N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meeting generally accepted scientific principles, acceptable for assessment.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Assessment of skin permeation through and sequestration in isolated mouse skin in vitro by measurement of radiolabelled test material uptake.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

14C

Species:

mouse

Strain:

other: hairless (HRS/hr hr)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: 6-14 weeks

Type of coverage:

open

Vehicle:

other: phosphate buffered saline (PBS)

Duration of exposure:

24 hours in permeation experiment; 16 hours in pre-treatment experiment

Doses:

- Actual doses: 16 mM
- Dose volume: 0.5 mL
- Rationale for dose selection: the concentration was chosen because it represented a similar chemical dose in µmoles as for C16BET, to which permeation of C12BET was compared.

No. of animals per group:

Not applicable, in vitro: eight separate measurements of penetration (using skin from a minimum of four mice)

Control animals:

no

Remarks:

not applicable

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions: dilution in PBS (pH 7.4)


APPLICATION OF DOSE:
0.5 ml of surfactant solution (spiked with 14C-labelled compound) in PBS was administered to the epidermal skin surface (0.95 cm²) exposed in the donor compartment of the diffusion cell.

VEHICLE
- Justification for use and choice of vehicle (if other than water): PBS is widely used as buffer in physiological systems


TEST SITE
- Preparation of test site: Immediately after euthanizing a hairless mouse by CO2 asphyxiation, full thickness dorsal skin was excised. Any subcutaneous fat adhering to the dermal surface was carefully removed. The skin was then mounted between the donor and the receptor compartments of glass permeation cells (Laboratory Glass Apparatus, Berkeley, CA). The accessible skin area for transport was 0.95 cm²; one mouse, therefore, typically provided sufficient skin for four cells.
The receptor compartment (volume = 3 ml) was perfused with pH 7.4 PBS at a rate of 3 mL/hr. The chamber temperature was regulated by a thermostat to maintain the skin surface temperature at 32 ± 1°C .
- Area of exposure: 0.95cm²
- Time intervals for shavings or clipplings: not applicable, in vitro


SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: not applicable, in vitro


SAMPLE COLLECTION
- Collection of perfusate samples: For 24 hours perfusate samples of 3 mL at each hour were collected on a fraction collector and then assayed for surfactant.
- Terminal procedure: At the end of the transport experiment, the skin membrane was washed quickly in fresh buffer solution and was then dissolved in Soluene (Packard, Downers Grove, IL) before liquid scintillation counting. In this way the amount of test substance sequestered within the skin during the penetration process was determined.


ANALYSIS
- Method type(s) for identification: Liquid scintillation counting (Searle Model 6880)


OTHER:
To assess the effects of surfactant pre-treatment on skin barrier function, hairless mouse skin was excised and mounted in the diffusion cell as described above. The receptor chamber was filled with PBS and maintained at 32 ± 1°C without perfusion. Non-Labelled surfactant solution (0.5 mL) was applied to the epidermal surface in the donor compartment for 16 hours (i.e. overnight). Concentrations applied were 16, 100 and 800 mM. At the end of the pre-treatment period, surfactant solution was removed and the skin gently washed three times with 0.5 ml of distilled water. The receptor compartment was drained, flushed with fresh PBS, and then perfused at 3 mL/hr. Nicotinamide solution (0.5 mL, concentration 100 µg/mL, spiked with 14C-labelled compound) was administered to the epidermal surface and hourly 3 ml samples of the receptor phase were collected for the subsequent 12 hours. Nicotimamide permeation was determined by analysing the fractions using liquid scintillation counting. Experiments were performed in quadruplicate. Parallel control measurements were performed in which the pre-treatment solution was PBS. Typically, skins from two mice were used such that half of the tissue from each mouse was pretreated with surfactant and the other half with PBS.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: hairless mice
- Type of skin: full thickness dorsal skin
- Preparative technique: excised after CO2 asphyxiation, any subcutaneous fat adhering to the dermal surface was carefully removed
- Thickness of skin (in mm): full thickness, not further specified
- Storage conditions: not stored


PRINCIPLES OF ASSAY
- Diffusion cell: glass permeation cells (Laboratory Glass Apparatus, Berkeley, CA)
- Receptor fluid: PBS pH 7.4
- Solubility of test substance in receptor fluid: yes, fully soluble
- Static system: donor compartment
- Flow-through system: receptor compartment, 3 ml/hr
- Test temperature: 32 ± 1°C
- Occlusion: no

Absorption in different matrices:

- Receptor fluid, receptor chamber, donor chamber (in vitro test system): 10.3 ± 3.5 % absorbed after 12 hours and 46.5 ± 8.2 % absorbed after 24 hours (corresponding to 220 ± 80 and 1010 ± 180 µg, respectively) (mean ± SD)
- Skin preparation (in vitro test system): approximately 25 % associated to the skin after 24 hours

Total recovery:

- Total recovery: nearly 75 %
- Recovery of applied dose acceptable: yes

Dose:

16 mM

Parameter:

percentage

Absorption:

ca. 10.3 %

Remarks on result:

other: 12 hours

Remarks:

mouse skin

Dose:

16 mM

Parameter:

percentage

Absorption:

ca. 46.5 %

Remarks on result:

other: 24 hours

Remarks:

mouse skin

Cumulative penetration of Nicotinamide (mean±SD) in 12 hours following pretreatment of hairless mouse skin with C12BET:

Surfactant	Applied concentration (mM)	Normalized concentration (C*)	Cumulative % dose absorbed	Number of replicates
Control	0	0	0.32 ± 0.26	42
C12BET	16	10	5.1 ± 1.7	4
	100	62.5	8.0 ± 3.1	4
	800	500	33.0 ± 7.4	4

C* = applied concentration divided by the CMC (1.6 mM)

Control pre-treated with pH 7.4 PBS containing no surfactant

Conclusion:

Pre-treatment with C12BET at all concentrations significantly (p < 0.05) promotes nicotinamide penetration across hairless mouse skin when compared to the controls.

Reference 2

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

read-across source

Absorption in different matrices:

- Receptor fluid, receptor chamber, donor chamber (in vitro test system): 10.3 ± 3.5 % absorbed after 12 hours and 46.5 ± 8.2 % absorbed after 24 hours (corresponding to 220 ± 80 and 1010 ± 180 µg, respectively) (mean ± SD)
- Skin preparation (in vitro test system): approximately 25 % associated to the skin after 24 hours

Total recovery:

- Total recovery: nearly 75 %
- Recovery of applied dose acceptable: yes

Dose:

16 mM

Parameter:

percentage

Absorption:

ca. 10.3 %

Remarks on result:

other: 12 hours

Remarks:

mouse skin

Dose:

16 mM

Parameter:

percentage

Absorption:

ca. 46.5 %

Remarks on result:

other: 24 hours

Remarks:

mouse skin

Cumulative penetration of Nicotinamide (mean±SD) in 12 hours following pretreatment of hairless mouse skin with C12BET:

Surfactant	Applied concentration (mM)	Normalized concentration (C*)	Cumulative % dose absorbed	Number of replicates
Control	0	0	0.32 ± 0.26	42
C12BET	16	10	5.1 ± 1.7	4
	100	62.5	8.0 ± 3.1	4
	800	500	33.0 ± 7.4	4

C* = applied concentration divided by the CMC (1.6 mM)

Control pre-treated with pH 7.4 PBS containing no surfactant

Conclusion:

Pre-treatment with C12BET at all concentrations significantly (p < 0.05) promotes nicotinamide penetration across hairless mouse skin when compared to the controls.

Key value for chemical safety assessment

Absorption rate - dermal (%):

46.5

Additional information

No toxicokinetic studies are available for the substance N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid.

In accordance with Annex VIII, Column 1, Item 8.8 of Regulation (EC) 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2012), assessment of the toxicokinetic behavior of the substance N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid was conducted to the extent that can be derived from the relevant available information on physicochemical and toxicological characteristics. Two studies focusing on dermal absorption are available for the substance CAS 683-10-3 used as read-across source substance following an analogue approach (Ridout et al., 1991; Buck et al., 1993).

N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid (molecular weight of 311.5 to 339.6 g/mol) is a white solid, which is soluble in water (> 470 g/L at room temperature). It has a log Pow of 2.73 at 23 °C and a vapour pressure of 131 Pa at 20 °C.

 

Absorption

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, the octanol/water partition coefficient (log Pow) value and the water solubility. The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2012).

 

Oral:

The smaller the molecule, the more easily it may be taken up. In general, molecular weights below 500 are favorable for oral absorption (ECHA, 2012). Since the molecular weight of N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid ranges between 311.5 and 339.6 g/mol, an absorption of the molecule in the gastrointestinal tract is in general anticipated.

Moreover, absorption after oral ingestion of N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid is also expected when the “Lipinski Rule of Five” (Lipinski et al., 2001; Ghose et al., 1999) is applied, as all rules are fulfilled.

As the substance is soluble in water, it will dissolve in gastrointestinal fluids. However, the log Pow of 2.73 suggests absorption of the substance via passive diffusion.

Additionally, there are experimental data available investigating the acute oral toxicity of the structural analogue substances CAS 68424-94-2 (Levenstein, 1979) and EC 931-700-2 (Huntigdon Research Centre, 1969). Both substances revealed LD50 values of the same order of magnitude (2640 mg/kg bw in mice and 3202 mg/kg bw in rats, respectively). Mortality was observed in both studies. For EC 931-700-2, necropsy of the deceased animals revealed gastrointestinal inflammation. Such effects were not noticed at necropsy of the surviving animals. Therefore, the mortality observed in this study had to be attributed to local irritant effects of the test substance rather than systemic toxicity.

Based on these results, no conclusions on the oral absorption potential of the substance are possible. 

