2,2,2-trifluoroethanol

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Acceptable, well documented publication which meets basic scientific principles.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Objective of study:

absorption

distribution

excretion

metabolism

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

no

Principles of method if other than guideline:

Radiolabelled Trifluoroethanol is injected by intraperitoneal route to male Whistar rats. At different time points after the injection, the radioactivity is quantified in different tissues and fluids.

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

14C

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Inc
- Age at study initiation: no data
- Weight at study initiation: 250-350g
- Fasting period before study: no data
- Housing: stainless steel metabolism cages (Lab products, Inc.)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATES: From: To: no data

Route of administration:

intraperitoneal

Vehicle:

water

Details on exposure:

not applicable

Duration and frequency of treatment / exposure:

single injection

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

12-15 µCi/kg

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

12-15 µCi/kg

No. of animals per sex per dose / concentration:

4 or 5 animals/dose/time point of sacrifice

Control animals:

no

Positive control reference chemical:

no data

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: plasma, urine, expired air, liver, lungs, kidneys and testes.
- Time and frequency of sampling: 15 min to 48 hours after intraperitoneal injection (at the moment of the sacrifice of the animal). Presence of TFE in expired air was measured during the 48h period of experiment.
- Other:
Plasma was obtained from heparinized blood collected during decapitation.
The liver, lungs, kidneys and testes of each animal were removed, rinsed with distilled water, blotted and weighed prior to counting.
Urine samples were collected in plastic containers under mineral oil. The mineral oil-water partition ratio was measured and found to be 0.004 +/- 0.001 at 20°C. Thus extraction of labelled TFE from urine by mineral oil is not of concern.
In order to measure the amount of radioactivity in expired air, several animals were placed individually into a 2.3 L glass chamber. Airflow through the chamber was 500 mL/min. The chamber exhaust passed through 2 glass impingers containing 20 mL of ice water to collect TFE and its derivatives, and then through an impinger containing 20 mL of ice cold 1N NaOH to collect CO2. Recovery by this trapping system of 1.2 µCi injections of 14C-TFE into the chamber was 72 (+/- 2) %. All expired radioactivity calculations were adjusted for this trapping inefficiency.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): Liver and testes
- Time and frequency of sampling: after the sacrifice of the animal 1 hour postinjection.
- From how many animals: 5
- Method type(s) for identification: Fractions of homogenates of Liver and testes were saved for scintillation counting, and for protein analysis using a modification of the Lowry method (1951).
- Limits of detection and quantification: no data
- Other: In order to study the binding of TFE to cellular proteins, rats were also pretreated by the addition of 0.2 % (w/v) sodium phenobarbital to their drinking water for 3 days in order to induce mixed function oxidase activity in their tissues. The animals were given tap water for 24 hrs before use. Control animals received tap water for all 4 days prior to use. Animals were then injected by intraperitoneal route with 100 mg TFE/kg (13.8 µCi/kg), and 1 hr later sacrificed using ether. The liver and testes were removed, rinsed with distilled water and then blotted. The tunica albuginea of the testis was removed, and both tissues were homogenized with 2 volumes of cold saline. Fractions of these homogenates were saved for scintillation counting, and for protein analysis using a modification of the Lowry method (1951).

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): not applicable

Statistics:

no data

Preliminary studies:

Not applicable

Type:

absorption

Results:

TFE was rapidly absorbed following ip administration. The disappearance of 14C activity from the plasma could be described by a biphasic curve, with half times of 2 hrs and 48 hrs.

Type:

distribution

Results:

The octanol-water partition ratio was determined to be Kow = 2.15 (+/- 0.07) at 20°C corresponding to a Log Kow of 0.33. TFE, therefore, is mildly lipophilic and may tend to concentrate in fatty tissue.

Type:

distribution

Results:

15 min following the administration of 14C-TFE (100 mg/kg bw) the percentage of the initial dose appearing in each organ was: 5.6 % for the liver, 1.4 % for the testes, 1.1 % for the kidneys, and 0.6 % for the lung.

Type:

distribution

Results:

No evidence for testicular concentration of TFE was obtained in the present study as the levels of radioactivity at all timepoints postinjection were found to be comparable to those in the liver, kidney, and lung.

Type:

excretion

Results:

Urinary excretion was the major route of elimination accounting for 60 percent of the initial dose after 2 days. Expired air samples were collected for only 6 hours, but accounted for 9% of the initial dose. Excretion of 14CO2 was not detected.

