Registration Dossier

Administrative data

Description of key information

Triethylamine (read-across)_Mouse Ear Swelling Test, not sensitizing

Triethylamine (read-across)_Intracutaneous in-vivo test with Guinea pigs, not sensitizing

Tributylamine (read-across)_ Study according to EPA guideline 798.4100, not sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
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), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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. When assessing the possible irritating properties of the substances, some differences have to be taken into account. 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 (induction of irritant dermatitis) of TMA/TEA and TBA can certainly be explained by the high concentration of hydroxyl anions in the solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. For example, the available data suggest additionally a corrosive potential for TMA (Skin Irrit. 2 and Eye Dam. 1), 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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins. However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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: Triethylamine (TEA, C6H15N, SMILES CCN(CC)CC, CAS 121-44-8, EC 204-469-4)
Source chemical: Tributylamine (TBA, C12H27N, SMILES CCCCN(CCCC)CCCC, CAS 102-82-9, EC 203-058-7)
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/Triethylamine/Tributylamine 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/TEA/TBA and TMA-HCl on their own. Nevertheless, the sp³-hybridized nitrogen with three methyl/ethyl/butyl groups can be regarded as a common structure / functional group of both TMA/TEA/TBA 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, all substances dissociate into either identical Trimethylammonium cations or to very similar Triethylammonium cations and Tributylammonium cations and exhibit very similar physico-chemical and toxicological properties, as outlined below.
In detail the dissociation constants of TMA/TEA and TBA allow the conclusion that virtually all molecules of - when dissolved in an excess of water - are present as the Trimethylammonium/Triethylammonium/Tributylammonium cation. Moreover, the available pka-data and the titration curve of TMA/TEA/TBA 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 Trimethylamine/Triethylamine/Tributylamine and Trimethylammonium chloride in water would result in solutions of the Trimethylammonium/ Triethylammonium/ Tributylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. Trimethylamine/Triethylamine/Tributylamine 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 for example 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. In detail, 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 / Triethylammonium / Tributylammonium cation.
The similar findings (refer to data matrix outlined below) for all substances support the conclusion that the identical/similar molecule will be formed from both substances when applied dermally, and this molecule, i.e. the Trimethylammonium cation, is responsible for the observed effects. In consequence, the Trimethylammonium/Triethylammonium /Tributylammonium cation is what is left to be considered and similar effects can be reasonably expected when testing TMA-HCl for the lacking endpoints, compared to the data obtained with TMA/TEA/TBA. Hence, TMA/TEA/TBA may serve as read-across substance for TMA-HCl. TEA and TBA were not sensitising in the skin sensitisation studies: Intracutaneous Test, Ear Swelling Test and in Buehler Test. So, the available data on TEA and TBA can be used to cover the skin sensitisation endpoint currently lacking from TMA-HCl, making further testing obsolete as it is unlikely that the target substance TMA-HCL possesses a skin sensitization potential.

4. DATA MATRIX
There is mainly data available on the toxicological properties of TMA/TEA/TBA, Data on TMA-HCl covers merely the toxicokinetic endpoint. Please refer to section: Overall remarks, attachments for a detailed summary of the available data for each of the source and target substances.
Reason / purpose for cross-reference:
read-across source
Positive control results:
80 % of the animals treated with DNCB exhibited a positive sensitization response (test ear at least 20 % thicker than control ear), at 24-hours and 100 % exhibited a positive response at 48 hours.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % TEA
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no ear swelling
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100% TEA. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no ear swelling.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 % TEA
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no ear swelling
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100% TEA. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no ear swelling.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 % TEA
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no irritation
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100% TEA. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no irritation.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 % TEA
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no irritation
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1005 TEA. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no irritation.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1 % DNCB
No. with + reactions:
4
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
1 % DNCB
No. with + reactions:
5
Total no. in group:
5

Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl  counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.

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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.

However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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).

