Reaction products of 4-amino-5-hydroxynaphthalene-2,7-disulfonic acid, coupled twice with diazotized 2-[(4-aminophenyl)sulfonyl]ethyl hydrogen sulfate, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic behaviour of the test substance was assessed in bacteria in vitro. In a GLP-compliant study according to OECD 471 no mutagenic effects have been noted in the plate incorporation tests and in the preincubation tests conducted both in presence and absence of an exogenous metabolic activation system. In addition, also data on one of the metabolic breakdown products of Reactive Black 5 are available showing also no mutagenic effects in the bacterial test system. Data investigating mutagenic behavior in mammalian cells in vitro are currently not available. However, Reactive Black 5 was found to have no clastogenic effect when exposed to human peripheral blood lymphocytes as per the OECD Guidleine 473.

Based on these results, Reactive Black 5 is judged to be not mutagenic and not clastogenic 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:

28. Aug. to 25. Oct. 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat (Ames) and hamster (Prival)

Test concentrations with justification for top dose:

Preliminary Test: 4, 20, 100, 500, 2500, 10000 µg/plate
Main Test: 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

aqua bidest

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

all strains with rat S9; TA 1535, TA 1537, and TA 100 with hamster S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains with rat S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: benzidine

Remarks:

TA98 with hamster S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 w/o S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 w/o S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 w/o S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 min (Prival)
- Expression time (cells in growth medium): 48 to 72 hours

DETERMINATION OF CYTOTOXICITY
- Method: thinning of bacterial lawn; reduced rate of spontaneously occurring colonies (surviving fraction)

Evaluation criteria:

Cytotoxicity: reduced growth rate or thinning of bacterial lawn
Positive:
- dose-related and reproducible increase in number of revertant colonies
- doubling of spontaneous mutation rate in at least one tester strains either with or without S9

Statistics:

-

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative

The test substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Executive summary:

The test item Reactive Black 5 was tested for mutagenicity with the strains TA100, TA1535, TA1537, TA98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of six different doses from 4 µg/plate to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains.

5000 µg/plate was chosen as top dose level for the mutagenicity study.

a) Ames test:

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the the test item

b) Prival Test:

In the presence of hamster Liver S9 using the preincubation method according to Prival the test item 'fl. getrocknet' did not induce a significant increase in the number of revertant colonies, with any of the tester strains.

Summarizing, it can be stated that the test item is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 June, 2019 to 08 January, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

A spacing factor of 3 instead of 3.16 was used in the preliminary cytotoxicity assay, which did not impact the study integrity.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name: FAT 40839/C
Batch Number: FAT 40839/C
Manufacturing Date: NA
Retest Date:12/04/2019
Expiry date: 26/02/2024
Physical Appearance: Dark color powder
Properties: Reactive chemical
Storage Conditions: Freezer (-15 ± 5 ºC)
Purity by Assay: 94.2 %

Target gene:

Human peripheral blood lymphocytes

Species / strain / cell type:

other: human peripheral blood

Metabolic activation:

with and without

Metabolic activation system:

Sodium phenobarbital & β-naphthaflavone induced rat liver microsomal enzymes (S9 homogenate), prepared in-house from male Wistar rats was used for the assay.

Test concentrations with justification for top dose:

500, 1000 and 2000 µg/ml for short term, both in the presence and absence of metabolic activation and 167.5, 335, 670 µg/ml for continuous exposure in the absence of metabolic activation.
Justification for top dose: The mitotic index of the highest concentration selected for the main study was between 40 and 50 % when tested in the preliminary cytotoxicity assay.

