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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation toxicity study was performed to determine the mutagenic nature of Solvent Red 1. The study was conducted as a part of range finding study using Salmonella typhimurium strains TA98, TA1538, TA100, and TA1535 with and without S9 metabolic activation system. The test chemical was dissolved in DMSO and used at six concentration from 0-800µg/plate. Three plates were used per concentration and the plates were observed for an increase in mutation frequency twice that observed in the controls.Solvent red 1 did not induce mutation in the reange finding study conducted usingSalmonella typhimurium strains TA98, TA1538, TA100, and TA1535 both in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

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 1
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 to determine the mutagenic nature of Solvent red 1
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: Solvent red 1 / 1 -((2Methoxyphenyl) azo-2-naphthol)
- Molecular formula: C17H14N2O2
- Molecular weight: 278.31 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: 99%
- Impurities (identity and concentrations): 1%
Target gene:
Histidine
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
Aroclor induced rat liver microsomes (S-9)
Test concentrations with justification for top dose:
Six concentration between 0-800 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The 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:
9-aminoacridine
2-nitrofluorene
benzo(a)pyrene
other: 2-aminoanthracene (2-AA)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- 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: No data
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other:

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for a doubling of revertants/plate
Statistics:
The average revertant frequency per plate was plotted against the concentration of the dyes and concentration response relationship determined for each dye for each S-9 concentration over the linear portion of the curve. To test the null hypothesis that the slope was not different from
zero, the data were first fit to a straight line by a least squares method, then the derived slope was tested using a Z-test with a linear dose axis and a
significance level of 0.05.
Species / strain:
other: TA98, TA1538, TA100, and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitate was observed
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

COMPARISON WITH HISTORICAL CONTROL DATA: No data

Concentration (µg/plate)

Strain

S9 (30µL/plate)

Background (X±SE)

Highest response (X±SE)

Slope±SE (revertants/µg)

0-800

TA98

-

30±3

32±1

0.002±0.003

 

+

36±3

43±2

0.008±0.003

TA1538

-

13±2

23±2

0.003±C 0.004

 

+

21±2

29±4

-0.001±0.005

TA100

-

114±3

134±6

0.010±0.006

 

+

121±5

170±10

0.01±0.02

TA1535

-

21±3

32±3

-0.004±0.006

 

+

10±1

15±5

0.002±0.002

Conclusions:
Solvent red 1 did not induce mutation in the range finding study conducted using Salmonella typhimurium strains TA98, TA1538, TA100, and TA1535 both in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of Solvent Red 1. The study was conducted as a part of range finding study using Salmonella typhimurium strains TA98, TA1538, TA100, and TA1535 with and without S9 metabolic activation system. The test chemical was dissolved in DMSO and used at six concentration from 0-800µg/plate. Three plates were used per concentration and the plates were observed for an increase in mutation frequency twice that observed in the controls.Solvent red 1 did not induce mutation in the reange finding study conducted usingSalmonella typhimurium strains TA98, TA1538, TA100, and TA1535 both in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Various data available for the target chemical was reviewed to determine the mutagenic nature of Solvent Red 1. The studies are as mentioned below:

Gene mutation toxicity study was performed by Brooks et al ( Environmental and Molecular Mutagenesis, 1989) to determine the mutagenic nature of Solvent Red 1. The study was conducted as a part of range finding study using Salmonella typhimurium strains TA98, TA1538, TA100, and TA1535 with and without S9 metabolic activation system. The test chemical was dissolved in DMSO and used at six concentration from 0-800µg/plate. Three plates were used per concentration and the plates were observed for an increase in mutation frequency twice that observed in the controls.Solvent red 1 did not induce mutation in the reange finding study conducted usingSalmonella typhimurium strains TA98, TA1538, TA100, and TA1535 both in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In the same study by Brooks et al, gene mutation toxicity study was performed to determine the mutagenic nature of Solvent Red 1. The study as per the preincubation protocol using Salmonella typhimurium strains TA98, TA1538, TA100, and TA1535 with 30% v/v hamster liver S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 50, 100, 250 or 500µg/plate.Solvent red 1 did not induce mutation preincubation assay conducted usingSalmonella typhimurium strains TA98, TA1538, TA100, and TA1535 in the presence S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study mentioned n the same publication of Brooks et al (1989), In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of Solvent red 1. Chinese Hamster ovary cells were exposed to the test chemical with and without S9 metabolic activation system at a dose level of 20µg/mL. The dye exposure duration was for 3 hrs. Two hours prior to harvest, colchicine was added to the culture to result in a final concentration of 10-5M. During and after the exposure the cells were exposed to 10µM bromodeoxyuridine (BrdU). All slides for induction of chromosome aberrations were evaluated at 9 hr after the start of the cultures.The frequency of aberrations was recorded on 200 cells per treatment. Solvent red 1 did not induce chromosomal aberration in Chinese Hamster ovary cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Also, In vitro mammalian cell gene mutation assay was performed (Brooks et al, 1989) to determine the mutagenic nature of Solvent red 1. The mutation assay was the Chinese hamster ovary/hypoxanthine guanine phosphoribosyl transferase (CHO/HGPRT). Chinese Hamster ovary cells were exposed to the test chemical with and without 1.25% and 2.5% S9 metabolic activation system. Solvent red 1 Concurrent positive control chemicals were also included in the study. The chemical was tested three times for their direct mutagenic effect without the addition of S-9. No indication of mutagenic activity was noted in the absence of S9. There was no significant increase in themutation frequency as a function of dye concentration for Solvent Red 1, P > 0.05. The mammalian cell mutation assay was also run for the dye with the addition of either 1.25% or 2.5% S-9. Adding S9 increased the background rate by a factor Of about two and increased the variability of the response. No significant increase in the slope of the concentration response for mutation frequency was observed for Solvent Red 1 using Student t-test to evaluate potential increases in the slopes above background, P> 0.05 for the null hypothesis.Solvent red 1 did not induce gene mutation in Chinese Hamster ovary cells in the presence and absence of 1.25% and 2.5% S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Sister chromatid exchange assay was also performed (Brroks et al, 1989) to determine the mutagenic nature of Solvent red 1. Chinese Hamster ovary cells were exposed to the test chemical with and without S9 metabolic activation system at a dose level of 20µg/mL. The dye exposure duration was for 3 hrs. Two hours prior to harvest, colchicine was added to the culture to result in a final concentration of 10-5M. During and after the exposure the cells were exposed to 10µM bromodeoxyuridine (BrdU). Because of mitotic delay, each dye and exposure concentration was evaluated for SCE frequency when adequate numbers of second division cells were first detected. For controls and Solvent Red 1, the cells were scored for the induction of SCE at 24 hr after dye exposure. Solvent red 1 produced an increase in the frequency of sister chromatid exchangesin the presence and absence of S9 metabolic activation system. The increased incidence of SCE after exposure to Solvent Red 1 may be related to its demonstrated cytotoxic effects at higher concentrations. It has been reported that delay of cells in the Go stage of the cell cycle results in repair of SCE lesions and that delays and prolongation of S phase of the cycle can increase the frequence of SCE and this may be the reason for the induction of SCE.

In another study by Harrington-Brock et al (Environmental and molecular mutagenesis, 1989) and Moore et al (USEPA< 1989) gene mutation toxicity study was performed to determine the mutagenic nature of Solvent Red 1. Mutagenic Screening of Solvent Red 1 was performed using L5178Y/TK+/- mouse lymphoma assay. The test chemical was dissolved in DMSO and used at varying concentration in two experiments without S9 and three experiments with S9. One 50-ml Corning polypropylene tube seeded with 6x106cells in 6.0 ml of medium with a reduced amount of serum (5% instead of 10%) was used for each dose. Four ml of serum-free Fischer's medium (FOP) were added to each tube. The compound was dissolved in DMSO at 100 times the highest concentration to be tested. Solvent Red 1was added to each appropriately labelled tube, the tubes were then regassed with 5% C02-in-air and incubated in a roller drum at 37°C for 4 hr. Following the 4-hr exposure period, the tubes were centrifuged for 10 min at 200 x g and the supernatant containing the test compound was discarded. The cells were then washed twice in 10 ml of F10P (2 x 10 min centrifugations at 200 x g), and resuspended in 20 ml of fresh F10P to a final cell concentration of 3x105cells/ml. The tubes were regassed with 5% C02-in-air and incubated in the roller drum at 37°C. Cell counts were determined with a Coulter Counter Model ZBI at 24 and 48 hr after exposure to the compound. Each culture was diluted daily to 2x105cells/ml. At the end of 48 hr, the cells were cloned. Fifteen ml of each culture were spun in a centrifuge at 200 x g for 10 min and the supernatant decanted. Approximately 1-2 ml of F1 0P were added to each culture for resuspension of the cell pellet. The cell pellet was vigorously resuspended to ensure a single cell suspension and placed in 100 ml of CM to give a cell concentration of 3x104cells/ml. The flasks were labelled with the appropriate culture number and selection agent to be used (TFT). Further the plates were incubated for 10-12 days. At the end of the incubation period the plates were scored for the number of colonies per plate using an Artek colony counter model 880. Also no consistent increase in the number of MN was observed. Solvent Red 1 did not induce mutation in TK+/- -3.7.2C heterozygote of L5178Y mouse lymphoma cells below a dose level of 6.1 ug/mL in experiment 1, 8.7 ug/ml in experiment 2 and 7.5 ug/ml in experiment 3 and hence it is not likely to classify as a gene mutant in vitro.

