Cobalt lithium dioxide

Carcinogenicity

Administrative data

Endpoint:

carcinogenicity: oral

Type of information:

experimental study planned (based on read-across)

Study period:

see attached justification for the study design

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out
Cobalt dichloride, being a representative highly bioaccessible and bioavailable substance for the cobalt category substances.
- Name of the substance for which the testing proposal will be used [if different from tested substance]

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
[please address all points below]:
- Available GLP studies
There are no GLP and/or guideline compliant studies on the carcinogenicity hazard following oral route of exposure available.
- Available non-GLP studies
There are no non-GLP and/or guideline compliant studies on the carcinogenicity hazard following oral route of exposure available.
- Historical human data
There are no human or epidemiological studies on the carcinogenicity hazard following oral route of exposure available.
- (Q)SAR
QSAR cannot be performed for inorganic substances to assess its oral/systemic carcinogenic hazard for animals and humans, since adequate models are not available.
- In vitro methods
In vitro experiments have already been performed in an integrated testing strategy (see the chapter on in vitro genotoxicity in the appendix of the CSR). Further in vitro models, predicting the dose-response relationship of systemic or gastro-intestinal tract genotoxic/carcinogenic events do not exist (see http://alttox.org/mapp/toxicity-endpoints-tests/organ-toxicity/).
- Weight of evidence
Not applicable due to the read-across approach for the cobalt category substances already used in the category substances dossiers. Weight of evidence not sufficient for evaluation of oral carcinogenicity hazard.
- Grouping and read-across
Cobalt dichloride has been selected as source substance for the bioavailable read-across group. Findings from this study will be read across to all other members of that group and subsequent conclusions for classification & labelling and DNEL derivation will be adopted accordingly.
- Substance-tailored exposure driven testing [if applicable]
Not applicable
- Approaches in addition to above [if applicable]
Not applicable
- Other reasons [if applicable]
Not applicable

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:
- The testing for oral carcinogenicity is not a formal data requirement according to Annex X, Section 8.9.1, Column 1, since inhalation carcinogenicity studies are available for the cobalt category substances.

Introduction
There are two lifetime inhalation studies with cobalt substances, cobalt metal powder (Co) and cobalt sulfate heptahydrate (CoSO4.7H2O, referred to as CoSO4 below), both conducted by the US National Toxicology Program (NTP). The applicability of these studies to the risk assessment and classification of cobalt compounds has been considered in detail.
In both NTP studies, incidence of lung tumours correlated with exposure concentrations in air as well as cobalt blood levels (where measured), and the substances meet the criteria for classification as carcinogenic by inhalation Cat 1B under CLP.
The CLP text includes a provision requiring that conclusive proof of absence of effect is needed to exclude a route of exposure from a hazard classification as carcinogenic. This has – despite absence of data suggesting that Co metal is carcinogenic by oral or dermal route – led to a recently (1st Q 2020) adopted harmonised classification of Co metal as Carc 1B by all routes of exposure. (RAC opinion, 22 September 2017: “There are no oral carcinogenicity studies available on cobalt or its compounds, which could provide evidence on the lack of cancer via other routes of exposure. Therefore, it is difficult to definitely exclude the possibility of induction of cancers via other routes of exposure.”)
The soluble inorganic cobalt salts are classified as inhalation carcinogens only. This stems from the fact that Co sulfate caused only lung tumours (local tumours) and pheochromocytomas (adrenal tumours secondary to lung toxicity and a common occurrence after exposure to inhalation toxicants).

Evaluation of systemic carcinogenicity based on inhalation data
In the Co metal study, in addition to the lung and the non-cobalt specific pheochromocytomas, systemic cancers such as mononuclear cell leukaemia (MCL), and tumours in pancreas and kidney were observed. Except for the pheochromocytomas, the systemic tumours occurred only in one sex (MCL in females only; remaining systemic tumours in males only) of one species (rat).
In the NTP study with Co metal powder, Co blood levels rose in proportion with the inhalation exposure concentrations, but there was no dose-response correlation between blood levels and incidence of other systemic tumours (kidney, mononuclear cell leukaemia, pancreas).
Co and Co sulfate act primarily via portal-of-entry effects in the lung, and the impact on the lung is thought to have resulted in the indirect morbidity and systemic findings in the test animals. A clear example of this are the pheochromocytomas. For other substances, pheochromocytomas were concluded to be a secondary consequence of respiratory distress, hypoxia or other primary lung effects {Greim, 2009 #333}.
Scientifically, it is not possible to conclude from the inhalation studies alone on the significance of the systemic tumour findings for the oral and dermal route of exposure.

