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Key value for chemical safety assessment

Additional information

There are only few experimental studies available in which the toxicokinetic properties of 2 -amino-2-(hydroxymethyl)-1,3-propanediol(TRIS AMINO) were investigated. Therefore, whenever possible, toxicokinetic behaviour was assessed taking into account the available information on physicochemical and toxicological characteristics of TRIS AMINO according to the “Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2009)”.

In its pure state, TRIS AMINO is a white crystalline solid (121.14 g/mol) and is highly soluble in water (684 g/L), but shows low lipid solubility. It has a very low vapour pressure (0.0003 Pa) and the relatively low partition coefficient (log Kow of -2.31) indicates a low potential to accumulate in biological systems. Moreover, TRIS AMINO is a highly alkaline substance with a pH of 10.4 of a 1% solution.

In general, TRIS AMINO is a biologically inert amino alcohol of low toxicity, which buffers carbon dioxide and acids in-vitro and in-vivo. As weak base with a pK of 7.8 at 37 °C, TRIS AMINO acts as proton acceptor.In-vivo, it supplements the buffering capacity of the blood bicarbonate system, accepting a proton and generatingbicarbonate (NH3+/HCO3-), but without forming CO2.

Due to the fact that TRIS AMINO is used in the therapeutic treatment of severe respiratory or metabolic acidosis and in other clinical indications for example salicylate and barbiturate intoxication, pharmacokinetic studies are available. However, since TRIS AMINO is most effective when administered intravenously, pharmacokinetic studies were performed predominantly via the intravenous route.

 

Absorption and distribution

Animal data

In the study of Brasch and Iven (1981), the pharmacokinetics of TRIS AMINO were investigated in rabbits after intravenous administration (121 mg/kg; pH 7.4). TRIS AMINO was infused in 2 min and blood samples were collected up to 24 h after the infusion. 2 min after the end of infusion, TRIS AMINO concentration in plasma was 550 µg/mL. Thereafter, TRIS AMINO levels declined steadily and equalled 2.9 µg/mL after 24 h. TRIS AMINO exhibited three-compartment characteristics with a half-life of 12.9 h. In erythrocytes, TRIS AMINO levels increased during the first hour after infusion. After 2 h, TRIS AMINO concentrations in erythrocytes were about 2.5 times higher than those in plasma and remained well above plasma levels during the rest of the observation period. The final volume of distribution (3700 mL/kg) was much larger than the volume of total body water (275 mL/kg), indicating accumulation of TRIS AMINO inside cells, but the equilibration between body compartments was slow.

When 14C labelled TRIS AMINO was intravenously administered to nephrectomised rats, the distribution of TRIS AMINO was determined between the extra- and the intracellular space as a function of time at constant plasma pH of 7.4 (Rothe and Heisler, 1984). The equilibrium in distribution of TRIS AMINO between the extra- and the intracellular space was not observed before 6-12 hours after administration. This indicates that TRIS AMINO permeates very slowly into the intracellular compartment. TRIS AMINO disappears from the extracellular space in a multi-exponential fashion, indicating that it equilibrates with the different body tissues at largely variable rates.

Human data

The pharmacokinetics of TRIS AMINO were also investigated in healthy volunteers (121 mg/kg bw; pH 7.4) and in patients (109-376 mg/kg; pH 10.9) suffering from metabolic acidosis (Brasch et al., 1982). Venous blood samples were collected during the 30 min infusion and after it had ended (5 min – 24 h). At the end of the infusion, in healthy volunteers TRIS AMINO concentrations in plasma averaged 565 µg/mL. There was a bi-exponential decline of plasma TRIS AMINO levels and after 24 h the level was only 3.8 µg/mL. Two-compartment characteristics were observed with a half-life of 6.6 h. TRIS AMINO concentration in erythrocytes rose more slowly, with a maximum 20 min after the end of infusion. After 2 h TRIS AMINO levels in erythrocytes were about 2.5 times higher than those in plasma, and they remained well above the corresponding plasma levels during the rest of the observation period. The final volume of distribution (894 mL/kg) was somewhat larger than the volume of total body water (580- 630 mL/kg), indicating uptake of TRIS AMINO into tissues, but equilibration between compartments was slow.

In contrast to healthy volunteers, the half-life in patients suffering from metabolic acidosis was much longer (16.3-45.6 h) and the final volumes of distribution were much larger (658-5247 mL/kg).

In conclusion, after intravenous administration, TRIS AMINO is rapidly distributed through the extracellular space and slowly penetrates the intracellular space, except for erythrocytes and hepatocytes.A half-life of 6.6 h was calculated for TRIS AMINO in healthy humans.

