Naphthenic acids, cobalt salts

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study. With respect to the purpose for which this study was conducted as a 90d inhalation toxicity study, it may be considered reliable without restriction. However, the study design and the reporting suffer from several major shortcomings which ultimately do not allow the identification of potential target organ for systemic effects as detailed below.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Inhalation toxicity studies of cobalt sulfate in F344/N rats and B6C3F1 mice

Author:

Bucher, J.R., et al.

Year:

1990

Bibliographic source:

Fund. Appl. Tox. 15, 357-372

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1991

Reference 3

Reference Type:

publication

Title:

Inhalation toxicity and carcinogenicity studies of cobalt sulfate

Author:

Bucher, J.R.; et al.

Year:

1999

Bibliographic source:

Toxicol. Sci. 49, 56-67

Materials and methods

Test guideline

Guideline:

other: No guideline stated, but the design, conduct and reporting of the study is generally in agreement with OECD

Principles of method if other than guideline:

Groups of 10 male and 10 female mice were exposed to aerosols containing 0, 0.3, 1.0, or 3.0, 10, 30 mg/m³ cobalt sulfate heptahydrate 6 hours per day, 5 days per week, for 13 weeks.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cobalt sulfate heptahydrate

IUPAC Name:

cobalt sulfate heptahydrate

Constituent 2

Reference substance name:

10026-24-1

Cas Number:

10026-24-1

IUPAC Name:

10026-24-1

Details on test material:

- Name of test material (as cited in study report): Cobalt sulfate heptahydrate (CoSO4 * 7H2O) (From Curtin Matheson Scientific inc. (Kansas City, MO)- Physical state: red, crystalline solid- Molecular weight: 281.13 g- Analytical purity: approx. 99 %; Elemental analyses for sulfur and hydrogen were in agreement with the theoretical values for cobalt sulfate heptahydrate. Karl Fischer water analysis indicated 44.6 % +/- 0.5% water- Impurities (identity and concentrations): Spark source mass spectroscopy indicated 140 ppm nickel present as impurity; all other impurities had a combined total of less than 175 ppm.- Lot No.: 412091- Storage condition of test material: To ensure stability, the bulk chemical was stored in its original shipping containers, metal cans, at room temperature.- Other: Cobalt sulfate heptahydrate is stable as a bulk chemical when stored protected from light at normal temperatures. The heptahydrate dehydrates to the hexahydrate at 41.5 °C and to the monohydrate when heated to 71°C, with no further changes expected below the decomposition temperature (708°C). Therefore, an accelerated stability study was not conducted. Stability was monitored during the study using elemental analysis by inductively coupled plasma/atomic emission spectroscopy (ICP/AES) normalized against a cobalt standard (National Institute of Standards and Technology, Gaithersburg, MD); no degradation of the bulk chemical was detected.

Test animals

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Taconic Farms, Inc.- Age at study initiation: approx. 7 weeks- Housing: Mice were housed individually- Diet (ad libitum except during exposure period): NIH-07 (Zeigler Brothers, Inc., Gardners, PA)- Water (ad libitum)- Quarantine period: 14 days before beginning of the studyENVIRONMENTAL CONDITIONS OF CHAMBERS FOR EXPOSURE- Temperature (°F): 71-78.7°F- Photoperiod (hrs dark / hrs light): 12 hours/day

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: 0.83-1.10 µm

Details on inhalation exposure:

Cobalt sulfate heptahydrate aerosol was generated from an aqueous solution by nebulisation using dried compressed air. The aerosol was heated to about 26° C to dry the particles partially and then was passed into a Nalgene® settling tank to eliminate large particles and water droplets. Further drying was accomplished by heating the aerosol to 45° C as it left the tank. The cobalt sulfate heptahydrate/air stream entered the distribution tube and was injected into each chamber (Hazleton 2000, Lab Products, Inc.) with air multiplier pumps. The aerosol was diluted to the desired concentration with air from the chamber air-conditioning system.Atmospheric concentrations are expressed in milligrams of cobalt sulfate per cubic meter of air rather than in milligrams of the heptahydrate.

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. The monitors were calibrated through the use of filter grab samples. Samples collected on filter paper were analyzed for cobalt by inductively coupled plasma analysis after extraction with dilute nitric acid.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours (plus T90: approx. 8 min) per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:0.300 mg/m³Basis:analytical conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:0.990 mg/m³Basis:analytical conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:2.93 mg/m³Basis:analytical conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:9.95 mg/m³Basis:analytical conc.

Doses / concentrations 5

Remarks:

Doses / Concentrations:30.0 mg/m³Basis:analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 16-day inhalation study with cobalt sulfate in rats and mice (doses: 0.1, 0.5, 5, 50, 200mg/m³)

Positive control:

No

Examinations

Observations and examinations performed and frequency:

Observed 2 X d; weighed initially and 1 X wk thereafterHematologic analyses performed; sperm morphology and vaginal cytology evaluated.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see any other information on material and methods)HISTOPATHOLOGY: Yes (see any other information on material and methods)

Statistics:

The analysis of organ weight, serum chemistry, hematologic, urinalysis, and male reproductive system data was carried out by using the nonparametric multiple comparison procedures of Dunn (1964) or Shirley (1977) to assess the significance of pair wise 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 pair wise comparisons. The proportion of time spent in each stage of the oestrous 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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

2 males in 30mg/m³ group died prematurely, no clinical signs

Mortality:

mortality observed, treatment-related

Description (incidence):

2 males in 30mg/m³ group died prematurely, no clinical signs

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

mean body weight decreased in males at 30mg/m³ and females at 10mg/m³ and above

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

absolute lung weight and the lung weight to body weight ratios increased in 10 and 30 mg/m³ groups, absolute testis weight and the testis weight to body weight ratio decreased in males at 30mg/m³, epididymal weight decreased in males at 30mg/m³

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see "details on results"

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see "details on results"

Details on results:

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. Data were not collected on mice exposed at lower concentrations. Compound-related microscopic lesions 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. No cardio toxic effects were observed.