Details on absorption:

The disappearance of 14C activity from the plasma could be described by a biphasic curve, with half times of 2 hrs and 48 hrs. From the plasma data, an initial apparent volume of distribution was calculated to be 318 mL.
Initially, red blood cells and plasma were found to contain similar levels of Carbon-14 activity (v/v comparison following the centrifugation of whole blood at 600*g for 15 min). By 24 hrs postadministration, the concentration of 14C in the red blood cells was twice that in the plasma. At 48 hrs postinjection the concentration of 14C in the red blood cells had decreased only to 1.5 times that in the plasma.

Details on distribution in tissues:

The octanol-water partition ratio was determined to be Kow = 2.15 (+/- 0.07) at 20°C corresponding to a Log Kow of 0.33. TFE, therefore, is mildly lipophilic and may tend to concentrate in fatty tissue. Since this preference towards lipid materials is very small in magnitude, it is suggested that bioconcentration of TFE in food-chains probably will not occur to any significant degree. The method used to determine the Log Kow did not meet the actual required criteria of standard methods.
Relative tissue concentrations 15 min following the ip injection of 5 or 100 mg/kg 14C-TFE were lung > liver >kidneys > testes. Tissue concentrations 15 min after a 100 mg/kg dose were estimated to be 1.5 mM in the liver and 1.2 mM in the testes.
The proportional amount of 14C-TFE reaching the organs closely resembled the relative tissue masses. 15 min following the administration of 14C-TFE (100 mg/kg) the percentage of the initial dose appearing in each organ was: 5.6 % for the liver, 1.4 % for the testes, 1.1 % for the kidneys, and 0.6 % for the lung. After 48 hours a similar ranking was observed, with each organ containing: 0.77 % in the liver, 0.14 % in the testes, and 0.12 % in both the kidneys and the lung.
One hour following the administration of 14C-trifluoroethanol to control rats, 3% of the radioactivity in the testis (on a milligram protein basis) was bound to proteins, while 5% of the radioactivity in the liver was associated with proteins (see details in Table 7.1.1/2). Pretreatment of animals with Phenobarbital did not significantly increase the levels of apparent binding in either organ. A repeat of this experiment with control animals (i.e., no Phenobarbital pretreatment) gave similar results, with only 1% of the radioactivity in the testis, and 3% of the radioactivity in the liver bound to cellular proteins. The radioactivity found in the final pellet and wash supernates accounted for 90% (+/- 15%) (Mean of 20 animals) of the organ counts. On the average, 74% of the radioactivity in both liver and testes was removed during the initial protein precipitation step. The 3 subsequent washes removed 7%, 2% and 0-1% of the initial organ activity, respectively.

No evidence for testicular concentration of TFE was obtained in the present study as the levels of radioactivity at all timepoints postinjection were found to be comparable to those in the liver, kidney, and lung. Thus, the fact that this organ (ie. testes) can be severely damaged by the compound reflects a genuine sensitivity of the testes to TFE. It should be noted however, that these experiments tell us nothing about the distribution of14C-TFE within the structural components of the organ, or among the different cells of the germinal epithelium. It is important to note that in the current study only the carbon-14 label of the trifluoroethanol molecule was monitored. Thus the compound with which the label is associated at any particular moment is unknown.

Details on excretion:

The excretion of radioactivity as percentage of the injected dose in rats following the administration of 14C-trifluoroethanol (100 mg/kg bw) is shown in Table 7.1.1/1.
Urinary excretion was the major route of elimination accounting for 60 percent of the initial dose after 2 days.
Expired air samples were collected for only 6 hours, but accounted for 9% of the initial dose. Excretion of 14CO2 was not detected, as measured by radioactivity in the sodium hydroxide trap. This would suggest that in vivo defluorination of trifluoroethanol did not occur.
Fecal excretion of radioactivity was minor, accounting for about 2% of the initial dose after 48 hrs. Due to the design of the metabolism cages, the feces were occasionally sprayed with urine. Thus it is possible that the true fecal excretion of radioactivity is less than that shown in table 7.1.1/1. The pattern of 14C excretion was: urine>>expired air>>feces.

Metabolites identified:

not measured

Table 7.1.1/1: Excretion of ¹⁴C-Trifluoroethanol when the animals are treated with 100 mg/kg by intraperitoneal injection.

 

Hours postadministration	Cumulative percentage of initial dose excreted
	Urine (n=4)	Feces (n=4)	Expired Air (n=5)
1	-	-	1.7 +/- 0.3
3	1.9 +/- 0.4	-	5.0 +/- 0.4
6	18.7 +/- 0.3	-	9.1 +/- 0.2
12	34.3 +/- 0.8	0.8 +/- 0.3	-
24	50.1 +/- 2.5	0.9 +/- 0.1	-
48	59.9 +/- 1.0	1.8 +/- 0.5	-

 n: number of animals

Table 7.1.1/2: Binding of 2-¹⁴C-Trifluoroethanol to liver and testis protein (100 mg/kg, 13.8 µCi/kg) (All values: mean +/- SD for n=5)

