Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1-Naphthol has been sufficiently investigated for the three types of mutations: gene mutation, structural and numerical chromosomal aberrations:

  • 1-Naphthol did not induce gene mutations in bacteria.
  • 1-Naphthol induced clastogenic effects in Chinese hamster ovary cells and gene mutations in the mouse lymphoma assay with mammalian cells (L5178Y) in the absence of metabolic activation. The increase in small colonies may indicate clastogenicity. These are in vitro studies.

1-Naphthol, itself, is not considered to have in vivo genotoxic potential.

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2004 - August 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
yes
Remarks:
The first trial and the third trial (in the presence of metabolioc activation) wereunacceptable due to excessive cytotoxicity. The second trial in the presence of metabolic activation was terminated due to a technical error.
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: n°batch I-38110
- Expiration date of the lot/batch: 14 Jan 2006
- Purity test date: No data
- Purity : 99.9%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No

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

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

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

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

NUMBER OF REPLICATIONS: one per concentration

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

NUMBER OF CELLS EVALUATED: 3 x 10E6

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

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

For the mutant Selection Data
The mutant selection data includes the total mutant colonies and the total viable colonies. These were used to calculate the mutant frequency for each culture. The ratio of cells seeded for mutant selection to cells seeded for cloning efficiency was 0.5 x 104. Therefore, the mutant frequency was: (total mutant colonies/total viable colonies) x 2 x 10-4. Mutant frequency was given in units of 10-6. Also included in this section was the absolute cloning efficiency of vehicle and positive controls calculated as (total number of viable colonies/600) x 100%.
Statistics:
Various models of calculators, computers, and computer programs were used to analyze data in this study. Because different models round off or truncate numbers differently, values in some tables (e.g., means, standard deviations, or individual values) may differ slightly from those in other tables, from individually calculated data, or from statistical analysis data. Neither the integrity nor the interpretation of the data was affected by these differences.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 90 µg/L, 4h exposure
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not affected.
- Effects of osmolality: not increased
- Evaporation from medium : not specified
- Water solubility : no specified
- Precipation : not affected
Other cofounding effects : none

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

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

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

Initial Nonactivation Mutation Assay (B3)

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

Initial Activation Mutation Assay (B4)

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

positive response.

Confirmatory Mutation Assay

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

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

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

Results

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

Conclusion

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

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2004
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:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Substance : GTS03979 (1-naphthol)
- Source and lot/batch No.of test material: Lot n°I-38110
- Expiration date of the lot/batch: 14 January 2006
- Purity test date: No Data
- Purity : 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: Yes
- Solubility and stability of the test substance in the solvent/vehicle: 9 days at -10 to -30°C (based on concentration analysis)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No

FORM AS APPLIED IN THE TEST Suspension
Target gene:
Histidine locus in the genome of several strains of Salmonella Typhimurium
Tryptophan locus of E. Coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Source : Dr Bruce Ames, Department of Biochemistry, University of California,

Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
Source : National Collection of Industrial and Marine Bacteria Torrey Research Station Scotland (United Kingdom)
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat livers (Aroclor TM induced)
Test concentrations with justification for top dose:
In the initial mutagenicity assay : 2.5 ; 5 ; 20 ; 50 ; 200 ; 500 ; 2000 ; 5000 µg/plate
In the confirmatory assay : 2.5 ; 10 ; 25 ; 50 ; 100 ; 200 ; 500 µg/ plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (Supplier Acros Organics)
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene (Purity >=97.3%) ; ICR-191 (Purity >=94%)
Details on test system and experimental conditions:
Initial Mutagenicity Assay
The initial mutagenicity assay was conducted by using the preincubation method. All five tester strains were tested concurrently against eight concentrations of the test article.The assay was performed with two plates prepared for each dose and all relevant controls for each strain. The vehicle control contained the same vehicle concentrations used in the test. Following approximately 48- to 72-hour incubation at 37 ± 2°C, revertant colonies per plate were determined. All other aspects of the test procedure were performed as outlined in the approved Test Facility SOPs.

Preincubation Method
This methodology has been shown to detect a wide range of classes of chemical mutagens. In the preincubation methodology, the S9 mix (or phosphate buffer, where appropriate), the tester strain, and the test article were preincubated prior to the addition
of molten agar. The S9 mix and dilutions of the test article were prepared immediately prior to their use. When S9 mix was required, 500 μL of S9 mix was added to 13 x 100 mm glass culture tubes, which were pre-heated to 37 ± 2°C. To these tubes,
100 μL of tester strain and 50 μL of vehicle or test article concentration, and 50 μL of positive control article were added. When S9 mix was not required, 500 μL of 0.1M phosphate buffer was substituted for the S9 mix. After the required components had been added, the mixture was vortexed and allowed to incubate for 20 ± 2 minutes at 37 ±2°C. Two mL of molten selective top agar was then added to each tube, and the mixture was then vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and incubated for 60 ± 12 hours at 37 ± 2°C. Following incubation, revertant colonies were counted.

