UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

MEMORANDUM 

Date: June 3, 201 5 

OFFICE OF 

CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

SUBJECT: Fosamine Ammonium. Review of 3 new DER.s and updating and writing 

executive swnmaries for three previously submitted studies. 

PC Codes: 106701 

Decision No.: 480859; 482631 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: 0057080 

MRID No.: See Table 

DP Barcode: D423543; 0414644; D413146 

Registration Nos.: NA 

Regulatory Action: Reviewing and updating DER.s 

Case No.: 2355 

CAS No.: 25954-13-6 

40 CFR: None 

FROM: Ayaad Assaad, D.V.M., Ph.D., Toxicologist ~~ THROUGH: Charles 

RAB IV, 

(B

HED 

illy) 

(7509P) 

Smith, Branch Chief(Acting

t___J 

v-=='i ~ 

TO: Russell Wasem/Thomas Moriarty RM 51 

Risk Management and Implementation Branch 1 

Pesticide Re-Evaluation Division (7508P) 

I. CONCLUSIONS 

RAB 4 has reviewed the following toxicology studies available for fosamine ammonium, 

and used in the hazard characterization of the chemical have been reviewed, and the Data 

Evaluation Records [DERs] and amended DER.s [Executive Summaries] are appended. 

The toxicology database on fosamine ammonium is considered complete. Data are 

sufficient for selecting endpoints for all exposure scenarios and for FQPA evaluation. 

There is also a complete mutagenicity battery. There are no data gaps at this time. 

II. ACTION REQUESTED 

Review 3 new studies submitted by the registrant: 

I . Immunotoxicity study in the mouse. 

2. 4-week inhalation toxicity study in rats. 

3. Prenatal toxicity study in rabbits. 

Page I of2 

Page 1 of 53
Page 2 of 2 

Update and generate new executive 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/guideline 

Page 2 of 53
Fosamine 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 test 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 perfused 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. Increases 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 statistically significant differences were 

observed in either forelimb or hindlimb grip strength, hindlimb foot splay or any other FOB 

1 

Page 3 of 53
Acute 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 any dose level when compared to controls. No 

differences were observed between the treated and control groups in any of the motor activity 

parameters. No treatment-related effects 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 acceptable/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 and it does alter the conclusions 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 provide

Page 4 of 53
Fosamine anunonium/106701 

Subchronic Neurotoxicity Study (rats) 1993 /Page l of.l, 

OPPTS 870.6200b/DACO 4.5.13/0ECD 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, 

09124193. 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 rats 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, 

Page 5 of 53
Subchronic Neurotoxicity Study (rats) 1993 / Page 2 of 2 

Fosamine ammonium/106701 OPPTS 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 with 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 mortality, clinical signs, body weight, brain weight or 

gross and histologic pathology or neuropathology. FOB and motor activity testing revealed no 

treatment-related effects. In addition, there were no statistically 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, erythrocytes, 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 neurotoxicity study in rats (870.6200b). 

COMMENTS: This is a revised Executive Summary only 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 current guidelines where NOAEL replaced the commonly 

used term NOEL 1990th. 

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality 

statements were provide

Page 6 of 53
Fosamine 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 Reproduction 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, Inc. 

EXECUTIVE SUMMARY: 

In a 90-day oral toxicity study (MRID # 00075736) Fosamine ammonium technical [(purity not 

specified); 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 assigned to the reproduction Phase (Phase II) of the study. The highdose animals received 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 signs in any group of either sex. However, 

individual animal data were not provided for clinical signs. There were slight, temporary 

decreases 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. 

However, by week 13, the body weight and food consumption of the high-dose rats was 

comparable to controls 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 

1 

Page 7 of 53
Subchronic (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 food consumption observed at the high-dose are 

considered non-adverse since only temporary 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 control and treated group individual 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 one 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 control males (none in control females), 0 in the low dose males or 

females, 5 mid-dose (3 males, 2 females), and 4 high-dose males (no females). The kidney 

histopathology also did not occur 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 slight in severity. There were no treatmentrelated changes in kidney weights, clinical chemistry or urinalysis to corroborate the kidney 

histopathology incidence. Therefore, based on: 1) the lack of severity of the kidney 

histopathology observed in a non-linear 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 changes 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 histopathological lesions. Adaptive hyperplasia 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 progress with dose or time, and the 

process is reversible. 

