Cobalt(2+) hydrogen citrate

Endpoint summary

Description of key information

Read-across with cobalt sulfate heptahydrate
No NOAECs were identified, neither for rats nor for mice.
LOAEC (local, rat): 0.5 mg/m³ cobalt hydrogen citrate (recalculated value)
LOAEC (local, mouse): 0.5 mg/m³ cobalt hydrogen citrate (recalculated value)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

- no urinalysis, no clinical chemistry; limited documentation of the results

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY, USA)
- Age at study initiation: 7 weeks
- Housing: individual housing
- Diet (e.g. ad libitum): NIH 07 rat and mouse ration (Zeigler Bros., Inc., Gardners, PA); ad libitium except during exposure periods

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 - 25.2
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The mass median aerodynamic diameter of the aerosol for all exposures ranged from 0.83 to 1.10 µm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton 2000, Lab Products, Inc.
- System of generating particulates/aerosols: Cobalt sulfate heptahydrate aerosol was generated from an aqueous solution by nebulisation using dried compressed air. The aerosol was diluted to the desired concentration with air from the chamber air-conditioning system.
- Method of particle size determination: Cascade impactor samples were taken to determine aerosol size distribution. The mass median aerodynamic diameter of the aerosol for all exposures ranged from 0.83 to 1.10 µm. Cobalt sulfate hydration in the aerosol distribution line was determined by ultraviolet/visible spectroscopy. Hydration ratios of 7.66 and 7.67 were determined for two samples taken during the studies.

TEST ATMOSPHERE
- Brief description of analytical method used: Three real-time aerosol monitors (Model RAM-1, GCA Environmental Instruments) were used to determine the concentration of the aerosol in the exposure chambers once every 20 minutes throughout the exposure period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three real-time aerosol monitors (Model RAM-1, GCA Environmental Instruments) were used to determine the concentration of the aerosol in the exposure chambers once every 20 minutes throughout the exposure period.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0.3, 1, 3, 10, 30 mg/m3
Basis:
other: nominal conc.; calculated as the anhydrous salt of cobalt(II)sulfate heptahydrate

Remarks:

Doses / Concentrations:
0.300±0.029; 0.990±0.087; 2.93±0.275; 9.95±0.579; 30.0±1.64 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

Animals distributed to weight classes and then assigned to cages and groups by a table of random numbers.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: weighed initially and once per week thereafter

HAEMATOLOGY: Yes
- Blood was obtained from the supraorbital sinus.

OTHER:
- estrous cyclicity
- sperm parameters (testis weight, sperm motility, sperm morphology)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The analysis of organ weight and male reproductive system data was carried out by using the non-parametric multiple comparison procedures of Dunn (1964) or Shirley (1977) to assess the significance of pairwise comparisons between dosed and chamber control groups. Jonckheere´s test (Jonckheere, 1954) was used to evaluate the significance of dose-response trends and to determine whether Dunn´s or Shirley´s test was more appropriate for pairwise comparisons.
The proportion of time spent in each stage of the estrous cycle was compared by using the Wilks criterion statistic (Wilks, 1932) of the multivariate analysis of variance procedure, which was performed after an arc sine transformation of the data.

Details on results:

CLINICAL SIGNS AND MORTALITY
Two of ten males exposed to 30 mg/m3 died before termination of the study. No observed clinical signs appeared to be related to the exposure, with exception of rapid breathing and skin discoloration in the two males that died during week 11.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of males and females exposed to 30 mg/m3 and females exposed to 10 mg/m3 were lower than those of controls throughout the study. The final mean body weight of mice at 30 mg/m3 was 14% lower than that of the controls for males and 22% lower for females.

HAEMATOLOGY
No consistent or dose-related hematologic effects were observed.

ORGAN WEIGHTS
The absolute lung weights and the lung weight to body weight ratios were significantly increased in the 10 and 30 mg/m3 exposure groups. The absolute testis weight and the testis weight to body weight ratios were significantly decreased for males exposed to 30 mg/m3.

