Registration Dossier

Administrative data

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer revewed publication

Data source

Reference
Reference Type:
publication
Title:
Chemical Mutagenesis Testing in Drosophila. V. Results of 53 Coded Compounds Tested for the National Toxicology Program
Author:
R.C. Woodruff, J.M. Mason, R. Valencia, and S. Zimmering
Year:
1985
Bibliographic source:
Environmental Mutagenesis 7:677-702 (1985)

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Direct blue 218 tested for mutagenicity in Drosophila melanogaster by adult feeding and, where results were negative, by adult injection for the induction of sex-linked recessive lethal mutations in meiotic and postmeiotic germ cell stages of Canton-S males.
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: amorphous powder
Details on test material:
Name of the test chemical: Tetrasodium [μ-[[3,3'-[(3,3'-dihydroxy[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonato]](8-)]]dicuprate(4-)
Common Name: C.I Direct Blue 218
IUPAC name: tetrasodium (3E)-5-amino-3-{2-[4-(4-{2-[(2E)-8-amino-1-oxo-3,6-disulfonato-1,2-dihydronaphthalen-2-ylidene]hydrazin-1-yl}-3- hydroxyphenyl)-2-hydroxyphenyl]hydrazin-1-ylidene}-4-oxo-3,4-dihydronaphthalene-2,7-disulfonate dicopper
Molecular Formula: C32H20Cu2N6Na4O16S4
Molecular Weight: 1087.84 g/mol
SMILES Notation: c12c3c(c(S(=O)(=O)[O-])cc1cc(S(=O)(=O)[O])cc2N)N=Nc1ccc(cc1O[Cu]O3)c1cc2c(N=Nc3c(cc4c(c3O[Cu]O2)c(cc(c4)S(=O) (=O)[O-])N)S(=O)(=O)[O-])cc1.[Na+].[Na+].[Na+].[Na+]
InChI: 1S/C32H24N6O16S4.2Cu.4Na/c33-19-11-17(55(43,44)45)5-15-9-25(57(49,50)51)29(31(41)27(15)19)37-35-21-3-1-13(7-23(21)39) 14-2-4-22(24(40)8-14)36-38-30-26(58(52,53)54)10-16-6-18(56(46,47)48)12-20(34)28(16)32(30)42;;;;;;/h1-12,39-42H,33-34H2,(H,43,44,45)(H,46,47,48)(H,49,50,51)(H,52,53,54);;;;;;/q;2*+2;4*+1/p-8/b37-35-,38-36-;;;;;;
Substance Type: Organic
Physical State: Solid Deep purple to dark blue amorphous powder
Specific details on test material used for the study:
- Name of test material: Direct blue 218
- IUPAC name: Copper,[tetrahydrogen-3,3'-[(3,3'-dihydroxy-4,4'-biphenylylene)bis(azo)]bis[5-amino-4-hydroxy-2,7-naphthalenedisulfonato](4-)]di-,tetrasodium salt (7CI)
- Molecular formula: C32H16Cu2N6O16S4.4Na
- Molecular weight: 1087.82 g/mol
- Substance type: Organic
- Physical state: No data

Test animals

Species:
Drosophila melanogaster
Strain:
other: Canton S (Basc (In(l)scSIL sc8R + S, scS1 sc8 waB))
Details on species / strain selection:
No data
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: No data available
- Age at study initiation:
Males: 1 day for adult feeding. 1-3 days for adult injections
Females: About 3-5 days
- Weight at study initiation: No data available
- Assigned to test groups randomly: [no/yes, under following basis: ] No data available
- Fasting period before study: No data available
- Housing: glass shell vials
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23-25 ⁰C
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

IN-LIFE DATES: From: To: No data available

Administration / exposure

Route of administration:
other: Feeding and injection
Vehicle:
- Vehicle(s)/solvent(s) used:
Adult feed: Solvent of choice was a sterile solution of 5 % sucrose in distilled water
Adult injection: Solvent of choice was a sterile solution of 0.7% NaCl in distilled water.
- Justification for choice of solvent/vehicle: No data
- Concentration of test material in vehicle: No data
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data
Details on exposure:
No data
Duration of treatment / exposure:
3 days
Frequency of treatment:
Daily
Post exposure period:
No data
Doses / concentrations
Remarks:
Adult feed: 0 or 10000 ppm
Adult injection: 0 or 1000 ppm
No. of animals per sex per dose:
No data
Control animals:
yes, concurrent vehicle
Positive control(s):
No data

