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EC number: 201-487-4 | CAS number: 83-56-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Boiling point
- Density
- Particle size distribution (Granulometry)
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- Additional physico-chemical properties of nanomaterials
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item naphthalene-1,5-diol was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Additionally, a study was performed to investigate the potential of naphthalene-1,5-diol to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, naphthalene-1,5-diol is considered to be non-mutagenic in this mouse lymphoma assay.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2004-03-12 to 2004-06-18
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- July 21, 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2000-05-19
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- The test item and the information concerning the test item were provided by the sponsor.
Identity: A 018
Henkel Code: SAT 030627
Batch No.: 820211/01
Aggregate State
at Room Temperature: solid (powder)
Colour: brown
Purity: 99.9 % (area %, HPLC)
Stability in solvent: not indicated by the sponsor
Storage: at approx 4° C, light protected
Expiration Date: July 31, 2008
On the day of the experiment, the test item A 018 was dissolved in DMSO (purity > 99 %, MERCK, D-64293 Darmstadt). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (4).
Precipitation of the test item was observed at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 5000 5000 2500, 5000 2500, 5000
TA 1537 5000 5000 2500, 5000 2500, 5000
TA 98 5000 / 2500, 5000 2500, 5000
TA 100 5000 5000 2500, 5000 2500, 5000
TA 102 5000 5000 2500, 5000 2500, 5000
/ no visible precipitation observed
The undissolved particles had no influence on the data recording. - Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-Naphthoflavone induced rat liver S9 was used as exogenous metabolic activation system
- Test concentrations with justification for top dose:
- Experiment I: 33-5000 µg/plate without and with S9-mix
Experiment II: 10-5000 µg/plate without and with S9-mix - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylenediamine, 2-aminoanthracene
- Details on test system and experimental conditions:
- Characterisation of the Salmonella typhimurium Strains
The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation (deletion of the uvrB gene) causes an inactivation of the excision repair system. The latter alteration also includes a deletion in the nitrate reductase and biotin genes. In the strains TA 98, TA 100, and TA 102 the R-factor plasmid pKM 101 carries umu DC analogous genes that are involved in error-prone repair and the ampicillin resistance marker. The strain TA 102 does not contain the uvrB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene (5).
In summary, the mutations of the TA strains used in this study can be described as follows
Strains Genotype Type of mutations indicated
TA 1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA 98 his D 3052; rfa-; uvrB-;R-factor " "
TA 1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA 102 his G 428; rfa-; uvrB+;R-factor " "
TA 100 his G 46; rfa-; uvrB-;R-factor " "
Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to B. Ames et al. (1) and D. Maron and B. Ames (5). In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535 and TA 1537 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 98 was obtained from E. Merck (D-64293 Darmstadt). The bacterial strains TA 100 and TA 102 were obtained from RCC Ltd (CH-4332 Stein).
Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.
Precultures
From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 µL tetracycline (2 µg/mL) was added to strain TA 102. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt) 5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.
Selective Agar
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.
Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121° C in an autoclave. - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2).
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- A statistical analysis of the data is not required (2).
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- This study was performed to investigate the potential of naphthalene-1,5-diol to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.
The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Experiment I: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Reduced background growth was observed without metabolic activation in strain TA 98 in experiment I. In experiment II, reduced background growth was observed with and without metabolic activation in all strains used (cf. tables of results, printed in bold).
Toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in nearly all strains used.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with A 018 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct in- crease of induced revertant colonies.
Conclusion
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, naphthalene-1,5-diol is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay both in the presence and absence of the specified metabolic activation system. - Executive summary:
The test item naphthalene-1,5-diol was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.
The assay was performed with and without liver microsomal activation. The pre-experiment with strain TA 98 had to be repeated due to a low rate of spontaneous revertants. The evaluation was terminated and no data obtained. The repeat experiment is reported as pre-experiment and experiment I. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:
Experiment I: 33; 100; 333; 1000; 2500; and 5000µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Reduced background growth was observed without metabolic activation in strain TA 98 in experiment I. In experiment II, reduced background growth was observed with and without metabolic activation in all strains used (cf. tables of results, printed in bold).
Toxic effects, evident as a reduction in the number of revertants were observed at the following concentrations (µg/plate):
Strain
Experiment I
Experiment II
without S9 mix
with S9 mix
without S9 mix
with S9 mix
TA 1535
2500, 5000
5000
/
/
TA 1537
5000
/
/
/
TA 98
5000
/
1000 - 5000
2500, 5000
TA 100
5000
5000
1000 - 5000
/
TA 102
2500, 5000
5000
2500, 5000
1000, 5000
/ no toxic effects observed
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with naphthalene-1,5-diol at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
In experiment I, in the presence of metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in in the solvent control of strain TA
100. Since this deviation is rather small, this effect is judged to be based upon biologically irrelevant fluctuations and has no detrimental impact on the outcome of the study.
The historical range of positive controls was exceeded in strains TA 100 and TA 102 (experiment I) without metabolic activation and with metabolic activation in strain TA 1537 (experiment I). These effects indicate the sensitivity of the strains rather than compromising theassay.