 

Page 8 of 53
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-day LOAEL was not established since the 

kidney histopathology is not considered adverse. 

This 90-day oral toxicity study in the rats predates the current 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 reproduction), six males and six females from each of 

the groups on the three-month feeding phase were used to establish groups of P1 animals. These 

animals were kept on their respective control and treatment 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 divided 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 significantly impaired up to the 

250/500 mg/kg/day dose. A second generation of reproduction parameters is not available nor is 

the offspring data as in current 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. 

 

Page 9 of 53
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): 

 

Page 10 of 53
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. Reproduction 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 for a reproduction study in rats.

COMMENTS: This is a revised Executive Summary and it does alter the conclusions 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. 

Page 11 of 53
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 Corporation 

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-11·014 

Page 12 of 53
FOSAMINE AMMONIUM /106701 

TXR#:0057080 

4-Week Inhalation Toxicity Study (rat) (2013) I Page 2 of 13 

OCSPP 870.3465; OECD 412 

DATA EVALUATION RECORD 

STUDY TYPE: 28-Day Inhalation Toxicity - Rat 

OCSPP 870.3465. 

PC CODE: 106701 DP BARCODE: 0414644 

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

SYNONYMS: Ammonium ethyl carbamoylphosphonate 

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. Unpublished. 

SPONSOR: Albaugh, 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 

exposure concentrations ofO, 0.1, 0.3, or 1 mg/L (actual exposure concentrations ofO, 0.10, 

0.30, or 1.0 mg/L). The MMADs (mass median aerodynamic diameters) were 2.5, 2.7, and 2.8 

μm for the low-, mid- and high concentrations, respectively. 

All animals survived until scheduled sacrifice. Yellow and/or brown material was observed 

around the anogenital area and limbs in males and females in the high exposure groups and in 

females in the mid-exposure group. No statistically significant effects were observed in body 

weight, body weight gain, or food consumption. There were no treatment-related effects on 

hematology or clinical chemistry parameters at any exposure concentration. Urine specific 

gravity was statistically higher in females at the high exposure concentration (p<0.05). No 

lesions were observed during the ophthalmoscopic examinations. Absolute (p<0.05) and relative 

(p<0.01) thymus 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 thymus weights were stated to be within the historical control range (data not 

provided). A higher incidence 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 with 

Page 13 of 53
Prenatal 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 treatment. 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/day group during GD 13-20 (24% lower; 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% lower, 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-adverse. 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 NOAEL 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, visceral and skeletal development. 

The developmental LOAEL for fosamine ammonium in rabbits was not determined, and 

the developmental NOAEL was greater than 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 developmental 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.

Page 14 of 53
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 testing laboratory on GD 1, 2 or 3 

Source: Covance Research Products, Inc. (Greenfield, IN) 

Housing: Individually in clean, suspended, stainless-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 animals 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, start 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), confirmation of mating, and verification of males as the same strain 

were not documented. 

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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 block design and assigned to dose groups as 

indicated in Table 1. Animals not assigned 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 toxicity study of fosamine ammonium in rabbits 

(WIL-925001). In that study, 6 time-mated rabbits/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 dosing 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 was oral (gavage) because this is a potential 

route of exposure for humans. Historically, this 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 amounts 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 room temperature storage for 10 days and 

remixing for a minimum of 30 minutes, samples were collected from the top and bottom 

strata of an aliquot of each formulation 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 performance liquid chromatography (HPLC). Mean 

measured concentrations were 9.98 mg/mL (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 following 10 days at room temperature ranged 

from 103% to 104% of target values. Concentrations 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 formulations, respectively. 