Vehicle / solvent:

DMSO- Vehicle(s)/solvent(s) used: DMSO;

- Justification for choice of solvent/vehicle: The test item was sparingly soluble in water. However, the test item was found to be soluble in DMSO. DMSO at 1% was found to be compatible with the survival of the cells and did not interfere with S9 activity. Hence, DMSO was selected as vehicle for this study.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Test System
Human venous blood from healthy young non-smoking individuals with no known illness or recent exposure to genotoxic agents (e.g. chemicals, ionizing radiations) was drawn into sterile sodium heparin coated tubes. In the preliminary cytotoxicity assay, the human venous blood from three healthy young male donors aged 20-27 years was collected and pooled before initiation of cultures. Similarly, human venous blood from one healthy young male donor aged 18years, was collected and used for the initiation of cultures for chromosome aberration assay.

Justification for the selection of test system
The in vitro chromosome aberration assay employs human peripheral blood as primary cell cultures derived from healthy human donors as one of the preferred test systems. The primary cell cultures of human whole blood were selected based on their ability to grow in culture, stability of karyotype, chromosome number, chromosome diversity and spontaneous frequency of chromosome aberrations.

Media and Cell Culture Conditions
Whole blood was cultured in 10 cm2 tissue culture tubes by adding 0.9 ml of heparinised blood to 8.9 ml of culture medium (RPMI-1640)containing 15 % FBS, penicillin-streptomycin solution(1 %) and 0.2 ml phytohaemagglutinin (PHA-M form).The cultures were incubated at 37 ± 2 °C in a humidified atmosphere of5 ± 0.5 % CO2 in air for approx., 48 h.

Dose Formulation Preparation
Formulations for the preliminary cytotoxicity assay were prepared freshly before treatment. A stock solution was prepared by dissolving 400.0 mg of test item in 2 ml DMSO, equivalent to 200 mg/ml. Subsequent serial dilutions were made from the stock solution to attain concentrations of 66.7, 22.2, 7.4, 2.5 and 0.8 mg/ml.
Formulations for the main study were prepared freshly before treatment. A stock solution was prepared by dissolving 800.02 mg of test item in 4.0 ml of DMSO equivalent to 200 mg/ml. Subsequently, dilutions were prepared from the stock solution to attain concentrations of 100, 50, 67, 33.5 and 16.75 mg/ml.

Main Study- Short and Continuous Exposure
The main study was conducted with an exposure time of approximately 4 h (short exposure), both in the presence and absence of metabolic activation system and with an exposure time of approximately 22 h (continuous exposure) in the absence of metabolic activation. Cultured cells were treated at 500, 1000 and 2000 µg/ml for short term both in the presence and absence of metabolic activation and 167.5, 335, 670 µg/ml for continuous exposure in the absence of metabolic activation.
Cultured human peripheral lymphocytes suspended in approximately 7.4 ml of serum free culture media (RPMI - 1640) for treatment with S9 mix and 7.9 ml of culture media supplemented with 15 % FBS for treatment without S9 mix. For presence of metabolic activation, 0.5 ml of S9 mix per culture was added. Cultures were then treated with 100µl of the test item, vehicle control and positive control at appropriate concentrations as specified in respective labels.
For cultures exposed to the test item for a short duration, treatment medium was replaced with fresh medium containing RPMI 1640 and 15% FBS after approximately 4h.
The pH of the culture medium used in all the treatment, vehicle and positive control groups were checked after the treatment period. No considerable change in the pH for the test concentrations was observed as compared to concurrent vehicle controls.
At the end of incubation period, cultures were washed with media to remove the test item, and fresh culture medium supplemented with 15% FBS was added. Incubation was continued until the time of harvest.
The pH of the treatment medium of all the test concentrations including the vehicle and positive control groups was checked before and after treatment.

