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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-04-05 to 1994-05-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(1,1,3,3-tetramethylbutyl)phenol
EC Number:
205-426-2
EC Name:
4-(1,1,3,3-tetramethylbutyl)phenol
Cas Number:
140-66-9
Molecular formula:
C14H22O
IUPAC Name:
4-(1,1,3,3-tetramethylbutyl)phenol
Constituent 2
Reference substance name:
4-(1,1,3,3-Tetramethylbutyl)-phenol
IUPAC Name:
4-(1,1,3,3-Tetramethylbutyl)-phenol
Details on test material:
- Name of test material (as cited in study report): Octylphenol PT
- Physical state: solid
- Stability under test conditions: > 1 year
- Storage condition of test material: in a tightly closed glass container at ambient temperature in the dark

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
H10: Ham`s F12 medium with 10% fetal calf serum (FCS), 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 mg/mL Streptomycin
H0: Ham`s F12 medium with 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 mg/mL Streptomycin
H6TG: Ham`s F12 medium with 10% FCS, 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 mg/mL Streptomycin, 10 µg/mL 6-thioguanine (does not contain hypoxanthine)
HAT: Ham`s F12 medium with 10% FCS, 2 mM L-Glutamine, 100 IU/mL Penicillin, 100 mg/mL Streptomycin, 200 µM glycin, 5 µM thymidine, 10 µM hypoxanthine, 3.2 µM aminopterin
- Properly maintained: not mentioned
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not mentioned
- Periodically "cleansed" against high spontaneous background: yes, in the beginning of the test with HAT medium for approx. 1 week
Metabolic activation:
with and without
Metabolic activation system:
microsomal liver enzymes (S9), activation system not mentioned
Test concentrations with justification for top dose:
RANGE-FINDING TEST:
pre-experiment for toxicity: 0 / 0.313 / 0.625 / 1.25 / 2.5 / 5 / 10 / 15 / 20 / 25 µg/mL without S9 mix
MAIN TEST: 0 / 0.25 / 0.625 / 1.25 / 2.0 / 2.5 µg/mL without S9 mix and 0 / 0.25 / 1.25 / 6.25 / 12.5 / 25 µg/mL with S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not stated
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
H0 medium containing 1% ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation

Migrated to IUCLID6: concentration in the test: 300 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
H0 medium containing 1% ethanol plus S9 mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
With metabolic activation

Migrated to IUCLID6: concentration in the test: 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days (subcultured after 3 days)
- Selection time (if incubation with a selection agent): 6 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS: none
Evaluation criteria:
The cloning efficiency of the negative control at the end of the expression period must be least 20%.
The mutation frequency of the negative control should not exceed the maximum spontaneous mutation frequency of approx. 20/10E6 cells.
The mutation frequencies of cell cultures treated with positive control substances must be significantly increased.
At least 4 dose levels must be examined, the highest dose being either significantly toxic (cloning efficiency approx. 20%) or being the solubility limit of the test substance. Concentrations higher than 5 mg/mL will not be tested.
Positive results must be reproducible in a second independent experiment.
Statistics:
The t-test was used (Two-sample analysis).

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
- With metabolic activation: not cytotoxic at tested concentrations - Without metabolic activation: 5 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Precipitation: not occurred
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: In the presence of S9 mix Octylphenol PT had no significant influence on the cloning efficiency of CHO cells. In the absence of S9 mix, however, test substance concentrations > 2.5 µg/ml resulted in complete cell loss.

COMPARISON WITH HISTORICAL CONTROL DATA: see below

GENOTOXIC EFFECTS:
- With metabolic activation: Both experiments were within the range (0-20 mutants/10E6 viable cells) normally obtained in the presence of exogenous metabolic activation. In both trials the positive control, MCA, induced mutant frequencies being significantly higher than the negative controls, thus demonstrated the sensitivity of the test system. In trial #1, Octylphenol PT did not induce mutant frequencies significantly higher than the concurrent or historical negative controls. At a concentration of 25 µg/mL in trial #2 the number of mutants was statistically different from the concurrent negative control (6±2 versus 2±1) but was well within the range of historical controls. As this increase was not observed in trial #1 it was considered to be of no biological relevance.
- Without metabolic activation:
The negative controls in both tests were clearly within the range (0-20 mutants/10E6 viable cells) normally obtained in the absence of exogenous metabolic activation. The positive control, EMS, in both trials induced mutant frequencies being significantly higher than the negative controls thus demonstrating the sensitivity of the test system. In trial #1, Octylphenol PT did not induce mutant frequencies significantly higher than the concurrent or historical negative controls. In trial #2, the highest test substance concentration of 2.5 µg/mL induced a mutant frequency of 10±4 mutants/10E6 viable cells (with a negative control of 1±2/10E6 cells). Although this difference is statistically significant, it is well within the range of historical controls for this cell system. In addition, this elevated mutant frequency was not observed in trial #1 of this experiment.
Remarks on result:
other: strain/cell type: K1
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table #1: Dose finding

