Registration Dossier

Administrative data

Description of key information

NOAEL (90-d oral, rat) >= 2015 mg/kg bw/day
NOAEL (90-d oral, mouse) >= 4342 mg/kg bw/day
LOAEC, local (90-d, inhalation, rat) = 21 mg/m³
NOAEC, systemic (90-d, inhalation, rat) = 210 mg/m³
NOAEL, dermal, systemic (extrapolation from 90-d oral rat) >= 2015 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
limited measurements and examinations
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services
- Age at study initiation: 6 weeks
- Weight at study initiation: 92 g (male), 93 g (female)
- Housing: 5 per cage
- Acclimation period: 11 - 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.9
- Humidity (%): 35 - 65
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
other: diet (irradiated NTP-2000 open formula diet)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): four times throughout the study
- Storage temperature of food: room temperature, protected from light (stability of the test substance under these conditions was at least 42 days)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
93 days
Frequency of treatment:
ad libitum
Remarks:
Doses / Concentrations:
80; 240; 2000; 10,000 or 50,000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
7, 19, 160, 805 and 4243 mg/kg bw (males)
Basis:
other: average daily doses
Remarks:
Doses / Concentrations:
6, 19, 162, 780 and 4254 mg/kg bw (females)
Basis:
other: average daily doses
No. of animals per sex per dose:
10 in core study and 10 per sex and dose and time point in special study (hematology and clinical chemistry analyses on days 3 and 21)
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly


BODY WEIGHT: Yes
- Time schedule for examinations: day 1, weekly throughout the study and at sacrifice


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food consumption was measured by cage covering a 7-day period
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 3, 21 and 93 (terminal sacrifice)
- Anaesthetic used for blood collection: Yes, CO2/O2
- Animals fasted: No data
- How many animals: 10
- Parameters checked: erythrocyte count, hemoglobin, packed cell volume, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, nucleated red blood cell count, total leukocyte count, differential leukocyte counts, red blood cell morphology and platelet morphology


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on days 3, 21 and 93 (terminal sacrifice)
- Animals fasted: No data
- How many animals: 10
- Parameters checked: sorbitol dehydrogenase, alkaline phosphatase, creatine kinase, creatinine, total protein, albumin, blood urea nitrogen, total bile acids and alanine aminotransferase


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights of liver, right kidney, heart, lungs, thymus and right testis
HISTOPATHOLOGY: Yes
complete (no further details given)
Statistics:
analysis of variance followed by the parametric multiple comparison procedures of Dunnett and Williams (organ and body weight), Kruskal-Wallis analysis of variance (hematology and clinical chemistry), Jonckheere's test (dose-response analysis), Cochran-Armitage and Fisher's exact test (prevalence of nonneoplastic lesions)
Dose descriptor:
NOAEL
Effect level:
>= 2 015 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects NOAEL converted from the tri-picolinate monohydrate to the basic acetate monohydrate
Critical effects observed:
not specified

No deaths occured.

No significant compound-related affects on body weight, body weight gain and food consumption were observed.

Administration of the test substance did not produce clinical signs of toxicity. However, the feces of high dose animals had a reddish colour, probably because of the reddish-colour of the test substance.

There were no statistically significant hematological and clinical chemistry changes observed.

Absolute organ weights showed slight alterations in exposed animals. Male heart and liver weights showed negative trends (not dose-related, 9% less than controls in high dose group). Female right kidney weights were significantyl increased in all dosed animals (up to 12% greater than controls). These effects were, however, not considered treatment-related.

Histopathological lesions were considered spontaneous or incidental and not related to the test substance.

Effects on reproduction are described in section 7.8.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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: The study follows the requirements of the relevant EU method B.29 / OECD TG 413. The detail level is typical for a published study and is lower than stipulated by the relevant testing guidelines.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
Reporting deficits (publication)
GLP compliance:
not specified
Remarks:
the test facility is known to be GLP certified, but no GLP compliance is claimed in the published report
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: MMAD (GSD): 4.2 (2.48), 4.2 (2.37), and 4.5 (2.50) µm for for the low-, mid- and high-exposure groups, respectively
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 h/day
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
3, 10, and 30 mg/m³ Cr(III)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
17, 54, 168 mg/m³ Cr(OH)(SO4)
Basis:
analytical conc.
No. of animals per sex per dose:
10 (main group)
5 (13-wk recovery group)
5 (5-day exposure, BAL)
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
Animals were observed daily prior to and following each exposure for clinical signs of toxicity, and were observed twice daily for morbidity and mortality during the recovery period and on weekends. Individual body weights were recorded weeltly during the exposure and recovery periods. All animals received an indirect ophthalmoscopic examination during the acclimation period and prior to terminal necropsy.
Sacrifice and pathology:
Animals found dead or euthanized by design at study termination were necropsied. At necropsy the heart, lungs, liver, spleen, kidneys, brain, adrenal glands, thyroid/parathyroid glands, testes, and ovaries were weighed. Tissues typically harvested for subchronic studies were also removed and preserved. All tissues were placed in 10% neutral buffered formalin, except eye tissue, which was fixed in Davidson's fixative. Microscopic evaluation was conducted on all hematoxylin and eosin-stained tissues from the control group and high-exposure-level groups of both test articles. The kidneys, livers, nasal tissues, trachea, lungs, larynx, mediastinal and mandibular
lymph nodes, and gross lesions from all animals in the low- and mid-exposure level groups for both test articles were also examined. A formal peer review of the histopathologic findings was performed.
Other examinations:
Bronchoalveolar lavage evaluation. Bronchoalveolar lavage (BAL) analyses were conducted on 5 animals per sex per group exposed for 5 consecutive days with the main study animals. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The lungs, heart, trachea, larynx, and tongue were removed en-block. Following tracheal cannulation, the airways were washed 3 successive times with warmed, physiological saline (30 µL per gram of body weight) and the resulting BALF was pooled. Nucleated cell counts were performed using a Neubauer hemocytometer, and cell differential counts were performed on Wright-Giemsa-stained smears. Chemical analyses performed spectrophotometrically included for lactate dehydrogenase (LDH), total protein, beta-glucuronidase, and glutathione reductase.

