CANCER ASSESSMENT DOCUMENT EVALUATION OF THE CARCINOGENIC POTENTIAL OF PHMB PC CODE 111801 TXR No. 0052040 FINAL July 16, 2003 CANCER ASSESSMENT REVIEW COMMITTEE HEALTH EFFECTS DIVISION OFFICE OF PESTICIDE PROGRAMS U. S. ENVIRONMENTAL PROTECTION AGENCY 1200 PENNSYLVANIA AVENUE, NW WASHINGTON, DC 20460 PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT DATA PRESENTATION: Jonathan Chen DOCUMENT PREPARATION: Jessica Kidwell, Executive Secretary COMMITTEE MEMBERS IN ATTENDANCE: ( Signature indicates concurrence with the assessment unless otherwise stated).

William Burnam Marion Copley Vicki Dellarco Kit Farwell Abdallah Khasawinah Nancy McCarroll Tim McMahon Linda Taylor Yintak Woo NON COMMITTEE MEMBERS IN ATTENDANCE ( Signature indicates concurrence with the pathology report and statistical analysis of data, respectively)

John Pletcher, Consulting Pathologist Lori Brunsman, Statistician OTHER ATTENDEES: None PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT ii TABLE OF CONTENTS EXECUTIVE SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 II. BACKGROUND INFORMATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 III. EVALUATION OF CARCINOGENICITY STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1. 2 Year Chronic Toxicity and Carcinogenicity Study in Rats ( 1996) . . . . . . . . . . . . . . . . . . 6 2. 2 Year Oncogenicity Study in Mice ( 1996) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 3. Dermal Carcinogenicity Study in Mice ( 1977) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 IV. TOXICOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 1. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 2. Mutagenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3. Structure Activity Relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4. Subchronic and Chronic Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 5. Mode of Action Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 V. COMMITTEE'S ASSESSMENT OF THE WEIGHT OF THE EVIDENCE . . . . . . . . . . . . 36 VI. CLASSIFICATION OF CARCINOGENIC POTENTIAL . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 VII. QUANTIFICATION OF CARCINOGENIC POTENTIAL . . . . . . . . . . . . . . . . . . . . . . . . . . 40 VIII. BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 1 EXECUTIVE SUMMARY On April 9, 2003, the Cancer Assessment Review Committee of the Health Effects Division of the Office of Pesticide Programs met to evaluate the carcinogenic potential of PHMB.

Dr. Jonathan Chen of the Antimicrobials Division presented the chronic toxicity/ carcinogenicity studies in Wistar rats and C57B1/ 10JfCD 1/ Alpk and Alderley Park mice by describing the experimental design; reporting on survival and body weight effects treatment related non neoplastic and neoplastic lesions, statistical analysis of the tumor data, and the adequacy of the dose levels tested; and presenting the weight of the evidence for the carcinogenicity of PHMB.

PHMB was administered in the diet: 1) to male and female Wistar rats ( 52 rats/ sex) at dose levels of 0, 200, 600, or 2000 ppm ( 0, 12.2, 36.3, or 126.1 mg/ kg/ day for males; 0, 14.9, 45.3, or 162.3 mg/ kg/ day for females) of PHMB for 105 weeks; and 2) to C57B1/ 10J f CD 1/ Alpk mice ( 55/ sex/ group) at 0, 400, 1,200 or 4,000 ppm ( equivalent to 55, 167, or 715 mg/ kg/ day for males and 69, 217, or 856 mg/ kg/ day for females) for 2 years.

In addition, four groups of specific pathogen free ( 50M + 50F) Alderley Park Mice received dermally 0.3 mL of test material at doses of 0 ( solvent in methanol), 0.6 mg ( 0.2% PHMB in ethanol), 6.0 mg ( 2% PHMB in ethanol), or 30.0 mg ( 10% PHMB in ethanol) per mouse per day for five days a week for 80 weeks. The treatment dosages are approximately equivalent to 0, 15, 150 and 750 mg/ kg/ day of 20% PHMB solution.

The CARC concluded that PHMB showed evidence of carcinogenicity based on the following:

Rat  Oral = There was no treatment related increase in any tumors in male Wistar rats.

= Vascular Tumors: Female Wistar rats from the Pathology Working Group ( PWG) diagnoses had significant increasing trends in liver hemangiomas and liver hemangiosarcomas, both at p< 0.05. There was also a significant increasing trend for hemangiomas and/ or hemangiosarcomas at all sites combined at p< 0.01. There were significant differences in the pair wise comparisons of the 2000 ppm dose group with the controls for liver hemangiomas and hemangiomas and/ or hemangiosarcomas at all sites combined, both at p< 0.05. The incidence of hemangiomas and hemangiosarcomas for all sites combined was 2/ 42 ( 5%), 1/ 42 ( 2%), 3/ 40 ( 8%), and 6/ 35 ( 17%) for the 0, 200, 600, and 2000 ppm dose groups, respectively. Historical control data regarding hemangiosarcomas shows the range of hemangiosarcomas by tissue in rats was 0 1.9%. The CARC considered the tumor response at the high dose ( 17% for the combined re read of liver and original diagnoses at other sites) to be treatment related and driven by the increase in hemangiomas when all sites are combined. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 2 = Adequacy of Dosing: The CARC concluded that dosing at the highest dose ( 2000 ppm) was considered to be adequate, but not excessive, for both male and female rats. In high dose females, this was based on increased mortality and a reduction in body weight of 5 8% throughout the study. In high dose males, body weights were significantly reduced ( 4 6%) through week 79. Food utilization ( g growth/ 100 g feed) for the first 12 weeks decreased significantly ( 97 8%) in both sexes at 2000 ppm.

Mouse  Oral = Vascular Tumors: Male C57B1/ 10J f CD 1/ Alpk mice from the PWG diagnoses had significant increasing trends in hemangiomas, hemangiosarcomas, and combined hemangiomas and hemangiosarcomas, all at p< 0.01. There were also significant differences in the pair wise comparisons of the 4000 ppm dose group with the controls, for hemangiomas ( p< 0.01), hemangiosarcomas ( p< 0.05), and combined hemangiomas and/ or hemangiosarcomas ( p< 0.01). The incidence of hemangiomas was 2/ 55 ( 4%), 3/ 55 ( 5%), 4/ 55 ( 7%), 11/ 53 ( 21%), for the 0, 400, 1200, and 4000 ppm dose groups, respectively. The incidence of hemangiosarcomas was 5/ 55 ( 9%), 4/ 55 ( 7%), 6/ 55 ( 11%), and 12/ 53 ( 23%) for the 0, 400, 1200, and 4000 ppm dose groups, respectively. The incidence of combined hemagiomas and hemangiosarcomas was 6/ 55 ( 11%), 6/ 55 ( 11%), 9/ 55 ( 16%), and 20/ 53 ( 38%) for the 0, 400, 1200, and 4000 ppm dose groups, respectively. For hemangiosarcomas, the tumor incidence in the 4000 ppm dose group ( 23%) exceeded the historical control range ( 1.8 18.3%). The CARC considered the increase in vascular tumors ( hemangiomas, hemangiosarcomas, and combined) at the high dose ( which is considered to be excessive) to be treatment related. Although not statistically significant, the tumor response ( 9/ 55 [ 16%] for the combined hemangiomas and hemangiosarcomas) in mice at the mid dose of 1200 ppm is considered treatment related since this tumor type was also seen in female mice ( orally and dermally) and female rats.

= Vascular Tumors: Female C57B1/ 10J f CD 1/ Alpk mice from the PWG diagnoses had significant increasing trends in hemangiomas ( p< 0.05), hemangiosarcomas ( p< 0.05), and combined hemangiomas and hemangiosarcomas ( p< 0.01). There was also a significant difference ( p< 0.05) in the pair wise comparison of the 4000 ppm dose group with the controls, for combined hemangiomas and/ or hemangiosarcomas. The incidence of combined hemangiomas and/ or hemangiosarcomas was 8/ 54 ( 15%), 5/ 53 ( 9%), 7/ 54 ( 13%), and 15/ 49 ( 31%) for the 0, 400, 1200, and 4000 ppm dose groups, respectively. For hemangiosarcomas, the tumor incidence in the 4000 ppm dose group ( 31%) exceeded the historical control range ( 0 10.9%). There are no historical control data for hemangiomas by tissue. The CARC considered the combined vascular tumors at the high dose ( which is considered to be excessive) to be treatmentrelated There were no increases in these tumors at doses which were not considered excessive.

= Rectal Anal Junction Tumors: In male mice there was a significant increasing trend at p < 0.01, and a significant difference in the pair wise comparison of the 4000 ppm dose group with the PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 3 controls at p < 0.05, for rectal anal junction squamous cell carcinomas. The incidence of rectalanal junction tumors was 0/ 45, 0/ 45, 0/ 45, and 5/ 48 for the 0, 400, 1200 and 4000 ppm dose groups, respectively. In female mice there was a significant increasing trend at p < 0.01 and a significant difference in the pair wise comparison of the 4000 ppm dose group with the controls at p < 0.01, for rectal anal junction squamous cell carcinomas. The incidence of rectal anal junction tumors was 0/ 42, 0/ 43, 0/ 43, 8/ 28 for the 0, 400, 1200 and 4000 ppm dose groups, respectively. The CARC considered the carcinomas at the high dose ( which is considered to be excessive) in both males and females to be treatment related but suggestive of a local irritant effect. These tumors did not contribute to the CARC's weight of the evidence analyses.

= Adequacy of Dosing: Dosing was considered to be excessive at the high dose of 4000 ppm based on decreased overall body weight gains of 50% in males and 32% in females at termination. In addition, decreased body weight gains of 33% in males and 19% in females were seen at 13 weeks. The study indicated that the animals at the 4000 ppm dose group had increased food consumption ( p< 0.05 or 0.01) from approximately week 12 through termination in males ( 87 29%) and females ( 87 26%).

Dosing was considered to be adequate, but not excessive, in both males and females at the middose of 1200 ppm based on decreased overall body weight gains ( males, 7%; females, 2%), increases in hematology parameters, and non neoplastic histopathological changes in the gall bladder, liver, and rectal anal junction.

Mouse  Dermal = Vascular Tumors: Female Alderley Park mice had significant increasing trends in liver angiosarcomas ( p < 0.01) and vascular tumors from all sites combined ( p < 0.05). There was also a significant difference in the pair wise comparison of the 30 mg dose group with the controls for liver angiosarcomas at p < 0.05. The incidence of liver angiosarcomas was 0/ 41, 0/ 45, 0/ 40, and 2/ 35 for the 0, 0.6, 6.0, and 30 mg dose groups, respectively. The CARC considered the tumor increase at the high dose to be equivocal since the tumors were only seen at an excessive dose. Vascular tumors were not seen at lower doses.

There was no treatment related increase in any tumors in male Alderley Park mice.

= Adequacy of Dosing: Dosing was considered to be excessive at the high dose ( 750 mg/ kg/ day of 20% PHMB equivalent) in both sexes due to increased mortality ( 78%, high dose, versus 33% and 28% in the male and female controls, respectively) and decreased body weight gain. Overall body weight gain decreases of 45% and 17% were seen in males and females, respectively. No treatment related effects were noted at the mid dose level of 150 mg/ kg/ day of 20% PHMB equivalent. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 4 Mutagenicity = There is no mutagenicity concern.

