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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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
The reliability of the read-across study was established to be R2: comparable to guideline study with acceptable restrictions.
Justification for type of information:
Justification for read-across is detailed at section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
yes
Remarks:
limited given data
GLP compliance:
yes
Remarks:
HAZLETON LABORATORIES AMERICA, INC.
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Reference substance name:
Similar Substance 03 - AB165:1
IUPAC Name:
Similar Substance 03 - AB165:1
Test material form:
not specified

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: adult- Weight at study initiation: 150 - 300 g
- Assigned to test groups randomly: yes
- Diet: Purina Certified Rodent Chow (Formula 5002); ad libitum.
- Acclimation period: minimum of 5 days prior to use.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: water
- Amount of vehicle (if gavage or dermal): 9 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
Fresh preparations of test article in vehicle were used for any testing purpose.
Duration of treatment / exposure:
4 h
Frequency of treatment:
single application
Post exposure period:
4 h
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
#1
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
#2
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
#3
Dose / conc.:
4 000 mg/kg bw/day (nominal)
Remarks:
#4
No. of animals per sex per dose:
3 animals/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Dimethylnitrosamine (DMN)
- Justification for choice of positive control: known to induce UDS in vivo in rat hepatocytes.
- Route of administration: intraperitoneal.
- Doses / concentrations: ca. 10 mg/kg bw

Examinations

Tissues and cell types examined:
hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION
In the preliminary study to determine dose and perfusion time, an attempt was made to gavage two animals with 5000 mg/kg, but the test material formed a sludge in CMC that could not be forced through a syringe. The highest dose that could be administered was between approximately 3200 mg/kg and 3400 mg/kg although one animal died due to gavage back up. The amount administered was not exact because the sludge was difficult to measure. One rat was sacrificed (liver perfusion) approximately 4.5 hours later and the other approximately 15 hours later and slides were prepared for UDS. Microscopic examination of the slides prepared at the two time points indicated that they were similar. It was decided that the UDS assay would be performed approximately 4 hours after administration of a single dose of the test material.

DETAILS OF SLIDE PREPARATION
UDS based on the procedures in rats described by Williams (1980) and Mirsalis, Tyson and Butterworth (1982): Briefly, isolated hepatocytes were cultured for 1.5 to 2 hours at 37 °C in a humidified atmosphere containing 5 % C02. Three of the replicate cultures from each animal were used for the UDS assay, thus labeld and fixed with acetic acid : ethanol (1:3) and dried for at least 24 hours.

METHOD OF ANALYSIS
The cells were examined microscopically at approximately 1500x magnification under oil immersion and the field was displayed on the video screen of an automatic counter. UDS was measured by counting nuclear grains and subtracting the average number of grains in three nuclear-sized areas adjacent to each nucleus (background count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting. The net nuclear grain count was determined for 50 randomly selected cells on each coverslip. Only nuclei with normal morphologies were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis occurred rather than repair synthesis. The mean net nuclear grain count was determined from the triplicate coverslips (150 total nuclei) for each treatment condition. Occasionally, a coverslip is recounted at a later date or by a different technician. Since a different cell population will generally be scored, the average count for 50 cells was used in the calculation of the mean for the triplicate treatment.
Evaluation criteria:
The test material is considered active in the UDS assay at applied concentrations that cause:
- an increase in the mean net nuclear grain count to at least six grains per nucleus after subtraction of the concurrent negative control value, and/or
- an increase in the percent of nuclei having 6 or more net grains to at least 10 % of the analyzed population after subtraction of the concurrent negative control value, and/or
- the percent of nuclei with 20 or more grains to reach or exceed 2 % of the analyzed population.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 3200 - 5000 mg/kg bw
- Solubility: material formed a sludge in CMC that could not be forced through a syringe at 5000 mg/kg bw.

Any other information on results incl. tables

Results:

Test condition Dose Level Animal UDS grains/nucleus Average* % nuclei with
>= 6 grains >= 20 grains
water - 1 0.45 ± 0.18 0.7 0.0
2 -0.43 ± 0.10 0.7 0.0
3 -0.163 ± 1.59 0.7 0.0
DMN 10 mg/kg bw 1 25.22 ± 8.79 94.7 64.7
2 15.47 ± 2.65 83.3 41.3
3 17.2 ± 7.8 84.0 41.3
Saeurebraun 6229 4000 mg/kg bw 1 0.36 ± 0.52 2.0 0.0
2 -0.08 ± 0.73 0.0 0.0
3 0.08 ± 0.66 0.7 0.0
2000 mg/kg bw 1 -1.32 ± 0.50 0.7 0.0
2 -1.00 ± 0.76 0.0 0.0
3 -1.03 ± 1.02 0.7 0.0
1000 mg/kg bw 1 -1.49 ± 0.57 0.0 0.0
2 -1.11 ± 0.88 5.3 0.0
3 -0.57 ± 0.60 0.0 0.0
5000 mg/kg bw 1 -0.20 ± 0.20 1.3 0.0
2 -1.18 ± 0.39 0.0 0.0
3 -0.92 ± 0.75 1.3 0.0

UDS: Average of net nuclear grain counts on triplicate coverslips (150 total cells), ± standard deviation between coverslips

*: Average values for triplicate coverslips

Applicant's summary and conclusion

Conclusions:
Inactive in the in-vitro/in-vivo Rat Epatocyte UDS Assay over a dosing range of about 500 to 4000 mg/kg.
Executive summary:

The substance was tested for genotoxicity following OECD 486.

The tets substance was inactive in the in-vitro/in-vivo Rat Epatocyte UDS Assay over a dosing range of about 500 to 4000 mg/kg.