Sperm evaluation. At necropsy, sperm samples from the left caudal epididymis of 10 males per group were used for automated evaluation of sperm motility, count, and morphology. The concentration and morphology of the sperm were evaluated using visual methods. Two hundred intact sperm were evaluated from each animal for morphology. Intact sperm were evaluated as normal or abnormal. The number of disarticulated sperm in each field was also assessed.

Clinical pathology. Standard hematology, clinical biochemistry, and urinalysis determinations were conducted on animals, 10 per sex per group, designated for necropsy at the end of exposures. Animals were fasted overnight prior to blood sampling, with water available. Blood samples were obtained from the orbital sinus plexus. At necropsy, bone marrow smears were prepared and differential cell counts were evaluated. All clinical procedures were performed using automated instrumentation except bone marrow smears, which were examined microscopically. Urinalysis determinations were conducted on samples collected overnight in stainless-steel metabolism cages.
Urinalysis determinations were performed by gross observation, microscopy, and automated clinical analyzer. Following urinalysis testing, aliquots of the remaining urine from 5 animals per sex from the control group, and the high-exposure level groups for both test articles were submitted for beta-microglobulin analysis.
Statistics:
Statistical analyses were performed of body weights, clinical pathology laboratory tests, BALF data, and organ weights using one-way analysis of variance. If the result was non-significant, no additional analysis was performed. If the result was significant, Bartlett's test for homogeneity of variance was performed. If Bartlett's test was non-significant, Dunnett's t-test was used for pairvise comparisons. If Bartlett's test was significant, the Welch t-test with Bonferonni correction was used for pairwise comparisons. The Kruskal-Wallis analysis of variance, followed where appropriate by the Mann-Whitney U test, was used for those parameters where parametric analysis was inappropriate. The level for statistical significance was set at p < 0.05.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No compound-related mortalities occurred during the conduct of this study. Six animals died on exposure day 1 as a direct result of the restraint tubes, and they were replaced. One male from the high-exposure (168 mg/m³) group that died on exposure day 4 was not replaced. Although the specific cause of death was not identified, this death was not considered related to exposure to basic chromium sulfate, since there were no significant signs of toxicity observed in any other animals from this group.
Clinical signs of toxicity were limited to sporadic labored breathing, noted during two of the weekly observations, in females exposed to the high concentration of basic chromium sulfate.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant, exposure-related reduced mean body weights were observed in the males of the mid- and high-exposure groups and the females of the high-exposure group during the 13-week exposure period. At the recovery sacrifice, the males from the same exposure groups continued to exhibit mean body weights that were significantly lower than the control group but body weight gains between the chromium-exposed and control groups were similar. The female mean body weights were comparable to the control group females in all treatment groups at the recovery sacrifice.Although quantitative food consumption data were not collected, no observable decreases in food consumption were noted during the study.


OPHTHALMOSCOPIC EXAMINATION
No exposure-related effects were noted in the ophthalmologic evaluations.

HAEMATOLOGY
Increased leukocytes associated with increased number of neutrophils, some statistically significant, were noted in the mid- and high exposure groups for males and females.

CLINICAL CHEMISTRY
Alkaline phosphatase was statistically elevated in high-exposure group females and serum cholesterol was statistically decreased in mid- and high-exposure group females.

URINALYSIS
No effects.

ORGAN WEIGHTS
At the terminal sacrifice, the males and females in all treatment groups demonstrated compound-related, statistically significant increases in mean absolute and relative lung/trachea weights. These organ-weight changes corresponded to changes seen microscopically. Statistically significant changes in absolute andor relative weights of some other organs, primarily in the high-level exposure group, were noted for brain (males), kidney (males), and thyroid/parathyroid (male and female). No microscopic tissue alterations were present to explain these organ weight changes.
Other statistically significant terminal-sacrifice organ-weight changes were noted in the high-exposure-level group for liver (males), spleen (males and females), and testes. These changes were generally very small with no corresponding microscopic pathology. Weight changes in these organs are typically observed with depressed body weights. At the recovery sacrifice, statistically significant increases in male and female mean absolute and relative lung weights continued for the mid- and high exposure level groups. Microscopic changes were present to explain the pulmonary weight increases. Other statistically significant organ weight changes not associated with microscopic changes included decreases in male mean absolute kidney weights and increased male mean relative brain/body weight ratios in the mid- and high-exposure level. Table 1 summarizes the organ weight changes.


