N-ethyl-N-[2-[1-(2-methylpropoxy)ethoxy]ethyl]-4-(phenylazo)aniline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30.08.-9.11.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

(see Overall remarks)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

gene for synthesis histidine or tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine requiring strain

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan requiring strain

Metabolic activation:

with and without

Metabolic activation system:

postmitochondrial fraction (S9)

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500, 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide for analysis (purity>99.9%), Merck, Lot No. K41063052 028

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(Dimethylsulfoxide)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylenediamine; 9-aminoacridine hydrochloride monohydrate; 2-aminofluorene; 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation)

NUMBER OF REPLICATIONS:
two series of experiments were performed with each strain - without and with metabolic activation, triplicate plating was used at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: total growth

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule which is compatible with the aplication of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.

Statistics:

For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods (2, 3).
2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240
3. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no toxicity with TA 100 (without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

ambiguous slight mutagenic effect

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The results obtained in most experiments did not show substantial (biologically significant) increases in the number of revertants versus solvent controls (Rt/ Rc < 2), and no experiment gave evidence of a rising trend in the number of revertants with increasing dose.

In some cases, experiments with increased values of revertants occurred, exclusively with metabolic activation. It concerns the following experiments:

- TA 100 +MAII, trend to 500 µg per plate Rt/Rc max =1.5;

- TA 1535 +MAI trend to 1500 µg per plate Rt/Rc max =1.7;

- TA 98 +MAI trend to 500 µg per plate ,max Rt/Rc 1.9 and experiment +MAII, max  Rt/Rc =1.6;

- E. coli + MAII, without any trend, max. Rt/Rc =1.5; increase caused rather by lower solvent control value.

 

In case of TA 100, TA 1535 and E.coli the results were neither confirmed by the parallel experiments nor Rt/Rt reached 2. In case of TA 98, increased values of revertants were observed in both experiments, while dose-response relationship was observed in the first experiment only. It could be caused relatively small enhancement of number of revertants and large variability of the biological system. Anyway, such increasing is not common in negative experiments.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
other: negative, but result of TA 98 with metabolic activation could be evaluated as ambiguous slight mutagenic effect

Under the above-described experimental design, the test substance Solvent Yellow 124 was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains in experiments without metabolic activation. It was nonmutagenic also for Salmonella typhimurium TA 100, TA 1535 and TA 1537 and for Escherichia coli WP2 uvrA with metabolic activation. The results in Salmonella typhimurium TA 98 with metabolic activation could be evaluated as ambiguous slight mutagenic effect.

Executive summary:

Test substance Solvent Yellow 124 was assayed for the mutagenicity by the Bacterial Reverse Mutation Test.

The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dissolved in dimethylsulfoxide and assayed in doses of 5-5000 µg, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Solvent Yellow 124 was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains in experiments without metabolic activation. It was nonmutagenic also for Salmonella typhimurium TA 100, TA 1535 and TA 1537 and for Escherichia coli WP2 uvrA with metabolic activation.

The results in Salmonella typhimurium TA 98 with metabolic activation could be evaluated as ambiguous slight mutagenic effect.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14.11.1988

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

other: Standards to be observed Mutagenity Testing Institutions

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese hamster lung (CHL/IU) cell line

Metabolic activation:

with and without

Metabolic activation system:

microsomal enzyme fraction (S9)

Test concentrations with justification for top dose:

156, 313, 625, 1250 , 2500, 5000 µg/ml preliminary test
consequently
156, 313, 625 µg/ml for continuous treatment
313, 625 and 1250 µg/ml for pulse treatment

Vehicle / solvent:

DMSO Dimethylsulfoxid

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium DMSO

DURATION
- Preincubation period: in principle 3 days

- Exposure duration: continuous experiment - 24 a 48 hours, pulse experiment - 6 hours followed 18 hours culture period

- Fixation time (start of exposure up to fixation or harvest of cells): 24 (or 48)+2 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2x10000

DETERMINATION OF CYTOTOXICITY
- Method: counting the number of metaphase cells of 600 cells ( with added colcemid)

Species / strain:

other: Chinese hamster lung (CHL/IU) cell line

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: metaphase cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Solvent Yellow-124 is considered to be negative in chromosome aberration test to CHL/IU cells in vitro.

Executive summary:

This study was designed to assess the potential chromosome aberration of Solvent yellow 124, on the metaphase chromosome analysis of Chinese hamster lung (CHL/IU) cell line.

