need to be considered during read-across. However, with regard to irritating properties, both OH- and H3O+ are known to exhibit irritating / corrosive properties. However, TMA-HCl solutions do not bear alkaline properties. Here, false positive effects are expected due to the strong irritating effects of these high-alkaline solutions. Taking into account this fact, the corrosive effects of TMA can certainly be explained by the high concentration of hydroxyl anions in TMA solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. TMA is legally classified as Skin Irrit. 2 and Eye Dam. 1, TMA-HCl has no legal classification. The available data suggest additionally a corrosive potential for TMA, dependent on its concentration, whereas TMA-HCl is only minor irritating / corrosive compared to TMA, only classifiable as irritating to the skin and Eye Irrit. 2 (60% aqueous solution). Consequently, the enhanced absorption of TMA compared to TMA-HCl due to damage of the skin barrier should be regarded when the substances are applied in corrosive concentrations or without pH neutralization.
So if irritating properties are observed it can be expected they do not result from the amine but rather the evolving ions.
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 Trimethylammonium cation.
The similar findings (refer to 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 Trimethylammonium cation, is responsible for the observed effects. In consequence, the Trimethylammonium cation is what is left to be considered in both cases and similar effects can be reasonably expected when testing TMA-HCl for the lacking endpoints, compared to the data obtained with TMA. Hence, TMA may perfectly serve as read-across substance for TMA-HCl and vice versa. So, the available data on TMA can be used to cover all systemic endpoints currently lacking from TMA-HCl, making further testing obsolete.

4. DATA MATRIX
There is mainly data available on the toxicological properties of TMA, Data on TMA-HCl covers merely the toxicokinetic endpoint. Hence, the identification and discussion of common properties of TMA and TMA-HCl will be mainly based on this and physicochemical data.
The different physical state of the two substances (TMA is as a pure substance, gaseous at room temperature, TMA-HCL is a solid tertiary 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 TMA 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 relevant for absorption into living organisms, are very similar. Both substances are small molecules with a molecular weight of 59.1103 (TMA) resp. 95.5712 (TMA-HCl), they are both very soluble in water (410 g/L at 19°C (TMA) and 758 g/L at 20°C (TMA-HCl)), have a small to negative logPow (- 0.245 (aqueous solution, 25°C, pH 10) / <-3.5 (neutralisation with hydrochloric acid, 25°C, pH 7) / ca. -2.9 (neutralisation with acetic acid, 25°C, pH 7) (TMA) and < -2.25 (Trimethylamine part) / < -3.4 (Chloride part) (TMA-HCl)), and at least TMA is readily biodegradable, making it very like for TMA-HCl to bear the same property. Although being expected to be hydrolytically stable in the natural environment, as a result, they both have a very low potential for bioaccumulation in aquatic and terrestrial organisms. Most importantly, TMA has a pKa of 9.78 - 9.8 at 20°C, which indicates that Trimethylamine exists almost entirely in the cationic form at pH values of 5 to 9.
For the following toxicological endpoints there is data derived from both substances available: Acute toxicity oral, Skin irritation / corrosion, Eye irritation /corrosion, Repeated dose toxicity oral and Toxicity to reproduction (fertility / developmental toxicity).
For the acute oral toxicity, the values for TMA are generally lower than the one of TMA-HCl, which trigger a classification as acute toxic cat. 4, i.e. LD50 values ranging from 397-820 mg/kg. Taking into account however the fact that TMA-HCl was applied as 58% solution, resulting in a LD50 of ca. 2 g/kg, and the generally higher molecular weight of TMA-HCl due to the additional HCl, the values for both substances can be considered as similar within normal biological variations.
Regarding Skin/Eye Irritation, TMA must be considered as (potentially) corrosive, whereas TMA-HCl is only irritating. These differences are likely attributable to differences in the pH value. The NOAELs derived for the endpoints Repeated dose toxicity oral and Toxicity to reproduction (fertility / developmental toxicity) also only differ marginally, which can be easily explained by the study design and the differences in molecular weight.
Reason / purpose:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The highest concentration to provide >10 % RS was 295.5 µg/mL, which gave 67 % and 54 % RS in the absence and presence of S-9, respectively.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table1: Trimethylamine (TMA-oplin water)Concentrations Tested

