Cobalt aluminate blue spinel

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The in vitro genetic toxicity of Cobalt aluminate blue spinel has been evaluated in a bacterial reverse gene mutation assay (acc. to OECD TG 471). No further in vitro genetic toxicity studies with the pigment Cobalt aluminate blue spinel are available.

The in vitro genetic toxicity of the analogues pigment Cobalt zinc aluminate blue spinel has been evaluated, in a mammalian cell gene mutation assay (acc. to OECD TG 476), and in a micronucleus test (acc. to OECD TG 487).

All the studies were performed according to the current guidelines and in compliance with GLP and were evaluated to be reliable without restrictions.

All the studies yielded negative results.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
According to the ECHA Guidance Read-Across Assessment Framework (RAAF, March 2017), the read-across hypothesis for systemic effects is based on the assumption that different compounds give rise to the same common moieties (“(Bio)transformation to common compound(s)”) to which the organisms are exposed.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance Cobalt aluminate blue spinel is a complex inorganic colored pigment (CICP) and considered to be a UVCB under REACH, following the sector specific guidance for complex inorganic colored pigments (Eurocolour, 2017). Cobalt and aluminium expressed as the respective oxides are the main contributing elements. It is noted, that during the manufacture of CICPs chemical bonds of the raw materials are broken, and atoms are homogeneously and ionically interdiffused to form a specific crystalline matrix. Therefore, metals and oxygen atoms occupy specific places within the crystalline lattice, and individual oxides do not exist.
A detailed characterisation of the target substance is given in the attached justification document, Table 1..

The source substance Cobalt zinc aluminate blue spinel is also a complex inorganic colored pigment (CICP) and considered to be a UVCB under REACH, following the sector specific guidance for complex inorganic colored pigments (Eurocolour, 2017). It consists of almost the same contributing elements (Cobalt, zinc and aluminium expressed, as oxides are the main constituent). It is noted, that during the manufacture of CICPs chemical bonds of the raw materials are broken, and atoms are homogeneously and ionically interdiffused to form a specific crystalline matrix. Therefore, metals and oxygen atoms occupy specific places within the crystalline lattice, and individual oxides do not exist.
A detailed characterisation of the sourcesubstance is given in the attached justification document, Table 2.

The common characteristic of the target substance and the source substance is that both substances crystallise in the spinel structure, which forms a strong and inert crystal lattice. Both substances consist of almost the same chemical constituents. Furthermore, both show a similar, very low solubility in different artificial and aqueous media which is used for (eco)toxicological predictions.

3. ANALOGUE APPROACH JUSTIFICATION
Inorganic pigments like the target and source substance are characterised by inertness as a consequence of the specific synthesis process (calcination at high temperatures, approximately 1000°C), rendering the substance to be of a unique, stable crystalline structure in which all atoms are tightly bound within the lattice.

Upon dissolution the target as well as the source substances liberate cobalt and aluminium cations to a similar very low extent.

According to the OECD Guidance on grouping of chemicals (2014) the following basis for grouping of metal compounds can be assumed:
“The main assumption underlying the grouping of metal compounds is that toxicological and ecotoxicological properties are likely to be similar or follow a similar pattern as a result of the presence of a common metal ion (or ion complex including a hydrated metal ion). It is the bioavailability of the metal ion (or a redox form of this ion) at target sites that, besides the toxicity potency, will determine the occurrence and severity of the effects to be assessed. This is a reasonable assumption for the majority of inorganic compounds and some organic compounds (e.g. metal salts of some organic acids), in the absence of demonstrated relative differences in bioavailability.”

According to the ECHA Guidance Read-Across Assessment Framework (RAAF, March 2017), the read-across hypothesis for systemic effects is based on the assumption that different compounds give rise to the same common moieties (“(Bio)transformation to common compound(s)”) to which the organisms are exposed.

Since the target substance and the source substance release the same (eco-)toxicological relevant units under environmental/physiological relevant conditions, the overall ecotoxicity/systemic toxicity of the CICPs can be interpolated by assessing the (eco-)toxicity of the individual dissolved contributing elements. The analogue hypothesis, i.e. release of the common (eco-)toxicological units, applies to the target substance and the source substances.

However, acc. to ECHA Guidance Chapter R.6 – QSAR and Grouping of chemicals (2008) some metal containing UVCB compounds may not be appropriate for consideration in a category approach, as their effects will not be expected to be adequately described by their metal content. In fact read-across based solely on the metal content of the inorganic pigment Cobalt aluminate blue spinel would be highly overestimating, due to the very low bioavailability.