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, triethylamine (C-1043) exhibited no potential to produce sensitiiation in mice.
These results can be used to fulfill the requirements for Trimethylamine hydrochloride as Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl  counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.
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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.
However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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:

The Mouse Ear Swelling Test was conducted to evaluate the allergic contact sensitization potential of Triethylamine (TEA) in mice. Albino Mice of CFL (BR) strain were used in all experiments. The concentrations of test material for induction and challenge phases were determined in a range-finding study. For induction, 1 % TEA in 70% ethanol was applied to abdomens of animals. For the challenge, 100 % TEA was applied topical, to the dorsal and ventral surface of the ear of the animals. 2,4- dinitrochlorobenzene (DNCB) was used as positive control. Freund's Complete Adjuvant (FCA) consisted from a mixture of paraffin oil and an emulsifier with mycobacteria. On the day 0 (Induction phase), a 50 % concentration of FCA in water was administered by injection to two clipped and stripped of horny layer sites of abdomen, one on the left side and one on the right side. 100 µL of whether test material, positive control or appropriate vehicle was administered directly to the test site, on the abdomen of the animals. This procedure was repeated on day 1, 2, and 3 (Induction phase). On the day 9 before the challenge administration, both ears of each animals were examined for the presence of irritation (erythema, edema). No pre-existing irritation was seen in any animal, therefore, all animals remained on study. On the day 10, test animals were challenged with whether 10 µL of 100 % TEA or DNCB on the dorsal and ventral surface of the left ear of each animal. 10 µL of appropriate vehicle was administered in the same manner on the right ear. In order to differentiate reactions produced by irritation from those produced by sensitization, five irritation control animals (previousiy treated with vehicle and FCA) were subjected to the same challenge procedure as the test animals. On the day 16, before pre-challenge administration, the ears were examined again for the presence of irritation. At re-challenge, the same procedures were followed as were followed at challenge (Day 10), except the ears were switched, i.e., only the right ear was dosed with the test material; the left ear was not treated. Irritation control animals, previously treated with vehicle and FCA only and not treated at challenge) were subjected to the same challenge procedure as the test animals. The first and the second readings (24 and 48 hours, respectively) were performed after challenge and re-challenge application.

Except for one animal which did not recover from the anesthesia at the 24-hour Challenge Response Measurement (DNCB irritation control animal , Group 1B), all animals survived throughout the study. 80 % of the animals treated with DNCB exhibited a positive sensitization response (test ear at least 20 % thicker than control ear) , at 24-hours and 100 % exhibited a positive response at 48 hours. There were no positive responders in the irritation control group. This positive response to a known sensitizer demonstrated the susceptibility of this group of animals to sensitization. Animals challenged with Triethylamine exhibited no positive responses at challenge or rechallenge in either the test group or irritation control groups.

These results can be used to fulfill the requirements for Trimethylamine hydrochloride as Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl  counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.

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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.

