4,4'-[1,3-phenylenebis(azo)]bisbenzene-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames - OECD TG 471 - Read-across - Positive

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his

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:

S-9 mix

Test concentrations with justification for top dose:

20 µg-5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: complete solubility

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: without S-9 mix:N-methyl-N'-nitro-N-nitroso-guanidine (MNNC) (in DMS0) for the strains TA 100, TA 98 and TA 1535/ 4-nitro-o-phenylenediamine (in DMSO) for the strain TA 1538/ 9-aminoacridine chloride monohydrate (in DMOS) for the strain TA1537

Remarks:

with S9-mix: aminoanthracene for the strains TA100,Ta98,TA1538,TA1537 and TA1535

Details on test system and experimental conditions:

METHOD OF APPLICATION: Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml aminoacid solution (minimal aminoacid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 450°C, and the remaining components are added in the following order: 0.1 ml test solution /0.1 ml bacterial suspension/0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation). After mixing, the samples are poured onto the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

DURATION
After incubation for 48 hours at 370°C in the dark, the bacterial colonies (his+ revertante) are counted.

Test system:
1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 1538, TA 98.
Doses: 0, 20, 100, 500, 2500 and 5000 µg/plate.
Solvent: DMSO.
Type of test: standard plate test with and without S9.
Test condition: S-9 Mix.
Number o f plates: 4 test plates per dose or per control.

2nd Experiment
Strain: TA 1538.
Doses: 0, 20, 100, 500, 2500 and 5000 µg/plate.
Solvent: DMSO.
Type of test: standard plate test.
test condition: standard plate test with S-9 mix.
Number of plates: 4 test plates per dose or per control.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Weakly positive reaction only with S9 mix at 2500 > ig - 5000 µg/plate (factor 2.5)

Cytotoxicity / choice of top concentrations:

not specified

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

from about 20 µg to 100 µg

Cytotoxicity / choice of top concentrations:

not specified

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

From about 500 µg/plate (TA 1537) onward with an increase in the number > of his+ > revertants by a factor of - 20 at - 500 > µg/plate.

Cytotoxicity / choice of top concentrations:

not specified

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Without S9 mix only slight increase in the rate at 2500 µg/plate (factor 2.0). After the addition of S9 mix mutagenicity was observed from ca.100 µg/plate onward with an increase in the number of revertant colonies by a factor > 20 at 2500-5000 µg/plate.

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

no bacteriotoxic effect was observed

Conclusions:

The substance was tested for in vitro gene mutation in bacteria following OECD 471. Under the experimental conditions the substance shows positive results with metabolic activation.

Executive summary:

The analogue substance 1 was tested for mutagenicity to bacteria with strains TA1535, TA100, TA1537, TA1538 and TA98 of Salmonella Typhimurium with and without metabolic activation. Positive reactions were detected with metabolic activation for all strains at low doses except for strain TA1535.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

OECD TG 474 - Micronucleus "In vivo" - Read-across - Negative

OECD TG 486 - UDS - Read-across - Negative

OECD TG 489 - TPE Comet assay - Read-across - in progress

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

other: read across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

not yet defined

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: analogue substance 03

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies: not available.
- Available non-GLP studies: not available.
- Historical human data: not available.
- (Q)SAR: not available.
- Weight of evidence: not available.
- Grouping and read-across:
• Mammalian Erythrocyte Micronucleus test (OECD TG 474) on analogue substance 02.
• Unscheduled DNA Synthesis (OECD TG 486) on analogue substance 01.
• In vitro gene mutation study in bacteria (OECD TG 471) on analogue substance 01.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Under Annex VIII Section 8.4., column 2 of REACH, further mutagenicity studies must be considered in case of a positive result in an in vitro gene mutation study in bacteria.
Guidance on information requirements R7a, section 7.7.6 (2017), states that regarding Annex VIII, when both the mammalian cell tests are negative but there was a positive result in the bacterial test, it will be necessary to decide whether any further testing is needed on a case-by-case basis. For example, suspicion that a unique positive response observed in the bacterial test was due to a specific bacterial metabolism of the test substance could be explored further by investigation in vitro. Alternatively, an in vivo test may be required.
The submitted dossier on EC: 213-888-1 [Solvent Brown 41] contains positive results for the in vitro gene mutation study in bacteria [in read across from analogue substance 01], following OECD TG 471, which raises the concern for in vivo gene mutation. No particular mechanism for the tested substance only related to bacteria are known as nowadays.
In particular, annex VIII, Column 2 requires the registrant to consider appropriate mutagenicity in vivo studies already at the Annex VIII tonnage level, which involves studies mentioned in Annex IX (among OECD TG 474. Mammalian Erythrocyte micronucleus test, OECD TG 488 Transgenic Rodent Mutation Assay, OECD TG 489 In vivo mammalian Alkaline Comet Assay and OECD TG 486 Unscheduled DNA Synthesis).

