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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, Prival modification with pre-incubation design, positive for WP2 uvrA with metabolic activation


test on going for OECD 489 In vivo alkaline Comet Assay on analogue substance

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his+/- and trp-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver S9 Homogenate
Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system is necessary.
Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation. Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. The protein concentration of the S9 preparation was 30.9 mg/mL (Lot. No.: 080721K) in experiment I.

Rat S9 Mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Hamster Liver S9 Homogenate
The S9 liver microsomal fraction was obtained from the liver of 7 - 8 weeks old male Syrian golden hamsters (not induced).
The S9 was prepared and stored according to the currently valid version of the SOP for hamster liver S9 preparation.
The protein concentration of the S9 preparation was 26.9 mg/mL (Lot. No.: 150621) in experiment II.
Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as congo red.

Hamster S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 30% v/v. The concentrated cofactor solution yielded the following concentrations in the S9 mix:
8.0 mM MgCl2
33.0 mM KCl
20.0 mM Glucose-6-phosphate
2.8 units/ml Glucose-6-phosphate-dehydrogenase
4.0 mM NADP
2.0 mM NADH
2.0 mM FMN
in 100 mM Sodium-Ortho-Phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al (2) and Prival and Mitchell (1).

S9 Mix Substitution Buffer
The S9 mix substitution buffer contained per litre:
700 mL 100 mM sodium-ortho-phosphate-buffer pH 7.4
300 mL KCl solution 0.15 M
During the experiment, the S9 mix substitution buffer was stored in an ice bath

Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate for the plate incorporation test (experiment I)

33; 100; 333; 1000; 2500; and 5000 µg/plate for the Prival modification (Experiment II)

The top dose was selected based on a preliminary toxicity test; In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades.
Vehicle / solvent:
On the day of the experiment, the test item was dissolved in deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
congo red
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable
- Test substance added in medium; in agar (plate incorporation) for experiment I and preincubation for experiment II




METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition or reduction of spontaneous revertants

METHODS FOR MEASUREMENTS OF GENOTOXICIY: count of reversal bacteria coloniesa by a validated computer system , which was connected to a PC with printer to print out the individual values, the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to the intense color of the test item the colonies were partly counted manually

- OTHER:
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
not mandatory according to the guidelines
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
follogin Prival modification and pre-incubatio design
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item in the overlay agar was observed neither in the test tubes nor on the incubated agar plates. Strong coloring of the overlay agar was observed on the incubated agar plates at 5000 µg/plate.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 with S9 mix at 5000 µg/plate in experiment I.

see attached file for individual tables of results

Conclusions:
The substance was tested for in vitro gene mutation toxicity to bacteria following OECD 471. Under the experimental conditions the substance induced gene mutation based on the result for strain WP2 uvrA with non-induced Hamster liver S9 mix (Prival modification).
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.


The assay was performed in two independent experiments both with and without liver microsomal activation. Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and Experiment II was performed with non-induced hamster liver S9 mix. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:


Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate


Experiment II:                                 33; 100; 333; 1000; 2500; and 5000 µg/plate


No precipitation of the test item in the overlay agar was observed neither in the test tubes nor on the incubated agar plates. Strong coloring of the overlay agar was observed on the incubated agar plates at 5000 µg/plate.


The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.


A single minor toxic effect, evident as a reduction in the number of revertants (below the indication factor of 0.5), was observed in strain TA 98 with S9 mix at 5000 µg/plate in experiment I.


In experiment I a minor increase in revertant colony count, neither reaching nor exceeding the threshold of twofold the revertant colony count of the corresponding solvent control, was observed following treatment with Reactive Orange 16 in strain WP2 uvrA in the presence of S9 mix. The historical control data of the negative and solvent control were exceeded from 2500 µg/plate onwards.
In experiment II a substantial increase in revertant colony numbers was observed following treatment with Reactive Orange 16 in strain WP2 uvrA in the presence S9 mix. The threshold of twofold the colony count of the corresponding solvent was exceeded at concentrations ranging from 1000 to 5000 µg/plate.