Assay Termination and Harvesting of the Cultures
About 0.1 ml Colchicine (50 µg/ml stock in sterile water) was added to all the cultures to arrest cells at the metaphase stage of mitosis, about 2h before harvesting. After mitotic arrest, at around 23 h, cultures were centrifuged at 1000 rpm for 10 minutes and the supernatant was discarded. The residual cell pellet was suspended in 5ml of 0.075 M potassium chloride by gentle vortexing and incubated for 20 minutes at 37 ± 2 °C for hypotonic treatment. Following hypotonic treatment, the cells were centrifuged, supernatant removed and were fixed in approximately 5 ml of chilled methanol:glacial acetic acid (3:1 v/v) solution, with gentle mixing. The fixation and removal steps were repeated thrice until the cell pellets appeared off white in colour.
After the last centrifugation, the supernatant was discarded from each culture tube, leaving the pellet in a small volume of supernatant. The resultant cell suspension was dropped onto a clean chilled slide from a height of approximately 30 to 60 cm and placed for drying on a slide warmer. Slides were prepared in duplicate for each culture.
After drying, the slides were stained with 5% Giemsa stain in Sorenson’s buffer (pH 6.8). All the slides were mounted with DPX and a quality control check of the slides was carried out before microscopic evaluation.
At least 1000 cells were analysed for mitotic index (per cent of cells in mitosis) from each culture in the test item, vehicle and positive control cultures. All the slides were coded before slide scoring to avoid bias during evaluation.
Cells were selected for scoring on the basis of well spread metaphases and only those cells with the number of centromeres equal to the modal number 46 ± 2 (44 - 48 chromosomes) were analysed. 300 metaphases per test concentration level (150 from each of the duplicate cultures) were read for structural chromosome aberrations. Percent polyploidy and endoreduplication were also noted simultaneously per culture and tabulated.
For each cell bearing an aberration, the microscope stage coordinates were recorded. The complete list of aberrations scored, and their definitions are provided in Appendix 6. Gaps were recorded separately and reported but were not included for analysis.
The experimental procedures of (culture initiation, test item exposure, harvesting of cultures and slide preparation) chromosome aberration assay for short term exposure and continuous exposure were performed together.
The cultures were harvested, processed for slide preparation and were observed under 100 X magnification for chromosome aberrations.

Evaluation criteria:

Assay Evaluation Criteria
 The test item is considered clastogenic, if a significant increase (p ≤ 0.05) in the number of cells with chromosome aberrations is observed at one or more test concentration levels tested. The linear trend test evaluates the dose-response. If a significant increase is seen at one or more concentration levels, a test concentration-response was performed.
 The test item is considered non-clastogenic, if it meets none of the above criteria under all experimental conditions.
 If the data set precludes making a definitive judgment about the activity of the test item, the test item is declared as equivocal or having a questionable response regardless of the number of times the assay is repeated.

Statistics:

The experimental unit was the cell, and therefore the percentage of cells with structural aberrations was the basis for evaluation. The statistical analysis was performed using GraphPad Prism version 4.0 applying chi square test for dose dependent significance among the treatment groups to compare the percentage of cells with aberrations in positive controls to the results obtained for the vehicle control. Statistical analysis was not performed for cells exhibiting polyploidy and/or endoreduplication.

Species / strain:

other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Results:

The mean percentage aberrations observed in cells with structural aberrations in the short-term exposure treatment without metabolic activation system, excluding gaps were 0.67, 0.33, 0.0, 1.00 with respect to vehicle control,500, 1000 and 2000 µg/ml of test item respectively. No significant increase in the frequency of percentage structural aberrations was observed. Similarly, the mean percentage aberrations in cells with structural aberrations for continuous exposure in the absence of metabolic activation was 0.67, 0.67, 0.34, 0.00, respectively for the vehicle control group and test item groups of 167.5, 335 and 670µg/ml and showed no statistically significance in increased percentage structural aberrations. The total structural aberrations in the short term treated cultures ranged from 0.0 to 1.67 % and for continuous exposure treated cultures from 0.0 to 0.67% in the absence of metabolic activation.

The mean percentage cells with structural aberrations in short term exposure treatment in the presence of metabolic activation system; excluding gaps were 0.34, 1.00, 0.34, 0.67 respectively for the vehicle control group and the test item groups at500, 1000 and 2000 µg/ml, thus indicating no statistical significance in increase in percentage structural aberrations. The total structural aberrations in the short term treated cultures with metabolic activation ranged from 0.33 to 2.0 %.

