UNITED STATES ENVIR ONMENTA L PROTECTION AG ENCY WASHINGTON, D .C. 20 460



### MEMORANDUM

Da te: June 3, 201 5 

### OFFI CE OF CHEMICAL SAFETY AND POLLUTION PREVEN TI ON

SUBJE CT: 

### Fosamine Ammonium. Review of 

### 3 new DER.s and upda ting and writing executi ve swnm aries for three previousl y s ubmitted studi es.



### PC Codes: 106 701 

### Decision No.: 480859; 482631 

### Petition No.: NA 

### Risk 

### Assessment Type: NA TXR 

### No.: 0057080 

### MRID No.: See Table DP 

### Barcode: D4 23543; 0414644; D413146 

### Registration Nos.: NA Regulatory Action: Reviewing and updating D ER.s 

### Case No.: 2355 

### CAS No.: 25954-1 3-6 40 

### CFR: None



### FROM : Ayaad Assaad, D.V .M., Ph.D., Toxicologist

# ~~

THROUGH: Charles (B ill y) 

### Smith, Branch Chief(Acting v-=='i

# ~ RAB IV, HED (7509P) 

### t___J

TO : Russ ell Wasem/ Thoma s Moriarty RM 51 Risk Management and Implementation Branch 1 Pesticide Re-Eval ua tion Division (7508P)

I. 

### CONCLUSIONS 

### RAB 4 

### has reviewed the following toxico logy studies available for fosamine ammonium, and used in the hazard charac terization of the chemical have been reviewe d, and the Data Eva luation Records [DERs] and amende d DER.s [E xe cutive Summaries] are appended. The tox icol ogy database on fosamine ammonium i s considered complete

. Da ta are sufficient for se l ecting endpoints for a ll exposure scenarios and for FQPA evaluation. There is also a complete mutagenicity battery

. There are no data gaps at this time.



### II. ACTION REQUESTED Rev iew 3 new studies submitted by the registrant: I

. Immunoto xi city study in the mouse. 2. 4-week i nhalation toxic i ty st udy in rats

. 3. Prenatal toxicity study in rabbits.

Page I of2Page 2 of 2 

### Update and generate new executiv e summaries for existing DERs: 1. Subchronic neurotoxicity study in rats. 2. 90-dayoral toxicity study in rats. 3. Acute neurotoxicity study in rats.

III. RESULTS/DISCUSSION (or MRID Summary Table, etc.)

MRID Summary Table Example Study Type MRID Comments Acute neurotoxicity study in rats (update). 42946501 Acceptable/non-guideline

Subchronic neurotoxicity study in rats (update) 42946501 & 42942201 Acceptable/guideline

90-day oral toxicity study in rats (update). 00075736 Acceptable/guideline

Immunotoxicity study in the mouse (new study) 49149902 Acceptable/guideline

4-week inhalation toxicity study in rats (new study). 49196901 Acceptable/guideline

Prenatal toxicity study in rabbits (new study). 49121701 Acceptable/guidelineFosamine ammonium/I 0670 I

EPA Reviewer: Ayaad Assaad, D.V.M, Ph.D. RAB IV, Health Effects Division (7509P)

TXR#: 0057080 Acute Neurotoxicity Study (rats) (1993) I Page l of2 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424
Signature:

# /)i}j!~<J Date:

## ~4'1~ 

# l7

Template version 03/12

DATA EVALUATION RECORD- Supplemental See TXR 

### # 00 l 0711 for root DER

STUDY TYPE: Acute Neurotoxicity

- Rats OPPTS 870.6200a [§81-8]; OECD 424.

PC CODE: 106701 DP BARCODE:**O'f,13~** 

### i

TEST MATERIAL (PURITY): Fosamine Ammonium (97.7%)

SYNONYMS: DPX-Rl 108-100

CITATION:**Christop~** G. (1993). Acute Neurotoxicity Study ofDPX-Rl 108-100 (Fosarnine Ammonium) in Rats. E. 

### I. du Pont de Nemours & Company, Haskell Laboratory for Toxicology and Industrial Medicine, Newark Delaware. HLR 431- 93. 09/22/93. 

### MR.ID# 42946501. Unpublished

SPONSOR: E. 

### I. du Pont de Nemours & Company, Agricultural Products

, Wilmington,Delaware.

EXECUTIVE SUMMARY:



### In an acute neurotoxicity study (MRID # 4294650 l ), groups of male and female Crl:CD®BR rats, 63 days old, (12/sex/dose) were given a single oral

. dose of fosamine ammonium (97.7%, Batch# IN Rll08-100 (Rll08-100} in sterile water at doses ofO, 500, 1000 or 2000 mg/kg bw and observed for 17-19 days. Neurobehavioral assessment (functional observational battery and
motor activity testing) was perfonned in 12 animals/sex/group. Motor activity (MA) assessments and the FOB was conducted prior to dosing (baseline), approximately 2 to 3 hours after dosing

on tes t day 1 and again on days 8 and 15. Cholinesterase activity (plasma, erythrocyte, and brain ) was not determined in thls study but measlll'ed in a separate study (MRID # 42942201). At study tennination, 6 animals/sex/group were euthanized and perfused 

### in situ for neuropathological examination. Of the perfus ed animals, only tissues from control and high dose rats were processed for histopathological evaluation of brain and peripheral nervous system tissues.

There were no mortalities at any dose. Increase s in the incidence of diarrhea were observed in both sexes in the 1000 (12/24) and 2000 (24/24) mg/kg dose groups. However, the diarrhea observed 

### in these animals did not correlate with other signs of physical stress and the diarrhea did not impact other parameters such as clinical chemistry (i.e., sodium/potassium levels), or body weights, or food consumed. There were no differences in body weights or food consumption for either sex at any dose level. No stat istically significant differences were observed in either forelimb or hindlimb grip strength, hindlimb foot splay or any other FOB


### 1Acute Neurotoxicity Study (rats) (1993) / Page 2 of 2 Fosamine ammonium/ 106701 OPPTS 870.6200a/ DACO 4.5.12/ OECD 424



### 2 parameter were observed in either sex at a ny dose level when compared to controls. No differences were observed between the treated an d control groups in any of the motor activity parameters. No treatment-related e ffects were observed in either the gross or the microscopic examinations of the brain and peripheral nerves.

Based on the lack of adverse effects seen in this study, the LOAEL was not observed and the NOAEL is 2000 mg/kg bw/day, the limit dose.

This neurotoxicity study is classified as accepta ble/non-guideline, and satisfies the guideline requirement for an acute neurotoxicity study in rats (870.6200; OECD 424).

COMMENTS : This is a revised Executive Summary a nd it does alter the c onclusions of the previous review which considered diarrhea as an observed effect ( NOEL) but not a (NOAEL). Since the Agency had changed its definitions of adverse vs. non-adverse effect, diarrhea by definition is not considered as an adverse effect.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provideFosamine anunonium/106701 Subchronic Neurotoxicity Study (rats) 1993 /Page l of.l, OPPTS 870.6200b/DACO 4.5.13/0E CD 424



### EPA Reviewer: Ayaad Assaad, D.V .M., Ph.D. 

### RAB IV, Health Effects Division (7509P)

TXR#: 0057080

DAT A EVALUATION RECORD

- Supplemental See TXR 

### # 0010711 for root DER



### STUDY TYPE: Subchronic Neurotoxicity, OPPTS 870.6200b [§82-7] [feeding] in rats; OECD guideline: N/ A.



### PC CODE: 10670 I



### TEST MATERIAL (PURITY): Fosamine ammonium, 97.7%.



### SYNONYMS: Ammonium ethyl (aminocarbonyl) phosphonate



### CITATION:

Christoph, G. R. (1993). Subchronic Neurotoxicity Study ofDPX-Rl 108-100 (Fosamine Ammonium) 

### in Rats. 

### E. I. du Pont de Nemours 

### & Company, Haskell Laboratory for Toxicology and Industrial Medicine, Newark, Delaware. LHLR 438:-93

, 

### 09 1241 93. MRID: 42946502. Unpublished.

MacKenzie, S. A. (1993). Cholinesterase Inhibition Study with 

### DPXR1108-100 (Fosamine Ammoniwn) in Rats. 

### E. I. du Pont de Nemours 

### & Company, Haskell Laboratory for Toxicology and Industrial Medicine, Newark, Delaware. HLR 599-93; Medical Research No. 9618-001, LHLR 438:-93, 09/215 /93

. MRID: 42942201. Unpublished.



### SPONSOR: 

### E. I. du Pont de Nemours 

### & Company, Agricultural Products, Wilmington, Delaware



### EXECUTIVE SUMMARY:

In 

### a subchronic neurotoxicity study (MRID # 42946502), Fosamine Ammonium, 97.7%, (batch #IN Rll08-IOO (RII08-l00) was administered to 12 Crl:CD®BR rats/sex/group at dose leyels ofO, 5000

, 10,000 or 20,000 ppm in the diet for 96-98 days (calculated to be 299

, 604 or 1193 mg/kg/day for males and 398,779 or 1567 mg/kg/day for females). Neurobehavioral assessment (functional observational battery and motor activity testing) were performed in 12 animals/sex/group prior to dosing (baseline), and again during study weeks 4, 8 and 13. Cholinesterase activity was determined 

### in a separate study 

### (MRlD # 42942201). In this study, fosamine ammoni wn was tested in two groups of I 0 rat s administered 10 ml/kg test solution 

### by gavage for 5 consecutive days with 

### either 0 or 2000 mg/kg test material. Plasma 

### (PChE), erythrocyte (RBCChE), and brain cholinesterase activities were determined on a Boehringer Mannheim/Hitachi 717 clinical chemistry analyzer using Boeluinger Mannheim reagents at immediately after. At termination of animals at the original study,Subchronic Neurotoxicity Study (rats) 1993 / Page 2 of 2 Fosamine ammonium/106701 OPPT S 870.6200b/DACO 4.5.13/OECD 424



### (MRID # 

### 42946502)

, 

### six rats/sex/group were euthanized by pentobarbital anesthesia and prepared for whole-body, in situ perfusion wi th appropriate fixative. Only tissues from control and high dose rats were processed for neuropathological examination of brain and peripheral nervous system tissues.

There were no treatment related effects on morta lity, clinical signs, body we ight, brain weight or gross and histologic pathology or neuropathology. FOB and motor activit y testing revealed no treatment-related effects. In a ddition, there were no statisticall y significant decreases in either plasma, erythrocyte, hippocampus, cerebellum or neo-cortex cholinesterase activities when compared to the control group.

Based on the effects seen in this study, the NOAEL 20,000 ppm, the highest dose tested. The LOAEL was not observed

The NOAEL for plasma, cholinesterase, ery throcytes, and brain inhibition was 2000 mg/kg. The LOAEL was not observed.

These two studies study are classified as acceptable, guideline, and satisfy the guideline requirement for a subchronic neurot oxicity study in rats (870.6200b).

COMMENTS : This is a revised Executive Summary onl y and does alter the conclusions of the previous review. The end points in the previous review were based on diarrhea which is not considered as an adverse effect in our curr ent guidelines where NOA EL replaced the commonly used term NOEL 1990 th



### .

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provideFosamine Ammonium/106701 Subchronic (90-day ) Oral Toxicity Study { rodents) ( 1969 ) I Page 1 off OPPTS 870.3100 / DACO 4.3.1/ OECD 408



### EPA Reviewer: Ayaad Assaa<i, D.V.M., Ph.D. 

### RAB IV, Health Effects 

### Division (7509P) 

### Signature: 

# P&:Z..)7

Date:

. '

### ,,,..~ 

# M..cJ-

'TempJate version 03/12



### TXR#: 0057080



### DATA EVALUATION RECORD

- Supplemental See TXR 

### # 0010711 for root DER

STUDY 

### TYPE: 90-Day Oral Toxicity Feeding (OPPTS 870.3100 (§82 -la]; OECD 408) with One-Generation Reproduction Study (OPPTS 870.3800)

- Rats;



### PC CODE: 106701

TEST 

### MATERIAL (PURITY): Fosamine ammonium technical.

SYNONYMS: R 1108 Technical (purity not specified)



### CITATION: Holsing, G 

### & Busey, W. 1969. Three-Month Dietary Feeding with OneGeneration Reproduct ion Study

- Rats. Hazleton Laboratories, Falls Church, VA. Hazleton Project No. 201-234. November 14, 1969. MRID 

### # 00075736. Unpublished.

SPONSOR: E. I. duPont de Nemours and Company, In c.



### EXECUTIVE SUMMARY:

In a 90-day oral toxicity study (MRID 

### # 000757 36) Fosamine ammonium technical [(purity not spec ified); batch# R 1108-5, R 1108-5, INR-1108-3, and R 1108-9,)] was administered to 16/sex/dose Charles River CD albino rats in the diet for 13 weeks at dose levels ofO. 200, 1000, and 5000/10,000 ppm (equivalent to 0, 10, 50, 250/500 mg/kg bw/day). In this study, 10 animals/sex/group were assigned to the main study (90-day feeding study). Another 6 animals/group / sex were assigne d to the reproduction Phase (Phase II) of the study

. The highdose animals receive d 5,000 ppm from week 0 through week 8; from week 9 through week 32 (throughout the remainder of the feeding phase and throughout the reproduction phase), the dietary level was increased to 10,000 ppm.