Examinations

Tissues and cell types examined:
No data
Details of tissue and slide preparation:
No data
Evaluation criteria:
Lethal mutation was observed;

A test result was considered positive if the P value was less than or equal to 0.01 and the mutation frequency in the tested group was greater than 0.10% or if the P value was less than or equal to 0.05 and the frequency in the treatment group was greater than 0.15%. A test was considered to be inconclusive if the P value was between 0.05 and 0.01 but the frequency in the treatment group was between 0.10% and 0.15% or if the P value was between 0.10 and 0.05 but the frequency in the treatment group was greater than 0.10%. A test was considered negative if the P value was greater than or equal to 0.10 or if the frequency in the treatment group was less than 0.10%.
Statistics:
Normal test. Calculation of lethal frequencies and statistical tests were performed after clusters were removed.

By use of the formula for the cumulative Poisson distribution with a 0.01 alpha value. All data from a parental male producing a cluster were excluded.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
Feeding and injection route
Toxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
not specified
Remarks on result:
other: No mutagenic potential
Additional information on results:
No data

Any other information on results incl. tables

Table: Results of Sex-Linked Recessive Lethal Mutation Tests

Dose

ROA

% mortality

% sterility

Lethals

Tests

Total lethals

Total tests

% lethals

Br1

Br2

Br3

Br1

Br2

Br3

1000

Injection

10

1

2

1

5

1784

1859

1885

8

5528

0.14

0

 

 

 

2

4

3

1802

1906

1939

9

5647

0.16

10000

Feeding

5

9

0

0

1

1919

1980

1852

1

5751

0.02

0

 

 

 

0

2

0

1952

1889

1866

2

5707

0.04

 

Applicant's summary and conclusion

Conclusions:
Direct blue 218 did not induce a significant increase in the frequency of SLRL mutations when administered by feeding and injection to male Canton S Drosophila melanogaster flies and hence it is not likely to classify as a gene mutant in vivo.
Executive summary:

Direct blue 218 tested for mutagenicity in Drosophila melanogaster by adult feeding and, where results were negative, by adult injection for the induction of sex-linked recessive lethal mutations in meiotic and postmeiotic germ cell stages of Canton-S males.

 

The test chemical was assayed in the SLRL test by feeding for 3 days to adult Canton-S wild-type maleDrosophila melanogasterno more than 24 hours old at the beginning of treatment. Because no response was obtained, it was retested by injection into adult males.

 

To administer direct blue 218 by injection, a glass Pasteur pipette was drawn out in a flame to a microfine filament, and the tip was broken off to allow delivery of the test solution. Injection was performed either manually, by attaching a rubber bulb to the other end of the pipette and forcing through sufficient solution (0.2 to 0.3 μL) to slightly distend the abdomen of the fly, or by attaching the pipette to a microinjector that automatically delivered a calibrated volume. Flies were anesthetized with ether and immobilized on a strip of tape. Injection into the thorax, under the wing, was performed with the aid of a dissecting microscope.

 

Canton-S males were allowed to feed for 72 hours on direct blue 218 at dose level of 0 or 10000 ppm. In the injection experiments, 24-to 72-hour old Canton-S males were treated with the test chemical at dose level of 0 or 1000 ppm and allowed to recover for 24 hours. A concurrent ethanol/saline control group was also included. Treated males were mated to three Basc females for 3 days and were given fresh females at 2-day intervals to produce three matings of 3, 2, and 2 days (in each case, sample sperm from successive matings was treated at successively earlier postmeiotic stages). F1 heterozygous females were mated with their siblings and then placed in individual vials.

 

Direct blue 218 did not induce a significant increase in the frequency of SLRL mutations when administered by feeding and injection to male Canton SDrosophila melanogaster flies and hence it is not likely to classify as a gene mutant in vivo.