Positive controls, Mitomycin C (0.25 µg/ml) in both short term and continuous exposure without metabolic activation and Cyclophosphamide (12.5 µg/ml) with metabolic activation showed a statistically significant induction (p ≤ 0.01) in the percentage of structurally aberrated cells viz., 8.33, 7.67 and 6.67% respectively when compared to vehicle control and hence demonstrated the sensitivity of the assay.Overall aberrations observed in the treatment groups included fragments, minutes, deletions, chromatid break and dicentric. No significant increase in the aberration rate was observed with treated concentrations in any of the treatment regimes.

Spontaneous aberrations observed in the vehicle controls were found to be comparable to the in-house historical vehicle control data (short term with S9: 0.87 ± 0.79, Short term without S9: 0.83 ± 0.69 and continuous exposure: 1.00 ± 0.79) and were regarded acceptable to include in the historical control range (95% confidence limits).

The frequency of numerical aberrations recorded was 0% in short term and from 0% in continuous exposure without the metabolic activation system.

The numerical aberration frequency for short term exposure with metabolic activation was from 0.0 to 0.34%.

AMENDMENTS AND DEVIATIONS

Three amendments were made to the study plan during conduct of the study. The first amendment was to correct the purity of the test item from 89.6 to 94.2% based on sponsor’s information. The second amendment was to extend the experiment completion date from August 2019 to September 2019.Third amendment was made based on sponsor’s suggestion to amend the Sponsor’s address.

One deviation was recorded to the study plan for using a spacing factor of 3 instead of 3.16 in the preliminary cytotoxicity assay, which did not impact the study integrity.

Conclusions:

FAT 40839/C is non-clastogenic to cultured human peripheral blood lymphocytes.

Executive summary:

FAT 40839/C was assessed for the ability to induce structural chromosome aberrations in cultured human peripheral blood lymphocytes, both in the absence and presence of an exogenous metabolic activation system (S9), according to OECD Guideline 473. The test item was soluble in Dimethyl Sulfoxide (DMSO) at 200 mg/ml. Based on the findings of preliminary cytotoxicity assay, human peripheral blood lymphocytes were exposed to 500, 1000 and 2000 µg/ml for short term, both in the presence and absence of metabolic activation and 167.5, 335, 670 µg/ml for continuous exposure in the absence of metabolic activation. For the presence of metabolic activation, cells were exposed for a short duration (approx. 4 h) whereas for absence of metabolic activations cells were exposed for short and continuous durations (approx. 4 h and 22 h). Concurrent vehicle control (DMSO) cultures were also maintained. Cyclophosphamide (12.5 µg/ml) and Mitomycin C with (0.25 µg/ml) were used as positive controls for both with and without metabolic activation respectively. All concentrations were tested in duplicate. Following treatment, the cells were arrested at metaphase stage, harvested and spread on microscope slides for the evaluation of metaphases. A total of 300 metaphases per test concentration (150 from each of the duplicate cultures) were evaluated microscopically for the presence of chromosome aberrations. No statistically significant or dose dependent increases in percent structural chromosome aberrations were observed in cultures treated with test item concentrations either in the short-term exposure or in the continuous exposures. Overall aberrations observed in the treatment groups included fragments, minutes, deletions, chromatid breaks and dicentrics. Spontaneous aberrations observed in the vehicle controls were found to be comparable to the in-house historical vehicle control data and were regarded acceptable to include in the historical control range (95 % confidence limits). The results of cultures exposed to positive controls revealed a significant (p ≤0.01) increase in the number of structural aberrations as compared to the vehicle control, thus demonstrating the sensitivity and validity of the test procedure.

