Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity by oral exposure : no data available

Repeated dose toxicity by dermal exposure : no data available

Repeated dose toxicity by inhalation: On reliable key study is available as described below.

In a 28-day repeated inhalation toxicity study (Tosoh, 2000) performed according to the OECD Guideline No. 412 and in compliance with GLP, 2,2,2 Trifluoroethanol (TFE) (purity 100%) was administered to male and female rats.

TFE was dispensed in vapor form and a whole body method exposure was used. The analytical concentrations of vapors of TFE in the inhalation chamber during exposure were 1.14 (low-dose group); 5.26 (medium-dose (1) group); 14.9 (medium-dose (2) group) and 51.1 ppm (high-dose group) corresponding to 4.7; 22; 62 and 213 mg/m3 respectively. These concentrations were chosen in accordance to the results of a previous 14-day repeated inhalation toxicity study.

The exposure was performed 6h/day, 7days/week for 4 weeks. An air control group was set up as the no treatment control group. Furthermore, 2 and 7-week recovery groups were added. The 2-week group consisted of a male and female air control group, a medium-dose (2) group, and a high-dose group. The 7-week group consisted of male air control group, a medium-dose (2) group and a high-dose group. The 7week recovery study involved only males and its main objective was to observe the recovery in male reproductivre organs.

Animals were observed every day for the presence of any clinical signs, and they were weighed at different time point during the exposure period and also during the recovery periods (2 or 7 weeks). In parallel the food consumption was also quantified during the exposure and the recovery periods. At the end of the exposure period or at the end of the recovery periods, animals were sacrificed and several parameters were analyzed such as hematology, clinical chemistry, urinanalysis, gross pathology (including organ weight), and histopathology.

At the highest concentration (213 mg/m3), several measured parameters were changed in comparison to the air control animals. Indeed, the animals presented a decrease in body weight and a change in the blood chemistry. Furthermore, at the highest concentration, TFE is highly toxic to male reproductive system since a decrease in testis and epididymis weights were observed during the exposure period and this decrease was maintained during the recovery period. This decrease of the reproductive organ weight was accompanied by a severe loss of germ cells such as spermatocytes , spermatids and spermatozoa while the seminiferous tubules contained only Sertoli cells.

At the middle concentration of 62 mg/m3, adverse effects were observed as a decrease of the thymus weight in the treated female rats and change in hematological parameters in both sexes suggesting an anemia reaction following TFE repeated dose exposure by inhalation.

Based on these results, the main effects due to 2,2,2 Trifluoroethanol were observed in the spermiogenesis inhibition of male reproductive organs and in the hematological changes that suggested hypochromic anemia.

The Lowest Observed Adverse Effect Concentration (LOAEC) is considered to be 51.1 ppm (approximately 213 mg/m3) based on the decrease in the mean corpuscular volume and the decrease in the mean corpuscular hemoglobin in the hematological analysis which were irreversible during the two-week recovery period. Moreover toxicity to the reproduction as spermiogenesis alteration was also observed at this concentration. At the middle level (14.9 ppm corresponding to 62 mg/m3), only hematological effects were observed which were reversible during the 2 -week recovery period . Hence the 62 mg/m3 exposure was considered as the No Observed Adverse Effect concentration (NOAEC) while the No Observed Effect Concentration (NOEC) was assumed to be 5.26 ppm (approximately 22 mg/m3) regarding the absence of any effects after 28-day repeated exposure.

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 412.

The effects on the male reproductive system observed after a repeated exposure by inhalation were already considered and taken into account in the self-classification for the toxicity to the reproduction (see § 7.8).

Regarding the hematological effects observed in the animal exposed by inhalation, they can be considered as relevant to human health following repeated exposure by inhalation. It is recognised that these effects which may be seen in humans are considered of minimal toxicological importance and do not justify classification (see Guidance on the application of the CLP criteria, § 3.9.2.5.2). However, considering the irreversibility of these effects during the two-week recovery period of the study, a classification is proposed as STOT-RE2 (H373) according to the CLP.

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-11-04 to 2000-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Report in japanese. Summary was translated in english by the sponsor.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 5 weeks old
- Weight at study initiation: 118.5 to 136.9 g
- Fasting period before study: no
- Housing: 2 per cage in stainless steel wire bottom cages (430 Wx300Dx187H mm)
- Diet (e.g. ad libitum): a pelleted diet (MF oriental Yeast Co., Ltd) was administered through a hand-down dispenser/bowl. The pellets were sterilized with an autoclave at 121C for 30 minutes. Both pellets and potable water impurities were analyzed and were verified safe for the study.
- Water (e.g. ad libitum): Water fed through an automatic water supply equipment from the Hita City waterworks was used as potable water (UV sterilized water). Furthermore, the potable water for urine collection was supplied by a water supply jar.
- Acclimation period: YEs but no data on the duration of this period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2°C
- Humidity (%): 55% +/- 10 %
- Air changes (per hr): 10-15 times / hour
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

