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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September to October 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Javanol
- Physical state: Pale yellow viscous liquid
- Analytical purity: 88.4% (GC, sum of two peaks)
- Lot/batch No.: 008825
Specific details on test material used for the study:
- Name of test material (as cited in study report): Javanol
- Physical state: Pale yellow viscous liquid
- Analytical purity: 88.4% (GC, sum of two peaks)
- Lot/batch No.: 008825

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
exogenous metabolic activation system (S9 mixture)
The S9 mixture was freshly prepared, kept in an ice bath. Its composition was:
Potassium chloride 0.165 M 0.2 ml perml mix Magnesium chloride 0.04 M 0.2 ml per mi mix Sodium phosphate buffered saline 0.2 M, pH 7.4 0.5 ml per ml mix NADP (Boehringer) 3.2 mg permi mix Glucose-6-phosphate (Boehringer) 1.53 mg per mi mix S9 fraction 0.1 ml perm! mix
In order to test the activity of the S9 mix used in the experiments positive controls requiring metabolic activation were tested concurrently.
The protein content of the S9 fraction was determined by the method of Lowry (Lowry et al., 1951) with bovine serum albumin (purity: at least 92 %) from SERVA (Control D) used as the internal standard.
The protein contents of the batches used for the main tests were 40.2 and 34.3 mg/ml (Batches K1/96 and 12/96) and 34.3 mg/ml (batch 11/96) for the toxicity test.
Test concentrations with justification for top dose:
in the preliminary experiment: 50µg/plate javanol
in the standard plate incorporation: 1.6 to 158 micrograms/plate (-S9) and 5 to 500 micrograms/plate (+S9) and 1.6 to 158 micrograms/plate (+S9) were selected
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
Test procedure:
Bacterial strains
The Salmonella typhimurium strains TA1535, TA97, TA98, TA100, and TA102 were obtained from B.N. Ames and are described elsewhere (Ames et al., 1975; Levin et al., 1982a; 1982b; Maron and Ames, 1983).
Nutrient broth cultures of each strain, supplemented with 9 % DMSO were stored in liquid nitrogen. Subcultures with a normal spontaneous frequency were stored in NB + 9 % DMSO at -80°C+ 20°C. The strain identities and characteristics were periodically checked by the recommended procedures (De Serres and Shelby, 1979; Zeiger et al., 1981; Maron and Ames, 1983).
For use in tests, cultures of the strains were grown overnight at 37°C in a shaking water bath in a NB liquid medium. The strain TA102 was incubated with 0.3 pg tetracycline per m! NB medium in order to ensure the presence of an adequate number of plasmids (Albertini and Gocke, 1988). The growth of the overnight cultures was controlled by measuring the optical density ona photometer (Lumetron Colorimeter) at 650 nm. Each bacterial strain was diluted 10° in 0.85 % sodium chloride, and 100 ul of the last dilution step was plated on a NB complete medium. Two replicate plates were incubated at 37°C, upside down, for two days. The number of colonies was registered and the number of cells plated on VB medium was calculated.
The sensitivity of the Salmonella typhimurium strains was verified using the following positive controls: sodium azide with strains TA1535 and TA100, ICR 191 with strain TA97, 2-nitrofluorene with strain TA98 and MMC with strain TA102. Moreover, 2-aminoanthracene was used with all strains with and without metabolic activation to examine the activity of the S9 mix.

Toxicity prescreen and dose selection
Toxicity of the test substance was assessed in a preliminary toxicity assay by evaluating the growth on Vogel-Bonner minimal agar plates (determination of the growth of the background lawn and/or frequency of spontaneous revertants). Each test substance dose, as well as the appropriate solvent control, was evaluated in duplicate in strain TA100 in the plate incorporation version of the assay. The highest test dose for the main experiments is chosen to either produce signs of toxicity (reduction in the revertant colony number and/or observation of thinning or absence of the background lawn) or to be evidently insoluble in the aqueous medium.
There is much debate about selection of highest dose for badly soluble compounds (Kirkland, 1994; Gatehouse et al., 1994). Homogeneous suspensions are included and only particulate aggregation is considered for limiting the dose range to be evaluated. If the compound is soluble and non toxic, then 5 mg/plate is chosen in general as the highest dose level. The solubility of the compound in the solvent, the occurrence of precipitation in the test tube after addition of the soft agar and on the agar plates after the incubation period are noted in the toxicity test with TA100. Since precipitation under the conditions of the preincubation version might differ from the plate incorporation E. Gocke, Report 167’801 -14-
version an examination of precipitation after addition of the phosphate buffer (as in the preincubation test) is included in the prescreen. In this case cells are not added to the mixture. The basis for dose selection is described in results and discussion.

