Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001
Reference Type:
secondary source
Title:
C.I. Pigment Brown 24
Author:
OECD
Year:
2002
Bibliographic source:
SIDS Initial Assessment Report for SIAM 15

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: Proposal for OECD guideline 487
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: Abl. Nr. 99-7135

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM medium with glutamine supplemented with 10% (v/v) fetal calf serum (FCS), 1 % (v/v) penicillin/streptomycin (10,000 IU/ 10,000 µg/mL) and 1 % (v/v) amphotericine B (250 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from 5 male Sprague-Dawley rats (200 - 300 g) which received a single i.p. injection of 500 mg Aroclor 1254 per kg body weight 5 days before sacrifice.
Test concentrations with justification for top dose:
Range-finding cytotoxicity tests:
Mixed Population Method
0.5, 10.0, 5.0, 10.0, 100.0, 250.0, 500.0, 750.0 and 900.0 µg/mL
Mitotic Shake Off Method
3.125, 6.250, 21.500, 25.000, 50 µg/mL

Main test:
Mixed Population Method
0.3125, 0.6250, 1.2500, 2.5000, 5.0000, 10.0000 and 15.0000 µg/mL (24 h exposure, 24 h harvest time, -/+S9)
Mitotic Shake Off Method
0.200, 0.390, 0.780, 1.560, 3.125, 6.250 and 12.500 µg/mL (24 h exposure, 27 h preparation time, -S9)
1.560, 3.125, 6.250, 12.500, 25.000, 50.000 and 75.000 µg/mL (4 hours exposure, 27 h preparation time, +S9)
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
with and without S-9 mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9-mix: 350 µg Ethylmethanesulfonate (EMS); +S9-mix: 2.5 µg Cyclophosphamide (CPP); given quantities were added to 1 mL (Mixed Population Method) or 5 mL (Mitotic Shake Off Method).
Details on test system and experimental conditions:
MIXED POULATION METHOD & MITOTIC SHAKE OFF METHOD
(according to Kallweit et al. 1999 and Seelbach et al. 1993, respectively)

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 6 hrs
- Exposure duration: without S9-mix 24 hrs; with S9-mix 4 hrs followed by a 20-hr period after replacing the S9-mix; treatment medium without fetal calf serum
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (MI)
A mitotic index based on 1,000 cells/culture was determined for all dose groups with and without S-9 mix both in the Mixed Population Method and Mitotic Shake Off Method
- Method: Proliferation Index (PI )
In addition to the evaluation of micronuclei and mitotic index in the Mixed Population Method, the proliferation index (PI) was determined as a measure of cytotoxicity. The PI, based on 1,000 cells per culture (2,000 cells per dose group), was determined for all test groups, with or without S-9 mix. The number of clones (packs) consisting of 1 cell, 2, 4 or 8 cells was recorded and the PI was calculated using the following formula:
PI = ((ncl-1) x 1 + (ncl-2) x 2 + (ncl-4) x 3 + (ncl-8) x 4)/total No . of clones; whereas
cl-1 = cell clone with 1 cell
cl-2 = cell clone with 2 cell s
cl-3 = cell clone with 3 or 4 cells
cl-4 = cell clone with 5, 6, 7 or 8 cells

OTHER
Cell morphology: About 3 - 4 hours after test substance treatment with S-9 mix and about 22 - 24 hours after treatment without S-9 mix, the cell morphology, which is an indication of attachment of the cells to the slides or flasks, was checked for each culture in all test groups using both modifications of the assays (with the exception of the positive controls) .
Fragmentation: During evaluation of 1,000 cells/culture the occurance of fragmentation (fragmented nuclei or multinucleated cells and cells with > 6 micronuclei) was recorded.
Evaluation criteria:
Definition of micronucleus
- the micronucleus consists of an area less than 1/3 of the area of the main nucleus
- the micronucleus and main nucleus retain the same staining
- the micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell
- for evaluation, only cells clearly surrounded by a nuclear membrane were scored

Evaluation criteria
The test chemical is to be considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of cells containing micronuclei.
- The proportion of micronucleus-containing cells exceeded both the concurrent negative control range and the negative historical control range.
A test substance is generally considered nonclastogenic in this test system if there was no significant increase in the number of micronucleus-containing cells at any dose above concurrent negative control frequencies and within the historical control data.
Statistics:
No statistical analysis due to the clear negative findings.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: Strong test substance precipitation in the cultures was observed at all test doses.

Any other information on results incl. tables

MICRONUCLEUS FREQUENCY  

Mixed population method

Substance

Dose (µg/mL)

Exposure (h)

Harvest (h)

S-9 mix

Frequency (%)

vehicle

solvent

24

24

-

0.65

solvent

24

24

+

0.65

test substance

1.25

24

24

-

1.20

1.25

24

24

+

0.40

2.50

24

24

-

0.60

2.50

24

24

+

0.70

5.00

24

24

-

1.15

5.00

24

24

+

0.65

10.00

24

24

-

0.75

10.00

24

24

+

0.65

15.00

24

24

-

0.70

15.00

24

24

+

0.65

Positive ctrl

EMS: 350

24

24

-

2.60

CPP: 2.50

24

24

+

10.15

Mitotic shake off method

Substance

Dose (µg/mL)

Exposure (h)

Harvest (h)

S-9 mix

Frequency (%)

vehicle

solvent

24

24

-

0.55

solvent

4

24

+

0.50

Test substance

0.78

24

24

-

0.60

1.56

4

24

+

0.35

1.56

24

24

-

0.70

3.13

4

24

+

0.35

3.13

24

24

-

0.20

6.25

4

24

+

0.45

6.25

24

24

-

0.35

12.50

4

24

+

0.45

12.50

24

24

-

0.25

25.00

4

24

+

0.20

Positive ctrl

EMS: 350

24

24

-

3.30

CPP: 2.50

4

24

+

4.45

 

MITOSIS

According to the results of the determination of the mitotic index, no suppression of the mitotic activity was observed under all experimental conditions.

CYTOTOXICITY (only Mixed Population Method)

According to the results of the determination of the PI, no cytotoxic response was observed under any of the experimental conditions.

CELL MORPHOLOGY

Cell attachment was not influenced at any dose evaluated for micronuclei.

PRECIPITATION

The test substance precipitation in the vehicle was observed at all test doses, in culture "obviously soluble up to 6.25 µg/mL”

Applicant's summary and conclusion