 

Tissue	Pretreatment	Whole homogenate activity (DPM/mg protein)	Pellet activity
			DPM/mg Dry weight	DPM/mg protein
Liver	-	556 +/- 20	19 +/- 0.6	30.2 +/- 1.0
	Phenobarbital	542 +/- 27	19 +/- 1.0	32.7 +/- 1.8
Testis	-	858 +/- 26	16.7 +/- 1.8	25.5 +/- 1.4
	Phenobarbital	899 +/- 28	17.6 +/- 1.3	28.0 +/- 2.1

 

DPM: Desintegration Per Minute

Conclusions:

Under the study conditions, it can be concluded that TFE is rapidly absorbed following ip administration.
Previous studies had shown that the volatile solvent 2,2,2-trifluoroethanol rapidly produces testicular damage following intraperitoneal administration or inhalation. Here, no evidence for testicular concentration of TFE was obtained in the present study as the levels of radioactivity at all timepoints postinjection were found to be comparable to those in the liver, kidney, and lung. Thus, the fact that this organ (ie. testes) can be severely damaged by the compound reflects a genuine sensitivity of the testes to TFE. It should be noted however, that these experiments tell us nothing about the distribution of 14C-TFE within the structural components of the organ, or among the different cells of the germinal epithelium. It is important to note that in the present study only the carbon-14 label of the trifluoroethanol molecule was monitored. Thus the compound with which the label is associated at any particular moment is unknown.
One consequence of the blood-testis barrier is that a permeant substance could be transformed inside the seminiferous tubules to a nonpermeant toxic metabolite. Because this metabolite could not escape from the tubules, it would tend to accumulate there in concentrations much higher than if the toxic metabolite itself were given to the animal. Toxic effects may thereby result at comparatively low doses of the precursor. The production of unique TFE metabolites by the testis may explain the high sensitivity of this organ to TFE.
The levels of protein binding observed in these experiments are so low as to be of questionable significance. Although care was taken to break up the protein pellets during each washing, it is possible that the radiolabel remained in the pellets due to physical entrapment.
The failure of Phenobarbital pretreatment to influence the levels of protein binding in either organ suggests that the metabolism of 14C-TFE by the mixed function oxidase system is not required for binding to occur.

Executive summary:

In this study, performed similarly to the OECD Guideline No. 417, 2,2,2-Trifluoroethanol (TFE) labelled with carbon 14 (TFE) was administered to male Srague-Dawley rats. The substance was administrated by a single intraperitoneal injection at dose levels of 5 or 100 mg/kg bw (4 or 5 animals per dose).

At different time points after the treatment (15 min, 1, 3, 6, 12, 24 and 48 hours postinjection), animals were sacrificed and the tissues and the body fluids were collected (plasma, urine, expired air, liver, lungs, kidneys and testes) to determine the absorption, distribution and excretion of TFE. Rats were also sodium phenobarbital pretreated via the drinking water for 3 days in order to induce mixed function oxidase activity in their tissue. Animals were then i.p. injected with 100 mg TFE/kg bw (13.8 µCi/kg bw), and 1 hr later sacrificed using ether. The liver and testes were removed and fractions of their homogenates were saved for scintillation counting, and for protein analysis using a modification of the Lowry method (1951).

Under the study conditions, it can be concluded that TFE is rapidly absorbed following ip administration.

Previous studies (75-89-8, Acute toxicity: inhalation, Wilkenfeld, 1981, W; 75-89-8, Acute toxicity:other route, intraperitoneal, Wilkenfeld, 1981; 75-89-8, Toxicity to reproduction, Wilkenfeld, 1981, W) showed that the volatile solvent 2,2,2-trifluoroethanol rapidly produced testicular damage following intraperitoneal administration or inhalation. Here, no evidence for testicular concentration of TFE was obtained in the present study as the levels of radioactivity at all timepoints postinjection were found to be comparable to those in the liver, kidney, and lung. Thus, the fact that this organ (ie. testes) can be severely damaged by the compound reflects a genuine sensitivity of the testes to TFE. It should be noted however, that these experiments tell us nothing about the distribution of¹⁴C-TFE within the structural components of the organ, or among the different cells of the germinal epithelium. It is important to note that in the current study only the carbon-14 label of the trifluoroethanol molecule was monitored. Thus the compound with which the label is associated at any particular moment is unknown.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Acceptable, well documented publication which meets basic scientific principles