Transformation/Dissolution data ( see also Table 5 and 6 of the attached document)
The target substance showed a solubility in the transformation/dissolution test acc. to OECD 29 below 0.5 µg/L for the element Co after 24h, 7 days and 28days at pH6 and pH8 and a 1 mg/L loading. Aluminium was soluble up to 2 µg/L after 28 days at pH8 and up to 1.2 µg/L after 28 days at pH6.
The source substance showed a higher solubility in the transformation/dissolution test acc. to OECD 29 for the element aluminium and a comparable lower dissolution for the elements cobalt and zinc below the LOD after 7 and 28 days at pH6 and a 1 mg/L loading (Grané, 2010).

Bioaccessibility data (see also Table 7 and 8 of the attached document)
Bioaccessibility has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation), as follows: 1.) Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions, 2.) phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum, 3.) artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating, 4.) artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and 5.) artificial gastric fluid (GST, pH 1.5), which mimics the very harsh digestion milieu of high acidity in the stomach.
The target as well as the source substance show a similarly low solubility of the individual elements in different artificial and aqueous media corroborating the inertness of the substances. The source substance shows slightly higher bioaccessibility for cobalt without showing any signs of systemic or local toxicity in various studies (acute oral and inhalation, skin/eye irritation, sensitisation, sub-acute oral toxicity). The target substance shows no toxicity in acute oral and skin and eye irritation studies. These studies are characterised as bridging studies, corroborating the read-across approach.

4. DATA MATRIX
A data matrix is presented in Appendix 1 of the read-across document (attached in IUCLID section 13.2).

A detailed read-across justification is provided in the read-across document (attached in IUCLID section 13.2) .

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

lymphocytes: human (females)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The clastogenic and aneugenic potential of the analogies pigment Cobalt zinc aluminate blue spinel was evaluated in an in vitro micronucleus test according to OECD TG 487 (2014) and under GLP. Human lymphocytes were exposed to at least three different concentration levels up to top concentrations selected in line with the OECD guideline. The first experiment (Experiment I) was conducted as a 4-hour pulse treatment with a 20-hour recovery time both with and without metabolic activation. The second experiment was a continuous 24-hour treatment in absence of metabolic activation. In Experiment I (4-hour pulse treatment) in the absence and presence of S9 mix and in Experiment II (24-hour continuous treatment) in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations (24.7, 74.1 and 66.7 µg/mL, respectively), which showed precipitation. Cobalt zinc aluminate blue spinel, tested up to precipitating concentrations did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2014). The study is considered to be reliable without restrictions [RL-1].
Due to the analogy of the target and the source substance, cobalt aluminate blue spinel can be considered to be non-clastogenic and non-aneugenic in the in vitro micronucleus test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
According to the ECHA Guidance Read-Across Assessment Framework (RAAF, March 2017), the read-across hypothesis for systemic effects is based on the assumption that different compounds give rise to the same common moieties (“(Bio)transformation to common compound(s)”) to which the organisms are exposed.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance Cobalt aluminate blue spinel is a complex inorganic colored pigment (CICP) and considered to be a UVCB under REACH, following the sector specific guidance for complex inorganic colored pigments (Eurocolour, 2017). Cobalt and aluminium expressed as the respective oxides are the main contributing elements. It is noted, that during the manufacture of CICPs chemical bonds of the raw materials are broken, and atoms are homogeneously and ionically interdiffused to form a specific crystalline matrix. Therefore, metals and oxygen atoms occupy specific places within the crystalline lattice, and individual oxides do not exist.
A detailed characterisation of the target substance is given in the attached justification document, Table 1..

The source substance Cobalt zinc aluminate blue spinel is also a complex inorganic colored pigment (CICP) and considered to be a UVCB under REACH, following the sector specific guidance for complex inorganic colored pigments (Eurocolour, 2017). It consists of almost the same contributing elements (Cobalt, zinc and aluminium expressed, as oxides are the main constituent). It is noted, that during the manufacture of CICPs chemical bonds of the raw materials are broken, and atoms are homogeneously and ionically interdiffused to form a specific crystalline matrix. Therefore, metals and oxygen atoms occupy specific places within the crystalline lattice, and individual oxides do not exist.
A detailed characterisation of the sourcesubstance is given in the attached justification document, Table 2.

