2,2,2-trifluoroethanol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Description of key information

Three studies are available for this endpoint and are used in a weight of evidence approach. A fourth study that is a secondary litterature with no details on test conditions and results was also considered as supporting study since results reported in this secondary litterature was quite similar to the 2 previous one.

In the 1st study (Wilkenfeld 1981), 2,2,2 trifluoroethanol (TFE; 99% purity) was administered to male Sprague-Dawley rats by inhalation for 6 hours per day, 5 days/week for 2 weeks at dose levels of 0, 100 and 200 ppm (nominal concentrations) corresponding to 415 and 830 mg/m3 respectively. The animals were exposed to TFE by whole body inhalation. After the exposure period, each week during 13 weeks, a serial mating was performed: each male was paired with a virgin female rat to examine the male fertility. Some male rats were sacrificed at the end of each exposure week and then at the end of the 1st, 3rd, 5th, 7th, 9th and 13th week post-exposure. Sperm production was evaluated by sperm counts in the 24 -hour urines twice weekly collected from animals subjected to surgical vasocystotomy. The testicular sorbitol (SDH) and malade deshydrogenase (MDH) activities were also measured.

Pregnant female rats were sacrificed prior post-partum corresponding to 17 days following mating. The implantation sites, the resorption sites, the live fetuses and the corpora lutea were checked.

Under the test conditions, repeated exposure to TFE by inhalation produced damages on the male reproductive system as necrosis of seminiferous tubules inducing a severe decrease in the fertility without a lack of libido or physical inabilities of male rats. Indeed, the copulatory behaviors were normal. It appeared that TFE affected predominantly the premeiotic germ cells of the testis (spermatogonia).

No NOEC was identified since effects were observed at the both tested concentrations of inhalation. However, the lowest observed adverse effect is determined at 100 ppm corresponding to 415 mg/m3of TFE. The results obtained in this study were consistent to those obtained in the repeated exposure study by inhalation of Tosoh (see 7.5.3). Indeed, severe damage effects in testis were observed in rats after a 28-day inhalation exposure period to 213 mg/m3TFE.

In the 2d study (Yoshitake, 2000), 2,2,2 trifluoroethanol (TFE) as a vapor was administrated to male and female rats by inhalation in a study performed in accordance to the OECD Guideline n° 412 and in complinace with GLP.

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).

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.

This study was used in this section as weight of evidence regarding TFE effects on testes.

At the highest concentration (213 mg/m3), TFE was found to be 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.

The Lowest Observed Adverse Effect Concentration (LOAEC) is considered to be 51.1 ppm (approximately 213 mg/m3) based on 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.

In a 3rd study (Lloyd, 1988), 2,2,2 trifluoroethanol (TFE; 99% purity) diluted in water was administered by gavage to wistar-derived male rats (10 males/dose) at single dose level of 0, 10 or 25 mg/kg bw. Three days after the TFE administration, testes were removed under anaesthesia in 5 animals per group and examined for histopathology. The remaining 5 animals were sacrificed to measure the testis and the body weights.

Trifluoroacetaldehyde (TFAld) and Trifluoroacetic acid (TFA) were administered at the same doses in parallel groups in order to compare the effects induced by these metabolites of TFE.

A significant decrease in testis relative weight was observed in the TFE groups at both doses. The effects on the spermiogenesis were also recorded including a partial loss of spermatogonia and early spermatocytes in stages IV and V. Similar effects were observed in the TFAld groups while no effect was noted in the TFA groups.

Under the test conditions, 2,2,2 trifluoroethanol administered by gavage at a single dose induced several damage effects on the male reproductive system from 10 mg/kg bw.

The 4th study (Silberstein, 1978) is a functional reproductive test derived from a repeated dose study inhalation. In this study male rats were exposed to 0, 10, 50 and 150 ppm concentations of TFE for 6h/day, 5d/week for 4 weeks. Some males of the treated male rats were allowed to recover from 2,2,2-trifluoroethanol expsoure for about 10 days, and then each was supplied with a different non exposed female each week for 5 weeks and then the conception rates were measured.