Structure Activity Relationship = No appropriate structural analogs could be located for comparison purposes. In general, with the exception of exposure via the inhalation route, high molecular weight ( MW) polymers are not expected to be of significant carcinogenic concern because of poor bioavailability. PHMB has indeed been shown to have low bioavailability. However, SAR consideration suggests that there is a likelihood that the lower MW fractions of PHMB may be metabolized to polyamines in analogy to the metabolism of arginine ( a guanidine containing amino acid) to form biogenic polyamines such as putrescine, spermidine and spermine. There is strong evidence that these polyamines are readily taken up by cells and may play an important role in promoting skin and colon carcinogenesis by serving as ingredients to support neoplastic growth. A metabolism study submitted by the company showed that low MW fractions of PHMB can indeed be metabolized in the rat; however, the metabolites have not yet been identified.

Mode of Action = No mode of action studies were available.

In accordance with the EPA Draft Guidelines for Carcinogen Risk Assessment ( July 1999), the CARC classified PHMB into the category " Suggestive Evidence of Carcinogenicity, but Not Sufficient to Assess Human Carcinogenic Potential" by the oral and dermal routes. The quantification of human cancer risk is not required. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 5 Figure 1: Chemical Structure of PHMB I. INTRODUCTION On April 9, 2003, the Cancer Assessment Review Committee ( CARC) of the Health Effects Division of the Office of Pesticide Program met to evaluate the carcinogenic potential of PHMB. This was the first time this compound was assessed for carcinogenicity by the CARC.

II. BACKGROUND INFORMATION PHMB, also known as Baquacil; Baquacil SB; Cosmoquil CQ; polihexanide; polyhexamethylbiguanide; poly( hexamethylenebiguanide) hydrochloride; Vantocil 1B; or polyhexamethylene biguanide. The CAS Number of this chemical is 32289 58 0. The PC Code of PHMB is 111801. PHMB is a group of polymers ( see Figure 1) that has been used as an antimicrobial agent in a wide variety of applications including oil in water and water in oil emulsions, industrial reagents, silicone systems, cellulose solutions and oil recovery systems. Now, PHMB is primarily used as a non chlorinated antimicrobial agent in swimming pool and spa facilities.

On 12/ 18/ 2000, 01/ 25/ 2001, 01/ 30/ 2003 and 02/ 06/ 2003, the Health Effects Division's Hazard Identification Assessment Review Committee ( HIARC) evaluated the toxicology data base of PHMB, established the Reference Doses ( RfDs) and selected the toxicological endpoints for dietary as well as occupational exposure risk assessments.

III. EVALUATION OF CARCINOGENICITY STUDIES PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 6 1. 2 Year Chronic Toxicity and Carcinogenicity Study in Rats ( 1996)

Reference:

Horner, S. A. ( 1996) Polyhexamethylene Biguanide: Two Year Feeding Study in Rats. Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK. Laboratory Project ID: Report No. CTL/ P/ 4663, Study No. PR0936, June 5, 1996. MRID 44059301. Unpublished.

Busey, W. M. ( 1996) Polyhexamethylene Biguanide ( PHMB): Two Year Feeding Study in Rats. Pathology Working Group Peer Review of Proliferative Vascular Lesions in Male and Female Rats. Zeneca CTL, Alderley Park, Macclesfield, Cheshire, UK. Study No. PR0936, Report No. CTL/ C/ 3172, May 13, 1996. MRID 44042801. Unpublished.

A. Experimental Design A combined chronic toxicity/ oncogenicity study in Alpk: APfSD Wistar rats was conducted by Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, England, for Zeneca Specialities, Wilmington, Delaware and dated June 5, 1996 ( Report No. CTL/ P/ 4663, Study No. PR0936, MRID No. 44059301). A Pathology Working Group ( PWG) peer review reevaluated the vascular lesions of this study in a report dated May 13, 1996 ( Report No. CTL/ C/ 3172, MRID No. 44042801). Both the original and the reread diagnoses are presented in this document.

The study design allocated groups of 52 rats per sex to dose levels of 0, 200, 600, or 2000 ppm ( 0, 12.2, 36.3, or 126.1 mg/ kg/ day for males; 0, 14.9, 45.3, or 162.3 mg/ kg/ day for females) of PHMB for 105 weeks. An additional 12 rats per sex per dose were designated for interim sacrifice at week 52.

B. Discussion of Tumor Data Survival Analysis The statistical evaluation of mortality indicated no significant incremental changes with increasing doses of PHMB in male Alpk: APfSD Wistar rats. Female Alpk: APfSD Wistar rats had a significant increasing trend in mortality with increasing doses of PHMB ( p< 0.01) and a significant difference in the pair wise comparison of the 2000 ppm dose group ( p < 0.05) with the control group ( Memo, L. Brunsman, 12/ 03/ 02, TXR No. 0051368). Tables 1 and 2 show the mortality results. Table 1. PHMB  1996 Alpk: APfSD Wistar Rat Study Male Mortality Rates+ and Cox or Generalized K/ W Test Results Weeks Dose 1 26 27 52 53i 53 78 79 105f Total ( ppm) PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 7 0 0/ 64 0/ 64 12/ 64 5/ 52 27/ 47 32/ 52 ( 0) ( 0) ( 10) ( 57) ( 62)

200 0/ 64 3/ 64 12/ 61 7/ 49 26/ 42 36/ 52 ( 0) ( 5) ( 13) ( 58) ( 69)

600 0/ 64 2/ 64 12/ 62 10/ 50 20/ 40 32/ 52 ( 0) ( 3) ( 19) ( 48) ( 62)

2000 3/ 64 2/ 61 10/ 59 5/ 49 22/ 44 32/ 54 ( 5) ( 3) ( 10) ( 48) ( 59)

+ Number of animals that died during interval/ Number of animals alive at the beginning of the interval.

iInterim sacrifice at week 53.

fFinal sacrifice at week 105.

( ) Percent. Note: Time intervals were selected for display purposes only.

Significance of trend denoted at control.

Significance of pair wise comparison with control denoted at dose level.

If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 8 Table 2. PHMB  1996 Alpk: APfSD Wistar Rat Study Female Mortality Rates+ and Cox or Generalized K/ W Test Results Weeks Dose 1 26 27 52 53I 53 78 79 105f Total ( ppm)

0 0/ 64 0/ 64 12/ 64 6/ 52 19/ 46 25/ 52 ( 0) ( 0) ( 12) ( 41) ( 48)**

200 0/ 64 0/ 64 12/ 64 5/ 52 15/ 47 20/ 52 ( 0) ( 0) ( 10) ( 32) ( 38)

600 1/ 64 1/ 63 12/ 62 7/ 50 14/ 43 23/ 52 ( 2) ( 2) ( 14) ( 32) ( 44)

2000 1/ 64 4/ 63 12/ 59 6/ 47 21/ 41 32/ 52 ( 2) ( 6) ( 12) ( 47) ( 62)*

+ Number of animals that died during interval/ Number of animals alive at the beginning of the interval.

iInterim sacrifice at week 53.

fFinal sacrifice at week 105.

( ) Percent.

Note: Time intervals were selected for display purposes only.

Significance of trend denoted at control.

Significance of pair wise comparison with control denoted at dose level.

If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 9 Tumor Analysis The study pathologist and study peer reviewer determined that there were 3 hemangiosarcomas of the liver in 2000 ppm females. Benign hemangiomas were not observed. The increased incidence of hemangiosarcomas in females gave positive results in trend analyses ( p< 0.05). A single observation of benign hemangioma was made in each of the control and high dose males; no hemangiosarcomas were observed in males. A Pathology Working Group ( PWG) was convened in 1996 to confirm the diagnoses of the vascular neoplasms ( MRID 44042801). The PWG determined that there were 2 hemangiomas and 1 hemangiosarcoma in 2000 ppm females and 2 hemangiomas in 2000 ppm males. The PWG concluded that the findings of vascular neoplasms in high dose females were incidental. However, the report of the PWG consensus indicated that no hemangiosarcoma or hemangioma had been observed in female controls in 18 studies with the same strain of rat. Dr. J. M. Pletcher, EPA's consulting pathologist, confirmed the validity of the PWG report ( TXR No. 0052000).

There were no compound related tumors observed in male rats. Female rats from the original study diagnoses had significant increasing trends in liver hemangiosarcomas and hemangiomas and/ or hemangiosarcomas at all sites combined, both at p < 0.05. There was also a significant difference in the pair wise comparison of the 2000 ppm dose group with the controls for liver hemangiosarcomas at p < 0.05.

Female rats from the PWG diagnoses had significant increasing trends in liver hemangiomas and liver hemangiosarcomas, both at p < 0.05. There was also a significant increasing trend for hemangiomas and/ or hemangiosarcomas at all sites combined at p < 0.01. There were significant differences in the pair wise comparisons of the 2000 ppm dose group with the controls for liver hemangiomas and hemangiomas and/ or hemangiosarcomas at all sites combined, both at p < 0.05 ( Memo, L. Brunsman, 12/ 03/ 02, TXR No. 0051368).

The statistical analyses of the female rats were based upon Peto's prevalence test. Tables 3 and 4 show the tumor analyses results.

Historical control data regarding hemangiosarcomas from studies using the Alpk: ApfSD Wistar rats and conducted by the Syngenta Central Toxicology Laboratory ( Alderley Park, UK) are provided ( MRID 457108 08). The total number of rats with hemangiosarcomas recorded, irrespective of site, are summarized in Table 5. The occurrence of hemangiosarcomas by tissue in rats is shown in Table 6. The historical control range for hemangiosarcomas was 0 1.9% for both male and female rats. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 10 Table 3. PHMB  1996 Alpk: APfSD Wistar Rat Study ORIGINAL STUDY DIAGNOSES Female Vascular Tumor Rates+ and Peto's Prevalence Test Results ( p values) Dose ( ppm)

0 200 600 2000 Liver Hemangiosarcomas 0/ 42 1/ 42 0/ 40 3a/ 35 (%) ( 0) ( 2) ( 0) ( 9) p = 0.0107* 0.1284  0.0338* Other Sites ( NOT LIVER) Hemangiomas 1/ 42 1/ 41 3/ 39 3b/ 34 (%) ( 2) ( 2) ( 8) ( 9) p = 0.0733 0.4208 0.1430 0.0748 Other Sites ( NOT LIVER)

Hemangiosarcomas 1c/ 40 0/ 40 0/ 35 0/ 28 (%) ( 2) ( 0) ( 0) ( 0) p =     Combined 2/ 42 2/ 42 3/ 40 6/ 35 (%) ( 5) ( 5) ( 8) ( 17) p = 0.0234* 0.3094 0.2804 0.0575 + Number of tumor bearing animals/ Number of animals examined, excluding those that died before observation of the first tumor.

aFirst liver hemangiosarcoma observed at week 91, dose 2000 ppm.

bFirst other sites ( NOT LIVER) hemangioma observed at week 92, dose 2000 ppm.

cFirst other sites ( NOT LIVER) hemangiosarcoma observed at week 95, dose 0 ppm.