Sterility Controls
For each assay Trial, all test article dilutions were checked for sterility by adding a 50 μL aliquot (the same volume used in the assay) to the overlay agar and then poured over the minimal agar. The S9 mix was checked for sterility by adding 0.5 mL to the overlay agar and then poured over the minimal agar.

Scoring the Plates
Plates which were not evaluated immediately following the incubation period were held at >0°C to 10°C until such time that colony counting and bacterial background lawn evaluation could take place.
Evaluation criteria:
Criteria for valid test : sterility controls, tester strain integrity, tester strain culture density, Positive control values in the absence of S9 mix, Positive control values in the presence of S9 mix, Cytotoxicity.

Evaluation of Test Results
- Tester Strains TA98, TA100, and WP2uvrA(pKM101). For a test article to be considered positive, at a peak of a dose-response it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
- Tester Strains TA1535 and TA1537. For a test article to be considered positive, at a peak of a dose response it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
Statistics:
The use of statistics was limited to the calculation of means and standard deviations where appropriate
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:

HISTORICAL CONTROL DATA : yes (Historical control data for tester strain WP2uvrA(pKM101) using the preincubation method was not available and therefore not reported. This deviation did not adversely impact the integrity of this study)

In the confirmatory mutagenicity assay, the increase inrevertantsobserved with tester strain TA98 in the presence of S9 mix in the initial mutagenicity assay was not reproduced in the confirmatory assay.

In the repeat initial and confirmatory mutagenicity assays, all control values were acceptable. No positive increases in the mean number ofrevertantsper plate were observed with tester strain TA98 in the presence of S9 mix.

The response observed with tester strain TA98 in the initial assay was not reproducible in the confirmatory assay, or in the repeat assay. In the repeat assay, there was no difference in the results obtained with the dose formulations used immediately after preparation or used 2 hours after preparation. Since the initial response was not reproducible, the increase observed in the initial mutagenicity assay is considered to be spurious. The overall evaluation is that the test article did not cause a positive increase with tester strain TA98 in the presence of S9 mix.

 

Table 1 – Mutagenicity Assay Results – Summary – Preincubation Method (initial test)

 

Dose/ plate

 µg

TA98

TA100

TA1535

TA1537

WP2uvrA(pKM101

BackgroundLawn

 

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

 

With activation

VehicleControl

17

1

81

25

11

3

6

1

191

54

N

 

Test Article

2.5

24

0

107

20

9

8

5

1

173

1

N

 

5

41*

29

96

3

12

6

5

1

182

19

N

 

20

84*

11

84

16

12

4

6

1

171

7

N

 

50

122*

9

95

6

11

3

5

2

136

23

N

 

200

21

4

67

19

9

2

7

1

97

18

N

 

500

0

0

0

0

0

0

4

5

62

1

R/A

 

2000

0

0

0

0

0

0

0

0

0

0

RP/AP

 

5000

0

0

0

0

0

0

0

0

0

0

AP

 

Positive Control

406

57

1264

192

101

7

183

21

1393

44

N

 

Without activation

VehicleControl

9

4

80

6

9

4

7

 

115

16

N

 

Test Article

2.5

12

1

74

9

10

2

3

1

95

10

N

 

5

14

4

77

13

13

1

7

2

100

4

N

 

20

12

1

73

1

7

4

4

1

86

0

N

 

50

12

2

83

7

7

2

5

4

120

19

N

 

200

10

0

77

13

11

4

8

2

96

11

N/R

 

500

0

0

0

0

0

0

0

0

0

0

R

 

2000

0

0

0

0

0

0

0

0

0

0

A

 

5000

0

0

0

0

0

0

0

0

0

0

A

 

Positive Control

319

9

1243

32

696

42

1909

205

2772

43

N

 

Background Lawn Evaluation Codes: N = normal R = reduced O = obscured A = absent P = precipitate

(*) An increase >2-fold the mean vehicle control value was observed.

  

Table 2 – Mutagenicity Assay Results – Summary – Preincubation Method (conformatory test)

 

Dose/ plate

 µg

TA98

TA100

TA1535

TA1537

WP2uvrA(pKM101

Background Lawn

 

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

 

With activation

Vehicle Control

19

3

93

15

12

5

6

2

198

23

N

 

Test Article

2.5

18

5

103

6

9

4

6

1

201

15

N

 

10

28

3

105

10

11

5

5

2

180

16

N

 

25

23

7

116

15

11

2

8

4

191

28

N

 

50

22

0

120

9

10

4

6

5

197

26

N

 

100

20

4

100

20

11

2

6

1

161

6

N

 

200

23

4

96

5

11

5

7

4

139

4

N

 

500

10

4

70

5

12

3

4

2

74

13

N/R

 

Positive Control

554

17

1607

108

163

7

187

23

1547

255

N

 

Without activation

Vehicle Control

12

5

68

3

11

5

6

4

95

41

N

 

Test Article

2.5

16

3

92

10

10

4

5

1

452

7

N

 

