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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2004 - August 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
yes
Remarks:
The first trial and the third trial (in the presence of metabolioc activation) wereunacceptable due to excessive cytotoxicity. The second trial in the presence of metabolic activation was terminated due to a technical error.
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
Light cream flakes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: n°batch I-38110
- Expiration date of the lot/batch: 14 Jan 2006
- Purity test date: No data
- Purity : 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: Yes
- Solubility and stability of the test substance in the solvent/vehicle: Yes
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No

FORM AS APPLIED IN THE TEST : solution

Method

Target gene:
Thymidine Kinase (TK) Locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr Donald Clive, Glaxo-Wellcome Inc. Research Triangel Park N.C.
- Methods for maintenance in cell culture if applicable: Yes (maintained in logarithmic growth by serial subculturing for up to 4 monthsand replaced by cells from the frozen stock - Cultures were grown in a shaker incubator at 35-38°C).

MEDIA USED
- Type and identity of media : RPMI 1640 (Amacher et al., 1980; Clive et al., 1987) supplemented with horse serum (10% by volume), Pluronic® F68, L-glutamine, sodium pyruvate, penicillin and streptomycin
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
NADP/ Isoitrate induced Rat liver S9
Test concentrations with justification for top dose:
With metabolic activation: 0.4, 0.6, 0.8, 1, 1.2, 1.3, 1.4, 1.6 μg/ml
Without metabolic activation: 10, 20, 40, 50, 60, 70, 80, 90, 100 and 150 μg/ml

Justification of the top dose : A preliminary toxicity test was conducted. In the presence of metabolic activation ten concentrations were tested ranging from 0.0313 to 4 μg/ml and in the absence of metabolic activation 10 concentrations ranging from 2.35 to 1200 μg/ml. Severe toxicity was observed > 2 μg/ml in the presence of metabolic activation and > 75 μg/ml in the absence of metabolic activation
Vehicle / solvent:
- Solvent used: DMSO (batch n° I-38110)
- Justification for choice of solvent/vehicle: solubility properties
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:; The cells were pelleted by centrifugation, the culture medium removed, and the cells resuspended to a final volume of 10 mL of treatment medium with a final serum concentration of approximately 3%. The tubes were placed in an orbital shaker incubator at 35-38°C and rotated at 70 ± 10 orbits per minute
- Cell density at seeding: 6*10E6 cells per tube

DURATION
- Preincubation period: none
- Exposure duration: 4h
- Expression time (cells in growth medium): 48h
- Fixation time (start of exposure up to fixation or harvest of cells): 12 days

NUMBER OF REPLICATIONS: one per concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: A total of 3 x 10E6 cells from each selected tube was suspended in selection medium in soft agar to recover TFT-resistant mutants. This sample was distributed into three 100 mm dishes. The absolute selection cloning efficiency was determined by seeding three dishes with a total of approximately 600 cells in agar cloning medium. All dishes were placed in an incubator at 35-38°C with 4-6% CO2:95% humidified air. After 12 days in the incubator, the colonies were counted with the Loats Associates, Inc. (LAI) High Resolution Colony Counter (HRCC) System for the Mouse Lymphoma Assay, version 2.3.1 build 1

NUMBER OF CELLS EVALUATED: 3 x 10E6

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

- OTHER:: size distribution of colonies
Rationale for test conditions:
-
Evaluation criteria:
Mutant frequency and cell survival (measured as relative total growth) were determined. In addition to the numbers of mutant colonies, the size of the colonies were determined and the ratio of small versus large colonies was calculated.

For the mutant Selection Data
The mutant selection data includes the total mutant colonies and the total viable colonies. These were used to calculate the mutant frequency for each culture. The ratio of cells seeded for mutant selection to cells seeded for cloning efficiency was 0.5 x 104. Therefore, the mutant frequency was: (total mutant colonies/total viable colonies) x 2 x 10-4. Mutant frequency was given in units of 10-6. Also included in this section was the absolute cloning efficiency of vehicle and positive controls calculated as (total number of viable colonies/600) x 100%.
Statistics:
Various models of calculators, computers, and computer programs were used to analyze data in this study. Because different models round off or truncate numbers differently, values in some tables (e.g., means, standard deviations, or individual values) may differ slightly from those in other tables, from individually calculated data, or from statistical analysis data. Neither the integrity nor the interpretation of the data was affected by these differences.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 90 µg/L, 4h exposure
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not affected.
- Effects of osmolality: not increased
- Evaporation from medium : not specified
- Water solubility : no specified
- Precipation : not affected
Other cofounding effects : none

RANGE-FINDING/SCREENING STUDIES: Yes. In the absence of metabolic activity, 1-naphthol induced no cytotoxicity to weak cytotoxicity up to and including 37.5 μg/mL, moderately high cytotoxicity at 75.0 μg/mL and excessive cytotoxicty at ≥150 μg/mL. In the presence of metabolic activity, 1-naphthol induced excessive cytotoxicity at all doses tested

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : Performed
- Positive historical control data: nonactivation studies - 13 µg/mL methy methanesulfonate (mean 344.3 +/-72.3x10E-6, range 227 to 487.4x10E-6 ; number of experiments 50) - nonactivation studies - 2 µg/mL 3-methylcholanthrene (mean 339.1 +/-60.8x10E-6, range 211.0 to 491 x 10E-6 ; number of experiments 50) - nonactivation studies - 4 µg/mL 3-methylcholanthrene (mean 365.2 +/-69.5 x10E-6, range 200.2 0 to 524 x 10E-6 ; number of experiments 50)
- Negative (solvent/vehicle) historical control data: nonactivation studies (mean 63 +/-24.5x10E-6, range 36.4 to 135.7x10E-6 ; number of experiments 50) - activation studies (mean 63.3 +/-18 x10E-6, range 34 to 123.9 x10E-6 ; number of experiments 50)

TEST ARTICLE/ VEHICULE MIXTUE CONCENTRTION ANALYSES
Results of frozen stability analyses indicated that the formulations were stable for 9 days at -10 to -30°C. All values were within 3.1% of initial concentration. Results of concentration verification analyses indicated that all formulations were within 7.5% of target.