Note: Significance of trend denoted at control. Significance of pair wise comparison with control denoted at dose level. If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 11 Table 4. PHMB  1996 Alpk: APfSD Wistar Rat Study Female Vascular Tumor Rates+ and Peto's Prevalence Test Results ( p values) Dose ( ppm) 0 200 600 2000 PWG RE READ OF LIVER VASCULAR TUMORS Hemangiomas 0/ 42 0/ 42 0/ 40 2a/ 35 (%) ( 0) ( 0) ( 0) ( 6) p = 0.0107*   0.0808*

Hemangiosarcomas 0/ 27 0/ 32 0/ 29 1b/ 20 (%) ( 0) ( 0) ( 0) ( 5) p = 0.0232*   0.1226 ORIGINAL DIAGNOSES OF VASCULAR TUMORS AT OTHER SITES Hemangiomas 1/ 42 1/ 41 3/ 39 3c/ 34 (%) ( 2) ( 2) ( 8) ( 9) p = 0.0733 0.4208 0.1430 0.0748 Hemangiosarcomas 1d/ 40 0/ 40 0/ 35 0/ 28 (%) ( 2) ( 0) ( 0) ( 0) p =     COMBINED PWG RE READ OF LIVER AND ORIGINAL DIAGNOSES AT OTHER SITES Combined 2/ 42 1/ 42 3/ 40 6/ 35 (%) ( 5) ( 2) ( 8) ( 17) p = 0.0079**  0.2869 0.0344* + Number of tumor bearing animals/ Number of animals examined, excluding those that died before observation of the first tumor. a First liver hemangioma observed at week 91, dose 2000 ppm. b First liver hemangiosarcoma observed at week 105, dose 2000 ppm. c First other sites ( NOT LIVER) hemangioma observed at week 92, dose 2000 ppm. d First other sites ( NOT LIVER) hemangiosarcoma observed at week 95, dose 0 ppm.

Note: Significance of trend denoted at control. Significance of pair wise comparison with control denoted at dose level. If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 12 Table 5. Historical control data regarding hemangiosarcomas from studies using the Alpk: ApfSD Wistar rats and conducted by the Syngenta Central Toxicology Laboratory ( Alderley Park, UK) PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 13 Table 6. Historical control data regarding hemangiosarcomas from studies using the Alpk: ApfSD Wistar rats and conducted by the Syngenta Central Toxicology Laboratory ( Alderley Park PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 14 C. Non Neoplastic Lesions In addition to liver hemangiomas and hemangiosarcomas, the liver is also the target organ in males and females. Plasma alkaline phosphatase activity was elevated significantly over controls ( p< 0.01) in females from the main study dosed at 2000 ppm ( 843 74%). In 2000 ppm males the enzyme was significantly increased by 36% and 27% at weeks 14 and 27, respectively. In females at 2000 ppm, absolute liver weight was reduced significantly ( p< 0.05) by 11%. In 2000 ppm males, microscopic observations of liver hepatocyte fat and spongiosis were increased 44% and 22% over controls, respectively. Corresponding increases in these lesions were not seen in females. There were no corroborating gross pathology findings of the liver abnormalities.

D. Adequacy of the Dosing for Assessment of Carcinogenicity The dosing was considered to be adequate, but not excessive, for both male and female rats. In the rat study at the high dose ( 2000 ppm) there was significantly increased mortality in females ( 62% vs. 48% in controls). Body weights were significantly reduced by 5 8% in high dose females throughout the study. In high dose males, body weights were significantly ( p< 0.01 or 0.05) lower than controls ( 94 6%) through week 79. Food utilization ( g growth/ 100 g feed) for the first 12 weeks decreased significantly ( p< 0.01) vs controls ( 97 8% in both sexes at 2000 ppm). Plasma alkaline phosphatase activity was significantly elevated 43 74% over controls in 2000 ppm females from the main study at the 27, 53, 79, and 105 week intervals. In males at 2000 ppm, alkaline phosphatase activity was increased significantly by 36% and 27% during weeks 14 and 27, respectively. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 15 2. 2 Year Oncogenicity Study in Mice ( 1996)

Reference:

Milburn G. M. ( 1996) Polyhexamethylene Biguanide: Two Year Oncogenicity Study in Mice. Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK SK10 4TJ. Laboratory Project ID: PM0937. June 21, 1996. MRID 44074201. Unpublished.

Mann, P. ( 2002) Polyhexamethylene Biguanide ( PHMB): Two Year Oncogenic Study in Mice. Pathology Working Group. Peer Review of Proliferative Vascular Lesions in Male and Female Mice. Report Identification CTL Study No. PM0937. EPL Project Number. 698 001. MRID 45710802. Unpublished.

A. Experimental Design In this mouse oncogenicity study ( MRID 44074201), poly( hexamethylenebiguanide) hydrochloride ( PHMB, 20.2% a. i.) was administered in the diet to C57B1/ 10J f CD 1/ Alpk mice ( 55/ sex/ group) at 0, 400, 1,200 or 4,000 ppm ( equivalent to 55, 167, or 715 mg/ kg/ day for males and 69, 217, or 856 mg/ kg/ day for females) for 2 years. A Pathology Working Group ( PWG) peer review reevaluated the vascular lesions of this study in a report dated June 27, 2002 ( MRID 45710802). Only the PWG re read diagnoses of the vascular tumors are presented in this document.

B. Discussion of Tumor Data Survival Analysis The statistical evaluation of mortality indicated a significant increasing trend with increasing doses of PHMB in female C57B1/ 10J f CD 1/ Alpk mice. There was no statistically significant incremental change in mortality with increasing doses of PHMB in male C57B1/ 10J f CD 1/ Alpk mice ( Memo, L. Brunsman, 12/ 03/ 02, TXR No. 0051368). Tables 7 and 8 show the mortality results. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 16 Table 7. PHMB  1996 C57B1/ 10J f CD 1/ Alpk Mouse Study Male Mortality Rates+ and Cox or Generalized K/ W Test Results Weeks Dose 1 26 27 52 53 78 79 105 f Total ( ppm)

0 0/ 55 0/ 55 5/ 55 24/ 50 29/ 55 ( 0) ( 0) ( 9) ( 48) ( 53)

400 0/ 55 2/ 55 6/ 53 18/ 47 26/ 55 ( 0) ( 4) ( 11) ( 38) ( 47)

1200 0/ 55 0/ 55 6/ 55 11/ 49 17/ 55 ( 0) ( 0) ( 11) ( 22) ( 31)* N 4000 0/ 55 5/ 55 7/ 50 17/ 43 29/ 55 ( 0) ( 9) ( 14) ( 40) ( 53)

+ Number of animals that died during interval/ Number of animals alive at the beginning of the interval.

f Final sacrifice at week 105.

( ) Percent.

N: Negative change from control.

Note: Time intervals were selected for display purposes only.

Significance of trend denoted at control.

Significance of pair wise comparison with control denoted at dose level.

If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 17 Table 8. PHMB  1996 C57B1/ 10J f CD 1/ Alpk Mouse Study Female Mortality Rates+ and Cox or Generalized K/ W Test Results Weeks Dose 1 26 27 52 53 78 79 105 f Total ( ppm)

0 1/ 55 0/ 54 7/ 54 17/ 47 25/ 55 ( 2) ( 0) ( 13) ( 36) ( 45)**

400 1/ 55 1/ 54 0/ 53 17/ 53 19/ 55 ( 2) ( 2) ( 0) ( 32) ( 35)

1200 1/ 55 0/ 54 5/ 54 14/ 49 20/ 55 ( 2) ( 0) ( 9) ( 29) ( 36)

4000 3/ 55 3/ 52 10/ 49 23/ 39 39/ 55 ( 5) ( 6) ( 20) ( 59) ( 71)**

+ Number of animals that died during interval/ Number of animals alive at the beginning of the interval.

f Final sacrifice at week 105.

( ) Percent.

Note: Time intervals were selected for display purposes only.

Significance of trend denoted at control.

Significance of pair wise comparison with control denoted at dose level.

If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 18 Tumor Analysis The study pathologist and study peer reviewer determined that there was an increase in vascular tumors, mainly hemangiosarcoma, in both sexes at the high dose ( 4000 ppm) when compared to controls. The reviewing pathologist agreed with the increase in vascular tumors, but not always with the diagnosis ( hemangioma vs. hemangiosarcoma). A pathologist working group ( PWG) was conducted in 2002 to confirm the diagnoses of the vascular neoplasms ( MRID 45710802). The PWG confirmed the study pathologist and peer reviewer's conclusion, that there was clear evidence of a treatment related increase in incidence of animals with either hemangioma or hemangiosarcoma in the high dose of both sexes. This increase was largely due to the increase in the number of vascular tumors in the liver. PWG suggested that it is more appropriate to consider the total number of animals with vascular neoplasms, rather than the individual organs with either primary or metastatic lesions. Dr. J. M. Pletcher, EPA's consulting pathologist, confirmed the validity of the PWG report ( TXR No. 0052033).

As shown in Table 9, male mice had significant increasing trends at p< 0.01, and significant differences in the pair wise comparisons of the 4000 ppm dose group with the controls, for hemangiomas ( p< 0.01), hemangiosarcomas ( p< 0.05) and combined hemangiomas and/ or hemangiosarcomas ( p< 0.01). There was also a significant increasing trend at p < 0.01, and a significant difference in the pair wise comparison of the 4000 ppm dose group with the controls at p < 0.05, for rectal anal junction squamous cell carcinomas.

Female mice had significant increasing trends for hemangiomas ( p< 0.05), hemangiosarcomas ( p< 0.05), and combined hemangiomas and hemangiosarcomas ( p< 0.01). There was also a significant difference in the pair wise comparison of the 4000 ppm dose group with the controls, for combined hemangiomas and/ or hemangiosarcomas, at p< 0.05. There was also a significant increasing trend at p< 0.01, and a significant difference in the pair wise comparison of the 4000 ppm dose group with the controls at p< 0.01 for rectal anal junction squamous cell carcinomas.

The statistical analyses of the male mice were based upon the Exact trend test and the Fisher's Exact test for pair wise comparisons. The statistical analyses of the female mice were based upon Peto's prevalence test ( Memo, L. Brunsman, 12/ 03/ 02, TXR No. 0051368). See Tables 9 through 12 for tumor analyses results.

Historical control data regarding hemangiosarcomas from studies using the C57BL/ 10J/ CD 1 Alpk mouse and conducted by the Syngenta Central Toxicology Laboratory ( Alderley Park, UK) are provided ( MRID 457108 04). The total number of rats with hemangiosarcoma recorded, irrespective of site are summarized in Table 13. There are no historical control data for hemangiosarcoma by tissue. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 19 Table 9. PHMB  1996 C57B1/ 10J f CD 1/ Alpk Mouse Study 2002 PWG Re Read Male Vascular Tumor Rates+ ( All Sites) and Fisher's Exact Test and Exact Trend Test Results ( p values) Dose ( ppm)

0 400 1200 4000 Hemangiomas 2/ 55 3/ 55 4/ 55 11a/ 53 (%) ( 4) ( 5) ( 7) ( 21)

p = 0.0007** 0.5000 0.3393 0.0063**

Hemangiosarcomas 5/ 55 4/ 55 6b/ 55 12/ 53 (%) ( 9) ( 7) ( 11) ( 23)

p = 0.0067** 0.5000 0.5000 0.0468*

Combined 6c/ 55 6c/ 55 9c/ 55 20d/ 53 (%) ( 11) ( 11) ( 16) ( 38)

p = 0.0000** 0.6195 0.2899 0.0010** + Number of tumor bearing animals/ Number of animals examined, excluding those that died before week 39.

a First hemangioma observed at week 39, dose 4000 ppm. b First hemangiosarcoma observed at week 59, dose 1200 ppm. c One animal in each of the 0, 400 and 1200 ppm dose groups had both an hemangioma and an hemangiosarcoma. d Three animals in the 4000 ppm dose group had both an hemangioma and an hemangiosarcoma Note: Significance of trend denoted at control.

Significance of pair wise comparison with control denoted at dose level.

