SRC TR - 12 - 073

HPV 2 TEST RUL E DATA ADEQUACY REVIEW : SEPTEMBER 2012 UPD ATE (FINAL )

1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS RN 4719 - 04 - 4

Prepared b y: Jennifer Ray

ner , Teresa Manyin, Justine Von Runnen, Ashlee Aldridge,

and William L . Richards Chemical, Biological and Environmental Center

SRC, Inc. 7502 Round Pond Road North Sy racuse, NY 13212 Contract No. EP - W - 09 - 027 Task FG007.2.009.00 1 Submitted to: OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460 Debra Milligan, Project Officer Meena Sonawane, Work Assignment Manager David Brooks, Alternate Work Assignment Manager

September 14, 2012**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

i **TABLE OF CONTENTS** A. SOURCE MATERIAL USED FOR ASSESSMENT ................................ .............................. 1 B. EVALUATING DATA ADEQUACY ................................ ................................ ..................... 1 C . SUMMARY OF DATA ADEQUACY FOR TESTI NG REQUIRED BY TEST RULE ........ 1 D. DATA FOR SIDS ENDPOINTS ................................ ................................ .............................. 5 D - 1. Acute Toxicity

to Fish ................................ ................................ ................................ ....... 5 D - 2. Acute Toxicity

to Aquatic Invertebrates ................................ ................................ .......... 30 D - 3. Toxicity

to Aquatic Plants ................................ ................................ ............................... 35 D - 4. Acute Toxicity ................................ ................................ ................................ .................. 46 D - 5. Repeated - Dose Toxicity ................................ ................................ ................................ ... 59 D - 6. Genetic Toxicity

– Gene Mutation ................................ ................................ ................... 68 D - 7. Genetic Toxicity

– Chromosomal Aberrations ................................ ................................ 80 D - 9. Reproductive Toxicity ................................ ................................ ................................ ...... 84 D - 10. Developmental Toxicity ................................ ................................ ................................ . 85 E. SUPPL EMENTAL DATA (NON - SI DS ENDPOINTS) ................................ ........................ 88 E - 1. Genetic Toxicity

– Other ................................ ................................ ................................ .. 88 E - 2. Skin Irritation ................................ ................................ ................................ .................... 93 E - 3. Ey e Irritation ................................ ................................ ................................ ..................... 99 E - 4. Skin Sensitization ................................ ................................ ................................ ........... 103 F. DA TA ADEQUACY SUMMARY TABLE ................................ ................................ ......... 107**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

1 **A. SOURCE MATERIAL USED FOR ASSESSMENT**  This document evaluates an algal inhibition test study report, which was submitted to EPA on July 3 0, 2012 by the MEA Triazine Consortium in response to the C6 testing requirement for 1,3,5 - triazine - 1,3,5(2H,4H,6H) - triethanol (CASRN 4719

– 04

–4) in the final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of Chemicals” (76 FR 1067, January 7, 2011; referred to here in as HPV2). The C6 requirement consists of Toxicity to Plants (Algae) testing, follo wing ASTM guideline E 1218 - 04 ε1

. The toxicity to algae study submitted by the MEA Triazine Consortium is evaluated for adequacy herein . This report also contains data for other ecological and toxicological endpoints that were evaluated previously by EPA and, therefore, provides an update of the data adequacy assessment previousl y compiled for CASRN 4719 - 04 - 4 in April 2011 . **B. EVALUATING DATA ADEQUACY** Data for the HPV2 test rule - required endpoint, as well as available data for other SIDS toxicity endpoints, are evaluated for adequacy in Section D . Each stud y was summarized according to a standard format and assigned a Klimisch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether data were adequate to satisfy SIDS endpoints, study

details were compared to current standard test guidelines to establish whether the study met current testing requireme nts. Studies for a common endpoint are grouped together in the document and an endpoint adequacy assessment is provided near the top of the first page of the summaries for that endpoint. If the data available for the endpoint were determined to be inadequate, a basis for that conclusion is supplied. Additional data for non - SIDS toxicity endpoints (“supplemental” data) are reviewed in Section E . A table summarizing data availability

/adequacy

for each SIDS endpoint is included on the last pa ge of this doc ument (Section F) . **C .**  **SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE** The submitted algae study is summarized and evaluated in detail on p p . 35 - 41 of this report. The following deficiencies in the algae stud y were noted: (1) T he stud y authors measured the concentrations of the primary degradation products (monoethanolamine and formaldeh yde) and did not measure the concentration of the parent test compound ( 1,3,5 - triazine - 1,3,5(2H,4H,6H) - triethanol ; CASRN 4719 - 04 - 4 ). This analy tical testing scheme differs from the proposed methodology in the s tud y plan submitted by SOCMA ( the MEA Triazine Consortium ) on January 6, 2012; th is study

plan indicated that verification of the test concentrations would be achieved b y measurements of the pare nt test item at 0 hours and measurement of the degradation products at 0 and 96 hours. EPA had approved this proposed plan for verification of test concentrations. Despite the fact that the algae test did not include measurements of the parent test com pound, the measured concentrations of the degradation products appear to suggest that complete degradation of CASRN 4719 - 04 - 4 was achieved quickl y. The measured concentrations of monoethanolamine and formaldeh yde at the initiation of exposure (time = 0 ho urs) were similar**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

2 to the expected concentrations of these degradation products based on nominal concentrations of CASRN 4719 - 04 - 4, assuming that CASRN 4719 - 04 - 4 degraded completely to form monoethanolamine and formaldeh yde . Tables 1 and 2 (below) show comparisons of the measured and nominal concentrations for monoethanolamine and formaldehyde, respectivel y. Table 1. Concentrations of monoethanolamine in test medium. Sample Nominal

Concentration of CASRN 4719 - 04 - 4 (mg a.i./L) 1 Nominal

Concentration of Mono ethanolamine (mg/L) 2 Measured

Concentration of Mono ethanolamine (mg/L) Measured

Concentration of Mono ethanolamine as Percent of Nominal Calculated

Concentration of CASRN 4719 - 04 - 4 Based on Measurement (mg/L) 3 0 hours 1.0 0.84 0.518 62 0.62

3.2 2.67 3.17 119 3.79 10 8.36 11.4 136 13.64 32 26.74 34.7 130 41.52 100 83.56 91.9 110 109.98 96 hours (opened every 24 h to obtain cell density samples) 1.0 0.84 0.343 41 0.41

3.2 2.67 2.82 105 3.37 10 8.36 7.6 91 9.09 32 26.74 19.6 73 23.45 100 83.56 56.8 68 67.97 96 hours (unopened) 1.0 0.84 0.373 45 0.45

3.2 2.67 2.68 100 3.21 10 8.36 7.61 91 9.11 32 26.74 19.8 74 23.69 100 83.56 57.7 69 69.05 1 a .i. = active ingredient (concentration adjusted for other components in test substance) 2 Nominal concentration of monoethanol amine is calculated from the nominal concentration of CASRN 4719 - 04 - 4 and assumes complete degradation of each mole of CASRN 4719 - 04 - 4 to produce 3 moles of monoethanolamine and 3 moles of formaldeh yd e. 3 Concentration of 4719 - 04 - 4 was calculated from the measured concentration of monoethanolamine and assumes that each mole of CASRN 4719 - 04 - 4 produced 3 moles of monoethanolamine.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

3 Table 2 . Concentrations of formaldeh yd e in test medium. Sample Nominal

Concentration of CASRN 4719 - 04 - 4 (mg a.i./L) 1 Nominal

Concentration of Formaldeh yd e (mg/L) 2 Measured

Concentration of F ormaldeh yd e (mg/L) Measured

Concentration of F ormaldeh yd e as Percent of Nominal Calculated

Concentration of CASRN 4719 - 04 - 4 Based on Measurement (mg/L) 3 0 hours 1.0 0.41 0.482 117 1.17

3.2 1.31 1.5 114 3.65 10 4.11 4.21 102 10.25 32 13.15 15 114 36.51 100 41.08 43.8 107 106.61 96 hours (opened every 24 h to obtain cell density samples) 1.0 0.41 0.11 27 0.27

3.2 1.31 0.429 33 1.04 10 4.11 2.96 72 7.20 32 13.15 10.2 78 24.83 100 41.08 30.5 74 74.24 96 hours (unopened) 1.0 0.41 0.129 31 0.31

3.2 1.31 0.511 39 1.24 10 4.11 2.95 72 7.18 32 13.15 8.53 65 20.76 100 41.08 27 66 65.72 1 a.i. = active ingredient (concentration adjusted for other components in test substance) 2 Nominal concentration of formaldeh yd e is calculated from the nominal concentration of CASRN 4719 - 04 - 4 and assumes complete degradation of each mole of CASRN 4719 - 04 - 4 to produce 3 moles of monoethanolamine and 3 moles of formaldeh yd e. 3 Concentration of 4719 - 04 - 4 was calculated from the measured concentration of formaldeh yde and assumes that each mole of CASRN 4719 - 04 - 4 produced 3 moles of formaldehyde . (2) The study

authors calculated the EC 50 values based on nominal test concentrations . In general, if measured concentrations indicate that exposure concentrations were not maintained within ± 20% of nominal values, the use of mean measured (rather than nominal) concentrations to calculate EC 50 values is preferred. At initiation of exposure (time = 0 hours) , the measured concentrations of monoethanolamine and formaldeh yd e were 62 - 136% and 102 - 117% of nominal (expected) concentrations, respectivel y. At the end of exposure (time = 96 hours), the measured concentrations of monoethanolamine and formaldeh yde wer e 41 - 105 % and 27 - 78 % of nominal (expected) concentrations, respectively . Thus, the measured concentrations of monoethanolamine and formaldeh yde both demonstrated variations outside the range of ± 20% of nominal values during the exposure period , with conc entrations of formaldehyde showing a higher degree of variability than those of monoethanolamine . The variation in concentrations could have been the result of uptake b y al gae cells and/or reactions between monoethanolamine and formaldehyde to form other chemical compounds.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

4 Nonetheless, the measured concentration s of monoethanolamine display ed a relative ly low degree of variation (measured concentrations were 91 - 1 36 % of nominal concentrations) at the nominal test concentration s of 3.2 and 10 mg a.i./L. Since the 96 - hour EC 50 values presented for growth rate, yi eld, and biomass (4.2, 3.2, and 3.3 mg/L, respectivel y) were within the nominal concentration range of 3.2 to 10 mg a.i./L , they are likely to represent reasonable approximations of the values that would be obtained if measured concentrations had been used to calculate EC 50 values (i.e., b y using calculated concentrations of CASRN 4719 - 04 - 4 that correspond to measured concentrations of degradation products as shown in Tables 1 and 2, abo ve) . (3) The complete composition of the test substance is not clearl y stated in the submitted study report. The “Certificate of Anal ys is” in Appendix 1 does not define the complete composition of the test substance . A purity of 77.5% is supplied on page 7 and Appendix 4 indicates that the test substance contained 22.5% water, suggesting that water was the onl y impurity were present in the test compound. The “Certificate of Analy sis” indicates that excess monoethan olamine (0.02 mole s ) was used in the production process; however, the excess monoethanolamine appears to amount to <1% of the total composition of the test substance. Since total impurit ies other than water appear to be <1%, the test substance is acceptab le. **Conclusions:** Despite the study deficiencies noted above, the a lgae toxicity data supplied on July 30 , 2012 by the MEA Triazine Consortium were considered adequate to satisfy the T oxicity to P lants ( Algae) C6 test requirement for CASRN 4719 - 04 - 4 in the HPV2 test rule . The re are no d ata gaps remain ing for an y S IDS endpoints.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

5 **D . DATA FOR SIDS ENDPOINTS**  **D -**  **1. Acute Toxicity to Fish**  **Summary of endpoint:** Adequate **Study 1: Acute toxicity study in rainbow trout (**  **Davis and Kernaghan, 1994) Title:**  Acute Toxicity

to Rainbow Trout, Oncorhynchus mykiss , Under Flow - through Test Conditions **This study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 83.8% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 16.2% unidentified **Remarks:** Also referred to as Triazine and Shaughnessy No. 083301 METHOD **Method/guideline followed:** OPP 72 - 1(A) **Test type:** Acute Toxicity Test for Freshwater Fish, Rainbow Trout ( Oncorhynchus mykiss ), flow - through **GL P compliant?:**  Not specified **Year study was performed:** 1994 **Test species:** Rainbow trout ( Oncorhynchus mykiss ), obtained from Mt. Lassen Trout Farms, Red Bluff, CA **Analytical monitoring:** Yes **Exposure duration:** 96 hours **Concentrations tested:** Nominal: 15.6, 25.9, 43.2, 72.0, and 120 mg a.i./L; mean measured: 13.8, 26.6, 44.5, 73.4, and 119 mg a.i./L **Test method/conditions remarks:** Rainbow trout were fed salmon starter and commercial flake food daily

throughout the holding period. During the 48 hours immedi atel y prior to initiation of the 96 - hour exposure, mortality was 0 and fish were not fed. Rainbow trout ranged from 40 to 55 mm standard length and from 0.81 to 2.21 g wet weight as measured from the control at test termination.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