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

Trifluoroethanol (TFE) or Trifluoroacetaldehyde (TFAld) were administered by intraperitoneal injection to male rats. Substances were also administered to rats pretreated with various inhibitors and inducers of drug metabolism. At different time points after each kind of treatment, animals were sacrificed and blood collected in order to determine the time course of metabolism of TFE. The mortality, leucocyte count and serum Trifluoroacetic acid (TFAA) and TFE concentration were measured to determine the mechanism of oxidation of TFE and the toxicity associated with intermediate metabolites. The effect of ethanol ingestion on TFE metabolism and toxicity was also investigated to determine the role of the ethanol-inducible cytochrome P-450 in the oxidation of TFE.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: from a colony maintained in the study laboratory
- Age at study initiation: no data
- Weight at study initiation: 250 g (+/- 30g)
- Fasting period before study: 24 hours before drug administration
- Housing: no data
- Individual metabolism cages: no data
- Diet (e.g. ad libitum): Purina Laboratory Rodent Chow, ad libitum except 24 hours before drug administration
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: To: no data

Route of administration:

intraperitoneal

Vehicle:

physiological saline

Details on exposure:

Not applicable

Duration and frequency of treatment / exposure:

single dose

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

TFE; diluted to 20% in sterile saline

Dose / conc.:

130 mg/kg bw/day (nominal)

Remarks:

TFE; diluted to 20% in sterile saline

Dose / conc.:

210 mg/kg bw/day (nominal)

Remarks:

TFE; diluted to 20% in sterile saline

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

TTAld; diluted to 20% in sterile saline

Dose / conc.:

240 mg/kg bw/day (nominal)

Remarks:

TFAld; diluted to 20% in sterile saline

No. of animals per sex per dose / concentration:

variable number of animals for each treatment. See details in Table 7.1.1/1

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

no data

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): no data

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion):
- Tissues and body fluids sampled (delete / add / specify): serum
- Time and frequency of sampling: at the sacrifice of the animal
- Other: no data

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): serum
- Time and frequency of sampling: at the sacrifice of the animal
- From how many animals: (samples pooled or not): n=5. No data regarding pooling of samples or not.
- Method type(s) for identification: GC-MS.
- Limits of detection and quantification: the limit of sensitivity for the TFAA concentration determination was 0.01 mM.
- Other: no data

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): not applicable

Statistics:

Treated and control groups were compared with Student's t-test.

Preliminary studies:

not applicable

Details on absorption:

The serum TFE concentration 1 hr after intraperitoneal injection of 0.21 g/kg TFE was 1.44 mM. After 2 hr this value was decreased by 17% and after 6 hr by 58%. At 24 hr after TFE administration, the serum TFE concentration was 0.06 mM.

Details on distribution in tissues:

no data

Details on excretion:

no data

Metabolites identified:

yes

Details on metabolites:

Under the study conditions, it was observed that trifluoroacetic acid (TFAA) is the major metabolite of TFE in Wistar rat. The conversion of TFE to TFAA had an apparent lag phase relative to the rate of disappearance of TFE from the blood. Maximal concentrations of TFAA were seen only 16 hr after TFE administration. In contrast, serum TFAA concentrations after trifluoroacetaldehyde (TFAld) hydrate administration were near maximal levels at 6 hr post-administration. Thus, the rate limiting step in the oxidation of TFE to TFAA is the oxidation of TFE to TFAld. TFAA was not rapidly eliminated since, at 24 hr after TFE administration, elevated concentrations of TFAA persisted. The toxic effects of TFE , which included decreased leukocyte count, bone marrow nucleated cell density, and dry weight of the small intestine, were observed 8 to 16 hr after the administration of the substance. This corresponds to the appearance of major concentrations of the TFAA metabolite in the blood.
A series of inhibitors has been used to determine the relationship between the metabolism and toxicity of TFE and to provide insights into these metabolic pathways. In accordance to the results obtained, the enzymes alcohol dehydrogenase, peroxidase and the cytochrome P450 participated in the metabolism of TFE to TFAA. However, there is a preponderance of evidence that cytochrome P450 is involved in the toxic metabolism of TFE. A pretreatment with ethanol stimulated the TFE metabolism which is in favor with the implication of an ethanol-inducible form of cytochrome P450.

Effects of a TFE treatment:1h after an intraperitoneal injection of TFE, the serum concentration of TFE was 1.44 mM, whereas this concentration was 0.06 mM 24 h postinjection. TFE treatment caused 75% (+/- 5%) mortality at 7 days and a severe decrease (about 90%) of leukocyte was observed compared to the control non-treated group of animals (see Tables 7.1.1/2 and 7.1.1/3).

Serum TFAA was first detected at very low concentration at 2 hr after TFE administration and levels increased with time up to 16 hr postinjection (At 16h, 0.98 mM of serum TFAA concentration) (see Table 7.1.1/3).