Concentration analysis: On two separate dates of preparation, the mean concentrations of the 

10, 30, and 70 mg/mL dosing formulations ranged 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. 

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The analytical data indicated that the mixing procedure was adequate 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. Individual clinical observations were recorded daily from the day 

of receipt through gestation day 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 data 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 performed blind to treatment group. Examinations at 

sacrifice consisted of the following: the thoracic, abdominal, and pelvic cavities were 

opened by a ventral mid-line incision, and the contents, including placenta, were examined. 

The uterus and ovaries were exposed and excised and the number of corpora lutea on each 

ovary was recorded. The trimmed uterus was weighed, and the number and location of all 

fetuses, early and late resorptions, and the total number of implantation sites were recorded. 

Uteri with no macroscopic evidence of implantation were opened and placed in 10% 

ammonium sulfide solution for detection of early implantation loss. Maternal tissues were 

preserved in 10% neutral-buffered formalin for possible future histopathologic examination 

as indicated by the gross findings. Representative sections of corresponding organs from a 

sufficient number of control animals were retained 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, individually 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 fetuses were examined by a midcoronal slice. All 

carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation, each fetus 

was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue, and 

then examined for skeletal malformations and developmental variations. External, visceral, 

and skeletal findings were recorded as developmental variations or malformations. The fetal 

developmental findings were summarized by: 1) presenting the incidence 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. 

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D. DATA ANALYSIS:

1. Statistical analyses: Analyses were conducted using two-tailed tests (except as noted 

otherwise) for minimum significance levels of 1% and 5%, comparing each test substancetreated group to the control group. Data obtained from non-gravid animals were excluded 

from statistical analyses. Where applicable, the litter was used as the experimental unit. 

Maternal body weights, body weight changes, and 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) intergroup variance, Dunnett’s t-test was used to 

compare the test substance-treated groups to the control group. Mean litter proportions 

(percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, 

total resorptions, pre- and post-implantation loss, and fetal sex distribution), total fetal 

malformations and developmental variations (external, visceral, skeletal, and combined), and 

each particular external, visceral, and skeletal malformation or variation were subjected to 

the Kruskal-Wallis nonparametric ANOVA test. If the ANOVA revealed significant 

(p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated 

groups to the control group. 

The Reviewer considers the analyses 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 (%) = Resorptions (Early/Late)/Litter x 100 

No. Implantation Sites/Litter 

Fetal Indices: 

Summation per Group (%) = Sum of Viable Fetuses Affected/Litter (%) 

No. Litters/Group 

Where: 

Viable Fetuses Affected/Litter (%) = No. Viable Fetuses Affected/Litter x 100 

No. Viable Fetuses/Litter 

3. Historical control data: Historical control data were provided to allow comparison with 

concurrent controls. Maternal and fetal data (e.g., number of viable fetuses/dam, pre- and 

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post-implantation loss, fetal body weights, external, 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 aborting, 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 observations 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, and similar clinical findings were not observed 

for other females in the same dose group, morbidity 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 the anogenital area, decreased defecation, soft stool, and small 

feces prior to abortion/death. The female that aborted 7 late resorptions on GD 24 had 

clinical findings of hypoactivity, a pale and cool body, decreased 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. All 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 minimal 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 control, 100, 300, and 700 mg/kg bw/day groups survived to the 

scheduled necropsy on GD 29. Although not statistically significant, test substance-related 

clinical findings included decreased defecation 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 corresponded to reduced mean food consumption noted 

in these groups. Other findings noted in the test substance-treated 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 conducted approximately 2 hours post-dosing. 

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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 data 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 effects on mean body weight gains during GD 7-

10 and/or 13-20 were not statistically significant and did not affect mean absolute body 

weights. Statistically significant (p<0.01 or p<0.05) differences in mean body weight gain 

were sporadically noted on individual gestation days throughout the treatment period (e.g., 

GD 11-12, 13-14, 16-17, and 21-22); however, these results were generally transient, 

discordant in direction of change, and/or did not occur in a dose-related 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, mean adjusted (net) 

terminal body weights, net body weight changes, and gravid uterine weights were similar to 

the control group. 