IN-LIFE DATES: From: To: no data
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: vapor generator ; model GEL-1A, Shibata Scientific Technology LTD). The test substance was introduced from the top part of the inhalation chamber through a pre-filter and HEPA filter after mixing and dilution with the air inside the breathing chamber and then ejected normally from the lower part (one pass method). The concentration of the test substance was adjusted by changing the supply quantity of 2,2,2 Trifluoroethanol to the chamber.
- Exposure chamber volume: 563 L (total volume), 384 L (air volume)
- Method of holding animals in test chamber: The rats were put in 10 individual cages housed inside a chamber. Food and water were not supplied during the exposure.
- Source and rate of air: Airflow, 100L/min
- Method of conditioning air: The ejected test substance was processed through an activated charcoal filter, diluted through a dilution tank and then released into the atmosphere.
- System of generating particulates/aerosols: no data
- Method of particle size determination: no data
- Treatment of exhaust air: no data
- Temperature, humidity, pressure in air chamber: The temperature and humidity was measured and continuously recorded everyday during the exposure period with a platinum resistance thermometer (dry bulb, wet bulb). See details in table 7.5.3/1.
TEST ATMOSPHERE
- Brief description of analytical method used: Sampling of the animal-breathing chamber was taken and the concentration was measured by equipment analysis (see details in Table 7.5.3/2). Sampling was conducted using a sampling rate if approximately 1.0 L/min at 60 and 80 minutes from the start of exposure.
- Samples taken from breathing zone: yes
VEHICLE (if applicable)
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See details in Table 7.5.3/3
Duration of treatment / exposure:
6 hours/day for 4 weeks (7 days/week)
Frequency of treatment:
daily
Dose / conc.:
1 ppm (nominal)
Remarks:
corresponding to 4.2 mg/m3
Dose / conc.:
5 ppm (nominal)
Remarks:
corresponding to 21mg/m3
Dose / conc.:
15 ppm (nominal)
Remarks:
corresponding to 62 mg/m3
Dose / conc.:
50 ppm (nominal)
Remarks:
corresponding to 208 mg/m3
Dose / conc.:
1.14 ppm (analytical)
Remarks:
Corresponding to 4.7 mg/m3
Dose / conc.:
5.26 ppm (analytical)
Remarks:
Corresponding to 22 mg/m3
Dose / conc.:
14.9 ppm (analytical)
Remarks:
Corresponding to 62 mg/m3
Dose / conc.:
51.1 ppm (analytical)
Remarks:
Corresponding to 213 mg/m3
No. of animals per sex per dose:
6 animals/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: This study is based on the results of the recent 14-day repeated inhalation toxicity study (Study code number: PIS-0008). The groups and their concentrations were determined after consulting with the sponsor of the study. They were the following: high-dose group 50 ppm, medium-dose (2) group 15 ppm, medium-dose (1) group 5 ppm, low-dose group 1 ppm. Also, an air control group was set up as the control group. Furthermore, two and seven week recovery groups were setup. The two-week group consisted of a male and female air control group, a medium-dose (2) group, and high-dose group. The seven-week group consisted of male air control group, a medium-dose (2) group and a high-dose group (see details in Table 7.5.3/4)
- Rationale for animal assignment (if not random): Following quarantine and habituation, the rats whose performance status was favorable and who developed strongly were assigned to groups where the average weight of each group was roughly equal using stratified random sampling technique for weight.
- Rationale for selecting satellite groups: Males had a two and seven week recovery period. Females had a two-week recovery period. Day 1 (recovery) started from the day after the final day of exposure and week 1 (recovery) started the week recovery began. The 7 week recovery study involved only males and its main objective was to observe the recovery of the effects on male sexual organs.
- Post-exposure recovery period in satellite groups: at the medium dose and high dose (15 and 50 ppm) a 2 week or a 7 week recovery period was tested. In parallel the same recovery period are followed for the air control group (see details in Table 7.5.3/4).
- Section schedule rationale (if not random):
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the exposure period all cases were observed everyday prior to exposure and one hour after the end of exposure. During the recovery period all cases were observed one time per day.
- Cage side observations checked in table 7.5.3/5 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the exposure period all cases were observed everyday prior to exposure and one hour after the end of exposure. During the recovery period all cases were observed one time per day.