10

15

3

92

12

12

2

7

2

153

17

N

 

25

18

3

93

4

13

3

7

1

177

22

N

 

50

16

4

97

11

14

6

7

1

163

18

N

 

100

17

2

97

6

19

5

4

2

158

22

N

 

200

13

4

95

14

13

8

7

1

135

15

N

 

500

0

0

0

0

0

0

0

0

4

4

N/A

 

Positive Control

369

15

1662

66

905

57

2595

116

1897

179

N

 

Background Lawn Evaluation Codes: N = normal R = reduced O = obscured A = absent P = precipitate

 

 

Table 3 – Mutagenicity Assay Results – Summary – Preincubation Method (Repeat of trial)

 

Dose/ plate

 µg

0Hour

BackgroundLawn

2Hour

BackgroundLawn

Mean

SD

Mean

SD

Repeat of Trial n°B1 (with activation)

VehicleControl

20

4

N

28

8

N

Test Article

2.5

24

9

N

34

8

N

5

19

4

N

29

7

N

20

17

3

N

27

13

N

50

15

8

N

29

5

N

200

10

1

R

21

2

N

500

0

0

R

13

1

R

2000

0

0

A

0

0

A

5000

0

0

AP

0

0

AP

Positive Control

482

92

N

503

100

N

Repeat of Trial n°C1 (with activation)

Vehicle Control

38

15

N

38

12

N

Test Article

2.5

28

2

N

36

2

N

10

28

3

N

22

4

N

25

28

1

N

27

3

N

50

18

8

N

29

8

N

100

25

5

N

26

2

N

200

21

3

R

19

2

N

500

9

10

R

9

5

R

Positive Control

414

81

N

487

83

N

Background Lawn Evaluation Codes: N = normal R = reduced O = obscured A = absent P = precipitate

 

 

 

Conclusions:
Under the test conditions used 1-naphthol did not induce gene mutations in bacteria.
Executive summary:

In the initial test, signs of toxicity were observed at ≥ 500 μg/plate in the presence of metabolic activation and at ≥ 200 μg/plate in the absence of metabolic activation. Except TA 98 with metabolic activation, there were no indications of an increase in the mutant frequency at any concentration in the tester strains either with or without metabolic activation. In TA 98 with metabolic activation, a clear concentration related increase in the mutant frequency until 50 μg/plate was observed with a 7-fold increase. A review of the raw data indicated that the dosing of tester strains TA 98 and TA 100 in the presence of metabolic activation was repeated in the initial test due to a technical problem with the initial dosing. This repeat dosing occurred two hours after the original dosing. In the confirmatory test, the dosing took place as soon as possible following dose formulation. In the confirmatory test there were no indications of an increase in the mutant frequency at any concentration in the tester strains either with or without metabolic activation. Thus, the

increase in mutant frequency with TA98 in the presence of metabolic activation was not reproduced. In order to investigate if the two-hour delay in using the dose formulations had any impact on the response observed with TA98 in the initial test, a repeat assay was performed with TA98 in the presence of S9 mix. In this repeat assay two dose formulations were prepared and one was dosed immediately after preparation and one was dosed two hours after preparation. This repeat assay showed no indications of an increase in the mutant frequency at any concentration and there was no difference in the results obtained

with the formulations used immediately after preparation or used two hours after preparation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2004 - August 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: n°batch I38110 - Test Material : GTSS03979
- Expiration date of the lot/batch: 14 January 2006
- Purity test date: No data
- Purity : 99.9%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING No

Target gene:
None
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-WBL
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Laboratory o Dr. S. Wolff, University of California, San Francisco.
- Cell cycle length, doubling time or proliferation index: 1.5 times a cell cycle time of approximately 13 hours
- Methods for maintenance in cell culture if applicable: The cells were subsequently subcloned in this laboratory and stock cultures stored in liquid nitrogen
- Modal number of chromosomes: chromosome number of 21
- Normal (negative control) cell cycle time: an average cycle time of 12 to 14 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: McCoy’s 5a culture medium which was supplemented with ~10% heat-inactivated fetal bovine serum (FBS), L-glutamine (2mM), penicillin G (100 units/mL), and streptomycin (100 μg/mL). Cultures were incubated with loose caps in a humidified incubator at 37°C ± 2°C in an atmosphere of 5% ± 1.5% CO2 in air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: No data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
NADP and Isocitric acid rat liver S9
Test concentrations with justification for top dose:
7.50 – 400 μg/mL without (4 hour)
1.25 – 120 μg/mL without (~20 hour)
0.500 – 4.00 μg/mL with metabolic activation

The Top dose is based on the results of the initial toxicity assay
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: one day
- Exposure duration: 4h or 20h (without activation) and 4h (with activation)
- Fixation time (start of exposure up to fixation or harvest of cells): 18h (after initiation of treatment)
- Selection time (if incubation with a selection agent): 2h

SELECTION AGENT : Colcemid

NUMBER OF REPLICATIONS: duplicate cultures

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slides were prepared by dropping the harvested cultures on clean, glass slides and air-dried. The slides were stained with 5% Giemsa solution for the analysis of mitotic index and chromosomal aberrations. All slides were then air-dried and mounted permanently.