For group exposed to 10 ppm, the conception rate was the same as for controls; for the males exposed to 50 ppm, it was 72% that of controls; and for the group exposed to 150 ppm, no conception occured. For the group exposed to 50 ppm, conception rates were lower than for controls during the first 3 weeks, reaching a minimum during the second week, and preimplantation losses followed an analogous pattern. Males were sacrificed 2 weeks later (for a total recovery period of 57 days) and their testes examined. Males exposed to 50 ppm showed normal spermatogenesis. Males exposed to 150 ppm also showed some recovery and some normal spermatogenesis.

 

Even if all these studies did not satisfy the requirements for a reproduction toxicity study (limited range endpoints evaluated), at least 2 of them were scientifically acceptable and all showed consistency of marked effects on the rat testis after TFE exposure by oral route and by inhalation, after a single or a repeated dose level.

Other studies reported in this dossier (Wilkenfeld 1981, rat-acute exposure via intraeritoneal route; Marshall 1983, dog-repeated dose exposure via inhalation route; Kim 1988, rat-repeated dose exposure via intraperitoenal route; Marshall 1981a, dog-repeated dose exposure via intravenous route; Marshall 1981b,c, Chinese hamster-repeated dose exposure via intraperitoneal route), although of low reliability, all reported deleterous effects of TFE on testes.

Moreover, the testicular damages obviously conducted to a decrease in the male fertility via a spermiogenesis alteration.

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Remarks:

Evaluation of TFE effects on the testis of rats and impact on the fertility

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

Remarks:

The study is not conducted according to guidelines for toxicity to reproduction and GLP. However, the study is focused on the male reproductive system. Details on the protocol and the results were available.

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male rats were exposed to the 2,2,2-Trifluoroethanol (TFE) by inhalation route at various concentrations for 2 weeks. Serial mating was used to assess fertility: exposed male rats were paired to normal female (ie. non exposed to the test item).

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Blue Spruce Farms, Inc.
- Age at study initiation: male rats were 70 days old at the start of the exposure period
- Weight at study initiation: the vasocystotomized (male) rats weighed 300-500 g, all other males rats weighed 250-350 g. Virgin females weighed 250-300 g
- Fasting period before study: no data
- Housing: no data
- Use of restrainers for preventing ingestion (if dermal): not applicable
- 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:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: no data

DIET PREPARATION: not applicable
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE: no data
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Details on mating procedure:

- M/F ratio per cage: trifluoroethanol exposed males were housed individually in wire mesh cages. Each week, for 13 weeks, each male was presented with a different pair of virgin females. Mating was evaluated daily, by checking for the presence of vaginal plugs in the cage excrement pans.
- Length of cohabitation: one week
- Proof of pregnancy: Throughout the thirteen week mating period, a sufficient number of seminal plugs were found under each cage to indicate that every male had mated with both of his females. Females were sacrificed 17 days following the first day of pairing with the males. The uterine horns and ovaries were removed and cheked for implantation sites, resorption sites, live fetuses, and corpora lutea. From these data, male fertility as well as preimplantation and postimplantation losses were evaluated.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility: no data
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): no data
- Any other deviations from standard protocol: not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Airborne concentrations of TFE in the exposure chambers were monitored using a Miran IA Infrared Analyser (Wilks Scientific). The cell pathlength was one meter, the slit width 1mm, and a response time of 10 s was used. The analysis wavelength of 8.6 µm corresponded to a sharp IR absorption peak of TFE, while being free of interfering infrared absorption by water, CO2 and ammonia. The exhaust air from the small 28 L chambers was piped directly through the analyser for determination of the airborne TFE concentration.

Duration of treatment / exposure:

6 hours/day, 5 days/week, for 2 weeks.

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until [...] weeks after selected from the F1 litters: no data
- Selection of parents from F1 generation when pups were [...] days of age: no data
- Age at mating of the mated animals in the study: [...] weeks: no data

Dose / conc.:

0 ppm (nominal)

Remarks:

Corresponding to 0 mg/m3

Dose / conc.:

100 ppm (nominal)

Remarks:

Corresponding to 415 mg/m3

Dose / conc.:

200 ppm (nominal)

Remarks:

Corresponding to 830 mg/m3

Dose / conc.:

0 ppm (analytical)

Remarks:

Corresponding to 0 mg/m3

Dose / conc.:

99 ppm (analytical)

Remarks:

Corresponding to 410 mg/m3

Dose / conc.:

202 ppm (analytical)

Remarks:

Corresponding to 840 mg/m3

No. of animals per sex per dose:

4 male rats /concentation for the mating study.
50 male rats / concentration in other analyses.
It is not clear how many animals are present in each group.