6 The flow - through test was conducted in 23 - L tanks which maintained a constant water volume of approximately

15.3 L and a water height of 13 cm. The diluter (proportional vacuum - siphon) cy cled at an average of 5.0 cycles per hour. Organism loading was calculated to be 0.14 g/L/da y, with a maximum loading at an y point in time of 1.6 g/L. Dilution water was municipal water, which was vigorousl y aerated to remove chlorine, then passed through activated carbon beds before being pumped into the laboratory . The water was re - aerated prior to use. The dilution water was a moderatel y hard freshwater with a mean hardness of 52 mg CaCO 3 /L , mean alkalinit y of 16 mg CaCO 3 /L , and a mean specific conductivity

of 428 μmhos/cm. A range - finding test was conducted to determine the concentration range for the definitive test. The nominal concentrations for the definitive test were: 15.6, 25.9, 43.2, 72.0, and 120 mg a.i./L . No solvent was utilized to solubilize the test material as Triazine is completely miscible in water. Twenty rainbow trout were randoml y distributed in the treatment and control chambers of the flow - through test s ys tem. Survival of the trout was monitored daily and any dead fish were removed. An y abnormalities in the behavior or phy sical appearance of rainbow trout were als o noted. Test solutions were gentl y aerated during the test. Test water quality

was monitored daily

during the test. Water temperature was measured in the dilution water control hourly during the test and ranged from 11.1 to 13.8 °C. Specific conductiv ity , total alkalinity , and total hardness of the dilution water were measured at test initiation and termination in the control container. Dissolved oxygen and pH were measured daily in all test solutions with surviving fish. Water samples were collected from the controls and all five test solutions at test initiation, day 2, and termination to verify

actual test concentrations. Mean measured concentrations were 13.8, 26.6, 44.5, 73.4, and 119 mg a.i./L. RESUL TS **Endpoint value(s):** The 96 - hour L C 50 for rainbow trout was > 119 mg a.i./L . The NOEC was 119 mg a.i./L. **Results remarks:** Mortality of rainbow trout exposed for 96 hours to Triazine was zero at all concentrations tested. No mortality occurred in the dilution water control. The 96 - hour LC 50 w as greater than 119 mg a.i./L . The NOEC was 119 mg a.i./L, the highest concentration tested, based on a lack of mortality and a lack of sublethal effects at this test concentration and all test concentrations. CONCLUSIONS The 96 - hour LC 50 for rainbow tr out was > 119 mg a.i./L, based on a lack of mortality and sublethal effects at the highest dose tested.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

7 STUDY RELIABILITY [1] Reliable without restrictions. This study was classified as “core” b y U.S. EPA’s Office of Pesticide Programs. REFERENCE Davis, J.W., and Kernaghan, N.J. 1994. Acute Toxicity to Rainbow Trout, Oncorhynchus mykiss , Under Flow - through Test Conditions. Performing Laboratory : Toxikon Environmental Sciences, Jupiter, FL. Submitted by the Triazine Joint Venture, c/o Buckman Laboratories International, In c., Memphis, TN. U.S. EPA MRI D No. 431431 - 01.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

8 **Study 2: Acute toxicity test in sheepshead minnow (Ward, 1994)**  **Title:** Acute Toxicity

to the Sheepshead Minnow ( Cyprinodon variegatus ), Under Flow - through Test Conditions **This study is:**  Adequate Not Adequate TEST SUBSTANCE **Identity:** 83.8% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 16.2% unidentified **Remarks:** Also referred to as Triazine and Shaughnessy No. 083301 METHOD **Method/guideline followed:** OPP 72 - 3(A) **Test type:** Acute Toxicity Test for Estuarine/Marine Fish, Sheepshead Minnow ( Cyprinodon variegatus ), flow - through **GL P compliant?:**  Yes **Year study was performed:** 1994 **Test species:** Juvenile s heepshead minnows ( C. variegatus ), obtained from Toxikon Sciences, Jupiter, FL; age: 8 weeks; length: 18 - 24 mm; wet weight: 0.14 - 0.61 g **Analytical monitoring:** Yes **Exposure duration:** 96 hours **Concentrations tested:** Nominal: 15.6, 25.9, 43.2, 72.0 and 120 mg a.i./L ; mean measured: 11.8, 23.8, 38.9, 70.9 and 118 mg a.i./L **Test method/conditions remarks:** Juvenile sheepshead minnows (8 weeks old), obtained from Toxicon Sciences, were initially raised on a diet of live brine shrimp nauplii and later transferred to a diet of flake flood and frozen brine shrimp. During the 48 hours prior to testing, the minnows were not fed. The minnows ranged from 18 to 24 mm in length and from 0.14 to 0.61 g wet weight, as determined from the control fish at test termination. The flow - through test was conducted in 23 - L tanks which maintained a constant water volume of approximately

15.3 L and a water height of 13 cm. The diluter (proportional vacuum - siphon) cy cled at an average of 5.7 cycles per hour. Organism loading was calculated to be 0.03 g/L/day with a maximum loading at an y point in time of 0.37 g/L.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

9 Dilution water was natural saltwater pumped from a shallow well. The saltwater was then filtered, carbon - treated, and adjusted to a salinity of approximately

20 ppt with carbon - treated aerated laboratory

freshwater. This dilution water was re - aerated prior to use . A range - finding test was conducted to determine the concentration range for the definitive test. The nominal concentrations for the definitive test were: 15.6, 25.9, 43.2, 72.0 and 120 mg a.i./L . No solvent was utilized to solubilize the test material as Triazine is completely miscible in water. Twenty sheepshead minnows were randoml y distributed in the treatment and control chambers of the flow - through test sy stem. Survival of the fish was monitored daily and any dead were removed. An y abnormalitie s in the behavior or phy sical appearance of the minnows were also noted. Test solutions were greatly aerated during the test. Test water quality

was monitored daily

during the test. Water temperature was measured in the dilution water control container hourl y during the test. Salinity was measured once daily . Dissolved oxygen concentrations and pH were measured daily in all test solutions with surviving fish. Water samples were collected from the controls and all five test solutions at test initiation , day 2, and termination to verify

actual test concentrations. Mean measured concentrations were 11.8, 23.8, 38.9, 70.9, and 118 mg a.i./L. RESUL TS **Endpoint value(s):** The 96 - hour L C 50 was > 118 mg a.i./L. The NOEC was 118 mg a.i./L . **Results remarks:** Mortality of sheepshead minnows exposed for 96 hours to Triazine was zero at all concentrations tested. No mortality occurred in the dilution water control. The 96 - hour LC 50 was greater than 118 mg a.i./L. The NOEC was 118 mg a.i./L , based on lack of mortalit y and sublethal effects at this test concentration and all lower test concentrations. CONCLUSIONS The 96 - hour LC 50 for sheepshead minnows was greater than 118 mg a.i./L , based on a lack of mortality and sublethal effects at the highest concentrat ion tested. STUDY RELIABILITY [1] Reliable without restrictions. This study was classified as “core” b y U.S. EPA’s Office of Pesticide Programs.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

10 REFERENCE Ward, G.S. 1994. “Acute Toxicity to the Sheepshead Minnow, Cyprinodon variegatus , Under Flow - thr ough Test Conditions”. Performing Laboratory : Toxikon Environmental Sciences, Jupiter, FL. Submitted by the Triazine Joint Venture, c/o Buckman Laboratories International, In c., Memphis, TN. U.S. EPA MRID No. 431431 - 02.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

11 **Study 3: Acute toxicity test i**  **n bluegill (LeBlanc and Sousa, 1982a) Title:**  Acute Toxicity

of Triadine 10 to Bluegill (Lepomis macrochirus) **This study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 63.6% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 6.4% sodium omadine, CAS No. not specified; 30% unidentified **Remarks:** Formulation, also referred to as Triadine 10 METHOD **Method/guideline followed:** Not specified **Test type:** Acute Toxicity for Freshwater Fish, Bluegill (Lepomis macrochirus) **GL P compliant?:**  Not specified **Year study was performed:** 1982 **Test species:** Bluegill (Lepomis macrochirus) **Analytical monitoring:** Not specified **Exposure duration:** 96 hours **Concentrations tested:** 36, 60, 100, 170, and 280 mg/L (not specified whether the concentrations were nominal or measured) **Test method/conditions remarks:**  Detailed methods for the exposure were not specified. The test procedure did not comply with the recommended EPA protocol of October 1982 (Part 158). The L C 50 was estimated using a binomial test. RESUL TS **Endpoint value(s):** The 96 - hour L C 50 v alue for bluegill was 77 mg/L. **Results remarks:** No mortality was observed at 36 and 60 mg/L. Mortality was 100% at 100, 170, and 280 mg/L. The 96 - hour L C 50 value for bluegill was 77 mg/L and the confidence interval was 60 to 100 mg/L.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

12 CONCLUSIONS This study

indicates that the test substance is slightly toxic to bluegill with an L C 50 of 77 mg/L. STUDY RELIABILITY [2] Reliable with restrictions. Deviations from the EPA protocol of October 1982 (part 158) included: (1) a formulated product was us ed, which was a mixture of two active ingredients; (2) the number of fish per concentration was not specified; and (3) dissolved oxygen after 96 hours in the control and 36 mg/L concentrations was 3.7 and 1.7 ppm, respectively, which is below 40% saturatio n. This study was classified as “supplemental” by U.S. EPA’s Office of Pesticide Programs. The stud y is considered to provide useful information, in support of core or key studies, and the results may be used as part of a weight - of - evidence approach to d escribe the acute toxicity

to fish. REFERENCE LeBlanc, G., and Sousa, J. 1982a. Acute Toxicity of Triadine 10 to Bluegill ( Lepomis macrochirus ); Report # BW - 82 - 2 - 1119. Performing Laboratory : EG&G Bionomics. Submitted by the Olin Corporation, New Haven, C T. U.S. EPA MRID No. 00105191.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

13 **Study 4: Acute toxicity study in rainbow trout (Raasina, 1973)**  **Title:** Four - day

Static Fish Toxicity

Studies with Milidin X - 2 Rainbow Trout and Bluegills **This study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; Purity : not specified. **Remarks:** Also referred to as Grotan and Milidin X - 2 METHOD **Method/guideline followed:** Not specified **Test type:** Acute toxicity test for Freshwater Fish, Rainbow Trout ( Oncorhynchus mykiss ), static **GL P compliant?:**  No **Year study was performed:**  1973 **Test species:** Juvenile rainbow trout ( O. mykiss ); length = 35 - 75 mm **Analytical monitoring:** No **Exposure duration:** 96 hours **Concentrations tested:** Nominal: 0, 18, 32, 56, 78 and 100 mg/L **Test method/conditions remarks:** Juvenile rainbow trout were observed for general health and suitability for a period not less than 14 day s prior to experimental use in preliminary screening studies. A 24 - hour acclimation period was conducted prior to the definitive test. Fish length ranged from 35 to 75 mm. This fish were fed either frozen brine shrimp or Purina Trout Chow #2 until 72 hours prior to test initiation. The water temperature was held at 12oC for a minimum of 14 day s prior to testing. The holding and dilution water cons isted of reconstituted deionized water, supplemented with 30 mg/L calcium sulfate, 30 mg/L magnesium sulfate, 48 mg/L sodium bicarbonate, and 2 mg/L potassium chloride. Stock solutions were prepared within one hour of dosing and were dispensed as a 10% (w/v) aqueous solution. A range - finding test was conducted to determine the concentration range for the definitive test. For the definitive test, nominal test concentrations were: 0 (control), 18, 32, 56, 78 and 100 mg/L. No solvent was utilized to solubi lize the test material. Ten fish per test concentration were placed in each test vessel under static conditions. The test vessels were lined with poly eth yl ene bags. There was one replicate per test concentration. The fish were not fed during**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

14 treatment. The untreated control consisted of dilution water and was treated similarly to the dosed groups. A positive control was included, in which fish were exposed to toxaphene in a 0.1% (w/v) solution in acetone. Observations were made during the first 6 hours and at 24, 48, 72, and 96 hours. Dissolved ox ygen and pH values were measured for the groups which experienced mortalit y and daily for the untreated control group. The Litchfield and Wilcoxon tests were used to determine the L C 50 . RESUL TS **Endpoint value(s):**  The 96 - hr L C 50 for rainbow trout was 74 mg/L. The NOEC was 18 mg/L. **Results remarks:** The untreated control had zero mortality . The mortalit y rates were 0, 0, 10, 50, and 100% at 18, 32, 56, 78, and 100 mg/L, respectively . Other toxic effects included dark discoloration and quiescence observed at all concentrations except 0 and 18 mg/L. Surfacing was observed at 78 and 100 mg/L. The measured pH range was 7.8 to 9.4. The control pH ranged from 7.1 to 7.3. The dissolved ox ygen concentration ranged from 7.7 to 9.3 ppm (62 to 74%). The control dissolved ox ygen ranged from 6.6 to 8.0 ppm (53 to 64%). The study authors reported a 96 - hr LC 50 for rainbow trout of 74 mg/L, with a 95% confidence interval of 61 - 89 mg/L, as well as a NOEC of 18 mg/ L, based on the absence of mortalit y and sublethal effects. An EPA OPP reviewer used EPA's TOXANAL program to verify the study author’s values. The L C 50 determined by TOXANAL was 74 mg/L, with a 95% confidence interval of 64.2 - 83.4 mg/L. The NOEC was no t available through the TOXANAL program. CONCLUSIONS The 96 - hr LC 50 for rainbow trout was 74 mg/L. The NOEC was 18 mg/L, based on the absence of mortality

and sublethal effects. STUDY RELIABILITY [2] Reliable with restrictions. Deviations from the EPA protocol OPPTS Guideline 850.1075 included the following: (1) test chemical concentrations were not monitored and recorded throughout the experiment; (2) the length of the longest fish was > 2x the shortest fish; (3) fish were fed until 72 hours prior to test initiation rather than the recommended 48 hours; (4) pH and dissolved oxygen (DO) were onl y measured daily for the control and if mortality occurred rather than daily for the control and all test groups; (5) some pH measurements (which ranged from 7.1 to 9.4) were outside of the acceptable range (guideline specifies 6.0 to 8.0); (6) the following information was not provided: L C 50 and 95% confidence limits for 24, 48, and 72 hours, range finding stud y data, purit y and physicochemical characteristics of test material, temperature of water during test period, water hardness and salinity , methods and data from water sample anal ys is verifying concentration and impurities, pretest mortality and disease treatment, description of acclimation and test facili ties, weight of fish, supplier of fish, whether fish were used in other tests prior to this study , data on fish placement in test chamber and whether distributed randomly , fish loading rate, construction materials used for the test tanks and covers over tanks, calibration or aeration of test sy stem and flow rate, photoperiod and light intensity ,**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