Effect levels

1

Dose descriptor:

NOAEC

Remarks on result:

not determinable

Remarks:

no NOAEC identified

2

Dose descriptor:

LOAEC

Effect level:

ca. 0.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Exposure of mice to aerosols of cobalt sulfate heptahydrate resulted primarily in severe necrotizing injury to the respiratory tract. The larynx appeared to be the most sensitive tissue, showing metaplastic and inflammatory lesions.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table: Numbers of mice with selected lesions in the 13-week inhalation study of cobalt sulfate heptahydrate (a)

Site/Lesion	Control	0.3mg/m³	1mg/m³	3mg/m³	10 mg/m³	30 mg/m³
MALE
Nose
Acute inflammation	0	--	0	1	**10	**9
Olfactory epithelium degeneration	0	--	0	0	**9	**8
Respiratory epithelium squamous metaplasia	0	--	0	0	**8	**8
Larynx
Inflammation	0	0	0	(b) 0	1	**9
Necrosis	0	0	0	(b) 0	0	3
Squamous metaplasia	0	**7	**10	*(b) 5	**9	**10
Trachea
Squamous metaplasia	0	--	--	--	0	2
Lung
Histiocytic infiltrates	0	**10	**9	**10	**10	**10
Chronic inflammation	0	0	0	0	1	**10
Bronchiolar epithelium regeneration	0	0	0	0	0	**10
Alveolar epithelium hyperplasia	0	0	0	0	3	**8
Mediastinal lymph nodes
Hyperplasia	0	--	--	--	(c) 0	**(b) 6
Testis
Atrophy	0	--	--	--	0	**9
Mineralization	0	--	--	--	0	*4
FEMALE
Nose
Acute inflammation	0	0	1	*4	**10	**10
Olfactory epithelium degeneration	0	0	1	*4	**104	**10
Respiratory epithelium squamous metaplasia	0	0	0	1	**9	**9
Larynx
Inflammation	0	0	0	(b) 0	**6	**(b) 8
Necrosis	0	0	0	(b) 0	0	**(b) 6
Squamous metaplasia	0	**8	**8	**(b)8	**9	**(b) 9
Trachea
Squamous metaplasia	0	--	--	--	0	3
Lung
Histiocytic infiltrates	0	--	--	--	0	3
Chronic inflammation	0	0	0	0	*5	**10
Bronchiolar epithelium regeneration	0	0	0	0	0	**10
Alveolar epithelium hyperplasia	0	0	0	0	**10	**10
Mediastinal lymph nodes
Hyperplasia	0	--	--	(d) 0	(e) 1	**7

(a) Ten mice were examined in each group unless otherwise specified; - indicates tissue not examined.

(b) Nine mice were examined.

(c) Seven mice were examined.

(d) Five mice were examined.

(e) Six mice were examined.

* P< 0.05 by Fisher exact test

**P< 0.05 by Fisher exact test

Applicant's summary and conclusion

Conclusions:

Exposure of mice to aerosols of cobalt sulfate heptahydrate resulted primarily in severe necrotizing injury to the respiratory tract. The larynx appeared to be the most sensitive tissue, showing metaplastic and inflammatory lesions after exposure at concentrations as low as 0.3 mg/m³ cobalt sulfate heptahydrate. Thus a LOAEC of 0.3mg/m³ for local effects in the respiratory tract is derived. Being designed as an inhalation toxicity study, it constitutes a reliable investigation of local effects in the lung. However, with respect to systemic toxicity, the study shows major shortcomings in design, conduct, dose/exposure selection and reporting, which do not allow the identification of potential target organs for systemic effects. Major deficiencies include that neither both species nor all exposure groups were chosen for histopathological examination, nor where presented the severity of such lesions is indicated. Because of this, for example the investigation of effects on the reproductive system, serum and thyroid function nor any other potential target organs for systemic toxicity or any other dose-response relationship can be determined. In addition, since the respiratory tract is particularly sensitive to soluble cobalt substances, this fact limits the maximum exposure/dose to a level at which systemic toxicity does not yet manifest itself. Other criticism in the design and conduct of the study are (i) full microscopic examination was only performed on the control and high dose group animals (ii) microscopic examinations of the reproductive organs in mice was only performed in the mid- and high-dose groups (3, 10, 30mg/m³) (iii) groups at 0.3 and 1mg/m³ were not examined (iv) authors were not able to determine a site of action related to the toxic effect in the reproductive organs of male mice. In conclusion, this study is not suitable to derive a NOAEC/LOAEC for systemic effects. Signs of general toxicity (decreased body weight) observed in males at 30mg/m³ and in females at 10 and 30mg/m³, are not suitable to identify target organs for systemic toxicity.