NUMBER OF CELLS EVALUATED: One hundred cells, if possible, from each replicate culture from at least four concentrations of the test article, negative, vehicle and one dose of the positive control cultures were analyzed for the different types of chromosomal aberrations

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE : Percent polyploidy and endoreduplication were also analyzed by evaluating at least 100 metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index.
- Any supplementary information relevant to cytotoxicity: visual observation

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Evaluation criteria:
Acceptable Controls
The vehicle control cultures must contain less than ~6% cells with aberrations and no single cell will have more than two aberrations (excluding gaps). The positive control result must be significantly higher (p ≤ 0.01) than the vehicle controls.

Acceptable High Dose
If the aberration results are negative and there is no significant reduction (~ ≥50%) in cell growth, the assay must include the highest applicable dose (a target dose of 10 mM or 5 mg/mL, whichever is lower) or a dose exceeding the solubility limit in culture medium.

Acceptable Number of Doses
The assay must include at least three analyzable concentrations.
Statistics:
A statistical analysis of the percent aberrant cells per treatment group compared to the control (vehicle) was made using a one-tailed Fisher's Exact test. The p values were adjusted to take into account multiple dose comparisons using a Bonferroni adjustment.

The average number of aberrations per cell was reported but no statistical analysis will be applied.

If there was a significant increase in the percentage of aberrant cells at one or more doses, then a trend test for the percent aberrant cells was performed to test for evidence of a dose response. A suggested test for trend is the Cochran-Armitage test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 60 µg/L - 4h (increase in cells with polyploidy)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 10 µg/l - 20h (increase in cells vith polyploidy)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
at 2.5µg/l - 4h (increase in cells with chomosomal aberrations)
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no effects
- Evaporation from medium: No data
- Precipitation: In the initial toxicity assay (4-hour treatment without metabolic activation), no precipitation was visible.
- Definition of acceptable cells for analysis: Cells were selected for good morphology and only cells with the number of centromeres equal to the modal number 21 ± 2 (range 19-23) were analyzed.
- Other confounding effects: none

HISTORICAL CONTROL DATA : Yes (positive and negative data)
Remarks on result:
other: structural and numerical aberrations

See data tables of results in attached background material.

Conclusions:
Under the test conditions used the 1-naphthol induced structural aberrations with and without metabolic activation. There was also an indication that the test article has an aneugenic potential and may inhibit cell cycle progression.
Executive summary:

Results

In the initial toxicity assay (4-hour treatment without metabolic activation), no cell growth ≥ 400 μg/ml and precipitation was visible. The reduction in cell growth at 120 μg/ml was 57%. In the presence of metabolic activation, precipitation was visible at 1200 μg/ml. The reduction in cell growth was 72% at 12 μg/ml. In the assay without metabolic activation with 20-hour treatment, precipitation was visible after dosing at 1200 μg/ml. At 120 μg/ml the reduction in cell growth was 99%.

Based on the results of the initial toxicity assay, the main assay was conducted using 11 concentrations between 7.5 and 400 μg/ml for 4-hours treatment without metabolic activation. Chromosomal aberrations were analysed from the cultures treated with 30, 60 and 90 μg/ml, giving reductions in cell growth of 28%, 38% and 50% respectively. A statistically significant increase in cells with polyploidy was observed in the cultures treated with 60 μg/ml. Moreover, a statistically significant increase in cells with endoreduplication at all three concentrations was observed in a concentration-related manner. Finally, a concentration-related increase in cells with chromosomal aberrations was observed and the increase was statistically significant at 90 μg/ml.

In the assay without metabolic activation with 20-hours treatment, precipitation was not observed. The assay was conducted using 12 concentrations between 1.25 and 120 μg/ml. Chromosomal aberrations were analysed from the cultures treated with 5, 10 and 20 μg/ml, giving reductions in cell growth of 9%, 17% and 55% respectively. A statistically significant increase was observed in cells with polyploidy in the cultures treated with 10 μg/ml. There were no indications of increases at any concentration in cells with chromosomal aberrations or endoreduplication.

In the assay with metabolic activation with 4-hours treatment precipitation was not visible. The assay was conducted using 11 concentrations between 0.5 and 4 μg/ml. Chromosomal aberrations were analysed from the cultures treated with 1, 2 and 2.5 μg/ml, giving a

reduction in cell growth of 54% at the highest concentration. There was a concentrationrelated increase in cells with chromosomal aberrations and the increase was statistically significant at 2.5 μg/ml. There were no indications of increases at any concentration in cells with endoreduplication or polyploidy.