Control animals:

yes

Details on study design:

no details

Positive control:

no data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations: no data

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OTHER: sperm production, testicular histology, specific activities of testicular enzymes, testis weight

Oestrous cyclicity (parental animals):

not applicable. Only male rats were treated with TFE

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations:
testis weight, sperm production, other: testicular histology, specific activities of testicular enzymes (sorbital dehydrogenase, malate dehydrogenase).

Litter observations:

Female were sacrificed 17 days following the first day of mating with the males. No birth occurred. The uterine horns and the ovaries were removed and checked for implantation sites, resorption sites, live fetuses and corpora lutea.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: animals were sacrificed using an overdose of sodium pentobarbital given intraperitoneally. Four of six animals from each exposure group were sacrificed at the end of the first and second week of exposure, and then at 1, 3, 5, 7, 9 and 13 weeks postexposure.
- Maternal animals: Females were sacrificed 17 days following the first day of pairing with the males.

GROSS NECROPSY: no data
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
Sections of lung, liver and kidney were fixed in 10% buffered formalin for histologic examination. Testes and epididymal sections were fixed with Bouin's solution. All sections were stained using a standard heamotoxylin-eosin stain.
Testis weight was determined.
Sperm production was evaluated using surgical vasocystotomy (bilateral diversion of the vasa diferentia to the urinary bladder). The surgical treatment was performed at least 2 weeks before the start of the dosing and allowed the quantification of sperm production in the collected rat urine. Sperm counts were done twice weekly on each animal using 24 hr urine samples.

Postmortem examinations (offspring):

Not applicable as no birth occured. The females were sacrificed prior post-partum.

Statistics:

no data

Reproductive indices:

no data

Offspring viability indices:

no data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

CLINICAL SIGNS (PARENTAL ANIMALS): During the two weeks exposure to airborne trifluoroethanol many animals in both exposure groups displayed increased levels of piloerection and sneezing, and a few animals exhibited dark mucous secretions at the nostrils.

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Description (incidence):

MORTALITY (PARENTAL ANIMALS): 2 deaths occured among the vasocystotomized rats during the second week of exposure to 200 ppm TFE.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

BODY WEIGHT (PARENTAL ANIMALS): Animal growth was temporarily depressed by exposure to TFE, but by the seventh week postexposure body weights were again comparable to those of the control group.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

PARENTAL ANIMALS: blood urea nitrogen was not significantly altered from control values.
Triglyceride analyses revealed a significant depression in the liver triglyceride levels of animals exposed to 200 ppm TFE for 2 weeks. The liver triglyceride levels had not returned to control values by the end of the first week postexposure.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

HISTOPATHOLOGY (PARENTAL ANIMALS):
Lung: a background of low grade pneumonitis was seen in both control and exposed rats. This was characterized by mild to moderate interstitial inflammatory cell infiltrates, perivascular mononuclear cell infiltrates and focal to multifocal areas of alveolar histocytosis. The incidence of these changes was not significantly different between animals exposed for 2 weeks and non-exposed animals. Two animals exposed to 200 ppm for 2 weeks and then sacrificed had evidence of pulmonary fibrosis, but it is questionable as to whether this fibrotic reaction could have developed within a two week period.
Liver: no effect
Kidneys: no effect
Brain: no effect
Testes: After one week exposure to 100 ppm TFE necrosis of spermatocytes and some spermatogonia was observed. Necrosis of all tubular cells but Sertoli cells, late stage (attached) spermatids, and spermatozoa was apparent in those animals exposed to 200 ppm TFE for one week. Occasional spermatidal giant cells were seen in the testes of both exposure groups. After one week postexposure early signs of testicular regeneration were observed in animals at both exposure concentrations. Generalized tubular atrophy and degenerating giant cells were still present, but most tubules now contained spermatogonia. By 3 weeks postexposure many of the seminiferous tubules of animals from both exposure groups had regenerated to the point where young spermatids were present. Regeneration of the seminiferous tubules of animals exposed to 100 ppm TFE was almost complete by 5 weeks postexposure. The tubules of animals exposed to 200 ppm TFE had recovered as far as intermediate stage spermatids by 5 weeks postexposure. By the seventh week postexposure approximately 80 to 95% of the seminiferous tubules of animals in the 100 ppm group had fully recovered. Only 50 to 90% of the tubules of animals exposed to 200 ppm TFE had fully recovered by this time. At the final sacrifice thirteen weeks postexposure, approximately 5 to 10% of the seminiferous tubules of animals in both TFE exposure groups were still atrophic. TFE did not damage Leydig cells or testicular capillaries.