15 method of stock preparation, test date and personnel, list of any protocol deviations occurring during test dates, quality

assurance and GLP compliance statements, and concentration - response and concentration - mortality curves. This study

was classified as “supplemental” by U.S. EPA’s Office of Pesticide Programs. The study is considered to provide useful information, in support of core or key

studies, and the resul ts may be used as part of a weight - of - evidence approach to describe the acute toxicity to fish. REFERENCE Raasina, G. 1973. Four - Day Static Fish Toxicity

Studies with Milidin X - 2 in Rainbow Trout and Bluegills; Report ID# IBT No. 665 - 03509. Performing Laboratory : Industrial Bio - test Laboratories, Inc. Submitted by the DeMille Chemical Corporation. U.S. EPA MRID No. 00073588.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

16 **Study 5: Acute toxicity study in bluegill (Raasina, 1973)**  **Title:** Four - day

Static Fish Toxicity

Studies with Milidin X - 2 Rainb ow Trout and Bluegills **This study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; Purity : not specified. **Remarks:** Also referred to as Grotan and Milidin X - 2 METHOD **Method/guideline followed:** Not specified **Test type:** Acute toxicity test for Freshwater Fish, Bluegill ( Lepomis macrochirus ), static **GL P compliant?:**  No **Year study was performed:** 1973 **Test species:** Juvenile bluegill ( L. macrochirus ); length = 35 - 75 mm **Analytical monitoring:** No **Exposure duration:** 96 hours **Concentrations tested:** Nominal: 0, 18, 32, 56, 78 and 100 mg/L **Test method/conditions remarks:** Juvenile bluegill were observed for general health and suitability for a period not less than 14 day s prior to experimental use in preliminary screening studies. A 24 - hour acclimation period was conducted prior to the definitive test. Fish length ranged from 35 to 75 mm. This fish were fed either frozen brine shrimp or Purina Trout Chow #2 until 72 hours prior to test initiation. The water temperature was held at 18oC for a minimum of 14 day s prior to testing. The holding and dilution water consisted of reconstituted de ionized water, supplemented with 30 mg/L calcium sulfate, 30 mg/L magnesium sulfate, 48 mg/L sodium bicarbonate, and 2 mg/L potassium chloride. Stock solutions were prepared within one hour of dosing and were dispensed as a 10% (w/v) aqueous solution. A range - finding test was conducted to determine the concentration range for the definitive test. For the definitive test, the nominal test concentrations were: 0 (control), 18, 32, 56, 78 and 100 mg/L. No solvent was utilized to solubilize the test materia l. Ten fish per test concentration were placed in each test vessel under static conditions. The test vessels were lined with poly eth yl ene bags. There was one replicate per test concentration. The fish were not fed during treatment. The untreated contr ol consisted of dilution water and was treated similarly to the**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

17 dosed groups. A positive control was included, in which fish were exposed to toxaphene in a 0.1% (w/v) solution in acetone. Observations were made during the first 6 hours and at 24, 48, 72, and 96 hours. Dissolved ox ygen and pH values were measured for the groups which experienced mortalit y and daily for the untreated control group. The Litchfield and Wilcoxon tests were used to determine the L C 50 . RESUL TS **Endpoint value(s):** The 96 - hr L C 50 for bluegill was 40 mg/L. The NOEC was 18 mg/L. **Results remarks:** The untreated control had zero mortality . The mortalit y rates were 0, 20, 90, 100, and 100% at 18, 32, 56, 78, and 100 mg/L, respectively . Other toxic effects included dark discolorat ion and quiescence observed at all concentrations except 0 and 18 mg/L. Surfacing was observed in bluegill at 56, 78, and 100 mg/L. The measured pH range was 8.7 to 9.3. The control pH ranged from 6.9 to 7.1. The dissolved oxygen concentration ranged f rom 6.5 to 8.5 ppm (52 to 68%). The control dissolved oxygen ranged from 7.4 to 7.9 ppm (59 to 63%). The study authors reported a 96 - hr LC 50 for bluegill of 40 mg/L , with a 95% confidence interval of 33 - 49 mg/L, as well as a NOEC of 18 mg/L, based on the absence of mortality and sublethal effects. An EPA OPP reviewer used EPA's TOXANAL program to verify the study author’s values. The LC 50 determined by TOXA NAL was 40 mg/L, with a 95% confidence interval of 32.2 - 48.3 mg/L. The NOEC was not available through the TOXANAL program. CONCLUSIONS The 96 - hr LC 50 for bluegill was 40 mg/L. The NOEC was 18 mg/L, based on the absence of mortality and sublethal effects. STUDY RELIABILITY [2] Reliable with restrictions. Deviations from OPPTS Guideline 850.1075 included the following: (1) test chemical concentrations were not monitored and recorded throughout the experiment; (2) the length of the longest fish wa s > 2x the shortest fish; (3) fish were fed until 72 hours prior to test initiation rather than the recommended 48 hours; (4) pH and dissolved ox ygen (DO) were onl y measured daily for the control and if mortality occurred rather than dail y for the control and all test groups; (5) some DO measurements (which ranged from 6.5 to 8.5 ppm [52 to 68%]) were outside of the acceptable range for a static test (guideline specifies > 60%); (6) some pH measurements (which ranged from 6.9 to 9.3) were outside of the acc eptable range (guideline specifies 6.0 to 8.0); (7) the following information was not provided: L C 50 and 95% confidence limits for 24, 48, and 72 hours, range - finding study data, purity

and phy sicochemical characteristics of test material, temperature of w ater during test period, water hardness and salinity , methods and data from water sample analy sis verifying concentration and impurities, pretest mortality

and disease treatment, description of acclimation and test facilities, weight of fish, supplier of f

ish, whether fish were used in other tests prior to this study, data on fish placement in test chamber and whether distributed randomly , fish loading rate, construction materials used for the test tanks and covers over tanks, calibration or aeration of tes t sy stem and**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

18 flow rate, photoperiod and light intensity , method of stock preparation, test date and personnel, list of any protocol deviations occurring during test dates, quality assurance and GLP compliance statements, and concentration - response and conc entration - mortality curves. This study

was classified as “supplemental” b y U.S. EPA’s Office of Pesticide Programs. The study is considered to provide useful information, in support of core or key studies, and the results may be used as part of a weight - of - evidence approach to describe the acute toxicity to fish. REFERENCE Raasina, G. 1973. Four - Day Static Fish Toxicity

Studies with Milidin X - 2 in Rainbow Trout and Bluegills; Report ID# IBT No. 665 - 03509. Performing Laboratory : Industrial Bio - test Lab oratories, Inc. Submitted by the DeMille Chemical Corporation. U.S. EPA MRID No. 00073588.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

19 **Study 6: Acute toxicity test in rainbow trout (Sousa and LeBlanc, 1982a)**  **Title:** Acute Toxicity

of Triadine 10 to Rainbow Trout ( Salmo gairdneri ) **This study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4, Purity : 63.6 - 65.9%; Impurit y: ~ 6.4% sodium omadine, CAS No. not specified; ≥ 30% unidentified **Remarks:** Also referred to as Grotan and Triadine 10; Industrial Microbiostat, L ot No. OD 9211 METHOD **Method/guideline followed:** Not specified **Test type:** Acute toxicity test in for Freshwater Fish, Rainbow Trout ( Oncorhynchus mykiss ), static **GL P**  **compliant?:** No **Year study was performed:** 1982 **Test species:** Juvenile rainbow trout, obtained from a commercial fish supplier in Montana (Bionomics lot no. 81A33); average wet weight: 0.63 g; average length: 43 mm **Analytical monitoring:** No **Exposure duration:** 96 hours **Concentrations tested:** Nominal: 7.8, 13, 22, 36, and 60 mg/L **Test method/conditions remarks:** Juvenile rainbow trout , obtained from a commercial fish supplier in Montana (Bionomics lot no. 81A33), were allowed to acclimate for 14 day s. The fish were held in well water that flowed through the holding tank at a rate of 13 - 15 tank volume replacements/day . The total hardness of the holding water was 30 mg CaCO 3 /L , the alkalinity was 24 - 30 mg CaCO 3 /L , the specific conductance was 100 μhos/cm, the pH ranged from 6.7 - 6.9, the temperature was 11 - 12 oC, the photoperiod was 16 hours light/8 hours dark, and the dissolved oxygen ranged from 83 - 94% of saturation. While being held, the fish were fed dry pellet food daily , ad libitum . Food was withheld for 48 hours prior to testing. The pretest mortality , observed for 48 hours prior to testing, was 0.2%. The dilution water consisted of soft water reconstituted from deionized water, using EPA recommended proc edures, and had a hardness of 42 mg CaCO 3 /Land specific conductance of 140 μhos/cm. The stock solution of Triadine 10 (15 mg/L) was prepared by adding distilled**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

20 water to 7.5 grams of Triadine 10 in a 500 - mLvolumetric flask, until an appropriate volume h ad been reached. The test vessels consisted of 19.6 - L glass jars with 15 Lof test solution. No evidence of a range - finding stud y was recorded. Ten juvenile rainbow trout per replicate were distributed randomly to each test jar within 10 minutes after the preparation of the test solution, and were exposed to nominal concentrations of 7.8, 13, 22, 36, or 60 mg/L under static conditions. The negative control jar contained the same dilution water as in the exposure jars. Three replicates were used for eac h concentration. The wet weight of the fish was ~0.63 g (ranging from 0.41 to 0.94 g), and the length was ~43 mm (ranging from 39 to 48 mm). All test temperatures were maintained at 12 ± 1 oC upon the addition of rainbow trout, were not aerated, and had a photoperiod of 16 hours light/8 hours dark. Biological observations were made every 24 hours. The temperature was recorded once for each exposure time during the definitive test. The pH and dissolved oxygen concentrations were measured at 0, 24, 48, and 96 hours in the control, high, middle and low test concentrations. The dose - related mortality

data from the definitive toxicity test was used to estimate the 24 - , 48 - , 72 - , and 96 - hour median lethal concentrations and 95% confidence intervals. Toxanol was used to perform the statistical analy sis, using the moving average angle method. It should be noted that this study was reviewed in two OPP documents, and there are some inconsistencies between these two reviews (authored by C. E. Laird [1983] and W. Erickson [2008]). In one OPP review (by W. Erickson [2008]), the purity of the test substance is reported as 65.9%, while in the second review (b y C. E. Laird [1983]), the purity of the test substance is stated as 63.6%, with sodium omadine as a component (6.4%). Additionally , it should be noted that only about 70% of this tested compound is accounted for b y sodium omadine and Triadine 10 in this study , leaving about 30% of the compound as “unknown substance.” RESUL TS **Endpoint value(s):** The 96 - hr L C 50 for rainbow trout was 42 mg/L. The LOEC was 7.8 mg/L, based on sublethal effects, and the NOEC was < 7.8 mg/L. **Results remarks:** The pH and dissolved oxygen concentrations and ranged from 6.8 to 9.1 and 6.9 to 10.6 mg/L , respectively . Mortalit y occurr ed at nominal concentrations of 36 and 60 mg/L at rates of 40 and 80%, respectivel y. No mortality was observed at concentrations ≤ 22 mg/L. Sublethal effects were observed at all treatment concentrations. Darkened pigment was noted at all concentrations and at all exposure times except 24 hours. Fish were respiring rapidly over all concentrations after 96 - h exposure, and at concentrations greater than 22 mg/L after 72 and 48 - h exposures. Using a moving average statistical method, the 96 - hr L C 50 for ra inbow trout was determined to be 42 mg/L, with a 95% confidence interval of 34 - 53 mg/L. The NOEC was < 7.8 mg/L , based on sublethal effects observed at the lowest concentration tested.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

