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Diss Factsheets

Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Test well described, but the test substance purity is not reported.

Data source

Reference
Reference Type:
publication
Title:
No information
Author:
Wilmer J. L. et al.
Year:
1997
Bibliographic source:
In Vitro Toxicology, 10(4), 429-436.

Materials and methods

Principles of method if other than guideline:
In vitro effects on proinflammatory cytokines and growth factors in human epidermal keratinocytes.
GLP compliance:
not specified
Type of method:
in vitro

Test material

Constituent 1
Chemical structure
Reference substance name:
Pyrocatechol
EC Number:
204-427-5
EC Name:
Pyrocatechol
Cas Number:
120-80-9
Molecular formula:
C6H6O2
IUPAC Name:
pyrocatechol
Details on test material:
Purchased from Sigma, purity unknown.

Test animals

Species:
human
Strain:
other: epidermal keratinocytes
Sex:
female
Details on test animals or test system and environmental conditions:
- Test system: Cryopreserved normal human keratinocytes (NHK) from breast skin of adult females were used.

Administration / exposure

Route of administration:
other: in vitro
Duration of treatment / exposure:
Exposure period: 4 hour(s)
Doses / concentrations
Remarks:
Doses / Concentrations:
11, 22, 44 µg/mL (100, 200 and 400 µM)
Basis:
nominal conc.
No. of animals per sex per dose:
Not applicable
Control animals:
yes, concurrent vehicle
Details on study design:
- Cell culture and chemical treatment:
Keratinocytes were cultured in Keratinocyte Growth Medium. The cells were trypsinized and subcultured in wells at a seeding density of 2.4 x 10E4 cells/cm2. After 24 h, at which time the cells were approximately 70% confluent, the complete medium was changed to growth medium without hydrocortisone, bovine pituitary extract and rh epidermal growth factor. 
Catechol was dissolved in phosphate buffered saline and sterile-filtered. Exposure of the cultures to catechol (11, 22, 44 mg/L), delivered in 10 mL aliquots, was for 4 hours in a humidified 5% CO2/95% air atmosphere. After exposure, the cultures were rinsed, and fresh growth factor-deficient medium in 4 mL aliquots was pipetted into each flask for a further 20-h culture period. The culture supernatants were removed, sterile-filtered into cryotubes and frozen at -70°C. Keratinocyte viability was assessed concurrently using a modified in situ trypan blue dye exclusion procedure. Four to six cultures were tested per experiment.

- Cytokine measurements:
* For IL-8, wells were coated with capture antibodies (mouse antihuman IL-8 monoclonal antibody) at a concentration of 2 µg/mL of coating buffer for 24 h at 4°C. After having blocked the free spaces, samples or standards were added in 0.1 mL aliquots and incubated for 2 h at 37°C. 0.1 mL of goat antihuman IL-8 polyclonal IgG antibody, diluted to 0.5 µg/mL, was added to each well for an additional 2 hours. The plates were incubated with peroxidase-conjugated rabbit antigoat IgG antibody, diluted 1:7500, for 1 hour. The cells were then incubated with peroxidase substrate for 20 minutes and the reaction was terminated by the addition of 50 µL of 2N sulfuric acid. Between each protocol phases, cells were washed with PBS.
* TGF-alpha concentrations were determined using a commercial enzyme-linked immunosorbent assay (ELISA). Absorbency was read at 450 nm using an UV computer reader.

- Statistical analysis:
The concentration-response was analysed initially by Bartlett's test for homogeneity of variances, and in some cases the data were transformed by the square-root to equalise variances prior to one-way analysis of variance (ANOVA). If the F-statistic was significant, post hoc comparisons were made using Tukey's honestly significant differences procedure to determine whether the individual treatment groups were significantly different (p < 0.05).

Results and discussion

Details on results:
* Catechol was not cytotoxic in NHK cells at the concentrations tested as assessed by trypan blue.
* Catechol did not induce TGF-alpha and had slight suppressive effects on both constitutive TGF-alpha and IL-8 secretions. 

- The secretion of TGF-alpha was 42 ± 1 pg/ml at the concentration of 200 µM catechol (not statistically different from the vehicle value: 48 ± 5 pg/ml) and 38 ± 5 pg/ml at the concentration of 400 µM (p < 0.05). Catechol was not tested at the concentration of 100 µM for TGF-alpha secretion. 
- The secretion of IL-8 was 1 ± 1 at the concentration of 100 and 200 µM catechol (p < 0.05, when compared to the vehicle value: 4 ± 1 pg/ml) and 0 ± 0 pg/ml at the concentration of 400 µM (p < 0.05).

Applicant's summary and conclusion

Conclusions:
Negative