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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
End of the study period: 25 August 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No long term treatment (exposure for 24h) was conducted in the absence of metabolic activation as recommended by the guideline OECD 473. Only short term treatments (3 h) with and without metabolic activation were conducted. Only 2 dose levels analysed for the genotoxicity had an acceptable cytotoxicity. The third dose analysed had a reduction of mitotic index by 80%.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
4-tert-butylpyrocatechol
EC Number:
202-653-9
EC Name:
4-tert-butylpyrocatechol
Cas Number:
98-29-3
Molecular formula:
C10H14O2
IUPAC Name:
4-tert-butylbenzene-1,2-diol

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: Chinese hamster ovary (CHO) cells
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rats liver induced with Aroclor 1254 (= S9)
Test concentrations with justification for top dose:
0, 2.5, 5, 7.5, 10 and 20 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (without S9); benzo(a)pyrene (with S9)
Details on test system and experimental conditions:
The assay using duplicate cultures was performed both with and without S9. A single sampling time was used: 24 hours, i.e. 1.5 cell cycle times from the beginning of treatment.

For each culture, approximately 5 x 10E5 cells were seeded in 3 mL DMEM (Eagle medium modified by Dulbecco) medium containing 10% fetal calf serum, 1% L. glutamine, penicillin, streptomycin and fungizon in flasks of 25 cm2. The flasks were then placed at 37°C in a humidified atmosphere of 5% CO2 in 95% air. After 24 hours, the cultures were exposed to the test subtance:
- for 3 hours at 37°C in culture medium without fetal calf serum for the assay without S9,
- for 3 hours at 37°C in the culture medium without fetal calf serum containing 15% S9, for the assay with S9.
After treatment, the cells were observed under a microscope to assess any morphological alterations, the treatment medium removed, the cells rinsed and fresh medium added. The cultures were then incubated for 21 hours at 37°C.
Two hours before cell collection, the cells were treated with a colcemid solution in order to stop mitosis at the metaphase-stage.
The cells were then rinsed, trypsinated, harvested and centrifuged. After a hypotonic treatment, they were fixed in a methanol/acetic acid mixture, spread on slides and stained with Giemsa. Two slides/culture were prepared.

Evaluation criteria:
- a test substance was considered as clastogenic if, at one or more concentrations, it induced a statistically significant increase in the incidence of cells with aberrations. This increase should exceed the normal range of historical negative control data.
- a test substance was considered as non-clastogenic if there was no significant increase in cells with aberrations frequency at any doses, above concurrent control frquencies.

Evaluation of the results:
The mitotic index was evaluated for all the cultures. The chromosomal abnormalities were scored on the slides corresponding to 3 treated cultures which were selected according to the mitotic index and to all the controls. 200 metaphases/concentration were analysed whenever possible. The structural aberrations (chromatid and chromosomal gaps, deletions and exchanges and others (multiple aberrations and pulverizations)) were recorded for each metaphase.
Statistics:
The incidence of cells wtih aberrations (excluding gaps) in treated cultures was compared to that of the solvent cultures using the "Chi-square" test.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 10 µg/mL and 20 µg/mL, a reduction of the mitotic index of 80% and 100% was observed, respectively. No significant reduction was noted at 7.5 µg/mL and below.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 10 and 20 µg/mL, a reduction of the mitotic index of 40% and 80% was observed, respectively. At 7.5 µg/mL and below, no significant reduction was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Cytotoxicity:
The mitotic index was, as compared to the controls:
- without S9: reduced by 100% at 20 µg/mL, by 80% at 10 µg/mL, by 13% at 7.5 µg/mL, by 40% at 5 µg/mL and by 27% at 2.5 µg/mL;
- with S9: reduced by 80% at 20 µg/mL, by 40% at 10 µg/mL and similar to that of controls at the lower concentrations (by 0% at 7.5 µg/mL, by 20% at 5 µg/mL and by 27% at 2.5 µg/mL)

Genotoxicity:
The chromosomal abnormalities were scored on the slides corresponding to the 3 following concentrations:
- without S9: 2.5, 5 and 10 µg/mL
- with S9: 5, 10 and 20 µg/mL.

