L NC MO S NU R Study s u mm ari es 

### SANITIZED

I n t he p r opo s ed S NU R, 75 Fed. Reg . 70665 (Nov . 18, 2010 ), t he A g ency de termi ned th at t he re su lt s o f

t he followi ng test s wou l d hel p c ha r ac teriz e th e hu m an hea lt h a nd en vir on m en t al e ff ects of LNCMO: 90-

day i nha lati on toxicity test (OPPTS Te st Guideli ne 870.3465 ); Fi sh a cu t e toxicity test

, fre shwa t er a nd

ma ri ne (OPPTS Te st Guide li n e 850. 1 075 ); A qu ati c invert eb rate ac ute toxicity test, fr e shwa t er d aph ni ds

(OPPTS Te st Guideli ne 850.1010 ); an d Alg al t ox icity test

, tier s I and II (OPPTS Te st Guide li ne

850.540 0)

. EPA ap prov ed t he p ro t oc ol s f or

e ach te st befor e tes ti ng be g an.

I n c omm un i ca ti on s w it h th e A g enc y

, pr opo s ed to co llect a dd i ti o nal

da ta that m ay provi de insig ht into t he potential pu lmonary and systemic t ox icity of LNC MO. Pulm ona r y endpo int s p rovid e ad ditio na l informati on on t he effects t o th e resp ir atory tr act bey ond h istopatholo gy

. T he val ue of t he se test s i s outli ned i n OECD Guideli ne 412, Sub a cu t e I nha lati on Toxi c it y: 28-D ay Stud y

, and su ch ap proa ches ha v e b een u s ed t o e valuate t he pu lm on ary t ox icity of m any i nha l ed particles 1,2

. U ri ne fr om e xpo s ed a nimal s was a nal yz ed f or

metal io ns as an i nd i ca ti on of bi oa vail ab ility

. These da ta

ar e pres en t ed i n th e 5- day R an ge- Find i ng Stud y.

I n a p revi ous comm un i ca ti on, sub m itt e d p relimi na ry informa ti on on pa rticl e si z e i n air

evaluat ed t he in ha lati on toxic it y of t he dry po w der as it oc curs in t he

workpl ace, as well as t he mill ed material. Th e inhala ti on toxic it y studie s s ho w ed that whil e bi oa vailabl e,

LNC MO does n ot elicit s ys temi c toxic it y.

5- day R an ge Findi ng In halati on Toxic it y Stud y d em on strat es ex pec t ed in fl a mm ator y r e spons e s i n

lun gs and bi oa vail ab ilit y.

I n t he 5- day st udy mal e rat s we r e exp osed t o

dust con c en tra ti on s of 2, 1 0 , or 50 mg/m 3 of LNC MO vi a a nos e- on ly expo sur e system

. T he du st w as

g ene rat ed u si ng a rota ti ng br ush f

ee d er of LNC MO that had b e en m ill e d to a respir ab le pa rticl e si ze ( 3.2

– 4.9 μm Ma ss M ed i an A erodynami c Dia met er (MMAD )) . Exp o su r es wer e c ond uct ed for 6 ho ur s p er d ay

f or

5 con s ec u ti v e da y s. Tw o da y s follo wi ng t he last ex posu r e, h alf t he nu m ber of an imal s i n e a ch gr oup

were euthaniz ed, bl ood coll ec te d for ser um che mis tr y, u ri n e c oll ec t ed f or

metal analysi s, and t he l un g s

exc ised f or

bron cho alv eolar lavag e p r oc e du re . M ajor ti ss ues were co ll ecte d and preserv ed f or

hist op atholo gy

. T he remaini ng an imal s we r e e ut ha ni ze d a ft er 21 day s of re co very.

T he re su lts indicate that a c onc e n tr ati on of 50 mg/m 3 w as lethal t o 16 of 22 an imals aft er 2 exp o su r e da y s.

At that point

, thi s gr oup wa s termi na ted

. Hist op athologi c finding s includ ed br onc hi al and nasal epit he lia l

de g ene ra ti o n, alveo lar da mag e, and sq uam ous met ap lasi a of t he nasal ep it heli um and lary nx.