Parameters evaluated for the three month feeding phase were survival

, clinical signs, body weight, food consumption. clinical pathology

, organ weights, and gross and microscopic pathology. There were no mortalities or clinical s igns in any group of either sex. However, individual animal data were not provided for clinical signs. There were sligh t , temporary d ecreases in body weight and food consumption (3-8%) in high-dose males at weeks 10, 11, and 12 as a result of the high-dose diets being increased from 5,000 to 10,000 ppm at week 9. Howev er, by week 13, the body weight and food consumption of the high-do se rats was comparable to control s indicating that the high -dose male rats had adapted to the I 0,000 ppm diets. There were no effects on body weight in the mid- and low-dose groups of both sexes. The

1Subchronic (90-day) Oral Toxicity Study (rodents) (1969) / Page 2 of 5 Fosamine Ammonium/106701 OPPTS 870.3100/ DACO 4.3.1/ OECD 408



### 2 temporary decreases in body weight and f ood consumption observed at the high-dose are considered non-adverse since only te mporary to the increase in dosage.

There were no treatment-related effects in any of the measured hematological or clinical chemistry parameters at 4, 8, and 13 weeks. All c ontrol and treated group i ndividual values were within the normal range of values for male and female rats. There were no treatment-related effects in urinalysis or organ weight findings in male or female rats at 4, 8, and 13 weeks.

Gross Pathology: Gross pathology of the kidney in the form of dilated kidney pelvis, kidney cyst, and/or enlarged kidney was observed in on e male and female of the control group. One female of the low dose group also had a dilated kidney pelvis.

Kidney Histopathology : The occurrence and severity of kidney histopathology findings are tabulated in Table 1. Kidney histopathology (in various forms) was observed in a non-linear pattern with occurrence in 2 cont rol males (none in control females), 0 in the low dose males or females, 5 mid-dose (3 males, 2 females), a nd 4 high-dose males (no females). The kidney histopathology also did not occu r consistently between sexes or in a dose-response pattern. Two control males were observed with slight focal regenerative epithelium of the kidney. Slight swelling of the deep convoluted tubules was noted in the mid-dose males (3/5) and high-dose males (4/5). Focal epithelial hyperplasia was also observed in 2 high-dose males. In all cases, the kidney histopathology were only minimal to s light in severity. Th ere were no treatmentrelated changes in kidney weights, clinical ch emistry or urinalysis to corroborate the kidney histopathology incidence. Therefore, based on: 1) the lack of severity of the kidney histopathology observed in a non-lin ear response across male groups; 2) the lack of response in kidney histopathology across the female dose-groups ; and 3) the lack of clinical chemistry, urinalysis, and kidney weight ch anges in either sex, the kidney histopathology is not considered adverse as these changes would not be expected to impact kidney function; 3) Two control males were observed with the same hist opathological lesions. Adaptive hype rplasia is the first form of hyperplasia to appear. It can be seen both in untreated controls and dosed animals. The distinguishing features are that in treated groups it does not progr ess with dose or time, and the process is reversible.Subchronic (90-day) Oral Toxicity Study (rodents) (1969) / Page 3 of 5 Fosamine Ammonium/106701 OPPTS 870.3100/ DACO 4.3.1/ OECD 408



### 3 TABLE 1: Incidence and Severity of Gross Pathology and Microscopic Histopathology of the Kidney of Fosamine Ammonium in Rats Males (n=5) Females (n=5) Dose in mg//kg/day 0 10 50 250/500 0 10 50 250/500 # of Animals with Gross Pathology Kidney 1 0 0 0 1 1 0 0

Dilated Kidney Pelvis 0 0 0 0 1 1 0 0 Kidney Cyst 1 0 0 0 0 0 0 0 Enlarged Kidney 1 0 0 0 0 0 0 0

Total # of animals with Kidney Histopathology 2 0 3 4 0 0 2 0

Minimal to Slight Interstitial Nephritis 1 0 2 1 0 0 0 0 Slight Swelling/Vacuolation of Deep Proximal Convoluted Tubules 0 0 3 4 0 0 2 0

Slight Focal Regenerative Epithelium 2 0 0 0 0 0 0 0 Focal Epithelial Hyperplasia 0 0 0 2 0 0 0 0

Epithelial hyperplasia Urinary Bladder 0 0 0 4 0 0 0 0 

### 90-day NOAEL is 250/500 mg/kg/day. The 90-d ay LOAEL was not established since the kidney histopathology is not considered adverse.

This 90-day oral toxicity study in the rats predates the curr ent OPPTS 870.3100 guideline and is acceptable/non-guideline; it satisfies the requirement for a 90-day oral toxicity study in rats.

In Phase II of the study (one-generation reproduc tion), six males and six females from each of the groups on the three-month feeding phase were us ed to establish groups of P1 animals. These animals were kept on their resp ective control and tr eatment diets. The reproductive indices evaluated were as follows: fertility index, number of pregnancies divided by the number of matings; gestation index, number of litters born divided by the number of pregnancies observed; live-birth index, number of pups born alive divide d by the number of pups born; lactation index, number of pups weaned divided by the number of pups nursed. From the summary table (Table 2) none of parameters examined for the F1A or F1b were significantl y impaired up to the 250/500 mg/kg/day dose. A second generation of repr oduction parameters is not available nor is the offspring data as in curre nt guideline reproduction studies. However, based on the summary information, there were no adverse treatment -related effects noted up to 10,000 ppm (250/500 mg/kg bw/day), the highest dose tested.Subchronic (90-day) Oral Toxicity Study (rodents) (1969) / Page 4 of 5 Fosamine Ammonium/106701 OPPTS 870.3100/ DACO 4.3.1/ OECD 408



### 4 Table 2. Summary of Breeding Data for male and female albino rats* P1 to F1A Parameter Controls 10 50 250/500 

### No. of Matings 6 6 6 6 No. of Pregnancies 6 4 4 6 Fertility Index 100% 66.7% 66.7% 100% No. Litter Born 6 4 4 6 Gestation Index 100% 100% 100% 100% No. Pups Born 56 45 50 61 No. of Pups Born Alive 56 45 50 61

Live Birth Index 100% 100% 100% 100% No of Pups Left to Nurse 42 32 32 47

No. of Pups Weaned 42 32 32 46 Lactation Index 100% 100% 100% 97.9% Mean Litter Size Born: No. Male Pups 4 4 5 5 No. of Female Pups 6 7 8 5 Mean Weight of Pups: Males at 24 hrs. (grams?) 7 8 7 8

Females at 24 hrs. (grams?) 7 8 7 7

Males at Weaning 55 55 55 57 Females at Weaning 52 56 54 54 P to F1B No. of Matings 6 6 6 6 No. of Pregnancies 6 5 5 6 Fertility Index 100% 83.3% 83.3% 100% No. Litter Born 6 5 5 6 Gestation Index 100% 100% 100% 100% No. Pups Born 81 62 52 77 No. of Pups Born Alive 81 62 43 77

Live Birth Index 100% 100% 82.7% 100% No of Pups Left to Nurse 48 35 34 48

No. of Pups Weaned 47 35 32 47 Lactation Index 97.9% 100% 94.1% 97.9% Mean Litter Size Born: No. Male Pups 6 7 5 7 No. of Female Pups 7 6 5 6 Mean Weight of Pups (g):Subchronic (90-day) Oral Toxicity Study (rodents) (1969) / Page 5 of 5 Fosamine Ammonium/106701 OPPTS 870.3100/ DACO 4.3.1/ OECD 408



### 5 Males at 24 hrs. 7 8 8 7 Females at 24 hrs. 7 7 7 7 Males at Weaning 60 57 61 56 Females at Weaning 56 56 56 54

Taken directly from page 50-51 of study report MRID 00075736.

Reproduction NOAEL is 250/500 mg/kg/day. Re production LOAEL > 250/500 mg/kg/day.

This 1-generation reproduction study in the rats predates the current OPPTS 870.3800 guidelines and is acceptable/non-guideline; it satisfies the requirement fo r a reproduction study in rats.

COMMENTS : This is a revised Executive Summary a nd it does alter the c onclusions of the previous review which was based on a NOEL of 200 ppm and LOEL of 1000 ppm. Based on the current review the 90-day NOAEL is 250/500 mg /kg/day with a LOAEL not established.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.DATA EVALUATION RECORD



### FOSAMINE AMMONIUM

STUDY TYPE: 4-WEEK INHALATION TOXICITY

- RAT (OCSPP 870.3465)



### Primary Reviewer: MRID 49196901


### Prepared for Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency One Potomac Yard 2777 South Crystal. 

### Drive

Arlington, VA 22202

Prepared 

### by Summitec Corporat i on 9724 Kingston Pike, Suite 602

Knoxville, Tennessee 37922

Task Order No. 6-77



### Thomas C .

. Marshall. Ph.D

.. DABT

Secondary Reviewers: H

. T. Borges. Ph

. D. MTCASCp). DABT



# . Robert Ross. M.S

.. Program**Man~ger** 

### Quality 

### Assurance; Angela Edmonds. B.S. Signature : 

# Al"e. 

# fAwvrvb

Date: \ll{?..(.1013

Disclaimer This review may han been altered subsequerit to the contractor's signllures above.

Summi1ec Corp. fottbe U

. S

. Environmental Protection Agency underConlnlet No.EP·W-11·014FOSAMINE AMMONIUM / 1 0670 1



### TXR#:0057080 4- Week Inhala tion Toxicity Study (rat) (2013) I Pag e 2 of 13 OCS PP 870.3465; OECD 412



### DATA EVALUATION RECORD

STUDY TYPE: 28-Day Inhalation Toxicity

- Rat O CSP P 870.3465.



### PC CODE: 106701 

### DP BARCODE: 04 1 4644



### TEST MATERIAL (PURITY): Fo samine Ammonium (95.3% a.i.)



### SYNONYMS: Ammonium ethyl carbamoylpho s phonate



### CITATION: Randazzo, J.M. (2013). A 4-week nose-only inhalation toxicity study of Fosamine Ammonium in Sprague-Dawley rats. WIL Reasearch, Ashland, 

### OH. Study ID: WIL-925003, August 7, 2013. MRID 49196901. Unpubli shed.



### SPONSOR: Alb augh, Inc

.



### EXECUTIVE SUMMARY:

In a nose-only inhalation toxicity study (MRID 49196901) Fosamine Ammonium (Batch No. 20120815; 95.3% purity) was administered by inhalation as an aerosol to male and female Sprague-Dawley rats (10/sex/concentration) for 6 hours/day, 

### 5 days/week for 4 weeks at

ex posure concentration s ofO, 0.1, 0.3, or 1 mg/L (actual exposure conc e ntrations ofO, 0.10, 0.30, or 1.0 mg/L)

. The MMADs (mass median aerodynamic diameters ) w ere 2.5

, 2.7, and 2.8 μm for the low-

, m id- and h i gh concentrations, respectively.

All animals survived until scheduled sacrifice. Yellow and/or brown material was observed around the anogenital ar e a and limbs in males and females in the high e xpos ure groups and in females in the mid-exposure group. No statistically significant effects w ere observed in body weight, body weight gain, or food consumption. There were no treatment-r e lated effects on hematology or clinical chemistry parameters at any exposure concentration. Urine specific gravity was statis tically higher in females at the high exposure concentra t ion (p <0. 05). No lesion s were observed during the ophthalmoscopic examinations. Abso l ute (p<0.05) and relative (p<0.01) thymu s weights were statistically higher in males at the high exposure concentration, and relative thymus weight was higher (p<0.05) at the low exposure concentration. The differences in thymu s weights were stated to be within the historical control range (data not provided). A higher incid e nce of dark red areas 

### in the thymus was observed in males (1110 controls; 0/10 at 0.1 mg/L; 4/10 at 0.3 mg/L; 2/10 at 1.0 mg/L) and females (0/10 controls; 0/10
at 0.1 mg/L; 1110 at 0.3 mg/L; 2/10 at 1.0 mg/L). Histologically, this finding correlated 

### withPrenatal Developmental Toxicity Study (Rabbit) (2013) / Page 3 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414


### Absolute body weight was not affected by treatmen t. Body weight gains were slightly lower in the 700 mg/kg bw/day group during GD 7-10 and GD 13-20 (32% and 35% lower, respectively; n.s.) and in the 300 mg/kg bw/d ay group during GD 13-20 (24% lowe r; n.s.). When evaluated over the entire treatment period (GD 7-29), mean body weight gains were slightly lower for the 300 and 700 mg/kg bw/day groups (18% and 16% lo wer, respectively; n. s.). Effects on food consumption corresponded with the effects on body weight gain and achieved statistical significance (p<0.05) on select days: at 700 mg/kg bw/day, GD 7-9 and GD 17-20; at 300 mg/kg bw/day, GD 17-20. The lower body weight gain and food consumption of these groups were considered test substance-related but non-adve rse. No test substance-related maternal macroscopic findings were noted at necropsy.

The maternal lowest-observed-adverse-effect level (LOAEL) for fosamine ammonium in rabbits was not determined. The maternal NO AEL was greater than or equal to 700 mg/kg bw/day.

There were no treatment-related effects on any developmental parameters, including cesarean section parameters and external, visc eral and skeletal development.

The developmental LOAEL for fosamine ammo nium in rabbits was not determined, and the developmental NOAEL was greater th an or equal to 700 mg/kg bw/day.