Conclusion

Under the test conditions used the 1-naphthol induced structural aberrations with and without metabolic activation. There was also an indication that the test article has an aneugenic potential and may inhibit cell cycle progression.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The genotoxic/clastogenic effects observed in the in vitro assays could not be confirmed in two in vivo assays. There were no signs of genotoxic effects of 1-naphthol tested in vivo, based on micronucleus test of bone marrow in mice and the unscheduled DNA synthesis (UDS) test in rats. 1-Naphthol, itself, is not considered to have in vivo genotoxic potential. These in vivo assays have several advantages over in vitro tests in that the metabolism, pharmacokinetics, and DNA repair processes are all taken into account.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2004 - August 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: n°batch I-38110 - Test material GTS03979 ou 1-naphthol
- Expiration date of the lot/batch: 14 January 2006
- Purity test date: No data
- Purity : 99,9%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: no Data
- Specific activity: 10 µCi/ mL
- Locations of the label: 3H-TdR
- Expiration date of radiochemical substance: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: Yes
- Solubility and stability of the test substance in the solvent/vehicle: Yes

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No

FORM AS APPLIED IN THE TEST : Solution
Species:
rat
Strain:
other:
Remarks:
Crl:CD®(SD)IGS BR
Details on species / strain selection:
This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles Rivers Laboratories, Raleigh, North Carolina
- Age at study initiation: 8-9 weeks old
- Weight at study initiation: 255 to 288 g (timepoint 2-4 hours) - 263 to 293g (timepoint 12-16)
- Assigned to test groups randomly: Yes on the basis of a computer program
- Housing: 2 per cage during acclimation and singly after randomization.
- Diet :ad libitum (PMI Certified Rodent Diet® #5002)
- Water : ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-4°C
- Humidity (%): 55 +/-15%
- Air changes (per hr): 10 or greater air changes/hour,
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Polyethylene Glycol 400 (PEG 400)
- Justification for choice of solvent/vehicle: selected by the Sponsor
- Concentration of test material in vehicle: 175 and 87.5 mg/mL
- Amount of vehicle : 10 mL
- Lot/batch no. (if required): X34621 (Mallickrodt) and 054K0063 (Sigma Chemical)
- Purity: No data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
2-4 and 12-16.4 hours
Dose / conc.:
875 mg/kg bw/day (nominal)
Dose / conc.:
1 750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control and low dose group: 3 males per dose group and per sacrifice time
High dose group: 5 males per sacrifice time
Control animals:
yes, concurrent vehicle
Positive control(s):
- N-dimethylnitrosamine
- Justification for choice of positive control(s): No data
- Route of administration: oral (gavage)
- Doses / concentrations: 10 mg/mL (time-point 2 to 4 hours) - 15 mg/mL (time-point 12 to 16 hours)
Tissues and cell types examined:
rat liver primary cell (hepatocyte)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The highest dose selected for the UDS assay was based on the results of the dose range finding assay and no differences in male and female were observed in toxicity, only males were treated in the UDS assay.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Each rat was anesthetized and a midventral incision was made to expose the liver. The animal were euthanized by exsanguination during the procedure.
- The liver was perfused with 0.5mM ethylene glycol-bis(β-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) solution followed by collagenase solution
- The liver was removed, transected, and shaken in a dilute collagenase solution to release the hepatocytes.
- The cells were pelleted by centrifugation, resuspended in complete Williams' medium E
- Approximately 5 x 105 viable cells were seeded into each of six 35mm tissue culture dishes.
- Plates used for the UDS assay contained 25mm coverslips and preconditioned complete WME
- A minimum of 6 cultures were set up for each rat. The hepatocyte cultures were maintained in a humidified atmosphere of 5±1% CO2 and 35 to 38°C.


DETAILS OF SLIDE PREPARATION:
- 90 to 180 minutes after plating, the cells were washed once with complete WME and refed with serum-free WME containing 10μCi 3H-TdR/mL.
- Four hours later, the radioactive medium was removed, the cultures were washed in serum-free WME containing 0.25 mM thymidine, and then refed with serum-free WME containing 0.25 mM thymidine and incubated for 17-20 hours.
- Seventeen to 20 hours after exposure to thymidine, the coverslips bearing cultures were washed once in serum-free WME. The cells were swelled in 1% sodium citrate solution and the cultures fixed in ethanol-glacial acetic acid fixative (3:1, v/v).
- The coverslips were allowed to air dry for at least 1 hour before mounting cell side up on glass slides.

METHOD OF ANALYSIS:
- All coded slides were read without knowledge of treatment group.
- The slides were viewed microscopically under a 100X oil immersion lens. Prior to scoring, slides were examined for cytotoxicity (irregularly shaped or very darkly stained nuclei , pyknosis,reduced labeling).
- An automated colony counter was interfaced with the microscope so that silver grains within each nuclei and the surrounding cytoplasm could be counted.
- If possible, 50 nuclei were scored from each of three replicate cultures for a total of 150 nuclei from each rat.

Evaluation criteria:
Criteria for a Valid Test
- Cell Culture Condition: The viability of the vehicle control hepatocytes collected from the perfusion process must be at least 50%.
- Acceptable Controls The average net nuclear labeling in the vehicle control cultures must be <1.00. In addition, less than 10% of the cells should be in repair (containing five or more net nuclear grains).