CONSIDERATIONS ON THE IN VIVO STUDIES INSERTED IN THE DOSSIER AND EXPERT ASSESSMENT ON TESTING PROPOSAL
In the submitted dossier on EC: 213-888-1 [Solvent Brown 41], an OECD TG 474 (Mammalian Erythrocyte Micronucleus test) in vivo study conducted on the analogue substance 02 is available and shows negative results. This study is adequate to cover the chromosomal aberration potential of the two substances and to waive the performance of an in vitro cytogenicity in mammalian cells, as laid down in Column II of Annex VIII of the REACH Regulation.
Moreover, an OECD TG 486 (in vivo UDS assay) study is also present. The study is conducted in read across from the analogue substance 01, which resulted negative and can be used as supporting information for the in vivo gene mutation properties assessment, since the cells analysed in the UDS assay involve only those of the liver.
However, in order to further and completely assess its gene mutation properties in different tissues of the animal, a Comet Assay, OECD TG 489, on analogue substance 03 was presented as testing proposal and it will be also used in read across for assessing the in vivo potential gene mutation properties of the target substance, Solvent Brown 41 [EC: 213-888-1].
Analogue substance 03 is, in fact, considered as representative of the mutagenic behaviour of Solvent Brown 41 [EC: 213-888-1]. See the read-across section.
OECD TG 489 allows to measure DNA strand breaks, that may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.
OECD TG 488 is not considered as the first choice for assessing the gene mutation in vivo for this substance, since preliminary data for gene mutation in vivo (OECD TG 486) already indicates negativity in the somatic cells of the liver. A confirmation by the Comet assay performed over other tissues (and for azo dyes the intestinal tract is the site of major metabolism and dye/metabolites absorption) would be sufficient to assess the genotoxic potential of the substance.
Finally, as reported in literature, from the analysis of 91 chemicals with published data from Comet Assay and Transgenic rodent mutation assay (TGR), the comet assay appears to yield similar results to the TGR assay in liver and gastrointestinal tract (predominantly stomach and colon data) and, hence, can be confidently performed to confirm in vivo gene mutation activity in terms of genotoxicity in general.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Route of administration:

oral: gavage

Sex:

not specified

Genotoxicity:

other: to be performed

Remarks on result:

other: the test is in read across from a submitted testing proposal still under evaluation

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

since March 11, 2005 to May 24, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Swiss: Naval Medical Reserch Institute

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland.
- Age at study initiation:5 - 8 weeks.
- Weight at study initiation: ~ 25 g.
- Housing: makrolon cages, separately according to sex. Before the start of the treatment the animals were transferred to Makrolon cages, type MI, and housed individually under the same conditions until the end of the test.
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland).
- Water drinking: water from bottles were available ad libitum.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%):30 - 70%
- Air changes (per hr):fully air-conditioned rooms in which central air conditioning guaranteed.
- Photoperiod: 12 hours cycle dark/light. (12 hours Iight from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours).