Appropriate reference mutagens were used as positive controls. They showed a distinct in- crease in induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2004-03-25 to 2004-08-03
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- February 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- thymidine kinase locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-Naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Experiment I:
15.0 - 60.0 μg/ml (without S9-mix)
0.25 - 2.0 μg/ml (with S9-mix)
Experiment II:
6.25 - 50.0 μg/ml (without S9-mix)
0.125 - 1.25 μg/ml (with S9-mix - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO (E. MERCK, D-64293 Darmstadt; purity 99.5 %). The final concentration of DMSO in the culture medium did not exceed 0.5 % (v/v). The solvent was chosen according to its solubility properties and its relative non-toxicity to the cell cultures.
The pH-values were determined in the solvent control and the maximum concentration without metabolic activation. The osmolarity was measured in the absence of metabolic activation in the solvent control (pre-experiment} and at the maximum concentration (experiment I):
Osmolarity mOsm ph-value
Pre-experiment
solvent control 371 7.32
A 018 (1600 µg/mL) * 7.30
Experiment I
solvent control 373 **
A 018 (60 µg/mL) 371 **
* could not be measured due to strong precipitation
** not determined
Test System
Reasons for the Choice of the Cell Line L5178Y
The L5178Y cell line has successfully been used in in vitro experiments for many years. L5178Y cells are characterised by a high proliferation rate (doubling time 10 - 12 h in stock cultures} and cloning efficiencies of untreated cells of usually more than 50 % both necessary for the appropriate performance of the study. The cells have a stable karyotype with a near diploid (40 ± 2) chromosome number (3).
Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays with cell cultures as many chemicals only develop a mutagenic potential when they are metabolised by the mammalian organism. However, metabolic activation of chemicals can at least partially be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
Cell Cultures
Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
hypoxanthine 1.0x10-4 M aminopterin 2.ox10·7 M thymidine 1.6x10·5 M
The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing:
hypoxanthine 1.0x10-4 M
thymidine 1.6x10-5 M
8.4 Mammalian Microsomal Fraction S9 Mix
8.4.1 S9 (Preparation by RCC-CCR)
Phenobarbital/β-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and J3-Naphthoflavone p.a. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80° C. Small numbers of the ampoules could be kept at -20°C for up to one week.
The protein concentration of the S9 preparation was 35.6 mg/ml (Lot. No.: 160104) in the pre-experiment and both main experiments.
S9 Mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to give a final protein concentration of 0.75 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCI
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath.
The S9 mix preparation was performed according to Ames et al. (1).
Pre-Test on Toxicity
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.
1x10 7 cells were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treatment the cells were washed twice by centrifugation (425 g, 10 min) and resuspended in "saline G". Subsequently the cells were resuspended in 30 ml complete culture medium for a 2-day growth period. The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3x105 cells/ml, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector (3).
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium}.
Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research allowing the repeated use of the same cell culture batch in many experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated in plastic flasks (GREINER, 72632 Frickenhausen} in RPMI 1640 complete culture medium (see page 18). The cells are subcultured at least three times a week. The cell cultures are incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % air. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: determined in pre-tests
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- In conclusion it can be stated that during the mutagenicity test described and under the ex perimental conditions reported the test item did not induce mutations in the mouse lym phoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, naphthalene-1,5-diol is considered to be non-mutagenic in this mouse lymphoma assay. - Executive summary:
The study was performed to investigate the potential of naphthalene-1,5-diol to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with a treatment period of 4 h with and without metabolic activation. In the second experiment the cells were treated for 24 h without and for 4 h with metabolic activation. The experimental part of the first experiment with metabolic activation was repeated in the second experiment since less than the required four analysable concentrations were available above the threshold of toxicity of 10 % of survivingcells.
Due to exceedingly toxic effects the second experiment with metabolic activation was repeated using lower concentrations (IIA). The results of this repeat experiment are included in experiment II.
The highest applied concentration in the pre-experiment (1600 µg/mL) was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM.
No biologically relevant and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments.
Appropriate reference mutagens were used as positive controls and showed a distinct in crease in induced mutant colonies, indicating that the tests were sensitive and valid.
The dose range of the main experiments was adjusted to toxicity data. The tested concentrations are described in table II (page 17). The evaluated experimental points and the results are summarised in table I (page 11).
Conclusion
In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, naphthalene-1,5-diol is considered to be non-mutagenic in this mouse lymphoma assay.
