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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not expected to be genotoxic in mammalian cells

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
(Q)SAR
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Justification for type of information:
SOFTWARE
QSAR DYES R&C
Report produced by version QSAR dyes R&C 2.0
Developed by Milano Chemometrics and QSAR Research Group, Dept. Earth and Environmental Sciences, University Milano-Bicocca, Italy.

Details about the tool are included into the attachment.
Principles of method if other than guideline:
QSAR Prediction. Details on the QSAR model used are reported in the attachment (i.e. (Q)SAR model reporting (QMRF)).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain:
other: (Q)SAR prediction
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Positive results in AMES test.
Executive summary:

The gene mutation potential on bacteria of the substance was investigated using a specific QSAR model, developed to predict the gene mutation potential in bacteria for dyes. The existing QSAR models have strong limitations to predict ionic complex structures as the organic dyes are, and consequently they provide unreliable results. The QSAR modelling was developped in accordance with the OECD principles (details in the documentation attached).

Based on the estimation, the substance is expected to be able to give positive results in AMES test system. The estimation resulted to be in the applicability domain of the model.

Conclusion

Positive results in AMES test.

Genetic toxicity in vivo

Description of key information

Not expected to be genotoxic

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
October 26 to November 18, 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
The read across approach is detailed into the document attached into the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Adopted 21st, July 1997
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
- Age at study initiation: approximately 7 weeks.
- Weight at study initiation: males mean 33.5 g (32.0 - 36.0 g); females mean 27.1 g (24.0 - 29.0 g).
- Assigned to test groups randomly: randomization schemes 98.0890 and 98.089.
- Housing: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate.
- Diet: rat/mice diet ssniff® R/M-H (V 1534), ad libitum.
- Water: tap water in plastic bottles, ad libitum.
- Acclimation period: 5 days under study conditions.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Relative humidity: 50 ± 20 %
- Photoperiod: 12 hours daily light.
Route of administration:
oral: gavage
Vehicle:
SOLVENT
- Solubility: suspension in Tylose HEC 4000 (0.5% w/v).
- Stability and homogeneity in the vehicle: confirmed over 5 hours in Tylose HEC 4000 (0.5% w/v)', signed October 26th, 1998
Details on exposure:
- Formulation of test compound: on the days of administration the test substance was suspended in Tylose HEC 4000 (0.5 % w/v) at the appropriate concentration. A magnetic stirrer was used to keep the prepaation homogeneous until dosing had been completed.

DOSE SELECTION
In a preliminary dose range finding study, oral administration of 2000 mg test item per kg body weight did not cause any toxic effects in male and female mice over an observation period of 7 days. This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.
Frequency of treatment:
Twice at an interval of 24 hours
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males and 5 females
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Endoxan containing cyclophosphamide, dissolved in distilled water.
- Dose: 50 mg/kg bw
Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow.
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION
Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Staining was performed as follows: 5 minutes in methanol; 5 minutes in May-Grünwald's solution; brief rinsing twice in distilled water; 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise); rinsing in distilled water; drying; and coating with Entellan®.

METHOD OF ANALYSIS
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
Evaluation criteria:
Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group.
A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All animals survived after treatment. No signs of toxicity were observed.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronudeated polychromatic erythrocytes in the dose group of test item was within the normal range of the negative control groups. No statistically significant increase of micronudeated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20 % of the control values.

Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Summary tables and statistics

Sex Dose mg/kg bw Killing time No animals No poly counted Poly/Ery Poly with MN
 mean mean SD mean (%) mean mean SD
male 0 - Control 24 hrs 5 2000 0.48 0.07 1.6 0.08 0.03
male 2000 test item 24 hrs 5 2000 0.48 0.04 2.2 0.11 0.11
male 50 - Endoxan 24 hrs 5 2000 0.42 0.04 60.6 3.03 0.69
female 0 - Control 24 hrs 5 2000 0.54 0.05 1.2 0.06 0.07
female 2000 test item 24 hrs 5 2000 0.53 0.06 2.4 0.12 0.06
female 50 - Endoxan 24 hrs 5 2000 0.44 0.05 53.8 2.69 0.59

Sex Dose mg/kg bw Killing time No animals No poly counted Poly/Ery Poly with MN Mu I.
 mean mean SD mean (%) mean mean SD
pooled 0 - Control 24 hrs 10 2000 0.51 0.07 1.4 0.1 0.05 1
pooled 2000 test item 24 hrs 10 2000 0.51 0.06 2.3 0.1 0.09 1.6
pooled 50 - Endoxan 24 hrs 10 2000 0.43 0.04 57.20* 2.9 0.63 40.9

Mut. I. = Mutagenic index

Control = Vehicle Tylose HEC 4000 (0.5 % w/v))

* significantly different from control (p < 0.05)

A cross comparison of individual data and pooled data may show discrepancies since the values are rounded.

