C.I. Reactive Yellow 161

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is non-mutagenic in the Salmonella typhimurium reverse mutation assay including the Prival modification for azo dyes. In an in-vitro chromosome aberration test in V79 hamster cells, the test item induced structural chromosome aberrations without metabolic activation after 28 hrs continuous treatment, only at a cytotoxic concentration and in the presence of precipitates. Hence it is considered unlikely that the test substance is clastogenic in this test system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only hamster S9, only Salmonella strains

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Hamster liver S9

Test concentrations with justification for top dose:

10.0; 100.0; 333.3; 1000.0; and 5000 μg/plate
based on a toxicity test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

other: 4-NOPD

Details on test system and experimental conditions:

Pre-incubation test
For each strain and dose level, including the controls, a minimum of three plates was used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl Test solution at each dose level,solvent control, negative control, or reference mutagen solution (positive control),
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
100 µl Bacteria suspension (cf. test system, pre-culture of the strains),
After pre-incubation 2.0 ml of molten 45 °C overlay agar was added to each tube. The mixture was poured an minimal agar plates.
After solidification the plates were incubated upside down for 72 hours at 37° C in the dark.

Evaluation criteria:

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 40'162/B to induce gene mutations according to the pre-incubation test for azo dyes using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was te-sted in triplicate. The test article was tested at the following concentrations:

10.0; 100.0; 333.3; 1000.0; and 5000.0 µg/plate.

Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in the test groups with and without metabolic activation at the highest investigated dose.

The plates incubated with the test article showed normal Background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, no significant and repro-ducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, FAT 40'162/B is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July to October 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

AMES, B.N., F.D. LEE, and W.E. DURSTON (1973), An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens. Proc. Natl. Acad. Sci. USA 70, 782-786.
AMES, B.N., W.E. DURSTON, E. YAMASAKI, and F.D. LEE (1973), Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection. Proc. Natl. Acad. Sci. USA 70, 2281-2285.
AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res. 31, 347-364.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomes and co-factors
In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added. 1 ml activation mixture contained: 0.3 ml S9 fraction of liver from rats (Tif:RAIf(SPF)) induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.7 ml of a solution of co-factors.

Test concentrations with justification for top dose:

The tests were performed with the following concentrations of the trial substance without and with microsomal activation:
First experiment: 20, 80, 320, 1280, 5120 µg/0.1 ml
Second experiment: 1481, 2222, 3333, 5000, 7500 µg/0.1 ml.

Vehicle / solvent:

The substance was dissolved in DMSO.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

cyclophosphamide

other: daunorubicin-HCl; N-methyl-N'-nitro-N-nitrosoguanidine; 2-amino-anthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate)
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: The plates were incubated for about 48 hours at 37 ± 1.5°C in darkness.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Counting of colonies

Evaluation criteria:

When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

No increase in mutation frequency was observed in the strains tested.

Executive summary:

FAT 40 162/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 µg/0.1 ml. In order to confirm the results, the experiments were repeated. An additional repetition was carried out with the concentrations of 1481, 2222, 3333, 5000 and 7500 µg/0.1 ml.

In none of the experiments a doubling rate of two or more were observed in any of the tested strains either with or without the metabolic activation system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 473 "In vitro Mammalian Chromosome Aberration Test".

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Commission Directive 2000/32/EC, L1362000, Annex 4A: "Mutagenicity — In vitro Mammalian Chromosome Aberration Test", dated May 19, 2000.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 — Environmental Agency"
"Eisei No. 127 — Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 Ministry of International Trade & Industry'.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Cytokinesis block (if used):

Colcemid was added (0.2 µg/mL culture medium) to the cultures 15.5 hrs and 25.5 hrs, respectively after the start of the treatment.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Preparation by RCC Cytotest Cell Research
- method of preparation of S9 mix
Phenobarbital/β-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D¬22335 Hamburg) and f3-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim), each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCI solution (1:3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80° C. Small numbers of the ampoules were kept at -20°C for up to one week.
The protein concentration was 32.6 mg/mL (Lot no. 021205). 8.4.2 S9 Mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCI
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath.