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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, rounded to the nearest whole number by the Reviewer. 

b Three females in the 700 mg/kg bw/day group had unscheduled 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 summarized in Table 4 and as follows. 

Although not statistically significant, at 700 mg/kg bw/day, maternal food consumption 

(g/animal/day or g/kg/day) was slightly lower than control during GD 7-10 (~15% lower). 

These results corresponded to slightly lower mean body weight gain (32% lower) during GD 

7-10. Differences in g/kg/day food consumption 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, slightly lower, not statistically significant, 

mean food consumption was noted during GD 13-20, corresponding to reduced mean body 

weight gains during GD 13-20. Differences were significant (<0.01 or p<0.05) during GD 

17-20 for both the 700 and 300 mg/kg bw/day groups, 20% and 18% lower, respectively. 

Significantly lower (19%, p<0.05) mean food consumption was noted in these groups during 

GD 22-24; however, these effects were transient and/or did not occur in a dose-related 

manner. Mean food consumption was similar to the control group throughout 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 consumption 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 were considered test 

substance-related; however, the effects were slight and the corresponding 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. 

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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 females 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 findings, 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. Macroscopic 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 were euthanized prior to scheduled necropsy. All other females 

survived to the scheduled necropsy on GD 29, and no test substance-related internal findings 

were observed in the surviving animals at any dosage levels up to 700 mg/kg bw/day. 

Macroscopic findings, such as cystic oviducts and accessory spleen, and other single 

incidence findings observed in the test groups occurred infrequently, at similar frequencies in 

the control group, and/or in a manner that was not dose-related. Therefore, no treatment 

related effects on gross pathology were noted.

5. Cesarean section data: Cesarean section data are summarized in Table 5 and as follows. 

Intrauterine growth and survival were unaffected by test substance administration at dosage 

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levels of 100, 300, and 700 mg/kg bw/day. Parameters evaluated included postimplantation 

loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora 

lutea and implantation sites and the mean litter proportions of pre-implantation loss were 

similar across all groups. Differences from the control 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. 

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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 litter 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 same respective groups, and were considered to be 

spontaneous in origin. When the total malformations and developmental variations were 

evaluated on a proportional basis, no statistically significant differences from the control 

group were noted. The mean (±SD) total percent per litter with variations (visceral and/or 

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skeletal) was 68±27%, 67±20%, 75±20%, and 70±28% in the 0, 100, 300, and 700 mg/kg 

bw/day dose groups, respectively. Fetal malformations and developmental variations, when 

observed in the test substance-treated groups, 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 

WIL historical control data ranges. Based on these data, no fetal malformations or 

developmental variations were attributed to the test substance.

1. External examination: External malformations were noted for 2 fetuses in the 300 mg/kg 

bw/day group: microphthalmia (right) was noted 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 proportions of these findings were not statistically significantly 

different from the control group and/or were within 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 external 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 variations included accessory spleen(s) and major 

blood vessel variation, among other less frequent variations; these occurred infrequently, at 

similar frequencies in the control group, and/or in a manner that was not dose-related. The 

mean (±SD) percent per litter with visceral variations was 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 statistically significantly different from the 

control group and were within the ranges of values in the WIL historical control data, with 

the following exception. The mean litter proportion 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 noted for 3 (2) fetuses (litters) and white 

precipitate contents of the kidneys were noted for 3 (1) fetuses (litter). These findings were 

not classified as either malformations or developmental variations, 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 (litter) in the 100 mg/kg bw/day group. In the 700 

mg/kg bw/day group, two fetuses in the same litter had vertebral anomaly without associated 

rib anomaly, consisting of absent thoracic arches and centra, extra lumbar 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/day 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 proportions of these findings were not statistically 

significantly different than the control group and were within the ranges of values in the WIL 

Page 25 of 53
Prenatal 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 skeletal malformations were noted in the 300 mg/kg 

bw/day group. Therefore, these malformations were not test substance related. Observed 

skeletal variations, including 13th rib(s) (full and rudimentary), ossified 5th and/or 6th