Conclusions:
Under the conditions of the present study, the results indicate that test item is not mutagenic in the micronucleus test.
Executive summary:

The micronucleus test was carried out with test substance, which was suspended in Tylose HEC 4000 (0.5 % w/v); it was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study). According to the test procedure, the animals were killed 24 hours after administration.

Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased.

The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with test item and was not less than 20 % of the control value.

Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Conclusion

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro AMES test assay, performed using QSAR approach and in vivo chromosomal aberration assay, performed on a structural analogues.

The estimation about in vitro bacteria gentic toxicity potential gave positive response and there are no data regarding mutagenic potential in mammalian cells of Disperse Red 073. Therefore, the available data on structural analogous Similar Substance 01 and Sikmilar Substance 02 have been taken into consideration in order to deeper investigate the genetic toxicity potential. The read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

IN VITRO GENE MUTATION ASSAY IN BACTERIA

The gene mutation potential on bacteria of Disperse Red 073 was investigated using a specific QSAR moled, developed to predict the gene mutation potential in bacteria for dyes. The existing QSAR models have strong limitations to predict ionic complex structures as the organic dyes are, and consequently they provide unreliable results. The QSAR modelling was developped in accordance with the OECD principles (details in the documentation attached).

Based on the estimation, the substance is expected to be able to give positive results in AMES test system. The estimation resulted to be in the applicability domain of the model.

It should be noted that mutagenicity of a number of nitroarenes has been proved (Sabbioni, 1994). A common metabolic fate of most nitro-aromatic compounds involves six-electron reduction to the corresponding aniline derivatives; the Salmonella typhimurium strains exhibit nitro-reductase enzyme activity (Aiub et al., 2006). More specifically, the “classical”, non-genetically modified Salmonella typhimurium Ames tester strains (TA 98, TA 100, TA 1535, TA 1538) express certain levels of endogenous nitro-reductase and O-acetyltransferase (Einisto et al, 1991).

IN VIVO CHROMOSOMAL ASSAY

The micronucleus test was carried out with the Similar Substance 02; it was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay. According to the test procedure, the animals were killed 24 hours after administration. The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with test item and was not less than 20 % of the control value. Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent. Therefore, under the conditions of the study, the results indicate that test item is not mutagenic in the micronucleus test.

Similar results were obtained also with Similar Substance 01. The micronucleus test was carried out to investigate the potential of test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. 5 males and 5 females per test group were evaluated for the occurrence of micronuclei. The following dose levels of the test article were investigated: at 24 h preparation interval 200, 670 and 2000 mg/kg b.w.; at 48 h preparation interval 2000 mg/kg b.w. The animals expressed slight toxic reactions. After treatment with the test article the mean number of NCEs was increased as compared to the corresponding vehicle controls thus indicating that test item had cytotoxic effectiveness. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

It was concluded that, during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

DISCUSSION AND CONCLUSION

The estimation of the gene mutation potential in baceria of Desperse Red 073 gave positive response, thus the available experiments conducted in mammalian cells using the structural analogous Similar Substance 02 have been taken into consideration, in order to clarify the potential genotoxicity of the subtsance under ivestigation. In addition, data on similar substance 01 have been also reported in supporting apporach.

The obtainement of positive responses in AMES tests in case of nitrous-compounds is not unusual; however, AMES-positive compounds are not necessarily positive in vivo; for example, they may be detoxified by the whole-body system. As anticipated, Salmonella bacteria are very efficient nitro-reduction. Mammalian cells do possess nitroreductases but these are usually inhibited by oxygen, and under normal conditions of mammalian cell culture, or in normal oxygenated tissues in vivo, nitroreduction of a chemical is likely to be inefficient or even absent (Kirkland et al, 2007). So, different results obtained in non-mammalian system and in mammalian cell tests may be addressed by considering possible differences in substance uptake and metabolism, or in genetic material organisation and ability to repair. In general, the results of mammalian tests are regarded as of higher significance.

Althought the positive response in AMES test (study report, 1994), Similar Substance 02 gave negative results in in vivo micronucleous test performed in mammalian cells. Similar results were obtained also in the case of Similar Substance 01, which gave positive response in AMES test (study report, 1997), but which resulted to be non mutagenic in both in vitro gene mutation assay in mammalian cells (study report, 1998) and in vivo micronucleous test performed in mammalian cells.

In conclusion, the review of all the available information suggests that test substance is not expect to be able to induce heritable mutations in mammalian cells.  

REFERENCE

Details in attachment

Justification for classification or non-classification

According to the CLP Regulation (EC ) No 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.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC) No 1272/2008.