After intravenous injection of TRIS AMINO, approximately 70% of the administered dose is available in the ionised form at physiological blood pH; if pH is decreased from pH 7.4, the ionised fraction of the drug is increased (Nahas, 1962, 1963; Nahas et al., 1998). While the ionised fraction reacts only with acids in the extracellular fluids, the fraction of the dose which remains unionised at physiologic pH is thought to be capable of penetrating the cell membrane (Nahas, 1962, 1963). TRIS AMINO is not bound to plasma proteins (Holmdahl and Nahas, 1962; Goldberg et al., 1962) and the low lipid solubility of ionised and unionised TRIS AMINO may be the factor that limits the rate of the intracellular uptake.

After oral intake TRIS AMINO is poorly absorbed from the gastro-intestinal tract and acts as a powerful osmotic laxative in humans. When given orally, TRIS AMINO is rapidly ionised by the acid content of the stomach and is not absorbed (Brinkman et al., 1960).

 

Absorption and distribution based on acute toxicity studies

Based on the lack of toxicokinetic data using the oral, inhalation or dermal exposure route,additional information on the physico-chemical and toxicological characteristics of TRIS AMINO will be given below.

Acute oral toxicity:

Acute oral toxicity studies have been conducted in rats, mice and rabbits. In a study performed by Kumar (2011), acute oral toxicity testing was carried out using an up and down procedure in rats, according to OECD 425. There was no mortality during the study period and no clinical signs were observed. Histopathological examinations revealed no substance-related findings. Therefore, the LD50 was determined to be > 5000 mg/kg bw. In another study, the acute oral toxicity of a 20% solution of TRIS AMINO was assessed in rats (Giroux and Beaulaton, 1961). The animals were administered 1000, 3000, 5000, 6000 and 7000 mg/kg bw of the test substance by gavage. The mortality was 0/10, 0/10, 3/10, 6/10 and 7/10, listed by increasing dose. The estimated LD50 was ~ 6000 mg/kg bw. Giroux and Beaulaton (1961) also administered TRIS AMINO to mice by gavage, using a 10% TRIS AMINO solution. For dose levels of 1000, 2000, 3000, 5000, 6000 and 7000 mg/kg bw, the mortality was 0/10, 0/10, 0/10, 0/10, 4/10 and 10/10, respectively. The animals that died had muscle tension and breathing difficulties, while the surviving mice were calm. The calculated LD50 was 6100 mg/kg bw. In the study performed by Rubenkoenig (1955), mice were administered 2000, 3500, 5000, 7000 and 10000 mg/kg bw TRIS AMINO. The mortality was 0, 2, 3, 9 and 10, respectively, by increasing dose. 10 animals per dose were used. The estimated LD50 was 5500 mg/kg bw. The effect of a 25% TRIS AMINO solution in water, either pH-neutralised or unchanged, was assessed in rabbits (Machle, 1940). In the animals that died, damage to the gastrointestinal tract associated with the alkalinity of the test substance was observed. The effect of the alkalinity was also apparent in the LD50 levels, which were 1000-2000 mg/kg bw for the unchanged solution and > 5000 mg/kg bw for the pH-neutralised solution.

The study of Darby and Anderson (1966) confirmed that TRIS AMINO orally administered caused diarrhoea in rats and dogs.In general, the findings of the acute oral toxicity studies evidenced that the main cause of acute toxicitywas most probably local irritation due to the highly alkaline properties of TRIS AMINO.With regard to the dose administered and the nature of effects observed, systemic bioavailability of the test substance is considered to be rather low via the oral route.Acute oral toxicity in rats, mice and rabbits showed low toxicity and confirmed the previous pharmacokinetic data of TRIS AMINO via the intravenous route in test animals and humans.

In conclusion, when given orally, TRIS AMINO is rapidly ionised by the acid content of the stomach and is therefore poorly absorbed from the gastrointestinal tract. After oral intake, it acts as a powerful osmotic laxative.

 

Acute inhalation toxicity:

No data on acute inhalation toxicity are available. However, as a consequence of the very low vapour pressure(0.0003 Pa at 20 °C)of TRIS AMINO, inhalation is not considered to be a significant route of exposure.The particle size distribution shows that the inhalable fraction, particles < 100 µm, constitutes 19% of the total(White and Woolley, 2011).Only 1.13% of the particles are < 5.5 µm. Most of the inhaled particles will therefore remain in the upper airways and/or will be swallowed, with few or no particles reaching the pulmonary alveoli.

Industrial and professional workers may be exposed via spray applications containing < 1% TRIS AMINO, meaning that the potential for acute toxicity via the inhalation route will be negligible. The general population is not exposed to the pure substance, but may be exposed to TRIS AMINO in spray applications. However, the concentration will be < 1% during normal handling and use of TRIS AMINO, and therefore the potential for acute toxicity via the inhalation route is considered to be negligible.