GROSS PATHOLOGY/HISTOPATHOLOGY:
Compound-related microscopic examinations were generally limited to the respiratory tract of mice of each sex. Lesions were concentration related and similar in incidence and severity in males and females. In the nose, degeneration of olfactory epithelium, squamous metaplasia of the respiratory epithelium, and an acute inflammatory cell exudate in the nasal cavity were seen primarily at the two highest exposure concentrations.
At the highest exposure concentration, necrosis, inflammation, and squamous metaplasia of the laryngeal epithelium were present in most mice. Some foci of necrosis in the laryngeal epithelium extended through the basement membrane into the underlying lamina propria. Squamous metaplasia of the respiratory epithelium in the trachea also occured in the higest exposure group in male and female mice. In all exposure groups below 30 mg/m3, inflammation and squamous metaplasia were observed.
In the lung of mice exposed to 10 or 30 mg/m3, there was regeneration of bronchiolar epithelium and hyperplasia of the alveolar epithelium. Infiltration of histiocytes (macrophages) into the alveolar spaces was also present. Chronic inflammation occurred primarily at the highest exposure concentration and consisted of fibrosis around bronchioles and in alveolar septae along with an inflammatory cell infiltrate. At the lower concentration, only a minimal increase in macrophages was seen in the alveoli.
Lymphoid hyperplasia was present in the mediastinal lymph nodes of mice at the 30 mg/m3 exposure concentration.
At the highest exposure concentration, atrophy of the testes was observed, which consisted of a loss of ferminal epithelium in the seminiferous tubules; more severely affected testes also contained foci of mineralisation.

OTHER:
REPRODUCTIVE FUNCTION: ESTROUS CYCLE
The estrous cycle was significantly longer in female mice exposed to 30 mg/m3.

REPRODUCTIVE FUNCTION: SPERM MEASURES
The number of abnormal sperm in mice exposed to 30 mg/m3 was significantly increased, and sperm motility was significantly reduced in mice exposed to 3, 10, or 30 mg/m3.
At the highest concentration, atrophy of the testis was observed, which consisted of a loss of germinal epithelium in the seminiferous tubules; more severly affected testes also contained foci of mineralisation.

Dose descriptor:

LOAEC

Effect level:

0.3 mg/m³ air

Sex:

male/female

Basis for effect level:

other: calculated as the anhydrous salt of cobalt(II)sulfate heptahydrate; the LOAEC based on squamous metaplasia in the larynx

Dose descriptor:

LOAEC

Effect level:

0.5 mg/m³ air

Sex:

male/female

Basis for effect level:

other: CoC6H6O7; recalculated value

Critical effects observed:

not specified

Lesions seen in the respiratory tract included degeneration of the olfactory epithelium, squamous metaplasia of the respiratory epithelium, and inflammation in the nose. The most sensitive tissue was the larynx, with squamous metaplasia observed also at the lowest exposure concentration. Thus, no NOAEC was identified.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

4.8 mg/m³

Study duration:

subchronic

Species:

mouse

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on the basic assumption that the cobalt ion is the determining factor for biological activity. Please refer to the endpoint discussion for further details.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

- limited documentation of the results

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY, USA)
- Age at study initiation: 6 weeks
- Housing: individual housing
- Diet (e.g. ad libitum): NIH 07 rat and mouse ration (Zeigler Bros., Inc., Gardners, PA); ad libitium except during exposure periods

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 - 25.2
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: The mass median aerodynamic diameter of the aerosol for all exposures ranged from 0.83 to 1.10 µm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton 2000, Lab Products, Inc.
- System of generating particulates/aerosols: Cobalt sulfate heptahydrate aerosol was generated from an aqueous solution by nebulisation using dried compressed air. The aerosol was diluted to the desired concentration with air from the chamber air-conditioning system.
- Method of particle size determination: Cascade impactor samples were taken to determine aerosol size distribution. The mass median aerodynamic diameter of the aerosol for all exposures ranged from 0.83 to 1.10 µm. Cobalt sulfate hydration in the aerosol distribution line was determined by ultraviolet/visible spectroscopy. Hydration ratios of 7.66 and 7.67 were determined for two samples taken during the studies.

TEST ATMOSPHERE
- Brief description of analytical method used: Three real-time aerosol monitors (Model RAM-1, GCA Environmental Instruments) were used to determine the concentration of the aerosol in the exposure chambers once every 20 minutes throughout the exposure period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three real-time aerosol monitors (Model RAM-1, GCA Environmental Instruments) were used to determine the concentration of the aerosol in the exposure chambers once every 20 minutes throughout the exposure period.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Remarks:

Doses / Concentrations:
0.3, 1, 3, 10, 30 mg/m3
Basis:
other: nominal conc.; calculated as the anhydrous salt of cobalt(II)sulfate heptahydrate

Remarks:

Doses / Concentrations:
0.300±0.029; 0.990±0.087; 2.93±0.275; 9.95±0.579; 30.0±1.64 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

Animals distributed to weight classes and then assigned to cages and groups by a table of random numbers.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: weighed initially and once per week thereafter

HAEMATOLOGY: Yes
- Blood was obtained from the retroorbital sinus.