Assay Evaluation Criteria
- A test article will be judged positive if it induces a dose-related increase in the group average mean net nuclear grain count with one dose group significantly above the negative control.
- A significant increase in the group average of the mean net nuclear grain count in both doses in the absence of a dose response is also considered positive.
Statistics:
Yes
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Perfusion
For the early UDS timepoint, the hepatocytes ranged in viability from 52.7% to 86.4% of the total cells collected in the perfusate. The attachment efficiency varied from 18.7% to 103.1%, and the viability of the attached cells was good, ranging from 89.9% to 99.7%.
For the 12- to 16-hour timepoint, the hepatocytes ranged in viability from 29.9% to 93.5% of the total cells collected in the perfusate. The attachment efficiency varied from 0.4% to 152.1%, and the viability of the attached cells was good, ranging from 8.3% to 100.0%.

- Slide Analysis
For the 2- to 4-hour timepoint, the mean net nuclear grain count for the vehicle control animals was 0.82, and that of the treatment groups was 0.53 at 875 mg/kg and 0.48 at 1750 mg/kg. None of the treatment groups yielded a mean net nuclear grain count that
was considered positive for UDS. Thus, no evidence for UDS was obtained at the early timepoint of 2 to 4 hours after treatment of the animals.
For the 12- to 16-hour timepoint, the mean net nuclear grain count for the vehicle control animals was 0.70, and that of the treatment groups was –0.07 at 875 mg/kg and –0.40 at 1750 mg/kg. None of the treatment groups yielded a mean net nuclear grain
count that was considered positive for UDS. Thus, no evidence for UDS was obtained at the timepoint of 12 to 16 hours after treatment of the animals.
The vehicle control results were well within the acceptable criteria for this study.

Table 1 – Summary of UDS Slide data

Treatment

Dose

(mg/kg)

Nb

Time

(hr)

Mean Nuclear

Grains

± SD

Mean Net

Nuclear Grains

± SD

Mean

Cytoplasmic

Grains ± S D

Mean % Cells

with ≥ 5 NNG

± SD

Vehicle

Control

0

3

2-4

4.33 ± 0.90

0.82 ± 0.82

3.50 ± 0.36

7.89 ± 9.66

0

3

12-16

4.38 ± 1.40

0.70 ± 0.76

3.68 ± 0.66

6.78 ± 4.67

Positive

Control

10

3

2-4

24.79 ± 4.15

20.23 ± 4.94

4.55 ± 0.92

95.11± 4.33

15

2*

12-16

13.37 ± 2.55

8.96 ± 0.98

4.40 ± 0.92

68.34 ± 3.30

Test

Material

875

3

2-4

5.18 ± 1.10

0.53 ± 1.01

4.65 ± 0.22

8.57 ± 9.16

3

12-16

4.24 ± 1.25

-0.07 ± 0.45

4.23 ± 0.84

5.00 ± 3.18

1750

3

2-4

3.59 ± 0.51

0.48 ± 0.59

3.11 ± 0.79

3.33 ± 2.00

3

12-16

371 ± 0.62

-0.40 ± 0.56

4.12 ± 0.25

3.64 ± 2.16

(*) One of the positive control animals at the 12-16 hour timepoint was considered inacceptable for evaluation due to the availability of a very small number of cells, and the poor morphology of the cells that were available. Evaluation of the remaining two animals in the group is sufficient to meet the requirements of this study

Survival and Clinical Observations

No mortality occurred. With the exception of one high dose animal at the 2-4 hour timepoint observed with salivation, all animals appeared normal immediately after dosing.

Prior to perfusion, all positive control animals and animals treated with 875 mg/kg of test article at the 2-4 hour timepoint were normal. All animals treated with 875 mg/kg of test article at the 12-16 hour timepoint were observed with soft feces. One vehicle control animal was observed with fecal stains, and another with soft feces. One high dose animal at the 2-4 hour timepoint was found to be slightly hypoactive with irregular respiration, piloerection and squinted eyes. All other high dose 2-4 hour timepoint animals were normal. Clinical observations seen in the high dose animals at the 12-16 hour timepoint included soft feces, slighthypoactivityand irregular respiration.

Bodyweight. No treatment related

Conclusions:
Under the test conditions used 1-naphthol is not considered genotoxic in the in vivo unscheduled DNA synthesis test.
Executive summary:

In a dose range-finding study, three male and three female rats per dose level were dosed once by oral gavage. The animals were dosed at 1500 or 2000 mg/kg bw and observed for up to 2 days after dosing for toxic signs and/or mortality. Clinical signs included salivation, irregular respiration, hypoactivity, piloerection, hunched posture, red nasal/oral crust, nonformed faeces and/or brown anal-genital stain. One female dosed at 2000 mg/kg bw was found dead one day after dosing. Based on the results of the dose range-finding assay, the test article was administered once in PEG 400 at doses of 875 and 1750 mg/kg bw to groups of three male rats per time-point in the low dose group. Five animals per time-point were treated in the high dose group. All animals from each group were perfused for the collection of hepatocytes and establishment of cultures. With the exception of the positive control at the 12-16 hour time point, cultures from three animals per group were evaluated for UDS (two were evaluated for the 12-16 hour positive control). For the early UDS time point, perfusions were initiated 2.6 to 3.0 hours after dose administration. For the 12- to 16-hour time point, perfusions were initiated 15.9 to 16.4 hours after dose administration. Dimethylnitrosamine was used as positive control.