Conclusions:

FAT 40839/C did not induce significant structural chromosome aberrations both in the presence and absence of metabolic activation up to the tested concentration of 2000 µg/ml. A confirmatory assay performed without metabolic activation at a continuous exposure revealed similar results up to a concentration of 670 µg/ml. Based on the results, it is concluded that FAT 40839/C is non-clastogenic to cultured human peripheral blood lymphocytes under the tested conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

To support these initial findings, mutagenic and clastogenic effects have been investigated in vivo. A GLP-compliant study according to OECD 475 investigated effects of the test substance on chromosomal level after single treatment of 5000 mg/kg bw. The test item was administered once orally by gavage in a single dose of 5000 mg/kg bodyweight to male and female Chinese hamsters. This dose had been shown in a preliminary study to be the maximum tolerated dose. As a result, no increase of chromosomal aberrations have been noted compared to control. Therefore, the test was judged negative (non-clastogenic) in this in vivo test system.

To test mutagenic behavior of the test item in vivo, the test substance was administered once by gavage as single doses of 0 (vehicle control), 2000 and 10000 mg/kg bodyweight to male and female NMRI mice (5 mice per sex and killing time point). 10000 mg/kg bw had been shown in a preliminary study to be the maximum feasible dose. A positive control group received Endoxan at an oral dose of 50 mg/kg body weight. Animals were killed 12, 24, or 48 hours after treatment. The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained. 1000 poly- and 1000 normochromatic erythrocytes were screened for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. Under the conditions tested, the test item caused no significant increase in the number of cells with micronuclei in the bone marrow cells of treated animals as compared with the control group. The results indicate that, under the conditions of the test, the test item is not mutagenic in the in vivo micronucleus test.

Based on these results in vivo, Reactive Black 5 is judged to be not mutagenic and not clastogenic in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09. to 19. July 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

data waiving: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: males: 25 to 33 g; females: 22 to 29 g
- Assigned to test groups randomly: yes, computer based randomization scheme
- Fasting period before study: -
- Housing: 5 animals/cage
- Diet: Altromin 1324 rat and mouse diet ad libitum
- Water: tap ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 45 to 65
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 09 to 19. July 1984

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: freshly 25 % suspension in water: 12500 mg TS mixed with water by means of a magnetic stirrer and filled up to 50 mL in a volumetric flask.

Dose level: 2000 and 10000 mg/kg bw
Dose concentration: 250 mg/mL
Dose volume: 8 and 40 mL/kg bw

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

Vehicle control and high dose: 24, 48, and 72 hours
Positive control and low dose: 24 hours

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Dose / conc.:

10 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 per time point

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (Endoxan - batch 0378)

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
maximum feasible dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
single administration
sampling 24, 48, and 72 hours after administration (vehicle control and high dose group)

DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum and centrifuged (5 min, 1000 rpm)
Air dried smears were stained with May-Grünwald-Giemsa:
- 5 minutes Methanol
- 3 minutes May-Grünwald-solution
- 2 minutes May-Grünwald-solution, diluted with aqua dest. 1 + 1
- 2x rinsed in aqua dest.
- 10 minutes stained with 1 part Giemsa-solution + 6 parts buffer solution pH 7.2 according to Weise
- rinsed in aqua dest.
- air dried
- coating with Entellan


METHOD OF ANALYSIS: (blinded scoring)
1000 polychromatic erythrocytes per animal were scored for incidence of micronuclei. The number of cells with micronuclei was recorded, not the number of individual micronuclei.
1000 mature erythrocytes were scored for incidence of micronuclei as inspection measure
the ratio of polychromatic to normochromatic erythrocytes was determined

Evaluation criteria:

statistically significant difference in number of cells with micronuclei in dose groups compared to respective vehicle control groups

Statistics:

incidence of cells with micronuclei (polychromatic or normochromatic) compared to the vehicle control groups by means of binominal distribution (computer program Diamant) with a level of significance of 95 %.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

diarrhea, narrowed lids, squatted posture, discolorations (eyes, extremities, muzzle, tail, ears, skin, urine, feces)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Necropsy findings: dark to light bluish staining of: skin, tongue, peritoneum, fatty tissue, muscles, stomach and intestines, urinary bladder, testes, ovaries, adrenals

Conclusions:

Interpretation of results: negative
No significant increase in number of cells with micronuclei at 2000 and 10000 mg/kg body weight test item. Administration of the test substance up to the maximum feasible dose did not result in any alterations.