CLINICAL CHEMISTRY: Yes

URINALYSIS: Yes

OTHER:
- estrous cyclicity
- sperm parameters (testis weight, sperm motility, sperm morphology)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

The analysis of organ weight and male reproductive system data was carried out by using the non-parametric multiple comparison procedures of Dunn (1964) or Shirley (1977) to assess the significance of pairwise comparisons between dosed and chamber control groups. Jonckheere´s test (Jonckheere, 1954) was used to evaluate the significance of dose-response trends and to determine whether Dunn´s or Shirley´s test was more appropriate for pairwise comparisons.
The proportion of time spent in each stage of the estrous cycle was compared by using the Wilks criterion statistic (Wilks, 1932) of the multivariate analysis of variance procedure, which was performed after an arc sine transformation of the data.

Details on results:

CLINICAL SIGNS AND MORTALITY
All rats survived to the end of the study.
Compound-related clinical signs included ruffled fur in rats and hunched posture in male rats exposed to 30 mg/m3.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of rats exposed to 30 mg/m3 were lower than those of controls throughout the study. The final mean body weight of male rats exposed to 30 mg/m3 was 14% lower than that of the controls. Mean body weights of exposed female rats were not related to the exposure.

HAEMATOLOGY
Polycythemia, seen at 10 and 30 mg/m3 for female rats and at 3, 10, 30 mg/m3 for male rats, was indicated by significant increases in erythrocytes, in the mean hemoglobin concentration, and in the hematocrit value. The reticulocyte count was significantly increased in female rats exposed to 30 mg/m3. No significant changes were found in the leukocyte or differential counts.

CLINICAL CHEMISTRY
Mean serum cholesterol values were significantly decreased for males exposed to 10 or 30 mg/m3 and for females exposed to 30 mg/m3. No consistent dose-related effects were seen on the glucose concentration, on total creatine kinase activity, or on serum triglyceride concentration.

URINALYSIS
Granular casts were observed in the urine from many exposed male rats compared to controls. A dose-related increase was seen in the number of epithelial cells in the urine from males that were exposed to 3 mg/m3 or more.
The amount of cobalt excreted in the urine over 16 hours varied from 2.5 µg at 0.3 mg/m3 to 105 µg at 30 mg/m3 for males and from 2.0 µg at 0.3 mg/m3 to 67 µg at 30 mg/m3 for females. The amount of cobalt excreted in the urine of rats exposed to 0.3 mg/m3 was approx. 10 fold increased compared to controls.
ORGAN WEIGHTS
The absolute lung weights and/or the lung weight to body weight ratio were significantly increased for male rats exposed to 0.3 mg/m3 or more and for female rats exposed to 1 mg/m3 or more. Relative kidney weights were increased in male rats at all exposure concentrations.

GROSS PATHOLOGY/HISTOPATHOLOGY:
Compound-related lesions were limited to the respiratory tract of rats of each sex exposed to the test item. Lesions were concentration related and similar in incidence and severity in males and females. In the nose, hyperplasia and squamous metaplasia of the respiratory epithelium were seen primarily at the two highest exposure concentrations. This was most prominent at the tips of the naso- and maxilloturbinates and on the lateral wall of the nasal cavity in the most anterior section of the nose. Degeneration of the olfactory epithelium was characterised by a thinning of the olfactory epithelial cell layer in the dorsal meatus and also on the nasal septum in the ethmoid region (degeneration was slightly more prominent in males).
At 10 and 30 mg/m3, inflammatory polyps were seen in the larynx of most rats. Polyps were consistently located at the base of the epiglottis and extended into the lumen of the larynx. These polyps had a fibrovascular stroma, which was covered by a well-differentiated squamous epithelium. Focal areas of necrosis and ulceration were frequently present in the epithelium of the polyp. Chronic inflammation and mineralisation were prominent in the stroma of the polyp. At 1 and 3 mg/m3, polyps did not occur, but squamous metaplasia of the laryngeal respiratory epithelium and chronic inflammation in the stroma were persisted. At 0.3 mg/m3, the severity of the metaplasia and inflammation was minimal to mild.
Regeneration of bronchiolar epithelium with dilation of bronchioles was observed in the lung of rats exposed to 30 mg/m3; distension or disruption of alveolar septa was also present. Fibrosis was present around bronchioles and within alveolar septae. Histiocytic infiltration, characterised by intraalveolar accumulation of macrophages and infiltration of alveolar septae with inflammatory cells also occurred at 30 mg/m3. At lower concentrations, only intraalveolar histiocytic infiltrates and subacute inflammation were present. Lymphoid hyperplasia was present in the mediastinal lymph nodes of exposed rats, but the incidence was not concentration related.

OTHER:
REPRODUCTIVE FUNCTION: ESTROUS CYCLE
The average estrous cycle of females exposed to 30 mg/m3 was longer (but not statistically) than that of the control animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES
No statistically significant effects on sperm motility, sperm counts, or the incidence of abnormal sperm were observed in exposed rats.