BODY WEIGHT: Yes
- Time schedule for examinations: All cases were measured prior to exposure on day-2 (when divided into groups), during exposure on days 1, 3, 8, 12, 17, 21, 26 and 28, during the recovery period for males on days 1 (recovery), 5, 10, 14, 15, 22, 29, 36, 43 and 49, and during the recovery period for females on days 1 (recovery), 5, 10 and 14. Also, the body weight was measured once prior to necropsy to calculate the relative weight of the organs.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes: All cases were measured one time prior to exposure and twice per week during exposure and during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (Ether anesthesia)
- Animals fasted: Yes: Blood was drawn from the abdominal aorta after a 21 hours fasting period after the end of exposure period or the recovery period.
- How many animals: no data
- Parameters checked in table 7.5.3/6 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: identical as the haematology analysis
- Animals fasted: Yes
- How many animals: no data
- Parameters checked in table 7.5.3/6 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: the urine was collected for 13-17 hours from the individual metabolic cages at the end of the exposure period or at the end of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes / No / No data
- Parameters checked in table 7.5.3/6 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.3/7)
HISTOPATHOLOGY: Yes (see table 7.5.3/7)
Other examinations:
no other examinations
Statistics:
The Bartlett's test was applied to the result for the body weight, food intake, the hematological test, the blood chemistry test, the volume of urine, and the organ weights to test for homogeneity of variance.
If no significant heterogeneity (5%) was detected, one-way analysis of variance was used. The parameters found to be significant in the analysis of variance were tested by Dunnett’s test (performed between the air control group and each dose group).
If significant heterogeneity was detected, the Kruskal-Wallis test was conducted. The parameters found to be significant in the Kruskal-Wallis’s test were test using the non-parametric Dunnett's test (performed between the air control group and each dose group).
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities were observed.
Mortality:
no mortality observed
Description (incidence):
No death occurred during the study period. No abnormalities were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No significant differences in the air control group were found. The weights of high concentration group test animals decreased after 3 days for males, and after 4 days for females. Even for recovery time, suppression of the weight was continued until 29 days (recovery) for male and 14 days (recovery) for female. The low body weights are believed to have gradually recovered because the exposure to TFE was stopped. See details in attached Tables 7.5.3_8, 7.5.3_9, 7.5.3_10.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No significant differences in all of the air control groups were found. Food consumptions were decreased after 4 days for both sexes. Even at the recovery duration, suppression of food consumption was continued 11 days (recovery) for male and 4 days (recovery) for female. Recovery was shown similar to that of the body weights. The changes were believed to be incidental and not caused by TFE exposure. Indeed, significant differences in body weight fluctuations were not observed in the males of the medium-dose (2) group whose food intake was low from days 4 to 21 and the changes were shown to be nearly identical to those of the air control group. See details in attached Tables 7.5.3_11, 7.5.3_12, 7.5.3_13.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of exposure test, males in the medium (2) concentration group and both sexes for high groups showed decrease of amount of average erythrocyte and decrease of amount of average erythrocyte hemoglobin, decrease of the number of platelet. For males in the high concentration group, the decrease of concentration of hemoglobin and decrease of hematocrit level were observed. For female rats in the high concentration group, increase of erythrocyte and elongation of prothrombin time were observed. Among these symptoms, decreases of concentration of hemoglobin and reduction of erythrocyte volume in male rats of high concentration group and decrease of the number of platelet in female rats were persisted until the end of recovery days (2 weeks). See details in attached Tables 7.5.3_14, 7.5.3_15.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the high concentration groups, decrease of total protein to both sexes, decrease of albumin and Alanine aminotransferase and severe decrease of sorbitol dehydrogenase to male rats, increase of A/G ratio to female rats were observed, respectively. Furthermore, a decrease in calcium and an increase in chlorine was observed in the high-dose group (male). However, since all of the changes were slight and within the ranges of the background data of this lab, the changes were incidental. At the end of 2 week recovery period only decrease of total protein to female rats in the high concentration remained. Changes to the blood protein composition of the high-dose group were found to be the main influence on the blood chemistry test and excluding the total protein decrease observed in the high-dose group (female) the other parameters would be favorable to recoverability. See details in attached Tables 7.5.3_16, 7.5.3_17, 7.5.3_18.
Urinalysis findings:
no effects observed
Description (incidence and severity):
no effect
Behaviour (functional findings):
not examined
Description (incidence and severity):
not examined
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Female: at the end of exposure, absolute and relative weight of thymus was severely decreased at the medium (2) group and high dose and the absolute and relative weight of pituitary gland was also severely decreased only at the high dose tested. At the both middle dose, an increase of the relative weight of adrenal gland was observed. These effects were reversible as the relative organ weights were normal (compared to the air control) during the recovery period.
Male: As for female, at the high concentration, a decrease of absolute and relative weight of pituitary gland and of thymus was observed. The male rats exposed to vapors of high concentrations of TFE presented also a decrease of relative weight of lungs, decrease of absolute and relative weight of testis, decrease of absolute and relative weight of epididymis and decrease of absolute weight of adrenal gland and brain (but for the two latter organs, there was no toxicological significance in the weights variances.
At the end of the 2 week recovery period, in the exposed animals to high concentrations of TFE, the increase relative weight of lungs remained. It was also the case for the severe decrease of relative weight of testis and the decrease of the relative weight of epididymis remained up to the 7 week recovery period. See details in attached Tables 7.5.3_19, 7.5.3_20, 7.5.3_21, 7.5.3_22, 7.5.3_23, 7.5.3_24, 7.5.3_25, 7.5.3_26.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of exposure, in male rats of the high concentration group, microgenesis of testes and microgenesis of epididymis were observed. These symptoms observed in all of the male tested rats continued until the end of the 7 week recovery period.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No histopathological abnormalities were observed for treated female rats. At the end of exposure period, male rats exposed to high concentrations of TFE vapors presented hyperplastic Leydig cells, disappearance of meiosis of spermatocyte from the Diplotene stage, disappearance of early and late elongated spermatids, loss of spermatocytes at Pachytene stage, disappearance of round spermatids, disappearance of spermatocytes at Zygotene stage, seminiferous tubule with only Sertoli cells, disappearance of spermatozoa in the lumen of epididymis and presence of germ cell residue in the lumen of epididymis. Therefore, the histopathological examination highlights the changes that inhibit spermiogenesis in male reproductive organs in the high dose group. These changes are believed to be characteristic of the effects of exposure to TFE under the test conditions and these changes showed a recovery trend at the end of the 7 week recovery period. The recovery implicated slow but reversible changes. See details in attached Tables 7.5.3_27, 7.5.3_28.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Description (incidence and severity):
no other findings
Key result
Dose descriptor:
NOEC
Effect level:
22 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
organ weights and organ / body weight ratios
Remarks on result:
other: Effects on organ weight observed at the high middle dose in female. Some low changes in haematology and blood chemistry parameters in both males and females. However all effects are reversible.
Key result
Dose descriptor:
NOAEC
Effect level:
62 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
213 mg/m³ air (nominal)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