T he remaini ng gr oups complet ed t he e xpos ure regim en. Hi st op athologi c finding s i n t he low er expo sur e

gr oups includ ed s li g ht nasal and bron ch i al ep itheli al degene rati on. I n t he 10 mg/m 3 gr oup, nec r os i s of th e

o lf actory ep ithe li um and p e ri br onc hial inflamma ti on wer e als o p resent

. Hematology and c li nic al

che mis tr y no t ed i nc r ea s ed r ed b lo od c ell cou nt s, he mogl ob i n and he mat oc ri t values , and increas ed

alkali ne ph o sph ata se activity. Br onchoa lveol ar lavag e endpo int s indicate d an ac ut e in fl ammatory

respo n se as evid enced by th e increa se i n n eutr op h il s

, a nd c ell lysi s a nd pha g oc ytosis ac tiv it y as evid enc ed

1 Kl e in CL, W ie nch K, W ie ma nn M, Ma-Hock L, va n R ave nz waay B, La nd si edel R. 20 12 , H aza rd i de ntif ic at io n of inha led

na no ma teri a ls making u se of s ho rt- term i nh a lati on st ud ie s. Ar ch. Tox ic ology 86, 1137-11 51

. 2 Ma-Hock L, Burkhardt S, S tra us s V, G ame r A O, W ie nch K, va n R ave nz w aa y B, La nd si edel R. 20 09 a. D eve l op ment of a s ho rtterm i nh a lati on te st in th e r at using nano-titanium dioxid e a s a m od el s ub st ance In ha l To xicol 21, 102-1 18

.This document is directed to USEPA pursuant the Toxic Substances Control Act (TSCA). This document is the property of the sponsor and may be used only for that purpose intended bythe sponsor and asauthorized under applicable law. Every other or additional use, exploitation, reproduction, publication or submission to other parties requires the written permission of the sponsor, except as otherwise required byapplicable law (e.g.,TSCA ). The use of these studies/documents for regulatory, registration or other commercial purposes [including but not limited to Registration, Evaluation, Authorization and Restriction ofChemical Substances (REACH))] by (i) private parties, or by (ii) authorities outside the US Government, without the express written permission of the sponsor is unauthorized and strictly prohibited.R eque s t t o A m end S NU R:

O c t ob e r 21, 2 013

Pa g e 2

by i nc r e a s ed a l b u m i n, and t he a c ti v i t i e s of l a c t a t e deh y d r o g enas e

, a l k a l i ne phos ph a t a s e, γ

- g l u t a m y l

tr a n s f e r a s e

, a nd β

- N

- ace t y l g l uco s a m i n i d a s e

. Al l fi n d i ng s were r e v e r s e d d u r i ng t he 21 day r eco v e r y

pe r i od.

A na l y s i s o f u ri n e f r om t he t op 2 exp o su r e g r oups f or c once n tr a t i ons o f li t h i u m , c o ba l t , and n i c k el w e r e

de t e c t a b l e and expo s u re

- r e l a t ed. The s e r e su l t s i n d i c a t e t h a t L N C MO ( o r t he d i s s oc i a t ed i on s ) w as

s y s t e m i ca l l y a v a il a b l e. T h e l e vel o f m an g anese w as b e l o w t he li m i t o f d e t e c t i on. L ung bu r den w a s

ca l c u l a t ed t o b e b e t w een 14 μ g ( ba s ed on L i ) t o 3 0 μ g ( ba s ed on C o or N i ) ba s ed o n m easu r e v a l u e s of

l i t h i u m , cob a lt

, n i c k e l , and m a n g anese i n t h e l u n g s of t he h i g h exp o su r e g r oup. T h e s e c once n t r a t

i ons w e r e

con s i de r ed t o be l o w e r t h an w hat m i g ht be ex p ec t ed b a s e d on con c en t r a ti on and m i nu t e v o l u m e. T he

h i g he s t expo s u r e con c en t r a t

i on s e l e c t ed f o r t he 9 0

- day s t udy w as 5 m g /

m 3 ba s ed o n t h e se r e s u l t s.

90

- day Inha l a t i on T o x i c i t y Study s ho w s pu l m on a ry i n fl a m m a ti on b u t no sy s t e m i c e ff e c t s.

I n a 9 0

- day sub c h r on i c i n ha l a

t i on t o x i c it y s t udy

m a l e and f e m a l e r a t s w e r e exp o s e d t o dust con c en t r a ti o n s o f 0, 0

. 2, 1.0, o r 5.0 m g / m 3 of

t he m il l ed L NC MO ( M M A D be t w een 2.77

– 3.87 μ m ) f or 6 hou r s p e r da y , 5 da y s per w e e k

, f or 13

w ee k s. Addi t i on a l a n i m a l s fr om t he h i g h expo s u r e and con t r ol g r o u ps w e r e a l l o w e d t o r eco v er f or 4

w ee k s f o l l o w i ng t h e l a s t e x posu r e. Ani m a l s w e r e o b s erv ed da il y f o r c li n i c a l s i g ns of t o x i c it y ; b ody

w e i g ht and f e e d co n su m p t i on w as m easu r ed wee k l y

. A ft e r 13 w ee k s o f ex p os u r e , an i m a l s w e r e e v a l ua t ed

f or n eu r o beha v i o r a l ch a n g es us i ng a Func t i o n al O b s e r v a ti on a l B a t t e r y ; m o t or a c t i v

it y i n an op e n a r ea w a s

a l so m eas u r ed. T h e e y es w e r e e xa m i ned f or o p h t h a l m o l o g i c c h a n g e s

. A n i m a l s w e r e e u t h an i z ed, b l ood

co l l e c t ed f or he m a t o l o g y and c l i n i c a l che m i s tr y , and a l l o r g ans p r e s e r v ed f o r h i s t opa t h o l o gy

.