The common characteristic of the target substance and the source substance is that both substances crystallise in the spinel structure, which forms a strong and inert crystal lattice. Both substances consist of almost the same chemical constituents. Furthermore, both show a similar, very low solubility in different artificial and aqueous media which is used for (eco)toxicological predictions.

3. ANALOGUE APPROACH JUSTIFICATION
Inorganic pigments like the target and source substance are characterised by inertness as a consequence of the specific synthesis process (calcination at high temperatures, approximately 1000°C), rendering the substance to be of a unique, stable crystalline structure in which all atoms are tightly bound within the lattice.

Upon dissolution the target as well as the source substances liberate cobalt and aluminium cations to a similar very low extent.

According to the OECD Guidance on grouping of chemicals (2014) the following basis for grouping of metal compounds can be assumed:
“The main assumption underlying the grouping of metal compounds is that toxicological and ecotoxicological properties are likely to be similar or follow a similar pattern as a result of the presence of a common metal ion (or ion complex including a hydrated metal ion). It is the bioavailability of the metal ion (or a redox form of this ion) at target sites that, besides the toxicity potency, will determine the occurrence and severity of the effects to be assessed. This is a reasonable assumption for the majority of inorganic compounds and some organic compounds (e.g. metal salts of some organic acids), in the absence of demonstrated relative differences in bioavailability.”

According to the ECHA Guidance Read-Across Assessment Framework (RAAF, March 2017), the read-across hypothesis for systemic effects is based on the assumption that different compounds give rise to the same common moieties (“(Bio)transformation to common compound(s)”) to which the organisms are exposed.

Since the target substance and the source substance release the same (eco-)toxicological relevant units under environmental/physiological relevant conditions, the overall ecotoxicity/systemic toxicity of the CICPs can be interpolated by assessing the (eco-)toxicity of the individual dissolved contributing elements. The analogue hypothesis, i.e. release of the common (eco-)toxicological units, applies to the target substance and the source substances.

However, acc. to ECHA Guidance Chapter R.6 – QSAR and Grouping of chemicals (2008) some metal containing UVCB compounds may not be appropriate for consideration in a category approach, as their effects will not be expected to be adequately described by their metal content. In fact read-across based solely on the metal content of the inorganic pigment Cobalt aluminate blue spinel would be highly overestimating, due to the very low bioavailability.


Transformation/Dissolution data ( see also Table 5 and 6 of the attached document)
The target substance showed a solubility in the transformation/dissolution test acc. to OECD 29 below 0.5 µg/L for the element Co after 24h, 7 days and 28days at pH6 and pH8 and a 1 mg/L loading. Aluminium was soluble up to 2 µg/L after 28 days at pH8 and up to 1.2 µg/L after 28 days at pH6.
The source substance showed a higher solubility in the transformation/dissolution test acc. to OECD 29 for the element aluminium and a comparable lower dissolution for the elements cobalt and zinc below the LOD after 7 and 28 days at pH6 and a 1 mg/L loading (Grané, 2010).

Bioaccessibility data (see also Table 7 and 8 of the attached document)
Bioaccessibility has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation), as follows: 1.) Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions, 2.) phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum, 3.) artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating, 4.) artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and 5.) artificial gastric fluid (GST, pH 1.5), which mimics the very harsh digestion milieu of high acidity in the stomach.
The target as well as the source substance show a similarly low solubility of the individual elements in different artificial and aqueous media corroborating the inertness of the substances. The source substance shows slightly higher bioaccessibility for cobalt without showing any signs of systemic or local toxicity in various studies (acute oral and inhalation, skin/eye irritation, sensitisation, sub-acute oral toxicity). The target substance shows no toxicity in acute oral and skin and eye irritation studies. These studies are characterised as bridging studies, corroborating the read-across approach.

4. DATA MATRIX
A data matrix is presented in Appendix 1 of the read-across document (attached in IUCLID section 13.2).

A detailed read-across justification is provided in the read-across document (attached in IUCLID section 13.2) .

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The gene mutation potential of the analogies pigment Cobalt zinc aluminate blue spinel was investigated in an in vitro mammalian cell gene mutation test at the Hprt locus according to OECD TG 476 (2015) and under GLP. Chinese hamster lung fibroblasts (V79) were exposed for 4 hours to Cobalt zinc aluminate blue spinel up to a of 2000 µg/mL both with and without metabolic activation (S9 fraction from phenobarbital/β-naphthoflavone induced rat livers). The outcome of this study was judged as clearly negative both in the absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system. The study is considered to be reliable without restrictions [RL-1].
Due to the analogy of the target and the source substance, cobalt aluminate blue spinel can be considered to be non-mutagenic in the HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-08-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

historical data was missing

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983-05-26

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9-mix: livers were obtained from young male Sprague-Dawley rats (180 - 200 g) injected i.p. with Aroclor® 1254 (500 mg/kg) 5 days before sacrifice by cervical transection.