Histopathological findings: neoplastic:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): oligospermia was noted 2 to 12 weeks postexposure in both groups of rats exposed to TFE. The 100 ppm group exhibited azoospermia during week 5 post-exposure, while the 200 pmm group had no detectable sperm in the urine between weeks 5 and 7 postexposure.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Males in the 100 ppm TFE exposure group were found to be infertile between weeks 4 and 5 postexposure. Animals in the 200 ppm TFE exposure group were infertile from 4 to 7 weeks postexposure. Decreased fertility, compared to the control group, was observed between 2 and 9 weeks postexposure in the 100 ppm, while the 200 ppm group displayed lowered fertility from the first week postexposure until 12 weeks postexposure.
During weeks 2 and 10 postexposure, decreased corpora lutea counts per pregnant females were correspondingly lower during these intervals, except for normal corpora lutea counts in the females paired with males in the 200 ppm group during the second week postexposure. Postimplantation losses in the 100 ppm group were greater than that in the control group during weeks 3 and 6 postexposure. Preimplantation losses (number of corpora lutea minus number of implantations per pregnancy) were significantly higher compared to the control group during week 3 postexposure in the 200 ppm group and during week 6 postexposure in the 100 ppm group. These increases in preimplantation loss may be attributes to the marked hypospermatogenesis produced by TFE exposure.

Key result

Dose descriptor:

LOAEC

Effect level:

415 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

415 other: mg/m3 air

System:

male reproductive system

Organ:

germ cells

seminiferous tubules

testes

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

not examined

Description (incidence and severity):

not applicable

Mortality / viability:

not examined

Description (incidence and severity):

not applicable as the fetuses were collected after 17 days of gestation.

Body weight and weight changes:

not examined

Description (incidence and severity):

not applicable

Sexual maturation:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings:

not examined

Description (incidence and severity):

not applicable

not applicable as no birth occured. Only Fetal observations (See detail below)

Key result

Remarks on result:

not measured/tested

Remarks:

fetuses were collected after 17 days of gestation.

Reproductive effects observed:

not specified

Observations of the fetuses: The average litter size (mean number of implantation sites) and the mean number of live fetuses per pregnancy of those females mated with males in the 100 ppm group were significantly less than that of females paried with control males during weeks 3 and 6 post-exposure. During weeks 2 and 10 post-exposure there was a similar decrease in litter size and the number of live fetuses for females paired with males in the 100 ppm group.

Testicular enzyme activities: The depletion of germ cells following TFE exposure was accompanied by concomitant changes in the specific activities of both sorbitol and malate dehydrogenase. The specific activity of sorbitol dehydrogenase was temporarily reduced by 72% in rats exposed to 100 ppm TFE, and by 86% in those animals exposed to 200 ppm. In contrast, the specific activity of malate dehydrogenase increased above control levels by 59% in the 100 ppm group and 108% in the 200 ppm group. The activity of both testicular enzymes had returned to control levels by the ninth week postexposure. Testicular protein content did not differ among the treatment groups over the course of the experiment.

Conclusions:

Under the test conditions, 2,2,2 trifluoroethanol produced necrosis of the testicular seminiferous tubules inducing a severe decrease of fertility in males rats exposed by whole-body inhalation at 100 ppm (420 mg/m3) for two weeks 6 hours/day, 5 days/week. TFE affected predominantly the premeiotic germ cells of the testis.