N o m o rt a l i t y occu r r ed du r i ng t he 13 wee k s, a n d no c l i n i c a l s i g ns of t o x i c i t y w e r e no t ed. M e an body

w e i g ht and f e e d co n su m p t i o n w e r e co m pa r ab l e a m ong g r oups, a lt h o u g h body w e i g ht g a i n w as l o w e r f or

t he 2 h i g he s t exp o su r e g r o u ps. No op h t h a l m o l o g i c ch a n g es w e r e o b s er v ed, and no e v i dence of

neu r o b eha v i o r al e f f e c t s w a s no t ed. No t r e a t m en t

- r e l a t e d chan g es w e r e o b s er v ed i n c l i n i c al pa t ho l o gy

. T he

l un g s were t he on l y t a r g et o r g an i de n t i f

i ed. Ab s o l u t e an d l un g

- t o

- body w e i g h t s we r e si g n i fi c an t l y h i g her

i n ex p os e d g r o u ps; t h e i n c r eas e s i n w e i g ht w e r e c o nc e n tr a t i o n

- depende n t . Le s i o n s i n t he l un g s i n c l ud ed

a l v eo l a r h i s t i oc y t o s i s , e p it h e l i al de g ene r a t

i on o f t h e a l v eo l i and b r o n ch i

, squ a m ous m e t ap l a s i a i n t he

b r onc h i o f f e m a l e r a t s, and ce l l u l ar d eb r i s and s i g ns of i n f l a m m

a ti on. T h e i nc i den c e and s e v e r i t y of

pu l m ona r y l e s i ons w e r e c o ncen t r a ti o n

- dependen t

. A NO EC f or p u l m ona r y e ff e c t s w as not i d e n t i f i ed, b ut

t he N O EC f o r s y s t e m i c e f fe c t s w a s 5 m g /

m 3

.

T h e se d a t a f r om t he i n h a l a

t i on t ox i c i t y s t u d i es de m ons tr a t e t h a t

, w h i l e b i o a v a il a b l e, L NC MO does not

e l i c i t s y s t e m i c t ox i c i t y

. A ny ri sk fr om occup a ti o nal e x posu r e can be m iti g a t ed v i a en g i ne e ri ng co n t r o l s o r

PPE.

Stud i e s o f t he t o x i c it y t o a q u eous s p ec i es s ho w no ef f e c t at l i m i t o f s o l u bi l i t y.

T he w a t er s o l u b i li t y of L N C MO is

I n a p r e v i ous c o m m un i ca ti on w it h t h e A g enc y

, i nd i c a t ed t hat i t had de t e r m i ned t h e a m ount o f L i

, N i

, C o and M n i n t h e aq u eous l e a ch a t e o f

L N C MO. T h e s e d i s so l u t i on t e s t s w e r e c a r r i ed o u t a c c o r d i ng t o O E C D G u i de l i ne 2 9 at pH 6 and a t t h r ee

d i f f e r

ent l o ad i n g s o f 1, 10 o r 100 m g /

L fo ll o w i ng 1 w e ek a g it a t i on. T he m easu r ed conc e n t r a t

i ons of N i

a f

t er 1 w eek w e r e 8 p g / L ( a t 1 ppm l oa d i n g ) , 50 μ g / L ( at 10 p pm l oad i n g ) a n d 220 μ g / L (at 100 ppm

l oa d i n g )

. .

I n an acu t e t ox i c i t y s t udy i n t he f a t head m i nnow fi s h

f a t h ead m i nnow w e r e exp o s e d t o a s at u r a t ed so l u t i on of L N C MO at a no m i nal w e i g htR eque s t t o A m end S NU R:

O c t ob e r 21, 2 013

Pa g e 3

conce n t r a t

i on o f 100 m g /

L. T h e s o l u t i on w a s p r ep a r ed i n a c co r dan c e wi t h EP A G u i dan c e 3 : ap p r ox. 2 g of

t e s t su b s t ance w as p l a c ed o n a c a r t ri d g e fi lt e r a n d 2 0

- L of t e s t w a t er w a s r e

- c ir c u l a t ed t h r o u g h t h i s f i l t er

f or 7 da y s t o g en e r a t e t he t e st s o l u ti o n. Al l t e st t r e a t m en t s w e r e v i s i b l y c l ear o v er t he e n t i r e e x pos u r e

pe r i od. T he con c en t r a ti o n o f i o n s was d e t e r m i ned t o be be l ow t he L e v el of Q u an t i f i c a ti o n ( L O Q ) o r 0.5

m

g /

L. A ft er 9 6 ho u r s, t he s u r v i val of t he f i sh was d e t e r m i ned; no m o r t a l it y w as fo und.

I n an acu t e t ox i c i t y s t udy i n t he w a t e r f l ea

D aphn i a m a g na w e r e ex po s e d t o a s a t u r a t ed so l u t i on o f L N C MO at a no m i nal w e i g ht conce n t r a t i on o f

100 m g /

L. T h e s o l u t i on w a s p r e pa r ed i n a c co r d ance wi t h EP A G u i d anc e : t he t e s t s o l u t i on w a s p r ep a r ed

by d ir e c tl y ad d i ng t e s t su b s t ance t o 2 L t es t m ed i u m

. T he t e s t s o l u t i on w as s t i r r ed on a

m

a g ne ti c s t i r r i ng p l a t e f or a bout 7 da y s a t t e s t t e m pe r a t u r e

. A f t e r t h i s ti m e, un d i s s o l v ed t e st sub s t an c e w a s

r e m o v ed by filt r a t i on wi t h a m e m b r ane f i l t er ( p o r e w i d t h 0.2 μ m )

. T he f i l t er w a s cond i t i oned w it h t e st

so l u ti o n b e f o r e c oll e c t i ng t he f i n a l s o l u t i on. The e x po s u r e was s t a r t ed a f t er s e p a r a t i on o f t h e un d i s s o l v ed

m

a t e ri a l

. Al l t e s t s o l u t i ons w e r e v i s i b l y c l ear o v er t h e expo s u r e p e r i od. T h e co n c e n tr a t i on o f i o ns was

de t e r m i ned t o be b e l ow t he L O Q or 0.2 m g /

L. A ft er 48 hou r s, t he su r v i v al o f t he D aphn i a was

de t e r m i ned; n o m o rt a l it y w as f o und.

T he a b i l it y of L N C MO t o i nh i b i t g r o w t h of G r een a l ga e was c o ndu c t ed

P

s eudo k i r chn e r i e l l a s ubcap i t a t

a w e r e e x pos e d t o a s a t u r a t e d s o l u t i on o f

L N C MO at a n o m i nal w e i g ht co n ce n t r a t

i on o f 100 m g /

L. T h e s o l u t i on w a s p r ep a r ed i n acc o r d a nce w it h

E P A G u i dan c e: t e s t s o l u ti o ns were p r e pa r ed by d i r e c t l y add i ng 200.39 m g of t e s t sub s t an c e t o 2 L t e s t

m

ed i u m

. T he t e s t s o l u ti o n w as s t i r r ed on a m a g ne ti c s t i r p l a t e f or abo u t 7 da y s a t r oom t e m pe r a t u r e. A f t er

t h i s t i m e, und i s so l v ed te s t s ubs t a nce w a s r e m o v ed by fil t r a t i on w it h a m e m b r ane f i lt e r ( po r e wi d t h 0.2

μ m )

. T he f i l t e r w as c on d i ti oned wi t h s e v e r al v o l u m es of t e s t s o l u t i on b e f o r e c o l l e c t i ng t he f i n a l s o l u ti o n.

T he co n tr o l w as t r e a t ed i n t he s a m e wa y

. T he expo s u r e w as s t a r t ed a f t er s e p a r a t

i on of t h e un d i s so l v ed

m

a t e ri a l

. Al l t e s t s o l u t i ons w e r e v i s i b l y c l ear o v er t h e expo s u r e p e r i od. T h e co n c e n tr a t i on o f i o ns i n

so l u ti o n w as d e t e r m i ned t o be b e l ow t h e L O Q or 0.2 m g /

L. A ft er 96 ho u r s, t he g r o w t h r a t e and y i e l d of

g r een a l g ae w e r e d e t e r m i n e d; no e f f e c t w as f ound.

3 U. S

. E n v ir on m e n tal P r o te c t io n A g e nc y O f f ice o f P r eve n t i o n

, P e st i c i d e s a n d T o x ic S u b st a n ce s ( 1 9 9 6 ), Ec o l o g ic a l Ef f ec ts T e st

G u i d e l in e O P P T S 8 50

. 1 0 0 0

, S p ec ial C on s i d e r a t ion s f o r C o n du c t i n g A qu a t i c L a bo r a t o ry S t ud ie s

, ( P u b l i c D r a f t )

, E P A 7 1 2

- C

- 96

-

11 3

.