No other information (Tables on Study results are in the attached document, see below).

Conclusions:
Based on the above results, the main effects due to 2,2,2 Trifluoroethanol were observed in the spermiogenesis inhibition of male reproductive organs and in the hematological changes that suggest anemia.
The No Observed Effect Concentration (NOEC) under the test conditions were assumed to be 22 mg/m3 due to a decrease in the mean corpuscular volume and a decrease in the mean corpuscular hemoglobin seen in the hematological test, and a decrease in the absolute and relative weight of the thymus in the medium-dose (2) group observed in organ weights. Therefore, the Lowest Observed Adverse Effect Concentration (LOAEC) is considered to be 62 mg/m3.
Executive summary:

In a 28-day repeated inhalation toxicity study performed in accordance to the OECD Guideline No. 412 and in compliance with GLP, 2,2,2 Trifluoroethanol (TFE) (purity 100%) was administered to Crj: CD (SD) IGS male and female rats (6 animals/sex/concentrations).

 

TFE was dispensed in vapor form and a whole body method exposure was used. TFE concentrations for the study were 1, 5, 15 and 50 ppm. The analytical concentrations of vapors of TFE in the inhalation chamber during exposure were 1.14 (named low-dose group); 5.26 (named medium-dose (1) group); 14.9 (named medium-dose (2) group) and 51.1 ppm (named high-dose group) corresponding to 4.7; 22; 62 and 213 mg/m3 respectively (using molecular mass of 100.06 g/mol for TFE and 24.05 L as the volume at 20°C of a mole of vapour when the pressure is at 1 atmosphere or 760 mm Hg). These concentrations were chosen in accordance to the results of a previous 14-day repeated inhalation toxicity study.

The exposure was performed for six hours per day for a repeated 28 day period. An air control group was set up as the no treatment control group. Furthermore, 2 and 7-week recovery groups were added. The 2-week group consisted of a male and female air control group, a medium-dose (2) group, and a high-dose group. The 7-week group consisted of male air control group, a medium-dose (2) group and a high-dose group. The 7‑week recovery study involved only males and its main objective was to observe the recovery of the effects on male sexual organs.

  

Animals were observed every day for the presence of any clinical signs, and they were weighed at different time point during the exposure period and also during the recovery periods (2 or 7 weeks). In parallel the food consumption was also quantified during the exposure and the recovery periods. At the end of the exposure period or at the end of the recovery period, animals were sacrificed and several parameters were analyzed such as hematology, clinical chemistry, urinanalysis, gross pathology (including organ weight), and histopathology.

 

At the highest concentration (213 mg/m3), several measured parameters were changed in comparison to the control air animals. Indeed, the animals presented a decrease in body weight and organ weight (thymus and hyphophysis only in females) a change in the blood chemistry in both sexes. Furthermore, at the highest concentration, TFE is highly toxic to male reproductive system since a decrease in testis and epididymis weights were observed during the exposure period and this decrease was maintained during the recovery period. This decrease of the reproductive organ weight was accompanied by a severe loss of germ cells such as spermatocytes, spermatids and spermatozoa while the seminiferous tubules contained only Sertoli cells.

