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Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-03-27 to 1995-10-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The study report was well documented and the study met basic scientific principles. The study was performed in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Three cell culture systems such as isolated leydig cell cultures, Sertoli cell only cultures and Sertoli-germ cell co-cultures obtained from male Sprague Dawley Rats, were exposed to the 2,2,2Trifluoroethanol (TFE). In parallel, cells were also treated with metabolites of TFE: Trifluoroacetaldehyde (TFAld) and Trifluoroacetic acid (TFA).Then, different cell parameters were analysed like the protein content, the lactate and pyruvate production, the hormonally stimulated testosterone production, the morphology depending on the type of cell cultures.
GLP compliance:
yes
Type of method:
in vitro

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,2,2 trifluoroethanol (TFE)
- Physical state: colorless liquid
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: assumed to be stable during the test (sponsor responsibility)
- Storage condition of test material: no data
- Other: The CTL reference number assigned to this sample of TFE was Y04314/008. The test item was purchased from Aldrich Chemical Company Limited (Gillingham, Dorset, UK).

Test animals

Species:
other: Cells in culture from male Sprague-Dawley or Alpk:APfSD (Wistar-derived) Rats were exposed to the test item
Strain:
other: not applicable
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK (Male Sprague Dawley (Crl:CD(SD)Br)) or Barriered Animal Breeding Unit (Zeneca, Alderley Park, Cheshire, UK ( Alpk:APfSD (Wistar-Derived)))
- Age at study initiation: 26-32 days for the isolation of Sertoli cells, 10 weeks for the isolation of Leydig cells
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: the rats were housed at up to 5 per cage
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: between 3 and 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C +/-2°C
- Humidity (%): 55% (+/- 15%)
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12hrs/12hrs

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
other: the cells were exposed to the test item in the culture medium
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
other: The test item was prepared in fresh culture medium used for the maintenance of the isolated cells
Details on exposure:
not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nuclear magnetic resonance (19F and 1H) analysis of this sample in-house confirmed its identity as TFE and its stated purity.
Duration of treatment / exposure:
approximately 5 hours (Leydig cells) or 24 hours (Leydic cells and Sertoli cells).
Frequency of treatment:
a single dose
Duration of test:
no data
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.05 µM-50 mM for the test on Leydig cells
Basis:
nominal conc.
Concentration of TFE
Remarks:
Doses / Concentrations:
0.1-20 mM for the test on Sertoli cells (SGCC and SCOC)
Basis:
nominal conc.
Concentration of TFE
Remarks:
Doses / Concentrations:
0.05 µM-50 mM for the test on Leydig cells
Basis:
nominal conc.
Concentration of TFAld
Remarks:
Doses / Concentrations:
5 µM-0.75 mM for the test on Sertoli cells (SGCC and SCOC)
Basis:
nominal conc.
Concentration of TFAld
No. of animals per sex per dose:
Not applicable
Control animals:
other: not applicable
Details on study design:
no details
Statistics:
The results were analysed by two-tailed Student's t-test and a p value of <0.05 was chosen to represent a statistically significant difference between values for treated samples compared with controls.

Results and discussion

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
5 other: mM
Based on:
not specified
Basis for effect level:
histopathology: non-neoplastic

Observed effects

Preliminary study and Dose selection rationale of 2,2,2 Trifluoroethanol:
A concentration of 100 mM of TFE for 24 hrs was cytotoxic to Leydig cells as indicating by a significant increase of LDH activity in the culture medium of cells treated in comparison with the culture medium control. TFE at concentrations up to and including 50 mM had little significant effects on extracellular LDH activity in Leydig cell cultures indicating that there was no cytotoxicity. Consequently, 50 mM was the maximum used concentration of TFE.
A concentration of 100 mM of TFE for 24 hrs was cytotoxic to Sertoli cells (SCOC) as indicating by a significant increase of LDH activity in the culture medium of cells treated in comparison with the culture medium control. A concentration up to and including 50 mM of TFE for 24 hrs was not cytotoxic to Sertoli cells (SGCC) as indicating by a level of LDH activity in the culture medium of cells treated equivalent to the one of the culture medium control.
Consequently a maximum concentration of 20 mM of TFE was used in subsequent experiments on SCOC and SGCC.