When ethanol was ingested for 10 days before TFE injection, the decrease of TFE in serum was accelerated whereas the production of TFAA was augmented and accelerated. It was noted that the pretreatment with ethanol had no effect on the toxicity of TFE in rat: the mortality rate was the same in ethanol preteated or not animals (see Tables 7.1.1/2 and 7.1.1/3).

Effects of a TFAld treatment:

24 hours after TFAld treatment a decrease in leukocyte number was observed in the same magnitude as after a TFE treatment (SeeTables 7.1.1/2 and 7.1.1/3). 6 hours after TFAld treatment a relatively small amount of TFE and a relatively large amount of TFAA were found in serum compared to these metabolite serum concentrations 6 hr after TFE administration (SeeTables 7.1.1/2 and 7.1.1/3).

Effects of a pre-treatment with inhibitors:

Pretreatment with Metyrapone and α-Naphthoflavone prior to TFE administration had no effect on the toxicity of TFE and on its metabolism.

First it was checked that the inhibitors used in the study caused no mortality when administered alone. Pyrazole and disulfiram are inhibitors of the alcohol dehydrogenase which is responsible for the conversion of toxic alcohols to aldehydes. Diethyldithiocarbamate is an inhibitor of the superoxide dismutase which is responsible for the dismutation of superoxide into oxygen and hydrogen peroxide. Pre-treatments with these 3 inhibitors prevented the toxic effects of TFE as the mortality was absent or low and the decrease in leukocyte number observed with only TFE treatment was much lower when rats were pretreated with inhibitor. Furthermore, after a pretreatment with these 3 inhibitors, serum TFAA formation is much lower in comparison to what was obtained in only TFE treated rats. These inhibitors prevented the metabolism of TFE.

Menadione and Allopurinol are stimulators of peroxidase activities. Pretreatment with these 2 inhibitors prior to TFE administration increased both TFE and TFAA serum concentrations. Therefore, the overall metabolism of TFE to TFAA was stimulated by these 2 substances.

SKF 525A, AIA and Isoniazid are Cytochrome P450 inhibitors. When these 3 inhibitors were used in pretreatment of rats prior to TFE administration, a severe decrease in the metabolism of TFE was observed. Indeed, SKF 525A decreased serum TFAA slightly at 6 hr after TFE (See Table 7.1.1/3) but not at 24 hr after TFE administration. However, AIA decreased metabolism by 53% at 6 hr and by 42 % at 24 hr after TFE administration. Isoniazid decreased TFE metabolism by 71% at 6 hr and by 40 % at 24 hr.

Table 7.1.1/2:Toxic effects of TFE and TFAld and TFE serum concentration after the different treatment conditions

Treatment	Dose of TFE or TFAld (g/kg)	% Mortality(a)	Leukocyte count (x10^3)(b)	TFE serum (mM)(c)	TFE serum concentration (% of TFE only)(d)
Vehicle only (saline)	-	0	11.2 +/- 0.6	-	-
TFE only	0.13	0	5.0 +/- 0.9	-	-
TFE only	0.21	75 +/- 5	1.5 +/- 0.3^e	0.6 +/- 0.03	100
Ethanol + TFE	0.13	0	5.3 +/- 0.4	-	-
Ethanol + TFE	0.21	80	1.8 +/- 0.2	-	-
Pyrazole + TFE	0.21	0	8.6 +/- 0.7^f	1.30 +/- 0.01^f	180^f
Disulfiram + TFE	0.21	0	5.6 +/- 1.0^ef	-	148^f
Diethyldithiocarbamate + TFE	0.21	30	3.4 +/- 0.7^e	-	143^f
Menadione + TFE	0.21	80	1.5 +/- 0.3^e	-	168^f
Allopurinol + TFE	0.21	100	1.8 +/- 0.4^e	-	158^f
SKF 525A + TFE	0.21	90	1.1 +/- 0.1^e	-	104
AIA + TFE	0.21	70	2.3 +/- 0.4^e	-	-
Metyrapone + TFE	0.21	80	-	-	-
α-Naphthoflavone + TFE	0.21	80	-	-	-
Isoniazid + TFE	0.21	-	8.3 +/- 0.9^f	-	-
TFAld only	0.24	-	2.6 +/- 0.4	0.19 +/- 0.02^h	-
Pyrazole + TFAld	0.24	-	7.4 +/- 0.8^g	0.40 +/- 0.08^h	-
Isoniazid + TFAld	0.24	-	6.2 +/- 0.6^g	-	-

 

(a)          : Cumulative mortality was determined 7 days after TFE in groups of 5-10 rats. Cumulative mortality for TFE alone is the average mortality of five groups of 9-10 rats.

(b)          : Leukocyte count was determined 24 h after TFE (groups of 3-9 rats) or TFAld (groups of 3-8 rats) administration.

(c)          : Blood for serum concentrations of TFE was taken at 6 hr after TFE and TFAld (groups of 4-5 rats).