 

At the middle concentration of 62 mg/m3, adverse effects were observed as a decrease of the thymus weight in the treated female rats and change in hematological parameters in both sexes suggesting an anemia reaction following TFE repeated dose exposure by inhalation.

Based on these results, the main effects due to 2,2,2 Trifluoroethanol were observed in the spermiogenesis inhibition of male reproductive organs and in the hematological changes that suggested hypochromic anemia.

The Lowest Observed Adverse Effect Concentration (LOAEC) is considered to be 51.1 ppm (approximately 213 mg/m3) based on the decrease in the mean corpuscular volume and the decrease in the mean corpuscular hemoglobin in the hematological analysis. Moreover toxicity to the reproduction as spermiogenesis alteration was also observed at this concentration. At the middle level (14.9 ppm corresponding to 62 mg/m3), only hematological effects were observed which were reversible during the 2 -week recovery period . Hence the 62 mg/m3 exposure was considered as the No Observed Adverse Effect concentration while the No Observed Effect Concentration (NOEC) was assumed to be 5.26 ppm (approximately 22 mg/m3) regarding the absence of any effects after 28-day repeated exposure.

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 412.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
62 mg/m³
Study duration:
subacute
Experimental exposure time per week (hours/week):
42
Species:
rat
Quality of whole database:
One reliable key study performed according to the OECD TG 412 is available.
Other available data are from secondary litterature or abstract but some of them have been considered as supporting studies.
System:
other: hematologic system and male reproductive organ
Organ:
blood
seminiferous tubules
testes

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-11-04 to 2000-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Report in japanese. Summary was translated in english by the sponsor.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 5 weeks old
- Weight at study initiation: 118.5 to 136.9 g
- Fasting period before study: no
- Housing: 2 per cage in stainless steel wire bottom cages (430 Wx300Dx187H mm)
- Diet (e.g. ad libitum): a pelleted diet (MF oriental Yeast Co., Ltd) was administered through a hand-down dispenser/bowl. The pellets were sterilized with an autoclave at 121C for 30 minutes. Both pellets and potable water impurities were analyzed and were verified safe for the study.
- Water (e.g. ad libitum): Water fed through an automatic water supply equipment from the Hita City waterworks was used as potable water (UV sterilized water). Furthermore, the potable water for urine collection was supplied by a water supply jar.
- Acclimation period: YEs but no data on the duration of this period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2°C
- Humidity (%): 55% +/- 10 %
- Air changes (per hr): 10-15 times / hour
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

IN-LIFE DATES: From: To: no data
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: vapor generator ; model GEL-1A, Shibata Scientific Technology LTD). The test substance was introduced from the top part of the inhalation chamber through a pre-filter and HEPA filter after mixing and dilution with the air inside the breathing chamber and then ejected normally from the lower part (one pass method). The concentration of the test substance was adjusted by changing the supply quantity of 2,2,2 Trifluoroethanol to the chamber.
- Exposure chamber volume: 563 L (total volume), 384 L (air volume)
- Method of holding animals in test chamber: The rats were put in 10 individual cages housed inside a chamber. Food and water were not supplied during the exposure.
- Source and rate of air: Airflow, 100L/min
- Method of conditioning air: The ejected test substance was processed through an activated charcoal filter, diluted through a dilution tank and then released into the atmosphere.
- System of generating particulates/aerosols: no data
- Method of particle size determination: no data
- Treatment of exhaust air: no data
- Temperature, humidity, pressure in air chamber: The temperature and humidity was measured and continuously recorded everyday during the exposure period with a platinum resistance thermometer (dry bulb, wet bulb). See details in table 7.5.3/1.
TEST ATMOSPHERE
- Brief description of analytical method used: Sampling of the animal-breathing chamber was taken and the concentration was measured by equipment analysis (see details in Table 7.5.3/2). Sampling was conducted using a sampling rate if approximately 1.0 L/min at 60 and 80 minutes from the start of exposure.
- Samples taken from breathing zone: yes
VEHICLE (if applicable)
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See details in Table 7.5.3/3
Duration of treatment / exposure:
6 hours/day for 4 weeks (7 days/week)
Frequency of treatment:
daily
Dose / conc.:
1 ppm (nominal)
Remarks:
corresponding to 4.2 mg/m3
Dose / conc.:
5 ppm (nominal)
Remarks:
corresponding to 21mg/m3
Dose / conc.:
15 ppm (nominal)
Remarks:
corresponding to 62 mg/m3
Dose / conc.:
50 ppm (nominal)
Remarks:
corresponding to 208 mg/m3
Dose / conc.:
1.14 ppm (analytical)
Remarks:
Corresponding to 4.7 mg/m3
Dose / conc.:
5.26 ppm (analytical)
Remarks:
Corresponding to 22 mg/m3
Dose / conc.:
14.9 ppm (analytical)
Remarks:
Corresponding to 62 mg/m3
Dose / conc.:
51.1 ppm (analytical)
Remarks:
Corresponding to 213 mg/m3
No. of animals per sex per dose:
6 animals/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: This study is based on the results of the recent 14-day repeated inhalation toxicity study (Study code number: PIS-0008). The groups and their concentrations were determined after consulting with the sponsor of the study. They were the following: high-dose group 50 ppm, medium-dose (2) group 15 ppm, medium-dose (1) group 5 ppm, low-dose group 1 ppm. Also, an air control group was set up as the control group. Furthermore, two and seven week recovery groups were setup. The two-week group consisted of a male and female air control group, a medium-dose (2) group, and high-dose group. The seven-week group consisted of male air control group, a medium-dose (2) group and a high-dose group (see details in Table 7.5.3/4)
- Rationale for animal assignment (if not random): Following quarantine and habituation, the rats whose performance status was favorable and who developed strongly were assigned to groups where the average weight of each group was roughly equal using stratified random sampling technique for weight.
- Rationale for selecting satellite groups: Males had a two and seven week recovery period. Females had a two-week recovery period. Day 1 (recovery) started from the day after the final day of exposure and week 1 (recovery) started the week recovery began. The 7 week recovery study involved only males and its main objective was to observe the recovery of the effects on male sexual organs.
- Post-exposure recovery period in satellite groups: at the medium dose and high dose (15 and 50 ppm) a 2 week or a 7 week recovery period was tested. In parallel the same recovery period are followed for the air control group (see details in Table 7.5.3/4).
- Section schedule rationale (if not random):
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the exposure period all cases were observed everyday prior to exposure and one hour after the end of exposure. During the recovery period all cases were observed one time per day.
- Cage side observations checked in table 7.5.3/5 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During the exposure period all cases were observed everyday prior to exposure and one hour after the end of exposure. During the recovery period all cases were observed one time per day.