If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 20 Table 10. PHMB  1996 C57B1/ 10J f CD 1/ Alpk Mouse Study Male Rectal Anal Junction Tumor Rates+ and Fisher's Exact Test and Exact Trend Test Results ( p values)

Dose ( ppm)

0 400 1200 4000 Squamous Cell Carcinomas 0/ 45 0/ 45 0/ 45 5a/ 48 (%) ( 0) ( 0) ( 0) ( 10)

p = 0.0011** 1.0000 1.0000 0.0329* + Number of tumor bearing animals/ Number of animals examined, excluding those that died before week 36.

a First squamous cell carcinoma observed at week 36, dose 4000 ppm.

Note: Significance of trend denoted at control.

Significance of pair wise comparison with control denoted at dose level.

If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 21 Table 11. PHMB  1996 C57B1/ 10J f CD 1/ Alpk Mouse Study 2002 PWG Re Read Female Vascular Tumor Rates+ ( All Sites) and Peto's Prevalence Test Results ( p values) Dose ( ppm) 0 400 1200 4000 Hemangiomas 6/ 51 2/ 53 5a/ 52 8/ 44 (%) ( 12) ( 4) ( 10) ( 18)

p = 0.0294*   0.1884 Hemangiosarcomas 6/ 54 4/ 53 4/ 54 10b/ 49 (%) ( 11) ( 8) ( 7) ( 20)

p = 0.0300*   0.1262 Combined 8c/ 54 5d/ 53 7e/ 54 15f/ 49 (%) ( 15) ( 9) ( 13) ( 31)

p = 0.0023**   0.0288* + Number of tumor bearing animals/ Number of animals examined, excluding those that died before observation of the first tumor.

a First hemangioma observed at week 69, dose 1200 ppm. b First hemangiosarcoma observed at week 54, dose 4000 ppm. c Four animals in the 0 ppm dose group had both an hemangioma and an hemangiosarcoma. d One animal in the 400 ppm dose group had both an hemangioma and an hemangiosarcoma. e Two animals in the 1200 ppm dose group had both an hemangioma and an hemangiosarcoma. f Three animals in the 4000 ppm dose group had both an hemangioma and an hemangiosarcoma.

Note: Significance of trend denoted at control. Significance of pair wise comparison with control denoted at dose level. If *, then p < 0.05. If **, then p < 0.0 PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 22 Table 12. PHMB  1996 C57B1/ 10J f CD 1/ Alpk Mouse Study Female Rectal Anal Junction Tumor Rates+ and Peto's Prevalence Test Results ( p values)

Dose ( ppm)

0 400 1200 4000 Squamous Cell Carcinomas 0/ 42 0/ 42 0/ 43 8a/ 28 (%) ( 0) ( 0) ( 0) ( 29)

p = 0.0000**   0.0002** + Number of tumor bearing animals/ Number of animals examined, excluding those that died before observation of the first tumor.

a First squamous cell carcinoma observed at week 86, dose 4000 ppm.

Note: Significance of trend denoted at control.

Significance of pair wise comparison with control denoted at dose level.

If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 23 Table 13. Historical control data regarding hemangiosarcomas from studies using the C57BL/ 10J/ CD 1 Alpk mouse and conducted by the Syngenta Central Toxicology Laboratory ( Alderley Park, UK). PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 24 C. Non Neoplastic Lesions At the 1,200 ppm level, mice were observed with decreased overall body weight gains ( males 97%; females 92%); increases in hematology parameters ( p< 0.05) including hemoglobin in females ( 86%), hematocrit in females ( 86%), and RBCs in females ( 85%); decreased absolute weight of the liver in males ( 915%) and females ( 921%); increased incidences of gross pathological changes including distended caeca in females ( 4% treated vs. 0% in controls); traumatized pinnae of ears in males ( 36% treated vs. 4% controls) and females ( 44% treated vs. 4% controls); increased incidences of nonneoplastic lesions including luminal dilatation of the gall bladder in females ( 9% treated vs. 2% controls), hepatocyte hypertrophy of the liver in males ( 13% treated vs. 0% controls) and females ( 35% treated vs. 0% controls), increased ploidy of the liver in males ( 13% treated vs. 0% controls) and females ( 36% treated vs. 0% controls), pigmentation of the liver in females ( 11% treated vs. 0% controls), and inflammation of the rectal anal junction in males ( 44% treated vs. 2% controls) and females ( 47% treated vs. 21% controls).

At the 4,000 ppm dose level, decreased overall body weight gain was observed in males ( 950%) and females ( 932%); increased food consumption ( p< 0.05 or 0.01) from approximately week 12 through termination in males ( 87 29%) and females ( 87 26%); decreased food utilization ( p< 0.01) during weeks 1 12 in males ( 940%) and females ( 920%); increased hemoglobin in males ( 87%) and females ( 817%), hematocrit in males ( 85%) and females ( 816%), and RBCs in males ( 810%) and females ( 817%); decreased weight of the liver in males ( 20%) and females ( 30%); decreased weight of the testes of males ( 915%) and in adrenals of females ( 922%); and increased incidences of gross pathological changes including swollen anuses in males ( 18% treated vs. 0% controls) and females ( 7% treated vs. 0% controls), distended caeca in males ( 9% treated vs. 0% controls) and females ( 13% treated vs. 0% controls), pinnae of ears traumatized in males ( 27% treated vs. 4% controls) and females ( 22% treated vs. 4% controls), distention of the gall bladder in males ( 49% treated vs. 11% controls) and females ( 47% treated vs. 9% controls), and liver mass in males ( 23% treated vs. 9% controls) and females ( 15% treated vs. 8% controls). Increased incidences of non neoplastic microscopic lesions were observed, including luminal dilatation of the gall bladder in males ( 48% treated vs. 20% controls) and females ( 39% treated vs. 2% controls), epithelial hyperplasia of the gall bladder in males ( 25% treated vs. 0% controls) and females ( 13% treated vs. 0% controls), hepatocyte hypertrophy of the liver in males ( 53% treated vs. 0% controls) and females ( 49% treated vs. 0% controls), ploidy of the liver in males ( 53% treated vs. 0% controls) and females ( 38% treated vs. 0% controls), pigmentation of the liver in males ( 36% treated vs. 0% controls) and females ( 42% treated vs. 0% controls), and inflammation of the rectal anal junction in males ( 82% treated vs. 2% controls) and females ( 74% treated vs. 21% controls).

Toxicity observed common to both sexes of the 1,200 and 4,000 ppm treatment groups included decreased overall weight gains, pinnae of ears traumatized, hepatocyte hypertrophy of the liver, increased ploidy of the liver, and inflammation of the rectal anal junction. There was also epithelial hyperplasia of the gallbladder common to females of both treatment groups, but occurring in males only at 4,000 ppm. No treatment related effects were seen in mice in the 400 ppm dose group. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 25 D. Adequacy of Dosing for Assessment of Carcinogenicity Dosing was considered to be excessive at the high dose of 4000 ppm based on decreased overall body weight gains of 50% in males and 32% in females at termination. In addition, decreased body weight gains of 33% in males and 19% in females were observed at 13 weeks. The study indicated that the animals at the 4000 ppm dose group had increased food consumption ( p< 0.05 or 0.01) from approximately week 12 through termination in males ( 87 29%) and females ( 87 26%).

Dosing was considered to be adequate, but not excessive, in both males and females at the mid dose of 1200 ppm based on decreased overall body weight gains ( males, 7%; females, 2%), and increases in hematology parameters, and non neoplastic histopathological changes in the gall bladder, liver, and rectal anal junction. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 26 3. Dermal Carcinogenicity Study in Mice ( 1977)

Reference:

Clapp, MJ. L.; Iswarn, T. J.; Major, P. ( 1977). Polyhexamethylene Biguanide: 80 Week Skin Painting Study in Mice: Report No. CTL/ P/ 331 ( Amended). ICI Americas, Inc., Washington, Del; CDL: 233269. MRID 00066475. Unpublished Data.

ICI Americas, Inc. ( 1975). Polyhexamethylene Biguanide: 80 Week Painting Study in Mice: Appendix B: Pathology Individual Animal Data: Report No. CTL/ P/ 331. CDL: 235604 A. MRID 00104796.

A. Experimental Design Four groups of specific pathogen free ( 50M + 50F) Alderley Park Mice received dermally 0.3 mL of test material at doses of 0 ( solvent in ethanol), 0.6 mg ( 0.2% PHMB in ethanol), 6.0 mg ( 2% PHMB in ethanol) and 30.0 mg ( 10% PHMB in ethanol) per day for five days a week for 80 weeks. The treatment dosages are approximately equivalent to 0, 15, 150 and 750 mg/ kg/ day of a 20% PHMB solution.

B. Discussion of Tumor Data Survival Analysis The statistical evaluation of mortality indicated a significant increasing trend with increasing doses of PHMB in male and female SPF Alderley Park mice ( Memo, L. Brunsman, 12/ 03/ 02, TXR No. 0051368). Tables 14 and 15 show the mortality results.

Tumor Analysis There were no compound related tumors observed in male mice. Female mice had significant increasing trends in liver angiosarcomas ( p < 0.01) and vascular tumors from all sites combined ( p < 0.05). There was a significant difference in the pair wise comparison of the 30 mg dose group with the controls for liver angiosarcomas at p < 0.05. The statistical analyses of the female mice were based upon Peto's prevalence test. See Table 16 for tumor analyses results. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 27 Table 14. PHMB  1977 SPF Alderley Park Mouse Study Male Mortality Rates+ and Cox or Generalized K/ W Test Results Weeks Dose 1 20 21 40 41 60 61 81 f Total ( mg/ animal) ( mg/ kg/ day of 20% PHMB equivalent)

0.0 0/ 49# 1/ 49 6/ 48 9/ 42 16/ 49 ( 0.0) ( 0) ( 2) ( 12) ( 21) ( 33)**

0.6 1/ 50 0/ 49 1/ 49 7/ 48 9/ 50 ( 15) ( 2) ( 0) ( 2) ( 15) ( 18)

6.0 1/ 50 0/ 49 6/ 49 6/ 43 13/ 50 ( 150) ( 2) ( 0) ( 12) ( 14) ( 26)

30.0 3/ 50 4/ 47 7/ 43 25/ 36 39/ 50 ( 750) ( 6) ( 9) ( 16) ( 69) ( 78)**

+ Number of animals that died during interval/ Number of animals alive at the beginning of the interval.

f Final sacrifice at week 80.

#: One animal missexed and removed from study at week 4.

( ) Percent.

Note: Time intervals were selected for display purposes only.

Significance of trend denoted at control.

Significance of pair wise comparison with control denoted at dose level.

If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 28 Table 15. PHMB  1977 SPF Alderley Park Mouse Study Female Mortality Rates+ and Cox or Generalized K/ W Test Results Weeks Dose 1 20 21 40 41 60 61 81f Total ( mg/ animal) ( mg/ kg/ day of 20% PHMB equivalent)

0.0 2/ 50 2/ 48 4/ 46 6/ 42 14/ 50 ( 0.0) ( 4) ( 4) ( 9) ( 14) ( 28)**

0.6 1/ 50 1/ 49 3/ 48 11/ 45 16/ 50 ( 15) ( 2) ( 2) ( 6) ( 24) ( 32)

6.0 1/ 50 2/ 49 7/ 47 8/ 40 18/ 50 ( 150) ( 2) ( 4) ( 15) ( 20) ( 36)

30.0 3/ 50 2/ 47 7/ 45 27/ 38 39/ 50 ( 750) ( 6) ( 4) ( 16) ( 71) ( 78)**

+ Number of animals that died during interval/ Number of animals alive at the beginning of the interval.

f Final sacrifice at week 80.

( ) Percent.

Note: Time intervals were selected for display purposes only.

Significance of trend denoted at control.

Significance of pair wise comparison with control denoted at dose level.