However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
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), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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. When assessing the possible irritating properties of the substances, some differences have to be taken into account. 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 (induction of irritant dermatitis) of TMA/TEA and TBA can certainly be explained by the high concentration of hydroxyl anions in the solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. For example, the available data suggest additionally a corrosive potential for TMA (Skin Irrit. 2 and Eye Dam. 1), 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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins. However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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: Triethylamine (TEA, C6H15N, SMILES CCN(CC)CC, CAS 121-44-8, EC 204-469-4)
Source chemical: Tributylamine (TBA, C12H27N, SMILES CCCCN(CCCC)CCCC, CAS 102-82-9, EC 203-058-7)
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/Triethylamine/Tributylamine 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/TEA/TBA and TMA-HCl on their own. Nevertheless, the sp³-hybridized nitrogen with three methyl/ethyl/butyl groups can be regarded as a common structure / functional group of both TMA/TEA/TBA 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, all substances dissociate into either identical Trimethylammonium cations or to very similar Triethylammonium cations and Tributylammonium cations and exhibit very similar physico-chemical and toxicological properties, as outlined below.
In detail the dissociation constants of TMA/TEA and TBA allow the conclusion that virtually all molecules of - when dissolved in an excess of water - are present as the Trimethylammonium/Triethylammonium/Tributylammonium cation. Moreover, the available pka-data and the titration curve of TMA/TEA/TBA 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 Trimethylamine/Triethylamine/Tributylamine and Trimethylammonium chloride in water would result in solutions of the Trimethylammonium/ Triethylammonium/ Tributylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. Trimethylamine/Triethylamine/Tributylamine 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 for example 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. In detail, 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 / Triethylammonium / Tributylammonium cation.
The similar findings (refer to data matrix outlined below) for all substances support the conclusion that the identical/similar molecule will be formed from both substances when applied dermally, and this molecule, i.e. the Trimethylammonium cation, is responsible for the observed effects. In consequence, the Trimethylammonium/Triethylammonium /Tributylammonium cation is what is left to be considered and similar effects can be reasonably expected when testing TMA-HCl for the lacking endpoints, compared to the data obtained with TMA/TEA/TBA. Hence, TMA/TEA/TBA may serve as read-across substance for TMA-HCl. TEA and TBA were not sensitising in the skin sensitisation studies: Intracutaneous Test, Ear Swelling Test and in Buehler Test. So, the available data on TEA and TBA can be used to cover the skin sensitisation endpoint currently lacking from TMA-HCl, making further testing obsolete as it is unlikely that the target substance TMA-HCL possesses a skin sensitization potential.

4. DATA MATRIX
There is mainly data available on the toxicological properties of TMA/TEA/TBA, Data on TMA-HCl covers merely the toxicokinetic endpoint. Please refer to section: Overall remarks, attachments for a detailed summary of the available data for each of the source and target substances.
Reason / purpose for cross-reference:
read-across source
Reading:
1st reading
Group:
test chemical
Dose level:
0.1 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
not specified, however it was concluded that no sensitization had taken place
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Group:
negative control
Dose level:
saline / deionised water
Remarks on result:
other: see 'Remarks'
Remarks:
no further details avialable
Reading:
1st reading
Group:
positive control
Remarks on result:
other: see 'Remarks'
Remarks:
no further details available

These results can be used to fulfill the requirements for Trimethylamine hydrochloride as Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.

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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.

However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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).

Interpretation of results:
GHS criteria not met
Conclusions:
Sensitization by the intracutaneous method was attempted with triethylamine on separate groups of male albino guinea pigs with negative results in all cases.
These results can be used to fulfill the requirements for Trimethylamine hydrochloride as Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl  counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.
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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.
However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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:

Groups of 350 to 450 g male albino guinea pigs were given the usual course of 8 sensitizing intracutaneous injections of 0.1 % solutions in 0.75 % saline made up in deionized water. Challenge doses were given 3 weeks after the last sensitizing dose. 8 out of 20 animals were killed or were infected during the study. The reason of bad conditions of animals was not related to treatment. The remaining 12 animals were participated till the end of the study. The guinea pigs used in this study were not a particularly healthy group although the survivors seemed to be in fair conditions. No animals were sensitized. It was concluded if the test compound was potential sensitizer it was very weak. This statement was made because of knowledge that subnormal animals were often refractory to sensitization.

In general, these results could be used to fulfill the requirements for Trimethylamine hydrochloride as Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl  counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.

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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.

However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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).