  Eperiment without S9 mix     Experiment with S9 mix    
 Concentration [µg/mL]  mean colony number ± SD  absolute C.E. [%]  mean colony number ± SD  absolute C.E. [%]
 0  147 ± 9  74  97 ± 9  49
 0.313  163 ± 21  81  91 ± 13  45
 0.625  160 ±6  80  91 ± 10  46
 1.25  142 ±5  71  76 ± 10  38
 2.50  140 ± 15  70  168 ± 15  84
 5.00  0 ± 0  0  161 ± 11  80
 10.0  0 ± 1  0  155 ± 8  77
 15.0  0 ± 0  0  161 ± 24  80
 20.0  0 ± 1  0  158 ± 8  79
 25.0  1 ± 1  0  144 ± 5  72

C.E. = cloning efficiency

Table #2: Cloning Efficiency at the end of exposure to Octylphenol PT

Cloning Efficiency               
concentration [µg/ml] test #1 -S9   test #2 -S9 concentration [µg/ml]  test #1 +S9  test #2 +S9
 Neg. control 183 ± 11 181 ± 8 Neg. control 169 ± 12 142 ± 10
 0.25 176 ± 9 175 ± 30 0.25 166 ± 4 146 ± 11
 0.625 161 ± 6 179 ± 7 1.25 162 ± 17 109 ± 4
1.25 168 ± 3 186 ± 3 6.25 179 ± 7 177 ± 15
2.0 190 ± 6 169 ± 22 12.5 165 ± 13 151 ± 9
2.5 163 ± 20 179 ± 10 25.0 158 ± 12 161 ± 5
Pos. control (EMS) 124 ± 6 105 ± 6 Pos. control (MCA) 181 ± 12 161 ± 13

Table #3: Mutant frequency

       Experiment without S9 mix   Experiment with S9 mix     
concentration[µg/mL] mutant frequency test #1   mutant frequency test #2  concentration[µg/mL]  mutant frequency test #1  mutant frequency test #2
solvent control   2 ± 2  1 ± 2 solvent control   5 ± 2  2 ± 1
0.25   2 ± 2  3 ± 2 0.25   2 ± 2  1 ± 1
0.625   3 ± 2  1 ± 1 1.25   3 ± 2  4 ± 3
1.25   0 ± 0  2 ± 1 6.25   4 ± 2  1 ± 1
2.00   4 ± 3  1 ± 2 12.5   6 ± 2  2 ± 1
2.50   1 ± 1  10 ± 4** 25.0   6 ± 2  6 ± 2**
Pos. control (EMS)  437 ± 34***  332 ± 30*** Pos. control (MCA)   124 ± 11***  103 ± 8***

** = difference statistically significant (0.01>P>0.001) *** = difference statistically significant (P>0.001)

Applicant's summary and conclusion

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

Treatment of CHO cells with Octylphenol PT in two independent experiments (each with and without metabolic activation by S9 mix) did not result in any reproducible statistically or biologically significant increase of the mutant frequency of the HPRT locus.
Executive summary:

In a mammalian cell HRPT gene mutation assay, CHO cells cultured in vitro were exposed to 4-(1,1,3,3-Tetramethylbutyl)-phenol (98,1%) in ethanol at concentrations of 0 / 0.25 / 0.625 / 1.25 / 2.0 / 2.5 µg/mL in the presence and absence of mammalian metabolic activation (S9). 

4-(1,1,3,3-Tetramethylbutyl)-phenol was tested up to insoluble concentrations. The highest concentration induced 10 +/-4 mutants/10-6vs 1 +/-2 mutants/10-6. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

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