GROSS PATHOLOGY
Exposure-related macroscopic findings at the terminal and recovery sacrifices were observed in the lungs and mediastinal lymph nodes of most animals in this study. Green lung discoloration was observed in animals exposed to chromic oxide at all exposure levels. Gray lung discoloration was commonly observed in animals exposed to basic chromium sulfate at the mid- and high-exposure levels.
The degree of discoloration with both materials increased with exposure level and was present both at the terminal and recovery sacrifices. Similar discoloration was observed in the mediastinal lymph nodes of animals exposed to or basic chromium sulfate (recovery sacrifice only). Mediastinal lymph-node enlargement was observed at the recovery sacrifice in animals exposed to basic chromium sulfate (all exposure levels). Tan focus/foci were observed in the lungs at the recovery sacrifice of a high percentage of males exposed to the high level of basic chromium sulfate. The tan focus/foci correlated well with the presence of alveolar macrophages, and mediastinal lymph node enlargement correlated well with histiocytosis described below. Additional macroscopic observations were few in number and considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
Terminal sacrifice
Chronic inflammation was observed involving the alveoli of all exposure-level groups, consisting of alveolar spaces filled with macrophages, neutrophils, lymphocytes, and cellular debris. Some foci exhibited quite intense inflammation and thickening of alveolar walls. Chronic interstitial inflammation was usually multifocally distributed and consisted of thickened alveolar septa caused by inflammatory cell infiltration and hyperplasia of alveolar septal cells (type II pneumocytes). Multifocal areas of granulomatous inflammation, characterized by infiltration of macrophages and multinucleated giant cells, was observed at all exposure levels and was closely associated with foreign material seen in the lung and presumed to be the test article. Trace to severe, multifocal to diffuse pulmonary infiltration of alveolar macrophages with foamy or granular appearing acidophilic cytoplasm was observed in the alveolar lumens and correlated with the gray discoloration of the lungs that was observed at necropsy.
These changes corresponded to the increased lung weights observed in all the exposure groups. Green refractile foreign material was present in the lamina propria and submucosa of the larynx of animals in all treatment groups and was associated with an infiltration of macrophages and multinucleated giant cells (granulomatous inflammation). Histiocytosis consisting of macrophages or histiocytes with abundant foamy
cytoplasm and lymphoid hyperplasia observed in the peribronchial lymphoid tissue and mediastinal lymph node correlated with lymph node enlargement observed at necropsy. Changes in nasal tissues considered to be test article-related, were observed in males and females, and included acute inflammation, suppurative exudate, and mucoid exudate. One or more of these changes were seen in all 4 examined levels of the nasal cavity but were slightly more prevalent in the mid-posterior portion. All other microscopic findings were incidental and/or
spontaneous and considered unrelated to the test article.

Recovery Sacrifice
Foreign material persisted in the lungs of some animals in all exposure groups, but with decreased incidence in most groups. Trace to moderate chronic inflammation of the alveoli, trace to moderate chronic interstitial inflammation, septal cell hyperplasia, and alveolar macrophages were approximately equal in incidence and severity at the mid- and high-exposure levels as compared to the terminal sacrifice
animals, with severity slightly reduced at the low exposure level. Peribronchial histiocytosis continued to be observed only in males and females of the mid- and high-exposure groups. The incidence and severity in the mid-exposure group was approximately equal to the terminal sacrifice animals while the incidence in the high-exposure group increased in both males and females, suggesting clearing of the
basic chromium sulfate via the reticuloendothelial system.
Granulomatous inflammation of the lung decreased in incidence except in the males and females of the high-exposure group where the incidence was approximately equal to that of the terminal sacrifice animals of this group. In the larynx, foreign material and granulomatous inflammation was either not detected at the low-exposure (both sexes) or mid-exposure (males) or markedly decreased in incidence and severity in mid-exposure females and high-exposure groups (both sexes) as compared to the terminal-sacrifice animals. The incidence
and severity of histiocytosis and lymphoid hyperplasia of the mediastinal lymph node generally remained at similar levels in both males and females of all treated groups in the terminal and recovery sacrifices. Nasal cavity findings were not detected except for trace suppurative exudate seen in the most anterior section in one or two animals from each treatment group. All other microscopic findings were incidental andor spontaneous and considered unrelated to the test article.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No findings.

