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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
Light cream flakes
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

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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)

Examinations

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

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.