Referenceopen allclose all
Controls
NegativeControls
Concurrent untreated and solvent controls were perform
Positive Control SubstancesWithout metabolicactivation
Strains: TA 1535, TA100
Name: sodium azide,NaN3
Supplier: SERVA, D-69042Heidelberg
CatalogueNo.: 30175
Purity: at least 99%
Dissolvedin: waterdeionised
Concentration: 10 µg/plate
Strains: TA 1537, TA98
Name: 4-nitro-o-phenylene-diamine, 4-NOPD
Supplier: SIGMA, D-82041Deisenhofen
CatalogueNo.: N9504
Purity: > 99.9%
Dissolvedin: DMSO (purity >99 %, MERCK, D-64293Darmstadt) Concentration: 10 µg/plate in TA 98, 50 µg/plate in TA1537
Strain: TA102
Name: methyl methane sulfonate,MMS
Supplier: MERCK-SCHUCHARDT, D-85662Hohenbrunn
CatalogueNo.: 820775
Purity: > 99.0%
Dissolvedin: waterdeionised
Concentration: 4.0 µL/plate
With metabolic activation
Strains: TA 1535, TA 1537, TA 98, TA 100, TA102
Name: 2-aminoanthracene, 2-AA
Supplier: SIGMA, D-82041Deisenhofen
CatalogueNo.: A1381
Purity: 97.5%
Dissolvedin: DMSO (purity >99 %, MERCK, D-64293Darmstadt) Concentration: 2.5 µg/plate(10.0 µg/plate in TA102)
The stability of the positive control substances in solution was unknown but a mutagenic response in the expected range is sufficient evidence of biological stability.
Mammalian Microsomal Fraction S9Mix
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
S9 (Preparation by R C C - C CR)
Phenobarbital/β-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar HanIbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) andβ-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week.
The protein concentration in the S9 preparation was 27.2 mg/mL (lot no. R 071103) in the pre-experiment and in experiment I, and 32.7 mg/mL (lot no. 141103) in experiment II
S9Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% v/v in the cultures. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.(1).
Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre- experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as the main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.
Dose Selection
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as part of experiment I. Based on the toxic effects observed in experiment I, seven concentrations were tested in experiment II and 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
Experiment I: 33; 100; 333; 1000; 2500; and 5000µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experimental Performance
For each strain and dose level, including the controls three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL Overlay agar
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark (2).
Data Recording
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben). The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to reduced background growth and precipitation, the colonies were partly counted manually.
Acceptability of the Assay
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
12.1 Pre-Experiment forToxicity
The following concentrations were tested for toxicity and mutation induction with each 3 plates.
Test Group |
Concentrationper plate [µg] |
Revertants per plate (mean value of three plates) |
|||||||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
TA 102 |
|||||||
- |
+ |
- |
+ |
- |
+ |
- |
+ |
- |
+* |
||
Negative control |
- |
14 |
12 |
6 |
10 |
24 |
31 |
85 |
103 |
275 |
278 |
Solvent control |
- |
17 |
11 |
7 |
10 |
26 |
27 |
79 |
94 |
253 |
278 |
4-NOPD |
50.0 |
/ |
/ |
52 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
4-NOPD |
10.0 |
/ |
/ |
/ |
/ |
108 |
/ |
/ |
/ |
/ |
/ |
MMS |
4.0 (µL) |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
1564 |
/ |
Sodium azide |
10.0 |
386 |
/ |
/ |
/ |
/ |
/ |
1218 |
/ |
/ |
/ |
2-aminoanthracene |
2.5 |
/ |
435 |
/ |
215 |
/ |
150 |
/ |
1561 |
/ |
/ |
2-aminoanthracene |
10.0 |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
/ |
854 |
Test item |
3 10 33 100 333 1000 2500 5000 |
13 10 14 15 15 12 6 3 |
9 8 8 6 9 8 9 0 |
8 9 10 9 6 9 4 0 |
8 10 7 12 11 9 10 21 |
28 23 23 20 21 25 15 5 |
30 26 18 23 28 30 27 29 |
79 86 83 81 81 84 55 16 |
96 87 90 76 71 68 54 35 |
285 296 278 305 250 228 81 11 |
287 344 364 335 222 218 136 43 |
* - = without S9 mix; + = with S9 mix, / = not performed; printed in bold = reduced background growth observed
12.1 Experiment I: Plate IncorporationTest
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 071103)
Teststrain: TA1535
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000 |
16 18 437 14 19 15 14 7 4 |
8 16 377 10 11 16 11 3 1 |
17 16 345 17 16 15 12 8 5 |
14 17 386 14 15 15 12 6 3 |
4.9 1.2 46.7 3.5 4.0 0.6 1.5 2.6 2.1 |