This developmental toxicity study in the rabbit is classified as Acceptable/Guideline and satisfies the guideline requirement for a de velopmental toxicity study (OSCPP 870.3700; OECD 414) in rabbits.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and No Claim of Data Confidentiality statements were provided.Prenatal Developmental Toxicity Study (Rabbit) (2013) / Page 4 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414



### I. MATERIALS AND METHODS:

A. MATERIALS:

1. Test material: Fosamine ammonium Description: Off-white, coarse powder
Batch #: 20120805 Purity: 95.3% a.i.
Compound stability: Stable at room temperature, with desiccation

CAS #of TGAI: 25954-13-6

Structure:



### 2. Vehicle: Deionized water, prepared on-site (Lot/Batch #N/A; Purity N/A).

3. Test animals: Species: Rabbits (females)

Strain: New Zealand White [Hra:(NZW)SPF] Age/weight at study initiation: Approximately 6 months old upon receipt; 2902 g to 3833 g on GD 0 ; time mated at supplier, received at testin g laboratory on GD 1, 2 or 3 Source: Covance Research Products, Inc. (Greenfield, IN) Housing: Individually in clean, suspended, stainl ess-steel cages; corncob bedding (PelO'Cobs ® ; The Andersons, Cob Products Division, Maumee, OH). Bedding was changed at least twice each week. Nesting material was not required

. Diet: PMI Nutrition International, LLC Certified Rabbit LabDiet ® 5322; Basal diet was offered in 25-g increments 3 times/day on the day of arrival and in increased amounts until the animal s gradually achieved ad libitum status prior to the dose administration; basal diet was offered ad libitum thereafter. Feeders were changed and sanitized weekly. One leaf of kale was provided to each animal 3 times/week for environmental enrichment and to aid in maintaining gastrointestinal health. Water: Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 18.4 to 19.3°C 50.6 to 57.9% 10/hr 12 hrs dark/ 12 hrs light Acclimation period: 4 to 6 days; Study exposure initiated GD 7



### B. PROCEDURES AND STUDY DESIGN

1. In life dates: Start: January 22, 2013 (GD 7, star t of dosing); End: February 15, 2013

2. Mating: The day of mating was designated as GD 0. Rabbits were received time-mated on GD 1, 2, or 3 from Covance Research Products, Inc. The mating technique (natural or artificial insemination), confir mation of mating, and verification of males as the same strain were not documented.Prenatal Developmental Toxicity Study (Rabbit) (2013) / Page 5 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414


### 3. Animal Assignment: Each animal judged to be in good health and meeting acceptable GD 0 body weight requirements was selected for use in the computerized randomization procedure based on body weight stratification in a bl ock design and assigned to dose groups as indicated in Table 1. Animals not as signed to the study were euthanized.

TABLE 1. Animal Assignment

Dose (mg/kg bw/day) 0 100 300 700 Number of Females 25 25 25 25 Data taken from page 14, MRID 449149902.



### 4. Dose selection rationale: The dosage levels were selected based on results from a previous dose range-finding prenatal developmental toxic ity study of fosamine ammonium in rabbits (WIL-925001). In that study, 6 time-mated ra bbits/group were dosed at 0, 100, 300, 700, or 1000 mg/kg bw/day from GD 7 through GD 28. In the 1000 mg/kg bw/day group, there was excessive maternal toxicity, including mean body weight loss, decreased body weight gain, and decreased food consumption during the dosi ng period. In addition, 1 female aborted and 2 females were euthanized in extremis due to persistent lack of appetite. Based on these results, 700 mg/kg bw/day was chosen as the high dosage level for the current study. The selected route of administration for this study wa s oral (gavage) because this is a potential route of exposure for humans. Historically, th is route has been used extensively for studies of this nature.

5. Dosage preparation and analysis: Test material-vehicle mixture was prepared approximately weekly by mixing appropriate am ounts of test substance with deionized water with storage at room temperature. Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 10, 30, and 70 mg/mL formulations. Following ro om temperature storage for 10 days and remixing for a minimum of 30 minutes, sample s were collected from the top and bottom strata of an aliquot of each fo rmulation for stability analyses . Concentration of the test mixture was evaluated weekly from the middle strata.

Results: Homogeneity analysis: The homogeneity of the 10 mg/mL, 30 mg/mL, and 70 mg/mL dosing formulations was assessed using high perfor mance liquid chromatography (HPLC). Mean measured concentrations were 9.98 mg/m L (99.8% of target, 0.41% RSD), 30.0 mg/mL (100% of target, 0.14% RSD), and 70.6 mg/mL ( 101% of target, 0.24% RSD), respectively. Test substance was not detected in the vehicle control formulation.

Stability analysis: Mean observed concentrations followi ng 10 days at room temperature ranged from 103% to 104% of target values. Concen trations of dosing formulations were 10.3 mg/mL (103% of target), 30.9 mg/mL (103% of target), and 73.0 (98.9% of mg/mL target) for the 10, 30, and 70 mg/mL fo rmulations, re spectively.

Concentration analysis: On two separate dates of preparat ion, the mean concentrations of the 10, 30, and 70 mg/mL dosing formulations ra nged from 10.1-10.4 mg/mL (101-104% of target), 30.0-31.2 mg/mL (99.9-104% of target), and 69.7-72.8 mg/mL (99.5-104% of target), respectively.Prenatal Developmental Toxicity Study (Rabbit) (2013) / Page 6 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414


### The analytical data indicated that the mixing procedure was ad equate and that the variance between nominal and actual dosage to the animals was acceptable.

6. Dosage administration: All doses were administered once daily by oral gavage, on GD 7 through 28, in a volume of 10 mL/kg of body weight. Dosing was based on the most recent body weight determination.

C. OBSERVATIONS:

1. Maternal observations and evaluations: The animals were checked for mortality or moribundity twice daily. Individu al clinical observations were recorded daily from the day of receipt through gestation da y 29; this was done prior to dose administration during the treatment period. Animals were also observed for signs of toxicity approximately 2 hours following dose administration. Body weight da ta were recorded on GD 0 (by supplier under non-GLP conditions), GD 4, and daily on GD 7-29. Food consumption data were recorded daily on GD 4-29. Dams were sacrificed on GD 29 by an intravenous injection of sodium pentobarbital. The examinations were performe d blind to treatment group. Examinations at sacrifice consisted of the following: the t horacic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the co ntents, including placenta, were examined. The uterus and ovaries were exposed and exci sed and the number of corpora lutea on each ovary was recorded. The trimmed uterus wa s weighed, and the number and location of all fetuses, early and late resorptions, and the tota l number of implantation sites were recorded. Uteri with no macroscopic evidence of impl antation were opened and placed in 10% ammonium sulfide solution for de tection of early implantation loss. Maternal tissues were preserved in 10% neutral-buffere d formalin for possible future histopathologic examination as indicated by the gross findings. Representa tive sections of corresponding organs from a sufficient number of control animals were re tained for comparison. The carcass of each female was then discarded.

2. Fetal evaluations: Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individu ally weighed, euthanized via a subcutaneous injection of sodium pentobarbital, and tagged for identification. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices. Crown-rump measurements, degrees of autolysis and gross examinations were recorded for late resorptions, if possible. Each viable fetus was subjected to a visceral examination to include the heart and major blood vessels. The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development. Heads from all fetuse s were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% et hyl alcohol. Following fixation, each fetus was macerated in potassium hydroxide and staine d with Alizarin Red S and Alcian Blue, and then examined for skeletal malformations and developmental variations . External, visceral, and skeletal findings were record ed as developmental variations or malformations. The fetal developmental findings were summarized by: 1) presenting the incide nce of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis.Prenatal Developmental Toxicity Study (Rabbit) (2013) / Page 7 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414



### D. DATA ANALYSIS:

1. Statistical analyses: Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of1% and 5%, comparing each test substancetreated group to the control group. Data obt ained from non-gravid animals were excluded from statistical analyses. Where applicable, th e litter was used as the experimental unit. Maternal body weights, body weight changes, an d food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA. If the ANOVA revealed significant (p<0.05) intergro up variance, Dunnett’s t- test was used to compare the test substance-treated groups to the control group. M ean litter proportions (percent per litter) of prenatal data (viable and nonviable fetu ses, early and la te resorptions, total resorptions, pre- and post-implantation lo ss, and fetal sex distribution), total fetal malformations and developmental variations (ext ernal, visceral, skelet al, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA te st. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was us ed to compare the test substance-treated groups to the control group.

The Reviewer considers the analys es used to be appropriate.

2. Indices: The following indices were calculated from cesarean section records of animals in the study:

Maternal Indices: Group Mean Litter Basis: Postimplantation Loss/Litter = No. Dead Fetuses, Resorptions (Early/Late)/Group No. Gravid Females/Group

Proportional Litter Basis: Summation per Group (%) = Sum of Postimplantation Loss/Litter (%) No. Litters/Group

Where: No. Dead Fetuses, Postimplantation Loss/Litter (%) = Resorp tions (Early/Late)/Lit ter x 100 No. Implantation Sites/Litter

Fetal Indices: Summation per Group (%) = Sum of Viable Fetu ses Affected/Litter (%) No. Litters/Group

Where: Viable Fetuses Affected/Litter (%) = No. Viable Fetuses A ffected/Litter x 100 No. Viable Fetuses/Litter

3. Historical control data: Historical control data were pr ovided to allow comparison with concurrent controls. Maternal and fetal data (e.g., number of viable fetuses/dam, pre- andPrenatal Developmental Toxicity Study (Rabbit) (2013) / Page 8 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414


### post-implantation loss, fetal body weights, extern al, visceral and skeletal malformations and variations) were obtained from female New Zealand White rabbits and their litters from 97 to 99 datasets conducted at WIL Research from 2006 to 2012.

II. RESULTS:

A. MATERNAL TOXICITY:

1. Mortality and clinical observations: In the 700 mg/kg bw/day group, 1 female was euthanized in extremis , 1 female was found dead after a borting, and 1 female aborted and was euthanized. The female euthanized in extremis on GD 10 displayed extreme anxiety and was not able to be dosed. Clinical observat ions of lying on side, body stiffened, increased respiration, and tonic convulsions were noted. Because this animal received only 2 doses, there were no notable macroscopic findings, an d similar clinical findi ngs were not observed for other females in the same dose group, morb idity was not considered test substancerelated. The female that was found dead on GD 27 after aborting 8 dead fetuses had clinical findings of brown material on th e anogenital area, decreased de fecation, soft stool, and small feces prior to abortion/death. The female th at aborted 7 late re sorptions on GD 24 had clinical findings of hypoactiv ity, a pale and cool body, decrea sed respiration, half-closed eyelids, brown material on the anogenital area , decreased defecation, soft stool, and small feces on the day of or prior to abortion. A ll three of these females consumed minimal amounts of feed and lost body weight prior to or during signs of morbidity.

In the 300 mg/kg bw/day group, one pregnant female was found dead on GD 29. Clinical findings of decreased defecation and soft stool were noted for this female prior to death. This female also lost weight and consumed mi nimal amounts of feed during the week prior to death. Similar severe body weight losses and reduced food consumption were not noted for any other animals in this group.

All other females in the cont rol, 100, 300, and 700 mg/kg bw/d ay groups survived to the scheduled necropsy on GD 29. Although not statistic ally significant, test substance-related clinical findings incl uded decreased defeca tion in the 300 and 700 mg /kg bw/day groups and soft stool and brown material on the anogenital area in the 700 mg/kg bw/day group. These findings were noted at the daily examinations, generally throughout the treatment period, and the findings of decreased defecation corresponde d to reduced mean food consumption noted in these groups. Other findings noted in the test substance-tr eated groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. No notable clinical findings were noted at any dosage level during the observations conduc ted approximately 2 hours post-dosing.Prenatal Developmental Toxicity Study (Rabbit) (2013) / Page 9 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414

TABLE 2. Maternal mortality and clinical observations a

Observation (Total occurrence/ # of animals) b Dose in mg/kg bw/day (# of Dams) Control (23) 100 (25) 300 (22-23) c 700 (20-23) c

Unscheduled death/sacrifice 0 0 1 3 Found dead 0 0 1 0 Found dead, aborted 0 0 0 1 Euthanized, aborted 0 0 0 1 Euthanized, in extremis 0 0 0 1 Decreased defecation 16/8 19/4 30/10 43/14 Soft stool 11/6 8/6 17/9 48/15 Feces small 14/7 8/5 18/6 8/5
Diarrhea 0/0 0/0 0/0 3/2 Wet brown material anogenital area 1/1 1/1 0/0 10/6 Dried brown material anogenital area 7/1 1/1 5/3 33/8 Hair loss, right forelimb 7/1 27/2 0/0 0/0 Hair loss, left forelimb 7/1 22/3 0/0 0/0

a Data obtained from MRID 49149902, pages 48-50. b Observations (excluding unscheduled deaths) are included that occurred 3 or more times and affected at least 2 animals in a
group.

c Three females in the 700 mg/kg bw/day group had unscheduled deaths at GD 10, GD 24, and GD 27; thus the number of does was 20-23 during the study. One female in the 300 mg/kg bw/day group was found dead on GD 29; thus the number of does was 22-23.



### 2. Body weight: Selected body weight da ta are given in Table 3. At 700 mg/kg bw/day, test substance-related, slightly lower mean body weight gains were noted during GD 7-10 and 13-20 (32-35% lower than control). At 300 mg/ kg bw/day, slightly lower, body weight gains were noted during GD 13-20 (24% lower than control). These decreases resulted in slightly lower mean body weight gains over the entire treatment period (GD 7-29) for both the 300 and 700 mg/kg bw/day groups, approximately 18% and 16% lower than control, respectively. Although test substance-related, the slight e ffects on mean body weight gains during GD 7- 10 and/or 13-20 were not stat istically significant and did not affect mean absolute body weights. Statistically signi ficant (p<0.01 or p<0.05) differences in mean body weight gain were sporadically noted on i ndividual gestation days thr oughout the treatment period (e.g., GD 11-12, 13-14, 16-17, and 21-22); however, thes e results were generally transient, discordant in direction of change, and/or di d not occur in a dose-re lated manner. Mean maternal body weight gains were unaffected by test substance administration at 100 mg/kg bw/day.