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 skeletal variations was 59±27%, 58±25%, 60±24%, and 66±27% in the 

0, 100, 300, and 700 mg/kg bw/day dose groups, respectively. The mean litter proportions of 

these findings were not statistically significantly different from 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 together. 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) incidence 

Page 26 of 53
Prenatal 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 anomaly with or without 

 associated rib anomaly 0 c

 1 (1) 0 2 (1) 

 Rib anomaly 0 1 (1) 0 1 (1) 

Variations: 

 13th full rib(s) 65 (18) 78 (20) 64 (20) 59 (18) 

 13th 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 more frequently observed variations (incidence >5) are listed. 

c

 Fetal (litter) incidence. 

III. DISCUSSION AND CONCLUSIONS:

A. INVESTIGATORS’ CONCLUSIONS: No test substance-related 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 euthanized and 1 female was found dead after 

aborting. In the 300 mg/kg bw/day group, 1 female was found dead. The abortions and/or 

mortality noted for these females was attributed 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/day 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 noted for this female, the morbidity of this 

animal was not considered test substance-related. Slightly lower mean body weight gains, 

with corresponding effects on mean food consumption, 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 substance-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. Therefore, these results were not considered 

adverse. 

Based on the lack of adverse effects at any 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. 

Page 27 of 53
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 with 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 (decreased 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 difference in mean absolute body weights. The 

Reviewer agrees that these effects were not adverse and that the 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 control 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, implantation sites, and litter 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 similar frequencies across groups and were 

considered to be spontaneous in origin. 

The developmental LOAEL for fosamine ammonium 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 examination of the internal structures of the head was done by midcoronal slice in all cases; whereas, according 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 sectioning 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. 

Page 28 of 53
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-11-014 

Page 29 of 53
FOSAMINE / 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 Project ID: WIL-925005, April 22, 2013. MRID 49121701. 

Unpublished. 

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 administered to 10 female Crl:CD-1 mice/dose by gavage once daily for 28 

consecutive days at dose levels 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 108 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 spleen was processed for 

shipment to the laboratory conducting the immunological evaluations. 

Treatment with up to 1000 mg/kg bw/day of fosamine anunonium for 28 days did not adversely 

affect clinical signs, body weight or body weight gain, or food consumption. Gross necropsy did 

not reveal any treatment-related 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 established Qimit dose tested). 

Page 30 of 53
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 euthanasia in this strain 

of rat (historical data not provided). Adrenal gland and kidney weights and relative-to-body 

weights were statistically increased in females from the high exposure group. Minimal to mild 

hypertrophy of the adrenal cortex that correlated with the increased adrenal weight was observed 

in 2/10 high concentration females. This microscopic lesion was not considered adverse, since 

the finding was stated to be associated with the stress of exposure, although no control animals 

had this lesion and historical data were not provided. Minimal to moderate 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. No metaplasia or degenerative changes were 

observed. Based on the scoring criteria from the pathologist report, the minimal and mild scoring 

are not considered adverse due to the lack of functional impairment, absence of metaplasia and 

degenerative changes, were observed. Additionally, 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 limited tissue or organ dysfunction. Therefore, the 

minimal and mild hyperplasia observed is not considered an adverse effect in this study. 

Based on the effects seen in this study, a NOAEL 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 inhalation 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. 

Page 31 of 53
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, PMI 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 exposure 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 noted in Table 1. Concentrations were selected based on the 

results of a 5-day inhalation toxicity study in that resulted in no microscopic findings. No 

details were provided. 

Page 32 of 53
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 concentration. An auger-type feeder (Accu-Rate) was 

used to deliver the test substance 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 article concentration. The animals were gradually 

acclimatized to the restraining devices for five days prior to the 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 filters. Results are in Table 1 and show that 

the achieved mean test substance concentrations were 100% of target values. Homogeneity 

of concentrations within the chamber 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 equilibrium was not reported. 