1.0 23.2 0.8 0.9 0.9 0.7 0.4 0.2 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000 |
10 11 421 8 6 9 4 9 0 |
10 11 432 8 5 7 7 10 0 |
15 11 452 9 8 12 13 7 0 |
12 11 435 8 6 9 8 9 0 |
2.9 0.0 15.7 0.6 1.5 2.5 4.6 1.5 0.0 |
1.0 39.5 0.8 0.6 0.8 0.7 0.8 0.0 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#sodium azide 10 µg/plate
##2-aminoanthracene 2.5 µg/plate
12.1 Experiment I: Plate IncorporationTest
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 071103)
Teststrain: TA1535
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000 |
16 18 437 14 19 15 14 7 4 |
8 16 377 10 11 16 11 3 1 |
17 16 345 17 16 15 12 8 5 |
14 17 386 14 15 15 12 6 3 |
4.9 1.2 46.7 3.5 4.0 0.6 1.5 2.6 2.1 |
1.0 23.2 0.8 0.9 0.9 0.7 0.4 0.2 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000 |
10 11 421 8 6 9 4 9 0 |
10 11 432 8 5 7 7 10 0 |
15 11 452 9 8 12 13 7 0 |
12 11 435 8 6 9 8 9 0 |
2.9 0.0 15.7 0.6 1.5 2.5 4.6 1.5 0.0 |
1.0 39.5 0.8 0.6 0.8 0.7 0.8 0.0 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#sodium azide 10 µg/plate
##2-aminoanthracene 2.5 µg/plate
Experiment I: Plate Incorporation Test
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 071103)
Teststrain: TA1537
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000 |
5 8 65 11 12 7 8 6 0 |
8 9 38 9 9 7 6 2 0 |
4 5 52 9 6 5 14 5 0 |
6 7 52 10 9 6 9 4 0 |
2.1 2.1 13.5 1.2 3.0 1.2 4.2 2.1 0.0 |
1.0 7.0 1.3 1.2 0.9 1.3 0.6 0.0 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000 |
9 12 227 12 16 13 8 15 20 |
13 6 191 5 11 10 9 8 19 |
9 13 227 5 9 9 9 8 25 |
10 10 215 7 12 11 9 10 21 |
2.3 3.8 20.8 4.0 3.6 2.1 0.6 4.0 3.2 |
1.0 20.8 0.7 1.2 1.0 0.8 1.0 2.1 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#4-nitro-o-phenylene-diamine 50 µg/plate
##2-aminoanthracene 2.5 µg/plate
Experiment I: Plate Incorporation Test
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 071103)
Teststrain: TA98
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000 |
23 29 103 25 24 17 28 15 5 |
25 22 117 25 19 22 27 10 6 |
25 28 104 19 17 24 21 19 4 |
24 26 108 23 20 21 25 15 5 |
1.2 3.8 7.8 3.5 3.6 3.6 3.8 4.5 1.0 |
1.0 4.1 0.9 0.8 0.8 1.0 0.6 0.2 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000 |
33 27 143 21 21 25 27 27 34 |
30 29 145 18 21 29 29 24 25 |
30 26 161 16 28 29 35 30 27 |
31 27 150 18 23 28 30 27 29 |
1.7 1.5 9.9 2.5 4.0 2.3 4.2 3.0 4.7 |
1.0 5.5 0.7 0.9 1.0 1.1 1.0 1.0 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#sodium azide 10 µg/plate
##2-aminoanthracene 2.5 µg/plate
printed in bold = reduced background growth observed
Experiment I: Plate Incorporation Test
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 071103)
Teststrain: TA100
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000 |
88 75 1223 85 74 96 87 59 19 |
71 82 1256 87 87 66 95 49 10 |
97 80 1174 76 83 82 69 58 18 |
85 79 1218 83 81 81 84 55 16 |
13.2 3.6 41.3 5.9 6.7 15.0 13.3 5.5 4.9 |
1.0 15.4 1.0 1.0 1.0 1.1 0.7 0.2 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000 |
100 72 1587 98 86 66 75 73 37 |
107 104 1549 81 80 61 61 63 33 |
101 106 1548 92 61 87 69 27 35 |
103 94 1561 90 76 71 68 54 35 |
3.8 19.1 22.2 8.6 13.1 13.8 7.0 24.2 2.0 |
1.0 16.6 1.0 0.8 0.8 0.7 0.6 0.4 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#sodium azide 10 µg/plate
##2-aminoanthracene 2.5 µg/plate
Experiment I: Plate Incorporation Test
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 141103)
Teststrain: TA102
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants / s.d. |
plate factor* |
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000 |
285 254 1426 265 308 224 232 77 5 |
278 245 1489 288 302 267 244 70 12 |
263 261 1778 282 305 260 207 95 15 |
275 253 1564 278 305 250 228 81 11 |
11.2 8.0 187.7 11.9 3.0 23.1 18.9 12.9 5.1 |
1.0 6.2 1.1 1.2 1.0 0.9 0.3 0.0 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants / s.d. |
plate factor* |
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000 |
272 288 750 352 308 224 212 135 53 |
278 270 835 379 325 219 215 138 48 |
283 276 978 360 371 222 226 136 27 |
278 278 854 364 335 222 218 136 43 |
5.5 9.2 115.2 13.9 32.6 2.5 7.4 1.5 13.8 |
1.0 3.1 1.3 1.2 0.8 0.8 0.5 0.2 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#methyl methane sulfonate 4 µL/plate
##2-aminoanthracene 10 µg/plate
12.2 Experiment II: Pre-IncubationTest
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 141103)
Teststrain: TA1535
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 10 33 100 333 1000 2500 5000 |
29 25 383 31 36 36 30 39 36 16 |
28 25 352 30 30 26 25 27 32 19 |
29 23 422 39 18 38 20 37 34 15 |
29 24 386 33 28 33 25 34 34 17 |
0.6 1.2 35.1 4.9 9.2 6.4 5.0 6.4 2.0 2.1 |