 

Acute dermal toxicity:

According to OECD 402, dermal acute toxicity was assessed by exposing rats skin to 5000 mg/kg bw TRIS AMINO under semi-occlusive conditions (Kumar, 2011). There was no mortality, and no signs of toxicity were observed during the study period. The necropsy and gross pathological examination revealed no substance-related findings. The test substance did not cause skin irritation effects on the application site. Therefore, the LD50 value is considered to be > 5000 mg/kg bw.

Also, a quantitative study of percutaneous absorption in-vitro was carried out on human abdominal skin placed in a FRANZ diffusion cell (Noel-Hudson, 1993). After 24 h, the percutaneous absorption of TRIS AMINO through human skin was very low regardless of the concentration of the TRIS AMINO solution (10% and 0.1% TRIS AMINO solutions were tested). Less than 1% of the applied dose was found. For both solutions, the maximum value of flux was reached after 4 h and remained essentially constant during the rest of the experiment (totally 24 h). However, the value of flux was about 150 times higher for the 10% solution (6.922 ± 6.179 µg/cm²/h) than for the 0.1% solution (0.039 ± 0.052 µg/cm²/h). After washing, the retention of TRIS AMINO in the epidermis and dermis was also less than 1% of the applied dose. Therefore, TRIS AMINO did not retain in the horny layer but was almost totally eliminated by washing the skin. The washing waters contained more than 90% of the applied dose.

For neat TRIS AMINO (i.e. a solid substance resulting in a basic solution), a QSAR based modelling published by Potts and Guy (1992), taking into account molecular weight and low Kow, estimated a dermalpermeability constant Kp of 7.54E-06 cm/h. Similar to the approach taken by Kroes et al. (2007), the maximum flux Imax (Imax = Kp [cm/h] x water solubility [mg/cm³]) was calculated, resulting in dermal absorption of 5.2µg/cm²/h TRIS AMINO.Usually, this value is considered as indicator for a dermal absorption of 40%(Mostert and Goergens, 2011).However, given the fact that experimental data are available, the information on dermal absorption generated from experiments is more relevant for the risk assessment compared to QSAR based calculations. Taking into account thatthe washing waters contained more than 90% of the applied dose (Noel-Hudson, 1993), adermal uptake of 10% has to be regarded as a worst case scenario.

 

In conclusion, all available data from the pharmacokinetic studies and from acute toxicity studies indicated low toxicity of TRIS AMINO. The effects observed were likely due to alkalinity of the test substance. Moreover, it can be assumed that administration of TRIS AMINO via the oral, inhalation or dermal exposure route yields only small quantities that can be absorbed based on the bio- and physico-chemical properties of the test substance. In addition,systemic bioavailability of TRIS AMINO is considered to be rather low. This is in accordance with the low toxicity observed in the acute and repeated dose toxicity studies.

 

Metabolism

After intravenous administration of TRIS AMINO to animals or humans, no evidence of a metabolism was found (Nahas, 1962, 1963; Brasch et al., 1982). Several studies confirmed that the protonated form of TRIS AMINO was solely detected in plasma and urine of test animals (rat, rabbits and dogs) and humans (Nahas, 1962; Holmdahl and Nahas, 1962; Christensen and Clifford, 1962; Nahas, 1963). According to the chemical structure of TRIS AMINO, it is also expected that the test substance will remain unmetabolised.Modelling of potential metabolites via OECD QSAR toolbox v.2.0 (2010) confirms this assumption.No relevant metabolites were generated by the liver metabolism simulator, by the skin metabolism simulator or by the microbial metabolism simulator. Based on this information, it seems to be very unlikely that TRIS AMINO will be metabolised by cytochrome P450 enzymes in-vivo.

Moreover, available studies on genetic toxicity in-vitro (Ames test, gene mutation in mammalian cells in-vitro, chromosome aberration in-vitro) were negative, indicating that there is no evidence of reactivity under in-vitro test conditions. With respect to skin sensitisation data, there was no evidence of direct protein reactivity which would cause skin sensitisation. Since no interactions with proteins were determined and no relevant metabolites were generated using QSAR modelling, reactivity of the test substance is considered rather unlikely under in-vitro and in-vivo conditions.

 

Excretion

TRIS AMINO is a highly water soluble substance and its elimination mainly occurs by glomerular filtration in the kidneys after intravenous injections (Linn and Roberts, 1961; Nahas and Reveillaud, 1961; Brown et al., 1961; Thompson et al., 1965; Bräunlich, 1975). Already 30 min after infusion of TRIS AMINO, 25% of the administered dose was found in human urines and after 24 h, 82% had been eliminated in this way (Brasch et al., 1982). However, this was less than the total elimination of TRIS AMINO, which, from the area under the curve, was calculated to be 97% during 24 h. Only insignificant amounts were found in gastric juice and bile (Brasch et al., 1982). No TRIS AMINO was detected in the expired air of nephrectomised cats and dogs, when intravenously given the test substance (Holmdahl and Nahas, 1962). TRIS AMINO is renally excreted in its ionised form at a rate that slightly exceeds creatinine clearance (Nahas et al., 1963; Brasch et al., 1982). The strong correlation observed between the clearance of creatinine and TRIS AMINO confirms that the kidney is the dominant excretory organ for test substance (Brasch et al., 1982). It may take between 24 to 72 h to achieve 80% elimination of TRIS AMINO in healthy humans after intravenous injection (Nahas, 1998; Brasch et al., 1982).