Executive summary:

In a fertility study 2,2,2 trifluoroethanol (TFE; 99% purity) was administered to male Sprague-Dawley rats by inhalation route for 6 hours per day, 5 days/week for 2 weeks at dose levels of 0, 100 and 200 ppm (nominal concentrations) coresponding to 415 mg/m3 and 830 mg/m3 respectively. The animals were exposed to TFE by whole-body inhalation.

After the exposure period, each week during 13 weeks, a serial mating was performed: each male was paired with a virgin female rat to examine the male fertility. Some male rats were sacrificed at the end of each exposure week and then at the end of the 1st, 3rd, 5th, 7th, 9th and 13th week post-exposure. Sperm production was evaluated by sperm counts in the 24 -hour urines twice weekly collected from animals subjected to surgical vasocystotomy. The testicular sorbitol (SDH) and malate deshydrogenase (MDH) activities were also measured.

Pregnant female rats were sacrificed prior post-partum corresponding to 17 days following mating. The uterine horns and the ovaries were removed and checked for implantation sites, resorption sites, live fetuses and corpora lutea.

Some organ weights were measured including kidney, lung, liver and testis.

Oligospermia was noted 2 to 12 weeks postexposure in both groups of rats exposed to TFE. The 100 ppm male group exhibited azoospermia during week 5 post-exposure, while the 200 pmm group had no detectable sperm in the urine between weeks 5 and 7 post-exposure.

Males in the 100 ppm TFE exposure group were found to be infertile between weeks 4 and 5 post-exposure. Animals in the 200 ppm TFE exposure group were infertile from 4 to 7 weeks post-exposure. Decreased fertility, compared to the control group, was observed between 2 and 9 weeks post-exposure in the 100 ppm, while the 200 ppm group displayed lowered fertility from the first week post-exposure until 12 weeks post-exposure.

During weeks 2 and 10 post-exposure, decreased corpora lutea counts per pregnant females were correspondingly lower during these intervals, except for normal corpora lutea counts in the females paired with males in the 200 ppm group during the second week post-exposure. Post-implantation losses in the 100 ppm group were greater than that in the control group during weeks 3 and 6 post-exposure. Pre-implantation losses (number of corpora lutea minus number of implantations per pregnancy) were significantly higher compared to the control group during the week 3 post-exposure in the 200 ppm group and during the week 6 post-exposure in the 100 ppm group. These increases in pre-implantation loss may be attributed to the marked hypospermatogenesis produced by TFE exposure.

Exposure to TFE at concentrations of 100 and 200 ppm did not produce pulmonary edema. Kidney and lung weights, lung wet-to-dry weight ratio were not significantly altered from control values following the two-week exposure to TFE. Liver weights were depressed during the first and second weeks of TFE exposure, but returned to the control levels by the end of the first week post-exposure. Three weeks following exposure to TFE, the relative testes weight was maximally depressed to 44% of control values in the 100 ppm group, and to 33% in the 200 ppm group. The relative testes weight had returned to control levels by the seventh week post-exposure for animals in the 100 ppm group, and by the thirteenth week post-exposure for animals in the 200 ppm group.

A background of low grade pneumonitis was seen in the lung of both control and exposed rats. This was characterized by mild to moderate interstitial inflammatory cell infiltrates, perivascular mononuclear cell infiltrates and focal to multifocal areas of alveolar histocytosis. The incidence of these changes was not significantly different between animals exposed for 2 weeks and non-exposed animals. Two animals exposed to 200 ppm for 2 weeks and then sacrificed had evidence of pulmonary fibrosis, but it is questionable as to whether this fibrotic reaction could have developed within a two week period.

No other effect was observed in the liver and kidney histopathological analysis.