In the same study by Moore et al (USEPA, 1989) gene mutation toxicity study was performed to determine the mutagenic nature of Solvent Red 1. Mutagenic Screening of Solvent Red 1 was performed using Salmonella Reversion Assay. The study was performed using variuos doses of the chemical being dissolved in DMSO using Salmonella typhimurium strains TA98, TA100, TA102, TA104, TA1537 and TA1538. The study was performed in the presence and absence of S9 metabolic activation system. The plates were incubated in -he dark at 37°C for 72 hr. The plates were examined for background growth, and the number of colonies per plate were counted using an Artek 880 automatic colony counter. Solvent Red 1 did not induce gene mutation in Salmonella typhimurium strains TATA1538, TA98 and TA1537 in the presence and absence of S9 metabolic activation system. Equivocal response was observed for strains TA102 and TA104 in the presence of S9 mix and a positive response was noted for Strain TA100 in the oresence of S9 metabolic activation system. However, the questionable responses could not be resolved due to the dense coloration of the plates (which created enumeration difficulties and obscured precipitation when it occurred), the potential overlapping of toxicity and mutagenicity, and the limits of solubility of the dye. Hence Solvenr Red 1 is considered to be negative for gene mutation in vitro.

Another study was also mentioned by Moore et al (USEPA, 1989). Gene mutation toxicity study was performed to determine the mutagenic nature of Solvent Red 1. Mutagenic Screening of Solvent Red 1 was performed using TLC/Salmonella assay. The study was performed using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 with and without metabolic activation system. The doses used for the study ranged between 2 µg to 1 mg per spot. Commercially available sterile glass-backed silica or cellulose HPTLC plates (10 cm x 10 cm) were developed (without sample application) in absolute ethanol. After development the plates were dried at room temperature under the hood for 5 min, and then dried an additional 20 min at 80°C. Samples were then spotted on the plate via a micropipette in varying volumes, depending on their mutagenic potency. Samples were evenly spaced along the width of the plate 1.5 cm from the bottom; ordinarily four sample volumes were applied across the plate. Plates were then lowered into a developing tank containing the appropriate solvent system at a depth of 1 cm. Chloroform was the first solvent used for TLC. The mobile phase was allowed to migrate up the plate at room temperature until the solvent front was about 1/2 cm from the top of the plate. Plates were then removed from the chamber and allowed to air dry under the hood. At this point, plates were examined under UV light for fluorescent activity. In order for the TLC plates to fit into the 150x150 mm disposable petri dishes, the upper two edges were removed with the aid of a glass cutter. Plates were placed into the dishes with the developed side facing up. To 30 ml of VBME agar at 45°C are added 500 M1 suspension (1 x 109bacteria/ml) of the tester strain and 1.5 ml of S9 mix. Contents were slowly poured into the space between the TLC plate and the edge of the petri dish, and the dish was rotated for even distribution. The agar was allowed to harden, and the plates were inverted and incubated at 37°C for 72 hours. Plates are then examined for clusters of colonies, toxic zones, and/or total revertant count. Solvent Red 1 did not induce gene mutation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the target chemical, Solvent Red 1 does not exhibit gene mutation in vitro. Hence the test chemical is not likley to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the data available for the target chemical, Solvent Red 1 does not exhibit gene mutation in vitro. Hence the test chemical is not likley to classify as a gene mutant in vitro.