21 CONCLUSIONS The 96 - hr LC 50 for rainbow trout was 42 mg/L. The NOEC was < 7.8 mg/L , based on sublethal effects including rapid respiration and dark coloration. STUDY RELIABILITY [2] Reliable with restrictions. Deviations from OPPTS Guideline 850.1075 included the following: (1) the test substance was a formulated prod uct, which was a mixture of more than one ingredient, and test chemical concentrations were not monitored and recorded throughout the experiment (guideline requires measurement at 0, 48, and 96 hours); (2) holding water and test water dilution came from tw o different sources and rainbow trout were not held in the test water dilution for a minimum of 7 day s during the acclimation period; (3) no evidence of a range finding test was provided as a basis for definitive test concentrations; (4) the number of repl icates for each concentration was not explicitly stated (three replicates assumed based on n = 30); (5) some pH measurements (range = 6.8 - 9.1) were outside the recommended range for freshwater species (6.0 - 8.0); and (6) the following information was not pr ovided: chemical anal ys is of dilution water, pretest mortality for the entire acclimation period, health of fish prior to test initiation, use and ty pe of cover for test vessels, dissolved oxygen data at 72 hours, hourl y temperatures in one replicate throu ghout the stud y, anal ys is of the stability of the test chemical, mortality at 6 hours, a graph of the concentration - mortality curve, and quality

assurance and GLP compliance statements. This study was classified as “supplemental” b y U.S. EPA’s Office of P esticide Programs. The study is considered to provide useful information, in support of core or key studies, and the results may be used as part of a weight - of - evidence approach to describe the acute toxicity

to fish. REFERENCE Sousa, J.V., and L eBlanc, G.A. 1982a. Acute Toxicity of Triadine 10 to Rainbow Trout ( Salmo gairdneri ); L aboratory

Report BW - 82 - 2 - 1117. Performing Laboratory : EG&G, Bionomics, Aquatic Toxicology

L aboratory , Wareham, MA. Submitted by the Olin Corporation, New Haven, CT. U.S. EPA M RID Nos. 247712 and 00105190.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

22 **Study 7: Acute toxicity study in rainbow trout**  **(Sousa and LeBlanc, 1982b) Title:**  Acute Toxicity

of Milidin X - 2 to Rainbow Trout ( Salmo gairdneri ) **This study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol , CAS No. 4719 - 04 - 4; Purity : not specified. **Remarks:** Also referred to as Grotan and Milidin X - 2. METHOD **Method/guideline followed:** Not specified **Test type:** Acute Toxicity Test for Freshwater Fish, Rainbow Trout ( Oncorhynchus mykiss ), static **GL P compliant?:**  No **Year study was performed:**  1982 **Test species:** Juvenile r ainbow trout; average wet weight: 0.28 g; average length: 35 mm **Analytical monitoring:** No **Exposure duration:** 96 hours **Concentrations tested:** Nominal: 0 (control), 17, 28, 46, 48, and 100 mg/L **Test method/conditions remarks:**  Juvenile rainbow trout were allowed to acclimate for 14 day s prior to experimental use. The holding water was well water with a flow - through rate of 13 tank volume replacements/day . The total hardness of the holding water was 30 - 34 mg CaCO 3 /L , alkalinity was 26 - 29 mg CaCO 3 /L , pH was 7.1 - 7.2, temperature was 11 - 12 oC, and dissolved oxygen ranged from 98 - 100% of saturation at 12 oC. The fish were fed dry pellets of food dail y, ad libitum . The pretest mortality rate, observed during the 48 - hour period prior to testing, was 0.3%. The dilution water consisted of soft water reconstituted from deionized water, using EPA recommended procedures. The dilution water had a hardness of 46 mg CaCO 3 /Land specific conductance of 140 μhos/ cm, measured at the start of the definitive study . The stock solution of Milidin X - 2 (15 mg/L) was prepared b y adding distilled water to 7.5 grams of Milidin X - 2 in a 500 - mLvolumetric flask. Test chambers consisted of 19.6 - L glass jars, each containing 15 L of test solution. No evidence of a range - finding study was provided.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

23 Ten juvenile rainbow trout were distributed randomly to each test chamber within 30 minutes after the preparation of the test solutions, and were exposed to nominal concentrations of 17 , 28, 46, 48, or 110 mg/L under static conditions. The negative control contained the same dilution water as in the treatment chambers. Three replicates were used per treatment level. Biological observations were made every 24 hours. The fish were 0.28 (0.18 - 0.42) g wet weight, and 35 (30 - 38) mm in length. Food was withheld for 48 hours before the definitive test began. All test solutions were maintained at 12 ± 1 oC and were not aerated . The pH and dissolved oxygen concentrations were measured at 0, 24, 48, and 96 hours in the control, high, middle and low test concentrations. The dose related mortality data from the definitive toxicity test was used to estimate the 24 - , 48 - , 72 - , and 96 - hour median lethal concentrations and 95% confidence int ervals. A computer program (Stephan, C. 1978. U.S. EPA, Environmental Research Laboratory , Duluth, MN) was used to perform the statistical anal ys is. An EPA OPP reviewer used EPA's Toxanol program to verify the LC 50 values using the moving average angle anal ys is and the probit method. RESUL TS **Endpoint value(s):** The 96 - hr L C 50 for rainbow trout was 71 mg /L. The LOEC was 28 mg/L, based on sublethal effects, and the NOEC was 17 mg/L. **Results remarks:** The range of test temperatures was 11 - 12 oC. The pH ranged from 7.0 to 9.4 and dissolved ox ygen from 8.6 to 11.6 mg/L (76 - 100% saturation). The measured pH values exceeded the acceptable range for freshwater fish testing. The mortality

rates at nominal concentrations of 68 mg/L and 110 mg/L were 20 an d 100%, respectivel y. No mortality was observed at concentrations ≤ 46 mg/L. Other effects observed included darkened pigmentation in the latter half of the study in the 28 mg/L and the 46 mg/L dose groups, as well as all timeframes of the 68 mg/L test group and at the 24 - hour mark in the 110 mg/L test group. Fish were found swimming at the surface of the test solution during the 24 - hour mark for the 110 mg/L test group, as well as at the 48 - , 72 - , and 96 - hour test marks for the 69 mg/L test group. Fish were observed respiring rapidly at the 96 - hour mark for the 46 mg/L test group. The study authors reported a 96 - hr LC 50 of 71 mg/L , with a 95% confidence interval of 46 - 110 mg/L. The 96 - hr LC 50 obtained using Toxanol was 79 mg/L, with a 95% confidence interval of 46 - 110 mg/L. The NOEC was 17 mg/L, based upon darkened pigmentation at 28 mg/L. CONCLUSIONS The 96 - hr LC 50 for rainbow trout was 71 mg/L. The LOEC was 28 mg/L, based on sublethal effects, and the NOEC was 17 mg/L.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

24 STUDY RELIABILITY [2] Reliable with restrictions. Deviations from OPPTS Guideline 850.1075 included the following: (1) purity

and phy sicochemical characteristics of the test material were not provided; (2) test chemical concentrations were not monitored through the experim ent (guidelines require measurements at 0, 48, and 96 hours); (3) holding water and test water dilution came from two different sources and fish were not held in the test water dilution for a minimum of 7 day s during the acclimation period; (4) the number of replicates for each concentration was not explicitly stated (assumed three replicates based on n = 30); (5) the pH of 7.0 to 9.4 did not alway s fall within the recommended range for freshwater species of 6.0 - 8.0 ; and (6) the following information was no t provided: chemical analy sis of dilution water, pretest mortality rates for the 14 - day acclimation period, photoperiod, background colors, light intensities, fish supplier, fish source population, breeding history , whether fish had been previously tested, biomass loading rate, use and ty

pe of cover for test chambers, dissolved oxygen concentration at 72 hours, data from a range - finding test, hourly temperatures in one replicate throughout the study , anal ys is of the stability

of the test chemical, mortality at 6 hours, a graph of the concentration - mortality curve, and quality

assurance and GLP compliance statements. This study was classified as “supplemental” b y U.S. EPA’s Office of Pesticide Programs. The stud y is considered to provide useful information, in support of core or key studies, and the results may be used as part of a weight - of - evidence approach to describe the acute toxicity to fish. REFERENCE Sousa, J.V. and L eBlanc, G.A. 1982b. Acute Toxicity of Milidin X - 2 to Rainbow Trout ( Salmo gairdne ri ); L aboratory

Report BW - 82 - 4 - 1145. Performing Laboratory : EG&G, Bionomics Aquatic Toxicology

L aboratory , Wareham, MA. U.S. EPA MRID No. 248883.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

25 **Study 8: Acute toxicity study in bluegill (Sousa and LeBlanc, 1982c)**  **Title:** Acute Toxicity

of Milidin X - 2 to Bluegill (Lepomis macrochirus) **This study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol , CAS No. 4719 - 04 - 4; Purity : not specified. **Remarks:** Also referred to as Grotan and Milidin X - 2. METHOD **Method/guideline followed:** Not specified **Test type:** Acute Toxicity Test for Freshwater Fish, Bluegill ( L. macrochirus ), static **GL P compliant?:**  No **Year study was performed:** 1982 **Test species:** Juvenile b luegill, obtained from a commercial fish supplier in Connecticut (Bionomics Lot No. 81A27), average wet weight = 0.34 g; average length = 34 mm **Analytical monitoring:** No **Exposure duration:** 96 hours **Concentrations tested:** Nominal: 0 (control), 13, 22, 36, 60, and 100 mg/L **Test method/conditions remarks:** Juvenile bluegill were allowed to acclimate for 14 day s prior to experimental use. The holding water was well water that flowed through the tank at a rate of 9 - 10 tank volume replacements/day . The total hardness of the holding water was 30 - 34 mg CaCO 3 /L , the alkalinity

was 26 - 29 mg CaCO 3 /L , the p H ranged from 6.8 - 7.0, the temperature was 20 - 21 oC, the specific conductance was 105 - 120 μmhos/cm, the photoperiod was16 hours light/8 hours dark, and the dissolved ox ygen was100% of saturation at 20 oC. The fish were fed dry pellet food dail y, ad libitu m . The pretest mortality rate, observed 48 hours prior to testing, was 1.0%. The dilution water consisted of soft water reconstituted from deionized water, using EPA recommended procedures. The stock solution of Milidin X - 2 (15 mg/L) was prepared b y ad ding distilled water to 7.5 grams of Milidin X - 2 in a 500 - mLvolumetric flask. No evidence of a range - finding stud y was provided. Ten juvenile bluegill per replicate were distributed randomly into 19.6 - L gl ass test jars containing 15 L of test solution w ithin 30 minutes after the preparation of the test solution. This**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

26 fish were exposed to nominal concentrations of 13 , 22, 36, 60, or 100 mg/L under static conditions. The negative control contained the same dilution water as in the exposure chambers. Thr ee replicates were used per concentration. Biological observations were made every 24 hours. The fish were 0.34 (0.20 - 0.52) g wet weight, and 34 (30 - 38) mm in length. Food was withheld for 48 hours before the definitive test began. All test temperature solutions were maintained at 21 - 22 oC and were not aerated . The pH and dissolved ox ygen concentrations were measured at 0, 24, 48, and 96 hours in the control, high, middle and low test concentrations. The dose related mortality data from the definiti ve toxicity test was used to estimate the 24 - , 48 - , 72 - , and 96 - hour median lethal concentrations and 95% confidence intervals. A computer program (Stephan, C. 1978. U.S. EPA, Environmental Research Laboratory , Duluth, MN) was used to perform the statisti cal anal ys is. An EPA OPP reviewer used EPA's Toxanol program to verify the LC 50 values using the moving average angle anal ys is and the probit method. RESUL TS **Endpoint value(s):** The 96 - hr L C 50 for bluegill was 39 mg/L. The LOEC was 13 mg/L, based on sublethal effects observed at the lowest concentration tested, and the NOEC was < 13 mg/L . **Results remarks:** The pH ranged from 6.8 to 9.4 and the dissolved oxygen concentration ranged from 1.2 - 11.4 mg/L(14 - 100% saturation). Some of the measured pH values exceeded the recommended range for freshwater fish testing. The total hardness, measured at the beginning of the test, was 46 mg CaCO 3 /L . The alkalinity was 31 mg CaCO 3 /L , specific conductance was 140 μmhos/cm, and the photoperiod was 16 hours light/8 hours dark . Mortality rates observed after 96 hours at nominal concentrations of 22, 36, 60, and 100 mg/L were 10, 30, 90, and 100%, respectively . Sublethal effects were observed in all test groups, except the high - dose group where the effects wer e 100% mortality . At the 72 - and 96 - hour marks for the 13 and 22 mg/L dose groups, cloudy test solutions, lethargy , rapid respiration, erratic swimming, and darkened pigmentation were observed. Cloudiness and rapid respiration were also observed at the 9 6 - hour mark in the 36 mg/L dose group. Six fish were lethargic at 24 hours in the 60 mg/L dose group, with two still lethargic at the 48 - and 72 - hour marks. Lethargy and erratic swimming were also seen at 96 hours in the 60 mg/L dose group. The study authors reported a 96 - hr LC 50 for bluegill of 39 mg/L , with a 95% confidence interval (C.I .) of 31 to 50 mg/L . The NOEC was < 13 mg/L, the lowest concentration tested, based on rapid respiration and darkened pigmentation at 13 mg/L. Using Toxanol, the 96 - hr LC 50 was 39 mg/L (95% C.I. = 31 - 50 mg/L) using the moving average method and the 96 - hr L C 50 was 40 mg/L (95% C.I. = 32 - 50 mg/L) using the probit method. CONCLUSIONS The 96 - hr LC 50 for bluegill was 39 mg/L. The NOEC was < 13 mg/L, based on sublethal effects at the lowest dose tested.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