1.0 15.8 1.4 1.2 1.4 1.0 1.4 1.4 0.7 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 10 33 100 333 1000 2500 5000 |
26 26 141 27 24 20 27 15 16 11 |
24 20 182 24 29 21 28 26 23 11 |
20 26 158 21 24 14 22 23 17 13 |
23 24 160 24 26 18 26 21 19 12 |
3.1 3.5 20.6 3.0 2.9 3.8 3.2 5.7 3.8 1.2 |
1.0 6.7 1.0 1.1 0.8 1.1 0.9 0.8 0.5 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#sodium azide 10 µg/plate
##2-aminoanthracene 2.5 µg/plate
printed in bold = reduced background growth observed
Experiment II: Pre-Incubation Test
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 141103)
Teststrain: TA1537
without S9 mix
Concentration µg/plate |
Plate 1 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 10 33 100 333 1000 2500 5000 |
10 8 8 11 79 85 8 7 7 4 9 7 9 11 11 8 36 21 n.a. n.a. |
10 9 80 4 5 9 7 6 1 n.a. |
9 9 81 6 5 8 9 8 19 -- |
1.2 1.5 3.2 2.1 1.5 1.2 2.0 2.5 17.6 -- |
1.0 8.7 0.7 0.6 0.9 1.0 0.9 2.1 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 10 33 100 333 1000 2500 5000 |
21 12 124 25 19 19 28 25 19 12 |
17 14 83 24 26 21 25 28 18 6 |
21 18 108 21 21 23 26 26 14 5 |
20 15 105 23 22 21 26 26 17 8 |
2.3 3.1 20.7 2.1 3.6 2.0 1.5 1.5 2.6 3.8 |
1.0 7.2 1.6 1.5 1.4 1.8 1.8 1.2 0.5 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#4-nitro-o-phenylene-diamine 50 µg/plate
##2-aminoanthracene 2.5 µg/plate
printed in bold = reduced background growth observed
n.a = not evaluable, no distinction possible between reverants and reduced background growth
Experiment II: Pre-Incubation Test
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 141103)
Teststrain: TA98
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 10 33 100 333 1000 2500 5000 |
27 27 262 21 22 18 17 5 1 1 |
19 21 280 20 24 25 18 3 0 2 |
27 24 306 21 22 24 19 0 1 1 |
24 24 283 21 23 22 18 3 1 1 |
4.6 3.0 22.1 0.6 1.2 3.8 1.0 2.5 0.6 0.6 |
1.0 11.8 0.9 0.9 0.9 0.8 0.1 0.0 0.1 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 10 33 100 333 1000 2500 5000 |
32 37 530 33 41 38 35 31 19 12 |
45 41 525 35 36 31 28 29 18 20 |
32 30 662 40 34 37 29 34 18 13 |
36 36 572 36 37 35 31 31 18 15 |
7.5 5.6 77.7 3.6 3.6 3.8 3.8 2.5 0.6 4.4 |
1.0 15.9 1.0 1.0 1.0 0.9 0.9 0.5 0.4 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#4-nitro-o-phenylene-diamine 10 µg/plate
##2-aminoanthracene 2.5 µg/plate
printed in bold = reduced background growth observed
Experiment II: Pre-Incubation Test
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 141103)
Teststrain: TA100
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 10 33 100 333 1000 2500 5000 |
144 122 610 123 107 137 126 54 12 5 |
151 131 628 136 119 134 130 50 14 3 |
169 134 654 127 131 130 128 64 7 18 |
155 129 631 129 119 134 128 56 11 9 |
12.9 6.2 22.1 6.7 12.0 3.5 2.0 7.2 3.6 8.1 |
1.0 4.9 1.0 0.9 1.0 1.0 0.4 0.1 0.1 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 10 33 100 333 1000 2500 5000 |
164 123 901 137 146 123 130 111 102 68 |
157 144 1014 121 124 139 115 125 117 98 |
161 121 853 125 141 114 141 119 119 63 |
161 129 923 128 137 125 129 118 113 76 |
3.5 12.7 82.7 8.3 11.5 12.7 13.1 7.0 9.3 18.9 |
1.0 7.1 1.0 1.1 1.0 1.0 0.9 0.9 0.6 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#sodium azide 10 µg/plate
##2-aminoanthracene 2.5 µg/plate
printed in bold = reduced background growth observed
Experiment II: Pre-Incubation Test
Testitem: NAPHTHALENE-1,5-DIOL
S9 mix from : Rat liver (Batch R 141103)
Teststrain: TA102
without S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Rev mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control# 10 33 100 333 1000 2500 5000 |
130 132 790 124 131 142 141 126 2 1 |
166 141 817 131 124 140 139 133 16 5 |
136 131 789 128 128 129 144 116 14 3 |
144 135 799 128 128 137 141 125 11 3 |
19.3 5.5 15.9 3.5 3.5 7.0 2.5 8.5 7.6 2.0 |
1.0 5.9 0.9 0.9 1.0 1.0 0.9 0.1 0.0 |
with S9 mix
Concentration µg/plate |
1 |
Plate 2 |
3 |
Reve mean |
rtants s.d. |
/ plate factor* |
Negative Control Solvent Control Positive Control## 10 33 100 333 1000 2500 5000 |
147 158 763 123 141 126 89 64 77 20 |
160 167 701 147 121 129 71 76 79 8 |
170 155 851 133 134 119 88 71 102 18 |
159 160 772 134 132 125 83 70 86 15 |
11.5 6.2 75.4 12.1 10.1 5.1 10.1 6.0 13.9 6.4 |
1.0 4.8 0.8 0.8 0.8 0.5 0.4 0.5 0.1 |
* enhancement factor=
Σ revertants / concentr. test item
Σ revertants / solvent control
#methyl methane sulfonate 4 µL/plate
##2-aminoanthracene 10 µg/plate
printed in bold = reduced background growth observed
12.3 Summary ofResults
Testitem: NAPHTHALENE-1,5-DIOL
|
with S9 Mix
Concentration µg/plate |
Revertants/plate mean from three plates TA1535 TA1537 TA98TA100TA 102 I II I II III I II I II |
