Alkali salt of sulfonated aryl azo amino sulfonyl aryl azo sulfonyl aryl azo aryl sulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No genotoxic effects were observed in studies conducted

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:

07 July 2014 to 20 Oct 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant with GLP and testing guideline, coherence among data, results and conclusions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Target gene:

The test item Reactive Brown DYHY0331/0334 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from induced rat (rat mixed induction) or uninduced Syrian hamster (Prival modification)

Test concentrations with justification for top dose:

In Main Assay I and II: 5000, 2500, 1250, 625 and 313 µg/plate.

Vehicle / solvent:

sterile water for injection

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, Trypan Blue

Details on test system and experimental conditions:

Toxicity and Main Assay I were performed using the plate incorporatin method; Main assay II using the pre-incubaton method

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

doubling rate (Chu et al 1981)
regression line

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No relevant increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of any S9 metabolic activation system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the test item, Reactive Brown DYHY0331/0334, does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in
the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Reactive Brown DYHY0331/0334 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation. In the preliminary toxicity test and in Main Assay I, treatments were performed using the plate incorporation method in the absence and presence of a cofactor-supplemented S9 metabolic fraction prepared from the livers of rats treated with phenobarbitone and b-naphthoflavone. Based on the chemical structure of the test item (azodyes), Main Assay II was performed using the preincubation method and a reductive metabolic activation system (Prival modification).

The test item was used as a solution in sterile water for injection.

The test item Reactive Brown DYHY0331/0334 was assayed in the toxicity test at a maximum concentration of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Plates treated with the test item presented a dose dependent black colour of the agar, which did not interfere with the scoring of colonies.

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate.

No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Plates treated with the test item presented a dose dependent black colour of the agar, which did not interfere with the scoring of colonies.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assy II was performed using the pre-incubation method in the presence of flavin mononucleotide and uninduced hamster liver S9.

The dose-range used was the same as in Main Assay I.

No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Plates treated with the test item presented a dose dependent black colour of the agar, which did not interfere with the scoring of colonies.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system metabolism.

It is concluded that, under the reported experimental conditions, the test item Reactive Brown DYHY0331/0334 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of the standard S9 metabolic activation or using a reductive metabolic activation, as described by Prival and Mitchell for azo-dyes.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2014 to 24 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

The test item Reactive Brown DYHY0331/0334 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants
in Chinese hamster V79 cells after in vitro treatment. 6-thioguanine can be metabolised by the enzyme hypoxanthine-guaninphosphoribosyl-transferase (HPRT) into nucleotides, which are used in nucleic acid synthesis resulting in the death of HPRTcompetent cells. HPRT-deficient cells, which are presumed to arise through mutations in the HPRT gene, cannot metabolise 6-thioguanine and thus survive and grow in its presence.

Species / strain / cell type:

mammalian cell line, other: Chinese hamster V79 cells

Details on mammalian cell type (if applicable):

- Type and identity of media: EMEM medium supplemented with 10% Foetal Calf Serum (EMEM Complete)
- Permanent stocks of the V79 cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use.
All incubations are at 37°C in a 5% carbon dioxide atmosphere (100% nominal relative humidity).
- Periodically checked for Mycoplasma contamination
- Periodically checked for karyotype, generation time, plating efficiency and mutation rates (spontaneous and induced)

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction - Species: Rat - Strain: Sprague Dawley - Tissue: Liver - Inducing Agents: Phenobarbital / 5,6-Benzoflavone - Producer: MOLTOX, Molecular Toxicology, Inc. - Batch Number: 3263

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed. at the following dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 μg/mL.

Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:
Assay No. S9 Dose level (μg/mL)
Main I + 5000, 2500, 1250, 625 and 313
Main I - 5000, 3570, 2550, 1820, 1300, and 930
Main II + 5000, 2500, 1250, 625 and 313
Main II - 3600, 3130, 2720, 2370 and 1790

Vehicle / solvent:

Test item solutions were prepared using EMEM minimal medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

A preliminary cytotoxicity test was undertaken in order to select appropriate dose levels for the mutation assays. Treatments were performed both in the absence and presence of S9 metabolism; a single culture was used at each test point and positive controls were not included.