27 STUDY RELIABILITY [2] Reliable with restrictions. Deviations from OPPTS Guideline 850.1075 included the following: (1) purity

and phy siochemical characteristics of the test material were not provided; (2) test chemical concentrations were not monitored and recorded through the experiment (guidelines require measurements at 0, 48, and 96 hours); (3) holding water and test dilution water came from two different sources and fish were not held in the dilution water for a minimum of 7 day s during the acclimation period; (4) the temperature in the holding tank during the 48 hours prior to testing was 20 oC, whereas protocol suggests 22 ± 1 oC; (5) measured dissolved oxygen concentrations (14 - 100% saturation) were not maintained at > 60% throughout the whole test; (6) some pH measurements (range = 6.8 to 9.4) were outside the recommended range (6.0 - 8.0) for freshwater species; and (6) the following information was not provided: phy sical condition and signs of disease in the fish, chemical analy sis of dilution water, pretest mortality rates for the entire 14 - day acclimation period; use and ty pe of cover for test vessels, dissolved oxygen concentration at 72 hours, range - finding test data, explicit st atement of the number of replicates for each concentration (assumed three replicates based on n = 30), hourl y temperatures in one replicate throughout the stud y, anal ys is of the stability of the test chemical, mortality at 6 hours, graph of the concentrati on - mortality curve, and quality

assurance and GLP compliance statements. This study was classified as “supplemental” b y U.S. EPA’s Office of Pesticide Programs. The study is considered to provide useful information, in support of core or key studies, and the results may be used as part of a weight - of - evidence approach to describe the acute toxicity

to fish. REFERENCE Sousa, J.V., and L eBlanc, G.A. 1982c. Acute Toxicity of Milidin X - 2 to Bluegill ( Lepomis macrochirus ); Laboratory Report BW - 82 - 4 - 1144. Pe rforming Laboratory : EG&G, Bionomics Aquatic Toxicology

L aboratory , Wareham, MA. U.S. EPA MRID No. 248883.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

28 **Study 9: Acute toxicity study in channel catfish (LeBlanc and Sousa, 1982b)**  **Title:** Acute Toxicity

of Triadine 10 to Channel Catfish ( Ictalurus punctatus ) **This study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 63.6% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol , CAS No. 4719 - 04 - 4; 6.4% sodium omadine, CAS No. not specified; 30% unidentified **Remarks:** Formulation, also referred to as Triadine 10 METHOD **Method/guideline followed:** Not specified **Test type:** Acute Toxicity Test for Freshwater Fish, Channel Catfish ( Ictalurus punctatus ) (static or flow - through not specified) **GL P compliant?:**  No **Year study was performed:**  1982 **Test species:** Channel catfish ( Ictalurus punctatus ) **Analytical monitoring:** Not specified **Exposure duration:** 96 hours **Concentrations tested:** 6, 10, 17, 28, and 46 mg/L (nominal or measured not specified) **Test method/conditions remarks:**  Channel catfish (10/dose) were exposed to concentrations of 6, 10, 17, 28, or 46 mg/L. Test procedures did not comply with the recommended EPA protocol of October 1982 (Part 158). An EPA OPP reviewer used a binomial probability test to estimate the LC 50 value. RESUL TS **Endpoint value(s):** The 96 - hr L C 50 for channel catfish was 36 mg/L . **Results remarks:** The mortality rate was 0, 10, 0, 0, and 100% at test substance concentrations of 6, 10, 17, 28, and 46 mg/L , respectivel y. Using the binomial probability method, the LC 50 was 36 mg/L, with a 95% confidence interval of 28 to 46 mg/L .**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

29 CONCLUSIONS The 96 - hr LC 50 for channel catfish was 36 mg/L, estimated by binomial probability . The test substance was found t o be slightly toxic to channel catfish. STUDY RELIABILITY [2] Reliable with restrictions. A formulated product was used instead of a technical grade chemical; the test substance was a mixture of two active ingredients. This study was classified as “supplemental” b y U.S. EPA’s Office of Pesticide Programs. The stud y is considered to provide useful information, in support of core or key studies, and the results may be used as part of a weight - of

- evidence approach to describe the acute toxicity to fis h. REFERENCE LeBlanc, G., Sousa, J. 1982b. Acute Toxicity of Triadine 10 to Channel Catfish ( Ictalurus punctatus ); Report # BW - 82 - 2 - 1120. Performing Laborotory : EG&G Bionomics. Submitted by the Olin Corporation, New Haven, CT. U.S. EPA MRI D No. 00105189.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

30 **D -**  **2. Acute Toxicity to Aquatic Invertebrates**  **Summary of endpoint:** Adequate **Study 1: Acute toxicity study in**  **Daphnia magna (Davis, 1994)**  **Title:** Triazine: Acute Toxicity to the Water Flea, Daphnia magna , Under Flow - through Test Conditions **This study is:**  Adequate Not Adequate TEST SUBSTANCE **Identity:** 83.8% Triazine, CAS No. 4719 - 04 - 4; 16.2% unidentified **Remarks:** Also referred to as Triazine and Shaughnessey No. 083301; Technical grade; Lot No. 0952309; clear yellow viscous liquid. METHOD **Method/guideline followed:** OPP 72 - 2(A) **Test type:** Freshwater Invertebrate Flow - Through Acute Toxic ity Test **GL P compliant?:**  Not specified **Year study was performed:** 1994 **Test species:** Water fleas, D. magna , (<24 hours old) taken from in - house cultures. **Analytical monitoring:** Yes **Exposure duration:** 48 hours **Concentrations tested:** Nominal: 0 (control), 7.78, 13.0, 21.6, 36.0, 60.0 and 100 mg a.i./L; mean measured: 0 (control), 7.49, 13.2, 19.7, 34.5, 59.0, and 95.2 mg a.i./L **Test method/conditions remarks:** For the definitive test, the nominal test concentrations were 100, 60.0, 36.0, 21.6, 13.0, and 7.78 mg a.i./Land a dilution water control. The definitive test was conducted under flow - through conditions in a modified proportional diluter sy stem constructed of glass, silicone adhesive, and silicone tubing. A total volume of 53.9 μLof te st substance was pumped into the chemical mixing chamber during each diluter cycle, providing a high nominal test concentration of 100 mg a.i./L . The test solution was proportionally diluted to provide the 5 lower test concentrations. A dilution water control was also maintained. The test dilution water was a moderatel y hard fresh water, with a mean hardness of 113 mg/L as CaCO 3 , mean alkalinity

of 32 mg/L , and a mean specific conductivity of 658 μmhos/cm.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

31 A test solution of ~ 220 mLwas delivered to e ach test chamber during every cy cle: the total volume was split in halves via a splitter box. Test tanks were 11.3 - L glass tanks positioned to provide a maximum depth of 6 cm. Retention chambers were used to prevent neonate floating. Each test container maintained a 450 - mLvolume of test solution; the diluter cy cled at an average rate of 2.9 cy cles/hour providing ~ 17 volume additions every 24 hours. Ten daphnids were impartially added to each chamber, with two chambers per concentration. All test containers were randomly positioned in a single water bath maintained at a temperature of 20 ± l° C. Fluorescent lighting provided a photoperiod of 16 hours light/8 hours dark, with a 15 - minute transition period. The light intensity ranged between 308 an d 375 lux. Survival of daphnids was monitored daily and any dead removed. Abnormalities were also noted. Daphnids were not fed during the test. Test solutions remained clear throughout the study . Test water quality

was monitored daily . Water samples were collected from the controls and all six test concentrations at initiation, day 1, and termination to verify concentrations. Samples were anal yz ed using HPLC. The median effective concentrations (EC 50 ) and associated 95% confidence intervals (C.I.) were calculated using a computer program (moving average angle, probit, logit, and nonlinear interpolation). RESUL TS **Endpoint value(s):** The 48 hour EC 50 value for D. magna was 26.1 mg a.i./L . The NOEC was 7.49 mg a.i./L based on mortality at the 13.2 m g a.i./Ltest concentration. **Results remarks:** The diluter functioned properly throughout the test. The mean measured concentrations ranged from 7.49 to 95.2 mg a.i./Land from 91 to 102% of nominal. The mean measured concentrations were 7.49, 13.2, 19. 7, 34.5, 59.0, and 95.2 mg a.i./L. Mortality was 0, 5, 0, 100, 100, and 100% at mean measured concentrations of 7.49, 13.2, 19.7, 34.5, 59.0, and 95.2 mg a.i./L , respectivel y. The slope of the concentration response curve could not be determined using th e binomial probabi1ity method. The study

author determined the 48 - hour EC 50 as 26.1 mg a.i./L , with 95% confidence limits of 19.7 to 34.5 mg a.i./L , and a NOEC of 19.7 mg a.i./L . An EPA OPP reviewer used EPA's Toxanal program to determine the 48 - hour EC 50 value as 26.1 mg a.i./L (95% C.I. = 19.7 - 34.5 mg a.i./L ). The NOEC was estimated to be 7.49 mg a.i./L , based on mortality

at the 13.2 mg a.i./L concentration. This NOEC value is more conservative than the value reported by the stud y author, which was 19.7 mg a.i./L . Initial alkalinit y, hardness, and conductivity of the dilution water as measured in the control were 32 mg/L, 130 mg/L, and 828 μmhos/cm, respectively . At test termination, they were 32 mg/L,**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

32 96 mg/L, and 488 μmhos/cm, respectivel y. During the test, the dissolved oxygen remained **≥** 7.8 mg/L **( ≥**  88% saturation). The pH was affected by the presence of triazine, with pH increasing with triazine concentration. The pH ranged from 7.7 to 7.8 in the control and from 8.5 to 9.6 in all test co ncentrations. CONCLUSIONS The 48 hour EC 50 for D. magna was 26.1 mg a.i./L. The NOEC was 7.49 mg a.i./L based on mortality at the 13.2 mg a.i./Ltest concentration. STUDY RELIABILITY [1] Reliable without restrictions. The study was scientificall y sound and generall y in accordance with SEP and ASTM guidelines. This study was classified as “core” b y U.S. EPA’s Office of Pesticide Programs. REFERENCE Davis, J. W. 1994. Triazine: Acute toxicity to the water flea, Daphnia magna , under flow - through test conditions. Project No. J9306004d. Performing Laboratory : Toxikon Environmental Sciences. Submitted by

Triazine Joint Venture. U.S. EPA MRID No. 431754 - 01.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

33 **Study 2: Acute toxicity study i**  **n Daphnia magna**  **(LeBlanc and Surprenant, 1982) Title:**  Acute Toxicity

of Triadine 10 to the Water Flea ( Daphnia magna ) **This study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 63.6% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol , CAS No. 4719 - 04 - 4; 6.4% sodium omadine, CAS No. not specified; 30% unidentified **Remarks:** Formulation, also referred to as Triadine 10 METHOD **Method/guideline followed:** EPA October 1982, Part 158 **Test type:** Freshwater Invertebrate Acute Toxicity Test (static or flow - through not specified) **GL P compliant?:**  Not specified **Year study was performed:** 1982 **Test species:** Water fleas, D. magna **Analytical monitoring:** Not specified **Exposure duration:** 48 hours **Concentrations tested:** 0.18, 0.3, 0.5, 0.8, 1.3, 2.2, 3.6, 6 and 10 mg/L (nominal or measured not specified) **Test method/conditions remarks:** The test procedure complied with the recommended EPA protocol of October 1982 (Part 158) except that a formulat ed product was used instead of a technical grade chemical and the test substance was a mixture of two active ingredients. Fifteen organisms were used per test concentration ( 0.18, 0.3, 0.5, 0.8, 1.3, 2.2, 3.6, 6, and 10 mg/L) . A computer program (Stephan, C. 1978. U.S. EPA, Environmental Research Laboratory , Duluth, MN) was used to perform the statistical analy sis. An EPA OPP reviewer used EPA's Toxanol program to verify

the EC 50 value using the moving average angle anal ys is and the probit method. RESUL TS **Endpoint value(s):** The 48 - hour EC 50 value for Daphnia magna was 5.2mg/L.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

34 **Results remarks:** Mortality observed at concentrations of 0.18, 0.3, 0.5, 0.8, 1.3, 2.2, 3.6, 6, and 10 mg/L was 0%, 0%, 0%, 13.3%, 26.7%, 20%, 53.3%, 33.3% and 73.3%, respectivel y. The study authors reported a 48 - hour EC 50 of 5.2 mg/L. Using Toxanol, the 48 - hour EC 50 , estimated by

the moving average angle method was 5.4 mg/L, with a 95% confidence inte rval of 3.6 - 10.79.4 mg/L. Using the probit method, the 48 - hour EC 50 was 5.2 mg/L, with a 95% confidence interval of 3.5 - 9.5 mg/L. CONCLUSIONS The 48 - hour EC 50 value for D. magna was 5.2 mg/L. STUDY RELIABILITY [2] Reliable with restrictions. A formulated product was used instead of a technical grade chemical; the test substance was a mixture of two active ingredients. This study was classified as “supplemental” b y U.S. EPA’s Office of Pesticide Programs. The stud y is considered to provide us eful information, in support of core or key studies, and the results may be used as part of a weight - of