If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 29 Table 16. PHMB  1977 SPF Alderley Park Mouse Study Female Vascular Tumor Rates+ and Peto's Prevalence Test Results ( p values) Dose  mg/ animal ( mg/ kg/ day of 20% PHMB equivalent) 0 0.6( 15) 6.0( 150) 30.0( 750) Liver Hemangioendotheliomas 0/ 37 0/ 40 0/ 37 1a/ 31 (%) ( 0) ( 0) ( 0) ( 3) p = 0.1059

Liver Angiosarcomas 0/ 41 0/ 45 0/ 40 2b/ 35 (%) ( 0) ( 0) ( 0) ( 6) p = 0.0025**   0.0401*

Other Sites ( NOT LIVER)

Hemangioendotheliomas 1c/ 41 0/ 44 0/ 40 0/ 33 (%) ( 2) ( 0) ( 0) ( 0) p =

Combined 1/ 41 0/ 45 0/ 40 3/ 35 (%) ( 2) ( 0) ( 0) ( 9) p = 0.0129*   0.1606 + Number of tumor bearing animals/ Number of animals examined, excluding those that died before observation of the first tumor.

a First liver hemangioendothelioma observed at week 68, dose 30.0 mg/ kg/ day.

b First liver angiosarcoma observed at week 60, dose 30.0 mg/ kg/ day.

c First other sites ( NOT LIVER) hemangioendothelioma observed at week 62, dose 0.0 mg/ kg/ day.

Note: Significance of trend denoted at control. Significance of pair wise comparison with control denoted at dose level. If *, then p < 0.05. If **, then p < 0.01. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 30 Historical control data regarding hemangiosarcomas from studies using the Alderley Park mice and conducted by the ICI Central Toxicology Laboratory are provided ( MRID 225025201) and are summarized in Table 17.

Table 17. Historical Control Data Regarding the Incidence of Liver Tumors in Control Alderley Park Mice in 80 Week Studies at the ICI Central Laboratory ( MRID 225025201)

1 2 3 4 5 6 1965 1065 1969 1969 1970 1972 M F M F M F M F M F M F Hepatosarcoma 4 1 3 0 1 0 3 0 0 0 0 0 Hepatoma 2 0 5 0 5 0 9 4 5 1 4 0 Hemangioendothel ioma 0 0 0 0 0 1 0 0 0 0 0 0 Total no. of tumors 6 1 8 0 6 0 12 4 5 1 4 0 No. of animal examined 41 39 35 39 30 31 45 50 32 39 45 49 % each sex with tumors 14.6 2.6 22.9 0 20 0 26.7 8 15.6 2.6 8.9 0 Combined % with tumors 8.75 12.5 9.8 16.8 8.6 4.3 Overall Incidence of Liver Tumors = 9.9% PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 31 C. Non Neoplastic Lesions The treatment dosages are equivalent to 0, 15, 150 and 750 mg/ kg/ day of 20% PHMB solutions. Mice that received the highest dose level showed a poorer condition, being very thin throughout the experiment. Death in both males and females in the highest dose group were slightly higher than in other groups during the first year. This pattern continued throughout the remainder of the study, resulting in a high mortality rate ( 75% in males and females) in the highest dose animals at termination, compared with approximately 30% in the other groups. The highest dose level of PHMB resulted in noticeable irritation to the skin of both males and females immediately after application. Erythema and some clumping of the growing fur were noticed during the first few weeks and after the 4th week; hyperkeratosis became evident, especially in males. No differences were apparent between the controls and those mice receiving 0, 0.6 or 6.0 mg PHMB per mouse per application. A significant reduction in mean body weight was observed for both male and female animals that received the highest dose level. There were no overall differences in food consumption between the control and treatment groups.

D. Adequacy of Dosing for Assessment of Carcinogenicity Dosing was considered to be excessive at the high dose ( 750 mg/ kg/ day of 20% PHMB equivalent) in both sexes due to increased mortality ( 78%, high dose, versus 33% and 28% in the male and female controls, respectively) and decreased body weight gain. Overall body weight gain decreases of 45% and 17% were seen in males and females, respectively. No treatment related effects were noted at the mid dose level of 150 mg/ kg/ day of 20% PHMB equivalent ( NOAEL).

IV. TOXICOLOGY 1. Metabolism Bioavailability of PHMB was investigated in male and female Sprague Dawley rats, fed diets of 200ppm or 2000ppm ( 10 and 100 mg/ kg nominal dose) for fourteen days followed by a single radiolabelled dose of either 0.08 mg/ kg or 0.8 mg/ kg ( MRID #' s 43567001 and 43599901), or after a single 100 mg/ kg dose ( MRID #' s 00077926 and 00086363). In both studies, feces represented the major route of excretion at all dose levels, comprising greater than 90% of the administered dose. A similar excretion in feces was observed in bile cannulated rats after a single radiolabelled dose of 20 mg/ kg ( MRID # 43567001). Thus, fecal excretion of PHMB derived radioactivity represents unabsorbed test material. The excretion pattern of low, mid, and high molecular weight fractions of PHMB was similar. Bioavailability was 4.7% and 3.9% for males and females, respectively, at the 10 mg/ kg dose, and 3.0% and 2.6% for males and females, respectively, at the 100 mg/ kg dose. Tissue distribution in rats given 10 mg/ kg PHMB showed concentrations in the liver and kidney of male rats to be 0.568 g/ g and 0.499 g/ g, respectively. In female rats, liver and kidney concentrations were 0.752 g/ g and 0.807 g/ g respectively. As a percentage of the dose, liver of male and female rats contained 0.18% and 0.19% of the dose respectively, while kidneys contained 0.03% and 0.04% PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 32 of the dose respectively. Metabolite analysis of pooled urine from rats administered a low molecular weight fraction of PHMB at 20 mg/ kg ( the fraction showing the greatest absorption) revealed the presence of more than one metabolite, but identification was not performed due to the small amount of sample available for analysis.

2. Mutagenicity In a battery of mutagenicity assays, PHMB was not mutagenic in bacteria or clastogenic in cultured human lymphocytes. There was also no evidence of in vivo clastogenicity or aneugenicity in mouse bone marrow or unscheduled DNA synthesis in rat liver. No mutagenic studies were found in the open literature. The following guideline studies were acceptable and satisfy the pre 1991 requirements for mutagenicity testing.

In two independently performed microbial gene mutation assays ( MRID No. 41687004), Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 were exposed to 0.32  500 µ g/ plate Vantocil IB ( 19.6% a. i.) in the absence or presence of S9 activation. Additional testing was carried out using comparable doses with and without S9 in TA1537 and TA98. The S9 fraction was derived from Aroclor 1254 induced rat livers and the test material was delivered to the test system in dimethyl sulfoxide. The utility of testing a bactericidal agent in a microbial mutation assay is questionable. Nevertheless, cytotoxicity was observed for the majority of strains at $ 200 : g/ plate +/ S9. All strains responded in the expected manner to the nonactivated and S9 activated positive controls. There was, however, no evidence that Ventocil IB induced a mutagenic response in any strain at any nonactivated or S9 activated dose.

In an in vitro mammalian cell cytogenetic assay ( MRID No. 41404501/ 42149905), human lymphocytes derived from male and female donors were exposed to Vantocil IB ( 19.6% a. i. in water) doses of 5, 25, or 50 µ g/ mL without S9 activation ( both donors) and levels of 25, 100 or 187.5 µ g/ mL + S9 ( male donor) or at 25, 100 or 250 µ g/ mL ( female donor) with S9 activation for approximately 2.5  3.5 hours. The S9 liver homogenate was derived from Aroclor 1254 induced Sprague Dawley rat livers. The test material was delivered to the test system in physiological saline. A 50% reduction in mitotic index occurred at 50 µ g/ mL  S9 ( both donors) and at 100 µ g/ mL + S0 ( male donor) or at 250 µ g/ mL + S9 ( female donor). The positive controls induced the expected high yield of chromosome aberrations in the lymphocytes derived from the male and female donors. There was, however, no evidence that Vantocil IB induced a clastogenic effect. This study is classified as Acceptable and satisfies the guideline requirement for an in vitro cytogenetic assay.

In a mouse micronucleus assay ( MRID No. 41096901/ 41404503), groups of five male and five female C57BL/ 6JfCD 1/ Alpk mice received single oral gavage administrations of 250 or 400 mg/ kg Vantocil IB ( 19.6% a. i.) prepared in deionized water. Bone marrow cells from mice in the highdose group were examined for the incidence of micronucleated polychromatic erythrocytes ( MPEs). Low dose animals were sacrificed at 24 hours. Two animals receiving 400 mg/ kg died prior to the scheduled sacrifice. There was also clear evidence of target cell cytotoxicity in the high dose males PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 33 and females at all sacrifice intervals. The positive control induced the expected high yield of MPEs in males and females. Vantocil IB did not, however, induce a clastogenic or aneugenic effect in either sex at any dose or sacrifice time. The study is classified as Acceptable and satisfies the requirements for FIFRA Test Guideline 84 2 for a micronucleus assay.

In two independently performed in vivo/ in vitro unscheduled DNA synthesis ( UDS) assays ( MRID No. 41404502/ 42149903), groups of two to three male rats were administered single oral gavage doses of 750 or 1500 mg/ kg Vantocil IB ( 19.6%) prepared in deionized water. Animals were sacrificed at 4 and 12 hours post treatment and recovered hepatocytes were scored for UDS. Clinical toxicity ( i. e., excessive salivation and subdued nature) was observed at 1500 mg/ kg; higher levels were lethal. No cytotoxicity for the target organ was seen at either level. The positive control induced the expected high yield of hepatocytes with net nuclear grains. There was, however, no evidence that the Vantocil IB induced a genotoxic response at either dose or sacrifice time. This study is classified as Acceptable and satisfies the guideline requirement for a UDS assay ( 84 4).

3. Structure Activity Relationship No appropriate structural analogs could be located for comparison purposes. In general, with the exception of exposure via the inhalation route, high MW polymers are not expected to be of significant carcinogenic concern because of poor bioavailability. PHMB has indeed been shown to have low bioavailability. However, SAR consideration suggests that there is a likelihood that the lower MW fractions of PHMB may be metabolized to polyamines in analogy to the metabolism of arginine ( a guanidine containing amino acid) to form biogenic polyamines such as putrescine, spermidine and spermine. There is strong evidence that these polyamines are readily taken up by cells and may play an important role in promoting skin and colon carcinogenesis by serving as ingredients to support neoplastic growth. The metabolism study submitted by the company showed that low MW fractions of PHMB can indeed be metabolized in the rat; however, the metabolites have not yet been identified. It would be important to carry out the identification study. If the metabolites can be identified to be polyamines, they could provide a potential mode of action to explain the carcinogenicity of PHMB. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 34 4. Subchronic and Chronic Toxicity a) Subchronic Toxicity Rats In a 90 day rat oral toxicity study, young adult specific pathogen free ( S. P. F) Wister rats ( 25M + 25 F) received levels of 0, 2500, and 5000 ppm ( 0, 310, 620 mg/ kg/ day) of poly( hexamethylenebiguanide) hydrochloride ( 25%) for 90 days in the diet. At the end of the 90 day test period all animals were killed with chloroform, and an immediate postmortem examination was made. All animals survived the 90 day test period. There were no specific adverse effects of the compound. Food consumption was comparable for the test groups and controls. Body weight was moderately reduced in males ( 13.2% less than controls), and females ( 16.6% less than controls) fed the compounds at the highest dietary level ( 5000 ppm). No abnormalities in hematological parameters were observed. No gross abnormalities were observed. No remarkable change in organ/ body weight ratios was detected. Microscopic evaluation revealed that the liver of some females given the compound at a level of 5000 ppm showed an unusual degree of iron pigment both within the liver cells and kupffer cells. Although the report states that no iron pigments were seen in animals fed 2500 ppm test material in the diet, the study does not include detailed histopathological results of the 2500 ppm animals. Therefore, the NOAEL cannot be determined in this study.