Executive summary:

The test item was administered once orally by gavage as single doses of 0 (vehicle control), 2000 and 10000 mg/kg bodyweight to male and female NMRI mice (5 mice per sex and killing time point). 10000 mg/kg bw had been shown in a preliminary study to be the maximum feasible dose. A positive control group received Endoxan at an oral dose of 50 mg/kg body weight.

Animals were killed 12, 24, or 48 hours after treatment by carbon dioxide asphyxiation. The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained. 1000 poly- and 1000 normochromatic erythrocytes were screened for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined.

Under the conditions of the present study, the test item caused no significant increase in the number of cells with micronuclei in the bone marrow cells of treated animals as compared with the control group. Endoxan, however, produced a marked increase in number of cells with micronuclei.

The results indicate that, under the conditions of the present study, the test item is not mutagenic in the in vivo micronucleus test.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21. to 24. March 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

hamster, Chinese

Strain:

other: Han: Chin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zentralinstitut fur Versuchstiere, Hannover
- Age at study initiation: 10 - 14 weeks
- Weight at study initiation: males: mean = 29.2 g (25 - 33 g)
females: mean = 27.29 g (24 - 31 g)
- Assigned to test groups randomly: yes, computer based randomization scheme
- Fasting period before study: no data
- Housing: one animal per cage
- Diet: Altromin 7010 hamster diet (Altromin GmbH, Lage/lippe), ad libitum
- Water: tap water in plastic bottles ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 45 to 65
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 21. to 24. March 1988

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 50 % suspension in sesame oil

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

12, 24 and 48 hours

Remarks:

Doses / Concentrations:
5000 mg/kg body weight
Basis:
other: nominal dose

Remarks:

Doses / Concentrations:
500 mg/mL
Basis:
nominal conc.

No. of animals per sex per dose:

Control and Reactive Black 5: 5 males and 5 females per killing time point
Positive control: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (Endoxan: batch 05459)
- Justification for choice of positive control(s): availability of historical data
- Route of administration: oral
- Doses / concentrations: 50 mg/kg body weight (5 mg/mL)

Tissues and cell types examined:

bone marrow cells from femur: 50 metaphases/animal

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: MTD based on preliminary study


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test substance was administered orally by gavage to the test animals at a single dose of 5000 mg/kg bodyweight. Sesame oil was administered in the same way to the negative control group. A positive control group, introduced after exactly 24 hours to run parallel with the negative control and the dose groups, received Endoxan at an oral dose of 50 mg/kg bodyweight. Two hours before killing by carbon dioxide asphyxation (12, 24, or 48 hours after treatment ), the hamsters each received an intraperitoneal injection of 3.3 mg demecolcin (Colcemid) per kg bodyweight.

DETAILS OF SLIDE PREPARATION:
After killing, both femora were removed and the bones completely stripped of muscle tissue. After removal of the epiphyses, the bone marrow was flushed in alternate directions out of the diaphysis into a centrifuge tube by means of a syringe containing Hanks solution (2 ml/femur) at the temperature of 37°C, mixed and centrifuged for 5 minutes at 1000 rpm after which all but a few drops of the supernatant was drawn off by pipette and sediment resuspended by tapping.
For hypotonic treatment, approximately 5 ml of 0.075 M potassium chloride solution at 37°C was quickly added and suspended. This suspension was then allowed to incubate for 10 minutes in a water bath at 37°C. Addition of 1.5 ml fixative (methanol : glacial acetic acid 3 + 1) and flow through with air. After re-centrifugation for five minutes at 1000 rpm, all but one drop of the supernatant was drawn off by pipette. The sediment was carefully covered with a layer composed of 2.5 ml fixative. After 20 minutes the fixation was removed carefully with a pipette and suspended in 2.5 ml fixative. After another 30 minutes, the mixture was centrifuged, after which the liquid was removed by pipette and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a refrigerator at 4°C.
After re-centrifuging for 5 minutes at 1000 rpm, all but one drop of the liquid was removed by pipette and a new suspension formed with a small quantity of freshly prepared fixative. A few drops of this suspension were placed with a Pasteur pipette onto clean microscopic slides which had been stored in distilled water at 4°C, the drops were then briefly passed through a Bunsen flame and air-dried for 24 hours. Staining was performed as follows:
- staining for 10 minutes in 2% orcein solution
- rinsing 3 times in distilled water
- rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetone/xylene
- 5 minutes in xylene
- 10 minutes in xylene
- embedding in Entellan or Eukitt