OTHER FINDINGS
No exposure-related effects were noted for sperm motility, morphology, or concentration.
Dose descriptor:
NOAEC
Effect level:
< 21 mg/m³ air (analytical)
Sex:
male/female
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Dose descriptor:
LOAEC
Effect level:
21 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: gross pathology; organ weights; histopathology; other: BAL analysis The LOAEC is converted from basic Cr(III) sulfate to basic Cr(III) acetate monohydrate
Dose descriptor:
NOAEC
Effect level:
>= 210 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: No systemic effects
Critical effects observed:
not specified

Table 1: Selected Organ Weight Changes at Terminal and Recovery Sacrifices of Rats Exposed to Basic Chromium Sulphate

Control

17 mg/m³

54 mg/m³

168 mg/m³

Males

Lung/trachea

abs. weight [g]

0.99 ±0.70

(1.32 ± 0.113)

1.26 ± 0.071**

(1.52 ± 0.132)

1.51 ± 0.088**

(1.95 ± 0.068**)

1.86 ± 0.89**

(2.67 ± 0.144**)

rel. weight [%×10]

4.42 ± 0.187

(3.89 ± 0.214)

5.60 ± 0.271**

(4.66 ± 0.373**)

7.15 ± 0.252**

(6.37 ± 0.298**)

10.69 ± 0.688**

(8.77 ± 0.274**)

Brain

abs. weight [g]

1.79 ± 0.087

1.82 ± 0.055

1.76 ± 0.061

1.71 ± 0.069*

rel. weight [%×10]

8.02 ± 0.380

8.12 ± 0.374

8.38 ± 0.473

9.83 ± 0.518**

Kidney

abs. weight [g]

1.54 ± 0.106

1.35 ± 0.049**

1.62 ± 0.085

1.64 ± 0.082

rel. weight [%×10]

7.62 ± 0.300

7.28 ± 0.207

7.30 ± 0.283

7.78 ± 0.350**

Liver

abs. weight [g]

5.48 ± 0.367

5.63 ± 0.271

5.17 ± 0.459

4.39 ± 0.146**

rel. weight [%]

2.45 ± 0.070

2.50 ± 0.050

2.45 ± 0.091

2.53 ± 0.120

Thyroid/parathyroid

abs. weight [mg]

14 ± 2.5

15 ± 2.9

14 ± 1.8

15 ± 3.5

rel. weight [%×103]

6.21 ± 1.052

6.64 ± 1.475

6.74 ± 1.021

8.76 ± 2.074*

Spleen

abs. weight [g]

0.45 ± 0.038

0.48 ± 0.036

0.40 ± 0.040*

0.32 ± 0.035**

rel. weight [%×10]

1.99 ± 0.149

1.91 ± 0.132

1.89 ± 0.125

1.84 ± 0.151

Testes

abs. weight [g]

2.36 ± 0.356

2.39 ± 0.261

2.22 ± 0.286

2.18 ± 0.215

rel. weight [%×10]

10.54 ± 1.315

10.65 ± 1.098

10.52 ± 1.049

12.53 ± 1.238**

Females

Lung/trachea

abs. weight [g]

0.81 ±0.081

(0.93 ± 0.079)

0.98 ± 0.094**

(1.08 ± 0.120)

1.29 ± 0.164**

(1.59 ± 0.120**)

1.66 ± 0.084**

(2.45 ± 0.120**)

rel. weight [%×10]

5.65 ± 0.418

(4.74 ± 0.384)

6.99 ± 0.619**

(5.75 ± 0.315**)

9.24 ± 1.036**

(8.02 ± 0.750**)

12.89 ± 1.134**

(13.34 ± 0.614**)

Thyroid/parathyroid

abs. weight [mg]

12 ± 1.9

11 ± 1.3

12 ± 1.8

14 ± 2.1*

rel. weight [%×103]

8.26 ± 1.493

7.96 ± 1.154

8.63 ± 1.265

10.77 ± 1.522**

Spleen

abs. weight [g]

0.33 ± 0.037

0.31 ± 0.033

0.30 ± 0.033

0.28 ± 0.033**

rel. weight [%×10]

2.32 ± 0.268

2.19 ± 0.212

2.17 ± 0.162

2.19 ± 0.273

Note. Organ weight changes, values given as mean ± SD; bw = body weight. Non-bracketed values = terminal sacrifice, bracketed values = recovery sacrifice. * p 0.05; **p 0.01.

Conclusions:
Local effects: A NOAEC was not established. The LOAEC for basic chromium sulphate was 17 mg/m³. This is equivalent to 21 mg/m³ basic chromium(III) acetate monohydrate.
Systemic effects: Adverse systemic effects were not observed. NOAEC >= 210 mg/m³ basic chromium(III) acetate monohydrate.
Executive summary:

The toxicity of basic chromium sulfate, was investigated in rats in a 13-week nose-only inhalation study that included a 13-week recovery period. Nose-only exposures to soluble basic chromium sulfate dust at 17, 54, or 168 mg/m³ (trivalent chromium equivalent concentrations of 3, 10, and 30 mg/m³) were carried out for 6 h/day, 5 days/week.

No compound-related mortality occurred.

General toxic effects, only observed with high-exposure levels of basic chromium sulfate, included sporadic signs of labored breathing and depressed body weights.

No apparent compound-related effects were noted for sperm motility or morphology, for any concentration of either test material. Bronchoalveolar lavage fluid evaluations showed increased neutrophils, protein, lactic dehydrogenase and cellular debris. The principle effects

were primarily to the respiratory tract.

Basic chromium sulfate produced severe and widespread effects in the nasal cavity, larynx, lungs, and mediastinal lymph node. Effects were characterized by accumulation of foreign material, infiltration of alveolar macrophages, septal cell hyperplasia, and granulomatous and chronic inflammation.

