Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Gene mutation toxicity study was performed for Direct blue 218 to evaluate its mutagenic nature. The study was performed as per the preincubation protocol and reduction of the test chemical using Salmonella typhimurium strain RA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system at doses of 0, 0.25 or 0.50 µmole/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Concurrent solvent and negative control chemicals were also included in the study. Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
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 reviewed publication
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed for Direct blue 218 to evaluate its mutagenic nature
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
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
- Purity: >98%
- Impurities (identity and concentrations): <2%
Target gene:
Histidine
Species / strain:
S. typhimurium TA 1538
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 was prepared from the livers of Aroclor-induced male Fisher rats and hamster S9 was prepared from uninduced female hamsters
Test concentrations with justification for top dose:
0, 0.25 or 0.50 µmole/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Negative controls:
not specified
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Benzidine congeners (Dimethoxybenzidine)
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 72 hr
- Expression time (cells in growth medium): 72 hr
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for revertants
Statistics:
No data
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: No mutagenic potential
Additional information on results:
No data

Table: Mutation data for the test chemical Direct blue 218 and the positive control chemical

Dose (µmole/plate)

Reduction system (activation system)

Bacterial (rat)

None (rat)

FMN (hamster)

None(hamster)

Bacterial (hamster)

Direct blue 218

 

 

 

 

 

0

43

35

33

-

-

0.25

40 (63)

14

27

-

-

0.50

47 (47)

16

21

-

-

Dimethoxybenzidine

 

 

 

 

 

0

43

35

33

27

26

0.25

790

843

235

169

108

0.50

1073

1203

316

125

167

1.0

1491

1287

366

170

124

Conclusions:
Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed for Direct blue 218 to evaluate its mutagenic nature. The study was performed as per the preincubation protocol and reduction of the test chemical using Salmonella typhimurium strain RA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system at doses of 0, 0.25 or 0.50 µmole/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Concurrent solvent and negative control chemicals were also included in the study. Direct blue 218 did notinduce gene mutation in theSalmonella typhimurium strain TA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Gene mutation in vivo:

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 melanogasterflies and hence it is not likely to classify as a gene mutant in vivo.

Link to relevant study records
Reference
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
Reference:
Composition 0
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 information:
Composition 1
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
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
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
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
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.
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

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

 

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene mutation in vitro:

Data available for the target chemical was reviewed to determine the mutagenic nature of 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)). The studies are as mentioned below:

Gene mutation toxicity study was performed by Reid et al (Environmental Mutagenesis, 1984) for Direct blue 218 to evaluate its mutagenic nature. The study was performed as per the preincubation protocol and reduction of the test chemical using Salmonella typhimurium strain RA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system at doses of 0, 0.25 or 0.50 µmole/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Concurrent solvent and negative control chemicals were also included in the study. Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA1538 both in the presence and absence of rat and hamster liver S9 metabolic activation system.

In another study by Zeiger et al (Environmental Mutagenesis, 1986), Gene mutation toxicity study was performed for Direct blue 218 to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of0, 33.0, 100.0, 333.0, 1000.0, 3333.0 or 10000.0 µg/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system.

 

Ames assay was also performed by Prival et al (Mutation Research, 1984) to determine the mutagenic nature of Direct blue 218. The test chemical was dissolved in DMSO and used at dose levels of 0, 0.03, 0.1, 0.3 or 1.0 µmoles/plate by the standard plate incorporation assay. Concurrent solvent and negative control chemicals were also included in the study. Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA98, TA100 and TA1537 both in the presence and absence of rat liver S9 metabolic activation system.

In the same study by Prival et al (Mutation Research, 1984), Gene mutation toxicity study was also performed for Direct blue 218 to evaluate its mutagenic nature by the FMN reduction protocol. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA98 both in the presence and absence of S9 metabolic activation system and FMN at doses of 0, 0.1, 0.3 or 1.0 µmoles/plate. DMSO was used at the vehicle. The plates were incubated after 30 mins preincubation before the evaluation of the revertant colonies could be made. Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA98 both in the presence and absence of FMN and hamster liver S9 metabolic activation system.

 

Gregory et al (Journal of Applied Toxicology, 1981) performed a set of modified Ames assay protocol for determining the mutagenic nature of Direct blue 218. Gene mutation toxicity study was performed for Direct blue 218 to evaluate its mutagenic nature. The study was performed as per the standard plate incorporation protocol using Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system. The plates were incubated for 72 hrs before the evaluation of the revertant colonies could be made. Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system.