Test concentrations with justification for top dose:

Since no relevant toxicity was observed in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. Therefore, 625.0 µg/mL was chosen as top treatment concentration in the absence of S9 mix and 156.3 µg/mL in the presence of S9 mix in Experiment I.
Dose selection of Experiment II was influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed alter 24 hrs exposure with 625.0 µg/mL and above. Therefore, 1250.0 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix 312.5 µg/mL was chosen as top treatment concentration with respect to the results obtained in Experiment I.

Doses applied in the Chromosome aberration test
Preparation Exposure
interval period Exp. Concentration in µg/mL
Without S9 mix
18 hrs 4 hrs I 19.5 39.1 78.1 156.3 312.5 625.0P
18 hrs 18 hrs ' II 39.1 78.1 156.3 312.5P 625.0P 1250.0P
28 hrs 28 hrs II 78.1 156.3 312.5P 625.0P 1250.0P
With S9 mix
18 hrs 4 hrs I 4.9 9.8 19.5 39.1. 76.1 156.3P
28 hrs 4 hrs II 9.8 19.5 39.1 78.1P 156.3P 312.5P

P: Precipitation occurred

Vehicle / solvent:

deionised water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Seeding of the Cultures
Exponentially growing stock cultures more than 50 % confluent are treated with trypsin-EDTA-solution at 37° C for approx. 5 minutes. Then the enzymatic treatment is stopped by adding complete culture medium and a single cell suspension is prepared. The. trypsin concentration for all sub-culturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution (Invitrogen GIBCO, D-76131 Karlsruhe).
Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution. The Ca-Mg-free salt solution is composed as follows (per litre):
NaCI 8000 mg
KCI 200 mg
KH2PO4. 200 mg
Na2HPO4 150 mg
The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau), which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 10E4 - 610E4 cells were seeded with regard to the preparation time. The medium was MEM with 10 % FCS (complete medium).

Treatment
Exposure period 4 hours
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
The "Saline G" solution was composed as follows (per litre):
NaCI 8000 mg
KCI 400 mg
Glucose x H20 1100 mg
Na2HPO4 x 7H20 290 mg
KH2PO4 150 mg
pH was adjusted to 7.2

Exposure period 18 and 28 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
All cultures were incubated at 37° C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Preparation of the Cultures
Colcemid was added (0.2 µg/mL culture medium) to the cultures 15.5 hrs and 25.5 hrs, respectively after the start of the treatment. The cells on the slides were treated 2.5 hrs later, in the chambers with hypotonic solution (0.4 % KCI) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).

Evaluation of Cell Numbers
For evaluation of cytotoxicity indicated by reduced cell numbers two additional cultures per test item and solvent control group, not treated with colcemid, were set up in parallel. These cultures were stained after 18 hrs and. 28 hrs, respectively, in order to determine microscopically the cell number within 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

Analysis of Metaphase Cells
Evaluation of the cultures was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic Index (% cells in mitosis) was determined. In addition; the number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed. A test item is classified as clastogenic if:
the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher's exact test (9) (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Fisher's exact test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

18h/4 h

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

18 h/18 h

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

28 h/28 h

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

8% aberrant cells only at cytotoxic conentration in the presence of precipitations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

28 h/4 h

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In Experiment I, at preparation interval 18 hrs, precipitation of the test item in culture medium was observed with 625 0 µg/mL and above in the absence of S9 mix and with 156.3 µg/mL and above in the presence of S9 mix. In Experiment II, in the absence of S9 mix, precipitation of the test item was observed with 312.5 µg/mL and above at preparation interval 18 hrs and 28 hrs. In the presence of S9 mix, precipitation was observed with 78.1 µg/mL and above.
In this study, no cytotoxicity was observed up to the highest evaluated concentrations in the absence and the presence of S9 mix, except for Experiment II, at the 28 hrs preparation. interval. In detail, in Experiment II in the absence of metabolic activation the mitotic Index was strongly reduced (47.7 % of control) after 28 hrs treatment with 312.5 µg/mL. However, in Experiment II, in the absence of S9 mix, at the 18 hrs preparation interval the highest applied concentration showing clear cytotoxicity was not evaluable for cytogenetic damage.
In Experiment II, a statistically significant increase in the number of cells carrying structural chromosome aberrations was observed (see Table 11, page 32). In detail, the aberration rate of the cells after 28 hrs continuous treatment with 312.5 µg/mL of the test item in the absence of metabolic activation was increased to 8.0 % aberrant cells, exclusive gaps. This response clearly exceeded the range of our historical control data (0.0 — 4.0 % aberrant cells, exclusive gaps), providing additional evidence of the biological relevant of the observation. In all other experimental parts, neither a statistically significant nor biologically relevant increase in the number of aberrant cells carrying chromosomal aberrations could be observed.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.1 — 2.8 %) as compared to the rates of the solvent controls (1.1 —2.5 %).
In both experiments, EMS (200, 300, and 400 µg/mL, respectively) and CPA (1.4 and 2.0 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