BODY WEIGHT: Yes
- Time schedule for examinations: All cases were measured prior to exposure on day-2 (when divided into groups), during exposure on days 1, 3, 8, 12, 17, 21, 26 and 28, during the recovery period for males on days 1 (recovery), 5, 10, 14, 15, 22, 29, 36, 43 and 49, and during the recovery period for females on days 1 (recovery), 5, 10 and 14. Also, the body weight was measured once prior to necropsy to calculate the relative weight of the organs.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes: All cases were measured one time prior to exposure and twice per week during exposure and during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (Ether anesthesia)
- Animals fasted: Yes: Blood was drawn from the abdominal aorta after a 21 hours fasting period after the end of exposure period or the recovery period.
- How many animals: no data
- Parameters checked in table 7.5.3/6 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: identical as the haematology analysis
- Animals fasted: Yes
- How many animals: no data
- Parameters checked in table 7.5.3/6 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: the urine was collected for 13-17 hours from the individual metabolic cages at the end of the exposure period or at the end of the recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes / No / No data
- Parameters checked in table 7.5.3/6 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7.5.3/7)
HISTOPATHOLOGY: Yes (see table 7.5.3/7)
Other examinations:
no other examinations
Statistics:
The Bartlett's test was applied to the result for the body weight, food intake, the hematological test, the blood chemistry test, the volume of urine, and the organ weights to test for homogeneity of variance.
If no significant heterogeneity (5%) was detected, one-way analysis of variance was used. The parameters found to be significant in the analysis of variance were tested by Dunnett’s test (performed between the air control group and each dose group).
If significant heterogeneity was detected, the Kruskal-Wallis test was conducted. The parameters found to be significant in the Kruskal-Wallis’s test were test using the non-parametric Dunnett's test (performed between the air control group and each dose group).
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities were observed.
Mortality:
no mortality observed
Description (incidence):
No death occurred during the study period. No abnormalities were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No significant differences in the air control group were found. The weights of high concentration group test animals decreased after 3 days for males, and after 4 days for females. Even for recovery time, suppression of the weight was continued until 29 days (recovery) for male and 14 days (recovery) for female. The low body weights are believed to have gradually recovered because the exposure to TFE was stopped. See details in attached Tables 7.5.3_8, 7.5.3_9, 7.5.3_10.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No significant differences in all of the air control groups were found. Food consumptions were decreased after 4 days for both sexes. Even at the recovery duration, suppression of food consumption was continued 11 days (recovery) for male and 4 days (recovery) for female. Recovery was shown similar to that of the body weights. The changes were believed to be incidental and not caused by TFE exposure. Indeed, significant differences in body weight fluctuations were not observed in the males of the medium-dose (2) group whose food intake was low from days 4 to 21 and the changes were shown to be nearly identical to those of the air control group. See details in attached Tables 7.5.3_11, 7.5.3_12, 7.5.3_13.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of exposure test, males in the medium (2) concentration group and both sexes for high groups showed decrease of amount of average erythrocyte and decrease of amount of average erythrocyte hemoglobin, decrease of the number of platelet. For males in the high concentration group, the decrease of concentration of hemoglobin and decrease of hematocrit level were observed. For female rats in the high concentration group, increase of erythrocyte and elongation of prothrombin time were observed. Among these symptoms, decreases of concentration of hemoglobin and reduction of erythrocyte volume in male rats of high concentration group and decrease of the number of platelet in female rats were persisted until the end of recovery days (2 weeks). See details in attached Tables 7.5.3_14, 7.5.3_15.