Experiment

S‑9

Concentration of treatment solution (mg/mL)

Final concentration (µg/mL)

 

 

 

 

Range-finder

-and +

0.1847

0.3694

0.7388

1.478

2.955

5.910

18.47

36.94

73.88

147.8

295.5

591.0

 

 

 

 

1

-and +

1.00

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

5.91

100

200

250

300

350

400

450

500

550

591.1

 

 

 

 

2

-

1.00

2.00

2.50

3.00

3.50

3.75

4.00

4.25

4.50

5.00

100

200

250

300

350

375

400

425

450

500

 

 

 

 

 

+

1.00

2.00

2.50

3.00

3.50

4.00

4.25

4.50

4.75

5.00

100

200

250

300

350

400

425

450

475

500

 

 

 

 

Experiment 1: ten concentrations, ranging from 100 to 591.1 µg/mL, were tested in the absence and presence of S‑9. Following the treatment incubation period, the highest three concentrations tested in the absence of S-9 (500 to 591.1 µg/mL) and the highest two concentrations tested in the presence of S-9 (550 and 591.1 µg/mL) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest remaining concentrations in the absence and presence of S‑9 (450 and 500 mg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, an intermediate concentration (300 mg/mL) in the absence of S-9 was not selected as there were sufficient concentrations to define an appropriate toxicity profile. All other concentrations in the absence and presence of S-9 were selected. The highest concentrations selected were 400 µg/mL in the absence of S‑9 and 450 µg/mL in the presence of S‑9, which gave 25 % and 5 % RS, respectively (seeTable 8). In the absence and presence of S-9, no concentration gave 10-20 % RS. In the absence of S‑9, cultures treated at 350 and 400 µg/mL, gave 50 % and 25 % RS, respectively and in the presence of S-9, cultures treated at 400 and 450 µg/mL, gave 38 % and 5 % RS, respectively. Both concentrations were therefore analysed under each treatment condition.

Experiment 2: ten concentrations, ranging from 100 to 500 µg/mL, were tested in the absence and presence of S‑9. Seven days after treatment, concentrations of 100 and 250 mg/mL in the presence of S-9 were not selected as there were sufficient concentrations to define an appropriate toxicity profile. All other concentrations in the absence and presence of S-9 were selected. The highest concentration selected was 500 µg/mL, which gave 20 % and 33 % RS in the absence and presence of S‑9, respectively (seeTable8). Cultures treated at 475 µg/mL in the presence of S-9 gave 23 % RS, which was sufficiently close to 10‑20 % RS to be considered acceptable.  

Table 2: Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

18.47

98

83

36.94

128

117

73.88

124

89

147.8

93

71

295.5

67

54

591

0

0

% RS               Percentage Relative Survival

Table 3: Experiment 1 (3-hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

2.93

 

0

 

100

4.91

 

100

 

90

5.47

NS

100

 

89

3.27

NS

200

 

79

3.91

NS

200

 

78

4.34

NS

250

 

55

4.08

NS

250

 

83

3.11

NS

350

 

50

2.69

NS

300

 

85

5.75

NS

400

 

25

5.03

NS

350

 

51

3.87

NS

 

 

 

 

 

400

 

38

5.30

NS

 

 

 

 

 

450

 

5

4.91

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

66

36.99

 

2

 

59

63.49

 

0.15

 

41

55.41

 

3

 

22

79.34

 

 

Table 4: Experiment 2 (3-hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

4.31

 

0

 

100

6.78

 

100

 

97

4.02

NS

200

 

90

4.26

NS

200

 

64

3.78

NS

300

 

75

5.01

NS

250

 

72

4.61

NS

350

 

71

3.65

NS

300

 

66

4.25

NS

400

 

59

4.95

NS

350

 

50

4.43

NS

425

 

54

6.38

NS

375

 

53

3.79

NS

450

 

30

5.62

NS

400

 

47

4.51

NS

475

 

23

3.43

NS

425

 

30

4.63

NS

500

 

33

6.92

NS

450

 

23

5.43

NS

 

 

 

 

 

500

 

20

4.72

NS

 

 

 

 

 

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

75

21.42

 