Test concentrations with justification for top dose:

10, 50, 100, 500, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (Lot: 34273443)
PREPARATION OF STOCK SOLUTION:
Dissolve H-21327 in DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: ICR-191 acridine

Remarks:

Without S9 activation; Positive controls: TA1535 and TA100 2 µg sodium azide/plate, TA97a 2 µgICR-191 acridine/plate and TA98 25 µg 2-nitrofluorene/plate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and S9 mix

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9 activation; Positive control: TA97 and TA100 1 µg/plate 2-aminoanthracene, TA98 and TA1535 2 µg/plate 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation
Aliquots of 0.1 mL containing at least 1 x 10^8 viable cells of each selected tester strain (grown overnight) and 0.1 mL of test material or control article will be added to the treatment medium with or without activation and then mixed and poured onto Davis minimal agar plates with dextrose (DMAD plates). After incubation at approximately 37°C fro approximately 48 hours, mutagenic activity at each treatment condition will be determined by counting the number of colonies on the plates.

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
Toxicity will be assessed simultaneously with mutagenicity testing in strain TA100. Doses used in strain TA100 will ordinarily be 5000, 2500, 1000, 500, 100, 50, 10, and 0 (negative control) µg/plate. Doses which are non-toxic or,if possible, slightly toxic, will be selected for testing in the remaining strains. Ideally, the highest dose of the test material should give approximately 50% of the negative control survival.

DATA ANALYSIS
Gross and microscopic observation: plates from each dose level will be examined grossly and microscopically. Appropriate information on the appearance of the colonies and the plate will be recorded.

Appearance of the colonies and background lawn: evidence for test material toxicity to the bacteria will be documented by recording the appearance of the colonies and background lawn using the following key:
TO = Background lawn appears normal
Tl = Background lawn is noticeably thinner and/or microcolonies are slightly larger than controls
T2 = Background lawn is markedly thinner and/or microcolonies are markedly larger than controls
T3 = Background lawn is severely reduced and/or microcolonies are greatly enlarged relative to controls
T4 = Background lawn is absent and/or microcolonies may be seen readily by the unaided eye
T5 = Reduction or absence of colony formation relative to controls

Presence of test material precipitate: formation of a precipitate by the test material will be documented using the following key:
PO = No precipitate observed
P1 = Microscopic precipitate present
P2 = Marked precipitate present which does not interfere with automated colony counting
P3 = Marked precipitate present which requires plate to be counted by hand
P4 = Heavy precipitate which prevents accurate colony counting and/or obscures the background lawn
N = None

Other Observations: other appropriate observations relevant to the appearance of the plates will be noted in the study records including:
N = None
R = Plate rejected

ACCEPTABILITY CRITERIA
An individual trial will include at least 5 dose levels of the test material (of which at least 4 must be acceptable), a negative control, and a positive indicator for each selected tester strain. Rejected data points, dose levels, or trials may be excluded from the mutagenicity analysis when either biological or statistical significance is questionable.
The acceptability criteria for data will be as follows:
- Culture density: the density of the overnight culture must be at least 1 x 10^9 cells/mL.
- Rejection of single data points: a single data point may be rejected if contamination or excess cytotoxicity is observed on a treatment plate or if precipitate on a treatment plate prevents accurate colony counting.
- Rejection of negative controls: a negative control data point may be rejected if it falls outside the historical data base for spontaneaus mutation.
- Rejection of a dose level: a dose level (or a negative control) will be rejected if there are less than 2 data points/dose level or if the values of data points are too divergent. This will be determined by the study director using scientific judgment and experience.
- Rejection of a trial: a trial (for an individual strain) will be rejected if the negative control is rejected, if mutagenic activity is absent on all positive indicator plates, or if the tester strains fail to exhibit the appropriate phenotypes.

Evaluation criteria:

A test material will be classified as POSITIVE (i.e., mutagenic) if:
- the average number of revertants in any strain at any test material concentration studied is at least two times greater than the average number of revertants in the negative control
AND
- there is a positive dose-response relationship in that same strain.