Histophatology of the testes showed necrosis of spermatocytes and some spermatogonia after one week exposure to 100 ppm TFE. Necrosis of all tubular cells but not Sertoli cells, late stage (attached) spermatids, and spermatozoa was apparent in the animals exposed to 200 ppm TFE for one week. Occasional spermatidal giant cells were seen in the testes of both exposure groups. After one week post-exposure, early signs of testicular regeneration were observed in animals at both exposure concentrations. Generalized tubular atrophy and degenerating giant cells were still present, but most tubules now contained spermatogonia. By 3 weeks post-exposure many of the seminiferous tubules of animals from both exposure groups had regenerated to the point where young spermatids were present. Regeneration of the seminiferous tubules of animals exposed to 100 ppm TFE was almost complete by 5 weeks post-exposure. The tubules of animals exposed to 200 ppm TFE had recovered as far as intermediate stage spermatids by 5 weeks post-exposure. By the seventh week post-exposure approximately 80 to 95% of the seminiferous tubules of animals in the 100 ppm group had fully recovered. Only 50 to 90% of the tubules of animals exposed to 200 ppm TFE had fully recovered by this time. At the final sacrifice thirteen weeks post-exposure, approximately 5 to 10% of the seminiferous tubules of animals in both TFE exposure groups were still atrophic. TFE did not damage Leydig cells or testicular capillaries.

Under the test conditions, repeated exposure to 2,2,2 trifluoroethanol by inhalation produced damages on the male reproductive system as necrosis of seminiferous tubules inducing a severe decrease in the fertility without a lack of libido or physical inabilities of male rats after the exposure to TFE. Indeed, the copulatory behaviors were normal. It appeared that TFE affected predominantly the premeiotic germ cells of the testis.

No NOAEC was identified as effects were observed at the both tested concentrations of inhalation. However, the lowest observed adversed effect is determined at 100 ppm corresponding to 415 mg/m3.

Reference 2

Endpoint:

reproductive toxicity, other

Remarks:

assessment of testicular toxicity following a single exposure.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Few information on study design but the method is relevant based on scientific principles.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the in vivo study an unique dose of 2,2,2-Trifluoroethanol (TFE) was administered to male rats by oral route. 3 days after the treatment, the testis were removed for histopathological observations.
Trifluoroacetaldehyde (TFAld) or trifluoroacetic acid (TFA) were administered at the same dose levels as TFE in parallel groups in order to compare the effects induced by these metabolites of TFE. .

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: Alpk/AP (Wistar derived)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: 10 wks old
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: rats were housed in polycarbonate and metal cages
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): PCD Diet (Special diet services, Essex, UK), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 50 (+/-10%)
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

IN-LIFE DATES: From: To: no data

Route of administration:

oral: unspecified

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the pH of the tested solution is adjusted to 7 using 1.0 M NaOH

DIET PREPARATION: not applicable

VEHICLE: no data

Details on mating procedure:

not applicable: no mating experiment was performed in this study

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

not applicable

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single dose

Details on study schedule:

not applicable

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

in 5 mL water/kg bw, adjusted to pH7 using 1.0 M NaOH

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

in 5 mL water/kg bw, adjusted to pH7 using 1.0 M NaOH

No. of animals per sex per dose:

10 males/dose

Control animals:

yes, concurrent vehicle

Details on study design:

no details

Positive control:

no data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations: the body weight was determined at the moment of the sacrifice of the animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:

Oestrous cyclicity (parental animals):

not applicable

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, stages of spermatogenesis (based on the classification of Leblond and Clermont)

Litter observations:

not applicable

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: 5 animals were anesthetized for removing of testis 3 days after the administration of the test item. The remaining animals were sacrificed (no data on the time of the sacrifice)
- Maternal animals:not applicable

GROSS NECROPSY: no data
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
Testes were removed and processed into glycol methacrylate. 2 µm sections were cut and stained with hematoxylin and eosin and with the periodic acid-Schiff technique for the demonstration of the spermatid acrosome. Definition of the stages of spermatogenesis was based on the classification of Leblond and Clermont.

Postmortem examinations (offspring):

not applicable

Statistics:

data were evaluated by analysis of variance followed by student's t test, taking p<0.05 as the level of significance.

Reproductive indices:

not applicable

Offspring viability indices:

not applicable

Clinical signs:

no effects observed

Description (incidence and severity):

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): over a period of 3 days after dosing the animal appeared healthy.

Mortality:

no mortality observed

Description (incidence):

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): over a period of 3 days after dosing the animal appeared healthy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

BODY WEIGHT (PARENTAL ANIMALS): significant reduction in body weight gain at the both doses (10 and 25 mg/kg). Final body weights were reduced compared to those of control after all treatments, being statistically significant after treatment with 25 mg/kg (83% of control).