Dose descriptor:

LOAEC

Effect level:

0.3 mg/m³ air

Sex:

male/female

Basis for effect level:

other: calculated as the anhydrous salt of cobalt(II)sulfate heptahydrate; the LOAEC based on squamous metaplasia in the larynx

Dose descriptor:

LOAEC

Effect level:

0.5 mg/m³ air

Sex:

male/female

Basis for effect level:

other: CoC6H6O7; recalculated value

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

0.5 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from a reference substance with similar structure and intrinsic properties. Read-across is justified based on the basic assumption that the cobalt ion is the determining factor for biological activity. Please refer to the endpoint discussion for further details.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are no data available on repeated dose toxicity for cobalt hydrogen citrate. However, there are reliable data for soluble cobalt compounds considered suitable for read-across using the analogue approach.

Cobalt hydrogen citrate is a metal-organic compound, which is water soluble and nearly completely dissociates in aqueous solutions. For identifying hazardous properties of cobalt hydrogen citrate concerning human health effects, the existing forms of the target chemical at physiological and very acidic pH conditions (e. g. in the stomach) are relevant for risk assessment. For cobalt hydrogen citrate, it can be assumed that cobalt cations are released under biological conditions that are considered to be toxicologically relevant. Furthermore it is anticipated that the cobalt cation released by the parent compound is the determining factor for toxicological effects, same as for other soluble cobalt compounds. Therefore, data originating from soluble cobalt compounds can be used in the derivation of toxicological endpoints for cobalt hydrogen citrate. For further details, please refer to the read-across justification attached in section 13 of the technical dossier.

In a 13-week inhalation study, groups of mice and rats were exposed to cobalt(II)sulfate heptahydrate aerosols at concentrations of 0.3, 1, 3, 10 and 30 mg/m³ (calculated as anhydrous salt and equivalent to 0.5, 1.6, 4.8, 16.1 and 48.2 mg cobalt hydrogen citrate/m³, respectively) for 6 hours/day and 5 days/week (Bucher, 1991). All rats, all female mice, and all but 2 male mice exposed at the top concentration survived to the end of the studies (10 animals per group). Rats and mice exposed to 48.2 cobalt hydrogen citrate/m³ lost weight during the first exposure week and then gained weight at the same rate as controls. Lung weights were increased over those of controls in rats exposed at concentrations as low as 0.5 mg cobalt hydrogen citrate/m³ and in mice exposed to 16.1 mg cobalt hydrogen citrate/m³ or more. Polycythemia was observed in rats exposed to the test substance but not in mice. Cobalt content in the urine of rats increased with increasing atmospheric cobalt exposure. In male mice, at ≥ 4.8 mg cobalt hydrogen citrate/m³, sperm motility was decreased and at a concentration of 48.2 mg cobalt hydrogen citrate/m³, testicular atrophy, increased abnormal sperm and decreased testis weight were observed (Reproductive functions were only examined in mice/rats exposed to 4.8, 16.1 or 48.2 mg cobalt hydrogen citrate/m³).

The estrous cycle was significantly longer in female mice exposed to the highest concentration than in the controls. In rats, no significant effects on the male or female reproductive systems were observed.

Both in rats and mice, lesions seen in the respiratory tract included degeneration of the olfactory epithelium, squamous metaplasia of the respiratory epithelium, and inflammation in the nose, inflammation, necrosis, squamous metaplasia, ulcers (rats), and inflammatory polyps (rats) of the larynx, squamous metaplasia of the trachea (mice); and histiocytic infiltrates, bronchiolar regeneration, peribronchiolar and septal fibrosis, and epithelial hyperplasia in the alveoli of the lung. The most sensitive tissue was the larynx, with squamous metaplasia observed in rats and mice at the lowest exposure concentration of 0.5 mg cobalt hydrogen citrate/m³. Thus, no NOAEC could be determined, neither in rats nor in mice.

 

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level.

Repeated dose toxicity: inhalation - systemic effects (target organ) urogenital: testes

Justification for classification or non-classification

Although the respiratory tract was damaged in rats and mice, soluble cobalt substances will not be classified in repeated dose toxicity since also carcinogenic effects in the respiratory tract were observed in a 2-year inhalation study (NTP, 1998, see carcinogenicity). Therefore, cobalt hydrogen citrate is classified as carcinogenic (DSD: Carcinogenicity category 2; R49; CLP: Carcinogenicity category 1B; H350). According to CLP, there should be no double classification for the same effect, thus no classification for repeated dose toxicity.

Based on an analogue approach, the available data on repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.