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the high concentration groups, decrease of total protein to both sexes, decrease of albumin and Alanine aminotransferase and severe decrease of sorbitol dehydrogenase to male rats, increase of A/G ratio to female rats were observed, respectively. Furthermore, a decrease in calcium and an increase in chlorine was observed in the high-dose group (male). However, since all of the changes were slight and within the ranges of the background data of this lab, the changes were incidental. At the end of 2 week recovery period only decrease of total protein to female rats in the high concentration remained. Changes to the blood protein composition of the high-dose group were found to be the main influence on the blood chemistry test and excluding the total protein decrease observed in the high-dose group (female) the other parameters would be favorable to recoverability. See details in attached Tables 7.5.3_16, 7.5.3_17, 7.5.3_18.
Urinalysis findings:
no effects observed
Description (incidence and severity):
no effect
Behaviour (functional findings):
not examined
Description (incidence and severity):
not examined
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Female: at the end of exposure, absolute and relative weight of thymus was severely decreased at the medium (2) group and high dose and the absolute and relative weight of pituitary gland was also severely decreased only at the high dose tested. At the both middle dose, an increase of the relative weight of adrenal gland was observed. These effects were reversible as the relative organ weights were normal (compared to the air control) during the recovery period.
Male: As for female, at the high concentration, a decrease of absolute and relative weight of pituitary gland and of thymus was observed. The male rats exposed to vapors of high concentrations of TFE presented also a decrease of relative weight of lungs, decrease of absolute and relative weight of testis, decrease of absolute and relative weight of epididymis and decrease of absolute weight of adrenal gland and brain (but for the two latter organs, there was no toxicological significance in the weights variances.
At the end of the 2 week recovery period, in the exposed animals to high concentrations of TFE, the increase relative weight of lungs remained. It was also the case for the severe decrease of relative weight of testis and the decrease of the relative weight of epididymis remained up to the 7 week recovery period. See details in attached Tables 7.5.3_19, 7.5.3_20, 7.5.3_21, 7.5.3_22, 7.5.3_23, 7.5.3_24, 7.5.3_25, 7.5.3_26.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of exposure, in male rats of the high concentration group, microgenesis of testes and microgenesis of epididymis were observed. These symptoms observed in all of the male tested rats continued until the end of the 7 week recovery period.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No histopathological abnormalities were observed for treated female rats. At the end of exposure period, male rats exposed to high concentrations of TFE vapors presented hyperplastic Leydig cells, disappearance of meiosis of spermatocyte from the Diplotene stage, disappearance of early and late elongated spermatids, loss of spermatocytes at Pachytene stage, disappearance of round spermatids, disappearance of spermatocytes at Zygotene stage, seminiferous tubule with only Sertoli cells, disappearance of spermatozoa in the lumen of epididymis and presence of germ cell residue in the lumen of epididymis. Therefore, the histopathological examination highlights the changes that inhibit spermiogenesis in male reproductive organs in the high dose group. These changes are believed to be characteristic of the effects of exposure to TFE under the test conditions and these changes showed a recovery trend at the end of the 7 week recovery period. The recovery implicated slow but reversible changes. See details in attached Tables 7.5.3_27, 7.5.3_28.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Description (incidence and severity):
no other findings
Key result
Dose descriptor:
NOEC
Effect level:
22 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
organ weights and organ / body weight ratios
Remarks on result:
other: Effects on organ weight observed at the high middle dose in female. Some low changes in haematology and blood chemistry parameters in both males and females. However all effects are reversible.
Key result
Dose descriptor:
NOAEC
Effect level:
62 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
213 mg/m³ air (nominal)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

