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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not expected to be genotoxic

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.
Qualifier:
according to guideline
Guideline:
other: AMES mutagenicity QSAR model, report N. 07
Version / remarks:
29/04/2016
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
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 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.

Conclusion

Positive result in AMES test.

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

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 / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From December 09th, 1997 to January 07th, 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
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 May 26, 1983
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf.
- Age at the acclimatization: 8-12 weeks.
- Weight at study initiation: males mean value 28.8 g (SD ± 2.6 g); females mean value 25.9 g (SD ± 1.8 g).
- Assigned to test groups randomly: yes.
- Fasting period before study: approximately 18 hours before treatment the animals received no food but water ad libitum.
- Housing: single, in Makrolon Type I, with wire mesh top.
- Diet: pelleted standard diet, ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.
- Quarantine: the animals were under quarantine in the animal house of testing facility for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 24 - 70 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
- Solvent: on the day of the experiment, the test article was formulated in PEG 400.
- Justification for choice of solvent: the vehicle was chosen due to its relative non-toxicity for the animals.
Details on exposure:
All animals received a single standard volume of 10 ml/kg body weight orally.

Pre-experiment for toxicity
A preliminary study on acute toxicity was performed with a small group of animals under identical conditions as in the mutagenicity study concerning: animal strain; vehicle; route, frequency, and volume of administration.
The animals were treated orally with the test article and examined for acute toxic symptoms at intervals of 1 h, 6 h, 24 h, and 48 h after administration of the test article.
Frequency of treatment:
Once and twice at an interval of 24 hours
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
at 24 hours
Dose / conc.:
670 mg/kg bw/day (actual dose received)
Remarks:
at 24 hours
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
at 24 and48 hours
No. of animals per sex per dose:
Six males and six females were assigned to each test group.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: cyclophosphamide.
Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow.
Details of tissue and slide preparation:
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Preparation of the animals
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x×g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Griinwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Analysis of cells
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
slight toxic reactions
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The animals expressed slight toxic reactions.
The mean number of normochromatic erythrocytes was increased after treatment with the test article as compared to the mean value of NCEs of the vehicle controls, indicating that test item had cytotoxic properties in the bone marrow.
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 and dose level after administration of the test article. The mean values of micronuclei observed after treatment with test item were close to the vehicle control value.

POSITIVE CONTROL
40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.
Conclusions:
The substance is considered to be non-mutagenic in the micronucleus assay.
Executive summary:

The study was performed to investigate the potential of test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in polyethylene glycol 400 (PEG 400). PEG 400 was used as vehicle control. The volume administered orally (gavage) was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE.

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 highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. 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.

Conclusion

In conclusion, it can be stated 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.

Therefore, the substance is considered to be non-mutagenic in the micronucleus assay.

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; in vivo chromosomal aberration assay, performed on a structural analogue.

The estimation about in vitro bacteria genetic toxicity potential gave positive response and there are no data regarding mutagenic potential in mammalian cells. Therefore, available data on the structural analogue Similar Substance 02 has 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 054 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 developed 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.

In addition, an experiment conduced with Disperse Red 054, following method and procedures described by AMES et al, is available. Test compound exhibited genetic activity with the strains TA-1538, TA-98 and TA-100 in the activation and the non-activation assays and was considered as mutagenic. Unfortunately, it was not possible to retrieve any detail about the actual composition of the lot/batch tested, thus it cannot be confirmed if the composition can be considered representative for the one of lots/batches currently in use.

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 (TA98, TA100, TA1535, TA1538) express certain levels of endogenous nitro-reductase and O-acetyltransferase (Einisto et al, 1991).

IN VIVO CHROMOSOMAL ASSAY

The study was performed to investigate the potential of Similar Substance 02 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in polyethylene glycol 400 (PEG 400) and the volume administered orally (gavage) was 10 ml/kg b.w.. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei in the experiment. The animals expressed slightly 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.

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 bacteria of Disperse Red 054 gave positive results, thus the available experiments conducted in mammalian cells using the structural analogue Similar Substance 02 have been taken into consideration, in order to clarify the potential genotoxicity of the substance under investigation.

Positive responses in AMES tests in case of nitro-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 in 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.

Similar Substance 02 gave positive response in an AMES test (study report 1997); however, it gave negative results in both in vitro gene mutation assay (study report 1998) and in vivo micronucleus test performed in mammalian cells.

Overall, test substance is not expect to be able to induce heritable mutations in mammalian cells.

 

REFERENCE

See the list of complete reference in attachment.

Justification for classification or non-classification

According to the CLP Regulation (EC 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 1272/2008).