(d)          : Blood for serum concentrations of TFE was taken 6 hr after TFE or TFAld treatment (groups of 3-9 rats).

e: significantly different from vehicle, p<0.01

f: significantly different from TFE only, p<0.01

g: significantly different from TFAld only, p<0.01

h: significantly different from TFE treated in each similar pretreatment group, p<0.01

Table 7.1.1/3:Toxic effects of TFE and TFAld and TFAA serum concentration after the different treatment conditions

Type of treatment	Dose of TFE or TFAld (g/kg)	% Mortality(a)	Leukocyte count (x10^3)(b)	TFAA serum (mM)(c)		TFAA serum concentration (% of TFE only)(d)
				6 hr	24 hr
Vehicle only (saline)	-	0	11.2 +/- 0.6	-	-	-
TFE only	0.13	0	5.0 +/- 0.9	-	-	-
TFE only	0.21	75 +/- 5	1.5 +/- 0.3^e	0.17 +/- 0.02	0.81 +/- 0.08	100
Ethanol + TFE	0.13	0	5.3 +/- 0.4	-	-	-
Ethanol + TFE	0.21	80	1.8 +/- 0.2	-	-	-
Pyrazole + TFE	0.21	0	8.6 +/- 0.7^f	0.03 +/- 0.01^f	0.06 +/- 0.01^f	18^f
Disulfiram + TFE	0.21	0	5.6 +/- 1.0^ef	-	-	0^f
Diethyldithiocarbamate + TFE	0.21	30	3.4 +/- 0.7^e	-	-	18^f
Menadione + TFE	0.21	80	1.5 +/- 0.3^e	-	-	147
Allopurinol + TFE	0.21	100	1.8 +/- 0.4^e	-	-	188^f
SKF 525A + TFE	0.21	90	1.1 +/- 0.1^e	-	-	65^f
AIA + TFE	0.21	70	2.3 +/- 0.4^e	-	-	47^f
Metyrapone + TFE	0.21	80	-	-	-	100
α-Naphthoflavone + TFE	0.21	80	-	-	-	78
Isoniazid + TFE	0.21	-	8.3 +/- 0.9^f	0.05 +/- 0.01^f	0.49 +/- 0.03^f	29^f
TFAld only	0.24	-	2.6 +/- 0.4	0.6 +/- 0.15^h	1.25 +/- 0.09^h	-
Pyrazole + TFAld	0.24	-	7.4 +/- 0.8^g	0.07 +/- 0.05^g	0.30 +/- 0.02^gh	-
Isoniazid + TFAld	0.24	-	6.2 +/- 0.6^g	-	0.59 +/- 0.02^g	-

 

(a)          : Cumulative mortality was determined 7 days after TFE in groups of 5-10 rats. Cumulative mortality for TFE alone is the average mortality of five groups of 9-10 rats.

(b)          : Leukocyte count was determined 24 h after TFE (groups of 3-9 rats) or TFAld (groups of 3-8 rats) administration.

(c)          : Blood for serum concentrations of TFAA was taken at 6 and 24 hr after TFE and TFAld administration (groups of 3-6 rats).

(d)          : Blood for serum concentrations of TFE was taken 6 hr after TFE or TFAld treatment (groups of 3-9 rats).

e: significantly different from vehicle, p<0.01

f: significantly different from TFE only, p<0.01

g: significantly different from TFAld only, p<0.01

h: significantly different from TFE treated in each similar pretreatment group, p<0.01

 

Conclusions:

Under the study conditions, it can be concluded that trifluoroacetic acid (TFAA) is the major metabolite of TFE in Wistar rat. The conversion of TFE to TFAA had an apparent lag phase relative to the rate of disappearance of TFE from the blood. Maximal concentrations of TFAA were seen only 16 hr after TFE administration. In contrast, serum TFAA concentrations after trifluoroacetaldehyde (TFAld) hydrate administration were near maximal levels at 6 hr post-administration. Thus, the rate limiting step in the oxidation of TFE to TFAA is the oxidation of TFE to TFAld. TFAA was not rapidly eliminated since at 24 hr after TFE administration elevated concentrations of TFAA persisted. The toxic effects of TFE , which included decreased leukocyte count, bone marrow nucleated cell density, and dry weight of the small intestine, were observed 8 to 16 hr after the administration of the substance. This corresponds to the appearance of major concentrations of the TFAA metabolite in the blood.
A series of inhibitors has been used to determine the relationship between the metabolism and toxicity of TFE and to provide insights into these metabolic pathways. In accordance to the results obtained, the enzymes alcohol dehydrogenase, peroxidase and the cytochrome P450 participated in the metabolism of TFE to TFAA. However, there is a preponderance of evidence that cytochrome P450 is involved in the toxic metabolism of TFE. A pretreatment with ethanol stimulated the TFE metabolism which is in favor with the implication of an ethanol-inducible form of cytochrome P450.