METHOD OF ANALYSIS:

After the slides had been coded, 50 metaphases per animal were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. The chromosomal aberrations were classified. The metaphases were examined for the following aberrations: gap (g), break (b), fragment (f), minute (m), deletion (d), exchanges including intrachanges (ex), dicentricity (di), chromosome disintegration (cd) ring (ri) and polyploidy (pp). In addition, metaphases with 5 and more aberrations were classified separatly as multiple aberrations (ma).
After the metaphases had been evaluated, the code was lifted. The values for the control group were compared at each killing time with the results from the dose groups and the positive control.

Evaluation criteria:

1. Structural aberrations
Gap: Non stained segment (achromatic gap) of chromatide without dislocation of the apparently separate part, irrespective of size of the non-stained area.
Break: A visible fracture of the chromatide structure where the broken piece is laterally dislocated or shifted in the longitudinal axis but can still be assigned to the corresponding centric part.
Fragment: Acentric part of a chromosome which may appear individually, regardless of their size.
Minute: Small chromatide body with a diameter smaller than the width of the chromatide.
Deletion: Terminal or interstitial losses of part of the chromatide.
Exchange: These are exchange aberrations, subdivided into intrachanges (the union of parts that can combine, within a chromosome) and interchanges (the union of parts that can combine from two or more chromosomes). Dicentric chromosomes and ring chromosomes are included in this group.

The chromatide aberrations specified above can also occur as iso-chromatide aberrations (e.g. isochromatid break)

2. Numerical aberrations
Aneuploidy: A deviation from the typical number of individual chromosomes in a set of chromosomes; a decrease in the number is known as hypoploidy an a increase as hyperploidy.
Polyploidy: More than two sets of chromosomes.

3. Additional criterion:
Chromosomal disintegration: where all or most of the chromosomes are irregular particles. If exchange figures occur in the metaphases, they are only included in this aberration group.

Statistics:

Comparison of no of aberrations of treated and control groups

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

feces black colored; urine greyblue colored

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw (highest applicable dose)
- Clinical signs of toxicity in test animals: no clinical signs, no deaths
- Evidence of cytotoxicity in tissue analyzed: -


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: not different from vehicle control

Conclusions:

Interpretation of results (migrated information): negative
The test item is not mutagenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.

Executive summary:

The test item was administered once orally by gavage in a single dose of 5000 mg/kg bodyweight to male and female Chinese hamsters. This dose had been shown in a preliminary study to be the maximum tolerated dose.

A positive control group, induced exactly 24 hours later to run parallel with the negative control and the dose group, received Endoxan in an oral dose of 50 mg/kg bodyweight.

Animals from each group were killed 12, 24 and 48 hours after treatment by carbon dioxide asphyxiation. 5 males and 5 females from each group were killed at each of these times.

The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained, after which 50 metaphases per animal were evaluated. The completeness in the number of chromosomes and the various chromatic and chromosomal aberrations were assessed.

Under the conditions of the present study, the test item caused no significant increase in the aberration rate in the bone marrow cells of the treated animals as compared with the control group.

Endoxan however produced a marked increase in the aberration rate in the test animals.

The results indicate that, under the conditions of the present study, the test item is not clastogenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available genotoxicity studies, Reactive Black 5 does not need to be classified for genotoxicity according to Directive 67/548/EEC and according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.