Standard Ames test procedure (Ames et al., 1975; Maron and Ames, 1983)
Test tubes containing 2 ml of 0.7 % agar medium were autoclaved and kept in a
prewarmed water bath at 42°C to 45°C. The following solutions were added in order:
¢ 0.2 ml of the histidine/biotin mixture corresponding to 21 ug L-histidine and 24.4 ug biotin
e 0.1 ml of the test compound at different concentrations or of the solvent or 0.05 ml of the different reference substances which were thawed shortly before use
e 0.1 ml of the overnight cultures of the bacterial strain
e 0.5 mi of the S9 mixture where metabolic activation was needed. The S9 mixture was replaced by 0.5 ml sodium phosphate buffered saline 0.2 M, pH 7.4 in the treatment without metabolic activation.
The contents of the tubes were mixed and poured immediately onto Vogel-Bonner minimal agar plates. Three replicate plates for the test compound and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.

Liquid preincubation assay (Yahagi et al., 1975; Matsushima et al., 1980)
In the modified procedure the following solutions are added in order:
¢ 0.1 ml of the test compound solutions or of the solvent or 0.05 ml of the different reference substances which were thawed shortly before use
• 0.5 ml of sodium phosphate buffered saline 0.2 M, pH 7.4 or 0.5 ml of the S9 mixture
e 0.1 ml of the overnight cultures of the bacterial strain.
The test tubes are incubated and shaken for 30 minutes at 37°C. 2.2 ml soft agar supplemented with 21 ug L-histidine and 24.4 ug biotin was added afterwards and the content of the tubes were mixed and poured on Vogel-Bonner minimal agar plates. Three replicate plates for the test compound and negative control or two replicate plates for the positive controls were incubated at 37°C, upside down, for 2 days.

Data reporting
Colonies are usually counted electronically using a DOMINO automatic image analysis system (Perceptive Instruments, Haverhill, Suffolk, England) after having inspected the E. Gocke, Report 167’801 -15-
background lawn for signs of toxicity. Plates exhibiting precipitate or contamination may be counted manually (for details see TABLES).
The microscopic examination of the bacterial background lawn, resulting from the trace of histidine added to the plates, is an aid to determine the toxicity of the test compound and is essential to the interpretation of results. The absence of a confluent lawn of bacteria was reported as BGR and the absence of bacterial lawn and/or presence of microcolonies as BGO. Inhibition of the growth was attributed to toxic effects by the substance (reported as t or T in the summary tables).
Evaluation criteria:
A positive result is defined as a reproducible, dose-related increase in the number of his* revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA1535 and TA98. For strains TA97, TA100 and TA102 a 1.5 - fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation (Claxton et al. 1987) and biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of his* revertant colonies.
Since it is impossible to define criteria that would apply to every configuration of data generated by the mutation assay, the study director is responsible for the ultimate decision in the evaluation of the results. The factors considered in making the decision are discussed in results and discussion.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity

Applicant's summary and conclusion

Conclusions:
No increase in the number of revertant colonies was apparent for any of the five tester strains after treatment with Javanol.
It can be concluded that neither Javanol per se, nor any of the metabolites formed under the described experimental conditions used is mutagenic in the Ames test.
Executive summary:

Javanol was evaluated for mutagenic activity in the Ames test. A standard plate incorporation and preincubation modification assay were performed in absence and in presence of an exogenous metabolic activation system (S9). Five Salmonella typhimurium tester strains (TA 1535, TA97, TA98, TA100, TA102) were employed. The activity of the S9 -mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment.

Javanol was dissolved in DMSO. Toxic effects were observed in a preliminary toxicity experiment starting at 50 micrograms/plate. For the standard plate incorporation assay the concentration ranges 1.6 to 158 micrograms/plate (-S9) and 5 to 500 micrograms/plate (+S9) and 1.6 to 158 micrograms/plate (+S9) were selected. Some turbidity was apparent at the two highest test concentrations. Toxicity of the compound was generally observed at the two highest test concentrations, with some variation depending on strain and test method.

No increase in the number of revertant colonies was apparent for any of the five tester strains after treatment with Javanol.

Thus it can be concluded that neither Javanol per se, nor any of the metabolites formed under the described experimental conditions used is mutagenic in the Ames test.