Dogs In a 90 day dog oral toxicity study, three groups of Beagle dogs ( 4M + 4F per group), 12.4  14.6 kg initial body weight, received poly( hexamethylenebiguanide) hydrochloride ( 25%) at dietary levels of 0, 5500 and 11,000 ppm ( 0, 137 or 275 mg/ kg/ day) ad libitum for 90 days. General observation, food consumption, body weight and the following clinico pathological studies were performed: hemoglobin, packed cell volume ( hematocrit), leucocyte count ( total), leucocyte count ( differential), blood urea, serum alkaline phosphatase, BSP ( liver function test) and urine analyses ( pH, specific gravity, glucose, protein, bilirubin, and microscopy of centrifuge deposits). At the end of the test period the animals were killed with an overdose of pentobarbitone administered intravenously. A full postmortem examination and microscopic examination were taken from the following: brain ( cerebrum, cerebellum and pons), spinal cord, pituitary, submaxillary gland, thyroid, thymus, heart, lung, aorta, stomach, duodenum, ileum, colon, liver, spleen, kidney, bladder, adrenal, ovary and uterus or testis and epididymis, and sciatic nerves.

Both treated and control animals maintained an excellent condition throughout and no adverse effects were noted. No food consumption data was submitted. Mean body weights were comparable between control and treated animals except for the 11,000 ppm female dogs which gained significantly less total weight than the control female dogs. Results of the hematological parameters were unremarkable. Clinical function tests showed no difference in retention of BSP PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 35 attributable to test material. Urine analyses did not appear to be influenced by treatment. Organ/ body weight ratios showed no significant variation from the normal as a result of treatment. No gross pathology attributable to test material was detected. Microscopic examination revealed slight hemosiderons in 2 out of 4 males at 11,000 ppm. No other microscopic abnormalities attributable to treatment were present at either dose level.

The NOAEL for this 90 day dog feeding study appears to be 5500 ppm ( low dose). The high dose treatment caused less total weight gain in female dogs and slight hemosiderons in 2 out of 4 male dogs.

b) Chronic Toxicity Dogs In a chronic toxicity study ( MRID # 43620501), poly( hexamethylenebiguanide) hydrochloride ( 25%) was administered to groups of 4 male and female Beagle dogs in the diet initially at dose levels of 0, 300, 1500, and 4500 ppm ( 0, 7.5, 37.5, and 112.5 mg/ kg/ day nominal dose) for one year. Following an unexpectedly severe reaction in 3 of 4 males at 4500 ppm ( scrotal skin lesions), the high dose was discontinued on week 9 or 10, reduced to 3000 ppm ( 75 mg/ kg/ day), and then recommenced on week 11 or 12. Up to and including the 3000 ppm dose, there were no consistent effects of PHMB on body weight, weight gain, food consumption, or hematological parameters. Plasma alanine aminotransferase ( ALT) activity was significantly increased in male and female dogs at the 3000 ppm dose level beginning at week 8, but there was variability in the response, and only one male dog was available for measurement after week 10. Testes weight was decreased 29% and 32% for the left and right testis of high dose male dogs, and testicular tubular degeneration was observed in the surviving male dog as well as in one dog sacrificed intercurrently. Liver weight in high dose male dogs was decreased 14%, and microscopic changes of the liver were also observed in male dogs at the high dose. In one female dog at the high dose, significant clinical signs ( decreased activity, stiff/ splayed gait, slight tremors) were observed which were not reversible. In addition, plasma alanine aminotransferase was increased almost 10 fold over the pre treatment level by week 35 of treatment. Plasma aspartate aminotransferase in this dog was almost doubled by week 35 of treatment. Marked dermatitis of the limbs was also observed in this dog. The LOAEL is 3000 ppm ( 75 mg/ kg/ day) for male and female dogs, based on changes in testis and liver weight and microscopic observations in male dogs, and based on clinical signs of toxicity and clinical chemistry alterations in the female dog. The NOAEL is 1500 ppm ( 37.5 mg/ kg/ day) for male and female dogs.

This study is classified as acceptable and satisfies the guideline requirement [ OPPTS 870.4100; OPP § 83 1] for a chronic oral toxicity study in dogs. 5. Mode of Action Studies There are no mode of action studies available at this time. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 36 V. COMMITTEE'S ASSESSMENT OF THE WEIGHT OF THE EVIDENCE 1. Carcinogenicity The CARC concluded that PHMB showed evidence of carcinogenicity based on the following:

Rat  Oral There was no treatment related increase in any tumors in male Wistar rats.

Vascular Tumors: Female Wistar rats from the PWG diagnoses had significant increasing trends in liver hemangiomas and liver hemangiosarcomas, both at p< 0.05. There was also a significant increasing trend for hemangiomas and/ or hemangiosarcomas at all sites combined, at p < 0.01. There were significant differences in the pair wise comparisons of the 2000 ppm dose group with the controls for liver hemangiomas and hemangiomas and/ or hemangiosarcomas at all sites combined, both at p < 0.05. The incidence of hemangiomas and hemangiosarcomas for all sites combined was 2/ 42 ( 5%), 1/ 42 ( 2%), 3/ 40 ( 8%), and 6/ 35 ( 17%) for the 0, 200, 600, and 2000 ppm dose groups, respectively. Historical control data regarding hemangiosarcomas shows the range of hemangiosarcomas by tissue in rats was 0 1.9%. The CARC considered the tumor response at the high dose ( 17% for the combined re read of liver and original diagnoses at other sites) to be treatment related and driven by the increase in hemangiomas when all sites are combined.

Adequacy of Dosing: The CARC concluded that dosing at the highest dose ( 2000 ppm) was considered to be adequate, but not excessive, for both male and female rats. In high dose females, this was based on increased mortality and a reduction in body weight of 5 8% throughout the study. In high dose males, body weights were significantly reduced ( 4 6%) through week 79. Food utilization ( g growth/ 100 g feed) for the first 12 weeks decreased significantly ( 97 8%) in both sexes at 2000 ppm.

Mouse  Oral Vascular Tumors: Male C57B1/ 10J f CD 1/ Alpk mice from the PWG diagnoses had significant increasing trends in hemangiomas, hemangiosarcomas, and combined hemangiomas and hemangiosarcomas, all at p< 0.01. There were also significant differences in the pair wise comparisons of the 4000 ppm dose group with the controls, for hemangiomas ( p< 0.01), hemangiosarcomas ( p< 0.05), and combined hemangiomas and/ or hemangiosarcomas ( p< 0.01). The incidence of hemangiomas was 2/ 55, 3/ 55, 4/ 55, 11/ 53, for the 0, 400, 1200, and 4000 ppm dose groups, respectively. The incidence of hemangiosarcomas was 5/ 55, 4/ 55, 6/ 55, and 12/ 53 for the 0, 400, 1200, and 4000 ppm dose groups, respectively. The incidence of combined hemagiomas and hemangiosarcomas was 6/ 55, 6/ 55, 9/ 55, and 20/ 53 for the 0, 400, 1200, and 4000 ppm dose groups, respectively. For hemangiosarcomas, the tumor PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 37 incidence in the 4000 ppm dose group ( 23%) exceeded the historical control range ( 1.8 18.3%). The CARC considered the increase in vascular tumors ( hemangiomas, hemangiosarcomas, and combined) at the high dose ( which is considered to be excessive) to be treatment related. Although not statistically significant, the tumor response ( 9/ 55 [ 16%] for the combined hemangiomas and hemangiosarcomas) in mice at the mid dose of 1200 ppm is considered treatment related since this tumor type was also seen in female mice ( orally and dermally) and female rats.

Vascular Tumors: Female C57B1/ 10J f CD 1/ Alpk mice from the PWG diagnoses had significant increasing trends in hemangiomas ( p< 0.05), hemangiosarcomas ( p< 0.05), and combined hemangiomas and hemangiosarcomas ( p< 0.01). There was also a significant difference ( p< 0.05) in the pair wise comparison of the 4000 ppm dose group with the controls, for combined hemangiomas and/ or hemangiosarcomas. The incidence of combined hemangiomas and/ or hemangiosarcomas was 8/ 54 ( 15%), 5/ 53 ( 9%), 7/ 54 ( 13%), and 15/ 49 ( 31%) for the 0, 400, 1200, and 4000 ppm dose groups, respectively. For hemangiosarcomas, the tumor incidence in the 4000 ppm dose group ( 31%) exceeded the historical control range ( 0 10.9%). There are no historical control data for hemangiomas by tissue. The CARC considered the combined vascular tumors at the high dose ( which is considered to be excessive) to be treatment related. There were no increases in these tumors at doses which were not considered excessive.

Rectal Anal Junction Tumors: In male mice there was a significant increasing trend at p < 0.01 and a significant difference in the pair wise comparison of the 4000 ppm dose group with the controls at p < 0.05, for rectal anal junction squamous cell carcinomas. The incidence of rectal anal junction tumors was 0/ 45, 0/ 45, 0/ 45, and 5/ 48 for the 0, 400, 1200 and 4000 ppm dose groups, respectively. In female mice there was a significant increasing trend at p< 0.01, and a significant difference in the pair wise comparison of the 4000 ppm dose group with the controls at p< 0.01, for rectal anal junction squamous cell carcinomas. The incidence of rectal anal junction tumors was 0/ 42, 0/ 43, 0/ 43, 8/ 28 for the 0, 400, 1200 and 4000 ppm dose groups, respectively. The CARC considered the carcinomas at the high dose ( which is considered to be excessive) in both males and females to be treatment related but suggestive of a local irritant effect. These tumors did not contribute to the CARC's weight of the evidence analyses.

Adequacy of Dosing Dosing was considered to be excessive at the high dose of 4000 ppm based on decreased overall body weight gains of 50% in males and 32% in females at termination. In addition, decreased body weight gains of 33% in males and 19% in females were seen at 13 weeks. The study indicated that the animals at the 4000 ppm dose group had increased food PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 38 consumption ( p< 0.05 or 0.01) from approximately week 12 through termination in males ( 87 29%) and females ( 87 26%).

Dosing was considered to be adequate, but not excessive, in both males and females at the mid dose of 1200 ppm based on decreased overall body weight gains ( males, 7%; females, 2%), increases in hematology parameters, and non neoplastic histopathological changes in the gall bladder, liver, and rectal anal junction.

Mouse  Dermal Vascular Tumors: Female Alderley Park mice had significant increasing trends in liver angiosarcomas ( p < 0.01) and vascular tumors from all sites combined ( p < 0.05). There was also a significant difference in the pair wise comparison of the 30 mg dose group with the controls for liver angiosarcomas at p < 0.05. The incidence of liver angiosarcomas was 0/ 41, 0/ 45, 0/ 40, and 2/ 35 for the 0, 0.6, 6.0, and 30 mg dose groups, respectively. The CARC considered the tumor increase at the high dose to be equivocal since the tumors were only seen at an excessive dose. Vascular tumors were not seen at lower doses.

There was no treatment related increase in any tumors in male Alderley Park mice.

Adequacy of Dosing Dosing was considered to be excessive at the high dose ( 750 mg/ kg/ day of 20% PHMB equivalent) in both sexes due to increased mortality ( 78%, high dose, versus 33% and 28% in the male and female controls, respectively) and decreased body weight gain. Overall body weight gain decreases of 45% and 17% were seen in males and females, respectively. No treatment related effects were noted at the mid dose level of 150 mg/ kg/ day of 20% PHMB equivalent ( NOAEL).

