Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards (similar to OECD 476 guideline), well-documented and acceptable for assessment.

Data source

Reference
Reference Type:
publication
Title:
Mouse lymphoma L5178Y thymidine kinase locus assay of 50 compounds
Author:
Wangenheim J, Bolcsfoldi G
Year:
1988
Bibliographic source:
Mutagenesis 3, 193-205

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Biphenyl
- Analytical purity: no numeral value reported, however test compounds were of highest available purity
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Stability under test conditions: no data
- Storage condition of test material: no data
- Other: Supplier: Aldrich-Chemie (FRG)

Method

Target gene:
TK+/- -> TK-/- forward mutation assay
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium containing 10% horse serum with additions (Clive et al., 1979). The pH of the culture medium was adjusted to 7.2 which was found to improve the growth rate of the cells compared with pH 6.8
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate (S9) from Aroclor 1254 pretreated male Sprague-Dawley rats (Garner et al, 1992), cofactors from Sigma Chemical Co.
Test concentrations with justification for top dose:
Without metabolic activation: 0; 0.987E-04; 1.970E-04; 2.960E-04; 3.450E-04; 3.950E-04 mol/L
With metabolic activation: 0; 0.501E-05; 1.000E-05; 2.000E-05; 4.000E-05; 6.000E-05 mol/L
Vehicle / solvent:
No data
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: substances tested positive were available
Remarks:
With and without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 48h
- Selection time (if incubation with a selection agent): no data
- Fixation time (start of exposure up to fixation or harvest of cells): no data


SELECTION AGENT (mutation assays): trifluorothymidine was added to the three undiluted cell suspensions to a final concentration of 1.0 µg/mL.


NUMBER OF REPLICATIONS: 6 replicates


NUMBER OF CELLS EVALUATED: 15 x E04 cells/mL (spontaneous mutation frequency was 76 +/- 25 without and 86 +/- 33 x E06 cells with metabolic activation (n = 35 and 20) respectively


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; total growth; other: mutation frequency, mutation index



Evaluation criteria:
Total survival was calculated from the suspension growth and cloning efficiency data as described by Clive et al. (1979) and the mutation frequency expressed as the number of mutant cells/E06 viable cells. Adopting a 2-fold or greater increase in mutation frequency, at 10% or higher total growth, was the criterian for a positive result.
Statistics:
The number of colonies formed on the six replicate control agar plates and the three replicate plates from each treated culture was tested for normal distribution according to Shapiro and Wilk (1965) and found to be normally distributed in 92% of the cases (n = 383). Further, the replicates were subjected to analysis of variance, which showed that the variance of the control and treated replicates was equal in 95% of the comparisons (n = 317). Therefore, a pairwise two-tailed Student's t-test was performed on each set of treated replicates versus the corresponding solvent control replicates. The application of linear, quadratic and cubic regression analysis to the results provided data (not reported in publication) which were in poor agreement with the subjective evaluation of the dose trends seen. The statistical analyses were performed using the SAS computer programming package (1985).

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at two highest concentrations tested (3.450E-04 and 3.950E-04 mol/L)
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
at concentrations resulting in a mutation index > 2.0 (4.000E-05 and 6.000E-05 mol/L)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at two highest concentrations tested (4.000E-05 and 6.000E-05 mol/L)
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: the pH of the culture medium was adjusted to 7.2 which was found to improve the growth rate of the cells compared with pH 6.8.



Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Total survival, mutation frequency and increase in mutation frequency relative to the solvent controls are shown in the following table:

 S9 Concentration (mol/L)  Total growth  Mutation frequency  Mutation index 
 -  0    66  
 -  0    57  
 -  0.987E-04  77  73  1.2
 -  1.970E-04  49  60  1.0
 -  2.960E-04  21  79*  1.3
 -  3.450E-04  14  105***  1.7
 -  3.950E-04  6  125***  2.0
 +  0    97  
 +  0    81  
 +  0.501E-05  102  93  1.0
 +  1.000E-05  75  98  1.1
 +  2.000E-05  35  123*  1.4
 +  4.000E-05  15  185***  2.1
 +  6.000E-05  12  319***  3.6

S9: - without metabolic activation; + with metabolic activation

Total growth: suspension growth x cloning efficiency

Mutation frequency: Mutants/E06 surviving cells

Mutation index: Mutation frequenc of treated culture/average mutation frequency of control cultures

Result of pairwise t-test of treated culture plated in triplicate against duplicate control cultures plated in triplicate: *0.01 < P < or = 0.05; **0.001< P < or = 0.01; ***0.0001 < P < or = 0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation at concentrations with mutation index > 2.0 (4.000E-05 and 6.000E-05 mol/L)

Biphenyl was found to be positive for genotoxicity in the presence of a metabolic activation system at concentrations resulting in a mutation index > 2.0 (4.000E-05 and 6.000E-05 mol/L). BIphenyl was cytotoxic at the two highest concentrations tested (3.450E-04 and 3.950E-04 mol/L without metabolic activation; 4.000E-05 and 6.000E-05 mol/L with metabolic activation) as reflected in low total growth at these concentrations.