OTHER:
- Feed analysis:
The feed used in the study was assayed for chemical and microbial contaminants. In view of the aim and duration of the study, contaminants occurring in commercial feed ought not to influence the test resuit.
- Water analysis:
The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and the Department of Water Chemistry and the Technical Services of BASF Aktiengesellschaft as well as for microorganisms by a contract laboratory. However, in view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water.
- Amount of vehicle: 20 ml/kg bw.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in purified water.
-The low dose group was given 2 500 mg test substance/kg body weight or 20 ml/kg body weight of a solution with a concentration of 12.5 g/100 ml.
- The intermediate dose group was given 5 000 mg test substance/kg body weight or 20 ml/kg body weight of a solution with a concentration of 25 g/100 ml.
- The top dose groups were given 10 000 mg test substance/kg body weight or 20 ml/kg body weight of a solution with a concentration of 50 g/100 ml.

Duration of treatment / exposure:

The animals were treated once and samples of bone marrow were taken 24 hours and 48 hours after the treatment.

Frequency of treatment:

one injection

Post exposure period:

24 - 48 hours

Remarks:

Doses / Concentrations:
2500, 5000, 10000 mg
Basis:
nominal conc.

No. of animals per sex per dose:

Sacrifice interval Test groups ml/kg or mg/kg b.w. Number of animals
male female
24 h Vehicle control 5 5
24 h 2500 mg Fastusol Braun PR 8195 A. S. 5 5
24 h 5000 mg Fastusol Braun PR 8195 A. S. 5 5
24 h 10000 mg Fastusol Braun PR 8195 A. S. 5 5
24 h 20 mg cyclophosphamide (CPP) 2 3
24 h 0.15 mg vincristine (VCR) 3 2
48 h Vehicle control 5 5
48 h 10 000 mg Fastusol Braun PR 8195 A. S. 5 5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Doses / concentrations:
cyclophosphamide (CPP) : 20 mg
vincristine (VCR): 0.15 mg

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

TREATMENT :
- The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur).
- The Suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 pl fresh FCS.
- 1 drop of this Suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.


DETAILS OF SLIDE PREPARATION:
- The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained in Giemsa solution (15 ml Giemsa, 185 ml purified water) for about 15 minutes.
- After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.


METHOD OF ANALYSIS:described by SCHMID, W. and SALAMONE, M. et al.

Evaluation criteria:

In general, 2000 polychromatic erythrocytes (PCEs) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
- The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuciei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes An alteration of this ratio indicates that the test substance actually reached the target
Individual animals with pathological bone marrow depression may be identified and excluded from the evaluation.
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)
The size of micronuclei may indicate the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect. Slides were coded before microscopic analysis.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: yes, pretest of acute toxicity
- Clinical signs of toxicity in test animals:In a pretest for the determination of the acute oral toxicity, at 10 000 mg/kg body weight (= 2 300 mg/kg dye component) all animals (male and female) survived. The clinical signs observed were coloured urin and feces (males and females), piloerection, squatting posture, irregular respiration and a poor general state (females only).

The single oral administration of the vehicle in a volume of 20 ml/kg body weight was tolerated by all animals without any signs or symptoms.

Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Conclusions:

Interpretation of results: negative
The analogue substance 2 was tested for in vivo genetic toxicity following OECD 474. Under the experimental conditions the substance does not show any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Executive summary:

The substance was tested for chromosomal damage (clastogenicity) and for its ability to induce spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in purified water was administered once orally to male and female animals at dose levels of 2500 mg/kg (~ 575 mg/kg dye component), 5000 mg/kg (~ 1150 mg/kg dye component) and 10000 mg/kg (~ 2300 mg/kg dye component) body weight in a volume of 20 ml/kg body weight in each case. As a negative control, male and female mice were administered merely the vehicle, purified water, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 10000 mg/kg body weight and in the vehicle controls. In the test groups of 5000 mg/kg and 2500 mg/kg body weight and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. According to the results of the present study, the single oral administration of the sdubstance did not lead to an increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the negative historical control data.

A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at the top dose of 10000 mg/kg body weight after 24 and 48 hours.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

15 Jul 1996 - 16 Oct 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Source: Karl Thomae GmbH, Biberach an der Riss, Germany.
Weight at study initiation: 254 g (mean)
Housing: makrolon cages, type III.
Diet: standardized pelleted feed (Kliba-Haltungsdiät/Provimi Kliba SA, Kaiseraugst, Switzerland) was available ad libitum.
Water: drinking water from bottles was available ad libitum.