Two mutation assays were performed including negative and positive controls, in the absence and presence of S9 metabolising system. Duplicate
cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. Following treatment, the cultures were incubated at 37°C for three hours. At the end of the incubation period, the treatment medium was removed and the cell monolayers
were washed with PBS. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere
(100% nominal relative humidity) to allow for expression of the mutant phenotype.
Determination of survival: The following day, the cultures were trypsinised and an aliquot was diluted and plated to estimate the viability of the cells
Subculturing: On Day 3 and Day 6, the cell populations were subcultured in order to maintain them in exponential growth.
Determination of mutant frequency: On Day 6 and Day 9, each culture was trypsinised, resuspended in complete medium and counted by microscopy. After dilution, an estimated 1 x 10^5 cells were plated in each of five 100 mm tissue culture petri dishes containing medium supplemented with
6-thioguanine. These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants.
After dilution, an estimated 200 cells were plated in each of three 60 mm tissue culture petri dishes. These plates were used to estimate Plating
Efficiency (P.E.).

Evaluation criteria:

For a test item to be considered mutagenic in this assay, it is required that:
– There is a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two consecutive doses of the test item.
If only the highest practicable dose level (or the highest dose level not to cause unacceptable toxicity) gives such an increase, then a single
treatment-level will sufficent.
– There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).

Statistics:

The results of these experiments were subjected to an Analysis of Variance in which the effect of replicate culture, expression time and dose level in
explaining the observed variation was examined. For each experiment, the individual mutation frequency values at each test point were transformed
to induce homogeneous variance and normal distribution. The appropriate transformation was estimated using the procedure of Snee and Irr (1981),
and was found to be y = (x + a)^b where a = 0 and b = 0.275. A three way analysis of variance was performed (without interaction) fitting to three
factors:
– Replicate culture: to identify differences between the replicate cultures treated.
– Expression time: to identify differences in response at the expression times used.
– Dose level: to identify dose-related effect in response, after allowing for the effects of replicate cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9 metabolism.

Species / strain:

mammalian cell line, other: Chinese hamster V79 cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: Chinese hamster V79 cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first experiment, in the absence of S9 metabolism, a severe toxic effect was observed at 5000 μg/mL (5% survival). At 3570 μg/mL, survival was reduced to 10%, while no relevant toxicity was observed at the remaining dose levels. In the presence of S9 metabolic activation, no relevant toxicity
was noted at any concentration tested.
In the second experiment, in the absence of S9 metabolism, a marked reduction in cell survival was observed at 3600 μg/mL (19%). The two next
lower concentrations (3130 and 2720 μg/mL ) yielded 35% and 36% relative survival, respectively; slight toxicity (RS = 51 and 62%) was noted at the
next two lower dose levels, while no relevant toxicity was observed at 1790 μg/mL. In the presence of S9 metabolism, no relevant toxicity was noted at any concentration tested.
At low survival levels the mutation data are prone to a variety of artefacts; accordingly, we have excluded from the statistical analyses, mutation data
obtained in Main Assay I at 5000 μg/mL, in the absence of S9 metabolism.
No relevant increases over the spontaneous mutation frequency were observed in any experiment, at any treatment level either in the absence or presence of S9 metabolic activation.

Conclusions:

Interpretation of results (migrated information):
negative

No reproducible five-fold or greater increase in mutant frequency was observed either in the absence or presence of metabolic activation at any test point. No concentration-related increase in mutant frequency was observed.
It is concluded that Reactive Brown DYHY0331/0334 does not induce gene mutation in Chinese hamster V79 cells in vitro.