||||
Negative Control Solvent Control Positive Control## 10 33 100 333 1000 2500 5000 |
12 23 11 24 435 160 / 24 8 26 6 18 9 26 8 21 9 19 0 12 |
10 20 10 15 215 105 / / 7 23 12 22 11 21 9 26 10 26 21 17 |
31 36 27 36 150 572 / 36 18 37 23 35 28 31 30 31 27 18 29 15 |
103 161 94 129 1561 923 / 128 90 137 76 125 71 129 68 118 54 113 35 76 |
278 159 278 160 854 772 / 134 364 132 335 125 222 83 218 70 136 86 43 15 |
#Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100
4-nitro-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate) Methyl methane sulfonate (4 µL/plate) strain TA 102
##2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100
2-aminoanthracene (10.0 µg/plate) strain TA 102 printed in bold = reduced background growth observed
n.a = not evaluable, no distinction possible between reverants and reduced background growth
/ = not performed
The assay was performed in two independent experiments, using two parallel cultures each.
The first main experiment was performed with a treatment period of 4 h with and without metabolic activation. The experimental part of the first experiment with metabolic activation was repeated in the second experiment since less than the required four analysable concentrations were available above the threshold of toxicity of 10 % surviving cells.
In the second experiment the cells were treated for 24 h without and for 4 h with metabolic activation.
Due to the exceedingly high toxicity the second experiment with metabolic activtion was terminated prematurely and repeated with lower concentrations (experiment IIA). The results of the repeated part are reported in experiment II. The raw data of all experiments will be archived under the RCC Project number of the present study.
The test item was evaluated at the following concentrations in experiment I:
Experiment I
without S9 mix: with S9 mix:
15.0; 20.0; 30.0; 40.0; and 60.0µg/ml
0.25; 0.5; 1.0; 2.0 µg/ml
Experiment II
without S9mix: with S9mix:
6.25; 12.5; 25.0; 37.5; and 50.0 µg/ml
0.125; 0.25; 0.50; 0.75; 1.0, and 1.25 µg/mL
In the second experiment without metabolic activation (24 h treatment) toxic effects occurred at 37.5 µg/ml and above. In the second experiment with metabolic activation relevant toxicity was already observed at 1.0 µg/ml and above.
No reproducible increase of the mutation frequency was observed in the first experiment in the absence of metabolic activation. The induction factor exceeded the limit of twice the mutation frequency of the solvent control in the second culture at 40 µg/ml. However, the absolute value of the mutation frequency still remained within the historical range of solvent controls and the increase of the induction factor is based on the relatively low solvent control. In the presence of metabolic activation a moderate increase of the mutation frequency just exceeding the threshold of 2.0 occurred in the second cultureat
2.0 µg/ml. Toxicity was severe at this concentration leading to a relative cloning efficiency
1 of only 5.1 and a relative total growth of 11.9. In the second experiment without metabolic activation an increase of the induction factor to values of 1.9 and 2.2 was observed at the maximum concentration of 50 µg/ml. However, toxicity was severe at this concentration leading to a corresponding relative total growth of 11.8 in culture I and 6.6in
culture II. So the threshold of 2.0 was only exceeded at a relative total growth below 10 %. In the presence of metabolic activation an isolated increase of the mutation frequency was recorded at the maximum concentration of 1.25 µg/ml in culture II (induction factor of 2.6). No comparable effect was observed in the parallel culture under identical conditions, so this isolated increase is considered as not reproduced and therefore, biologically irrelevant. In general, the increases of the mutation frequency observed in this experiment coincide with severe toxic effects. Since these increases were not really reproduced in the parallel culture under identical conditions, secondary toxic effects are judged as the reason for these effects. It is known, that secondary effects like selection phenomena or endonucleases released from lysosomes at highly toxic concentrations may lead to seemingly enhanced mutation frequencies. Severe toxic effects often trigger intracellular signalling pathways leading to apoptosis. The DNA of L5178Y cells however, contains two mutated P53 alleles leading to impaired apoptosis (9). The failure of apoptosis renders such cells susceptible to ill defined genetic rearrangements that may lead toartefacts.