Food consumption and compound intake (if feeding study):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

HISTOPATHOLOGY (PARENTAL ANIMALS): Histological examination of testes from animals treated with 10 mg/kg of TFE revealed specific loss of late pachytene and dividing spermatocytes in spermatogenic stages X-XIV, extending to include loss of rounds spermatids in stages I-III. After 25 mg/kg TFE, the same profile of testicular damage was seen as after 10 mg/kg, but in addition there was also partial loss of spermatogonia and early spermatocytes in stages IV and V.

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Description (incidence and severity):

not applicable

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

not applicable

Reproductive performance:

not examined

Description (incidence and severity):

not applicable

Key result

Dose descriptor:

NOAEL

Sex:

male

Remarks on result:

not determinable

Remarks:

Single exposure, therefore NOAEL not applicable.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

germ cells

seminiferous tubules

testes

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

not examined

Description (incidence and severity):

not applicable

Mortality / viability:

not examined

Description (incidence and severity):

not applicable

Body weight and weight changes:

not examined

Description (incidence and severity):

not applicable

Sexual maturation:

not examined

Description (incidence and severity):

not applicable

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

not applicable

Gross pathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings:

not examined

Description (incidence and severity):

not applicable

not applicable

Key result

Remarks on result:

not measured/tested

Remarks:

Effects on the testis of parent animals was performed. No mating was pareformed.

Reproductive effects observed:

not specified

no other information

Conclusions:

Under the test conditions, 2,2,2 trifluoroethanol administered by gavage in a single dose induced several damage effects on the male reproductive system.

Executive summary:

2,2,2 trifluoroethanol (TFE; 99% purity) diluted in water was administered once by gavage to wistar-derived male rats (10 males/dose) at dose levels of 0, 10 and 25 mg/kg bw.  Three days after the TFE administration, testes were removed under anaesthesia in 5 animals per group and examined for histopathology. The remaining 5 animals were sacrificed and organ and body weights determined.

Trifluoroacetaldehyde (TFAld) and Trifluoroacetic acid (TFA) were administered at the same doses in parallel groups in order to compare the effects induced by these metabolites of TFE.

A significant decrease in testis relative weight was observed in the TFE groups at both doses. The effects on the spermatogenesis were also recorded including a partial loss of spermatogonia and early spermatocytes in stages IV and V. Similar effects were observed in the TFAld groups while no effect were noted in the TFA groups.

Under the test conditions, 2,2,2 trifluoroethanol administered by gavage in a single dose induced several damage effects on the male reproductive system.

 

This study is scientifically acceptable even if there is a lack of information on the study design and on the results.

Reference 3

Endpoint:

reproductive toxicity, other

Remarks:

Assessment of TFE effect on rat testis

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1999-11-04 to 2000-06-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study for sub-acute reapeated dose toxicity study. However this study is used here to report effect of TFE on rat testes. Report is in japanese but the summary was translated in english by the sponsor.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 412

Principles of method if other than guideline:

The study is an sub-acute repeated inhalation study performed in accordance to OCDE 412 and in compliance with BPL. This study is used is this section as supporting evidence of the TFE on male reproductive organs.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system 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 handdown
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 (if applicable):

whole body

Remarks on MMAD:

MMAD / GSD: not applicable

Vehicle:

air

Details on 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

Details on mating procedure:

No mating was performed in this study

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

Details on study schedule:

not applicable

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.:

14.6 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 determin
ed 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 mediumdose
(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 techniq
ue 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 exposur
e 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 5
0 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).

Positive control:

no

Parental animals: Observations and examinations:

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 nec
ropsy 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 m
etabolic 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

Oestrous cyclicity (parental animals):

not applicable

Sperm parameters (parental animals):

not appicable

Litter observations:

not applicable

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes (see table 7.5.3/7)
HISTOPATHOLOGY: Yes (see table 7.5.3/7)

Postmortem examinations (offspring):

not applicable

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).

Reproductive indices:

not applicable

Offspring viability indices:

not applicable

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities were observed.

Dermal irritation (if dermal study):

no effects observed

Mortality:

no mortality 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.

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 decre ased after 4 days for both sexes. Even at the recovery duration, suppression of food consumption wa s 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.

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).

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 wereslight and within the ranges of the background data of this lab, the changes were incidental. At theend 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.