In conclusion, when given via the oral, inhalation or dermal exposure route, TRIS AMINO will only be absorbed in small amounts based on the fact that predominantly the unionised form of TRIS AMINO can permeate the cell membrane and based on its physico-chemical properties. As TRIS AMINO acts as proton acceptor in-vivo, it is rapidly ionised e.g. by the acid content of the stomach and is therefore not absorbed. The limited amounts of TRIS AMINO that are systemically bioavailable are not bound to plasma proteins and exist in the ionised form. At physiological blood pH, TRIS AMINO supplements the buffering capacity of the blood bicarbonate system, accepting a proton. Bioavailable levels of TRIS AMINO are rapidly eliminated by the kidneys and found not metabolised in the urine. No relevant metabolism is expected, based on experimental data and QSAR modelling. Therefore, the potential to accumulate in biological systems is also expected to be low if TRIS AMINO is ingested using the oral, inhalation or dermal exposure route. This is also confirmed by the low toxicity observed in acute and repeated dose toxicity studies.                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  

References (not cited in the IUCLID)

Rothe, K.F. and Heisler, N. (1984) Kinetik der Verteilung von THAM (TRIS AMINO) auf Intra- und Extrazellulärraum. Anästh. Intensivther. Notfallmed. 19:24-26

Nahas, G.G. (1962) The pharmacology of tris(hydroxymethyl)aminomethane during CO2 load. Am. J. Physiol. 204:113-118

Nahas, G.G. (1963) The clinical pharmacology of THAM (tris(hydroxymethyl)-aminomethane). Clin. Pharmacol. Ther. 4:784-803

Nahas, G. et al. (1998) Guidelines for the treatment of acidaemia with THAM. Drugs 55(2): 191-224

Holmdahl, M.H. and Nahas, G.G. (1962) Volume of distribution of C14 labeled tris(hydroxymethyl)aminomethane. Am. J. Physiol. 202:1011-1014

Goldberg, A.R. et al.(1962) Equilibrium dialysis of tris(hydroxymethyl)aminomethane (THAM) in serum and albumin. Fed. Proc. 21:173

Brinkman, G.L. et al. (1960) The treatment of respiratory acidosis with THAM. Amer. J. med. Sci. 239:341-346

Christensen, H.N. and Clifford, J. (1962) Test for metabolic attack on tris(hydroxymethyl)aminomethane and alpha-Hydroxymethylserine in the rat. Proc. Soc. exp. Biol. Med. 111:140-142

Nahas, G.G. and Reveillaud, R.J. (1961) Elimination du 2-amino-2-hydroxyméthyl-1,3-propanediol par le rein. C. R. Acad. Sci. 253:721-723

Brown, E.S. et al. (1961) Effects of 2-amino-2-hydroxymethyl-1,3-propanediol on CO2 elimination and production in normal man. Ann. N. Y. Acad. Sci. 92:508-520

Thompson, S.W. et al. (1965) Toxicity studies with tris(hydroxymethyl)aminomethane . U.S. Army Med. Res. and Nutrional Laboratory Rep. 285

Linn, S. and Roberts, M. (1961) Microassay of tris(hydroxymethyl)aminomethane applicable to blood and urine.Ann. N.Y. Acad. Sci. 92: 419-426

Bräunlich, H. (1975) Altersabhängigkeit und Stimulierbarkeit der renalen Ausscheidung von tris(hydroxymethyl)aminomethan (Tham).Arch. int. Pharmacodyn. 216:144-159

Potts, R. and Guy, R. (1992)Predicting skin permeability.Pharm. Res. 9(5): 663-669

Kroes, R. et al. (2007) Application of the threshold of toxicological concern (TTC) to the safety evaluation of cosmetic ingredients. Food Chem. Toxicol. 45, 2533–2562

Mostert, V. and Goergens, A. (2011) Dermal DNEL setting: using QSAR predictions for dermal absorption for a refined route-to-route extrapolation. Society of Toxicology, Annual Meeting, ISSN 1096-6080 (http://www.toxicology.org/AI/PUB/Toxicologist11.pdf), 120(2): 107

Discussion on bioaccumulation potential result:

See toxicokinetics, metabolism and distribution.