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Toxicity to reproduction: other studies

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Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1995-03-27 to 1995-10-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study report is well documented and the study meets basic scientific principles. The study was performed in compliance with GLP.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
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 Trifluoroacetic acid (TFA). In parallel, cells were also treated with 2,2,2Trifluoroethanol (TFE) and a metabolite of TFE: Trifluoroacetaldehyde (TFAld).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

Constituent 1
Chemical structure
Reference substance name:
Trifluoroacetic acid
EC Number:
200-929-3
EC Name:
Trifluoroacetic acid
Cas Number:
76-05-1
Molecular formula:
C2HF3O2
IUPAC Name:
trifluoroacetic acid
Details on test material:
- Name of test material (as cited in study report): Trifluoroacetic acid (TFA)
- 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 TFA was Y08565/001. 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
Details on test animals or test system 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
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) analysis of this sample in-house confirmed its identity as TFA and its stated purity.
Duration of treatment / exposure:
approximately 5 hours (Leydig cells) or 24 hours (Leydig cells and Sertoli cells).
Frequency of treatment:
a single dose
Duration of test:
no data
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1 µM to 25 mM for the test on Leydig cells
Basis:
nominal conc.
Concentration of TFA
Remarks:
Doses / Concentrations:
0.1 to 15 mM for the test on Sertoli cells (SGCC and SCOC)
Basis:
nominal conc.
Concentration of TFA
Remarks:
Doses / Concentrations:
0.05 µM-50 mM for the test on Leydig cells
Basis:
nominal conc.
Concentration of TFE and Concentration of TFAld
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:
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

Dose descriptor:
NOEL
Remarks:
effect on testosterone production in Leydig cells under hCG stimulation
Effect level:
1 other: mM
Based on:
not specified

Observed effects

Preliminary study and Dose selection rationale of Trifluoroacetic acid:
Concentrations of 50 or 100 mM of TFA 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. TFA at concentrations up to and including 20 mM had no significant effects on extracellular LDH activity in Leydig cell cultures indicating that there was no cytotoxicity. Consequently, 20 mM was the maximum used concentration of TFA for the study on Leydig cells.
A concentration of 25 mM and above of TFA for 24 hrs was cytotoxic to Sertoli cells (SCOC and SGCC) as indicating by a significant increase of LDH activity in the culture medium of cells treated in comparison with the culture medium control. Consequently a maximum concentration of 15 mM of TFA was used in subsequent experiments on SCOC and SGCC.

Effect of TFA on Leydig cells:
The addition of TFA to isolated Leydig cells resulted in a small but statistically significant increase in basal testosterone production at 5 hours compared with culture medium controls. Nevertheless, this effect was small in magnitude and occurred at high concentration (10 mM and greater) of TFA and is therefore considered to be of little biological relevance. Exposure of Leydig cell cultures to TFA for 24 hours under basal conditions resulted in statistically significant changes in testosterone production compared with culture medium control values in 2 out of 3 experiments. However, these effects were small in magnitude and the direction of change was inconsistent (increase or decrease).Therefore these effects were considered to be of little biological relevance.
Testosterone production by hormonally-stimulated Leydig cells exposed to TFA for 5 hours was statistically significantly decreased compared with hCG control values in 2 out of 3 experiments. This effect was dose related. The no-effect level for this effect was less than 1 mM TFA. After a 24 hour incubation period with TFA, the effects on hormonally-stimulated testosterone production were small and inconsistent within 3 experiments. Therefore these statistically significant changes were considered to be of little biological relevance.

Effect of TFA on Sertoli cells:
Exposure of SGCC and SCOC to TFA for 24 hours had no effect on the morphology of the cultures.
The presence of TFA, at concentrations up to 15 mM, in the culture medium of SCOC incubated in the absence of hormone for 24 hours, had little significant inconsistent effect (increase or decrease) on lactate production compared with culture medium controls. Under the stimulation of Sertoli cells (SCOC) by dbcAMP, incubation for 24 hours with TFA enhanced the lactate production in a significant and dose-related manner. TFA had no effect under basal conditions on lactate production in Sertoli germ cell co-culture (SGCC). However under the stimulation of Sertoli cells (SGGC) by dbcAMP, incubation for 24 hours with TFA the lactate production was decreased in a statistically significant manner but this result was considered to be of little biological relevance.
The presence of TFA in SCOC under basal conditions or after dbcAMP stimulation had no significant effect on the pyruvate production in the culture medium. On the contrary, under basal conditions, the addition of TFA to SGCC for 24 hours resulted in a statistically significant increase in pyruvate production compared with culture medium controls. However, this increase was small in magnitude and occurred only at the highest used concentration of TFA. Therefore, it was considered to be of little biological relevance. Under the stimulation by dbcAMP, TFA had no effect on pyruvate production.
The addition of TFA to SGCC under basal conditions for 24 hours resulted in a statistically significant decrease in the number of cells detached into the culture medium compared with culture medium controls. However the relevance of this observation is unclear since an adverse effect of a compound on SGCC would be expected to result in an increase in the loss of cells and not a decrease. The same observation was made under hormone stimulation.
The exposure of SGCC to TFA for 24 hours up to and including 15 mM had no significant effect on LDHX activity in the culture medium compared with culture medium control values.

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 the test conditions, an incubation of Sertoli cells (SCOC) for 24 hours and under the stimulation by dbcAMP with trifluoroacetic acid enhanced the lactate production in a significant and dose-related manner. However the relevance of this single effect is unknown. The test item affected Leydig cell function by inhibiting testosterone output but only in the presence of hormone at 5 hours. The no observed-effect level (NOEL) for this effect was less than 1 mM TFA. To resume, TFA showed only a small effect on Leydig cell function and essentially no effect on Sertoli cells.
Executive summary:

In an in vitro fertility study performed in compliance with GLP, the intrinsic potential of Trifluoroacetic acid (TFA) to alter the function and/or integrity of the principal cell types of the testis was determined. TFA is the end metabolite of Trifluoroethanol (TFE). The TFE and the intermediate product between TFE and TFA (trifluoroacetaldehyde, TFAld) 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.

TFA (99.9% purity) was added for 5 or 24 hours to cells cultured in 24 well tissue culture plates. The TFA concentration range was 1 µM to 25 mM to expose Leydig cells and 0.1 to 15 mM to expose Sertoli cells. Moreover, the hormone, human chorionic gonadotrophin (hCG) was added to the LCC to stimulate testosterone production.

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

After this in vitro TFA incubation 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, an incubation of Sertoli cells (SCOC) for 24 hours and under the stimulation by dbcAMP with trifluoroacetic acid enhanced the lactate production in a significant and dose-related manner. However the relevance of this single effect is unknown. The TFA affected also Leydig cell function by inhibiting testosterone output but only in the presence of hormone at 5 hours. The no observed-effect level (NOEL) for this effect was less than 1 mM TFA. The results were the same for TFE. 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.

To resume, TFA showed only a small effect on Leydig cell function and essentially no effect on Sertoli cells.

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.