5. Particle size determination: Particle size for each exposure concentration was determined at 

least four times during the study using 7-stage stainless steel cascade impactors. The mass 

median aerodynamic diameter (MMAD) and geometric standard deviation (σg) were 2.5 μm 

(2.20), 2.7 μm (2.42), and 2.8 μm (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 consumption, organ weight, and clinical pathology 

were subjected to a one-way parametric analysis of variance (ANOVA) to determine 

intergroup differences. When significant differences 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, 

Page 33 of 53
4-Week Inhalation Toxicity Study (rat) (2013) / Page 6 of 13 

FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412 

abnormal behavior, morbidity, and mortality. 

 1b. Clinical examinations: Clinical examinations were conducted prior to exposure, at 

the midpoint of daily exposure, and at the end of exposure. Detailed clinical 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 Hemoglobin distribution width 

 (Clotting time) X RBC distribution width 

X (Prothrombin time) Mean platelet volume 

* Recommended for subchronic inhalation studies based on Guideline 870.3465

Page 34 of 53
4-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 Serum protein electrophoresis 

 Lactic acid dehydrogenase (LDH) X Albumin/Globulin ratio 

X Alanine aminotransferase (ALT/also SGPT)* X Appearance 

X Aspartate aminotransferase (AST/also SGOT)* 

X Sorbitol dehydrogenase* 

X Gamma glutamyl transferase (GGT)* 

 Glutamate dehydrogenase 

* Recommended for subchronic inhalation studies based on Guideline 870.3465

c. Cholinesterase determination: Not conducted. 

6. Urinalysis: Urine was collected from all animals 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 exsanguination while under isoflurane 

anesthesia. Any gross lesions were recorded and tissues were collected from animals in all 

exposure groups and processed for microscopic 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. 

Page 35 of 53
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, thoracic* 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 Epididymides*+ 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 around 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). 

 

Page 36 of 53
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 effects on hematology parameters at any 

exposure concentration. Lower mean corpuscular hemoglobin concentration was 

observed in high exposure males relative to control, but the investigators stated that 

neither the individual values nor mean value were outside the historical 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 higher in females at the high exposure 

concentration (p<0.05). No other treatment-related 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 areas 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. 

 

Page 37 of 53
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 Table 3. Adrenal gland and 

kidney weights and relative-to-body weights were statistically increased in females from 

the high exposure group. Minimal to mild hypertrophy 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 thymus weight was higher at the low exposure 

concentration (p<0.05). The differences in thymus and kidney weights were stated to be 

within the historical control range (historical data not provided). 

Table 3. Changes in selected 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 changes 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 highest concentration. No metaplasia or 

degenerative changes were observed at any concentration. The pathologist report and 

scoring definitions are included 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. The incidence and severity of 

the thymus lesions do not follow a consistent dose-response pattern. The investigators 

stated that this finding was a perimortem anomaly that occurs at the time of euthanasia, 

Page 38 of 53
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 concluded 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. 

Page 39 of 53
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’ conclusions. The thymus and adrenal 

hypertrophy are not considered adverse 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 scoring definitions from the pathologist 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 NOAEL 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 inhalation 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). 

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 microscopic findings were noted in the nasal cavity in all test substancetreated male and female groups. Mucous cell hyperplasia was observed within the nasal cavity of 

the 0.1, 0.3, and 1 mg/L group males and females at several levels. The change was characterized 

by increased numbers of mucous-containing cells within the respiratory epithelium lining the 

nasal turbinates (in levels II and IV) and nasopharyngeal duct (in levels V and VI). A doseresponse was present for both sexes and the change 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 transitional 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 correlated with higher adrenal gland weights noted in the test 

substance-exposed female groups. This change was not observed in any of the 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 within normal limits, under the conditions of 

the study and considering the age, sex, and strain of the animal concerned. 

Page 40 of 53
4-Week Inhalation Toxicity Study (rat) (2013) / Page 13 of 13 

FOSAMINE AMMONIUM /106701 OCSPP 870.3465; OECD 412 

B. Present: A non-gradable histologic change for which no additional information would 

be gained by assigning a severity 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 change that is easily identified but of limited severity. 