ENVIRONMENTAL CONDITIONS:
Temperature (°C): 20-24°C
Humidity (%): 30-70%
Photoperiod (hrs dark/hrs light): 12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours). During the time between treatment and perfusion the day/night rhythm was not followed.

Route of administration:

oral: unspecified

Vehicle:

- Vehicle/solvent used: CMC (carboxymethyl cellulose).
- Justification for choice of solvent/vehicle: due to the Iimited solubility of the test substance in water, a 0.5% CMC formulation was selected as the vehicle.
- Concentration of test material in vehicle: test group 3 and 4: 5 g/100mL; Test group 5 and 6: 10 g/100mL.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: to achieve homogeneity of the test substance in the vehicle, the test substance formulation was stirred constantly during removal and administration.

Duration of treatment / exposure:

12 and 18h

Frequency of treatment:

The treatment consisted of a single oral administration.

Remarks:

Doses / Concentrations: 500 mg/kg Basis:nominal conc.dose group 3 and 4

Remarks:

Doses / Concentrations: 1000 mg/kgBasis:nominal conc.dose group 5 and 6

No. of animals per sex per dose:

3

Control animals:

yes, historical

Positive control(s):

2-acetylaminofluorene;
- Justification for choice of positive control(s): 2-acetylaminofluorene is a well-established UDS-inducing agent.
- Route of administration: oral.
- Doses/Concentrations: 50 mg/kg.

Tissues and cell types examined:

Isolated primary rat hepatocytes.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
Culture conditions (seeding and attachment period): the isolated hepatocytes were seeded on coverslips on 1.9 cm² well containing 2 ml of attachment medium (WMEC).- about 400,000 viable cells were seeded per well.- 6 wells/per animal were used for the UDS assay.After an attachment period of about 2 hours with 5% CO2 at 37°C and > 90% humidity, the medium (WMEC) was replaced by fresh medium (WMF 1) to remove non-adherent celIs.LabelingThe medium (WMEI) was replaced by 2 ml labeling medium, and the cells were incubated with 5% CO2 at 37°C and > 90% humidity for 4 hours. After the labeling period, cells were washed with HBSS or WMEI (about 37°C); then fresh medium containing 0.25 mM unlabeled thymidine was added and the cells were incubated for another 14 hours. The cells on the coverslips were then fixed with ethanol/acetic acid (3 :1, v/v) for at least 30 minutes, rinsed 2 - 4 times with aqua dest. and air-dried. The dried coverslips were mounted ceIl side up on glass slides using Corbit-Balsam and dried overnight.
Autoradiography: the slides were coated with KODAK NTB-2 photographic emulsion (about 37°C) for about 5 - 10 seconds. After drying at room temperature in the dark (Iightproof boxes) for about 16 hours, the coated slides were started in the dark with a desiccant (in the presence of a drying agent) at - 20°C for 2 -10 days. Thereafter, the slide boxes were left at room temperature for at least 3 hours. The photographic emulsion was then developed with KODAK D-19 (about 15°C), fixed in KODAK Acidofix for about 5 minutes, washed in water for about 5 -10 minutes and stained with methyl green-pyronine Y. After rinsing with water and ethanol and air-drying, the slides were covered with a second coverslip using Corbit-Balsam.

METHOD OF ANALYSIS:
Quantification of UDS/Microscopic evaluation: the quantification of UDS was performed microscopically generally using 3 slides per test group. 25 - 50 cells in good morphological conditions were randomly selected per slide and examined to achieve a total number of 100 cells/animal. For each celI, the following counts were performed with an automatic image analyzer (ARTEK):
- The nuclear grain (NG) count (= number of silver grains overlying the nucleus).
- The cytoplasmic grain (GG) count (= number of grains in two or three nucleusequivalent areas adjacent to the nucleus).
The following parameters were calculated:
- The net nuclear grain (NNG) count of each cell (= nuclear grain count minuscytoplasmic grain count; NG - GG).
- The mean nuclear grain (NG) count- the mean cytoplasmic grain (GG) count.
- The mean net nuclear grain (NNG) count.
- The percentage of cells in repair (cells showing net nuclear grain counts of > 0).
- The percentage of cells in repair (cells showing net nuclear grain counts of > 5).
Slides were coded before microscopic evaluation.