Pigment was still present in basic chromium sulfate-treated animals after the 13-week recovery period, with partial recovery of the pathological lesions.

A NOAEC for effects on the respiratory system was not established.

Adverse systemic effects were not observed. The observed organ weights effects can be considered secondary to the stress imposed on the respiratory tract and the resulting impairment of body weight gain. The highest test concentration is considered to represent the systemic NOAEC.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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: The study follows the requirements of the relevant EU method B.29 / OECD TG 413. The detail level is typical for a published study and is lower than stipulated by the relevant testing guidelines.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
Reporting deficits (publication)
GLP compliance:
not specified
Remarks:
the test facility is known to be GLP certified, but no GLP compliance is claimed in the published report
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: MMAD (GSD): 4.2 (2.48), 4.2 (2.37), and 4.5 (2.50) µm for for the low-, mid- and high-exposure groups, respectively
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
6 h/day
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
3, 10, and 30 mg/m³ Cr(III)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
17, 54, 168 mg/m³ Cr(OH)(SO4)
Basis:
analytical conc.
No. of animals per sex per dose:
10 (main group)
5 (13-wk recovery group)
5 (5-day exposure, BAL)
Control animals:
yes, sham-exposed
Observations and examinations performed and frequency:
Animals were observed daily prior to and following each exposure for clinical signs of toxicity, and were observed twice daily for morbidity and mortality during the recovery period and on weekends. Individual body weights were recorded weeltly during the exposure and recovery periods. All animals received an indirect ophthalmoscopic examination during the acclimation period and prior to terminal necropsy.
Sacrifice and pathology:
Animals found dead or euthanized by design at study termination were necropsied. At necropsy the heart, lungs, liver, spleen, kidneys, brain, adrenal glands, thyroid/parathyroid glands, testes, and ovaries were weighed. Tissues typically harvested for subchronic studies were also removed and preserved. All tissues were placed in 10% neutral buffered formalin, except eye tissue, which was fixed in Davidson's fixative. Microscopic evaluation was conducted on all hematoxylin and eosin-stained tissues from the control group and high-exposure-level groups of both test articles. The kidneys, livers, nasal tissues, trachea, lungs, larynx, mediastinal and mandibular
lymph nodes, and gross lesions from all animals in the low- and mid-exposure level groups for both test articles were also examined. A formal peer review of the histopathologic findings was performed.
Other examinations:
Bronchoalveolar lavage evaluation. Bronchoalveolar lavage (BAL) analyses were conducted on 5 animals per sex per group exposed for 5 consecutive days with the main study animals. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The lungs, heart, trachea, larynx, and tongue were removed en-block. Following tracheal cannulation, the airways were washed 3 successive times with warmed, physiological saline (30 µL per gram of body weight) and the resulting BALF was pooled. Nucleated cell counts were performed using a Neubauer hemocytometer, and cell differential counts were performed on Wright-Giemsa-stained smears. Chemical analyses performed spectrophotometrically included for lactate dehydrogenase (LDH), total protein, beta-glucuronidase, and glutathione reductase.

Sperm evaluation. At necropsy, sperm samples from the left caudal epididymis of 10 males per group were used for automated evaluation of sperm motility, count, and morphology. The concentration and morphology of the sperm were evaluated using visual methods. Two hundred intact sperm were evaluated from each animal for morphology. Intact sperm were evaluated as normal or abnormal. The number of disarticulated sperm in each field was also assessed.

Clinical pathology. Standard hematology, clinical biochemistry, and urinalysis determinations were conducted on animals, 10 per sex per group, designated for necropsy at the end of exposures. Animals were fasted overnight prior to blood sampling, with water available. Blood samples were obtained from the orbital sinus plexus. At necropsy, bone marrow smears were prepared and differential cell counts were evaluated. All clinical procedures were performed using automated instrumentation except bone marrow smears, which were examined microscopically. Urinalysis determinations were conducted on samples collected overnight in stainless-steel metabolism cages.
Urinalysis determinations were performed by gross observation, microscopy, and automated clinical analyzer. Following urinalysis testing, aliquots of the remaining urine from 5 animals per sex from the control group, and the high-exposure level groups for both test articles were submitted for beta-microglobulin analysis.
Statistics:
Statistical analyses were performed of body weights, clinical pathology laboratory tests, BALF data, and organ weights using one-way analysis of variance. If the result was non-significant, no additional analysis was performed. If the result was significant, Bartlett's test for homogeneity of variance was performed. If Bartlett's test was non-significant, Dunnett's t-test was used for pairvise comparisons. If Bartlett's test was significant, the Welch t-test with Bonferonni correction was used for pairwise comparisons. The Kruskal-Wallis analysis of variance, followed where appropriate by the Mann-Whitney U test, was used for those parameters where parametric analysis was inappropriate. The level for statistical significance was set at p < 0.05.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No compound-related mortalities occurred during the conduct of this study. Six animals died on exposure day 1 as a direct result of the restraint tubes, and they were replaced. One male from the high-exposure (168 mg/m³) group that died on exposure day 4 was not replaced. Although the specific cause of death was not identified, this death was not considered related to exposure to basic chromium sulfate, since there were no significant signs of toxicity observed in any other animals from this group.
Clinical signs of toxicity were limited to sporadic labored breathing, noted during two of the weekly observations, in females exposed to the high concentration of basic chromium sulfate.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant, exposure-related reduced mean body weights were observed in the males of the mid- and high-exposure groups and the females of the high-exposure group during the 13-week exposure period. At the recovery sacrifice, the males from the same exposure groups continued to exhibit mean body weights that were significantly lower than the control group but body weight gains between the chromium-exposed and control groups were similar. The female mean body weights were comparable to the control group females in all treatment groups at the recovery sacrifice.Although quantitative food consumption data were not collected, no observable decreases in food consumption were noted during the study.