This change probably does not produce any functional impairment. 

E. Grade 3 (Moderate): A histologic change that is a prominent feature of the tissue but 

not of maximal severity. Limited tissue or organ dysfunction is possible. 

F. Grade 4 (Severe): A histologic change that is an overwhelming feature of the tissue 

and that may cause significant tissue or organ dysfunction. 

Page 41 of 53
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 

Summitec Corporation 

9724 Kingston Pike, Suite 602 

Knoxville. Tennessee 37922 

Task Order No. 6-73 

. ,. 

Dare: 

Signature:-'J. _______:______013 ~ 

Signature: VU'ff""'- , 

't.'\n ........ .,.~~IE 

Date: 11 I an I J; 

15 

Robert H. Ross, M.S .• Program Manager 

Date: 

Signature: ~~~ ~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.S. Environmental Protection Agency under Contract No.EP-W-11-014 

Page 42 of 53
FOSAMINE/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 

. 0413146 

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/dose 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 lutea counts, and 

the numbers and positions of live and dead fetuses, and early and late resorptions were recorded, 

and all fetuses were weighed and examined for external anomalies. Live fetuses were sexed 

internally and subjected to visceral examination by fresh dissection (with heads examined by 

mid-coronal slice) then processed for and subjected skeletal examination. 

In the 700 mg/kg bw/day group, 3 females experienced unscheduled mortality: 1 female was 

euthanized in extremis on GD 10 due to extreme 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 findings consisted of 

decreased defecation, soft stool, and brown material on the anogenital area in the 700 mg/kg 

bw/day group, with similar but less frequent effects in the 300 mg/kg bw/day group; these 

findings were not statistically significant. 

Page 43 of 53
Immunotoxicity (2013) Page 3 of 12 

FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None 

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/106

 spleen cells) or 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 established (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. 

Page 44 of 53
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 Laboratories, 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 body weight stratification in a block design. 

Test substance was administered orally by gavage in a dose volume of 10 mL/kg once daily 

for 28 consecutive days; a cyclophosphamide (CPS) positive control group received four 

daily intraperitoneal (IP) injections of 50 mg/kg bw/day of cyclophosphamide on days 24-27. 

The first day of fosamine ammonium dosing was designated as day 0. Individual doses were 

Page 45 of 53
Immunotoxicity (2013) Page 5 of 12 

FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None 

based on the most recently recorded body weights to provide the correct mg/kg bw/day 

dosage. 

 The Spleen Antibody-Forming Cell Response (AFC Assay) was used to measure humoral 

immunity via the T-cell dependent antibody response to sheep red blood cells (sRBC). All 

mice in the AFC assay (Groups 1-5) were administered 0.2 mL of 1 x 108 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 (cyclophosphamide monohydrate) once daily at 50 mg/kg bw/day on study days 24-27 by 

iv injection. 

3. Dose selection 

The dose levels were selected based on the results from dose range-finding study where 

groups of 4 female Crl:CD-1 mice were administered 100, 500, or 1000 mg/kg/bw/day of 

fosamine ammonium by oral gavage daily for 5 consecutive days. No observable effects 

were observed in mortality, morbidity, clinical signs, physical examination, or body weight. 

4. Dose formulation preparation and analysis

The vehicle for administration to the vehicle control 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 substance formulations were stirred continuously 

throughout the preparation, sampling, and dose administration 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 absorbance detection: once before the initiation of 

dosing, and once during the fourth week of dosing. The author stated that test substance 

homogeneity and stability following 10 days of room temperature storage were assessed and 

verified in formulations ranging in concentration 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 previous study; the data were not provided. 