Bioaccumulation potential cannot be judged based on study results

Executive summary:

In a metabolism study Trifluoroethanol (TFE) or Trifluoroacetaldehyde (TFAld) was administered to male Whistar rats. The number of treated animals for each condition of treatment was not always clearly specified in the publication. The substance was administrated by an intraperitoneal injection at dose levels of 0, 130 or 210 mg/kg bw for TFE and 0 or 240 mg/kg bw for TFAld. TFE or TFAld was also administered to rats pretreated with various inhibitors and inducers of drug metabolism.

At different time points after the treatment, animals were sacrificed and the blood was collected in order to determine the time course of metabolism of TFE. The mortality, the leucocyte count and the serum TFE and Trifluoroacetic acid (TFAA) concentrations were measured to determine the mechanism of oxidation of TFE and the toxicity associated with intermediate metabolites. The effect of ethanol ingestion on the TFE metabolism and its toxicity was also investigated to determine the role of the ethanol-inducible cytochrome P-450 in the oxidation of TFE.

Under the study conditions, it can be concluded that TFAA was the major metabolite of TFE in Wistar rats. The conversion of TFE to TFAA had an apparent lag phase relative to the rate of disappearance of TFE from the blood. Serum TFAA was first detected at very low concentrations at 2 hrs after TFE administration and levels increased with time up to 16 hrs after TFE administration. In contrast, the serum TFAA concentrations after TFAld hydrate administration were near maximal levels at 6 hr post-administration. Thus, the rate limiting step in the oxidation of TFE to TFAA is the oxidation of TFE to TFAld. TFAA was not rapidly eliminated since at 24 hr after TFE administration elevated concentrations of TFAA persisted in serum. The toxic effects of TFE, which included decrease in leukocyte count, bone marrow nucleated cell density, and dry weight of the small intestine, were observed 8 to 16 hr after the administration of the substance. This corresponded to the appearance of major concentrations of the TFAA metabolite in the blood.

A series of inhibitors has been used to determine the relationship between the metabolism and the toxicity of TFE and to provide insights into these metabolic pathways. In accordance to the results obtained, the enzymes alcohol dehydrogenase, peroxidase and the cytochrome P450 participated in the metabolism of TFE to TFAA. However, there was a preponderance of evidence that cytochrome P450 is involved in the toxic metabolism of TFE. In fact, a rat pretreatment with ethanol stimulated the TFE metabolism which is in favor with the implication of an ethanol-inducible form of cytochrome P450.

 

This metabolism study in the rat is considered as acceptable based on basic scientific principles as it is well conducted even though it did not satisfy any guideline.

Description of key information

Short description of key information on bioaccumulation potential result: 
Trifluoroethanol (TFE) is rapidly absorbed, metabolised to trifluoroacetaldehyde by cytochrome P4502E1 followed by oxidation to trifluoroacetic acid. Urinary excretion is the main route of elimination followed by the excretion in the expired air and the feces.
Short description of key information on absorption rate: 
Dermal absorption of TFE is likely to occur during skin contact.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Trifluoroethanol (TFE) is rapidly absorbed after intraperitoneal injection or by inhalation , metabolised by liver cytochrome P450 (evidence for the CYP2E1 implication) to trifluoroacetaldehyde followed by the oxidation to trifluoroacetic acid . TFE is distributed in the liver, the kidney, the lungs and the testes. Urinary excretion is the major route of elimination followed by the excretion in the expired air and the feces.

Regarding its partition coefficient of log Kow (as 0.36-0.41) together with its molecular mass (100 g/mol), dermal absorption of TFE is likely to occur during skin contact. Moreover, TFE is water-miscible and therefore is not considered as bioaccumulable.

Discussion on bioaccumulation potential result:

Data were available in the literature on the toxicokinetics of Trifluoroethanol (TFE) including two studies used according to a weight of evidence approach (Fraser, 1987 and Wilkenfeld, 1981).

 

Absorption: In two studies where TFE is administered intraperitoneally to rats, the test substance is rapidly absorbed (Fraser, 1987 and Wilkenfeld, 1981). The serum TFE concentration 1 hr after intraperitoneal injection of 210 mg/kg bw TFE was 1.44 mM. After 2 hr and 6hr this value was respectively decreased by 17% and 58%. At 24 hr after TFE administration, the serum TFE concentration was 0.06 mM (Fraser, 1987). The disappearance of¹⁴C activity from the plasma could be described by a biphasic curve, with half times of 2 hrs and 48 hrs after the intraperitoneally injection of radiolabelled TFE (Wilkenfeld, 1981).