For all groups, mean absolute body weights throughout gestation, m ean adjusted (net) terminal body weights, net body weight changes, and gravid uterine weig hts were similar to the control group.Prenatal Developmental Toxicity Study (Rabbit) (2013) / Page 10 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414

TABLE 3. Mean (±SD) maternal body weight changes (g) a

Interval Dose in mg/kg bw/day (# of Dams) Control (23) 100 (25) 300 (22-23) b 700 (20-23) b
Pretreatment: Days 0-4 10±63 3±47 18±56 12±83 Pretreatment: Days 4-7 92±92 80±74 80±57 95±76 Treatment: Days 7-10 41±67 45±51 40±30 28±66 Treatment: Days 10-13 56±46 57±40 39±34 61±50 Treatment: Days 13-20 130±63 119±78 99±60 84±93 Treatment: Days 20-29 112±80 147±72 109±69 92±173 Treatment: Days 7-29 340±165 368±115 280±89 287±169 Gestation: Days 0-29 c 443±170 451±145 374±122 399±197
Gravid uterus weight 491±108 537±93 486±103 493±98 Corrected BW gain: Days 0-29 -48±146 -86±142 -112±157 -94±185

a Data obtained from MRID 49149902, pages 57-63, rounde d to the nearest whole number by the Reviewer. b Three females in the 700 mg/kg bw/day group had unschedul ed deaths/sacrifices at GD 10, GD 24, and GD 27; thus the number of does was 20-23 during the study. One female in the 300 mg/kg bw/day group was found dead on GD 29; thus the number of does was 22-23.

c BW gain ± S.D. from G 0-29 was calculated by the Reviewer using individual animal data. The study reported mean initial and terminal body weights ± S.D., but not the mean BW gain ± S.D. No statistically significant differences from the control for BW gain during these time intervals.



### 3. Food consumption: Food consumption data are summari zed in Table 4 and as follows. Although not statistically significant, at 700 mg/kg bw/d ay, maternal food consumption (g/animal/day or g/kg/day) was slightly lowe r than control during GD 7-10 (~15% lower). These results corresponded to slightly lowe r mean body weight gain (32% lower) during GD 7-10. Differences in g/kg/day food consump tion were significant during GD 7-9 (15-20% lower, p<0.01 or p<0.05).

At 700 mg/kg bw/day and 300 mg/kg bw/day, sligh tly lower, not statis tically significant, mean food consumption was noted during GD 13-20, corresponding to reduced mean body weight gains during GD 13-20. Differences were significa nt (<0.01 or p<0.05) during GD 17-20 for both the 700 and 300 mg/kg bw/day gr oups, 20% and 18% lower, respectively. Significantly lower (19%, p<0.05) mean food c onsumption was noted in these groups during GD 22-24; however, these effects were transien t and/or did not occu r in a dose-related manner. Mean food consumption was similar to the control group th roughout the remainder of the treatment period (GD 20-29) and when the entire treatment period (GD 7-29) was evaluated.

The effects noted on mean food consumpti on in the 700 mg/kg bw/day group during GD 7- 10 and in the 300 and 700 mg/kg bw/day group s during GD 13-20 were considered test substance-related; however, the effects we re slight and the co rresponding body weight changes did not affect mean body weights. Maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 100 mg/kg bw/day group was unaffected by test substance administration.Prenatal Developmental Toxicity Study (Rabbit) (2013) / Page 11 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414

TABLE 4. Mean (±SD) food consumption (g/kg/day) a

Interval Dose in mg/kg bw/day (# of Dams) Control (23) 100 (25) 300 (22-23) b 700 (20-23) b
Treatment: Days 4-7 43±8.9 43±10.1 43±7.3 45±6.9 Treatment: Days 7-10 50±11.7 46±8.1 45±5.9 43±8.7 Treatment: Days 10-13 40±8.7 38±7.8 37±6.4 38±7.2 Treatment: Days 13-20 40±7.1 39±8.7 36±8.2 35±11.1 Treatment: Days 20-29 32±4.7 30±5.1 29±5.1 30±6.5 Treatment: Days 7-29 38±5.1 36±5.7 35±5.2 35±5.7 Day 7-8 52±9.7 48±11.7 46±8.5 44±9.1* (-15) c
Day 8-9 50±15.5 44±7.9 44±5.8 40±12.6** (-20) Day 17-18 46±6.0 42±10.2 38±9.4* (-17) 36±13.0** (-22) Day 18-19 47±5.5 41±9.7 39±7.9* (-17) 38±11.3** (-19) Day 19-20 46±5.7 40±12.7 37±7.6* (-20) 37±11.8* (-20) Day 20-21 42±7.0 37±9.6 37±7.7 36±12.2 Day 21-22 41±6.1 36±9.2 35±11.4 33±16.0 Day 22-23 37±6.4 33±7.9 31±7.9 30±12.4* (-19) Day 23-24 36±7.2 32±8.3 29±8.7* (-19) 31±11.1

a Data obtained from MRID 49149902, pages 70-75. b Three females in the 700 mg/kg bw/day group had unscheduled deaths at GD 10, GD 24, and GD 27; thus the number of does was 20-23 during the study. One female in the 300 mg/kg bw/day group was found dead on GD 29; thus the number of does was 22-23.

c Numbers in parentheses are percent change from control, calculated by the Reviewer. * Statistically different (p <0.05) from the control. ** Statistically different (p <0.01) from the control.



### 4. Gross pathology: In the 700 mg/kg bw/day group, 3 fe males were euthanized prior to scheduled necropsy. The female that was euthanized in extremis on GD 10 had cystic oviducts and a distended, gas filled ileum at necropsy. Because this animal received only 2 doses and there were no notable macroscopic fi ndings, morbidity was not considered test substance-related. No significant macroscopic findings were noted for the female that was found dead on GD 27 after aborting. Macroscopi c findings for the female that aborted on GD 24 were limited to red matting of the skin on the urogenital area. The mortality and abortion noted in this group were not considered treatment related. In the 300 mg/kg bw/day group, the 1 female found dead on GD 29 had no significant macroscopic findings, thus, the mortality of this female was not attributed to test substance administration. No fetal malformations were noted in any fetus from the females in the 700 or 300 mg/kg bw/day groups that aborted or that we re euthanized prior to schedu led necropsy. All other females survived to the scheduled necropsy on GD 29, an d no test substance-related internal findings were observed in the surviving animals at a ny dosage levels up to 700 mg/kg bw/day. Macroscopic findings, such as cystic oviduc ts and accessory spleen, and other single incidence findings observed in th e test groups occurred infrequen tly, at similar frequencies in the control group, and/or in a manner that wa s not dose-related. Th erefore, no treatment related effects on gross pathology were noted.

5. Cesarean section data: Cesarean section data are summar ized in Table 5 and as follows. Intrauterine growth and surviv al were unaffected by test subs tance administration at dosagePrenatal Developmental Toxicity Study (Rabbit) (2013) / Page 12 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414


### levels of 100, 300, and 700 mg/kg bw/day. Para meters evaluated included postimplantation loss, live litter size, mean fetal body weights, an d fetal sex ratios. M ean numbers of corpora lutea and implantation sites a nd the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the c ontrol group were slight and not statistically significant, with the following exceptions. Mean fetal weights (combined or male only) in the 300 mg/kg bw/day group were significantly lower (9%, p<0.05, or 12%, p<0.01, respectively) than the concurrent control group; however, these results did not occur in a dose-related manner and the values were within the ranges of values in the WIL historical control data.Prenatal Developmental Toxicity Study (Rabbit) (2013) / Page 13 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414

TABLE 5. Cesarean section observations a

Observation Dose (mg/kg bw/day) 0 100 300 700 No. Animals assigned (mated) 25 25 25 25 No. Animals pregnant 23 25 23 23 Pregnancy rate (%) 92 100 92 92 No. Nonpregnant 2 0 2 2 Maternal wastage 0 0 1 3 No. died pregnant 0 0 1 0 No. sacrificed in extremis pregnant 0 0 0 1
No. aborted 0 0 0 2 b
Total No. corpora lutea Corpora lutea/Dam 233 10.1±1.98 267 10.7±2.01 233 10.6±2.28 200 10.0±1.72 Total No. implantations (Implantations/Dam) 205 8.9±1.88 243 9.7±2.19 215 9.8±2.62 190 9.5±1.64 Total No. litters 23 25 22 20
Total No. live fetuses (Live fetuses/Dam) 191 8.3±1.79 230 9.2±2.12 198 9.0±2.27 174 8.7±1.84
Total No. dead fetuses (Dead fetuses/Dam) 0 0±0.0 0 0±0.0 0 0±0.0 0 0±0.0 Total No. resorptions 14 13 17 16 Early 10 8 9 16
Late 4 5 8 0 Resorptions/Dam 0.6±0.94 0.5±0.71 0.8±1.02 0.8±1.06 Early 0.4±0.84 0.3±0.56 0.4±0.73 0.8±1.06
Late 0.2±0.39 0.2±0.50 0.4±0.66 0±0.0 Litters with total resorptions 0 0 0 0

Mean fetal weight (g) 42.2±4.07 42.1±4.37 38.6±5.05* (-8.5) c 41.0±4.69

Males 42.9±5.42 42.8±5.14 37.9±4.24** (-11.7) 41.3±4.38

Females 41.7±3.57 41.6±4.37 38.2±5.75 40.8±5.42
Sex ratio (% male) d 44.5 50.4 49.5 44.8 Preimplantation loss (%) 11.3±12.4 8.9±12.2 7.6±17.0 4.8±6.4 Postimplantation loss (%) 6.4±9.5 5.1±7.2 7.1±8.5 8.6±12.2

a Data obtained from MRID 49149902, pages 77-80. b One died following abortion, and one was euthanized following abortion.

c Numbers in parentheses are percent change from control, calculated by the Reviewer. d Calculated by Reviewer from total number of viable fetuses and total number of males. Data were presented in the report as the mean % male for each lit ter in each treatment group. * Statistically different (p <0.05) from the control. ** Statistically different (p <0.01) from the control.



### B. DEVELOPMENTAL TOXICITY: The numbers of fetuses (litters) available for morphological evaluation were 191 (23), 230 (25) , 198 (22), and 174 (20) in the control, 100, 300, and 700 mg/kg bw/day groups, respectively. Malformations were observed in 3 (3), 2 (1), 2 (2), and 3 (2) fetuses (litters) in the sa me respective groups, and were considered to be spontaneous in origin. When the total malformations and de velopmental variations were evaluated on a proportional basis, no statis tically significant differe nces from the control group were noted. The mean (±SD ) total percent per lit ter with variations (visceral and/orPrenatal Developmental Toxicity Study (Rabbit) (2013) / Page 14 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414



### skeletal) was 68±27%, 67±20%, 75±20%, a nd 70±28% in the 0, 100, 300, and 700 mg/kg bw/day dose groups, respectively. Fetal malfor mations and developmental variations, when observed in the test substance-treated groups, oc curred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the WIL historical control data ranges. Base d on these data, no fetal malformations or developmental variations were at tributed to the test substance.

1. External examination: External malformations were not ed for 2 fetuses in the 300 mg/kg bw/day group: microphthalmia (right) was not ed for one fetus, and meningoencephalocele (portion of brain and meninges protruded through an opening in the cranium) and open eyelid (bilateral) were noted for the second fetus. These findings did not occur in a dose-related manner, and the mean litter propo rtions of these findings were not statistically significantly different from the control group and/or were with in the ranges of values in the WIL historical control data. Therefore, these findings were not considered test substance-related. No other external malformations were noted. No exte rnal variations were noted in this study.

2. Visceral examination: Three fetuses in the control group had lobular agenesis of the lungs (right accessory lobe absent), while no visceral malformations were noted for fetuses in the test substance-treated groups. Visceral vari ations included accessory spleen(s) and major blood vessel variation, among other less frequent variations; thes e occurred infrequently, at similar frequencies in the cont rol group, and/or in a manner that was not dose-related. The mean (±SD) percent per litter with visceral variations wa s 32±21%, 23±17%, 32±25%, and 24±22% in the 0, 100, 300, and 700 mg/kg bw/day dose groups, respectively. The mean litter proportions of these findings were not st atistically significantly different from the control group and were within th e ranges of values in the WIL historical control data, with the following exception. The mean litter pr oportion of accessory spleen(s) in the 100 mg/kg bw/day group (~9%) was significantly (p<0.05) lower than the control group (23.2%); however, this did not occur in a dose-related manner and the value was within the range of values in the historical control data.

One fetus in the 700 mg/kg bw/day group had a distended, gas filled stomach. In the 100 mg/kg bw/day group, cystic oviducts were not ed for 3 (2) fetuses (litters) and white precipitate contents of the kidneys were noted for 3 (1) fetu ses (litter). These findings were not classified as either malf ormations or developmental vari ations, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently, in single litters, and/or in a manner that was not dose-related.

