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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Manganese, 4-[(4-chloro-5-methyl-2-sulfophenyl)azo]-3-hydroxy-2-naphthalenecarboxylic acid complex
EC Number:
235-471-3
EC Name:
Manganese, 4-[(4-chloro-5-methyl-2-sulfophenyl)azo]-3-hydroxy-2-naphthalenecarboxylic acid complex
Cas Number:
12238-31-2
Molecular formula:
C18H11N2O6SClMn
IUPAC Name:
manganese, 4-[(4-chloro-5-methyl-2-sulfophenyl)azo]-3-hydroxy-2-naphthalenecarboxylic acid complex
Test material form:
solid: nanoform
Details on test material:
- Name of test material (as cited in study report): LITHOL Fast Maroon L 4763, Test substance No.: 06/0430-1
- Physical state: Solid (powder), red
- purity and composition: see confidential details on test item
- Impurities (identity and concentrations):
- Lot/batch No.: Standardmuster 030009P040 vom 15.07.2003
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle DMSO and in water each over a period of 4 hours were determined analytically.
- Storage condition of test material: Room temperature
- Composition of test material, percentage of components: CAS-Nr. 12238-31-2: 54% (Pigment Red 52 Mn) (The dose used for testing was adjusted for the low concentration.)

Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.
Specific details on test material used for the study:
- Name of test material (as cited in study report): LITHOL Fast Maroon L 4763, Test substance No.: 06/0430-1
- Physical state: Solid (powder), red
- purity and composition: see confidential details on test item
- Impurities (identity and concentrations):
- Lot/batch No.: Standardmuster 030009P040 vom 15.07.2003
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle DMSO and in water each over a period of 4 hours were determined analytically.
- Storage condition of test material: Room temperature
- Composition of test material, percentage of components: CAS-Nr. 12238-31-2: 54% (Pigment Red 52 Mn) (The dose used for testing was adjusted for the low concentration.)

Method

Target gene:
his, trp
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
20 µg - 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2-AA), with S9 mix ; N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), without S9 mix; 4-nitro-o-phenylendiamine (NOPD), without S9;
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.


Toxicity detected by a
− decrease in the number of revertants
− clearing or diminution of the background lawn (= reduced his- or trp- background growth)
− reduction in the titer
is recorded for all test groups both with and without S-9 mix in all experiments.
Statistics:
The data were not statistically analyzed

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Weak bacteriotoxic effect in the standard plate test depending on the strain and test conditions from about 4630 μg/plate onward. In the preincubation assay bacteriotoxicity was observed depending on the strain and test conditions from about 926 μg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 926 μg/plate onward.

RANGE-FINDING/SCREENING STUDIES:
In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st experiment.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data or above

Any other information on results incl. tables

Standard plate test (20 - 5000 µg/plate) 1.Exp

 

Strain

Metabolic activation system

mean revertants in Controls

maximum revertant factor

dose dependency

Assessment

TA 98

no

29

0.9

no

negative

yes

38

0.8

no

negative

TA 1537

no

10

0.7

no

negative

yes

10

0.9

no

negative

TA 1535

no

16

1.0

no

negative

yes

18

0.8

no

negative

TA 100

no

103

1.0

no

negative

yes

118

1.0

no

negative

E.coli WP2 uvrA

no

31

1.1

no

negative

yes

34

0.9

no

negative

Preincubation test (20 - 5000 µg/plate) 2. Exp.

 

Strain

Metabolic activation system

mean revertants in Controls

maximum revertant factor

dose dependency

Assessment

TA 98 

no

32

0.9

no

negative

yes

31

1.0

no

negative

TA 1537 

no

9

0.9

no

negative

yes

10

1.0

no

negative

TA 1535 

no

17

1.0

no

negative

yes

17

0.8

no

negative

TA 100 

no

110

1.0

no

negative

yes

113

1.1

no

negative

E.coli WP2 uvrA 

no

32

1.3

no

negative

yes

40

1.0

no

negative

Applicant's summary and conclusion