Urinalysis findings:

no effects observed

Description (incidence and severity):

no effect.

Behaviour (functional findings):

not examined

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.
No histopathological abnormalities were observed for treated female rats.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

62 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

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 other: mg/m3 air (nominal)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Remarks on result:

not measured/tested

Remarks on result:

not measured/tested

Remarks on result:

not measured/tested

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

213 mg/m³ air (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

62 mg/m³

Study duration:

subacute

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

In an in vitro study performed in compliance with GLP (William, 1997), the intrinsic potential of 2,2,2 trifluoroethanol (TFE) to alter the function and/or integrity of the principal cell types of the testis, was determined. The TFE metabolites such as trifluoroacetaldehyde (TFAld) and trifluoroacetic acid (TFA) were also included in the study. Isolated Leydig cell cultures (LCC), Sertoli cell only cultures (SCOC) and Sertoli-germ cell co-cultures (SGCC) were obtained from the testis of Sprague Dawley rat.

Several positive controls (ketoconazole, 1,3 -dinitrobenzene, methoxyacetic acid) were used to validate the study.

Various cell parameters including LCC and SGCC culture protein content, LDH activity, LCC medium culture secreted testosterone, LDH-X (specific isoenzyme of LDH of spermatocyte in pachytene stage) in SGGC culture medium, SGCC medium culture lactate and pyruvate concentrations were measured. Moreover, the morphological cell appearance was analysed in order to evaluate the potential adverse effect of TFE on reproductive system cells.

Under the test conditions, TFE showed only a small effect on the Leydig cell function and essentially no effect on Sertoli cells. Indeed, at high concentrations, TFE affected Leydig cell function by inhibiting testosterone output but only in the presence of hCG hormone . However, TFAld induced marked effects on the Leydig cell as decreased testotesterone production, on the SCOC and SCCC as altered morphology, decreased lactate and pyruvate production in SCOC, increased cell loss and LDHX leakage in SGCC. This study doesn't satisfy the requirements for a reproduction toxicity study but this study is scientifically acceptable as it was well conducted and focused on the target cells of the testis in order to explain the effects observed in vivo on the rat testis after TFE exposure. This study provides the evidence that the adverse effect on reproduction observed in vivo were not a secondary non-specific consequence of other toxic effects.

Under general considerations, the negative effects observed with TFE on the reproductive cells indicated a possible limitation of the in vitro techniques as used in this study for chemicals which require metabolism to produce an active species. In fact, TFAld as direct TFE metabolite, showed significant positive results in this study. The whole results postulated the hypothesis that TFE must be metabolised to TFAld to induce damages on the testicular cells.

 

Justification for classification or non-classification

According to the existing toxicokinetic data, TFE is directly metabolised in vivo in TFAld via a cytochrome P450 pathway (see § 7.1). In addition, in vivo studies showed severe adverse effects on male reproductive system with a decrease of fertility after an exposure to TFE. These effects were observed both after oral exposure or after inhalation, and after a single or a repeated exposure (14 or 28 days). Furthermore, in vitro studies showed direct adverse effects of TFAld (the TFE metabolite) on male germ cells but almost no effect was observed after an in vitro exposure to TFE. The in vitro study provided the evidence that the adverse effects on reproduction observed in vivo were not a secondary non-specific consequence of other toxic effects. TFE induced an alteration of spermiogenesis by the intermediate of TFAld.

 

All of these informations are relevant and can be extrapolated to human. Indeed, the rat germ cell types damaged in these studies are comparable to the human germ cells. Moreover, the cytochrome P450 metabolism route, showed to be involved in the oxidation of TFE to TFAld, is also a well characterized metabolism pathway in human. TFE is therefore considered as a presumed human reproductive toxicant.

 

Harmonized classification:

No harmonized classification is available according to the Regulation No 1272/2008.

Self classification:

Based on the obvious testicular damages interfering with fertility in rat after 2,2,2 Trifluoroethanol exposure by inhalation and by the oral route, TFE is self-classsified for the fertility as Presumed human reproductive toxicant, May damage fertility (Repr. 1B; H360F) according to the Regulation No 1272/2008 (CLP).

No self-classification is proposed for the development.