3. Skeletal examination: Skeletal malformations were noted for 3 (2) fetuses (litters) in the 700 mg/kg bw/day group and 2 (1) fetuses (li tter) in the 100 mg/kg bw/day group. In the 700 mg/kg bw/day group, two fetuses in the same litte r had vertebral anomaly without associated rib anomaly, consisting of absent thoracic arch es and centra, extra lu mbar arches, and/or thoracic or lumbar arches, and centra located more anterior or posterior than normal. A fetus from a different litter in the 700 mg/kg bw/d ay group had a rib anomaly consisting of fused ribs. In the 100 mg/kg bw/day group, one fetus had rib anomaly consisting of fused ribs and another fetus from the same litter had vertebral anomaly without associated rib anomaly consisting of extra lumbar arches and lumbar arches and centra located more anterior or posterior than normal. The mean litter proportio ns of these findings were not statistically significantly different than the control group and we re within the ranges of values in the WILPrenatal Developmental Toxicity Study (Rabbit) (2013) / Page 15 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414



### historical control data. Furthermore, no skel etal malformations were noted in the 300 mg/kg bw/day group. Therefore, these malformations were not test substance related. Observed skeletal variations, including 13 th 

### rib(s) (full and rudi mentary), ossified 5 th 

### and/or 6 th 

### sternebra(e), and 27 presacral vertebrae, occurred infrequently, at similar frequencies in the treated and control groups, and/ or in a manner that was not dose-related. The mean (±SD) percent per litter with skel etal variations was 59±27%, 58± 25%, 60±24%, and 66±27% in the 0, 100, 300, and 700 mg/kg bw/day dose groups, respectiv ely. The mean litter proportions of these findings were not statisti cally significantly different fr om the control group and were within the ranges of values in the WIL historical control data. Therefore, these findings were not test substance related.

TABLE 6a. External examinations a

Observations b Dose (mg/kg bw/day) 0 100 300 700 No. Fetuses(litters) examined 191 (23) 230 (25) 198 (22) 174 (20) Malformations: No. Fetuses(litters) affected 0 0 2 (2) 0 Microphthalmia and/or anophthalmia 0 c 0 1 (1) 0 Meningoencephalocele 0 0 1 (1) 0 Open eyelid 0 0 1 (1) 0

a Data obtained from MRID 49149902, page 81. b Some observations may be grouped toge ther. No external variations observed.

c Fetal (litter) incidence

TABLE 6b. Visceral examinations a

Observations b Dose (mg/kg bw/day) 0 100 300 700 No. Fetuses(litters) examined 191 (23) 230 (25) 198 (22) 174 (20) Malformations: No. Fetuses(litters) affected 3 (3) 0 0 0 Lungs- lobular agenesis 3 (3) c 0 0 0 Variations: Accessory spleen 41 (17) 17 (11) 32 (17) 20 (9) Major blood vessel variation 19 (9) 18 (10) 17 (6) 9 (4) Heart-extra papillary muscle 9 (6) 7 (4) 1 (1) 6 (5)
Gallbladder-absent or small 2 (2) 10 (7) 18 (7) 5 (3)

a Data obtained from MRID 49149902, pages 81 and 86. b Some observations may be grouped together; only the more frequently observed variations (incidence >5) are listed.

c Fetal (litter) incidencePrenatal Developmental Toxicity Study (Rabbit) (2013) / Page 16 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414

TABLE 6c. Skeletal examinations a

Observations b Dose (mg/kg bw/day) 0 100 300 700 No. Fetuses(litters) examined 191 (23) 230 (25) 198 (22) 174 (20) Malformations: No. Fetuses(litters) affected 0 2 (1) 0 3 (2) Vertebral anomal y with or without associated rib anomaly 0 c 1 (1) 0 2 (1)
Rib anomaly 0 1 (1) 0 1 (1) Variations:
13 th full rib(s) 65 (18) 78 (20) 64 (20) 59 (18)
13 th rudimentary rib(s) 28 (14) 44 (20) 38 (14) 36 (15) Sternebra(e) #5 and/or #6 unossified 18 (8) 25 (14) 25 (9) 22 (10)
27 Presacral vertebrae 20 (11) 10 (6) 7 (4) 15 (8) Hyoid arch(es) bent 4 (4) 1 (1) 5 (5) 5 (4) Sternebra(e) with threadlike attachment 2 (2) 1 (1) 1 (1) 6 (5)

a Data obtained from MRID 49149902, pages 81 and 86. b Some observations may be grouped together; only the mo re frequently observed variations (incidence >5) are listed.

c Fetal (litter) incidence.



### III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS: No test substance-relate d effects on survival or abortion were noted in the 100, 300, and 700 mg/kg bw/day groups. In the 700 mg/kg bw/day group, 1 female aborted and was euth anized and 1 female was found dead after aborting. In the 300 mg/kg bw/day group, 1 fema le was found dead. The abortions and/or mortality noted for these females was attribut ed to the severely reduced body weight and minimal food consumption noted for these individual females, as similar severe body weight and food consumption effects were not noted for other animals in these groups. A single female in the 700 mg/kg bw/d ay group was euthanized in extremis due to extreme anxiety and inability to dose; however, because this animal received only 2 doses and there were no notable post-dosing or macroscopic findings no ted for this female, the morbidity of this animal was not considered test substance-relate d. Slightly lower mean body weight gains, with corresponding effects on mean food consum ption, were noted in the 700 mg/kg bw/day group during GD 7-10 and in the 300 and 700 mg/kg bw/day groups during GD 13-20. These results were considered test substanc e-related; however, the differences from the control group were slight and mean absolute body weights in these groups remained similar to the control group throughout the study. Theref ore, these results were not considered adverse.

Based on the lack of adverse effects at a ny dosage level, a dosage level of 700 mg/kg bw/day, the highest dosage level evaluated, was considered to be the no-observed-adverseeffect level (NOAEL) for maternal toxicity and prenatal development when fosamine ammonium was administered orally by gavage to time-mated New Zealand White rabbits.Prenatal Developmental Toxicity Study (Rabbit) (2013) / Page 17 of 17 FOSAMINE/106701 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414



### B. REVIEWER COMMENTS:

1. Maternal toxicity: The Reviewer in general agrees w ith the Author’s conclusion with respect to maternal toxicity. Test substance -related effects were seen at 300 and 700 mg/kg bw/day and included clinical findings (decr eased defecation, soft stool, and/or brown material on the anogenital area) and lower mean body weight gains with correlated decreases in food consumption but no corresponding differe nce in mean absolute body weights. The Reviewer agrees that these effects were not adverse and that th e deaths/unscheduled sacrifices and abortions in these groups could not be attributed to treatment.

Therefore, under the conditions used in this study, the maternal LOAEL for fosamine ammonium in rabbits was not determined, and the maternal NOAEL was greater than or equal to 700 mg/kg bw/day.

2. Developmental toxicity: Observed fetal malformations and developmental variations occurred infrequently or at a frequency similar to that in the control group, did not occur in a dose-related manner, and/or were within the historical contro l data ranges. Therefore, no fetal malformations or developmental variations were attributed to the test substance.

a. Deaths/resorptions: There was no mortality in fetuses of does surviving to scheduled termination. Resorptions, corpora lutea, impl antation sites, and litte r proportions of preimplantation loss were similar across all groups.

b. Altered growth: Intrauterine growth and survival were unaffected by test substance administration up to 700 mg/kg bw/day.

c. Developmental variations: The total percent per litter with visceral or skeletal variations was similar across all groups. No external variations were observed.

d. Malformations: Malformations occurred in simila r frequencies across groups and were considered to be spontaneous in origin.

The developmental LOAEL for fosamine ammo nium in rabbits was not determined. The developmental NOAEL was greater than or equal to 700 mg/kg bw/day.

B. STUDY DEFICIENCIES:

 

### In this study, visceral examin ation of the internal structures of the head was done by midcoronal slice in all cases; whereas, accord ing to OCSPP 870.3700, an adequate evaluation of the internal structures of the head, including the eyes, brain, nasal passages, and tongue, should be conducted (e.g. by serial sectio ning technique) for at least half of the fetuses.

 

### The mating technique (natural or artificial insemination), confirmation of mating, and verification of males as the same strain were not documented.

### DATA EVALUATION RECORD

FOSAMINE

OCSPP 870. 7800 STUDY TYPE: JMMUNOTOXICOLOGY -MOUSE



### Primary 

### Reviewer; Clau<lia Troxel. Ph.D.
Secondary Reviewers: MRID 49121701

Prepared for



### Health Effects Division Office of Pesticide Programs U.S. Environmental 

### Protection Agency 

### One Potomac Yard 2777 South Crystal 

### Drive



### Arlington, VA 

### 22202

Prepared by

Swnmitee Corporation 9724 Kingston Pike, Suite 602 

### Knoxville, Tennessee

Task Order No.: 6-73

Signature: Date:

H.T. Borges. Ph.D

.. MT(ASCP). D.A.B.T.



### Robert H

.. Ross. M.S. Program Manager

Quality Assurance: Angela M. Edmonds. B.S.

Disclaimer 

### Signature: Date:

Signature: Date:

This review may have been altered subsequent to the contractor's signatures above. Summitec Corp. for the U.S. Environmental Protection Agency under Contract No. EP·W-11-014FOSAMIN E / 106701 lmmunotoxicity (2013) Page 2 of 12 OCSPP 870.7800 I DACO 4.8 / OECD None



### EPA Reviewer: 

### Ayaad Assaad, D. V

. M,, Ph.D. Signature:

# /};~ o)

Risk Assessment Branch IV, Health Effects Division (7509P) 

# Daa.

EPA Secondary Reviewer: Marquea King, Ph.D.



### _ Signatu

. ·

# ~' Risk Assessment Branch IV, Health Effects Division (7509P)

# Date:~~ plate version 09/11 

### TXR #: 

### 0057080 

# I DATA EVALUATION RECORD 

# I

STUDY TYPE: I;mmunotoxicity (gavage)

- [mouse}; OCSPP 870.7800



### PC CODE: 106701 

### DPBARCODE: D413146



### TEST MATERIAL <PURITY): Fosamine anunonium (95.3% 

### ai.)

SYNONYMS: None given



### CITATION: Bultman

, 

### J. (2013). A 28-day oral gavage immunotoxicity study offosamine ammonium in CD-I mice. WIL**Researc~** 1407 George Road, Ashland, OH 44805-8946 and ImmunoTox®, Inc., Virginia BioTechnology Research Park, 800 East Leigh St., Suite 209, Richmond

, VA 23219. Laboratory Proje ct ID: WIL-925005

, April 22, 2013. MRID 49121701

. Unpublish ed

.



### SPONSOR: Albaugh, Inc., 1525 NE 36th St., Ankeny, IA 50021



### EXECUTIVE SUMMARY:

In an immunotoxicity study (MRID 49121701), fosamine ammonium (95.3% a.i., Lot# 20120805) was administer ed to 10 female Crl :CD-1 mice/do se by gavage once daily for 28 consecutive days at dose l evels ofO, 100, 500, and 1000 mg/kg bw/day. In addition, 10 mice in a positive control group were administered 50 mg.lkg bw/day of cyclophosphamide monohydrate (CPS) via intraperitoneal (IP) injection once daily for 4 consecutive days before sacrifice (days 24 through 27). The effects of test substance on antibody response to antigen were determined using the Spleen Antibody-Forming Cell Response (AFC Assay). On day 24, all mice were immwtlzed with a single 0.2 mL injection of I x 10 8 sheep red blood cells (SRBC) via the lateral tail vein. On day 28, animals were sacrificed; blood samples were obtained from the inferior

vena cava; the liver, spleen and thymus were weighed; and the sp leen was processed for shipment to the laboratory conducting the immunological evaluatio ns.

Treatment with up to 1000 mg/kg bw/day of fosamine anun onium for 28 day s did not adversely affect clinical signs, body weight or body weight gain, or food consumption. Gross necropsy did
not rev eal any treatment-related effects, and l iver, spleen, and thymus weights were unaffected

.



### The systemic toxicity NOAEL for fosamine ammonium in female Crl:CD-1 mice was 1000 mg/kg bw/day; the LOAEL was not established Qimit dose tested).4-Week Inhalation Toxicity Study (rat) (2013) / Page 3 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412



### minimal to moderate hemorrhage, which was stated to be an anomaly of euth anasia in this strain of rat (historical data not provided). Adrena l gland and kidney weights and relative-to-body weights were statistically increased in female s from the high exposure group. Minimal to mild hypertrophy of the adrenal cortex that correlated with the increased adre nal weight was observed in 2/10 high concentration females. This micros copic lesion was not cons idered adverse, since the finding was stated to be associated with th e stress of exposure, al though no control animals had this lesion and historical data were not provided. Minimal to moderate mucous cell hyperplasia was observed in a dose-dependent ma nner in the nasal cavities of both males and females at all three test substance exposure levels . No metaplasia or degenerative changes were observed. Based on the scoring criteria from the pa thologist report, the mi nimal and mild scoring are not considered adverse due to the lack of functional impairme nt, absence of metaplasia and degenerative changes, were observed. Additiona lly, no nasal discharge or respiratory distresses were observed in these animals. Only one female in the high dose group had a moderate mucous cell hyperplasia score which was defined as lim ited tissue or organ dysfunction. Therefore, the minimal and mild hyperplasia observed is not cons idered an adverse effect in this study.

Based on the effects seen in this study, a NO AEL in male and female Sprague-Dawley rats was the aerosol concentration of 1 mg/L (achieved mean concentration = 1.0 mg/L). A LOAEL was not identified.

This 4-week subchronic inhalati on toxicity study in the rat is acceptable/Guideline; and satisfies the Guideline requirement for a subchronic inhalation study in the rat (OCSPP 870.3465; OECD 413).