Due to the high mortality on test animals in this study, the results are considered Klimisch 3 - not reliable, and will not be used for Classificaiton and Labelling purposes.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
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), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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. When assessing the possible irritating properties of the substances, some differences have to be taken into account. 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 (induction of irritant dermatitis) of TMA/TEA and TBA can certainly be explained by the high concentration of hydroxyl anions in the solution, which are likely to occur even when the gas gets in contact with skin moisture or other body fluids. For example, the available data suggest additionally a corrosive potential for TMA (Skin Irrit. 2 and Eye Dam. 1), 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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins. However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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: Triethylamine (TEA, C6H15N, SMILES CCN(CC)CC, CAS 121-44-8, EC 204-469-4)
Source chemical: Tributylamine (TBA, C12H27N, SMILES CCCCN(CCCC)CCCC, CAS 102-82-9, EC 203-058-7)
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/Triethylamine/Tributylamine 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/TEA/TBA and TMA-HCl on their own. Nevertheless, the sp³-hybridized nitrogen with three methyl/ethyl/butyl groups can be regarded as a common structure / functional group of both TMA/TEA/TBA 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, all substances dissociate into either identical Trimethylammonium cations or to very similar Triethylammonium cations and Tributylammonium cations and exhibit very similar physico-chemical and toxicological properties, as outlined below.
In detail the dissociation constants of TMA/TEA and TBA allow the conclusion that virtually all molecules of - when dissolved in an excess of water - are present as the Trimethylammonium/Triethylammonium/Tributylammonium cation. Moreover, the available pka-data and the titration curve of TMA/TEA/TBA 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 Trimethylamine/Triethylamine/Tributylamine and Trimethylammonium chloride in water would result in solutions of the Trimethylammonium/ Triethylammonium/ Tributylammonium cation (common "breakdown product"). One must only regard the physico-chemical properties of the respective counterion. Trimethylamine/Triethylamine/Tributylamine 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 for example 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. In detail, 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 / Triethylammonium / Tributylammonium cation.
The similar findings (refer to data matrix outlined below) for all substances support the conclusion that the identical/similar molecule will be formed from both substances when applied dermally, and this molecule, i.e. the Trimethylammonium cation, is responsible for the observed effects. In consequence, the Trimethylammonium/Triethylammonium /Tributylammonium cation is what is left to be considered and similar effects can be reasonably expected when testing TMA-HCl for the lacking endpoints, compared to the data obtained with TMA/TEA/TBA. Hence, TMA/TEA/TBA may serve as read-across substance for TMA-HCl. TEA and TBA were not sensitising in the skin sensitisation studies: Intracutaneous Test, Ear Swelling Test and in Buehler Test. So, the available data on TEA and TBA can be used to cover the skin sensitisation endpoint currently lacking from TMA-HCl, making further testing obsolete as it is unlikely that the target substance TMA-HCL possesses a skin sensitization potential.

4. DATA MATRIX
There is mainly data available on the toxicological properties of TMA/TEA/TBA, Data on TMA-HCl covers merely the toxicokinetic endpoint. Please refer to section: Overall remarks, attachments for a detailed summary of the available data for each of the source and target substances.
Reason / purpose for cross-reference:
read-across source
Positive control results:
6/10 test sites were erythematous 24 h after challenge, 4/10 were clear. At the 48 h reading there were 8 sites with erythema and 2 without effect. Scores ranged from 0.5 (very faint, nonconfluent) to 3 (strong).
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
4 %
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 4%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
4 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 4%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.08 % DNCB
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.08% DNCB. No with. + reactions: 6.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.08 % DNCB
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.08% DNCB. No with. + reactions: 8.0. Total no. in groups: 10.0.

All animals appeared active and healthy and gained weight during the induction phase and in the period to challenge.

Induction phase: The occurence of generall sporadic and transient very faint non-confluent erythema (score 0.5) was observed during the first two weeks of the induction phase. One animal had eschar at the test site between the 5th and 6th induction treatment which did not re-occur after change of the test site. One animal showed a faint erythema after the 9th induction.

Challenge: Two sites challenged with the test substance showed very faint, non-confluent erythema 24 h after challenge (score 0.5; remaining animals: score 0). No reaction (score 0) was observed in all animals 48 h after application.

These results can be used to fulfill the requirements for Trimethylamine hydrochloride as Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl  counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.

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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.