Hazard assessment
The consistency of the lung tumour findings in experimental animals has led the CoRC /the Co industry to apply a self-classification to Co metal as Carc 1B (H350i).
Based on the known secondary nature of the pheochromocytomas, the lack of consistency and dose-response in the systemic tumours (rats only, single sex) and supported by the negative findings in recent (2017) epidemiological studies (32,354 workers from three companies in 17 manufacturing sites in five countries) of cobalt-exposed workers, the self-classification as H350i has not been expanded to include all routes, and instead has resulted in the present proposal by the CoRC/the Co industry to generate relevant hazard data for the endpoint cancer following a non-inhalation route of exposure.

Risk assessment
Hazard as well as robust risk assessment for cancer following oral and dermal exposure is crucial based on the multitude of consumer uses of cobalt containing mixtures, i.e. alloys and materials containing cobalt as an impurity. Affected sectors and uses include the use of stainless steel in food contact surfaces, where oral (pots and pans; use of stainless steel as material for water pipes) as well as dermal (cutlery) exposure to Co may occur on a daily, chronic time scale. Further included is the use of stainless steel and cobalt-chrome alloys in medical and dental applications, where there are numerous systemic or oral exposure pathways, e.g. through the use of hypodermic needles, surgical devices and medical and dental implants. Another potential source of exposure stems from the use of Co containing alloys as touch surfaces (door handles and pads), as are common in many public spaces, e.g. in public transport.
All these areas require a robust risk assessment for oral, dermal and systemic exposure routes for effects that occur in the absence of the portal of entry effects following inhalation.

Proposal for oral study
In view of the extremely large population exposed by oral and dermal route (but not by inhalation) to Co from alloys on a daily and chronic basis, it is necessary to generate data for the relevant exposure routes. The oral route is proposed as a route for chronic dosing, since there are good range finding data indicating that the same systemic levels of cobalt can be achieved as in the Co metal inhalation study, without the local lung toxicity effects. This allows the study of systemic effects in animals that do not suffer from morbidity following lung injury. In an oral study, Co will be dosed to Maximum Tolerated peroral Dose and the relevant systemic endpoints such as haematological changes and body weight will be measured to demonstrate that Maximum Tolerated Dose (MTD) was achieved. Since there are toxicokinetic data on cobalt blood levels following inhalation exposure to Co metal powder, it will be possible to estimate and apply oral exposures that generate blood Co levels matching the levels observed in the NTP study. In other words, the Co blood and tissue levels achieved in the Co metal powder inhalation study can be reproduced or potentially exceeded by oral exposure to Co, if the form of Co is orally highly bioavailable.
Co toxicity following oral exposure is expected to have a different mode of action, as compared to inhalation exposure. While potential portal-of-entry effects (GI tract) are unknown, oral exposure is not predicted to cause lung injury as observed following inhalation exposure. Therefore, if the systemic tumours observed in the Co metal study are secondary to lung injury, they will not be reproduced in an oral study with a cobalt compound, even with comparable systemic cobalt levels.
The notion that there is potentially a different mode of action of Co by the oral versus the inhalation route, that high oral doses will be required for a potential carcinogenic effect, and that there is a data gap in the hazard assessment for chronic local (GI) effects is supported by the RAC opinion of September 2017: “However, when taking into account the potential mechanisms of action of cobalt and the fact that these systemic cancers occurred mainly at the highest dose level, which is considered to exceed the MTD, they are likely to exert a threshold. At the second highest dose level, only the incidence of pheochromocytomas was increased. The mechanism for these pheochromocytomas is unclear but may be related to local lung effects and HIF-1 activation as has been postulated also in the case of nickel metal that has caused similar effects. Therefore, it is very likely that high doses are needed to induce systemic cancers by the oral route of exposure. On the other hand, local carcinogenicity in the gastrointestinal tract after oral exposure cannot be excluded, especially when taking into account that repeated dose toxicity studies with cobalt and cobalt chloride affect the gastro-intestinal tract and Kirkland et al. (2015) demonstrated nuclear anomalies (apoptotic changes) in the gastrointestinal-tract after single dose oral exposure (see ‘RAC evaluation of germ cell mutagenicity’).”
It needs to be considered that following a 90-day oral exposure to CoCl2-hexahydrate adverse effects were observed that do not match any of the endpoints observed following inhalation exposure to cobalt. While inhalation exposure results in local (portal-of-entry toxicity), oral exposure did not have local effects, but caused as most sensitive endpoint systemic effects on the haematological system. The key effect of Co by oral exposure is an increase in red blood cells and haemoglobin, matched by an increase in red blood cell production in the bone marrow. These hallmark effects are currently the basis for DNELs in the risk assessment for Co substances by oral exposure; the potential further outcome of these effects on a chronic timescale is however not known.
In conclusion, data on potential carcinogenic or non-carcinogenic effects following administration by the oral route of exposure are required for hazard and risk assessment of systemic exposure to the cobalt ion and will address the knowledge gap for hazard classification of cobalt substances for non-inhalation routes of exposure. Further, these data will enable robust risk assessment of exposure to Co for any cancer or non-cancer endpoints following oral and dermal exposure.


FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
The “cobalt category” is comprised of all substances within the portfolio of the Cobalt REACH Consortium. The cobalt category is assessed for the human health hazards within the read-across concept. The substances of the cobalt category are grouped into three read-across groups for the oral route of exposure, i.e. Group 1: Bioavailable Co substances group (BCoS), Group 2: Inorganic poorly soluble (IPS), Group 3: Poorly soluble in aqueous solutions with organic ligand (PS-OL). Of relevance to this Testing Proposal is the BCoS group. The BCoS substances are characterised by the same physico-chemical characteristics considered relevant for this assessment:
1. all represent inorganic salts, oxides/hydroxides or elemental cobalt
2. all liberate divalent cobalt cations upon dissolution in biological media
3. the cobalt cation is considered the toxicologically relevant species
4. anionic species liberated upon dissolution are considered to contribute to a much lesser extent to the overall toxicity compared to the cobalt cation

The toxicity of Co compounds is conferred by the toxic unit, the cobalt ion. It has been shown that the higher the release of Co ion, the higher the toxicity. Under the assumption of the common toxic unit (the Co ion), a grouping and read across paradigm has been developed in accordance with the RAAF (cf. Appendix 1.1 of the CSR). In this grouping paradigm, Co metal and the five soluble inorganic Co salts (Co dichloride, Co sulfate, Co di(acetate), Co carbonate, Co dinitrate) are in the same read across group, referred to “Bioavailable Co substances” (BCoS) and results obtained from any of these substances will be read across to the whole group.
For the oral carcinogenicity study CoCl2.6H2O is proposed as the test substance representing the Group 1 bioavailable cobalt substances.
Co dichloride is considered the most suitable candidate as source substance for the oral carcinogenicity study, as it is the most studied and best understood for its oral toxicity, in comparison to other group members. One reason for the preferred use of Co dichloride in oral toxicology studies is the ease of application (soluble in water), and the lack of side effects from the counter ions at doses up to maximum Tolerated Dose (MTD), based on significant weight reductions and significant haematological changes exceeding 20%. Also, at the dose levels required to achieve systemic LOAELs and above, there have been no reports of local toxicity or site-of-contact irritation.
Co metal powder has less commonly been used in toxicity studies, except for inhalation, where the fine powder can easily be manipulated to generate respirable dusts. Co metal powder does not have good solubility at neutral pH, which has made its use in oral toxicity studies problematic. It can be applied as a suspension in methyl cellulose gel; however, the dissolution of Co metal involves a “corrosion” step, potentially generating reactive oxygen species, as well as release of Co ion. It has been less easy to orally apply Co metal powder and generate a good dose response, as studies so far appear to generate either “no effect” at low doses or, at higher doses, very strong effects including mortality based on portal-of-entry toxicity (e.g. aspiration of test item and lung oedema or GI bleeding). These effects would preclude survival of animals in a 2-year study and would therefore preclude conclusions on the endpoint of interest, cancer.
Considering the database, it is recommended to use Co dichloride as test substance for an oral carcinogenicity study, representing the soluble and bioavailable cobalt compounds with an inorganic ligand. The results of the study will be read across to these compounds and to Co metal for hazard and risk assessment purposes. Co dichloride is one of the substances with the highest bioavailabilities in gastric and intestinal juice, and represents the most orally bioavailable form of Co, and thereby a worst-case scenario when reading across to the other group members, including Co metal.
CoCl2 is not representative of poorly soluble cobalt substances with inorganic ligands (Group 2), nor of Co compounds with a long-chain organic ligand (Group 3).

Test:
Oral carcinogenicity study with Co dichloride hexahydrate applied by daily gavage. Dose levels of between 1 and 20 mg test substance/kg bw as high dose are being considered. Due to uncertainties about the chronic dose response following oral exposure, one control group and 3 or 4 dose groups (50 animals each group) are being proposed. It is recommended to conduct the study in CD® / Crl:CD (SD) rats only (not mice), males and females, based on findings of systemic tumours of unknown relevance following inhalation exposure to Co metal in rats only. Additional endpoints are recommended (haematological markers, including erythropoietin, Co levels in blood and urine) at four timepoints throughout the study, and at sacrifice. It is proposed to conduct Co level tissue analysis in all predicted target organs (adrenal medulla, kidney, pancreas, bone marrow) and in liver and brain at sacrifice. Additional screening of thyroid function is proposed.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cobalt dichloride

EC Number:

231-589-4

EC Name:

Cobalt dichloride

Cas Number:

7646-79-9

Molecular formula:

Cl2Co

IUPAC Name:

cobalt(2+) dichloride

Test material form:

solid

Test animals

Species:

rat

Strain:

other: CD® / Crl:CD (SD)

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

No. of animals per sex per dose:

see attached justification for the study design