2. Mutagenicity There is no mutagenicity concern.

3. Structure Activity Relationship No appropriate structural analogs could be located for comparison purposes. In general, with the exception of exposure via the inhalation route, high molecular weight ( MW) polymers are not expected to be of significant carcinogenic concern because of poor bioavailability. PHMB has indeed been shown to have low bioavailability. However, SAR consideration suggests that there is a likelihood that the lower MW fractions of PHMB may be metabolized to polyamines in analogy to the metabolism of arginine ( a guanidine containing amino acid) to form biogenic polyamines such as putrescine, spermidine and spermine. There is strong PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 39 evidence that these polyamines are readily taken up by cells and may play an important role in promoting skin and colon carcinogenesis by serving as ingredients to support neoplastic growth. A metabolism study submitted by the company showed that low MW fractions of PHMB can indeed be metabolized in the rat; however, the metabolites have not yet been identified.

4. Mode of Action No mode of action studies were available.

VI. CLASSIFICATION OF CARCINOGENIC POTENTIAL In accordance with the EPA Draft Guidelines for Carcinogen Risk Assessment ( July 1999), the CARC classified PHMB into the category " Suggestive Evidence of Carcinogenicity, but Not Sufficient to Assess Human Carcinogenic Potential" by the oral and dermal routes. The weightof the evidence considerations for this classification are as follows:

( i) A treatment related statistically significant increase ( trend and pair wise) in vascular tumors ( mainly benign) was seen in female rats at an oral dose that was considered to be adequate, but not excessive. This was considered as the strongest evidence in the CARC's evaluation of PHMB.

( ii) Oral exposure to male and female mice also resulted in treatment related vascular tumors seen at an excessive dose. However, at the next highest dose level, which was considered adequate but not excessive, there was a slight but not statistically significant increase in this same tumor which added to the CARC's concern for this tumor type.

( iii) It is noted that dermal exposure to female mice resulted in an equivocal increase in vascular tumors seen at only an excessive dose.

( iv) No treatment related increase in any tumors was seen in male rats via the oral route or in male mice via the dermal route of exposure.

VII. QUANTIFICATION OF CARCINOGENIC POTENTIAL The quantification of human cancer risk is not required. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 40 VIII. BIBLIOGRAPHY MRID No. CITATION 00066475 Clapp, MJ. L.; Iswarn, T. J.; Major, P. ( 1977). Polyhexamethylene Biguanide: 80 Week Skin Painting Study in Mice: Report No. CTL/ P/ 331 ( Amended). ICI Americas, Inc., Washington, Del; CDL: 233269. Unpublished Data. ( Cited in in Report No. 003810, 1978. Section C 16).

00104796 ICI Americas, Inc. ( 1975). Polyhexamethylene Biguanide: 80 Week Painting Study in Mice: Appendix B: Pathology Individual Animal Data: Report No. CTL/ P/ 331. CDL: 235604 A. Unpublished Data.

225025201 ICI Americas, Inc. ( 1981). Polyhexamethylene Biguanide: 80 Week Painting Study in Mice: Incidence of Tumors in the Liver. Unpublished Data.

43620501 Horner S. A. 1995. Polyhexamethylene Biguanide: 1 Year Dietary Toxicity Study in Dogs. Zeneca Central Toxicology Laboratory, Cheshire, UK. Laboratory Report No. CTL/ P/ 4488; Study No. PD0947.

44042801 Busey, W. M. ( 1996) Polyhexamethylene Biguanide ( PHMB): Two Year Feeding Study in Rats. Pathology Working Group Peer Review of Proliferative Vascular Lesions in Male and Female Rats. Zeneca CTL, Alderley Park, Macclesfield, Cheshire, UK. Study No. PR0936, Report No. CTL/ C/ 3172, May 13, 1996. Unpublished.

44059301 Horner, S. A. ( 1996) Polyhexamethylene Biguanide: Two Year Feeding Study in Rats. Zeneca Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK. Laboratory Project ID: Report No. CTL/ P/ 4663, Study No. PR0936, June 5, 1996. Unpublished.

44074201 Milburn G. M. ( 1996) Polyhexamethylene Biguanide: Two Year Oncogenicity Study in Mice. Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, UK SK10 4TJ. Laboratory Project ID: PM0937. June 21, 1996. Unpublished.

45710801 Piccirillo, V. J. 2002. Evaluation of the Weight  of Evidence for the Carcinogenic Potential of PHMB. Avecia, Inc. Willimington, DE . July 1, 2002. Unpublished Data.

45710802. Mann, P. ( 2002) Polyhexamethylene Biguanide ( PHMB): Two Year Oncogenic Study in Mice. Pathology Working Group. Peer Review of PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 41 Proliferative Vascular Lesions in Male and Female Mice. Report Identification CTL Study No. PM0937. EPL Project Number. 698 001. Unpublished.

45710803 Freemantle, M. R. 2002. Polyhexamethylene Biguanide ( PHMB): Two Yeat Oncogenic Study in Mice. Statistical Analysis of the Results from the Pathology Working Group Peer Review of Vascular Lesions in Male and Female Mice. Supplemental Information for MRID 44074201. Syngenta Central Toxicology Lab. Avecia, Inc. Wilmington, DE . June 28, 2002. Unpublished Data.

45710804 Brown, E. A. 2002. Historical Control Data for Occurrence of Hemangiosarcoma ( Angiosarcoma) in C57BL/ 10J/ CD 1 Alpk Mice. Avecia, Inc. Wilmington, DE. July 1, 2002. Unpublished Data.

45710805 Brown E. A. 2002. Exceedance of the Maximum Tolerated Dose at the Highest Tested Dose of PHMB in Mice Supplementary Information to MRID 44074201. Avecia, Inc. Wilmington, DE. June 28, 2002. Unpublished Data.

45710807 Brown E. A. 2002. Potential Carcinogenicity from Dermal Application of PHMB. Avecia, Inc. Wilmington, DE. June 28, 2002. Unpublished Data.

45710808 Brown E. A. 2002. Historical Control Data for Occurrence of Hemangiosarcoma ( Angiosarcoma) in Alpk: ApfSD Wistar Rats at the Syngenta Central Toxicology Laboratory Alderley Park Macclesfield, Cheshire, UK. Avecia, Inc. Wilmington, DE. June 28, 2002. Unpublished Data.

 Brunsman, L. L. 2002. PHMB Qualitative Risk Assessment Based On 1977 SPF Alderley Park Mouse Dermal Study, and 1996 C57B1/ 10JfCD 1/ Alpk Mouse and 1996 Alpk: APfSD Wistar Rat Dietary Studies. TXR No. 0051368.

 Dykstra, William ( 1978) 10182 EUP 11. Baquacil Swimming Pool Sanitizer ( containing Poly ( hexamethylene biguanide hydrochloride). Experimental Use Permit Application for the Evaluation of Baquacil in Recreational Swimming Pools. Caswell # 676. Report No. 003801.

 McMahon, T. 1995. Polyhexamethylene Biguanide ( PHMB, Baquacil): Review of a Mutagenicity Study Submitted by the Registrant. Report No. 011380. PHMB CANCER ASSESSMENT DOCUMENT FINAL REPORT 42  McMahon, T. 1997. Vantocil IB ® ( Baquacil): Review of Mutagenicity Data. DP Barcode: D240678.

 Megosh, LC, Hu, J, Geroge K, O'Brien TG. 2002. Genetic control of polyamine dependent susceptibility to skin carcinogenesis. Genomics 79: 505 512.

 Milovic V, Turchanowa L. 2003. Polyamines and colon cancer. Biochem Soc. Trans. 31: 381 383.

 Pletcher, J. M. 2002. Memorandum on Pathology Working Group Peer Review of Proliferative Vascular Lesions in Male and Female Mice. October 28, 2002. TXR No. 0052033.

 Pletcher, J. M. 2003. Memorandum on Pathology Working Group Peer Review of Proliferative Vascular Lesions in Male and Female Rats. July 16, 2003. TXR No. 0052000.


[Federal Register Volume 83, Number 43 (Monday, March 5, 2018)]
[Notices]
[Pages 9351-9354]
[FR Doc No: 2018-04420]

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SECURITIES AND EXCHANGE COMMISSION

[Release No. 34-82790; File No. SR-NASDAQ-2018-013]

Self-Regulatory Organizations; The Nasdaq Stock Market LLC;
Notice of Filing and Immediate Effectiveness of Proposed Rule Change To
Amend Rule 7018(a)

February 28, 2018.
    Pursuant to Section 19(b)(1) of the Securities Exchange Act of 1934

(``Act''),\1\ and Rule 19b-4 thereunder,\2\ notice is hereby given that
on February 13, 2018, The Nasdaq Stock Market LLC (``Nasdaq'' or
``Exchange'') filed with the Securities and Exchange Commission
(``Commission'') the proposed rule change as described in Items I, II,
and III below, which Items have been prepared by the Exchange. The
Commission is publishing this notice to solicit comments on the
proposed rule change from interested persons.
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    \1\ 15 U.S.C. 78s(b)(1).
    \2\ 17 CFR 240.19b-4.
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I. Self-Regulatory Organization's Statement of the Terms of Substance
of the Proposed Rule Change

    The Exchange proposes to amend the Exchange's transaction fees to
modify the credits provided for displayed Designated Retail Orders
under Rules 7018(a)(1)-(3).
    While these amendments are effective upon filing, the Exchange has
designated the proposed amendments to be operative on February 1,
2018.\3\
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    \3\ The proposed fees were initially filed with the Commission
as an immediately effective and operative rule change on February 1,
2018. See SR-NASDAQ-2018-009. The Exchange is withdrawing SR-NASDAQ-
2018-009 and replacing it with this filing, which makes a technical
correction and descriptive changes to the proposal.
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    The text of the proposed rule change is available on the Exchange's
website at http://nasdaq.cchwallstreet.com/, at the principal office of
the Exchange, and at the Commission's Public Reference Room.

II. Self-Regulatory Organization's Statement of the Purpose of, and
Statutory Basis for, the Proposed Rule Change

    In its filing with the Commission, the Exchange included statements
concerning the purpose of and basis for the proposed rule change and
discussed any comments it received on the proposed rule change. The
text of these statements may be examined at the places specified in
Item IV below. The Exchange has prepared summaries, set forth in
sections A, B, and C below, of the most significant aspects of such
statements.