OPHTHALMOSCOPIC EXAMINATION
No exposure-related effects were noted in the ophthalmologic evaluations.

HAEMATOLOGY
Increased leukocytes associated with increased number of neutrophils, some statistically significant, were noted in the mid- and high exposure groups for males and females.

CLINICAL CHEMISTRY
Alkaline phosphatase was statistically elevated in high-exposure group females and serum cholesterol was statistically decreased in mid- and high-exposure group females.

URINALYSIS
No effects.

ORGAN WEIGHTS
At the terminal sacrifice, the males and females in all treatment groups demonstrated compound-related, statistically significant increases in mean absolute and relative lung/trachea weights. These organ-weight changes corresponded to changes seen microscopically. Statistically significant changes in absolute andor relative weights of some other organs, primarily in the high-level exposure group, were noted for brain (males), kidney (males), and thyroid/parathyroid (male and female). No microscopic tissue alterations were present to explain these organ weight changes.
Other statistically significant terminal-sacrifice organ-weight changes were noted in the high-exposure-level group for liver (males), spleen (males and females), and testes. These changes were generally very small with no corresponding microscopic pathology. Weight changes in these organs are typically observed with depressed body weights. At the recovery sacrifice, statistically significant increases in male and female mean absolute and relative lung weights continued for the mid- and high exposure level groups. Microscopic changes were present to explain the pulmonary weight increases. Other statistically significant organ weight changes not associated with microscopic changes included decreases in male mean absolute kidney weights and increased male mean relative brain/body weight ratios in the mid- and high-exposure level. Table 1 summarizes the organ weight changes.


GROSS PATHOLOGY
Exposure-related macroscopic findings at the terminal and recovery sacrifices were observed in the lungs and mediastinal lymph nodes of most animals in this study. Green lung discoloration was observed in animals exposed to chromic oxide at all exposure levels. Gray lung discoloration was commonly observed in animals exposed to basic chromium sulfate at the mid- and high-exposure levels.
The degree of discoloration with both materials increased with exposure level and was present both at the terminal and recovery sacrifices. Similar discoloration was observed in the mediastinal lymph nodes of animals exposed to or basic chromium sulfate (recovery sacrifice only). Mediastinal lymph-node enlargement was observed at the recovery sacrifice in animals exposed to basic chromium sulfate (all exposure levels). Tan focus/foci were observed in the lungs at the recovery sacrifice of a high percentage of males exposed to the high level of basic chromium sulfate. The tan focus/foci correlated well with the presence of alveolar macrophages, and mediastinal lymph node enlargement correlated well with histiocytosis described below. Additional macroscopic observations were few in number and considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
Terminal sacrifice
Chronic inflammation was observed involving the alveoli of all exposure-level groups, consisting of alveolar spaces filled with macrophages, neutrophils, lymphocytes, and cellular debris. Some foci exhibited quite intense inflammation and thickening of alveolar walls. Chronic interstitial inflammation was usually multifocally distributed and consisted of thickened alveolar septa caused by inflammatory cell infiltration and hyperplasia of alveolar septal cells (type II pneumocytes). Multifocal areas of granulomatous inflammation, characterized by infiltration of macrophages and multinucleated giant cells, was observed at all exposure levels and was closely associated with foreign material seen in the lung and presumed to be the test article. Trace to severe, multifocal to diffuse pulmonary infiltration of alveolar macrophages with foamy or granular appearing acidophilic cytoplasm was observed in the alveolar lumens and correlated with the gray discoloration of the lungs that was observed at necropsy.
These changes corresponded to the increased lung weights observed in all the exposure groups. Green refractile foreign material was present in the lamina propria and submucosa of the larynx of animals in all treatment groups and was associated with an infiltration of macrophages and multinucleated giant cells (granulomatous inflammation). Histiocytosis consisting of macrophages or histiocytes with abundant foamy
cytoplasm and lymphoid hyperplasia observed in the peribronchial lymphoid tissue and mediastinal lymph node correlated with lymph node enlargement observed at necropsy. Changes in nasal tissues considered to be test article-related, were observed in males and females, and included acute inflammation, suppurative exudate, and mucoid exudate. One or more of these changes were seen in all 4 examined levels of the nasal cavity but were slightly more prevalent in the mid-posterior portion. All other microscopic findings were incidental and/or
spontaneous and considered unrelated to the test article.