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.4-Week Inhalation Toxicity Study (rat) (2013) / Page 4 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412



### I. MATERIALS AND METHODS:

A. MATERIALS:

1. Test material: Fosamine Ammonium
Description: White powder
Lot/batch #: 20120815 Purity: 95.3% a.i.
CAS # of TGAI: 25954-13-6
Structure:



### 2. Vehicle: Filtered air

3. Test animals: Species: Rat Strain: Crl:CD(SD) Age/weight at study initiation: ~5 weeks/ Males 183-229 g; Females 156-189 g Source: Charles River Ltd., Raleigh, NC Housing: Individually in stainless steel wire mesh cages Diet: Certified Rodent LabDiet® 5002, PM I Nutrition International, LLC, ad libitum except during
exposure. Water: Reverse osmosis-treated tap water, ad libitum except during exposure. Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 21.4-21.7 o C 44.3-48.2% At least 10/hour 12 hours light/dark

Acclimation period: 14 days and 5 days for e xposure system acclimation



### B. STUDY DESIGN:

1. In life dates: Start: February 19, 2013; End: March 18, 2013

2. Animal assignment and treatment: Animals were stratified by weight and randomly assigned to the test groups not ed in Table 1. Concentrations were selected based on the results of a 5-day inhalation toxicity study in that resulted in no mi croscopic findings. No details were provided.4-Week Inhalation Toxicity Study (rat) (2013) / Page 5 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412

TABLE 1. Study Design

Experimental parameter Fosamine Ammonium Concentrations (mg/L)
0 0.1 0.3 1 Number of Rats Total no. of animals assigned 20 (10/sex) 20 (10/sex) 20 (10/sex) 20 (10/sex) Sacrifice and necropsy 20 (10/sex) 20 (10/sex) 20 (10/sex) 20 (10/sex) Measured Concentration Achieved aerosol concentration (mg/L ±s.d .) NA 0.10 ±0.027 0.30 ±0.032 1.0 ±0.11 Data obtained from page 34 (MRID 49196901)



### 3. Generation of the test atmosphere / chamber description: Chamber atmospheres were generated using a jet mill air micronizer (Jet-O -Mizer Model 00) coupled with a distribution system that delivered a controlled amount of powder aerosol and filtered compressed air to the inlet of each 7.9 L stainless steel nose- only exposure chamber (WIL), which was metered to deliver the appropriate atmospheric concen tration. An auger-type f eeder (Accu-Rate) was used to deliver the test subs tance to the generators. Nose- only exposure chambers (one for each exposure level) were fitted with air flow meters, pressure gauges, and other devices to measure temperature, humidity, and test arti cle concentration. The animals were gradually acclimatized to the restraining de vices for five days prior to th e commencement of exposures.

4. Test atmosphere concentration: The exposure concentrations in each exposure chamber were measured gravimetrically at least six times per day. Samples were taken from the breathing zone of the animals using pre-weighed f ilters. Results are in Table 1 and show that the achieved mean test substan ce concentrations were 100% of target values. Homogeneity of concentrations within the ch amber was tested at the low- and high exposure concentrations and the range of variability relative to the reference point was -2.2% to +15.4%. Rotation of animals was used to reduce the variability of exposure and assure that the target range of ±10% was met. Time to e quilibrium was not reported.

5. Particle size determination: Particle size for each exposure concentration was determined at least four times during the study using 7-stag e stainless steel cascad e impactors. The mass median aerodynamic diameter (MMAD) and geometric standard deviation ( σ g) were 2.5 μ m (2.20), 2.7 μ m (2.42), and 2.8 μ m (2.16) for the low, mid, and high concentration groups, respectively.

6. Statistics: All analyses were conducted comparing each test substance-exposed group to the control group by sex. The overall level of significance for intergroup differences was p ≤ 0.05. Body weight, body weight change, food consump tion, organ weight, a nd clinical pathology were subjected to a one-way parametric an alysis of variance (ANOVA) to determine intergroup differences. When significant differen ces were detected, Dunnett's test was used to compare the test substance-exposed groups to the control group.

The Reviewer considers the statistical analyses used to be appropriate.

C. METHODS:

1. Observations:

1a. Cageside observations: Animals were observed twice daily for clinical signs,4-Week Inhalation Toxicity Study (rat) (2013) / Page 6 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412



### abnormal behavior, morbid ity, and mortality.

1b. Clinical examinations: Clinical examinations were c onducted prior to exposure, at the midpoint of daily exposure, and at the end of exposure. Detailed c linical examinations were conducted weekly.

1c. Neurological evaluations: Neurological evaluations were not conducted.

2. Body weight: Animals were weighed weekly during the experimental exposure period and at study termination prior to necropsy.

3. Food consumption: Food consumption was recorded weekly.

4. Ophthalmoscopic examination: Eyes of all rats were examined by an ophthalmoscope prior to the first day of exposure and during week 4.

5. Hematology and clinical chemistry: Blood was collected from all surviving animals at termination for hematology and clinical chemistry; the animals were fasted overnight prior to blood collection. Blood was collected from the retro-orbital sinus while under isoflurane anesthesia. Blood used to measure coagulation parameters was collected from the vena cava at the time of euthanasia while under isoflurane anesthesia. The CHECKED (X) parameters were examined.

a. Hematology: X Hematocrit (HCT)* X Leukocyte differential count* X Hemoglobin (HGB)* X Mean corpuscular HGB (MCH)* X Leukocyte count (WBC)* X Mean corpusc. HGB conc.(MCHC)* X Erythrocyte count (RBC)* X Mean corpusc. volume (MCV)* X Platelet count* X Reticulocyte count Blood clotting measurements* X Red cell morphology X (Thromboplastin time) X He moglobin distribution width (Clotting time) X RBC distribution width X (Prothrombin time) Mean platelet volume
* Recommended for subchronic inhalati on studies based on Guideline 870.34654-Week Inhalation Toxicity Study (rat) (2013) / Page 7 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412



### b. Clinical chemistry:

X ELECTROLYTES X OTHER
X Calcium X Albumin* X Chloride X Creatinine* Magnesium X Urea nitrogen* X Phosphorus X Total Cholesterol* X Potassium* X Globulins X Sodium* X Glucose*

X ENZYMES (more than 2 hepatic enzymes eg., *) X Total bilirubin

X Alkaline phosphatase* X Total serum protein (TP)* Cholinesterase (ChE) X Triglycerides Creatine phosphokinase Se rum protein electrophoresis Lactic acid dehydrogenase (LDH) X Albumin/Globulin ratio X Alanine aminotransferase (ALT/also SGPT)* X Appearance X Aspartate aminotransfera se (AST/also SGOT)* X Sorbitol dehydrogenase* X Gamma glutamyl transferase (GGT)* Glutamate dehydrogenase
* Recommended for subchronic inhalati on studies based on Guideline 870.3465



### c. Cholinesterase determination: Not conducted.

6. Urinalysis : Urine was collected from all anim als during overnight fasting at study termination. The CHECKED (X) parameters were examined.

X Appearance* X Glucose* X Volume* X Ketones X Specific gravity / osmolality* X Bilirubin X pH* X Blood / blood cells* X Sediment (microscopic) X Nitrites X Protein* X Urobilinogen * Optional for inhalation toxicity studies



### 7. Sacrifice and pathology: Rats were killed by exsangui nation while under isoflurane anesthesia. Any gross lesions we re recorded and tissues were collected from animals in all exposure groups and processed for microscopi c evaluation. Microscopic examination was conducted on all rats from the control and high exposure groups. The CHECKED (X) tissues were collected and the (XX) organs were weighed.4-Week Inhalation Toxicity Study (rat) (2013) / Page 8 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412

X DIGESTIVE SYSTEM X CARDIOVASC./HEMAT. X NEUROLOGIC X Tongue X Aorta, t horacic* XX Brain*+ X Salivary glands* XX Heart*+ X Peripheral nerve* X Esophagus* Bone marrow* X Spinal cord (3 levels)* X Stomach* X Lymph nodes* X Pituitary* X Duodenum* XX Spleen*+ X Eyes (optic nerve )* X Jejunum* XX Thymus*+ X GLANDULAR
X Ileum* XX Adrenal gland*+
X Cecum* X UROGENITAL X Lacrimal gland
X Colon* XX Kidneys*+ X Parathyroid* X Rectum* X Urinary bladder* X Thyroid*
XX Liver*+ XX Testes*+ X OTHER

Gall bladder* (not rat) XX Epididymid es*+ X Bone (sternum and/or femur) Bile duct* (rat) X Prostate* X Skeletal muscle X Pancreas* X Seminal vesicles* X Skin
X RESPIRATORY XX Ovaries*+ X All gross lesions and masses*
X Trachea* XX Uterus*+ X Blood smear XX Lung* X Mammary gland* X Harderian gland X Nose* X Cervix Carina X Pharynx* X Oviduct X Peyer’s patches X Larynx* X Vagina
* Recommended for subchronic rodent studies based on Guideline 870.3465

+ Organ weights required



### II. RESULTS:

A. OBSERVATIONS:

1. Clinical signs and Mortality: All animals survived until scheduled sacrifice. Yellow and/or brown material was observed ar ound the anogenital area and limbs in males and females in the high exposure groups and in females in the mid-exposure group. This finding was not considered adverse.

B. BODY WEIGHT AND BODY WEIGHT GAIN: No statistically significant effects were observed in body weight or body weight gain (Table 2).4-Week Inhalation Toxicity Study (rat) (2013) / Page 9 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412

TABLE 2. Mean body weight and body weight gain during 4 weeks of exposure a

Analytical concentration (mg/L) Body weights (g ±SD) Total weight gain
Day 0 Day 7 Day 14 Day 25 c g % from control b

Male

0 229 ±14.0 266 ±18.1 297 ±26.4 338 ±35.1 +109 NA

0.1 230 ±14.2 262 ±24.7 292 ±31.8 336 ±40.2 +107 -2

0.3 229 ±15.0 265 ±20.3 293 ±25.4 337 ±33.9 +108 -1

1.0 230 ±11.3 267 ±10.7 298 ±12.0 339 ±15.0 +109 0

Female

0 187 ±8.0 209 ±8.6 225 ±9.1 246 ±9.9 +60 NA

0.1 186 ±8.3 209 ±10.2 223 ±11.9 247 ±12.0 +61 +2

0.3 183 ±5.7 204 ±7.0 220 ±8.8 237 ±11.1 +53 -13

1.0 185 ±6.6 206 ±8.6 224 ±10.9 243 ±15.4 +58 -3

a Data obtained from pages 65-74 of MRID 49196901.

b Calculated by reviewer.

c Day 26 for females.



### C. FOOD CONSUMPTION : There were no statistically significant or treatment-related effects on food consumption at any exposure concentration.

D. BLOOD ANALYSES:

1. Hematology: There were no treatment-related eff ects on hematology parameters at any exposure concentration. Lower mean cor puscular hemoglobin concentration was observed in high exposure males relative to c ontrol, but the inves tigators stated that neither the individual values nor mean value were outside the hist orical control ranges (historical data not provided).

2. Clinical chemistry: There were no treatment-related effects on clinical chemistry parameters at any exposure concentration.

E. URINALYSIS: Specific gravity was statistically hi gher in females at the high exposure concentration (p<0.05). No other treatment-rela ted effects on urinalysis parameters were observed.

F. OPHTHALMOSCOPIC EXAMINATION: No lesions were observed during the ophthalmoscopic examinations.

G. SACRIFICE AND PATHOLOGY:

1. Gross pathology: A higher incidence of dark red ar eas in the thymus was observed in males (1/10 controls; 0/10 at 0.1 mg/L; 4/10 at 0.3 mg/L; 2/10 at 1.0 mg/L) and females (0/10 controls; 0/10 at 0.1 mg/L; 1/10 at 0.3 mg/L; 2/10 at 1.0 mg/L). Histologically this correlated with minimal to mild hemorrhage.4-Week Inhalation Toxicity Study (rat) (2013) / Page 10 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412



### 2. Organ weight: Selected organ weight data are presented in Ta ble 3. Adrenal gland and kidney weights and relative-to- body weights were statistically increased in females from the high exposure group. Minimal to mild hype rtrophy of the adrenal cortex correlated with the increased adrenal weight observed in high concentration females. Absolute (p<0.05) and relative (p<0.01) thymus weights were statistically higher in males at the high exposure concentration, and relative thym us weight was higher at the low exposure concentration (p<0.05). The differences in thym us and kidney weights were stated to be within the historical control range (historical data not provided).