Executive summary:

The test item Reactive Brown DYHY0331/0334 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using EMEM minimal medium. A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 5000 μg/mL and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 μg/mL. In the absence of S9 metabolism, survival was reduced to 10% of the concurrent negative control value at the highest dose level, while no relevant toxicity was noted over the remaining concentrations tested. In the presence of S9 metabolism, no relevant toxicity was observed at any concentration tested. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Assay No. S9 Dose level (μg/mL)

Main I + 5000, 2500, 1250, 625 and 313

Main I - 5000, 3570, 2550, 1820, 1300, and 930

Main II + 5000, 2500, 1250, 625 and 313

Main II - 3600, 3130, 2720, 2370 and 1790

No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that Reactive Brown DYHY0331/0334 does not induce gene

mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No genotoxic effects were observed in studies conducted

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 03 Oct to 09 Dec 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy
- Age at study initiation: 7 to 8 weeks (day of allocation)
- Weight at study initiation: (P) Males: 251 g; Females: 213 g
- Fasting period before study: no
- Housing: 5 animals/sex/cage
- Diet (e.g. ad libitum): ad libitum except for clinical pathology
- Water (e.g. ad libitum): ad libitum except for clinical pathology
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2°C
- Humidity (%): 55%+/-15%
- Air changes (per hr): approximately 15 to 20 air change per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: from 03 October (day of allocation) to 09 December 2014 (last day of necropsy)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: up to 7 days interval

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: 6.25, 25, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): n/a
- Purity: n/a

Duration of treatment / exposure:

Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter
until the day before necropsy for a total of 48 days.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and
post partum periods until at least up to, and including, Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for
each animal according to the last recorded body weight. One female, no. X0010033 (Group 2), was erroneously dosed on Day 4 post partum.
During the gestation period, dose volumes were calculated according to individual body weights on Days 0, 7, 14 and 20 post coitum and on Day 1
post partum. Thereafter individual dose volumes remained constant.

Positive control group
Animals received a single dose (Mitomicyn-C) approximately 24 hours before sacrifice by intraperitoneal injection.

Frequency of treatment:

once a day

No. of animals per sex per dose:

Main groups
3 groups of 10 males and 10 females each dosed at 62.5, 250 and 1000 mg/kg/day bw. One control group (group 1) of 10 males and 10 females
received the vehicle alone.

Recovery groups
1 group of 5 males and 5 females dosed at 1000 mg/kg/day bw. One control group (group 5) of 5 males and 5 females received the vehicle alone.

Positive control group
One positive control of 5 males only was administered with Mitomycin-C (2 mg/kg) once by intraperitoneal injection at the dose volume of 10 mL/kg
body weight.

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Justification for choice of positive control(s): to induce known genotoxic effects
- Route of administration: intraperitoneal
- Doses / concentrations: 2 mg/kg/ 0.2 mg/mL

Tissues and cell types examined:

Bone marrow from one femur of male animals only.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Samples of bone marrow were collected approximately 24 hours following the final treatment and approximately 48 hours following the second last
treatment from 5 males of the main groups randomly selected and from all animals of the positive control group (Group 7). One femur of each animal
was removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared
to make smears on slides. These slides were air-dried and then stained with haematoxylin and eosin solutions and mounted with Eukitt.
Three slides were made from each animal.
METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power and one
slide from each animal was selected according to staining and quality of smears. Two thousand polichromatic erythrocytes (PCEs) per animal were
examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time, the numbers of normal and
micronucleated normochromatic erythrocytes (NCEs) were also recorded.

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
•The incidence of micronucleated PCEs of the vehicle control group fell within the historical negative control range.
•The positive control item induced a significant increase in the frequency of micronucleated PCEs.
• At least 5 animals per group are available for slide analysis.

Evaluation criteria
The test item was considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes
(at P<0.05) was observed in any treatment group.
Where statistically significant increases in the incidence of micronucleated PCEs were observed, but fell within the range of negative control values
within this laboratory, then historical control data were used to demonstrate that these increases did not have any biological significance.
Historical controls are included.

Statistics:

Only counts obtained from polychromatic cells were subjected to statistical analysis and the original observations (and not micronucleus frequencies
per 1000 cells) were used. The variation between individual animals within each treatment group was assessed by chi^{2} calculation.
In case of no significant heterogeneity within either group, the chi^{2} test was employed to compare treated groups with the control.
If at least one of the groups was not homogeneous, the variance ratio (F) value was calculated from the between-group and within-group
chi^{2} values to show the significance of any difference between treated and control groups.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information): negative
No signs of genotoxicity were detected by measuring the induction of micronuclei in polychromatic erythrocytes from the bone marrow.