Evaluation criteria:

The test substance was considered positive if a dose-related increase was demonstrated in both of the following:
- The mean number of net nuclear grain (NNG) counts, which must exceed zero at one of the test points.
- The percentage of cells in repair (NNG > 5) when > 20. A dose-related increase in % cells in repair > 5 outside the values of both the concurrent negative control and the historical control data base (> 5 < 20) and a dose-related increase in the mean number of NNG counts near to but without exceeding zero is considered to be an indication for a marginal response which needs to be confirmed/clarified in a further experiment. A test article producing both NNG counts and % cells in repair in the range of the negative control data is considered to be negative in the in vitro UDS assay.

Statistics:

Due to very clear negative findings, a statistical evaluation was not carried out.

Sex:

male

Genotoxicity:

negative

Remarks:

(500 mg/kg and 1000 mg/kg)

Toxicity:

no effects

Remarks:

no cytotoxicity noted (trypan blue exclusion technique)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

After the single oral administration of the test substance preparation, all animals showed symptoms in form of:
- Piloerection
- Orange discoloration of for- and hindlimbs, tail and ears-yellow to brownish discoloration of the lymphatic organs, the adiposal tissue and the liver.
- Anogenital region smeared with feces and test substance.
The urine of the animals was discolored orange/darkbrown.Furthermore, 5 out of 11 animals in the 1.000 mg/kg group died.

DNA repair activity

 

Perfusion 12 hours after treatment (mean of 3 animals ± standard deviation)

Test Group	Vehicle control	500 mg/kg	1000 mg/kg	positive control
NG counts	6.53± 1.84	7.06 ±0.70	8.19±1.89	18.11 ±4.44
CG counts	10.17±2.04	10.73±0.77	10.41 ±1.26	11.23 ±1.60
NNG counts	-3.64 ±0.47	-3.67 ±0.62	-2.21 ±2.45	6.88 ±2.88
% cells in repair NGG > 0	9.33 ± 4.51	7.00 ± 4.36	20.00 ± 18.19	78.67 ±7.57
% cells in repair NGG > 5	0.00 ± 0.00	0.00 ± 0.00	5.67 ± 8.96	54.00 ±11.53

NG = nuclear grains

CG = cytoplasmic grains

NNG = net nuclear grains

 

Perfusion 18 hours after treatment (mean of 3 animals ± standard deviation)

Test Group	Vehicle control	500 mg/kg	1000 mg/kg	positive control
NG counts	6.35 ± 0.36	6.54 ± 0.54	7.48±0.88	20.43 ± 1.63
CG counts	11.08 ±0.30	9.90 ± 0.85	10.94±1.16	11.88 ±0.56
NNG counts	-4.73 ±0.65	-3.36 ± 0.59	-3.45 ±0.53	8.56 ±1.34
% cells in repair NGG > 0	7.67 ± 4.04	15.00 ± 4.00	14.67 ±4.73	91.67 ±4.04
% cells in repair NGG > 5	0.00 ± 0.00	0.33 ±0.58	0.67 ±0.58	65.67 ±9.50

NG = nuclear grains

CG = cytoplasmic grains

NNG = net nuclear grains

mean per group (mean of 3 animals)

Conclusions:

Interpretation of results: negative.
The test substance did not cause a relevant increase in unscheduled DNA synthesis, as measured by an increase in net nuclear grain counts, and it was concluded that the test substance was negative in this in vivo assay using primary rat hepatocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

See the read-across justification in section 13

Justification for classification or non-classification

According to the CLP Regulation (EC n. 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

According to the ECHA Guidance R7.a Table R.7.7-5, in the presence of a positive result in Gene Mutation on bacteria test, a negative result in the “Cytotoxicity in vitro” test and a negative result in “Gene mutation in vivo” test, the substance can be stated as non genotoxic.

A new evaluation of the present endpoint will be made when results of the COMET assay (OECD TG 489) in read-across on analogue substance 03 will be available.