In this study the range of the negative and solvent controls was from 72 up to 151 mutant colonies per 106cells; the range of the groups treated with the test item was from 92 up to 386 mutant colonies per 106cells.
MMS (13 µg/ml) and CPA (3.0 µg/ml in experiment I, 4.5 µg/ml in experiment II) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus largecolonies.
Conclusion
In conclusion it can be stated that during the mutagenicity test described and under the ex perimental conditions reported the test item did not induce mutations in the mouse lym phoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, naphthalene-1,5-diol is considered to be non-mutagenic in this mouse lymphoma assay.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
A study was performed to investigate the potential of naphthalene-1,5-diol to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Therefore, naphthalene-1,5-diol is considered to be non-mutagenic in this micronucleus assay.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2004-07-14 to 2005-04-25
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1997
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian bone marrow chromosome aberration test
- Species:
- mouse
- Strain:
- NMRI
- Details on species / strain selection:
- The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Strain: NMRI
Source: RCC Ltd., Animal Breeding Services; CH-4414 Füllinsdorf
Number of animals: 90 (54 males/36 females)
Initial age at start of acclimatization: Males: 5 - 8 weeks
Females: 7 - 10 weeks
Acclimatisation: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 36.0 g (SD ± 1.8 g)
females mean value 36.0 g (SD ± 1.9 g)
According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.
The animals were distributed into the test groups at random and identified by cage number.
Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding
(Harlan Winkelmann GmbH, D-33178 Borchen)
Feed: pelleted standard diet, ad libitum
(Harlan Winkelmann GmbH, D-33178 Borchen)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
Environment: temperature 22 ± 3 °C
relative humidity 30 - 83 % artificial light 6.00 a.m. - 6.00 p.m. - Route of administration:
- intraperitoneal
- Vehicle:
- The test item was formulated in aqueous DMSO (30%). The vehicle was chosen to its relative non-toxicity for the animals.
- Details on exposure:
- Pre-Experiment for Toxicity
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain; vehicle; route, frequency, and volume of administration.
The animals were treated i.p. with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.
Dose Selection
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.
Study Procedure
Test Groups:
Six males and six females were assigned to each test group.
Treatment:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time.
Analysis of the Test Item Concentration in Blood
In order to quantify the concentration of the test item in blood 3 additional males per sampling interval were treated with 50 mg test item/kg b.w. intraperitoneally. 20 and 40 minutes as well as 1 and 4 hours after the treatment, the animals were sacrificed and their blood was collected and stored at – 80 °C until shipment on dry ice to the sponsor (Henkel KGaA,VTF-HSA, Henkelstraße 67, D-40191 Düsseldorf). Due to a technical error (no anti- coagulant was added to the blood samples), this part of the experiment had to be repeated.
The test item concentration in the blood samples was analysed in a separate study by the sponsor. - Duration of treatment / exposure:
- The animals received the test item, the vehicle or the positive control substance once.
- Frequency of treatment:
- The animals received the test item, the vehicle or the positive control substance once.
- Post exposure period:
- The animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test item.
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively. - Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Remarks:
- On the basis of 8 pre-exepriments, 50 mg/kg b.w. were estimated to be suitable as the high dose. The volume administered was 10 mL/kg b.w.
- Dose / conc.:
- 25 mg/kg bw/day (nominal)
- Remarks:
- The volume administered was 10 mL/kg b.w.
- Dose / conc.:
- 12.5 mg/kg bw/day (nominal)
- Remarks:
- The volume administered was 10 mL/kg b.w.
- No. of animals per sex per dose:
- In the main experiment for the highest dose group 24 animals (12 males, 12 females) received i.p. a single dose of 50 mg/kg b.w. of naphthalene-1,5-diol formulated in aqueous DMSO (30%) .
For the mid dose group 12 animals (6 males, 6 females) received i.p. a single dose of 25 mg/kg b.w. naphthalene-1,5-diol formulated in aqueous DMSO (30%).
For the low dose group 12 animals (6 males, 6 females) received i.p. a single dose of 12.5 mg/kg b.w. naphthalene-1,5-diol formulated in aqueous DMSO (30%) .
The volume administered was 10 mL/kg b.w.. - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Name: CPA; Cyclophosphamide
Supplier: Sigma-Aldrich Vertriebs GmbH
82041 Deisenhofen
Catalogue no.: C 0768 (purity: > 98%)
Dissolved in: deionised water
Dosing: 40 mg/kg b.w.
Route and frequency of administration: intraperitoneally, once
Volume administered: 10 mL/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is sufficient. At 25°C only 3.5 % of its potency is lost after 24 hours (7). - Tissues and cell types examined:
- The animals were anaesthetised with CO2 and sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded.
- Details of tissue and slide preparation:
- A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.