- evidence approach to describe the acute toxicity to daphnids. REFERENCE LeBlanc, G., and Surprenant, D. 1982. Acute Toxicity of Triadine 10 to the Wat er Flea ( Daphnia magna ); Report # BW - 82 - 2 - 1122. Performing Laboratory : EG&G Bionomics. Submitted by the Olin Corporation, New Haven, CT. U.S. EPA MRID No. 00105192.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

35 **D -**  **3 . Toxicity to**  **Aquatic Plants**  **Summary of endpoint:** Adequate **Study 1: Toxicity to**  **Pseudokirchneriella subcapitata (**  **Vryenhoef and Mullee, 2012 )**  **Title:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - T riethanol (C AS No. 4719 - 04 - 4 ): Algal Inhib i tion Test **This study is:**  Adequate Not Adequate TEST SUBSTANCE **Identity:** 1,3,5 - triazine - 1,3,5(2H,4H,6H) - triethanol , CAS RN 4719 - 04 - 4 **Remarks:** Phy sical state: yellow slightl y viscous liquid ; Composition: 1 ,3,5 - Triazine 1,3,5(2H,4H,6H) - T riethanol (77.5%) and w ater (22.5%) ; Supplier: Q 2 Technologies; Batch # 3951 - 91 METHOD **Method/guideline followed:** OECD TG 201 (2006); OPPTS 850.5400 (1996); ASTM E1218 - 04 (2004) **Test type:** Static t oxicity test to freshwater algae **GL P compliant?:**  Yes **Year study was performed:** 201 2 **Test species:** Freshwater green algae ( Pseudokirchneriella subcapitata strain CCAP 278/4 , formerl y Selenastrum capricornutum ) ; culture supplied by the Culture Collection of Algae and Protozoa ( CCAP ) , SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotlan d . **Analytical monitoring:** Yes (degradation products only) **Exposure duration:** 96 hours **Concentrations tested:**  Range - finding test (nominal): 0, 0.10, 1.0, 10, 100, and 1000 mg active ingredient ( ai ) /L ; mean measured concentrations were not provided. Definitive test (n ominal ) : 1.0, 3.2, 10, 32 , and 100 mg ai /L ; mean measured concentrations were not provided. **Test method/conditions remarks :**  After a range - finding test at nominal concentrations ranging from 0.1 to 1000 mg active ingredient (ai)/L , a definitive growth inhibition test was conducted.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

36 T he test substance was dissolved directly in culture medium at nominal concentrations of 0, 1.0, 3.2, 10, 32, and 100 mg ai/L . The culture medium was prepared according to ASTM E 1218 - 04 ε1 using reverse osmosis purified deionised water as dilution water. Additional sodium bicarbonate (500 mg/L) was added to culture medium prior to the definitive test “to provide a sufficient supply

of CO 2 and to counteract the increase in pH due to algal growth in an enclosed sy stem . ” [ Vry enhoef and Mullee 2012, p. 8] The test substance had a water content of 22.5%, and all nominal test concentrations were corrected for the water content and expressed as mg ai/L . “Amounts of test item (258 and 82 mg) were each separately dissolved in culture medium and the volume adjusted to 2 litres to give 100 and 32 mg ai /l stock solutions respectively . A series of dilutions was made from these stock solutions to give further stock solutions of 10, 3.2, and 1.0 mg ai /l . An aliquot (1800 m l ) of each of the stock solutions was separatel y inoculated with algal suspension (37 m l ) to give the required test concentrations of 1.0, 3.2, 10, 32, and 100 mg ai /l . The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity . The concentration and stability of the test item in the test preparations were verified b y chemical anal ys is at 0 and 96 hours...” [ Vry enhoef and Mullee 2012 , p. 10] “Six [250 mLglass conical ] flasks each completely filled with test preparation were use d for the control and three flasks each completel y filled were used for each treatment group. The control group was maintained under identical conditions but not exposed to the test item. Pre - culture conditions gave an algal suspension in log phase growth characterised by a cell density of 4.95 x 10 5 cells per m l . Inoculation of 1800 m l of test medium with 37 m l of this algal suspension gave an initial nominal cell density of 1 x 10 4 cells per m l and had no significant dilution effect on the final test concentration.” [ Vry enhoef and Mullee 2012 , p. 10] “The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron® Version 2 incubator) at 24 ± 1 ° C under continuous illumination ( intensity approximately 7000 lux) provided b y warm white lighting (380 - 730 nm) and constantly shaken at approximately 150 rpm for 96 hours. Samples were taken at 0, 24, 48, 72, and 96 hours and the cell densities determined using a Coulte r® Multisizer Particle Counter” [ Vry enhoef and Mullee 2012 , p. 11] “The pH of the control and each test concentration was determined at initiation of the test and after 48 and 96 hours exposure... The temperature within the incubator was recorded hourl y using the incubators’ internal pt - 100 temperature probe and Multicom Software Package.” [ Vry enhoef and Mullee 2012 , p. 11]**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

37 “A re - growth test was performed after 96 hours to determine the algicidal or algistati c effect o f the test item. Aliquots were removed from each replicate control and test culture (0.25 m l and 0.50 m l respectivel y) and the replicate s pooled. Fresh sterile culture medium (100 m l ) was added to each to ensure that the test concentration was reduced to below the inhibiting level. The flasks were plugged with poly urethane foam bungs are [ sic ] incubated ...at 24 ± 1°C under continuous illumination (intensity approximately

7000 lux) and constantly shaken at approximately

150 rpm for 168 hours.” [ Vry enhoef and Mullee 2012 , p. 11] “Samples were taken from the control (replicate s R 1 - R6 pooled) and each test group (replicate s R 1 - R3 pooled) at 0 and 96 hours for quantitative analysi s. Given the volatile nature of the test item, an additional test replicate was prepared for each test concentration at 0 hours and incubated alongside the study remaining unopened. Samples of these addi tional replicate s were taken for unopened vessel anal ys is at 96 hours.” [ Vry enhoef and Mullee 2012 , p. 1 1 ]

“ [A] t the request of the Sponsor all test concentrations were corrected for the [22.5%] water content. [According to the study authors:] The test i tem was known to degrade immediatel y in water to form ethanolamine (0.84 mg/l ethanolamine ≡ to 1 mg ai /l ) and formaldehy de (0.41 mg/l formaldeh yd e ≡ to 1 mg ai /l ). Therefore analy sis of the test preparations was [ performed ] for the 2 degradation products only . The ethanolamine concentration in the test samples was determined b y high performance liquid chromatography coupled to mass spectrometry (HPL C MS) using an external standard. The formaldeh yd e concentration in the test samples was determined b y derivatisation with 2,4 - dinitrophenylh ydrazine phosphoric acid solution with high performance liquid chromatograph y (HP LC) using an external standard. The ethanolamine and th e formaldeh yd e gave a chromatographic profile consisting of a single peak ... A volume of test sample was diluted (if required) with test medium to give a theoretical concentration of 1 mg ai/1. To 100 ml of this was added 1 ml of 2,4 - dinitrophenylhydrazi ne phosphoric acid solution* [* Fluka

– 0.2M derivatising agent] and 50 μl of pentafluoropropionic acid. The test sample was then shaken to mix and allowed to stand for 15 minutes before anal ys is for both ethanolamine and formaldehyde. ” [ Vry enhoef and Mul lee 2012 , p. 40] Statistical Analy sis: “One way anal ys is of variance incorporating Bartlett’s test for homogeneit y of variance...and Dunnett’s multiple comparison procedure for comparing several treatment with a control...was carried out on the growth rate, yield and biomass integral data after 72 and 96 hours for the control and all test concentrations to determine an y statistically significant differences between the test and control group. All statistical analy ses were performed using the SAS computer software package...” [ Vry enhoef and Mullee 2012 , p p . 14 - 15]

“ For each individual test vessel ... , percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation usi ng the Xlfit software package (ID BS). ECx values were then determined from the equation for the fitted line. Where appropriate 95% confidence limits for the EC 50 values were calculated, using the**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

38 simplified method of evaluating dose - effect experiments of L itchfield and Wilcoxon (1949). ” [ Vry enhoef and Mullee 2012, p. 14 ] Validation Criteria: The results of the test were considered valid by the study authors if the following performance criteria were met: the cell con centration of the control cultures must increase b y a factor of at least 16 afte r 72 hours and by a facto r of at least 100 after 96 hour s ; the mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (0 - 24, 24 - 48, and 48 - 72 hours) must not exceed 35 % ; the coefficient of variation of the average specific growth rate in replicate control cultures after 72 hours must not exc eed 7%; and the coefficient of variation of the cell density values in replicate control cultures must not exceed 20% after 72 and 96 hour s ). A positive control study

was conducted using zinc chloride as the reference item. RESUL TS **Endpoint value(s ):**  **“** Given that toxicity cannot be attributed to a single degradation product or mixture of degradation products it was considered appropriate [by the study authors] to calculate the results based on nominal test concentrations only . ” [ Vry enhoef and Mullee 2012 , p. 6] The following results were determined from the data based on nominal test concentrations of CASRN 4719 - 04 - 4 : 72 - h EC 50 (growth): 5.2 mg ai/ L * 96 - h EC 50 (growth): 4.2 mg ai/L ; 95% con fidence limits 3.9

– 4.6 mg ai/L 96 - h NOEC (growth): 1.0 mg ai/ L 72 - h EC 50 (yield): 3.5 mg ai /L ; 95% confidence limits 3.2

– 3 .8 mg ai /L 96 - h EC 50 ( yi eld): 3 .2 mg ai /L ; 95% confidence limits 3. 0

– 3. 4 mg ai /L 96 - h NOEC (yield) = 1.0 mg ai /L 72 - h EC 50 (biomass): 3. 6 mg ai /L ; 95% confidence limits 3.3

– 3.9 mg ai /L 96 - h EC 50 (biomass): 3.3 mg ai /L ; 95% confidence limits 3.1

– 3.6 mg ai /L 96 - h NOEC (biomass) = 1.0 mg ai /L

“ * Itwas not possible to calculate 95% confidence limits for the E r C 50 value as the data generated did not fit the models available for the calculation of confidence limits.” [ Vry enhoef and Mullee 2012 , p. 18] **Results remarks:** Range - Finding Test: “The results showed no effect on growth at the [nominal] test concentrations of 0.10 and 1.0 mg ai /l . However, growth was observed to be reduced at 10, 100 and 1000 mg ai /l . Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg ai /l were selected for the definitive test. ” [ Vryenhoef and Mullee 2012 , p. 16]**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

39 Measurements of degradation products : E thanolamine (HPL C - MS) concentrations were 0.519, 11.0, and 88.6 mg/L at nominal concentrations of 1.0, 10, and 100 mg ai/L , respectivel y, at 0 hours and 0.379, 11.0, and 85.2 mg/L at nominal concentrations of 1.0, 10, and 100 mg ai/L , respectivel y, at 96 hours . F ormaldeh yd e (HPLC) concentrations were 0.498, 3.95, and 44.2 mg/L at nominal concentration s of 1.0, 10, and 100 mg ai/L, respectivel y, at 0 hours and 0.0964, 3.13, and 35.6 mg/L at nominal concentrations of 1.0, 10, and 100 mg ai/L , respectivel y, at 96 hours. “ Onl y the test concentrations spanning the range of concentrations to be employ ed in the definitive test were anal ysed. ” [ Vry enhoef and Mullee 2012, p. 16] Definitive Test: Growth, Yield, and Biomass Integral Data: After 96 hours of exposure, the mean cell densit y was 1.29, 1.43, 0. 639, 0.00 930 , 0.00 731, and 0.0 114 x 10 6 cells/mLat nominal concentrations of 0, 1.0, 3.2, 10, 32, and 100 mg ai/L, respectivel y. The mean inhibition of growth rate was - 1, 15, 102, 107, and 98% at nominal concentrations of 1.0, 3.2, 10, 32, and 100 mg ai/L, respectivel y. The mean inhibition of yield was - 11, 51, 100, 100, and 100% at nominal concentrations of 1.0, 3.2, 10, 32, and 100 mg ai/L , respectivel y. The mean inhibition of biomass was - 18, 43, 100, 100, and 100%, respectively , at nominal concentrations of 1.0, 3.2, 10, 32, and 100 mg ai/L, respectivel y.