Page 46 of 53
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 preparation; 101-103% of nominal for the 50 mg/mL 

formulation; and 101-103% of nominal for 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 standard deviation (S.D.), standard error 

(S.E.), and the number of animals (N) used to calculate 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 statistical tests were performed using WTDMSTM 

unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted 

otherwise) at minimum significance levels of 1% and 5%, comparing each test substancetreated group to the vehicle control group. 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 (Group 1). The positive control 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 definitive phase of this study were analyzed by 

ImmunoTox®, Inc. The AFC data were expressed as both Specific Activity, IgM antibodyforming cells per million spleen cells (AFC/106

 spleen cells), and as IgM total spleen activity 

(AFC/spleen). Data were first tested for homogeneity of variances using the Bartlett’s ChiSquare Test. Homogenous data were evaluated using parametric one-way ANOVA. When 

significant differences occurred, the treatment groups were compared to the vehicle control 

group using Dunnett’s test. Non-homogenous data were evaluated using a nonparametric 

analysis of variance. When significant differences occurred, the treatment groups were 

compared to the vehicle control group using the Gehan-Wilcoxon 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 animals 1 week prior to 

randomization, on the day of randomization, weekly during the dosing period, and on the day 

of scheduled necropsy. 

Page 47 of 53
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 collected 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 surface, all orifices, and the cranial, thoracic, abdominal, and 

pelvic cavities, including viscera. The liver, 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 oral 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 (study days 24 through 27). All mice were 

immunized with a single injection of 0.2 mL of 1 x 108 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 sacrificed and spleens were aseptically 

removed and weighed. Spleens were then placed into tubes containing appropriate 

media (EBSS in HEPES medium with gentamicin 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 described 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, and warm agar (0.5%). After thoroughly 

mixing, each test tube mixture was plated onto 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 samples using a Model Z1 Coulter Counter. 

The cells/spleen, AFC/106

 spleen cells, and AFC/spleen were determined. The 

plaques that developed were counted using a Bellco plaque viewer. The data are 

Page 48 of 53
Immunotoxicity (2013) Page 8 of 12 

FOSAMINE / 106701 OCSPP 870.7800 / DACO 4.8 / OECD None 

expressed as Specific Activity (AFC/106 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 end of the tail or on the left and right ear; 

and missing the end of tail or tail sheath. 

2. Mortality: All animals survived to the scheduled termination date. 

B. Body weight and weight gain: No statistically significant, 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 weight gain data are summarized in Table 2 

and Figure 1. 

TABLE 2. Average body weights and body weight 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 reviewer 

Page 49 of 53
Immunotoxicity (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 related 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 did not affect absolute or relative (to 

body weight) liver weight, thymus weight or spleen weight. 

Organ weights affected in CPS positive control animals included reduced absolute and 

relative (to body weight) spleen weight (-37% for both) and thymus weight (-68 and -67%, 

respectively). Liver weight was not affected. 

2. Histology: Histological examination was not performed. 

Page 50 of 53
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 trend or distribution that would 

demonstrate significant suppression 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 significantly alter the IgM 

antibody-forming cell response to the T-dependent antigen, sheep erythrocytes. 

Sensitivity of the assay was adequately demonstrated with the positive control responses. 

Treatment with the positive control CPS resulted in statistically significant decreases in 

spleen cell numbers, Specific Activity, and Total 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 107) 

Specific activity 

 (IgM AFC/106 spleen cells) 

Total Spleen activity 

(IgM AFC/spleen (x 103)) 

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. 

Page 51 of 53
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 signs, body weight or body weight gain, or food consumption. 

Gross necropsy did not reveal any treatment-related 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 established (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/106

 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 established (limit dose tested). 

 

Page 52 of 53
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 LOAEL was not identified, the 

limit dose was tested. It is noted that stability and homogeneity data could not be verified 

because they were determined in a previous study and the data were not provided. 

D. REFERENCE: 

Jerne, N.K. and Nordin, A.A. (1963). Plaque 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 Erythrocyte T-Dependent 

Antibody Response (TDAR). In Immunotoxicity Testing: Methods and Protocols, 1st ed.; 

Dietert, R.R., ED.; Methods in Molecular BiologyTM, Volume 598, Part 4; Humana Press: 

New York, NY, 2010; pp. 173-184. 

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