2

 

37

25.78

 

0.15

 

69

24.06

 

3

 

19

82.32

 

§                     6TG resistant mutants/106viable cells 7 days after treatment

% RS               Percent relative survival adjusted by post treatment cell counts

NS                   Not significant

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the mammalian cell gene mutation assay (hprt test in mouse lymphoma cells). Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

Conclusions:
Interpretation of results: negative
In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Trimethylamine (TMA-opl in water) at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. Under both treatment conditions, concentrations giving approximately 10-20 % RS (or less) were analysed in both experiments and there was no evidence of mutagenic activity at any concentration analysed in the absence and presence of S-9 in either experiment. Furthermore, the increases in pH observed in the absence and presence of S-9 in both experiments were not associated with increases in mutant frequency, therefore they were not considered biologically relevant.
CONCLUSION
It is concluded that Trimethylamine (TMA-opl in water) did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).
Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the mammalian cell gene mutation assay (hprt test in mouse lymphoma cells).Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).
Executive summary:

Trimethylamine (TMA-opl in water) was assayed by Covance (2010) for its potential to induce mutation at the hypoxanthine‑guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post‑mitochondrial fraction (S‑9).

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S‑9, ranging from 18.47 to 591.0 mg/mL (equivalent to approximately 10 mM at the highest concentration tested). The highest concentration to provide >10 % relative survival (RS) was 295.5 mg/mL, which gave 67 % and 54 % RS in the absence and presence of S‑9, respectively.

Accordingly, for Experiment 1 ten concentrations, ranging from 100 to 591.1 µg/mL, were tested in the absence and presence of S‑9. Seven days after treatment, the highest concentrations selected to determine viability and 6TG resistance were 400 µg/mL in the absence of S‑9 and 450 mg/mL in the presence of S-9, which gave 25 % and 5 % RS, respectively. In the absence and presence of S-9, no concentration gave 10‑20% RS (in the absence of S‑9, cultures treated at 350 and 400 µg/mL, gave 50 % and 25 % RS, respectively and in the presence of S-9, cultures treated at 400 and 450 µg/mL, gave 38 % and 5 % RS, respectively). Both concentrations were therefore analysed under each treatment condition. In Experiment 2 ten concentrations, ranging from 100 to 500 µg/mL, were tested in the absence and presence of S‑9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance was 500 µg/mL, which gave 20 % and 33 % RS in the absence and presence of S‑9, respectively. Cultures treated at 475 µg/mL in the presence of S-9 gave 23 % RS, which was sufficiently close to 10‑20 % RS to be considered acceptable. In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Trimethylamine at any concentration tested in the absence and presence of S‑9 and there were no significant linear trends. It is concluded that Trimethylamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S‑9).

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the mammalian cell gene mutation assay (hprt test in mouse lymphoma cells).Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No genetic toxicity studies are available for Trimethylamine hydrochloride. Therefore read-across from its nearest analogue Trimethylamine (CAS 75 -50 -3) is performed (please refer to read-across statement, Chemservice S.A., 2015).

Bacterial reverse mutation assays

The gene mutation properties of Trimethylamine were investigated in a bacterial reverse mutation assay (Ames test) conducted (Key study 1, 2001). This test was performed according to the OECD guideline 471 with Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 as well as with Escherichia coli strain WP2 uvr A. The concentrations of the test substance ranged from 0 to 5000 µg and the test result was negative in all tested strains, with and without metablic activation.

Another bacterial reverse mutation assay (Ames test) was performed to investigate trimethylamine for gene mutation (Key study 2, 1986), in accordance with OECD guideline 471 (not under GLP conditions). Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 were investigated with and without metabolic activation, whereas Salmonella typhimurium strain TA97 was tested only with metabolic activation system, which was rat / hamster S9 - mix (Aroclor 1254 included). A preincubation assay was done and the test concentration were 10, 33, 100, 333, and 1000 µg/plate, as vehicle water was used. The genotoxicity test showed for all strains under all circumstances a negative result.