It should be noted that the dose levels of 10 µg/mL in the absence of metabolic activation, and 20 µg/mL in the presence of metabolic activation, showed a very severe cytotoxicity (reduction of the mitotic index of 80%). Indeed, these 2 tested dose levels should not have been selected for a reliable genotoxicity analysis. According to the recent guideline OECD 473, the highest tested concentration should achieve 55 ± 5% cytotoxicity using the recommended cytotoxicity parameters (i.e. reduction in Mitotic Index), and care should be taken in interpreting positive results only to be found in the higher end of this 55 ± 5% cytotoxicity range.
In addition, no analysis was conducted on the dose level of 7.5 µg/mL (probably because the mitotic index is the less reducted at this dose).
The absence of the analysis at the dose of 7.5 µg/mL did not affect the reliability of the study. The analysed dose range was narrow with a concentration interval of 2 fold between the analysed doses (2.5, 5 and 10 µg/mL without S9 mix and 5, 10 and 20 µg/mL with S9 mix). In addition, no cytotoxicity was observed at 7.5 µg/mL and below, and no genotoxicity was noted over the analysed range.

The analysis of the chromosomal aberrations gave the following results:
- With S9, the incidence of cells with aberrations in the cultures treated with TBC was similar to that of the negative controls, whatever the analysed dose.
- Without S9, at 2.5 and 5 µg/mL, the incidence of cells with aberrations was similar to that of the controls.
At 10 µg/mL, a statistically significant increase (p<0.05) was observed when compared to the solvent control and out of the the historical data. but this increase was not statistically significant when compared to the untreated cultures. In addition, as discussed above, this positive effect was observed at the dose level of 10 µg/mL showing a mitotic index reduced by 80%, i.e. very above the maximum cytotoxicity recommended by the guideline. This severe cytotoxicity of 80% does not allow a reliable genotoxicity analysis, and the genotoxicity analysis should not be considered.
Over the analysed range from 2.5 to 5 µg/mL (showing an acceptable cytotoxicity), no increase in the cells with aberrations was observed.

The incidence of cells with aberrations in the negative and solvent controls was within the range of the historical data.

The incidence of cells with aberrations in the positive controls was significantly higher (p<0.001) than that in the control cultures, indicating the sensitivity of the test system.

In conclusion, according to the recent guideline OECD 473 (adopted on 29 july 2016), no increase in the cells with aberrations was observed in the tested dose levels with an acceptable cytotoxicity, either in absence or in presence of metabolic activation.
Remarks on result:
other: A weak positive effect was observed at 10 µg/mL. The cytotoxicity was very severe at this dose (reduction of the mitotic index by 80%). According to the recent guideline, this dose level should not have been selected for a reliable genotoxicity analysis.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions, 4-tert-butylpyrocatechol did not showed any increase in the chromosomal aberrations test performed in Chinese Hamster Ovary (CHO) cells treated up to the limit of an acceptable cytotoxicity and under an exposure time of 3 hours, either in the absence or in the presence of metabolic activation.
Executive summary:

In a mammalian cell cytogenetics assay, CHO (Chinese Hamster Ovary) cell cultures were exposed for an exposure time of 3 hours to 4 -tert-butylpyrocatechol (TBC) (purity: 99%) in DMSO at concentrations of 0, 2.5, 5, 7.5, 10 and 20 µg/mL with and without metabolic activation (rat liver S9 fraction).

In the absence of metabolic activation, the test substance showed severe cytotoxic effects as evaluated by mitotic index which was reduced by 80% compared to the control at 10 µg/mL and by 100% at 20 µg/mL. The dose selected for the genotoxicity effect were 10, 5 and 2.5 µg/mL. At 10 µg/mL, a statistically significant increase was observed when compared to the solvent control and it was out of the the historical data.

But this increase was not statistically significant when compared to the untreated cultures. In addition, this positive effect was only observed at the dose level of 10 µg/mL showing a mitotic index reduced by 80%, i.e. very above the maximum cytotoxicity recommended by the guideline. This severe cytotoxicity of 80% does not allow to retain this tested dose for the genotoxicity analysis.

In absence of S9, the genotoxicity analysis over the range showing an acceptable cytotoxicity (2.5 to 5 µg/mL) did not show any increase in the cells with aberrations.

In the presence of metabolic activation, the test substance showed cytotoxic effects at 20 µg/mL as seen by a decrease in mitotic index of 80% compared to the control. The analysis of the clastogenic effect was done at 5, 10 and 20 µg/mL. No increase in the cells with aberrations was noted.

Positive controls induced the appropriate response.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data (In vitro chromosomal analysis in CHO cells).

Under the experimental conditions, 4-tert-butylpyrocatechol did not showed any increase in the chromosomal aberrations test performed in Chinese Hamster Ovary (CHO) cells treated up to the limit of an acceptable cytotoxicity according to the recent guideline OECD 473, either in the absence or in the presence of metabolic activation after 3 hours of incubation.