Table 3. Changes in sel ected organ weights following 4 weeks of exposure. a

Organ Exposure concentration (mg/L)
0 0.1 0.3 1.0

MALES Absolute weight (g)

Adrenals 0.0536 ±0.0105 0.0525 ±0.0062 0.0566 ±0.0085 0.0574 ±0.0048
Kidney 2.27 ±0.173 2.22 ±0.315 2.19 ±0.201 2.28 ±0.285
Thymus 0.4105 ±0.0955 0.5172 ±0.1267 0.4639 ±0.1299 0.5571 ±0.0624*
Relative to body weight (g/100g body weight)

Adrenals 0.018 ±0.0033 0.017 ±0.0029 0.019 ±0.0017 0.019 ±0.0015
Kidney 0.749 ±0.0436 0.735 ±0.0663 0.725 ±0.0374 0.748 ±0.0841
Thymus 0.134 ±0.0244 0.169 ±0.0281* 0.153 ±0.0399 0.184 ±0.0209**

FEMALES Absolute weight (g)

Adrenals 0.0648 ±0.0065 0.0722 ±0.0085 0.0713 ±0.0051 0.0757 ±0.0141
Kidney 1.66 ±0.121 1.67 ±0.131 1.69 ±0.111 1.74 ±0.083
Thymus 0.449 ±0.0757 0.430 ±0.102 0.450 ±0.072 0.456 ±0.115
Relative to body weight (g/100g body weight)

Adrenals 0.028 ±0.0025 0.032 ±0.0033 0.033 ±0.0030* 0.034 ±0.0062*
Kidney 0.726 ±0.532 0.739 ±0.0433 0.777 ±0.0227* 0.780 ±0.0392*
Thymus 0.197 ±0.034 0.190 ±0.045 0.207 ±0.035 0.204 ±0.045 Data obtained from pages 110-124 in MRID 49196901.

a Values expressed as group means ± s.d. *p<0.05 ; * *p<0.01



### 3. Microscopic pathology: Minimal to mild mucous cell hyperplasia was observed in a dose-dependent manner in the nasal cavities of both males and females at all three test substance exposure levels (Table 4). It is noted that these change s were not observed in Nasal Levels I, III, but noted for Levels II , IV, V, and VI. One female was observed with moderate mucosal hyperplasia at the highe st concentration. No metaplasia or degenerative changes were observed at any concentration. The pa thologist report and scoring definitions are incl uded in the appendix. Table 5 shows the incidence of hemorrhage observed in the thymus of both sexes of control and exposed rats, which correlated with dark red areas during gross examination. Th e incidence and severity of the thymus lesions do not follow a consistent dose-response patter n. The investigators stated that this finding was a perimortem anom aly that occurs at the time of euthanasia,4-Week Inhalation Toxicity Study (rat) (2013) / Page 11 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412



### but historical data were not provided. Females (2/10) at the high concentration had minimal to mild diffuse adrenal cortical hypertrophy

Data obtained from page 41 in MRID 49196901.

TABLE 5. Incidence of thymus lesions

Lesion Treatment group (mg/L)

Male Female

0 0.1 0.3 1 0 0.1 0.3 1 Thymus hemorrhage
-minimal
-mild
-moderate 0/10
1/10
0/10 NE
NE
NE 1/4
2/4
1/4 1/10
1/10
0/10 0/10
1/10
0/10 NE
NE
NE 1/1
0/1
0/1 1/10
1/10
0/10

NE = None examined Data obtained from pages 139 and 153 in MRID 49196901.



### III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS: The investigators conclude d that the no-observedadverse-effect level (NOAEL) for both males and females was the high aerosol concentration of 1 mg/L (achieved mean concentration = 1.0 mg/L).

B. REVIEWER COMMENTS:

TABLE 4.4-Week Inhalation Toxicity Study (rat) (2013) / Page 12 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412



### The reviewer generally agrees with the investigators’ conclu sions. The thymus and adrenal hypertrophy are not considered a dverse since no dose-response was observed and may have been an artifact of euthanasia. The minimal to mild hyperplasia observed is not considered an adverse effect due to the specific sc oring definitions from the path ologist report and the lack of functional impairment, absence of metaplasia and other degenerative changes. Additionally, no nasal discharge or respiratory distresses were observed in these animals. The one high dose female with the moderate score is not considered robust to define an adverse effect level for the study.

Based on the effects seen in this study, a NO AEL in male and female Sprague-Dawley rats was the aerosol concentration of 1 mg/L (achieved mean concentration = 1.0 mg/L). A LOAEL was not identified.

This 4-week subchronic inhalati on toxicity study in the rat is acceptable/Guideline; and satisfies the Guideline requirement for a subc hronic inhalation st udy in the rat (OCSPP 870.3465; OECD 413).

C. STUDY DEFICIENCIES:

 The time to equilibrium in the exposure chamber was not reported (minor).

Appendix (taken from page 248-252 & 255-258) of study report: 1. MICROSCOPIC EXAMINATION: Test substance-related microscopi c findings were noted in the nasa l cavity in all test substancetreated male and female groups. Mucous cell hyperpl asia was observed within the nasal cavity of the 0.1, 0.3, and 1 mg/L group males and females at several levels. The cha nge was characterized by increased numbers of mucous-c ontaining cells within the resp iratory epithelium lining the nasal turbinates (in levels II and IV) and nas opharyngeal duct (in levels V and VI). A doseresponse was present for both sexes and the chan ge was observed at a higher incidence in females. In males, the change was limited to the nasopharyngeal duct in nasal levels V and VI. Mucous cell metaplasia of the olfactory or tran sitional epithelium was not observed in any of the animals examined. No degenerative changes were observed within the affected respiratory epithelial cells.

Two of the 1 mg/L group females (nos. 6709 and 6721) had minimal to mild diffuse adrenal cortical hypertrophy. This correl ated with higher ad renal gland weights noted in the test substance-exposed female groups. This change wa s not observed in any ofthe test substanceexposed males or in any of the 0.1 and 0.3 mg/L group females.

2. Histopathology Grading system: Nasal levels (I to VI) of the nasal cavity in males and females were identified as target organs and examined in the low and mid-exposure groups. Stained histologic sections were examined by light microscopy and observations were entered in the WIL Toxicology Data Management System (WTDMSTM) by the pathologist.

A. Unremarkable: Tissue considered to be w ithin normal limits, under the conditions of the study and considering th e age, sex, and strain of the animal concerned.4-Week Inhalation Toxicity Study (rat) (2013) / Page 13 of 13 FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412



### B. Present: A non-gradable hist ologic change for which no additional information would be gained by assigning a se verity grade (e.g. cyst).

C. Grade 1 (Minimal): A histologic change that barely exceeds that which is considered to be within normal limits. This change is unlikely to produce any functional impairment.

D. Grade 2 (Mild): A histologic ch ange that is easily identified but of limited severity. This change probably does not produce any functional impairment.

E. Grade 3 (Moderate): A histolog ic change that is a prominent feature of the tissue but not of maximal severity. Limited tissu e or organ dysfunction is possible.

F. Grade 4 (Severe): A histologic change that is an overwhelming feature of the tissue and that may cause significan t tissue or orga n dysfunction.

### DATA EVALUATION RECORD

FOSAMJNE



### STUDY TYPE : PRENATAL DEVELOPMENTAL TOXICITY STUDY -RABBIT OCSPP 870.3700b (§83..JbJ; OECD 414



### Primary Reviewer : Stephanie J

. GarciL 

### Ph.D.

Secondary 

### Reviewers : Donna L 

### Fefee. D.V.M. MRID 49149902


### PreJNQ"eC1 for Health Effects Division 

### Office of Pesticide Programs U

. S

. 

### Environmenta1 Protection 

### Agency One 

### Potomac Yard 2777 South Crystal Drive

Arlington, VA 22202

Prepared 

### by 

### Summ i tec Corporation 9724 

### Kingston Pike, 

### Su i te 602 

### Knoxville. Tennessee 37922

Task Order No

. 6-73



# . 

# ,. Signature:-'J.**~** Dare: 

### _______:______013

Signature: 

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, 't.'\n

........

## .,.~~IE Date : 

### 11 

# I an I 

# J; 15 Robert H. 

### Ross, 

### M.S .• Program Manager Signature :

# ~~~**~** Date:

### ~1£io Signature:

..

# ~ ll 

### Date:

### l~l.3 Quality Assmance: 

### Jennifer Goldbqg. B.S

.

Disclaimer



### This review 

### may have 

### been altered 

### subsequent to the contractor ' s signatures above.



### Swnmitec Corp

. for the U

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. Environmental Protection Agency under Contract No

. EP-W

- 11-014FOSAMINE/l 0670 I Prenatal Developmental Toxicity Study (Rabbit) (2013) I Page 2 of 17 OCSPP 870.3700b/ DACO 4.5.3/ OECD 414



### EPA Reviewer : Ayaad Assaad. D .V.M. Ph.D. 

### Signature: Risk Assessment Branch IV, Health Effects Division (7509P) Date:**~~h~~~--**



### TXR#:0057080

DATA EVALUATION RECORD

STUDY TYPE: Prenatal Developmental Toxicity Study

- Rabbit ; OCSPP 870.3700b [§83-3b]; OECD 414.



### PC CODE: l 06701 

### DPBARCODE:

TEST MATERIAL (PURITY): Fosamine ammonium (95.3% a.i.)



### SYNONYMS: Ammonium ethyl carbamoylphosphonate . 041 3146



### CITATION: Kappeler, C. (2013) 

### An oral (gavage) prenatal developmental toxicity study of fosamine ammonium in rabbits. WIL Research, Ashland, Ohio

. Laboratory report number WIL-925002, June 5, 2013. MRID 49149902. Unpublished.



### SPONSOR: Albaugh, Inc.

, Ankeny, 

### Iowa.

EXECUTIVE SUMMARY:

In a developmental toxicity study (MRID 49149902), fosamine ammonium (95.3% a.i., batch# 20120805) was administered once daily by oral gavage to 25 time-pregnant New Zealand White [Hra:(NZW)SPF] rabbits/do se in deionized water at dose levels of 0, 100, 300, or 700 mg/kg bw/day (10 mL/kg bw dosing volume) on gestation days (GDs) 7 through 28, inclusive. On GD 29, the does were sacrificed and necropsied. Gravid uterine weights, corpora l utea counts, and the numbers and positions of live and dead fetuses, and early and late resorptions were recorded, and all fetuses were weighed and examin ed for external anomalies. Live fetuses were sexed internally and subjected to visceral examination 

### by fresh dissection (with heads exami ned by mid-coronal slice) then processed for and subjec ted ske letal examination.

In the 700 mg/kg bw/day group, 3 female s experienced unscheduled mortality: 1 female was euthanized in extremis on GD 10 due to extrem e anxiety and inability to dose, 1 female aborted

on GD 24, and 1 female was found dead after aborting on GD 27. 

### In the 300 mg/kg bw/day
group, l female was found dead on GD 29. The abortions and/or mortality noted for these females were attributed to the severely reduced body weight and minimal food consumption noted for these females just prior to death

. Test substance related clinical findin gs consisted of decreased defec ation, soft stool, and brown material on the anogenital area in the 700 mg/kg bw/day group, with si milar but less frequent effects in the 300 mg/kg bw/day group; these findings were not statistically sign ificant.Immunotoxicity (2013) Page 3 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None


### Fosamine ammonium did not suppress the humor al immune response using the sRBC Assay. The CPS positive control res ponded appropriately, exhibiting statistically significant decreases in absolute and relative spleen and thymus wei ghts, spleen cell numbers, Specific Activity (AFC/10 6 

### spleen cells) or Total Spleen Activity (AFC/Spleen).

The immunotoxicity toxicity NOAEL for fosa mine ammonium in female Crl:CD-1 mice was 1000 mg/kg bw/day; the LOAEL was not establ ished (limit dose tested).

This immunotoxicity study is classified Acceptable/Guideline and satisfies the guideline requirement for an immunotoxicity study (OCSPP 870.7800) in mice.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.Immunotoxicity (2013) Page 4 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS:

1. Test material: Fosamine ammonium Description: Off-white, coarse powder Lot/Batch #: 20120805 Purity: 95.3% a.i. Compound Stability: Not reported CAS # of TGAI: 25954-13-6



### 2. Vehicle: Deionized water (prepared on site)

3. Positive control: Cyclophosphamide monohydrate (CPS) in phosphate-buffered saline, Lot # 120M1253V; Purity 100%

3 Test animals: Species: Mice Strain: Crl:CD-1 Age/weight at study initiation: Females, ~7 weeks, 19.9-26.6 g Source: Charles River Laboratorie s, Inc., Raleigh, NC Housing: Individually in clean, stainless steel, wire-mesh cages suspended above cageboard Diet: PMI Nutrition International, LLC, Certified LabDiet ® 5002 ad libitum Water: Reverse-osmosis-treated (on-site) drinking water ad libitum Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 22±3°C (actual: 20.8-21.4oC) 50±20% (actual: 43.2%-51.7%) Minimum 10/hr 12 hrs dark/12 hrs light Acclimation period: Minimum of 14 days



### B. STUDY DESIGN:

1. In life dates - Start: September 25, 2012 End: November 6, 2012

2. Animal assignment : Animals were assigned to the test groups noted in Table 1 based on a computerized randomization procedure using bod y weight stratificati on in a block design. Test substance was administered orally by ga vage in a dose volume of 10 mL/kg once daily for 28 consecutive days; a cyclophosphamide (C PS) positive control group received four daily intraperitoneal (IP) inje ctions of 50 mg/kg bw/day of cyclophosphamide on days 24-27. The first day of fosamine ammonium dosing wa s designated as day 0. Individual doses wereImmunotoxicity (2013) Page 5 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None


### based on the most recently recorded body wei ghts to provide the correct mg/kg bw/day dosage.

The Spleen Antibody-Forming Cell Response (A FC Assay) was used to measure humoral immunity via the T-cell dependent antibody re sponse to sheep red blood cells (sRBC). All mice in the AFC assay (Groups 1-5) were administered 0.2 mL of 1 x 10 8 

### sRBC via intravenous (IV) administration once on study day 24, four days before sacrifice. The sRBC immunizations were administered prior to CPS dose administration on study day 24.

TABLE 1: Study design a

Test group Group number Dose to animal (mg/kg bw/day) # Female

Control 1 0 10 Low 2 100 10 Mid 3 500 10 High 4 1000 10
CPS Positive control b 5 50 10

a On study day 24, all mice were immunized with injection of sRBC.
b Mice administered CPS (cyclophospha mide monohydrate) once dail y at 50 mg/kg bw/day on study days 24-27 by iv injection.