 


Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

test on-going

Link to relevant study records
Reference
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:
guideline study
Justification for type of information:
NON-CONFIDENTIAL NAME OF SUBSTANCE:

Name of the substance on which the test has been performed: refer to the attached document

This test will be used in read-across on the target substance Reactive Orange 16 (EC: 701-348-2).


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

a) This part refers to the available studies on the target substance Reactive Orange 16 (EC: 701-348-2):

- 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:

b) This part refers to the available studies on the analogue substance that were used in read across to cover the endpoint of genotoxicity of Reactive Orange 16 (EC: 701-348-2):
In vivo alkaline Comet Assay (OECD 489)


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, the performance of an appropriate in vivo somatic cell genotoxicity study must be considered if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII.
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 choice of the appropriate in vivo study is triggered by the availability of reliable cytogenicity studies, either in vitro or in vivo, covering the endpoint. In fact,
a. If the test results of the available cytogenicity test(s) are negative, an in vivo mammalian alkaline comet assay (test method: OECD TG 489) in rats, or if justified, in other rodent species, oral route, on the following tissues, liver, glandular stomach and duodenum needs to be performed
b. If the test results of the available cytogenicity test(s) are positive, an in vivo mammalian alkaline comet assay (test method: OECD TG 489) combined with in vivo mammalian erythrocyte micronucleus test (test method: OECD TG 474) in rats, or if justified, in other rodent species, oral route needs to be performed. For the In vivo mammalian alkaline comet assay the following tissues shall be analysed: liver, glandular stomach and duodenum
The submitted dossier Reactive Orange 16 EC 701-348-2 contains results for the in vitro gene mutation study in bacteria, following OECD 471, which raises the concern for in vivo gene mutation, since it shows positive results (i.e., an increment of the number of revertants for strain WP2 uvrA with S9 mix at concentrations > 1000 ug/plate with Prival modification and pre-incubation).
An in vitro mammalian chromosome aberration test OECD 473, performed on an analogue substance is inserted, which was considered to fulfill the information requirement. The tested substance resulted non-clastogenic to cultured human peripheral blood lymphocytes, under the experimental conditions. Moreover, an in vivo chromosomal aberration OECD 475 on the same analogue substance, is submitted and the substance resulted not mutagenic in bone marrow cells of the Chinese hamster under the experimental conditions. Based on the considerations reported in the read across justification, same results can be applied also to Reactive Orange 16 and cytogenicity can be considered overall negative.
Therefore, by summarizing all the above available information, an in vivo mammalian alkaline Comet Assay needs to be performed on analogue substance 3 in order to elucidate the in vivo gene mutation potential. The information requirements is fulfilled by applying read across approach from analogue substance 3 , for which an in vivo Comet Assay testing proposal was accepted and will be performed, to target substance Reactive Orange 16.


Both target and analogue substance show positivity to the OECD471 test. Both substances rely for cytogenicity to analogue substance 2, which, as reported in the read across section, in particular based on the submitted toxicokinetics studies, can be used for predicting this genotoxicity potential. Meanwhile, an in vivo alkaline Comet Assay, OECD 489, was requested by the Authorities involving stomach, liver and duodenum as target organs for analogue substance 3.
Altogether the above information on genotoxicity and the considerations reported in the read across document, justify the use of the results of the OECD489 for assessing the potential in vivo gene mutation properties of the target substance Reactive Orange 16.

OECD 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 recognizes 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.

CONCLUSIONS
In order to completely assess the in vivo genotoxic potential of Reactive Orange 16, triggered by positive results in an vitro gene mutation study in bacteria and negative results in an in vitro cytogenicity test, a Comet Assay performed on analogue substance is used in read across. The two substances share chemical similarities, phys-chem and toxicological properties that allow the use of the read across methodology.
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Sex:
male
Route of administration:
oral: gavage
Vehicle:
water
No. of animals per sex per dose:
5 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
yes
Sex:
male
Genotoxicity:
other: in progress
Remarks on result:
other: the test is in read-across from an on going OECD 489 on a similar substance
Conclusions:
study in progress
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Justification for classification or non-classification

A final assessment on genotoxicity classification following the requirements of Regulation 1272/2008 is not possible since the in vivo alkaline Comet assay is in progress. An update on the classification will be performed once the results are available.