In summary, according to its physico-chemical properties, an absorption of N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid is possible. However, as the substance is classified as corrosive according to Regulation (EC) No 1272/2008 and EU Directive 67/548/EEC, the local effects will be probably more predominant than the systemic toxicity.

 

Dermal:

The smaller the molecule, the more easily it may be taken up. In general, a molecular weight below 100 favours dermal absorption; above 500 the molecule may be too large. Log Pow values between 1 and 4 favour dermal absorption particularly if water solubility is high (ECHA, 2012). As N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid has a molecular weight of 311.5 and 339.6 g/mol and a log Pow of 2.73 paired with a high water solubility, dermal absorption is likely.

If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration (ECHA, 2012). As the substance is considered as skin corrosive in humans, an enhanced penetration of the substance due to local skin damage cannot be excluded.

Additionally, QSAR prediction for the skin absorption potential of N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid resulted in a high dermal absorption potential of about 80% (Mostert, V. and Goergens, A., 2011).

Furthermore, there is one publication on permeation through and sequestration in mouse skin in vitro available for the substance CAS 683-10-3,used as read-across source substance (Ridout et al., 1991). While 46.5% had penetrated the skin after 24 hours, approximately 25% of the applied dose was sequestrated within the skin. Total recovery of radioactivity was nearly 75% of the applied dose, indicating a high dermal absorption potential of the test substance. Additionally, this publication demonstrated a significant impairment of skin barrier function after pre-treatment with the test substance.

Published data of an in vivo dermal penetration study in human volunteers with the same substance demonstrated a maximum uptake of only 0.4% of the applied dose, which was primarily localized in the outer layers of the stratum corneum (Bucks et al., 1993). However, the investigation of the dermal absorption was conducted only 30 minutes after application of the test substance. Systemic availability of the applied test substance was not tested.

Acute dermal toxicity of the analogue substance CAS 61789-40-0 was investigated in male and female rats (Kao, 1987). No systemic toxicity was observed up to the highest dose tested (LD50 > 620 mg/kg bw a.i.). However, local irritation of the treated skin sites was noticed in all animals.

In summary, based on all available data, a dermal absorption of N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid is likely.

 

Inhalation:

N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid has a vapour pressure of 131 Pa at 20 °C thus being of low volatility. However, if the substance is sprayed, it may be available for respiratory absorption after inhalation of aerosols. In humans, particles with aerodynamic diameters below 100 µm have the potential to be inhaled. Particles with aerodynamic diameters below 50 µm may reach the thoracic region and those below 15 µm the alveolar region of the respiratory tract (ECHA, 2012).

Since the substance is marketed only in a non-solid or granular form and as it is not used in spray applications, there is no availability for absorption via the inhalation route.

 

Distribution

Distribution within the body through the circulatory system depends on the molecular weight, the lipophilic character and water solubility of a substance. In general, the smaller the molecule, the wider is the distribution. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues (ECHA, 2012).

Distribution of N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid within the body cannot be excluded. Due to the fact that the test substance has a molecular weight of 311.5 to 339.6 g/mol and a water solubility of > 470 g/L it is assumed, that it will be distributed in aqueous compartments of the body. On the other hand, as the test substance has a log Pow of 2.73, it is likely to distribute into cells and into fatty tissues, too. However, with a log Pow < 4 and a water solubility of > 470 g/L, an accumulation of the substance in the fatty tissue is unlikely.

 

Metabolism

Based on the chemical structure of N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid, metabolism into chemically reactive compounds under in vivo conditions is unlikely. This assumption is confirmed by calculation of potential metabolites via OECD QSAR toolbox v.3.1. The simulators for liver and skin metabolism generated 6 and 4 potential metabolites (N,N-dimethyl-alkyl-1-amines and alkali hydroxide). None of them had chemically reactive structures. The microbial metabolism simulator revealed 117 potential metabolites, which are considered to be of minor relevance due to the fact that absorption from the gastrointestinal tract mainly takes place in the small intestine.

 

Excretion

Characteristics favourable for urinary excretion are low molecular weight (below 300 in the rat), good water solubility, and ionization of the molecule at the pH of urine.

In the rat, molecules that are excreted in the bile are amphipathic (containing both polar and nonpolar regions), hydrophobic/strongly polar and have a high molecular weight. In general, for organic cations with a molecular weight below 300 it is unlikely that more than 5-10% will be excreted in the bile. For organic anions this cut off value may be lower. Substances excreted via bile may potentially undergo enterohepatic circulation (ECHA, 2012).

As N,N-dimethyl-alkyl-1-amines, reaction products with alkali hydroxide and chloroacetic acid has a molecular weight of 311.5 to 339.6 g/mol and contains polar and non-polar regions it is assumed that one part of the substance burden will be excreted via the bile and faeces. However, as the substance is good water soluble (> 470 g/L), the fraction of the substance burden which is not excreted via the faeces will be excreted via urine.