However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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).

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study tributylamine was not sensitising in a Buehler guinea pig sensitisation test.
These results can be used to fulfill the requirements for Trimethylamine hydrochloride as Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl  counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.
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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.
However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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:

In this study according to EPA guideline 798.4100 a highest non-irritating concentration of a 4 % dilution of Tributylamine (89 % purity) in 95 % ethanol was determined in a pre-test. 10 young adult male guinea pigs were treated ten times for 6 h each with this concentration during a 3.5 week induction phase. Another group of 10 males were treated equally with the positive control substance (0.08 % dinitrochlorobenzene in 95 % ethanol). The occurrence of general sporadic and transient very faint non-confluent erythema was observed during the first two weeks of the induction phase. One animal developed transient eschar at the test site which did not re-occur after change of the test site. One animal showed a faint erythema after the 9th induction. Challenge: Fourteen days after the last induction a challenge with the same concentration of test substance or the positive control substance was performed at a naive site of each animal and the reaction was scored 24 and 48 h later. 5 animals per each group served as naive controls and were treated with the test material or control material only at challenge. Two animals challenged with Tributylamine showed very faint, non-confluent erythema 24 h after challenge (Score 0.5, remaining animals: score: 0). No reaction was observed in all animals 48 h after application. Based on these results Tributylamine was judged to be not sensitising. The positive control substance produced a positive response (Pennwalt/PSL, 1988). This study was judged to be reliable (RL2) and selected as weight of evidence.

These results can be used to fulfill the requirements for Trimethylamine hydrochloride as Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl  counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.

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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.

However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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 (not sensitising)
Additional information:

Skin sensitization:

No data is available for the target substance Trimethylammonium hydrochloride. However, there is data for the structural analogues Triethylamine and Tributylamine.

The Mouse Ear Swelling Test was conducted to evaluate the allergic contact sensitization potential of Triethylamine (TEA) in mice. Albino Mice of CFL (BR) strain were used in all experiments. The concentrations of test material for induction and challenge phases were determined in a range-finding study. For induction, 1% TEA in 70% ethanol was applied to abdomens of animals. For the challenge, 100% TEA was applied topical, to the dorsal and ventral surface of the ear of the animals. 2,4- dinitrochlorobenzene (DNCB) was used as positive control. Freund's Complete Adjuvant (FCA) consisted from a mixture of paraffin oil and an emulsifier with mycobacteria. On the day 0 (Induction phase), a 50% concentration of FCA in water was administered by injection to two clipped and stripped of horny layer sites of abdomen, one on the left side and one on the right side. 100 µL of whether test material, positive control or appropriate vehicle was administered directly to the test site, on the abdomen of the animals. This procedure was repeated on day 1, 2, and 3 (Induction phase). On the day 9 before the challenge administration, both ears of each animals were examined for the presence of irritation (erythema, oedema). No pre-existing irritation was seen in any animal, therefore, all animals remained on study. On the day 10, test animals were challenged with whether 10 µL of 100% TEA or DNCB on the dorsal and ventral surface of the left ear of each animal. 10 µL of appropriate vehicle was administered in the same manner on the right ear. In order to differentiate reactions produced by irritation from those produced by sensitization, five irritation control animals (previousiy treated with vehicle and FCA) were subjected to the same challenge procedure as the test animals. On the day 16, before pre-challenge administration, the ears were examined again for the presence of irritation. At re-challenge, the same procedures were followed as were followed at challenge (Day 10), except the ears were switched, i.e., only the right ear was dosed with the test material; the left ear was not treated. Irritation control animals, previously treated with vehicle and FCA only and not treated at challenge) were subjected to the same challenge procedure as the test animals. The first and the second readings (24 and 48 hours, respectively) were performed after challenge and re-challenge application.