The test item induced structural chromosome aberrations without metabolic activation after 28 hrs continuous treatment only at a cytotoxic concentration and in the presence of precipitates. Hence it is considered unlikely that the test substance is clastogenic in this test system.

Executive summary:

The test item, suspended (pre-experiment and Experiment II) or dissolved (Experiment I) in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsterin vitroin two independent experiments. The following study design was performed:

 

	Without S9 mix			With S9 mix
	Exp. I	Exp. II		Exp. I	Exp. II
Exposure period              .	4 hrs	18 hrs	28 hrs	4 hrs	4 hrs
Recovery	14 hrs			14 hrs	24 hrs
Preparation interval	18 hrs	18 hrs	28 hrs	18 hrs	28 hrs

 

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In this study, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, except for Experiment II, at the 28 hrs preparation interval. However, in Experiment II, in the absence of S9 mix, at the 18 hrs preparation interval the highest applied concentration showing clear cytotoxicity was not evaluable for cytogenetic damage.

In Experiment II, clastogenicity was observed after 28 hrs continuous treatment with 312.5 µg/mL in the absence of metabolic activation (8.0 aberrant cells exclusive gaps).

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Consequently, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro without metabolic activation only at a cytotoxic concentration and in the presence of precipitates. Hence it is considered unlikely that the test substance is clastogenic in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance did not exhibit any sign of cytogenicity in the mouse micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CF-1

Remarks:

non-consanguineous OF-1 albino mice

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IFFA-CREDO, L'Arbresle, France (Charles River)
- Age at study initiation:
- Weight at study initiation: ca 25 g
- Assigned to test groups randomly: yes
- Housing: in groups of 5 separated by sex
- Diet (ad libitum): Aliment Rats-Souris Charles River, produced by U.A.R., Villemoisson, France
- Water (ad libitum): tap water
- Acclimation period: 1 week

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

We first carried out a preliminary range finding test by treating three groups of three male mice with doses of 500, 1000 and 2000 mg/kg given by intra-gastric intubation. As no death occurred, the dose of 2000 mg/kg was chosen for the micronucleus test. This concentration was the maximum quantity of the test substance which can be suspended in water.
For the micronucleus test, each treatment group was comprised of five male and five female mice receiving one intragastric intubation using 0.5 ml per 25 g body weight (20 mL/kg bw). Animals were sacrificed at regular intervals after treatment.

Duration of treatment / exposure:

1. Control (distilled water): 0.5 ml - sacrificed at 44 hours
2. Positive control (Thio-TEPA): 20 mg/kg - sacrificed at 44 hours
3. FAT 40162/B: 2000 mg/kg - Sacrificed at 20 hours
4. FAT 40162/B: 2000 mg/kg - Sacrificed at 44 hours
5. FAT 40162/B: 2000 mg/kg - Sacrificed at 68 hours.

Frequency of treatment:

single

Post exposure period:

20, 44, 68 h

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Thio-TEPA (N, N', N"-triethylenethiophosphoramide), reference N° 509-227, made by Lederle Laboratories Ltd.

Tissues and cell types examined:

bone marrow - erythrocytes
Two types of erythrocytes were observed in the bone marrow smears: normo-chromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells).
The proportion of polychromatic erythrocytes containing one or more micronuclei was compared with the total number of polychromatic erythrocytes, and statistical comparisons were made between these ratios for the different groups. A minimum of 500 polychromatic erythrocytes were counted per smear (two smears per animal).

Details of tissue and slide preparation:

The method of preparation of mouse bone marrow smears for use in the micro-nucleus test was described in detail by Schmid (1977).
After sacrifice of the animals the femurs were taken and broken open at one end. Bone marrow cells were suspended in foetal calf serum using a small syringe, and the cells were centrifuged at 120 x g for 5 minutes. The super-natant was removed with a Pasteur pipette, cells were spread on a microscope slide and the smears allowed to dry in air. The following day smears were stained with Giemsa (1:6 in water) and mounted with a coverslip after drying.