- Evaluation criteria:
- Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining 6th animal of each sex is usually evaluated in case an animal dies in its test group spontaneously. - Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. - Executive summary:
The test item naphthalene-1,5-diol was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
The test item was formulated in aqueous DMSO (30%) , which was also used as vehicle control. The volume administered i.p. was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.
Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.
To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.
The following dose levels of the test item were investigated:
24 h preparation interval: 12.5, 25 and 50 mg/kg b.w.. 48 h preparation interval: 50 mg/kg b.w..
As estimated by pre-experiments 50 mg naphthalene-1,5-diol per kg b.w. was the highest applicable dose without significant effects on the survival rates. At this dose the animals showed clear signs of toxicity. At a higher dose (75 mg/kg) one male and one female animal died.
The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that A 018 had no cytotoxic properties in the bone marrow. The analysis of the blood samples of the males (see annex 2) treated with 50 mg test item /kg b.w. showed, that the test item could be quantified in the blood of the treated animals only in the blood samples taken 20 minutes after the treatment (2.5, 1.0 and 0.5 µg/mL). The samples from the later time points did not contain any quantifiable levels of the test item (data of the 1 and 4 h samplings not included). Thus, the bioavailability of the test item could be confirmed.
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with naphthalene-1,5-diol were below or near to the value of the vehicle control group.
40 mg/kg b.w. cyclophosphamide administered i.p. was used as positive control which showed a statistically significant increase of induced micronucleus frequency.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Reference
Toxic Symptoms in the Main Experiment
In the main experiment for the highest dose group 24 animals (12 males, 12 females) received i.p. a single dose of 50 mg/kg b.w. A 018 formulated in aqueous DMSO (30%) . The volume administered was 10 mL/kg b.w..
The animals treated with 50 mg/kg b.w. expressed toxic reactions as shown in the table:
Toxic
Reactions |
hours post-treatment hours post-treatment male / female |
||||
1 h |
2-4 h |
6 h |
24 h |
48 h* |
|
reduction of spontaneous activity |
12/12 |
12/12 |
12/12 |
12/9 |
3/2 |
abdominal position |
12/12 |
12/12 |
12/12 |
0/0 |
0/0 |
ruffled fur |
12/12 |
12/12 |
12/12 |
12/5 |
2/1 |
apathy |
1/2 |
0/0 |
0/0 |
0/0 |
0/0 |
*: data only from 6 animals per sex.
For the mid dose group 12 animals (6 males, 6 females) received i.p. a single dose of 25 mg/kg b.w. A 018 formulated in aqueous DMSO (30%) . The volume administered was 10 mL/kgb.w..
The animals treated with 25 mg/kg b.w. expressed toxic reactions as shown in the table:
toxic reactions |
hours post-treatment male / female |
|||
1 h |
2-4 h |
6 h |
24 h |
|
reduction of spontaneous activity |
6/6 |
6/6 |
6/6 |
4/3 |
abdominal position |
1/2 |
4/5 |
3/2 |
0/0 |
ruffled fur |
5/6 |
6/6 |
6/6 |
4/3 |
For the low dose group 12 animals (6 males, 6 females) received i.p. a single dose of
12.5 mg/kg b.w. A 018 formulated in aqueous DMSO (30%) . The volume administered was 10 mL/kg b.w..
The animals treated with 12.5 mg/kg b.w. expressed toxic reactions as shown in the table:
toxic reactions |
hours post-treatment male / female |
|||
1 h |
2-4 h |
6 h |
24 h |
|
reduction of spontaneous activity |
2/3 |
6/6 |
6/6 |
4/3 |
ruffled fur |
3/2 |
3/3 |
1/2 |
2/1 |
Summary of Micronucleus Test Results
test group |
dose mg/kg b.w. |
sampling time (h) |
PCEs with micronuclei (%) |
range |
PCE per 2000 erythrocytes |
vehicle |
0 |
24 |
0.105 |
0 -4 |
1099 |
test item |
12,5 |
24 |
0.080 |
0 -4 |
1124 |
test item |
25 |
24 |
0.105 |
0 -4 |
1096 |
test item |
50 |
24 |
0.080 |
0 -4 |
1082 |
positive control |
40 |
24 |
2.270 |
20 -76 |
1035 |
test item |
50 |
48 |
0.090 |
0 -4 |
1089 |
Biometry
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Vehicle control versus test group |
Significance |
p |
12.5 mg A 018/kg b.w.; 24 h |
n.t. |
- |
25 mg A 018/kg b.w.; 24 h |
n.t. |
- |
50 mg A 018/kg b.w.; 24 h |
n.t. |
- |
40 mg CPA/kg b.w.; 24 h |
+ |
< 0.0001 |
50 mg A 018/kg b.w.; 48 h |
n.t. |
- |
- = not significant
+ = significant;
n.t = not tested, as the mean micronucleus frequency was not above the vehicle controlvalue
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. These changes may involve a single gene or gene segment, a block of genes or chromosomes.
According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:
-substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans (Category 1) or
-substances, which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans (Category 2).
Based on results of in vitro and in vivo gene mutation studies performed, it is concluded that the test item did not induce gene mutations in any of the three studies.
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