Effect of TFE on Leydig cells:
The addition of TFE to isolated Leydig cells resulted in a small but statistically significant increase in basal testosterone production at 5 hours and 24 hours compared with culture medium controls. Nevertheless, this small increase is not considered to be biologically relevant since isolated Leydig cells have a much larger capacity to produce testosterone.
Testosterone production by hormonally-stimulated Leydig cells exposed to TFE for 5 hours was statistically significantly decreased compared with culture medium control values in all 3 experiments. This effect was dose related. The no-effect level for this effect was less than 5 mM TFE. After a 24 hour incubation period with TFE, the effects on hormonally-stimulated testosterone production were small and inconsistent within 2 experiments. Therefore these statistically significant changes are considered to be of little biological relevance.

Effect of TFE on Sertoli cells:
Exposure of SGCC to TFE had no effect on the morphology of the cultures. It was not possible to critically evaluate the effects on SCOC as the morphological appearance of the control samples was of poor quality.
The presence of TFE, at concentrations up to and including 20 mM, in the culture medium of SCOC incubated in the absence of hormone for 24 hours, had no effect on lactate production compared with culture medium controls. However at the same concentration in the culture medium of SGCC, a statistically significant increase in lactate production was observed. Nevertheless, these observations were considered to be of little biological relevance. In the presence of dbcAMP, TFE had no significant effect on lactate production by SCOC and SGCC compared with dbcAMP controls.
The presence of TFE in SCOC and SGCC culture in the absence or presence of dbcAMP had no significant effect on the pyruvate production in the culture medium.
The addition of TFE to SGCC under basal conditions resulted in small but statistically significant effects on cell loss from the cultures compared with culture medium controls. These effects were inconsistent between the 3 experiments. Therefore these changes are considered to be of little biological relevance. Under hormonally-stimulated conditions, TFE addition to SGCC resulted in a significant increase in cell loss from the monolayer in 2 out of 3 experiments. In the third experiment, TFE had no effects on this parameter. Overall, is considered that TFE had little effect on cell loss from SGCC in the presence of hormone.
The exposure of SGCC to TFE for 24 hours up to and including 20 mM had no significant effect on LDHX activity in the culture medium compared with culture medium control values.


Effect on a TFAld exposure:
The addition of TFAld to isolated Leydig cells resulted in a statistically significant decrease in basal testosterone production compared with culture medium controls at 5 hours and 24 hours which was dose-related.
TFAld is highly cytotoxic to Sertoli cells. Therefore, a maximum concentration of 0.75 mM TFAld was used. TFAld affected Sertoli cells in altering their morphology, in inducing a significant decrease in basal lactate and pyruvate production.

Any other information on results incl. tables

The cell cultures used in this study were demonstrated to be responsive to known positive control compounds (KTZ, 1,3 DNB, MAA and TFAA) and the effects produced were used as benchmarks to aid in the assessment of the test compounds.

Applicant's summary and conclusion

Conclusions:
Under general considerations, the negative results 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.
Executive summary:

In an in vitro fertility study performed in compliance with GLP, 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 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.

TFE (99.9% purity) was added for 5 or 24 hours to cells cultured in 24 well tissue culture plates. The TFE concentration range was 0.05 µM to 50 mM to expose Leydig cells and 0.1 to 20 mM to expose Sertoli cells. Moreover, the hormone, human chorionic gonadotrophin (hCG) was added to the LCC to stimulate testosterone poduction.

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

After the in vitro TFE exposure period, the cells were harvested and the medium was also collected to further evaluations and observations. 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.

Under general considerations, the negative results 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.

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.