Statistics:

The proportion of polychromatic erythrocytes containing one or more micronuclei was compared with the total number of polychromatic erythrocytes, and statistical comparisons were made between these ratios for the different groups using BMPD computer programme 7D.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 2000 mg/kg bw compared to negative control animals. In animals treated with Thio-TEPA there is a statistically significant increased number of micronucleated cells.
The ratio of polychromatic to normochromatic erythrocytes is markedly decreased in mice treated with Thio-TEPA. There is no statistical difference between the animals treated with the test substance and the negative control for this ratio.

Conclusions:

The test substance did not exhibit any sign of cytogenicity in the mouse micronucleus test.

Executive summary:

Reactive Yellow 161 was assayed for mutagenicity using the micronucleus test (EEC protocol). The compound was administered orally to mice at a concentration of 2000 mg/kg body weight. No mutagenic effect was observed in bone marrow smears taken 20, 44 and 68 h after administration of the test substance. A positive control (Thio-TEPA) administered at a concentration of 20 mg/kg showed pronounced evidence of mutagenicity 44 h after administration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Kirkland et al (2005) demonstrated an extremely high false-positive rate for in-vitro clastogenicity tests, particularly in mammalian cell tests, when compared to rodent carcinogenicity study results (Poth, A (2008)). “Certain characteristics of the commonly used rodent cell lines (CHO, CHL, V79, L5178Y, etc.), such as their p53 status, their karyotypic instability and their DNA repair deficiencies, are recognized as possibly contributing to the high rate of irrelevant positives. Also the need for exogenous metabolism with the cell systems is expected to contribute to these irrelevant positive findings, as metabolites produced by S9 used as a metabolic source in cell culture may be quite different from those produced by normal human liver metabolism.” Accordingly, “the high false-positive rate of the established in-vitro mammalian cell tests means that an increased number of compounds are subjected to earlier and additional in-vivo genotoxicity testing.”

Additional information

Reactive Yellow 161 was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed on strains TA 98, TA 100, TA 1535 and TA 1537 with the following concentrations of the trial substance without and with microsomal activation: 20, 80, 320, 1280 and 5120 µg/0.1 ml. In order to confirm the results, the experiments were repeated. An additional repetition was carried out with the concentrations of 1481, 2222, 3333, 5000 and 7500 µg/0.1 ml.

In none of the experiments a doubling rate of two or more were observed in any of the tested strains either with or without the metabolic activation system.

 

A second study was performed to investigate the potential of Reactive Yellow 161 to induce gene mutations according to the pre-incubation test for azo dyes using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

10.0; 100.0; 333.3; 1000.0; and 5000.0 µg/plate.

Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in the test groups with and without metabolic activation at the highest investigated dose.

The plates incubated with the test article showed normal Background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

 

The test item, suspended (pre-experiment and Experiment II) or dissolved (Experiment I) in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments.  

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (5000 µg/mL) was chosen with regard to the solubility properties of the test item in an appropriate solvent with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In this study, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations, except for Experiment II, at the 28 hrs preparation interval. However, in Experiment II, in the absence of S9 mix, at the 18 hrs preparation interval the highest applied concentration showing clear cytotoxicity was not evaluable for cytogenetic damage.

In Experiment II, clastogenicity was observed after 28 hrs continuous treatment with 312.5 µg/mL in the absence of metabolic activation (8.0 aberrant cells exclusive gaps).

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Consequently, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro without metabolic activation only at a cytotoxic concentration and in the presence of precipitates. Hence it is considered unlikely that the test substance is clastogenic in this test system.

Reactive Yellow 161 was assayed for mutagenicity using the micronucleus test (EEC protocol). The compound was administered orally to mice at a concentration of 2000 mg/kg body weight. No mutagenic effect was observed in bone marrow smears taken 20, 44 and 68 h after administration of the test substance. A positive control (Thio-TEPA) administered at a concentration of 20 mg/kg showed pronounced evidence of mutagenicity 44 h after administration.

 

Justification for classification or non-classification

No relevant genotoxic effects were observed in the studies conducted.N