Any other information on results incl. tables

Initial Nonactivation Mutation Assay (B3)

In the initial nonactivation mutation assay, the average background mutant frequency (142.5x10-6) was outside the acceptable range specified in the protocol (20-130 x10-6); therefore, a repeat trial was performed using the same doses as the initial trial. The repeat nonactivation mutation assay with a treatment period of approximately 4 hours was initiated with twelve concentrations at 2.50, 5.00, 10.0, 20.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100 and 150 μg/mL. Treatments at and below 5.00 μg/mL were terminated because a sufficient number of higher concentrations were available and treatments above 90.0 μg/mL were terminated due to excessive cytotoxicity. The remaining eight treatments induced no cytotoxicity to high cytotoxicity (93.6% to 10.5% relative total growths). Mutant frequencies for the treatments analyzed ranged from 59.8 to 169.5 x 10-6. The background mutant frequency for the vehicle control in the initial nonactivation assay was 49.8 x 10-6, therefore a mutant frequency greater than or equal to 139.8 x 10-6 was required for a treatment to be evaluated as exhibiting a positive response. Treatments at 60.0, 70.0 and 90.0 μg/mL induced a mutant frequency that met criteria for a positive response.

Initial Activation Mutation Assay (B4)

Four mutation assays in the presence of metabolic activation were initiated, but the first trial was unacceptable due to excessive cytotoxicity. The second mutation assay in the presence of metabolic activation was terminated due to a technical error during dosing. The third mutation assay in the presence of metabolic activation was also terminated due to excessive cytotoxicity. The fourth mutation assay was considered acceptable and described in this report. The fourth activation assay with a treatment period of approximately 4 hours was initiated with twelve treatments at 0.200, 0.400, 0.600, 0.800, 1.00, 1.20, 1.30, 1.40, 1.60, 1.80, 2.00, and 2.40 μg/mL. The treatment at 0.200 μg/mL was terminated because a sufficient number of higher concentrations were available and treatments above 1.60 μg/mL were terminated due to excessive cytotoxicity. The remaining eight treatments induced no cytotoxicity to high cytotoxicity (106.3% to 15.9% relative total growth). Mutant frequencies for the treatments analyzed ranged from 95.8 to 50.0 x 10-6. The average background mutant frequency for the vehicle control in the initial activation assay was 53.3 x 10-6, therefore a mutant frequency greater than or equal to 143.3 x 10-6 was required for a treatment to be evaluated as exhibiting a positive response (Table 5). None of the treatments induced dose-responsive increases in mutant frequencies that met the criteria for a

positive response.

Confirmatory Mutation Assay

A confirmatory assay is not needed when clear positive results are observed in the initial definitive mutagenicity assay (OECD Guideline 476, 1997 and ICH S2A, 1996). In this study, clear positive results were obtained in the presence of S9.

Applicant's summary and conclusion

Conclusions:
Under the test conditions used 1-naphthol is considered mutagenic in the absence of metabolic activation.
Executive summary:

The test substance was examined for its mutagenic activity in the L5178Y tk+/- mouse lymphoma test in the absence and presence of metabolic activation. A preliminary toxicity test was conducted. In the presence of metabolic activation ten concentrations were tested ranging from 0.0313 to 4 μg/ml and in the absence of metabolic activation 10 concentrations ranging from 2.35 to 1200 μg/ml. As positive controls MMS (methyl methanesulfonate) for testing without metabolic activation and MCA (methylcholanthrene) for testing with metabolic activation were used.

Results

In the preliminary toxicity test, severe toxicity was observed > 2 μg/ml in the presence of metabolic activation and > 75 μg/ml in the absence of metabolic activation. Based on these data the main experiment was performed between 0.4 to 1.60 μg/ml in the presence of metabolic activation. In the absence of metabolic activation, 10 to 90 μg/ml were tested. In the presence of metabolic activation, toxicity ranged from no cytotoxicity to high cytotoxicity (100% to 16% relative total growth). A concentration related increase was observed in the mutant frequency. However, it was weak and the highest induction of mutant frequency was observed at the highest concentration (1.6 μg/ml) tested with a mutant frequency of 32 x 10-6 greater than the control. This increase was much less than the required 90 induced mutants per 106 clonable cells above the control value for obtaining a positive response. In the absence of metabolic activation toxicity levels ranged from 94% relative total growth to 11% at the highest concentration. A clear concentration related increase in mutant frequency was observed. Treatments of 60, 70, and 90 μg/ml induced a mutant frequency that met the criteria for a positive response. Mutant colonies from the positive controls MMS (methyl methanesulfonate for testing without metabolic activation) and MCA (methylcholanthrene for testing with metabolic activation) treated cultures showed both small and large colonies. The test article doses (60, 70 and 90 μg/ml without metabolic activation) which exhibited a positive response, showed a preferential increase in small colonies indicating larger mutations.

Conclusion

Under the test conditions used 1 -naphthol is considered mutagenic in the absence of metabolic activation.