“ There were no statistically significant differences between the control and 1.0 mg ai / l test concentration (P ≥ 0.05); however all other test concentrations were significantly different (P < 0.05) and, therefore the “ N o O bserved E ffect C oncentration” (NOEC) based on [ growth rate, yi eld and biomass integral ] was 1.0 mg ai / l . Correspondingl y the “Lowest Observed Effect Concentration” (LOEC) based on [growth rate, yi eld, and biomass integral] was 3.2 mg ai/l. ” [Vry enhoef and Mullee 2 0 12 , p p . 18 - 19 ] Validation Criteria: “[T]he cell concentration of the control cultures increased b y a factor of 73 after 72 hours and by a factor of 150 after 96 hours ... The mean coefficient of variation for section by section specific growth rate for the control cultures over the test period (0 - 72 h ours ) was 15 % ... The coefficient of variation for average specific growth rate for the control cultures over the test period (0 - 72 h) was 3 % ... The coefficient of variation of th e cell densit y values in replicate control cultures was 14% after 72 hours and 7% after 96 hours ... ” [Vry enhoef and Mullee 2007, p. 1 7 ] Obse rv ations on Cultures: “All test and control cultures were inspected microscopically at 96 hours. T here were no abnormalities detected in any of the control or test cultures.” [Vry enhoef and Mullee 20 12 , p. 1 9 ] Obse rv ations on test material solubili ty : “At the start of the test all control and test cultures were observed to be clear colo urless solutions. After the 96 - H our test period all control and 1 . 0 mg ai / l test cultures were observed to**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

40 be bright green dispersions . The 3.2 mg ai/l test cultures were observed to be green dispersions, the 10 mg ai/l test cultures were observed to be extremel y pale green dispersi ons whilst the 32 and 100 mg ai / l test cultures were observed to be clear colorless solutions.” [Vry enhoef and Mullee 2007, pp. 19

– 2 0 ] Phy sico - chemical measurements: The range of pH measurements in all cultures was 7.7 to 9.1 at 0 hours, 8.1 to 9.2 at 48 hours, and 8.2 to 10.3 at 96 hours. “ The pH value of the control cultures ... was observed to increase from pH 7.7 at 0 hours to pH 8.8 at 48 hours and pH 10.2 at 96 hour s. This increase was considered to be due to the amount of carbon dioxide required by the large number of algal cells in the log phase of growth ... Due to the need to test in completely filled and sealed test vessels there was minimal transfer of CO 2 from th e gaseous phase to the aqueous phase and hence CO 2 required for photos ynthesis and growth would be derived from bicarbonate in solution which results in an increase in the pH of the culture. Despite the addition of 500 mg/l of sodium bicarbonate to the cu lture medium the increase in pH after 96 hours was in excess of that recommended in the Test Guidelines (1.5 pH units after 96 hours). This was considered to have had no adverse effect on the results of the study given that the increase in cell concentrat ion in the control cultures exceeded the validation criterion given in the Test Guidelines.” [ Vry enhoef and Mullee 2012 , p. 20] Verification of test concentrations: Measured ethanolamine (HPL C - MS) concentrations were 0.518, 3.17, 11.4, 34.7, and 91.9 mg/L at nominal concentrations of 1.0, 3.2, 10, 32, and 100 mg ai/L, respectivel y, at 0 hours and 0.343, 2.82, 7.60, 19.6, and 56.8 mg/L at nominal concentrations of 1.0, 3.2, 10, 32, and 100 mg ai/L , respectivel y, at 96 hours. Measured formaldeh yd e (HPL C) co ncentrations were 0.482, 1.5, 4.21, 15.0, and 43.8 mg/L at nominal concentrations of 1.0, 3.2, 10, 32, and 100 mg ai/L, respectivel y, at 0 hours and 0.110, 0.429, 2.96, 10.2, and 30.5 mg/L at nominal concentrations of 1.0, 3.2, 10, 32, and 100 mg ai/L , res pectivel y, at 96 hours. At initiation of exposure (time = 0 hours), the measured concentrations of monoethanolamine and formaldeh yd e were 62 - 136% and 102 - 117% of nominal (expected) concentrations, respectivel y, assuming complete degradation of each mole o f CASRN 4719 - 04 - 4 to produce 3 moles of monoethanolamine and 3 moles of formaldehyde. At the end of exposure (time = 96 hours), the measured concentrations of monoethanolamine and formaldeh yde were 41 - 105% and 27 - 78% of nominal (expected) concentrations, respectivel y. Therefore, a decline in the concentrations of both degradation products was evident after 96 hours. In unopened test vessels, measured ethanolamine concentrations were 0.373, 2.68, 7.61, 19.8, and 57.7 mg/L at nominal concentrations of 1.0, 3.2, 10, 32, and 100 mg ai/L, respectivel y, at 96 hours. Measured formaldehy de concentrations in unopened test vessels were 0.129, 0.511, 2.95, 8.53, and 27.0 mg/L at nominal concentrations of 1.0, 3.2, 10, 32, and 100 mg ai/L, respectivel y, at 96 hours. “ These results were similar to the results obtained from the test samples which had been opened on a dail y basis and hence it was considered [by the study authors] that the decline in concentration observed over the test duration was due to instability rather than volatility . ” [ Vry enhoef and Mullee 2012 , p 21]**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

41 A mass balance calculation for the data obtained for the 100 mg/L, 0 - hour test sample was provided. “The data shows that equimolar concentrations of formaldeh yd e and ethanolamine were foun d, and... the s e were then used independently to calculate and confirm that the concentration of the 1,3,5 - triazine active ingredient at 0 hours was within the expected limits for the 100 mg/ l test sample” [Vry enhoef and Mullee 2012, p. 50] Re - Growth Experiment: “ A re - growth test was performed which showed the test item to be algistatic in effect.” [Vry enhoef and Mullee 20 12, p. 21 ] Positive Control: “The results from the positive control with zinc chloride were within the normal range s for this reference item .” [Vry enhoef and Mullee 20 12 , p . 38 ] CONCLUSIONS

“ The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 96 - Hour period and gave the following results based on nominal test concentrations : ...” [Vry enhoef and Mullee 2007, p . 2 1 ] The 72 - hr EC 50 values were 5 .2 mg ai/L for growth rate , 3.5 mgai/L for yi eld , and 3.6 mg ai/L for biomass integral . The 96 - hr EC 50 values were 4.2 mg ai /Lfor growth rate , 3.2 mg ai /Lfor yi eld , and 3.3 mg ai/Lfor biomass integral . The 96 - hour NOEC and LOEC were 1.0 and 3.2 mg ai/L , respectivel y , for growth rate , yi eld , and biomass integral . STUDY RELIABILITY [2] Reliable with restrictions. The following s tudy deficiencies were noted : (1) The stud y authors measured the concentrations of the primary degradation products (monoethanolamine and formaldeh yd e) but did not measure the concentration of the parent test compound (1,3,5 - triazine - 1,3,5(2H,4H,6H) - triethanol; CASRN 4719 - 04 - 4); (2) the study authors calculated the EC 50 values based on nominal, rather than measured, test concentrations; (3) the complete composition of the test substance is not clearly stated in the submitted study report; (4) the loss of monoethanolamine and form aldeh yde from solution during the algae test is not clearly explained; and (5) 95% confidence limits were not calculated for the 72 - hour ErC 50 value because the data generated did not fit the models available for the calculation of confidence limits. REFERENCE Vry enhoef, H., and Mullee, D.M. 2012. 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - T riethanol (C AS No. 4719 - 04 - 4 ): Algal Inhibtion Test; Project No. 41103115. Performing Laboratory : Harlan Laboratories Ltd., Shardlow, Derb ys hire, UK. Sponsor: Society of Chemical Manufacturers & Affiliates, Washington, DC, USA. Text in quotes is taken directl y from Vry enhoef and Mullee , 2012.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

42 **Study 2**  **: Toxicity to**  **Selenastrum capricornutum**  **( Oldersma and Bowmer** 

**, 2002) Title:**  Determination of the effect of KM200 on the growth of the freshwater green alga Selenastrum capricornutum (OECD 201 and EU C.3 Guidelines) **This study is:** Adequate Not Adequate The study is invalid because: (1) No anal yt ical measures of the test substance were obtained during testing. An appropriate method for performing anal yt ical measurements of the test substance was not used. The test needs to be redone using an appropriate method. (2) The purit y of the test substance was less than 100% and information on the composition of the remaining percentage of the test substance was not provided. (3) It i s not clear that the reported nominal concentrations take into account the purit y of the test substance. TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4 **Remarks:** Purit y: 70 - 90%; trade name: KM200; clear liqui d; Batch no. L11381 METHOD **Method/guideline followed:** OECD Guideline 201 and EU C.3 Guidelines **Test type:** Static toxicity test to freshwater algae **GL P compliant?:**  Yes **Year study was performed:** 2002 **Test species:** Green algae ( Pseudokirchneriella subcapitata , formerl y Selenastrum capricornutum ); culture supplied by CCAP, The Freshwater Biological Association, Cumbria, England. **Analytical monitoring:** No (Anal yt ical monitoring was attempted, but results were not reported.) 1 **Exposure duration:** 7 0.5 hours

1 Analytical monitoring was attempted by the study authors. How ever, due to inconsistent results from the chemical analyses, the measured concentrations of test substance were not reported by the study authors. The measurements were considered to be unreliable. See section on Study Reliability for more information.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

43 **Concentrations tested:** Nominal: 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L 2 ; measured concentrations were not reported. However, no indication is provided as to whether the concentrations take into account the 70 - 90% purity of the test substance. **Test method/conditions remarks:** After a range - finding test, a definitive growth inhibition test was conducted. The test medium was prepared in ultra pure water and was sterilized by micropore filtration. The prepared medium contained 150 mg NaHCO 3 /Land had a hardness of 24.3 mg CaCO 3 /L . Test solutions were prepared in medium at nominal concentrations of 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L. At test initiation, an inoculum of 1.0 mLof an algal suspension containing 8 x 10 5 cells/mL was added to 200 - mLflasks containing 100 mL of test solution, yielding an initial cell density of 8 x 10 3 cells/mL . The test was carried out in duplicate, with four controls containing algae onl y and a single set of flasks containing test substance witho ut algae (blanks) at each nominal concentration. All flasks were incubated at 23 ± 2 °C and shaken at approximately 200 rpm on an orbital shaker. The flasks were illuminated using fluorescent lamps at a light intensity of 60 - 120 μmol/s/m 2 (measured with a Bottemanne Weather Instruments Photosynthetic Radiometer RA200 Q). The initial cell density

was calculated from the measured density in the algal pre - culture used for the inoculation, divided by the appropriate dilution factor. Algal densities (cells /mL ) and algal biovolume (μm3/mL ) were determined after 23.5, 47, and 70.5 hours using an electronic particle counter (Coulter Multisizer IIe). Measured values were corrected for background values in the appropriate blanks. The mean measured values were used for calculation of effect levels. The pH was measured at the start (in medium without algae) and after 70.5 hours in selected cultures. The morphology of the algae was examined visually with the aid of a microscope at the start and end of the test. The light intensity at two different culture positions was measured at the start of the test. Samples for chemical analy sis of test substance concentrations were taken at the start of the test from control series without algae and at the end of the test from series with algae. Chemical anal ys is was based on an HPL C method using a fluorescence detector and an external standard. The test substance was fluorogenicall y labeled with a mixed o - phtalaldehyde/mercaptoethanol reagent before HPL C analy sis. The EC values with respect to growth rate (E r C values) were calculated by means of a parametric model, assuming exponential growth and a constant error per measurement. The EC values with respect to area under the growth curve (E b C values) were calculated b y the method provided in OECD Guideline 201. The values were calculated by linear interpolation of a plot of the percentage reduction in growth (I A ) against the log concentration of the test substance. The NOEC was determined as the highest concentration at which no statistically significant inhibition

2 The nominal concentrations were reported as 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L in section 2.5.3 and Annex D of the study report, but were reported as 0, 3.3, 10, 18, 33, 57, 102, and 326 mg/L in section 2.5.1 of the study report.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

44 was observed. The effect was considered significant if the average inhibition of the growth rate was higher than 5%. Statistical significance was determined with a one - tailed t - test (α = 5%). RESUL TS **Endpoint value(s):** Based on nominal concentrations reported: E r C 50 = 62 mg/L ; E b C 50 = 33 mg/L ; NOEC (growth rate) = 18 mg/L; LOEC (growth rate) = 32 mg/L **Results remarks:** The temperature and light intensity were measured to be 23.9 - 24.6 °C (average of 24.0 °C) and 87 - 115 μmol/s/m2 , respectively. At the start of the test, the pH of each flask was found to be 8.4. At the end of the test, the pH range was 8.2 - 8.3 in the ab sence of algal cells and 8.2 - 9.1 in the presence of algal cells. Due to inconsistent results from the chemical analyses of test substance concentrations, the measured concentrations in test solutions were not reported b y the stud y authors. Therefore, the effect levels were determined using nominal concentrations. After 70.5 hours of exposure, the mean cell density was 76.6, 80.0, 80.1, 76.3, 34.6, 12.4, 1.8, and 1.3 x 10 4 cells/mLat nominal concentrations of 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L , re spectivel y. The mean percent reduction in growth rate was - 7, - 8, - 4, 49, 80, 98, and 99% at nominal concentrations of 3.2, 10, 18, 32, 56, 101, and 322 mg/L , respectively . Calculated biomass values were 65.8, 65.7, 64.7, 59.4, 36.9, 7.0, 0.8, and 0.3 x 10 6 μm 3 /mLat nominal concentrations of 3.2, 10, 18, 32, 56, 101, and 322 mg/L , respectivel y. Microscopic inspection of the morphology

of algal cells revealed no abnormal cells in treated cultures at the end of the test. Based on nominal concentrations, the E r C 50 was found to be 62 mg/L, with a 95% confidence interval of 55 to 70 mg/L. The E r C 10 and E r C 90 were 31 and 120 mg/L, respectivel y. The E b C 50 was found to be 33 mg/L, with a 95% confidence interval of 32 to 56 mg/L . The E b C 10 and E b C 90 were 21 and 79 mg/L , respectivel y. Based on inhibition of growth rate, the NOEC was found to be 18 mg/L. CONCLUSIONS A significant reduction in the growth rate of P. subcapitata was observed at a nominal test substance concentration of 32 mg/L. The EC 50 was found to be 62 mg/Lbased on inhibition of growth rate and 33 mg/L based on reduction in biomass. STUDY RELIABILITY [3] Not reliable. The study is not reliable for the following reasons: (1) No reliable analy tical measures of the test substance were obtained during testing. The anal yt ical method attempted for measuring test substance concentrations during the toxicity test was not appropriate because it relied on rapid h ydrol ysis of the test substance and, therefore, analytical testing using an**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

45 appropriate method was not conducted; 3 (2) the composition of 10 - 30% of the test substance (described as 70 - 90% pure) is not defined, suggesting that the test substance may contain impurities; (3) it is not clear if the nominal concentrations have been adjusted to account for purity of the tes t substance, such that the concentrations of the test substance reported in the study may be overestimated by

as much as 30%; hence, the EC 50 s, NOECs, and LOECs may be overestimated b y as much as 30%; and (4) the definitive test used only two replicates pe r test concentration, whereas OECD Guideline 201 recommends three replicates per test concentration. REFERENCE Oldersma, H. and C.T. Bowmer. 2002. Determination of the effect of KM200 on the growth of the freshwater green alga Selenastrum capricornutum (OECD 201 and EU C.3 Guidelines). TNO Study

Number 02 - 4064/02. Submitted by Troy Corporation.