A. Self-Regulatory Organization's Statement of the Purpose of, and
Statutory Basis for, the Proposed Rule Change

1. Purpose
    The purpose of the proposed rule change is to amend the credits
provided for displayed Designated Retail Orders \4\ under Rules
7018(a)(1)-(3) by: (1) Reducing the $0.0034 per share executed credit
to $0.0033 per share executed; (2) requiring members to have a ratio of
at least 85% liquidity provided through one or more of its [sic] Nasdaq
Market Center MPIDs to all volume (adding and removing liquidity)
through one or more of its [sic] Nasdaq Market Center MPIDs during the
month to qualify for the proposed $0.0033 per share executed credit;
and (3) adding a new $0.00325 per share executed credit tier.
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    \4\ As defined by Rule 7018.
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    Rule 7018 sets forth the fees and credits for use of the order
execution and routing services of Nasdaq for securities priced at $1 or
more. Rule 7018(a)(1) sets forth the fees and credits for the execution
and routing of orders in Nasdaq-listed securities (``Tape C
Securities''); Rule 7018(a)(2) sets forth the fees and credits for the
execution and routing of securities listed on the New York Stock
Exchange LLC (``NYSE'')(``Tape A Securities''), and Rule 7018(a)(3)
sets forth the fees and credits for the execution and routing of
securities listed on exchanges other than Nasdaq and NYSE (``Tape B
Securities'').
    A Designated Retail Order is an agency or riskless principal order
that meets the criteria of FINRA Rule 5320.03 and that originates from
a natural person and is submitted to Nasdaq by a member that designates
it as such, provided that no change is made to the terms of the order
with respect to price or side of market and the order does not
originate from a trading algorithm or any other computerized
methodology. An order from a ``natural person'' can include orders on
behalf of accounts that are held in a corporate legal form--such as an
Individual Retirement Account, Corporation, or a Limited Liability
Company--that has been established for the benefit of an individual or
group of related family members, provided that the order is submitted
by an individual. Members must submit a signed written attestation, in
a form prescribed by Nasdaq, that they have implemented policies and
procedures that are reasonably designed to ensure that substantially
all orders designated by the member as Designated Retail Orders comply
with these requirements. Orders may be designated on an order-by-order
basis, or by designating all orders on a particular order entry port as
Designated Retail Orders.
    Currently, under Rules 7018(a)(1)-(3) the Exchange provides a
$0.0034 per share executed credit to members for displayed Designated
Retail Orders in securities of all three Tapes. There is no
qualification criteria that must be met to receive the credit under
Rules 7018(a)(1)-(3). The Exchange is proposing to lower the $0.0034
per share executed credit to $0.0033 per share executed for displayed
Designated Retail Orders under Rules 7018(a)(1)-(3). The Exchange is
also proposing to adopt new qualification criteria for each of the
proposed $0.0033 per share executed credits under Rules 7018(a)(1)-(3).
Specifically, the Exchange is proposing to require a member to have a
ratio of at least 85% liquidity provided through one or more of its
Nasdaq Market Center MPIDs to all volume (adding and removing
liquidity) through one or more of its Nasdaq Market Center MPIDs during
the month to qualify for the $0.0033 per share executed credit under
Rules 7018(a)(1)-(3). Last, the Exchange is proposing to add a new
credit of $0.00325 per share executed for displayed Designated Retail
Orders in securities of all three Tapes under Rules 7018(a)(1)-(3).
Like the current $0.0034 per share executed credit, the Exchange is not
proposing any qualification criteria that must be met to receive the
proposed $0.00325 per share executed credit under Rules 7018(a)(1)-(3).
2. Statutory Basis
    The Exchange believes that its proposal is consistent with Section
6(b) of the Act,\5\ in general, and furthers the objectives of Sections
6(b)(4) and 6(b)(5) of the Act,\6\ in particular, in that it provides
for the equitable allocation of reasonable dues, fees and other charges
among members and issuers and other persons using any facility, and is
not designed to permit unfair discrimination between customers,
issuers, brokers, or dealers.
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    \5\ 15 U.S.C. 78f(b).
    \6\ 15 U.S.C. 78f(b)(4) and (5).
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    The Exchange believes that the proposed $0.0033 per share executed
credit is reasonable because it is competitive with the credits of
other exchanges. For example, NYSE Arca provides a $0.0033 per share
credit for Retail Orders that provide liquidity to the NYSE Arca
book.\7\
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    \7\ The credit is available to ETP Holders, including Market
Makers that execute an Average Daily Volume of Retail Orders that
provide liquidity during the month that is 0.15% or more of the US
CADV. US CADV is defined as ``US CADV means United States
Consolidated Average Daily Volume for transactions reported to the
Consolidated Tape, excluding odd lots through January 31, 2014
(except for purposes of Lead Market Maker pricing), and excludes
volume on days when the market closes early and on the date of the
annual reconstitution of the Russell Investments Indexes.
Transactions that are not reported to the Consolidated Tape are not
included in US CADV.'' See https://www.nyse.com/publicdocs/nyse/markets/nyse-arca/NYSE_Arca_Marketplace_Fees.pdf.

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    Currently, members are provided a credit of $0.0034 per share
executed; under the proposal, the credit will be $0.0033 per share
executed.
    The Exchange believes that the proposed $0.0033 per share executed
credit and the proposed qualification criteria required to receive the
credit is [sic] an equitable allocation and is not unfairly
discriminatory because the Exchange will apply the same credit to all
similarly situated members that meet the qualification criteria. The
proposed $0.0033 per share executed credit and the proposed
qualification criteria will reduce the cost of the incentive to the
Exchange while also improving market quality by applying a
qualification requirement that a member provide a significant share of
its volume in providing liquidity on the Exchange, namely, a ratio of
at least 85% liquidity provided through one or more of its Nasdaq
Market Center MPIDs to all volume (adding and removing liquidity)
through one or more of its Nasdaq Market Center MPIDs. The Exchange has
limited funds to apply in the form of incentives, and thus must deploy
those limited funds to incentives that it believes will be the most
effective at improving market quality in areas that the Exchange
determines are in need of improvement. In this instance, reducing the
amount of credit provided and applying new qualification criteria,
which not all members that currently qualify for the $0.0034 per share
executed credit will likely satisfy, should reduce the cost of
providing credits for Designated Retail Orders. In turn, the Exchange
would be able to apply any funds realized by the proposed changes to
other incentives that may improve market quality.
    The Exchange believes that the proposed $0.00325 per share executed
credit is reasonable because it is competitive with the credits of
other exchanges. As noted above, NYSE Arca provides a $0.0033 per share
credit for Retail Orders that provide liquidity to the NYSE Arca
book.\8\ Like the Exchange's proposed $0.0033 per share executed
credit, NYSE Arca has qualification criteria required of its
participants to receive its Retail Order credit. The proposed $0.00325
per share executed credit will not have any such requirements. Thus,
the lower credit reflects the absence of additional market-improving
behavior required to receive the credit.
---------------------------------------------------------------------------

    \8\ Id.
---------------------------------------------------------------------------

    The Exchange believes that $0.00325 per share executed credit is an
equitable allocation and is not unfairly discriminatory because the
Exchange will apply the same credit and criteria to all similarly
situated members. Like the current credit, the Exchange will not
require a member to meet any qualification criteria to receive the
credit. As a consequence, members will continue to have the opportunity
to receive a significant credit for such orders, which the Exchange
believes will also continue to provide incentive to members to enter
such beneficial orders. In this regard, the Exchange notes that
displayed liquidity promotes price discovery and retail orders often
represent investors with long-term investment horizons.
    Last, the Exchange notes that the proposed credits, like the
current credits provided for Designated Retail Orders, are available
for orders that have originated from natural persons only. The Exchange
believes that limiting the credit to Designated Retail Orders is an
equitable allocation and is not unfairly discriminatory because the
credit is designed to attract retail order flow to the Exchange, which
also benefits other market participants (including non-natural persons)
by providing additional liquidity to the Exchange with which such other
market participants may interact. As noted above, displayed liquidity
promotes price discovery and retail orders often represent investors
with long-term investment horizons, both of which benefit all market
participants by providing more liquid markets and a more diverse group
of market participants.

B. Self-Regulatory Organization's Statement on Burden on Competition

    The Exchange does not believe that the proposed rule change will
impose any burden on competition not necessary or appropriate in
furtherance of the purposes of the Act. In terms of inter-market
competition, the Exchange notes that it operates in a highly
competitive market in which market participants can readily favor
competing venues if they deem fee levels at a particular venue to be
excessive, or credit opportunities available at other venues to be more
favorable. In such an environment, the Exchange must continually adjust
its fees and credits to remain competitive with other exchanges and
with alternative trading systems that have been exempted from
compliance with the statutory standards applicable to exchanges.
Because competitors are free to modify their own fees and credits in
response, and because market participants may readily adjust their
order routing practices, the Exchange believes that the degree to which
fee changes in this market may impose any burden on competition is
extremely limited.
    In this instance, the proposed changes to the credits available to
members for execution of securities in securities of all three Tapes do
not impose a burden on competition because the Exchange's execution
services are completely voluntary and subject to extensive competition
both from other exchanges and from off-exchange venues. The proposed
credits are reflective of the Exchange's desire to allocate credits and
rebates to their most efficient use. The Exchange does not believe that
proposed changes will reduce the level of Designated Retail Orders
provided to the Exchange, but may reduce costs incurred by the Exchange
in supporting the incentive. Thus, the proposed changes reflect a
balance of targeting the correct level of incentive for the behavior
sought. As discussed above, the proposed credits are consistent [sic]
the credits provided by other exchanges for retail orders. In sum, if
the changes proposed herein are unattractive to market participants, it
is likely that the Exchange will lose market share as a result.
Accordingly, the Exchange does not believe that the proposed changes
will impair the ability of members or competing order execution venues
to maintain their competitive standing in the financial markets.

C. Self-Regulatory Organization's Statement on Comments on the Proposed
Rule Change Received From Members, Participants, or Others

    No written comments were either solicited or received.

III. Date of Effectiveness of the Proposed Rule Change and Timing for
Commission Action

    The foregoing rule change has become effective pursuant to Section
19(b)(3)(A)(ii) of the Act.\9\
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    \9\ 15 U.S.C. 78s(b)(3)(A)(ii).
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    At any time within 60 days of the filing of the proposed rule
change, the Commission summarily may temporarily suspend such rule
change if it appears to the Commission that such action is: (i)
Necessary or appropriate in the public interest; (ii) for the
protection of investors; or (iii) otherwise in furtherance of the
purposes of the Act. If the Commission takes such action, the
Commission shall institute proceedings to determine whether the
proposed rule should be approved or disapproved.

IV. Solicitation of Comments

    Interested persons are invited to submit written data, views, and
arguments concerning the foregoing,

including whether the proposed rule change is consistent with the Act.
Comments may be submitted by any of the following methods:

Electronic Comments

     Use the Commission's internet comment form (http://www.sec.gov/rules/sro.shtml); or
     Send an email to [email protected]. Please include
File Number SR-NASDAQ-2018-013 on the subject line.

Paper Comments

     Send paper comments in triplicate to Secretary, Securities
and Exchange Commission, 100 F Street NE, Washington, DC 20549-1090.

All submissions should refer to File Number SR-NASDAQ-2018-013. This
file number should be included on the subject line if email is used. To
help the Commission process and review your comments more efficiently,
please use only one method. The Commission will post all comments on
the Commission's internet website (http://www.sec.gov/rules/sro.shtml).
Copies of the submission, all subsequent amendments, all written
statements with respect to the proposed rule change that are filed with
the Commission, and all written communications relating to the proposed
rule change between the Commission and any person, other than those
that may be withheld from the public in accordance with the provisions
of 5 U.S.C. 552, will be available for website viewing and printing in
the Commission's Public Reference Room, 100 F Street NE, Washington, DC
20549 on official business days between the hours of 10:00 a.m. and
3:00 p.m. Copies of the filing also will be available for inspection
and copying at the principal office of the Exchange. All comments
received will be posted without change. Persons submitting comments are
cautioned that we do not redact or edit personal identifying
information from comment submissions. You should submit only
information that you wish to make available publicly. All submissions
should refer to File Number SR-NASDAQ-2018-013, and should be submitted
on or before March 26, 2018.

    For the Commission, by the Division of Trading and Markets,
pursuant to delegated authority.\10\
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    \10\ 17 CFR 200.30-3(a)(12).
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Eduardo A. Aleman,
Assistant Secretary.
[FR Doc. 2018-04420 Filed 3-2-18; 8:45 am]
 BILLING CODE 8011-01-P