No other information (Tables on Study results are in the attached document, see below).

Conclusions:
Based on the above results, the main effects due to 2,2,2 Trifluoroethanol were observed in the spermiogenesis inhibition of male reproductive organs and in the hematological changes that suggest anemia.
The No Observed Effect Concentration (NOEC) under the test conditions were assumed to be 22 mg/m3 due to a decrease in the mean corpuscular volume and a decrease in the mean corpuscular hemoglobin seen in the hematological test, and a decrease in the absolute and relative weight of the thymus in the medium-dose (2) group observed in organ weights. Therefore, the Lowest Observed Adverse Effect Concentration (LOAEC) is considered to be 62 mg/m3.
Executive summary:

In a 28-day repeated inhalation toxicity study performed in accordance to the OECD Guideline No. 412 and in compliance with GLP, 2,2,2 Trifluoroethanol (TFE) (purity 100%) was administered to Crj: CD (SD) IGS male and female rats (6 animals/sex/concentrations).

 

TFE was dispensed in vapor form and a whole body method exposure was used. TFE concentrations for the study were 1, 5, 15 and 50 ppm. The analytical concentrations of vapors of TFE in the inhalation chamber during exposure were 1.14 (named low-dose group); 5.26 (named medium-dose (1) group); 14.9 (named medium-dose (2) group) and 51.1 ppm (named high-dose group) corresponding to 4.7; 22; 62 and 213 mg/m3 respectively (using molecular mass of 100.06 g/mol for TFE and 24.05 L as the volume at 20°C of a mole of vapour when the pressure is at 1 atmosphere or 760 mm Hg). These concentrations were chosen in accordance to the results of a previous 14-day repeated inhalation toxicity study.

The exposure was performed for six hours per day for a repeated 28 day period. An air control group was set up as the no treatment control group. Furthermore, 2 and 7-week recovery groups were added. The 2-week group consisted of a male and female air control group, a medium-dose (2) group, and a high-dose group. The 7-week group consisted of male air control group, a medium-dose (2) group and a high-dose group. The 7‑week recovery study involved only males and its main objective was to observe the recovery of the effects on male sexual organs.

  

Animals were observed every day for the presence of any clinical signs, and they were weighed at different time point during the exposure period and also during the recovery periods (2 or 7 weeks). In parallel the food consumption was also quantified during the exposure and the recovery periods. At the end of the exposure period or at the end of the recovery period, animals were sacrificed and several parameters were analyzed such as hematology, clinical chemistry, urinanalysis, gross pathology (including organ weight), and histopathology.

 

At the highest concentration (213 mg/m3), several measured parameters were changed in comparison to the control air animals. Indeed, the animals presented a decrease in body weight and organ weight (thymus and hyphophysis only in females) a change in the blood chemistry in both sexes. Furthermore, at the highest concentration, TFE is highly toxic to male reproductive system since a decrease in testis and epididymis weights were observed during the exposure period and this decrease was maintained during the recovery period. This decrease of the reproductive organ weight was accompanied by a severe loss of germ cells such as spermatocytes, spermatids and spermatozoa while the seminiferous tubules contained only Sertoli cells.

 

At the middle concentration of 62 mg/m3, adverse effects were observed as a decrease of the thymus weight in the treated female rats and change in hematological parameters in both sexes suggesting an anemia reaction following TFE repeated dose exposure by inhalation.

Based on these results, the main effects due to 2,2,2 Trifluoroethanol were observed in the spermiogenesis inhibition of male reproductive organs and in the hematological changes that suggested hypochromic anemia.

The Lowest Observed Adverse Effect Concentration (LOAEC) is considered to be 51.1 ppm (approximately 213 mg/m3) based on the decrease in the mean corpuscular volume and the decrease in the mean corpuscular hemoglobin in the hematological analysis. Moreover toxicity to the reproduction as spermiogenesis alteration was also observed at this concentration. At the middle level (14.9 ppm corresponding to 62 mg/m3), only hematological effects were observed which were reversible during the 2 -week recovery period . Hence the 62 mg/m3 exposure was considered as the No Observed Adverse Effect concentration while the No Observed Effect Concentration (NOEC) was assumed to be 5.26 ppm (approximately 22 mg/m3) regarding the absence of any effects after 28-day repeated exposure.

This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 412.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
One reliable key study performed according to the OECD TG 412 is available.
Other available data are from secondary litterature or abstract, but some of them have been considered as supporting studies

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) N°1972/2008.

Self classification:

Oral route:

no information is available. However, considering the only industrial uses of TFE (oral route not relevant) together with the self classification as Repr. 1B classification (see § 7.8), no classification is proposed for repeated dose toxicity by oral route.

Dermal route:

no information.

However, considering the absence of systemic effects in the acute dermal toxicity study and the absence of local effect in the skin irritation study, it is unlikely that TFE induces effects by dermal route after chronic exposure. Therefore, no classification is proposed for repeated dose toxicity by dermal route. Moreover, considering the self classification as Repr. 1B classification (see § 7.8), it is assumed that the Risk Management Measures are protecting from human exposure occurring by dermal route.

Inhalation route:

Based on the hematological effects observed in the animal which are relevant to human health following repeated exposure, TFE is self-classified as Specific Target Organ Toxicants following Repeated Exposure, May cause damage to hematological system through prolonged or repeated exposure by inhalation (STOT-RE 2 ; H373) according the Regulation (EC) No 1272/2008 (CLP).

The effects on the male reproductive organ observed after a repeated exposure by inhalation are already considered and taken into account in the self-classification for the toxicity to the reproduction in cat 1B (see § 7.8).