### 3. Dose selection

The dose levels were selected based on th e results from dose range-finding study where groups of 4 female Crl:CD-1 mice were ad ministered 100, 500, or 1000 mg/kg/bw/day of fosamine ammonium by oral gavage daily fo r 5 consecutive days. No observable effects were observed in mortality, morbidity, clini cal signs, physical examination, or body weight.

4. Dose formulation preparation and analysis

The vehicle for administration to the vehicle contro l group and for preparation of the test substance formulations was prepared weekly, divided into aliquots for daily dispensation, and stored at room temperature. The test subs tance formulations were stirred continuously throughout the preparation, sampling, and dose ad ministration procedures. During the study, samples of the 0, 10, 50, and 100 mg fosamine ammonium/mL dosing formulations were analyzed twice for concentration using UV absorb ance detection: once befo re the initiation of dosing, and once during the fourth week of dosi ng. The author stated that test substance homogeneity and stability following 10 days of room temperature storage were assessed and verified in formulations ranging in c oncentration from 10 to 100 mg fosamine ammonium/mL in a previous study. The data were not provided.

Results Homogeneity analysis: Homogeneity was verified in a previous study; the data were not provided.

Stability analysis: Stability was verified in a previ ous study; the data were not provided.Immunotoxicity (2013) Page 6 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None


### Concentration analysis: The mean concentrations of fosamine ammonium ranged from 100- 103% of nominal for the 10 mg/mL prepara tion; 101-103% of nominal for the 50 mg/mL formulation; and 101-103% of nominal fo r the 100 mg/mL dose formulations.

The analytical data indicated that the variance between nominal and actual dosage to the animals was acceptable. Stability and homogeneity data could not be verified because they were determined in a previous study and the data were not provided.

5. Statistics: Each mean was presented with the st andard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to ca lculate the mean. Statistical analyses for the dose range-finding phase data were not conducted due to lack of a concurrent vehicle control group. For the definitive phase data, all stat istical tests were performed using WTDMSTM unless otherwise noted. Analyses were conducte d using two-tailed tests (except as noted otherwise) at minimum significance levels of1% and 5%, comparing each test substancetreated group to the vehicle control gr oup. Body weight, body weight change, food consumption, and liver weight data were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett’s test was used to compare the test substance-treated groups (Groups 2-4) to the vehicle control group (Gr oup 1). The positive co ntrol group data (Group 5) were evaluated using the two-sample t-test and compared to the vehicle control group (Group 1).

The terminal body weight, thymus, and spleen weights (absolute and relative to terminal body weight), and data obtained during the defi nitive phase of this study were analyzed by ImmunoTox®, Inc. The AFC data were expr essed as both Specific Activity, IgM antibodyforming cells per million spleen cells (AFC/10 6 

### spleen cells), and as IgM total spleen activity (AFC/spleen). Data were first tested for homogeneity of vari ances using the Bartlett’s ChiSquare Test. Homogenous da ta were evaluated using para metric one-way ANOVA. When significant differences occurred, the treatment groups were compared to the vehicle control group using Dunnett’s test. N on-homogenous data were eval uated using a nonparametric analysis of variance. When significant diffe rences occurred, the treatment groups were compared to the vehicle cont rol group using the Gehan-Wilc oxon Test, as appropriate. The Jonckheere’s Test was used to test for dose- related trends across the vehicle control and treatment groups. The positive control data were evaluated using the Student’s t-Test and compared to the vehicle control group. The criteria for accepting the results of the positive control group was a statistically significant (p<0.05) decrease in the response compared to that of the vehicle control group.

C. METHODS:

1. Observations: Animals were inspected twice daily for morbidity, mortality, and clinical signs; clinical examinations were performed once daily on nondosing days for the positive control group. Detailed physical examinations were conducted on all anim als 1 week prior to randomization, on the day of randomization, week ly during the dosing period, and on the day of scheduled necropsy.Immunotoxicity (2013) Page 7 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None


### 2. Body weight: Animals were weighed 1 week prior to randomization, on the day of randomization, on study day 0 (prior to dosing) , and twice weekly during the study. Final body weights were recorded from nonfasted animals on the day of necropsy.

3. Food/water consumption: Individual food weights were recorded 1 week prior to randomization, on the day of randomization, on study day 0 (prior to dosing), and twice weekly during the study.

4. Sacrifice and pathology

Animals (nonfasted) were sacrificed by carbon dioxide inhalation followed by exsanguination on day 28. Blood samples were colle cted from the inferior vena cava for IgM antibody analysis.

a. Gross necropsy: A complete necropsy was performed on all animals and included examination of the external su rface, all orifices, and the cran ial, thoracic, abdominal, and pelvic cavities, including viscera. The liv er, thymus and spleen were weighed.

b. Tissue preparation/histopathology: The liver and thymus were collected and placed in 10% neutral-buffered formalin for possible microscopic examination while the spleen was collected and placed in EBSS/HEPES buffer and prepared for the AFC assay.

5. Immunotoxicity:

a. Antibody plaque-forming cell (AFC) assay: Female mice were exposed to fosamine ammonium or vehicle control by or al gavage daily for 28 days. In addition, 10 female mice in the CPS positive control group (Group 5) were administered 50 mg/kg bw/day of cyclophosphamide (CPS) via IP injection once daily for 4 consecutive days before sacrifice (s tudy days 24 through 27). All mice were immunized with a single in jection of 0.2 mL of 1 x 10 8 

### sheep red blood cells (sRBC) via the lateral tail vein on study day 24; in the positive control group the sRBC immunizations were administered prior to CPS dose administration. On day 28 (peak day of IgM response), the animals were sa crificed and spleens were aseptically removed and weighed. Spleens were then placed into tubes containing appropriate media (EBSS in HEPES medium with gent amicin added as a bacteriostat) and shipped on ice to the laboratory performing the immunotoxicity studies. Upon arrival at the laboratory the following day, the spleen cells were prepared and the immunological evaluations performed. The primary IgM response to sheep erythrocytes was measured using a modification of the original hemolytic plaque assay of Jerne (1963, 1974) and as describe d by White et al. (2010) . After isolation and resuspension of the spleen cells, 1: 30 and 1:120 dilutions were prepared. An aliquot of spleen cells from each suspension was added to separate test tubes, each containing guinea pig complement, sRBC, a nd warm agar (0.5%). After thoroughly mixing, each test tube mixture was plated on to a separate petri dish, covered with a microscope cover slip, and incubated at approximately 36.4°C for 3 hours. Spleen cell counts were performed on the 3 mL sa mples using a Model Z1 Coulter Counter. The cells/spleen, AFC/10 6 

### spleen cells, and AFC/spl een were determined. The plaques that developed were counted usi ng a Bellco plaque viewer. The data areImmunotoxicity (2013) Page 8 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None


### expressed as Specific Activity (AFC/10 6 

### Spleen Cells) and Total Spleen Activity (AFC/Spleen).

II. RESULTS:

A. OBSERVATIONs:

1. Clinical signs of toxicity: No treatment-related clinical signs were noted. Clinical signs that were observed occurred in low incidence and/or were not related to dose and included an abnormal pupil position in the eye; scabbing on the e nd of the tail or on th e left and right ear; and missing the end of tail or tail sheath.

2. Mortality : All animals survived to th e scheduled termination date.

B. Body weight and weight gain: No statistically significan t, treatment-related effects on body weight were observed in mice treated with fosamine ammonium or with the positive CPS control. Selected body weight and body we ight gain data are summarized in Table 2 and Figure 1.

TABLE 2. Average body weights and body we ight gains during 28 days of treatment a

Dose rate

(ppm) Body weights (g±SD) Total weight gain Week 1 (Day 7) Week 2 (Day 14) Week 3 (Day 21) Week 4 (Day 28) g % of control b

0 23.8 ± 1.9 24.0 ± 1.9 25.0 ± 2.0 26.7 ± 1.9 3.3 ± 1.0

- Low 23.6 ± 1.8 24.2 ± 0.8 25.3 ± 1.9 26.7 ± 2.1 3.6 ± 1.4 ↑ 9% Mid 24.3 ± 1.9 24.5 ± 1.9 25.9 ± 3.6 27.4 ± 2.0 4.4 ± 1.6 ↑ 33% High 24.6 ± 2.0 24.6 ± 2.2 25.9 ± 2.5 27.4 ± 2.7 4.0 ± 1.9 ↑ 21% CPS Positive

Control 24.1 ± 1.5 24.2 ± 1.8 25.5 ± 2.1 26.5 ± 2.6 3.4 ± 1.3 ↑ 3%

a Data obtained from MRID 49121701, pp. 57-62 in the study report.
b Percentage of controls; calculated by reviewerImmunotoxicity (2013) Page 9 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None



### C. FOOD CONSUMPTION:

1. Food consumption: Food consumption was not affected by treatment with fosamine ammonium or by CPS.

2. Food efficiency: Food efficiency was not calculated.

D. GROSS NECROPSY: No treatment-related findings were observed during necropsy. Findings were of low incidence and not relate d to dose, and included a clear fluid in the uterus and ovarian cysts.

Gross necropsy findings in the CPS positive control group were limited to a small thymus in 5/10 animals.

1. Organ weight: Treatment with fosamine ammonium di d not affect absolute or relative (to body weight) liver weight, thymus weight or spleen weight.

Organ weights affected in CPS positive cont rol animals included reduced absolute and relative (to body weight) spleen weight (-37% for both) and thymus weight (-68 and -67%, respectively). Liver we ight was not affected.

2. Histology : Histological examination was not performed.Immunotoxicity (2013) Page 10 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None



### E. IMMUNOTOXICITY TESTS:

a. Antibody plaque-forming cell (PFC) assay: Immunotoxicity findings for the antibody plaque-forming cell assay are summarized in Table 3 and Figure 2. A high interindividual variability was noted in all the treatment groups as well as in the control group. Evaluation of individual animal data did not show any tre nd or distribution that would demonstrate significant suppre ssion of SRBC antibody response. The data suggest that under the conditions of this study, fosamine ammonium did not suppress the humoral immune response in a dose-dependent manner in that it did not signi ficantly alte r the IgM antibody-forming cell response to the T-de pendent antigen, sheep erythrocytes.

Sensitivity of the assay was adequately demonstr ated with the positive control responses. Treatment with the positive control CPS resulte d in statistically signi ficant decreases in spleen cell numbers, Specific Activity, and To tal Spleen Activity, indicating there was a suppression of the antibody- forming cell response.

TABLE 3: Antibody plaque-forming cell (AFC) assay (a) Test group

(n = 10) Spleen cells (x 10 7 ) Specific activity (IgM AFC/10 6 spleen cells) Total Spleen activity (IgM AFC/spleen (x 10 3 ))

Control 14.37 ± 0.57 1209 ± 205 168 ± 27 Low 15.68 ± 1.30 978 ± 176 155 ± 34 Mid 15.57 ± 1.04 1482 ± 380 233 ± 64 High 19.46 ± 2.25 1609 ± 377 307 ± 67 CPS Positive Control 7.27** ± 0.55 23** ± 15 b 1** ± 1 b

a Data obtained from MRID 49121701, p. 169.
b n=9; one animal was an outlier according to Q-test for outliers * Statistically different (p <0.05) from the control. ** Statistically different (p <0.01) from the control.Immunotoxicity (2013) Page 11 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None



### Figure 2. Taken from MRID 49121701, p. 165.

III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS: Based upon the results of this phase, fosamine ammonium administered via oral gavage for 28 consecutive days to female CD-I mice at dosage levels of 100, 500, and 1000 mg/kg bw/day resulted in a no-observed-effect level (NOEL) for the AFC assay (humoral immune response) of 1000 mg/kg/day, the highest dosage level evaluated.

B. REVIEWER COMMENTS:

Treatment with up to 1000 mg/kg bw/day of fosamine ammonium for 28 days did not adversely affect clinical si gns, body weight or body weight gain, or food consumption. Gross necropsy did not reveal any treatment-re lated effects, and liver, spleen, and thymus weights were unaffected.

The systemic toxicity NOAEL for fosamine ammonium in female Crl:CD-1 mice was 1000 mg/kg bw/day; the LOAEL was not estab lished (limit dose tested).

Fosamine ammonium did not suppress the humoral immune response using the sRBC Assay. The CPS positive control responded appropriately, exhibiting statistically significant decreases in absolute and relative spleen and thymus weights, spleen cell numbers, Specific Activity (AFC/10 6 

### spleen cells) and Total Spleen Activity (AFC/Spleen).

The immunotoxicity toxicity NOAEL for fosamine ammonium in female Crl:CD-1 mice was 1000 mg/kg bw/day; the LOAEL was not establ ished (limit dose tested).Immunotoxicity (2013) Page 12 of 12 FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None



### C. STUDY DEFICIENCIES:

No major study deficiencies were identified. Although a L OAEL was not identified, the limit dose was tested. It is noted that stabilit y and homogeneity data could not be verified because they were determined in a previ ous study and the data were not provided.

D. REFERENCE:

Jerne, N.K. and Nordin, A.A. (1963). Pl aque Formation in Agar by Single AntibodyProducing Cells. Science, 140:405.

Jerne, N.K., Henry, C., Nordin, A.A., Fuji, H., Koros, A.M., and Lefkovits, I. (1974). Plaqueforming cells: Methodology and theory. Trnspl. Rev. 18:130-191.

White, K.L., Musgrove, D.L., Brown, R.D. (2010). The Sheep Eryt hrocyte T-Dependent Antibody Response (TDAR). In Immunotoxicity Te sting: Methods and Pr otocols, 1st ed.; Dietert, R.R., ED.; Methods in Molecular BiologyTM, Volume 598, Part 4; Humana Press: New York, NY, 2010; pp. 173-184.