Except for one animal which did not recover from the anesthesia at the 24-hour Challenge Response Measurement (DNCB irritation control animal , Group 1B), all animals survived throughout the study. 80% of the animals treated with DNCB exhibited a positive sensitization response (test ear at least 20% thicker than control ear), at 24-hours and 100% exhibited a positive response at 48 hours. There were no positive responders in the irritation control group. This positive response to a known sensitizer demonstrated the susceptibility of this group of animals to sensitization. Animals challenged with Triethylamine exhibited no positive responses at challenge or rechallenge in either the test group or irritation control groups.

Furthermore, an intracutaneous in-vivo test with Guinea pigs was performed. Groups of 350 to 450 g male albino guinea pigs were given the usual course of 8 sensitizing intracutaneous injections of 0.1% solutions in 0.75% saline made up in deionized water. Challenge doses were given 3 weeks after the last sensitizing dose. 8 out of 20 animals were killed or were infected during the study. The reason of bad conditions of animals was not related to treatment. The remaining 12 animals were participated till the end of the study. The guinea pigs used in this study were not a particularly healthy group although the survivors seemed to be in fair conditions. No animals were sensitized. It was concluded if the test compound was potential sensitizer it was very weak. This statement was made because of knowledge that subnormal animals were often refractory to sensitization.

Last but not least a study according to EPA guideline 798.4100 was conducted with Tributylamine. The highest non-irritating concentration of a 4% dilution of Tributylamine (89% purity) in 95% ethanol was determined in a pre-test. 10 young adult male guinea pigs were treated ten times for 6 h each with this concentration during a 3.5 week induction phase. Another group of 10 males were treated equally with the positive control substance (0.08% dinitrochlorobenzene in 95% ethanol). The occurrence of general sporadic and transient very faint non-confluent erythema was observed during the first two weeks of the induction phase. One animal developed transient eschar at the test site which did not re-occur after change of the test site. One animal showed a faint erythema after the 9th induction. Challenge: Fourteen days after the last induction a challenge with the same concentration of test substance or the positive control substance was performed at a naive site of each animal and the reaction was scored 24 and 48 h later. 5 animals per each group served as naive controls and were treated with the test material or control material only at challenge. Two animals challenged with Tributylamine showed very faint, non-confluent erythema 24 h after challenge (Score 0.5, remaining animals: score: 0). No reaction was observed in all animals 48 h after application. Based on these results Tributylamine was judged to be not sensitising. The positive control substance produced a positive response (Pennwalt/PSL, 1988). This study was judged to be reliable (RL2) and selected as weight of evidence.

All of these results can be used to fulfill the requirements for Trimethylamine hydrochloride as Trimethylamine (and Trimethylammonium chloride), Triethylamine and Tributylamine belong to the group of tertiary aliphatic amines with a sp³-hybridized nitrogen with three methyl / ethyl / butyl groups as a common structure / functional group attached to the nitrogen atom (R-N(R1)-R2). TMA-HCl is a tertiary ammonium salt. 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 Trimethylamine and Trimethylammonium chloride, Triethylamine and Tributylamine in water results in solutions of the Trimethylammonium, Triethylammonium and Tributylammonium cation (common "breakdown product"). No skin sensitisation potential can be attributed to the hydroxyl counterion (OH-), but causes a decrease of the pH, resulting in alkaline solutions, whereas the other anion (Chloride anion, Cl-, 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.

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 / Triethylammonium / Tributylammonium cation, which might further interact with skin proteins.

However, Triethylammonium and Tributylammonium cation have been shown not to bind to proteins, thereby not producing a hapten eliciting a skin sensitisation response and are therefore classified as not skin sensitising. So, information on skin sensitisation of Triethylamine and Tributylamine allows to strengthen the read-across approach. 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).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The classification and labelling for Trimethylammonium hydrochloride is based on the results of three recent skin sensitisation studies with the structural analogues Triethylamine and Tributylamine.

In accordance with EU CLP Regulation (EC) No. 1272/2008, no classification is required for Trimethylammonium hydrochloride.