Registration Dossier
Registration Dossier
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EC number: 201-487-4 | CAS number: 83-56-7
Genetic toxicity: in vitro
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 004
- Report date:
- 2004
Materials and methods
Test guidelineopen allclose all
- 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
Test material
- Reference substance name:
- Naphthalene-1,5-diol
- EC Number:
- 201-487-4
- EC Name:
- Naphthalene-1,5-diol
- Cas Number:
- 83-56-7
- Molecular formula:
- C10H8O2
- IUPAC Name:
- naphthalene-1,5-diol
- Reference substance name:
- unknown
- Molecular formula:
- none
- IUPAC Name:
- unknown
Constituent 1
impurity 1
- 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.
Method
Species / strain
- 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
Controls
- 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).
Results and discussion
Test resultsopen allclose all
- 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
Any other information on results incl. tables
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
Applicant's summary and conclusion
- 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.
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