Solvent Yellow-124 demostrated no increase in both the frequency of structural and numerical chromosome aberration with and without metabolic activation method.  Solvent Yellow-124 is therefore considered to be negative under this experiment design.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.9.-17.10.2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was carried out in accordance with internationally valid GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: M & B A/S, DK-8680 Ry
- Age at study initiation: 7 weeks
- Weight at study initiation: 28-33g
- Assigned to test groups randomly: yes, using a randomisation scheme
- Fasting period before study: no data
- Housing: groups of 2 or 5 in transparent polycarbonate (macrolone type III) cages ( floor area: 810 cm2)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum, domestic drinking quality acified with HCl to pH 2.5 in order to prevent microbial growth
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C±3°C
- Humidity (%): 55% ±15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

sezame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions were prepared in warm sezame oil aprox. 37°C at concentrations 50, 100, and 200 mg/ml fresly before use. These solutions were used to dose the mice at 500, 1000, and 2000 mg/kg using a fixed dose volume of 10 ml/kg.

Duration of treatment / exposure:

24,48 hours

Remarks:

Doses / Concentrations:
2000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 males (24 hours) and 5 males (48 hours)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 20 mg/kg

Tissues and cell types examined:

• A bluish mauve strongly coloured uniform circular particle in the cell
• The particle should have a certain size (not being punctiform) and it should be located in the same plane as the cell (cell and the micronucleus should be in focus at the same time)
• During focusing, the particle should stay uniform in colour, light refraction and shape within a relatively large interval
• Cells with 2 or more micronuclei were counted as single micronucleated cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In the preliminary toxicity test, no adverse reactions to treatment was observed at 500, 1000, and 2000 mg/kg body weight. Accordingly 2000 mg/kg was selected for the main test as the maximum dose level required by OECD quideline for materials of low toxicity.

DETAILS OF SLIDE PREPARATION:
The cell suspension bone marow from each femur in 2.5 ml of foetal calf serumwas centrifuged for 10 minutes at 1000 rpm and most of the supernatant was removed. The cells were resuspesed in the remainder and smeared on clean glass slides. The slide prepartion was fixed in methanol and stained with Giemsa. The slides were dried and coverslips were aplied using Dammarxylen® mountant

Evaluation criteria:

• increases in the frequency of micronucleated polychromatic erythrocytes were observed in treated animals compared to the corresponding negative controls
• the increases were reproducible between the animals of each group
• the increases were statistically significant
• the increases exceeded historical control range for this laboratory

Statistics:

SAS® procedures (v 6.12) described in “SAS®/STAT User´s Guide, Version 6, Fourth Edition, Vol. 1+2”, 1989, SAS Institute Inc., Cary, North Carolina 27513, USA

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that Solvent Yellow 124 showed no evidence of mutagenic or clastogenic activity in this mouse micronucleus test after administration by oral gavage.

Executive summary:

Solvent Yellow 124 was tested in the Mouse Micronucleus Test performed in accordance with the OECD quideline “Mammalian Erythrocyte Micronucleus Test”, No 474 (1997).

 

In the main test, a group of male mice was treated with a solution of the test article in sesame oil at a dose level of 2000 mg/kg. A negative control group was dosed with sesame oil, and a positive control group was dosed with Cyclophosphamide at 20mg/kg. All the mice were dosed by oral gavage on one occasion using a dose volume of 10 ml/kg body weight.

 

Bone marrow samples were taken from five mice from each of the three groups 24 hours after dosing and from five mice of the test article treatment and negative control groups 48 hours after dosing. Bone marrow smears were prepared on glass slides, stained, and scored using a microscope.

 

No adverse clinical sings were observed in the nice. All mice treated with the test article lost weight during the treatment period. The urine of mice treated with the test article was dark yellow two hours after dosing. The internal organs of these mice were also coloured yellow after 48 hours after they had been dosed. These observations demonstrate systemic distribution of the test article and/or its metabolite s, and hence, exposure of the bone marrow. No reduction in the frequency of polychromatic erythrocytes among total erythrocytes was observed after treatment with Solvent Yellow 124, indicating that there was no toxic effect on the bone marrow.

 

No biologically or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in mice treated with Solvent Yellow 124.

 

A large statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed in the positive control group, demonstrating the sensitivity of the test system.

 

It is concluded that Solvent Yellow 124 showed no evidence of mutagenic or clastogenic activity in this mouse micronucleus test after administration by oral gavage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

It is concluded that Solvent Yellow 124 showed no evidence of mutagenic or clastogenic activity in this mouse micronucleus test after administration by oral gavage.

Justification for selection of genetic toxicity endpoint
The Mouse Micronucleus Test performed in accordance with the OECD quideline “Mammalian Erythrocyte Micronucleus Test”, No 474 (1997) is recent in vivo study.