Executive summary:

The objective of this study was to assess the potential genotoxic effect of the test item by examining the induction of micronuclei in bone marrow erythrocytes of treated and control animals. This study was conducted to OECD, EU and EPA test guidelines in compliance with GLP and reported with a valid GLP certificate.

This genotoxicity endpoint was included in the “Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test” (OECD Guideline 422).

A reproductive/developmental toxicity study, including evaluation of systemic toxicity after repeated dose of Reactive Brown DYHY 0331/0334, was conducted in Sprague Dawley SD rats up to Day 4 post partum. A genotoxicity assessment for potential clastogenic/aneugenic effects was also investigated in males of the main groups. The evaluation was performed in males only, as no gender-specific differences were observed so far in any toxicity tests. Furthermore, the males of this study were not effected by the reproductive endpoint of this study.

The animals received the test item, dissolved in softened water, at the dosages of 62.5, 250 and 1000 mg/kg body weight/day. Three groups of 10 males and 10 females each were administered orally by gavage with the test item at constant volume of 10 mL/kg body weight. A control group of the same number of animals/sex was administered with softened water only. In addition, two groups (Groups 5 and 6) of 5 animals/sex were included and administered for 4 consecutive weeks at the same treatment conditions to serve as recovery groups. A 4-week treatment-free period was allowed for these two additional groups, in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase. In addition, a positive control group comprising of 5 male rats were included, receiving 2 mg//kg of Mitomycin –C.

The micronucleus test was carried out in 5 males of the main groups by measuring the presence of micronuclei in polychromatic erythrocytes from the bone marrow.

 

Incidence of micronucleated cells

Following treatment with the test item, no increase in the number of micronucleated PCEs was observed in any group. The incidences of micronucleated PCEs were comparable to our historical control data for negative control animals.

The validity of the test system was confirmed by increases in the frequency of micronucleated PCEs observed in the positive control group. The proportion of immature erythrocytes (PCEs) among total erythrocytes (PCEs + NCEs) was acceptable at all dose levels of the test item compared to the vehicle control value (more than 20%) suggesting validity of micronucleus testing.

 

Bone marrow cell toxicity

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

 

Validity of the assay

The incidence of micronucleated PCEs of the negative control group fell within the historical control range. Statistically significant increases in the incidence of micronucleated PCEs over the control values were seen in the positive control group, demonstrating the laboratory proficiency in the conduct of the test.

Five animals per groups were available for micronucleus slide analysis.

Based on the stated criteria the assay was therefore accepted as valid.

 

Analysis of results

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated PCEs over the control value was observed in any treatment group.

A summary of the results obtained is presented in the following table:

 

Dose level(mg/kg/day)	Incidence in micronucleated PCEs			PCE/(PCE+NCE)% over the mean control value
	Mean	SE	Range
0.00	0.7	0.1	0.5 -1.0	100
62.5	1.1	0.3	0.0 - 2.0	85
250	1.2	0.2	1.0 - 2.0	88
1000	1.6	0.3	0.5 - 2.5	90
Mitomycin-C 2.00 mg/kg	10.5	1.4	7.0 - 15.0	87

 

 

Conclusions

On the basis of the results obtained, it is concluded that Reactive Brown DYHY 0331/0334 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test item Reactive Brown DYHY 0331/0334 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation. In the preliminary toxicity test and in Main Assay I, treatments were performed using the plate incorporation method in the absence and presence of a cofactor-supplemented S9 metabolic fraction prepared from the livers of rats treated with phenobarbitone and b-naphthoflavone. Based on the chemical structure of the test item (azodyes), Main Assay II was performed using the preincubation method and a reductive metabolic activation system (Prival modification).

The test item was used as a solution in sterile water for injection.

The test item Reactive Brown DYHY 0331/0334 was assayed in the toxicity test at a maximum concentration of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Plates treated with the test item presented a dose dependent black colour of the agar, which did not interfere with the scoring of colonies.

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate.

No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Plates treated with the test item presented a dose dependent black colour of the agar, which did not interfere with the scoring of colonies.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the pre-incubation method in the presence of flavin mononucleotide and uninduced hamster liver S9.

The dose-range used was the same as in Main Assay I.

