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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to OECD Guideline 473 with acceptable restrictions (limited documentation on cytotoxicity; unusual harvest time after addition of colcemid; no data about osmolarity at high dose levels; no data about historical control values of this lab)

Data source

Reference
Reference Type:
publication
Title:
Chromosomal aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. IV. Results with 15 chemicals
Author:
Ivett JL, Brown BM, Rodgers C, Anderson BE, Resnick MA, Zeiger E
Year:
1989
Bibliographic source:
Environ Mol Mutagen 14: 165-187

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 99.9%
- Impurities (identity and concentrations): no data
- Purity test date: no data, but analysis performed by Radian Corp., Austin, TX.
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data, but stability in aqueous solutions have been shown in other studies
- Storage condition of test material: no data
- Source: Fluka

Method

Target gene:
Target: chromosome
Species / strain
Species / strain:
Chinese hamster Ovary (CHO)
Details on mammalian cell lines (if applicable):
CHO-WBL
Metabolic activation:
with and without
Metabolic activation system:
S9 obtained from livers of Aroclor 1254-treated male Sprague-Dawley rats
Test concentrations with justification for top dose:
The top dose (TD) was based on toxicity; doses used were generally the TD, 0.75 TD, 0.50 TD, 0.25 TD, 0.1 TD, 0.075 TD, 0.05 TD, 0.025 TD.
The highest three doses with sufficient number of cells analyzed for chromosomal aberrations.
Without metabolic activation (MA): 600, 700, 800 µg/ml
With MA: 2000, 2500, 3000 µg/ml

Vehicle:
dimethyl sulfoxide (DMSO)
Controls
Solvent controls:
yes
Positive controls:
yes
Remarks:
Mitomycin C without MA and cyclophosphamide with MA
Remarks:
concurrent controls
Details on test system and conditions:
The pH of the test chemical solution diluted in culture media was in the range of 7.0-7.5. Positive results verified with additional testing in repeat trials; the dose with the positive response was bracketed with a higher and lower dose.
Without MA cells exposed to phenol for 8 hr, chemical washed off, and cells treated with 0.1 ug/ml Colcemid for 2-2.5 hr, followed by further incubation (see below). With MA cells were exposed to phenol plus the metabolic activation mixture for 2 hr, washed, incubated for 8 h, and then treated with Colcemid for 2-2.5 h. A delayed harvest was used (cell cycle delay): cell growth period extended to about 20 hr. After cells were harvested air-dried slides were coded and stained with Giemsa. 100-200 cells from each of the three highest scorable doses analyzed. All aberrations individually classified (e.g., chromatid breaks, chromosome breaks, triradials) combined as the percent of cells with simple (deletions), complex (exchanges), and total (simple, complex and other) aberrations. Only total percent cells with aberrations considered in the statistical evaluation. Gaps and endoreduplications recorded but not included.
Evaluation criteria:
Trials with two or more significant doses considered positive, and trials with one significant dose and a significant trend judged as weak positive (+ W). Trials with a significant response at one dose and no significant trend, and trials with no significant responses but having a significant trend were considered equivocal.
Statistics:
A significant increase in aberration based on a binomial sampling assumption; the P values were adjusted according to Dunnett's method to take into account multiple dose comparisons. The trend test for both assays used a linear regression analysis: the percentage of cells with aberrations vs . the log dose.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
slight reduction in cell confluency at the two top doses with and without S-9 mix; no further data
Vehicle controls valid:
yes
Positive controls valid:
yes
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
slight reduction in cell confluency at the two top doses with and without S-9 mix; no further data
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Significant increases in chromosomal aberrations were observed with MA and delayed harvest time.

Any other information on results incl. tables

Aberrations in CHO cells after exposure to phenol without metabolic activation

 Dose level in µg/ml  % aberrations
 0  2
 600  4
 700  6
 800  7
 positive control  48

In parallel experiments the range for aberration of negative controls was 1 - 4%

Aberrations in CHO cells after exposure to phenol with metabolic activation

 Dose level in µg/ml  % aberrations
 0  2
 2000  18
 2500  14
 3000  17
 positive control  34

In parallel experiments the range for aberration of negative controls was 1 - 4%

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

In CHO cells phenol was mutagenic in the chromosome aberration assay with metabolic activation.
Executive summary:

The study is comparable to OECD Guideline 473 with acceptable restrictions (limited data on cytotoxicity; unusual harvest time after addition of colcemid; no data about osmolarity at high dose levels; no data about historical control values of this lab).

Chinese hamster ovary cells were exposed without metabolic activation (MA) to 600, 700, 800 µg/ml (exposure duration 8 hours) and with MA to 2000, 2500, 3000 µg/ml (exposure duration 2 hours) (solvent DMSO). 100-200 cells from each of the three highest scorable doses were analyzed and cytotoxicity evaluated by cell confluency. No increase in aberrations were recorded without MA. However, increased incidence of chromosome aberration were found with MA. A slight reduction in cell confluency at the two top doses were found with and without MA.

Conclusion: Phenol was mutagenic in the chromosome aberration assay in CHO cells with metabolic activation.