LNCMO SNUR Study summaries

SANITIZED

In the proposed SNUR, 75 Fed. Reg. 70665 (Nov. 18, 2010), the Agency determined that the results of 

the following tests would help characterize the human health and environmental effects of LNCMO: 90- 

day inhalation toxicity test (OPPTS Test Guideline 870.3465); Fish acute toxicity test, freshwater and 

marine (OPPTS Test Guideline 850.1075); Aquatic invertebrate acute toxicity test, freshwater daphnids 

(OPPTS Test Guideline 850.1010); and Algal toxicity test, tiers I and II (OPPTS Test Guideline 

850.5400). EPA approved the protocols for each test before testing began.

In communications with the Agency, proposed to collect additional 

data that may provide insight into the potential pulmonary and systemic toxicity of LNCMO. Pulmonary 

endpoints provide additional information on the effects to the respiratory tract beyond histopathology. 

The value of these tests is outlined in OECD Guideline 412, Subacute Inhalation Toxicity: 28-Day Study, 

and such approaches have been used to evaluate the pulmonary toxicity of many inhaled particles1,2. 

Urine from exposed animals was analyzed for metal ions as an indication of bioavailability. These data 

are presented in the 5-day Range-Finding Study. 

In a previous communication, submitted preliminary information on particle size in air 

evaluated the inhalation toxicity of the dry powder as it occurs in the 

workplace, as well as the milled material. The inhalation toxicity studies showed that while bioavailable, 

LNCMO does not elicit systemic toxicity. 

5-day Range Finding Inhalation Toxicity Study demonstrates expected inflammatory responses in 

lungs and bioavailability.

In the 5-day study male rats were exposed to 

dust concentrations of 2, 10, or 50 mg/m3 of LNCMO via a nose-only exposure system. The dust was 

generated using a rotating brush feeder of LNCMO that had been milled to a respirable particle size (3.2 – 

4.9 μm Mass Median Aerodynamic Diameter (MMAD)). Exposures were conducted for 6 hours per day 

for 5 consecutive days. Two days following the last exposure, half the number of animals in each group 

were euthanized, blood collected for serum chemistry, urine collected for metal analysis, and the lungs 

excised for bronchoalveolar lavage procedure. Major tissues were collected and preserved for 

histopathology. The remaining animals were euthanized after 21 days of recovery. 

The results indicate that a concentration of 50 mg/m3 was lethal to 16 of 22 animals after 2 exposure days. 

At that point, this group was terminated. Histopathologic findings included bronchial and nasal epithelial 

degeneration, alveolar damage, and squamous metaplasia of the nasal epithelium and larynx.

The remaining groups completed the exposure regimen. Histopathologic findings in the lower exposure 

groups included slight nasal and bronchial epithelial degeneration. In the 10 mg/m3 group, necrosis of the 

olfactory epithelium and peribronchial inflammation were also present. Hematology and clinical 

chemistry noted increased red blood cell counts, hemoglobin and hematocrit values, and increased 

alkaline phosphatase activity. Bronchoalveolar lavage endpoints indicated an acute inflammatory 

response as evidenced by the increase in neutrophils, and cell lysis and phagocytosis activity as evidenced

1 Klein CL, Wiench K, Wiemann M, Ma-Hock L, van Ravenzwaay B, Landsiedel R. 2012, Hazard identification of inhaled 

nanomaterials making use of short-term inhalation studies. Arch. Toxicology 86, 1137-1151. 2 Ma-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, van Ravenzwaay B, Landsiedel R. 2009a. Development of a shortterm inhalation test in the rat using nano-titanium dioxide as a model substance Inhal Toxicol 21, 102-118. 

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by increased albumin, and the activities of lactate dehydrogenase, alkaline phosphatase, γ-glutamyl 

transferase, and β-N-acetyl glucosaminidase. All findings were reversed during the 21 day recovery 

period.

Analysis of urine from the top 2 exposure groups for concentrations of lithium, cobalt, and nickel were 

detectable and exposure-related. These results indicate that LNCMO (or the dissociated ions) was 

systemically available. The level of manganese was below the limit of detection. Lung burden was 

calculated to be between 14 μg (based on Li) to 30 μg (based on Co or Ni) based on measure values of 

lithium, cobalt, nickel, and manganese in the lungs of the high exposure group. These concentrations were 

considered to be lower than what might be expected based on concentration and minute volume. The 

highest exposure concentration selected for the 90-day study was 5 mg/m3 based on these results.

90-day Inhalation Toxicity Study shows pulmonary inflammation but no systemic effects.