A test material will be classified as NEGATIVE (i.e., nonmutagenic) if:
- there are no test material concentrations with an average number of revertants which is at least two times greater than the average number of revertants in the negative control
AND
- there is no positive dose-response relationship.

Results not meeting these criteria for positive or negative assessments will be evaluated on a case by case basis using scientific judgment and experience. If one trial is positive and the other is negative, a third trial may be conducted.

Statistics:

Trials will be evaluated independently. Foreach selected tester strain, the average number of revertants and the standard deviation at each dose level with and without S9 activation will be calculated. When deemed appropriate by the study director, additional statistical analyses may be performed to detect linear dose-response relationships. Such analyses will be documented in the study records and described in the final report.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not examined

Positive controls validity:

not specified

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Evidence for test susbtance toxicity to the bacteria was documented as follows:
TA1535 (with and without activation): 2500 and 5000 µg/plate
TA97a (with activation): 1000, 2500 and 5000 µg/plate

Conclusions:

No evidence of mutagenicity was detected. In this study, the test substance is negative.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gen mutation in bacteria

The bacteria reverse mutation assay (Reference D'Amico (1995) ) yielded a negative result. However, tests on the mutagenic potential of cobalt aluminate blue spinel in bacteria are considered dispensable for principal considerations, since inorganic metal compounds are frequently negative in this assay due to limited capacity for uptake of metal ions (Guidance on information requirements and chemical safety assessment, Chapter R.7a, p. 387; HERAG facts sheet mutagenicity, Chapter 2.1).The evaluation of this reference is therefore primarily based on the method description.

The Ames test (Reference D'Amico (1995)) performed according to OCED 471 was rated reliable without restrictions and used as a supportive study. The results indicate that the test substance under the experimental conditions described, is not mutagenic to Salmonella typhimurium strains TA1535, TA97a, TA100, and TA98 in the presence and absence of a metabolising system.

In vitro gene mutation in mammalian cells:

The gene mutation potential of the analogies pigment Cobalt zinc aluminate blue spinel was investigated in an in vitro mammalian cell gene mutation test at the Hprt locus according to OECD TG 476 (2015) and under GLP. Chinese hamster lung fibroblasts (V79) were exposed for 4 hours to Cobalt zinc aluminate blue spinel up to a of 2000 µg/mL both with and without metabolic activation (S9 fraction from phenobarbital/β-naphthoflavone induced rat livers). The outcome of this study was judged as clearly negative both in the absence and presence of S9 metabolic activation. Appropriate positive controls demonstrated the activity of the metabolic activation system and the sensitivity of the test system. The study is considered to be reliable without restrictions [RL-1].

 

In vitro clastogenicity and aneugenicity:

The clastogenic and aneugenic potential of the analogies pigment Cobalt zinc aluminate blue spinel was evaluated in an in vitro micronucleus test according to OECD TG 487 (2014) and under GLP. Human lymphocytes were exposed to at least three different concentration levels up to top concentrations selected in line with the OECD guideline. The first experiment (Experiment I) was conducted as a 4-hour pulse treatment with a 20-hour recovery time both with and without metabolic activation. The second experiment was a continuous 24-hour treatment in absence of metabolic activation. In Experiment I (4-hour pulse treatment) in the absence and presence of S9 mix and in Experiment II (24-hour continuous treatment) in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentrations (24.7, 74.1 and 66.7 µg/mL, respectively), which showed precipitation. Cobalt zinc aluminate blue spinel, tested up to precipitating concentrations did not induce biologically relevant increases in the micronucleus formation frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. All validity criteria were met. The study was fully compliant with OECD 487 (2014). The study is considered to be reliable without restrictions [RL-1].

 

 

Overall conclusion:

Cobalt aluminate blue spinel is not considered to be genotoxic, since Cobalt aluminate blue spinel has shown no evidence for a potential to induce gene mutation in bacteria nor has the analogues pigment Cobalt zinc aluminate blue spinel shown evidence for a potential to induce chromosome, or genome mutations in the test systems used.

Justification for classification or non-classification

Genotoxicity studies with Cobalt aluminate blue spinel and the analogue pigments Cobalt zinc aluminate blue spinel did not show any effects in a bacterial reverse mutation test, in a mammalian cell gene mutation test (HPRT assay), or in a clastogenicity/aneugenicity study (micronucleus test).

 

The classification criteria acc. to regulation (EC) 1272/2008 as germ cell mutagen are not met, thus no classification is required.