Results

For the 2-4 hours UDS time-point, the hepatocytes ranged in viability (determined by trypan blue dye exclusion) from 52.7 to 86.4% of the total cells collected in the perfusate. The attachment efficiency varied from 18.7 to 103.1%, and the viability of the attached cells was good, ranging from 89.9% to 99.7%. For the 12- to 16-hour time-point, the hepatocytes ranged in viability from 29.9% to 93.5% of the total cells collected in the perfusate. The attachment efficiency varied from 0.4% to 152.1%, and the viability of the attached cells ranged from 8.3% to 100.0%. For the 2- to 4-hour time point, there were no indications of an increase in the mean net nuclear grain count compared to the control at both dose levels. For the 12- to 16-hour time-point, there were no indications of an increase in the mean net nuclear grain count compared to the control at both dose levels. There were no difference between the control values and the two doses tested in the percentage of cells in repair (cells with ≥ 5 net nuclear grains) for both the 2- to 4-hour and 12- to 16-hour time-point.

Conclusion

Under the test conditions used 1-naphthol is not considered genotoxic in the in vivo unscheduled DNA synthesis test.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2004 - August 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: n°Batch I-38110 - Test Substance GTS03979 ou 1-naphthol
- Expiration date of the lot/batch: 14 January 2006
- Purity test date: No data
- Purity : 99.9%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No

FORM AS APPLIED IN THE TEST : Solution

Species:
mouse
Strain:
CD-1
Remarks:
(ICR)BR
Details on species / strain selection:
This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, Norhh Carolina
- Age at study initiation: 6 weeks old
- Weight at study initiation: 27.6 to 33.9 g
- Assigned to test groups randomly: Yes (computer system - CDMS)
- Housing: separated by gender (DRF) and by full dose group/ harvest timepoint.
- Diet : ad libitum (PMI Certified Rodent Diet® #5002)
- Water : ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 27°C
- Humidity (%): 30-70%
- Air changes (per hr): 10 or greater air changes/ hour
- Photoperiod (hrs dark / hrs light): 12 hlight/ 12h dark cycle

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Polyethylene Glycol 400 (PEG400)
- Justification for choice of solvent/vehicle: based on information provided by the Sponsor
- Concentration of test material in vehicle: 125, 250 and 500 mg/kgbw
- Amount of vehicle : 10 mL/kg
- Lot/batch no.: 054K0063
- Purity: No data
Details on exposure:
Route : oral (gavage)

PREPARATION OF DOSING SOLUTIONS:
- Based on the results of the dose range-finding assay, the highest dose being 500 mg/kg bw (estimated maximum tolerated dose) and since no relevant differences in toxicity between the sexes were observed, only males were used in the micronucleus assay.
- Test material was prepared by adding the appropriate volume of the vehicle, PEG 400, to a pre-weighed quantity of the test article and mixed, forming a solution. Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature prior to dosing and stirred during the dosing procedure.
- Results of stability analyses indicated that the formulations were stable for 7-9 days at -10 to -30°C and all values were within 2% of initial concentration.
- Results of concentration verification analyses indicated that all formulations were within 8.2% of target.-
- Homogeneity analysis was not conducted due to the dose formulations forming solutions.

Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
24h and 48 hours (high dose only)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per dose and per sacrifice time
Control animals:
yes, concurrent vehicle
Positive control(s):
- Yes : cyclophosphamide (CP)
- Justification for choice of positive control(s): No data
- Route of administration: oral (gavage)
- Doses / concentrations: 8 mg/mL and 10 mL/kgbw
Tissues and cell types examined:
Bone marrow was extracted and at least 2000 PCEs per animal were subsequently microscopically analyzed for the frequency of micronuclei.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: a dose range-finding study was conducted

TREATMENT AND SAMPLING TIMES :
- The hind limb bones (femurs) were removed for marrow extraction from the surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).

DETAILS OF SLIDE PREPARATION:
- Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in May-Grünwald solution and Giemsa, and protected by mounting with coverslips.