Recovery Sacrifice
Foreign material persisted in the lungs of some animals in all exposure groups, but with decreased incidence in most groups. Trace to moderate chronic inflammation of the alveoli, trace to moderate chronic interstitial inflammation, septal cell hyperplasia, and alveolar macrophages were approximately equal in incidence and severity at the mid- and high-exposure levels as compared to the terminal sacrifice
animals, with severity slightly reduced at the low exposure level. Peribronchial histiocytosis continued to be observed only in males and females of the mid- and high-exposure groups. The incidence and severity in the mid-exposure group was approximately equal to the terminal sacrifice animals while the incidence in the high-exposure group increased in both males and females, suggesting clearing of the
basic chromium sulfate via the reticuloendothelial system.
Granulomatous inflammation of the lung decreased in incidence except in the males and females of the high-exposure group where the incidence was approximately equal to that of the terminal sacrifice animals of this group. In the larynx, foreign material and granulomatous inflammation was either not detected at the low-exposure (both sexes) or mid-exposure (males) or markedly decreased in incidence and severity in mid-exposure females and high-exposure groups (both sexes) as compared to the terminal-sacrifice animals. The incidence
and severity of histiocytosis and lymphoid hyperplasia of the mediastinal lymph node generally remained at similar levels in both males and females of all treated groups in the terminal and recovery sacrifices. Nasal cavity findings were not detected except for trace suppurative exudate seen in the most anterior section in one or two animals from each treatment group. All other microscopic findings were incidental andor spontaneous and considered unrelated to the test article.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No findings.

OTHER FINDINGS
No exposure-related effects were noted for sperm motility, morphology, or concentration.
Dose descriptor:
NOAEC
Effect level:
< 21 mg/m³ air (analytical)
Sex:
male/female
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Dose descriptor:
LOAEC
Effect level:
21 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: gross pathology; organ weights; histopathology; other: BAL analysis The LOAEC is converted from basic Cr(III) sulfate to basic Cr(III) acetate monohydrate
Dose descriptor:
NOAEC
Effect level:
>= 210 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: No systemic effects
Critical effects observed:
not specified

Table 1: Selected Organ Weight Changes at Terminal and Recovery Sacrifices of Rats Exposed to Basic Chromium Sulphate

Control

17 mg/m³

54 mg/m³

168 mg/m³

Males

Lung/trachea

abs. weight [g]

0.99 ±0.70

(1.32 ± 0.113)

1.26 ± 0.071**

(1.52 ± 0.132)

1.51 ± 0.088**

(1.95 ± 0.068**)

1.86 ± 0.89**

(2.67 ± 0.144**)

rel. weight [%×10]

4.42 ± 0.187

(3.89 ± 0.214)

5.60 ± 0.271**

(4.66 ± 0.373**)

7.15 ± 0.252**

(6.37 ± 0.298**)

10.69 ± 0.688**

(8.77 ± 0.274**)

Brain

abs. weight [g]

1.79 ± 0.087

1.82 ± 0.055

1.76 ± 0.061

1.71 ± 0.069*

rel. weight [%×10]

8.02 ± 0.380

8.12 ± 0.374

8.38 ± 0.473

9.83 ± 0.518**

Kidney

abs. weight [g]

1.54 ± 0.106

1.35 ± 0.049**

1.62 ± 0.085

1.64 ± 0.082

rel. weight [%×10]

7.62 ± 0.300

7.28 ± 0.207

7.30 ± 0.283

7.78 ± 0.350**

Liver

abs. weight [g]

5.48 ± 0.367

5.63 ± 0.271

5.17 ± 0.459

4.39 ± 0.146**

rel. weight [%]

2.45 ± 0.070

2.50 ± 0.050

2.45 ± 0.091

2.53 ± 0.120

Thyroid/parathyroid

abs. weight [mg]

14 ± 2.5

15 ± 2.9

14 ± 1.8

15 ± 3.5

rel. weight [%×103]

6.21 ± 1.052

6.64 ± 1.475

6.74 ± 1.021

8.76 ± 2.074*

Spleen

abs. weight [g]

0.45 ± 0.038

0.48 ± 0.036

0.40 ± 0.040*

0.32 ± 0.035**

rel. weight [%×10]

1.99 ± 0.149

1.91 ± 0.132

1.89 ± 0.125

1.84 ± 0.151

Testes

abs. weight [g]

2.36 ± 0.356

2.39 ± 0.261

2.22 ± 0.286

2.18 ± 0.215

rel. weight [%×10]

10.54 ± 1.315

10.65 ± 1.098

10.52 ± 1.049

12.53 ± 1.238**

Females

Lung/trachea

abs. weight [g]

0.81 ±0.081

(0.93 ± 0.079)

0.98 ± 0.094**

(1.08 ± 0.120)

1.29 ± 0.164**

(1.59 ± 0.120**)

1.66 ± 0.084**

(2.45 ± 0.120**)

rel. weight [%×10]

5.65 ± 0.418

(4.74 ± 0.384)

6.99 ± 0.619**

(5.75 ± 0.315**)

9.24 ± 1.036**

(8.02 ± 0.750**)

12.89 ± 1.134**

(13.34 ± 0.614**)