In a 90-day subchronic inhalation toxicity study

male and female rats were exposed to dust concentrations of 0, 0.2, 1.0, or 5.0 mg/m3 of 

the milled LNCMO (MMAD between 2.77 – 3.87 μm) for 6 hours per day, 5 days per week, for 13

weeks. Additional animals from the high exposure and control groups were allowed to recover for 4 

weeks following the last exposure. Animals were observed daily for clinical signs of toxicity; body

weight and feed consumption was measured weekly. After 13 weeks of exposure, animals were evaluated 

for neurobehavioral changes using a Functional Observational Battery; motor activity in an open area was 

also measured. The eyes were examined for ophthalmologic changes. Animals were euthanized, blood

collected for hematology and clinical chemistry, and all organs preserved for histopathology.

No mortality occurred during the 13 weeks, and no clinical signs of toxicity were noted. Mean body 

weight and feed consumption were comparable among groups, although body weight gain was lower for 

the 2 highest exposure groups. No ophthalmologic changes were observed, and no evidence of 

neurobehavioral effects was noted. No treatment-related changes were observed in clinical pathology. The 

lungs were the only target organ identified. Absolute and lung-to-body weights were significantly higher

in exposed groups; the increases in weight were concentration-dependent. Lesions in the lungs included 

alveolar histiocytosis, epithelial degeneration of the alveoli and bronchi, squamous metaplasia in the

bronchi of female rats, and cellular debris and signs of inflammation. The incidence and severity of

pulmonary lesions were concentration-dependent. A NOEC for pulmonary effects was not identified, but 

the NOEC for systemic effects was 5 mg/m3

.

These data from the inhalation toxicity studies demonstrate that, while bioavailable, LNCMO does not

elicit systemic toxicity. Any risk from occupational exposure can be mitigated via engineering controls or

PPE.

Studies of the toxicity to aqueous species show no effect at limit of solubility.

The water solubility of LNCMO is

In a previous communication with the Agency, 

indicated that it had determined the amount of Li, Ni, Co and Mn in the aqueous leachate of

LNCMO. These dissolution tests were carried out according to OECD Guideline 29 at pH 6 and at three

different loadings of 1, 10 or 100 mg/L following 1 week agitation. The measured concentrations of Ni 

after 1 week were 8 pg/L (at 1 ppm loading), 50 μg/L (at 10 ppm loading) and 220 μg/L (at 100 ppm 

loading). .

In an acute toxicity study in the fathead minnow fish

fathead minnow were exposed to a saturated solution of LNCMO at a nominal weight
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concentration of 100 mg/L. The solution was prepared in accordance with EPA Guidance

3

: approx. 2 g of 

test substance was placed on a cartridge filter and 20-L of test water was re-circulated through this filter 

for 7 days to generate the test solution. All test treatments were visibly clear over the entire exposure 

period. The concentration of ions was determined to be below the Level of Quantification (LOQ) or 0.5 

mg/L. After 96 hours, the survival of the fish was determined; no mortality was found.

In an acute toxicity study in the water flea

Daphnia magna were exposed to a saturated solution of LNCMO at a nominal weight concentration of

100 mg/L. The solution was prepared in accordance with EPA Guidance: the test solution was prepared 

by directly adding test substance to 2 L test medium. The test solution was stirred on a

magnetic stirring plate for about 7 days at test temperature. After this time, undissolved test substance was

removed by filtration with a membrane filter (pore width 0.2 μm). The filter was conditioned with test 

solution before collecting the final solution. The exposure was started after separation of the undissolved 

material. All test solutions were visibly clear over the exposure period. The concentration of ions was 

determined to be below the LOQ or 0.2 mg/L. After 48 hours, the survival of the Daphnia was 

determined; no mortality was found.

The ability of LNCMO to inhibit growth of Green algae was conducted

Pseudokirchneriella subcapitata were exposed to a saturated solution of 

LNCMO at a nominal weight concentration of 100 mg/L. The solution was prepared in accordance with 

EPA Guidance: test solutions were prepared by directly adding 200.39 mg of test substance to 2 L test 

medium. The test solution was stirred on a magnetic stir plate for about 7 days at room temperature. After 

this time, undissolved test substance was removed by filtration with a membrane filter (pore width 0.2 

μm). The filter was conditioned with several volumes of test solution before collecting the final solution. 

The control was treated in the same way. The exposure was started after separation of the undissolved 

material. All test solutions were visibly clear over the exposure period. The concentration of ions in 

solution was determined to be below the LOQ or 0.2 mg/L. After 96 hours, the growth rate and yield of 

green algae were determined; no effect was found.

3 U.S. Environmental Protection Agency Office of Prevention, Pesticides and Toxic Substances (1996), Ecological Effects Test

Guideline OPPTS 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies, (Public Draft), EPA 712- C-96-

113.