Registration Dossier

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

Referenceopen allclose all

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000-05-19
Deviations:
no
GLP compliance:
yes (incl. certificate)
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

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

 

 

Concentration

µg/plate

Revertants/plate mean from three plates

TA1535       TA1537         TA98          TA100         TA 102 I        II        I        II        I        II        I        II        I         II

Negative Control Solvent Control Positive Control#

10

33

100

333

1000

2500

5000

14      29

17      24

386    386

/       33

14      28

15      33

15      25

12      34

6       34

3       17

6        9

7        9

52      81

/        6

10       5

9        8

6        9

9        8

4       19

0      n.a.

24      24

26      24

108    283

/       21

23      23

20      22

21      18

25       3

15       1

5        1

85     155

79     129

1218   631

/      129

83     119

81     134

81     128

84      56

55      11

16       9

275    144

253    135

1564   799

/      128

278    128

305    137

250    141

228    125

81      11

11       3

 

S9 mix from: Rat liver (Batch R 071103 and R 141103) without S9 mix

 

 

 

 

 

 

 

 

 

 

 

 

 

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

 

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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
February 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
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. 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 experiment I relevant toxicity (relative cloning efficiency 1 and/ or relative total growth of less than 50 %) was detected at 60.0 µg/ml in the absence of metabolic activation. In the presence of metabolic activation the test item induced severe toxic effects below the limit of 10% survival at 2.0 µg/ml and above.

 

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.

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.

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
Reference
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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
GLP compliance:
yes (incl. certificate)
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 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

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


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.

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.