Thyroid/parathyroid

abs. weight [mg]

12 ± 1.9

11 ± 1.3

12 ± 1.8

14 ± 2.1*

rel. weight [%×103]

8.26 ± 1.493

7.96 ± 1.154

8.63 ± 1.265

10.77 ± 1.522**

Spleen

abs. weight [g]

0.33 ± 0.037

0.31 ± 0.033

0.30 ± 0.033

0.28 ± 0.033**

rel. weight [%×10]

2.32 ± 0.268

2.19 ± 0.212

2.17 ± 0.162

2.19 ± 0.273

Note. Organ weight changes, values given as mean ± SD; bw = body weight. Non-bracketed values = terminal sacrifice, bracketed values = recovery sacrifice. * p 0.05; **p 0.01.

Conclusions:
Local effects: A NOAEC was not established. The LOAEC for basic chromium sulphate was 17 mg/m³. This is equivalent to 21 mg/m³ basic chromium(III) acetate monohydrate.
Systemic effects: Adverse systemic effects were not observed. NOAEC >= 210 mg/m³ basic chromium(III) acetate monohydrate.
Executive summary:

The toxicity of basic chromium sulfate, was investigated in rats in a 13-week nose-only inhalation study that included a 13-week recovery period. Nose-only exposures to soluble basic chromium sulfate dust at 17, 54, or 168 mg/m³ (trivalent chromium equivalent concentrations of 3, 10, and 30 mg/m³) were carried out for 6 h/day, 5 days/week.

No compound-related mortality occurred.

General toxic effects, only observed with high-exposure levels of basic chromium sulfate, included sporadic signs of labored breathing and depressed body weights.

No apparent compound-related effects were noted for sperm motility or morphology, for any concentration of either test material. Bronchoalveolar lavage fluid evaluations showed increased neutrophils, protein, lactic dehydrogenase and cellular debris. The principle effects

were primarily to the respiratory tract.

Basic chromium sulfate produced severe and widespread effects in the nasal cavity, larynx, lungs, and mediastinal lymph node. Effects were characterized by accumulation of foreign material, infiltration of alveolar macrophages, septal cell hyperplasia, and granulomatous and chronic inflammation.

Pigment was still present in basic chromium sulfate-treated animals after the 13-week recovery period, with partial recovery of the pathological lesions.

A NOAEC for effects on the respiratory system was not established.

Adverse systemic effects were not observed. The observed organ weights effects can be considered secondary to the stress imposed on the respiratory tract and the resulting impairment of body weight gain. The highest test concentration is considered to represent the systemic NOAEC.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
21 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

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

Subchronic oral administration of the source substance Cr(III) picolinate to mice and rats (10 animals/sex/dose) in a study performed similar to OECD 408 showed no substance-related effects regarding body weight, food consumption, haematology, clinical chemistry, gross pathology, organ weight determinations and histopathology (Rhodes et al., 2005). No death occured. The only clinical sign observed was reddening of the faeces of the high dose animals which was due the the reddish colour of the test susbtance. The NOAELs in rats and mice were ≥ 2015 and ≥ 4342 mg/kg bw/day, respectively, when corrected for the molecular weight of basic Cr(III) acetate monohydrate. The lack of systemic effects even at very high dietary levels are reflective of the poor oral bioavailability of Cr(III).

Subchronic inhalation exposure of rats (10 animals/sex/dose) to the source substance soluble basic chromium sulphate dust at 17, 54, or 168 mg/m³ in a study performed similar to OECD 413 produced effects on the respiratory tract without indications for systemic effects (Derelanko et al. 1999). Basic chromium sulphate produced severe and widespread effects in the nasal cavity, larynx, lungs, and mediastinal lymph node. Effects were characterized by accumulation of foreign material, infiltration of alveolar macrophages, septal cell hyperplasia, and granulomatous and chronic inflammation. Pigment was still present in basic chromium sulphate-treated animals after the 13-week recovery period, with partial recovery of the pathological lesions. An NOAEC for effects on lung was not established; The LOAEC for basic chromium sulphate was 17 mg/m³. This is equivalent to 21 mg/m³ basic chromium(III) acetate monohydrate.

These effects can be considered as local effects on the respiratory tract. Substance-related systemic effects were not observed. The observed organ weights effects can be considered secondary to the stress imposed on the respiratory tract and the resulting impairment of body weight gain. Therefore, the systemic NOAEC is 210 mg/m³.

A subchronic dermal study is not deemed necessary, since the dermal absorption of Cr(III) salts can be predicted to be very low, judging from the very low (<1%) gastrointestinal absorption and the lack of acute percutaneous toxicity. Because of the skin-sensitizing properties of basic Cr acetate, skin contact must be avoided by wearing appropriate protective clothing. As a result, dermal exposure will be insignificant.

The oral NOAEL can be extrapolated to the dermal route, since dermal absorption is expected to be in the same order of magnitude as oral absorption (<1%).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The selected study is the most adequate and reliable study with the longest duration and the lowest dose descriptor.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
There is only one study available.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
There is only one study available.

Justification for classification or non-classification

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 is therefore conclusive but not sufficient for classification.