Other genotoxicity tests

Trimethylamine (TMA-dissolved in water) was assayed for its potential to induce mutation at the hypoxanthine‑guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells (according to OECD guideline 476, under GLP conditions) (Key study 3, 2010a). The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post‑mitochondrial fraction (S‑9). In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S‑9, ranging from 18.47 to 591.0 mg/mL (equivalent to approximately 10 mM at the highest concentration tested). The highest concentration to provide >10 % relative survival (RS) was 295.5 mg/mL, which gave 67 % and 54 % RS in the absence and presence of S‑9, respectively. Accordingly, for Experiment 1 ten concentrations, ranging from 100 to 591.1 µg/mL, were tested in the absence and presence of S‑9. Seven days after treatment, the highest concentrations selected to determine viability and 6TG resistance were 400 µg/mL in the absence of S‑9 and 450 mg/mL in the presence of S-9, which gave 25 % and 5 % RS, respectively. In the absence and presence of S-9, no concentration gave 10‑20 % RS (in the absence of S‑9, cultures treated at 350 and 400 µg/mL, gave 50 % and 25 % RS, respectively and in the presence of S-9, cultures treated at 400 and 450 µg/mL, gave 38 % and 5 % RS, respectively). Both concentrations were therefore analyzed under each treatment condition. In Experiment 2, ten concentrations, ranging from 100 to 500 µg/mL, were tested in the absence and presence of S‑9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance was 500 µg/mL, which gave 20 % and 33 % RS in the absence and presence of S‑9, respectively. Cultures treated at 475 µg/mL in the presence of S-9 gave 23 % RS, which was sufficiently close to 10‑20 % RS to be considered acceptable. In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Trimethylamine at any concentration tested in the absence and presence of S‑9 and there were no significant linear trends. It is concluded that Trimethylamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S‑9).

Trimethylamine (TMA-dissolved in water) was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments (according to OECD 473 under GLP conditions; Key study 4, 2010b). Treatments covering a broad range of concentrations were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The highest concentration used in the main experiments, 591.1 mg/mL(equivalent to 10 mM) was determined following a preliminary cytotoxicity Range-Finder Experiment. The test article concentrations for chromosome analysis were selected by evaluating the effect of Trimethylamine on mitotic index. Treatment of cultures with Trimethylamine in the absence and presence of S-9 resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within normal ranges. No increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were generally observed in cultures treated with Trimethylamine in the absence and presence of S-9. The only exception to this was observed in Experiment 2 in the absence of S-9 at one intermediate concentration (320.0 µg/mL), where the frequency of cells with numerical aberrations marginally exceeded the 95th percentile of the normal range (but fell within the observed normal range) in both cultures. The increases were almost entirely attributable to hyperdiploid cells but they were small and there was no evidence of a concentration-related response, therefore they were not considered biologically relevant.

It is concluded that Trimethylamine did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of cytotoxicity for 20+0 hours in the absence of S-9 and up to a maximum of 10 mM for 3+17 hours in the absence and presence of S-9.

Trimethylamine and Trimethylammonium chloride belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl groups (common structure / functional group). The solvation of both Trimethylamine and Trimethylammonium chloride in water results in solutions of the Trimethylammonium 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 Trimethylammonium cation. Genetic toxicity of the respective counterion is not applicable (Cl-, OH-), so primarily the trimethylammonium cation has to be evaluated. The Trimethylammonium cation is believed to act and to be metabolised by the same mechanisms by microorganisms and by other classes of living organisms and not to bind to biomolecules or DNA. Trimethylamine has been shown not to be a genetic toxicant in the Ames Test, HPRT test and in human lymphocytes (chromosome aberration test).

Therefore both substances are expected to follow the same pattern of genetic toxicity. For the detailed procedure of the read-across principle and justifications, please refer to the analogue approach justification depicted below and the separate Read-Across Statement (Chemservice S.A., 2015).


Justification for selection of genetic toxicity endpoint
No study is selected since all studies were negative.

Short description of key information:
The source substance Trimethylamine (CAS 75-50-3) was negative in all in vitro tests: Ames Test (OECD 471), gene mutation (OECD 476) and chromosome aberration test (OECD 473).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The source substance Trimethylamine (CAS 75 -50 -3) does not possess genetic toxicity potential in in vitro studies in bacterial and in mammalian cells. Therefore, TMA HCl is considered to be not genotoxic and classification is not warranted according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.