No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Plates treated with the test item presented a dose dependent black colour of the agar, which did not interfere with the scoring of colonies.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of any metabolic activation system metabolism.

It is concluded that, under the reported experimental conditions, the test item Reactive Brown DYHY0331/0334 does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of the standard S9 metabolic activation or using a reductive metabolic activation, as described by Prival and Mitchell for azo-dyes.

The test item Reactive Brown DYHY 0331/0334 was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using EMEM minimal medium. A preliminary cytotoxicity assay was performed. The test item was assayed at a maximum dose level of 5000 μg/mL and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 μg/mL. In the absence of S9 metabolism, survival was reduced to 10% of the concurrent negative control value at the highest dose level, while no relevant toxicity was noted over the remaining concentrations tested. In the presence of S9 metabolism, no relevant toxicity was observed at any concentration tested. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Assay No. S9 Dose level (μg/mL)

Main I + 5000, 2500, 1250, 625 and 313

Main I - 5000, 3570, 2550, 1820, 1300, and 930

Main II + 5000, 2500, 1250, 625 and 313

Main II - 3600, 3130, 2720, 2370 and 1790

No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system. It is concluded that Reactive Brown DYHY0331/0334 does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

The test item Reactive Brown DYHY 0331/0334 was examined for the potential to induct micronuclei in bone marrow erythrocytes of treated and control animals. This genotoxicity endpoint was included in the “Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test” (OECD Guideline 422) instead conducting the in vitro chromosome aberration or micronucleus assay. This decision was made because the test item is a reactive vinyl-sulphone dye. It is well known that vinyl-sulphone compounds result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warra TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. As in the currently assessed dye, vinyl sulphone moieties are used in fibre-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in‑vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amount, as could be shown in plenty of tests with vinyl sulphone dyes (internal data DyStar). Hence, the in-vivo test produces more reliable data for this kind of substance.

A reproductive/developmental toxicity study, including evaluation of systemic toxicity after repeated dose of Reactive Brown DYHY 0331/0334, was conducted in Sprague Dawley SD rats up to Day 4 post partum. A genotoxicity assessment for potential clastogenic/aneugenic effects was also investigated in males of the main groups. The evaluation was performed in males only, as no gender-specific differences were observed so far in any toxicity tests. Furthermore, the males of this study were not effected by the reproductive endpoint of this study.

The animals received the test item, dissolved in softened water, at the dosages of 62.5, 250 and 1000 mg/kg body weight/day. Three groups of 10 males and 10 females each were administered orally by gavage with the test item at constant volume of 10 mL/kg body weight. A control group of the same number of animals/sex was administered with softened water only. In addition, two groups (Groups 5 and 6) of 5 animals/sex were included and administered for 4 consecutive weeks at the same treatment conditions to serve as recovery groups. A 4-week treatment-free period was allowed for these two additional groups, in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase. In addition, a positive control group comprising of 5 male rats were included, receiving 2 mg//kg of Mitomycin –C.

The micronucleus test was carried out in 5 males of the main groups by measuring the presence of micronuclei inpolychromatic erythrocytes from the bone marrow.

 

Incidence of micronucleated cells

Following treatment with the test item, no increase in the number of micronucleated PCEs was observed in any group. The incidences of micronucleated PCEs were comparable to our historical control data for negative control animals.

The validity of the test system was confirmed by increases in the frequency of micronucleated PCEs observed in the positive control group. The proportion of immature erythrocytes (PCEs) among total erythrocytes (PCEs + NCEs) was acceptable at all dose levels of the test item compared to the vehicle control value (more than 20%) suggesting validity of micronucleus testing.

 

Bone marrow cell toxicity

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

 

Validity of the assay

The incidence of micronucleated PCEs of the negative control group fell within the historical control range. Statistically significant increases in the incidence of micronucleated PCEs over the control values were seen in the positive control group, demonstrating the laboratory proficiency in the conduct of the test.

Five animals per groups were available for micronucleus slide analysis.

Based on the stated criteria the assay was therefore accepted as valid.

 

Analysis of results

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated PCEs over the control value was observed in any treatment group.

 

Conclusions

On the basis of the results obtained, it is concluded that Reactive Brown DYHY 0331/0334 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Justification for classification or non-classification

not classified