 

Distribution: After an intraperitoneally injection of radiolabelled TFE, radioactivity is found in lung, liver, kidneys and testes (Wilkenfeld, 1981). In this study, the octanol-water partition ratio was determined to be 0.33 at 20°C. Other data found a Log Kow comprised between 0.36 and 0.41 (see 4.7). In all cases, TFE is very mildly lipophilic and since this preference towards lipid materials is very small in magnitude, it can be considered that TFE is not bioaccumulable.

Tissue concentrations 15 min after a 100 mg/kg bw dose level were estimated to be 5.6% of the initial dose in the liver, 1.4% in the testes, 1.1 % in the kidneys and 0.6% in the lung. After 48 hours a similar ranking was observed, with each organ containing: 0.77 % in the liver, 0.14 % in the testes, and 0.12 % in both the kidneys and the lung.

Previous studies (75-89-8, Acute toxicity: inhalation, Wilkenfeld, 1981, W; 75-89-8, Acute toxicity:other route, intraperitoneal, Wilkenfeld, 1981, S; 75-89-8, Toxicity to reproduction, Wilkenfeld, 1981, W) have shown that the volatile solvent 2,2,2-trifluoroethanol rapidly produces testicular damage following intraperitoneal administration or inhalation. Here, no evidence for testicular concentration of TFE was obtained in the present study as the levels of radioactivity at all timepoints postinjection were found to be comparable to those in the liver, kidney, and lung. Thus, the fact that this organ (ie. testes) can be severely damaged by the compound reflects a genuine sensitivity of the testes to TFE.

 

Metabolism: After an intraperitoneally injection of Trifluoroethanol, Trifluoroacetaldehyde (TFAld) and Trifluoroacetic acid (TFA) are rapidly found in the fluids and tissues of rats. Indeed, TFE is rapidly biotransformed in other metabolites and TFA is the end product (TFE-TFAld-TFA) (Fraser, 1987). TFA was first detected in the serum at very low concentration at 2 hr after TFE administration and levels increased with time up to 16 hr postinjection. Six hours after TFAld treatment a relatively small amount of TFE and a relatively large amount of TFA were found in serum compared to these metabolite serum concentrations 6 hr after TFE administration. The conversion of TFE to TFA had an apparent lag phase relative to the rate of disappearance of TFE from the blood. Thus, the rate limiting step in the oxidation of TFE to TFA is the oxidation of TFE to TFAld. TFA was not rapidly eliminated since at 24 hr after TFE administration elevated concentrations of TFA persisted. The toxic effects of TFE, which included decreased leukocyte count, bone marrow nucleated cell density, and dry weight of the small intestine, were observed 8 to 16 hr after the administration of the substance. This corresponds to the appearance of major concentrations of the TFA metabolite in the blood.

A serie of inhibitors has been used to determine the relationship between the metabolism and toxicity of TFE and to provide insights into these metabolic pathways. The results obtained with inhibitors showed that there is a preponderance of evidence that cytochrome P450 is involved in the toxic metabolism of TFE. A pretreatment with ethanol stimulated the TFE metabolism which is in favor with the implication of an ethanol-inducible form of cytochrome P450.

We can conclude that TFE is metabolized in TFAld by liver cytochrome P450 enzymes following by a biotransformation to TFA by acetaldehyde-oxidase. The intermediate TFAld is stable in serum and can be transported to the target tissues such as bone marrow and small intestine (Fraser, 1988).

The study of Kaminsky et al. (1992), brought some additional information on the enzymes involved in the TFE metabolism pathway. TFE and TFAld or their deuterated analogs and coenzymes (NAD coenzyme family) were incubated in vitro with subcellular fractions prepared from rat liver (microsomes, cytosol or mitochondria). In some reactions, pyrazole or inhibitor of microsomal reactions were added prior to initiation of the reaction. The role of ethanol-inducible cytochrome P4502E1 in rat liver microsomal metabolism was probed using a monoclonal antibody to cytochrome P4502E1. The results showed that the hepatic P4502E1 was the primary source for metabolism of TFE on a pathway leading to toxicity. TFAld was also metabolized by P450 but not by P4502E1.

 

Excretion: Urinary excretion is the major route of elimination accounting for 60% of the initial dose after 2 days (Wilkenfeld, 1981). A lower percentage of the initial dose of TFE is collected in the expired air (9% of the initial dose during the first 6hrs postinjection).

Fecal excretion of radioactivity is minor, accounting for about 2% of the initial dose after 48 hrs.

Discussion on absorption rate:

Regarding its partition coefficient of log Kow (as 0.36-0.41) together with its molecular mass (100 g/mol), dermal absorption of TFE is likely to occur during skin contact.