 

In another experiment, the study was performed as per the standard plate incorporation protocol using Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system and 1 mM riboflavin. The plates were incubated for 72 hrs before the evaluation of the revertant colonies could be made. Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system and 1mM riboflavin.

 

Ames assay was performed in the same study of Gregory et al (Journal of Applied Toxicology, 1981) as per the preincubation protocol using Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system and 1 mM riboflavin. The plates were incubated for 72 hrs before the evaluation of the revertant colonies could be made. Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system and 1mM riboflavin.

 

In the same study, Ames assay was performed as per the preincubation protocol using Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system. After preincubation, the poured plates were incubated in an N2 atmosphere for 24 h, followed by incubation in air for an additional 43 h. The plates were incubated for a total of 72 h before the evaluation of the revertant colonies could be made. Direct blue 218 did not induce gene mutation in the Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system (in the nitrogen atmosphere) and hence the chemical is not likely to classify as a gene mutant in vitro.

 

Gene mutation toxicity study (Journal of Applied Toxicology, 1981) was performed for Direct blue 218 to evaluate its mutagenic nature. The study was performed as per the standard plate incorporation protocol using Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system with reduction with sodium dithionate. The plates were incubated for 72 hrs before the evaluation of the revertant colonies could be made. Direct blue 218induced gene mutation in the Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system upon reduction with sodium dithionate.

 

In another study by Prival et al (Environmental Mutagenesis, 1982), Gene mutation toxicity study was performed for Direct blue 218 to evaluate its mutagenic nature. The study was performed as per the modified Ames “Salmonella typhimurium” assay using Salmonella typhimurium both in the presence and absence of uninduced S9 metabolic activation system. Direct blue 218 did notinduce gene mutation in the modified preincubation protocol usingSalmonella typhimurium in the presence and absence of uninduced hamster liver S9 metabolic activation system.

 

In vitro mammalian chromosome aberration study (NTP, 1994) was performed for Direct blue 218 to evaluate its mutagenic nature. The study was performed using Chinese hamster ovary (CHO) cells both in the presence and absence of S9 metabolic activation. In the Abs test without S9, cells were incubated in McCoy's 5A medium with Direct Blue 218 for 10 hours; Colcemid was added and incubation continued for 2 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with Direct Blue 218 and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with Colcemid present for the final 2 hours. Cells were harvested in the same manner as for the treatment without S9. Distilled water was used at the vehicle. Fifty-five to 100 first-division metaphase cells were scored at each dose level. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). Direct blue 218 did not induce chromosome aberration in the Chinese hamster ovary (CHO) cells both in the presence and absence of S9 metabolic activation system.

 

In the same NTP report (1994), Sister chromatid exchange assay in mammalian cells was performed for Direct blue 28 to evaluate its mutagenic nature. The study was performed using Chinese hamster ovary (CHO) cells. In the SCE test without S9, CHO cells were incubated for 26 hours with Direct Blue 218 in McCoy's 5A medium supplemented with fetal bovine serum, I-glutamine, and antibiotics. Bromodeoxyuridine (BrdU) was added 2 hours after culture initiation. After 26 hours, the medium containing Direct Blue 218 was removed and replaced with fresh medium plus BrdU and Colcemid, and incubation was continued for 2 hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with Direct Blue 218, serum-free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing serum and BrdU and no Direct Blue 218, and incubation proceeded for an additional 26 hours, with Colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9. All slides were scored blind and those from a single test were read by the same person. Fifty second-division metaphase cells were scored for frequency of SCEs/cell from each dose level. Direct blue 218 did not induce sister chromatid exchange in the Chinese hamster ovary (CHO) cells in the presence of S9 metabolic activation system. It however induced a small but significant increase in sister chromatid exchanges in Chinese hamster ovary cells at the highest dose tested without S9.

Gene mutation in vivo:

 

Direct blue 218 tested for mutagenicity by Woodruff et al (Environmental Mutagenesis, 1985) 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 male Drosophila melanogaster no 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 mating 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 S Drosophila melanogaster flies and hence it is not likely to classify as a gene mutant in vivo.

 

Based on the existing data available for the target chemical, 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))) does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant in vitro and in vivo as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the existing data available for the target chemical, Direct blue 218 (CAS no: 28407 -37 -6 (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))) does not exhibit gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant in vitro and in vivo as per the criteria mentioned in CLP regulation.