METHOD OF ANALYSIS:
The criteria for the identification of micronuclei were those of Schmid (1976) and the staining procedure permitted the differentiation by color of PCEs and NCEs

OTHER: the animal group historical vehicle control and postive control data are also used
Evaluation criteria:
Criteria for a Valid Essay
- Acceptable Controls : The vehicle control group mean incidence of micronucleated polychromatic erythrocytes must not exceed 10/2000 (0.5%)
- Acceptabe High Dose

Assay Evaluation Criteria
Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 1000 total erythrocytes for each animal
Statistics:
Assay data analysis was performed using an analysis of variance.
If the analysis of variance was statistically significant (p ≤ 0.05), Dunnett's t-test was used to determine which dose groups, if any, were statistically significantly different from the vehicle control.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei : no indications of an increase at any concentration tested (24 or 48 hours)
- Ratio of PCE/NCE : No signs of cytotoxicity, expressed as a decreased PCE:NCE ratio observed in any dose group.
- Appropriateness of dose levels and route: Bioanalytical evaluation of plasma samples collected from high dose group animals showed the presence of test material and confirmed target organ exposure. The peak mean plasma concentration (~648 ng/ml) was observed at approximately 1 hour after test material administration.
- Statistical evaluation:
The vehicle control group had less than approximately 0.4% micronucleated PCEs and the group mean was within the historical control range.
The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard error of 0.97 ± 0.20%.

Table 1 –MicronuleusAssay – Summary Table

Treatment

Dose

Harvest Time

% micronucleatedPCEs

Mean of 2000 per animal ± SE

Males

% PCE

Mean ± SE

Males

Control

Vehicle

PEG400 10 mL/kg

24hr

0.04 ± 0.02

36.92 ± 2.19

48hr

0.07 ± 0.03

36.22 ± 2.19

Control Positive

CP 80 mg/kg

24hr

0.97 ± 0.20*

36.89 ± 1.49

Test article

125 mg/kg

24hr

0.04 ± 0.01

33.67 ± 2.16

250 mg/kg

24hr

0.04 ± 0.02

33.67 ± 1.53

500 mg/kg

24hr

0.07 ± 0.01

31.83 ± 1.14

48hr

0.03 ± 0.02

39.46 ± 2.62

(*) Significantly greater than the corresponding vehicle control, p≤0.01

 

Other

·        All animals in all the dose groups, vehicle and positive control groups appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint.

·        Individual and mean group body weights. None of the test article dose groups had a mean body weight less than 80% of the corresponding vehicle control group.

Conclusions:
Under the test conditions used, 1-naphthol is not considered clastogenic in the bone marrow micronucleus test in mice.
Executive summary:

A dose range-finding study was conducted and the test article was formulated in PEG 400 and administered once by oral gavage to three males and three females per dose level. The animals were dosed at 500, 1000, or 2000 mg/kg bw and observed for up to 2 days after dosing for toxic signs and/or mortality. Mortality was observed in 1/3 males in the 500 mg/kg bw dose group, 1/3 males and 1/3 females in the 1000 mg/kg bw dose group, and in 1/3 males and 1/3 females in the 2000 mg/kg bw. Clinical signs of toxicity observed in these animals prior to being found dead included slight hypoactivity, ataxia, and/or irregular

respiration. Based on the results of the dose range-finding assay, 1 -naphthol was administered once in PEG 400 at three doses, the highest dose being 500 mg/kg bw (estimated maximum tolerated dose) to groups of five male mice per bone marrow sampling time. An additional group of six male mice were dosed at 500 mg/kg bw for use as possible replacements in the event of mortality. Five animals from all the groups were sacrificed 24 hours after dosing and from the vehicle control and high dose group at 48 hours after dosing. Plasma samples were collected at 1, 2, 4, 6, 8, 24 and 48 hours after dosing for detection of the test article (satellite group). For all groups at 24 hours and vehicle and high dose group only at 48 hours, bone marrow was extracted and at least 2000 PCEs per animal were subsequently microscopically analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 1000 total erythrocytes for each animal. Cyclophosphamide (CP) was used as positive control.

Results

There were no signs of clinical toxicity in the animals treated at concentration levels up to 500 mg/kg bw. There were no indications of an increase in micronucleated PCEs at any concentration tested, 24 or 48 hours after the mice were sacrificed. There were no signs of cytotoxicity, expressed as a decreased PCE:NCE ratio observed in any dose group. However bioanalytical evaluation of plasma samples collected from high dose group animals showed the presence of test material and thus confirmed target organ exposure. The peak mean plasma concentration (~648 ng/ml) was observed at approximately 1 hour after test material administration.

Conclusion

Under the test conditions used, 1 -naphthol is not considered clastogenic in the bone marrow micronucleus test in mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

1-Naphthol has been sufficiently investigated for the three types of mutations: gene mutation, structural and numerical chromosomal aberrations. 1-Naphthol did not induce gene mutations in bacteria. 1-Naphthol induced clastogenic effects in Chinese hamster ovary cells and gene mutations in the mouse lymphoma assay with mammalian cells (L5178Y) in the absence of metabolic activation. The increase in small colonies may indicate clastogenicity. The genotoxic/clastogenic effects observed in the in vitro assays could not be confirmed in two in vivo assays. There were no signs of genotoxic effects of 1-naphthol tested in vivo, based on micronucleus test of bone marrow in mice and the unscheduled DNA synthesis (UDS) test in rats.

1-Naphthol, itself, is not considered to have in vivo genotoxic potential.