3 Although the study report states that the test substance is unstable in water and, therefore, the test solutions were undefined mixtures of the test substance and hydrolysis products from the onset of testing, the U.S. EPA Reregistration Eligibility Decision for Grotan (HHT) indicates that the hydrolysis half - life of CAS No. 4719 - 04 - 4 i s 50 and 302 days at pH 7 and 9, respectively. Therefore, the hydrolysis of the test substance is not expected to be rapid under the test conditions described for the algae study.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

46 **D -**  **4. Acute Toxicity**  **Summary of endpoint:** Adequate **Study 1: Acute oral toxicity study in rats (Rush, 1989)**  **Title:** Acute Oral Toxicity Study in Rats with Busan 1060 **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 79.4% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 20.6 % unidentified **Remarks:** Clear y ellow liquid, referred to in the reviews as Busan 1060, HHT, hexahydro 1,3,5 - tris(2 - hydrox yeth yl ) - s - triazine, and dihydro - 5 - (2 - hydrox yeth yl ) - 1,3,5 - triazine - l,3(2H,4H) - dipropanol METHOD **Method/guideline followed:** OPPTS 870.1100 **Test type:** Acute Oral -- Rat **GL P compliant?:**  Not indicated **Year study performed:** 1989 (issue y ear) **Test species:** Sprague - Dawley rats, 182 to 219 grams (males) and 173 to 203 grams (females) at study start. **Number of animals/sex/dose:** 5 **Route of administration:** Oral gavage **Vehicle:** Distilled water **Doses/concentrations tested:** 500, 1000, or 1500 mg/kg - bw (males) and 500, 750, or 1000 mg/kg - bw (females). **Statistical methods used:**  Mortality data were analyzed separately for male and female rats by probit analy sis. LD 50 values and 95% confidence intervals were calculated by the method of Litchfield and Wilcoxon (J. Pharmacol. Exp. Ther., 96:99 - 113, 1949). Body weight means and standard deviations were calculated.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

47 **Test method/conditions remarks:** Different doses were test ed in males and females because a rangefinding stud y was thought to indicate sex - related differences in lethality . RESUL TS All deaths occurred b y Day 2 after dosing. Incidences of mortalit y were 0/5, 2/5, and 3/5 in males at respective doses of 500, 1000 , and 1500 mg/kg - bw and 0/5, 2/5, and 5/5 in females at respective doses of 500, 750, and 1000 mg/kg - bw. Frequently

observed clinical signs included underactivity , breathing difficulties, prostration, soft stools, “cool - to - touch”, decreased fecal produc tion, fecal/urine stains, and dark material around the facial area. In male survivors, mean weight gain was slightly depressed at the mid - and high doses compared with animals at the low dose. Gross pathology

revealed no adverse findings in survivors at any dose level, with the single exception of one 500 mg/kg male that exhibited a dilated renal pelvis. Gross necrops y findings for animals found dead included: dark red adrenal glands, reddened mucosa of the digestive tract, dark red l ymph nodes, and colored fluid/mucoid contents in the digestive tract. **Endpoint value(s):** LD 50 (male rats) = 1250 (840 - 1858) mg/kg - bw; LD 50 (female rats) = 763 (701 - 832) mg/kg - bw **Results remarks:** None specified CONCLUSIONS The LD 50 was 1250 (840 - 1858) mg/kg - bw for male rats and 763 (701 - 832) mg/kg - bw for female rats. STUDY RELIABILITY [1] Reliable without restrictions. REFERENCES Locke, R.K. (1995). H exahydro - 1,3,5 - tris(2 - hydroxyeth yl ) - s - triazine . Review of Toxicity Data. DP Barcode: D212071. Submission #: S481738. Document No. 011557. Rush, R.E. (1989). Acute oral toxicity study in rats with Busan 1060. Springborn Laboratories Study No. 3138.47. EPA/OPP MRI D 41675206 as cited in L ocke ( 1995).**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

48 **Study 2 :**  **Acute oral toxicity study in mice ( Consultox Laboratories, 1973)**  **Title:** Glokill 77: Acute Oral Toxicity **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% unidentified (Chen and Tao 2008) or purit y unstated (Consultox L aboratories 1973). **Remarks:** None METHOD **Method/guideline followed:** OPPTS 870.1100 **Test type:** Acute oral -- mouse **GL P compliant?:**  Not indicated **Year study performed:** 1973 **Test species:** Tyler’s original strain white mice, 20 ± 2 g **Number of animals/sex/dose:** 5 **Route of administration:** Oral gavage **Vehicle:** Distilled water **Doses/concentrations tested:** 0.75, 1, 1.5, 1.75, or 2 mL/kg - bw **Statistical methods used:** Not stated **Test method/conditions remarks:**  Glokill 77 of unstated purity was diluted to 20% in distilled water and administered by oral gavage to fasted mice. Food and water were available ad libitum. A preliminary

range - finding study , carried out using 2 mice/dose, te sted doses of 0.5, 1, 2, 3, 4, or 5 mL/kg - bw. Animals were observed for 1 week after dosing. Based on the results of the preliminary

study (all mice administered ≥ 2 mL/kg - bw died), a final assay was performed on 5 groups of mice using 10 mice (5/sex/dose ). Doses used in the main study were 0.75, 1, 1.5, 1.75, or 2 mL/kg - bw. Animals were observed for mortality and/or signs of toxicity

for 14 day s.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

49 RESUL TS In the main study , mortality was 0/10, 2/10, 7/10, 8/10, and 10/10 at respective doses of 0.75, 1, 1.5, 1.75, or 2 mL/kg - bw. All deaths occurred within 24 hours. Signs of toxicity that occurred within 30 minutes of dosing included disoriented locomotion, loss of activity , and coma. **Endpoint value(s):** LD 50 = 0.75, 1, 1.5, 1.75, or 2 mL/kg . **Results remarks:** None specified CONCLUSIONS The oral (gavage) LD 50 for mice was 0.75, 1, 1.5, 1.75, or 2 mL/kg in both sexes combined. STUDY RELIABILITY [2] Reliable with restrictions. Animal weights were not recorded, animals were not observed dail y for signs of toxicity , surviving animals were not examined and necropsied, the LD 50 was not provided for each sex, the method used to calculate the LD 50 was not reported, and the doses in mL/kg - bw were not converted to mg/kg - bw. REFERENCES Chen, J.; Tao, J. (2008). Hexahydro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. C onsultox Laboratories. (1973). Glokill 77: Acute Oral Toxicity . Acc. No. 260195. Unpublished study , 4 p. MRID No. 00155959 As cited in Stewart (1986) and Chen and Tao (2008) . Stewart, J.E. (1986). Memorandum, Subject: Hexahydro - 1.3.5 - tris(2 - hydroxyeth y1 ) - s - triazine; Bioban GK; Glokill 77, EPA File Sy mbol 48301 - RU. Document 005165, Caswell No. 481C, Project No. 902.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

50 **Study 3: Acute inhalation toxicity study in rats**  **(BASF Corporation, 2010) Title:**  Acute Inhalation Study in Wistar Rats (RccHan:WI ST(SPF)) with hexahy dro - 1,3,5 - tris(2 - hydrox ye thyl) - s - triazine (CAS No. 4719 - 04 - 4) **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 73.3% aqueous 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; not indicated if test substance contains other chemicals **Remarks:** None specified METHOD **Method/guideline followed:** OECD TG 403 **Test type:** Acute Inhalation Toxicity **GL P compliant?:**  Not Indicated **Year study performed:** Not Indicated **Test species:** RccHan:WIST(SPF) Wistar rats **Number of animals/sex/dose:** 5 **Route of administration:**  Inhalation, nose - onl y **Vehicle:** Air **Doses/concentrations tested:** 0.26, 0.485, or 1.07 mg/L (measured concentrations) **Statistical methods used:** Not stated **Test method/conditions remarks:** Groups of 5 rats/sex were exposed nose - only to a liquid aerosol of the test substance for 4 hours. The surviving animals were observed for clinical signs of toxicity

for 14 day s. RESUL TS Within day

1 of the study, 3 of 5 male and all 5 females exposed to 1.07 mg/L died. The surviving animals in this group were in poor condition (apathy or laid on lateral position) and were sacrificed moribund at study day 1. At 0.485 mg/L, 3 of 5 males and 3 of 5 females died within 3 day s after exposure. At 0.26 mg/L , 1 of 5 males and 2 of 5 females died during**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

51 exposure or by

study day 2. Clinical signs of toxicity included gasping, respiration sound, abdominal respiration, nose discharge, red encruste d nose, salivation, distended abdomen, piloerection, and yellow - discolored and/or substance - contaminated fur. Dilated stomach and/or small intestine with gaseous content were seen at necrops y. Histological examination, performed only at 1.07 mg/L, showed moderate to severe diffuse erosion and ulceration of the nasal cavity and lary nx with moderate to severe diffuse erosion and ulceration. **Endpoint value(s):** LC 50 = 0.371 mg/L for both sexes (0.4 mg/L for males and 0.338 mg/L for females) **Results remarks:**  No additional details were available from this study abstract. CONCLUSIONS The L C 50 (both sexes combined) was 0.371 mg/L. The L C 50 values for males and females were 0.4 and 0.338 mg/L, respectivel y. STUDY RELIABILITY [2] Reliable with restrictions. Information is needed on the composition and stability of the test substance. REFERENCE TSCATS submission (8EHQ - 10 - 18183; 88110000067): BASF Corporation, November 18, 2010. Notice in Accordance with Section 8(e): Results of an Acute Inhalati on Study in Wistar Rats with hexahydro - 1,3,5 - tris(2 - hydrox yeth yl ) - s - triazine (CAS No. 4719 - 04 - 4) as L iquid Aerosol. [This is an abstract; the full study was not available]**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

52 **Study 4: Acute sensory irritation and toxicity study in mice (**  **Krystofiak, 1994) Title:**  Evaluation of the Respiratory Effects from Components of a Metalworking Fluid in Mice **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; purity and composition not indicated **Remarks:** Amber liquid thicker than corn oil, referred to in the study as Triazine, CAS No. 4719 - 04 - 4. METHOD **Method/guideline followed:** Unclear. The study in the TSCATS submission suggests that an ASTM method (ASTM 1984) was used but does not explicitly state what method was followed. **Test type:** Acute sensory irritation from inhalation of airborne chemicals **GL P compliant?:**  Not Indicated **Year study performed:** Not Indicated **Test species:** Pathogen - free Swiss - Webster mice, 24 - 28 g **Number of animals/sex/dose:** 4 males/concentration **Route of administration:**  Inhalation, head - onl y **Vehicle:** Air **Doses/concentrations tested:** 55, 103, 204, 332, or 1619 mg/m 3 (measured concentrations); the mass median aerod yn amic diameter for the aerosol particles of triazine was 1.41 ± 2.33 μm. **Statistical metho ds used:**  The maximum change in respiratory frequency

(f) that occurred during the 180 - minute exposure period was evaluated with respect to control. Significant responses were plotted as a function of the logarithm of exposure concentration. L east - squares regres sion anal ys is was then conducted to establish concentration - response relationships (i.e., testing that the slope of the line from regression analy sis was significantly different from zero, p < 0.05). These relationships were used to calculate the exposure concentration that resulted in a 50% decrease in respiratory frequency

(RD 50 ) in the exposed animals. **Test method/conditions remarks:** Concentrations of test substance generated by an aerosol generator were determined at least 5 times during each exposure . Aerosol samples were drawn**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

53 from the test chamber onto glass fiber filters and the filters were weighed before and after sampling to determine test substance concentrations gravimetricall y. For each concentration of aerosolized test substance, each of 4 male rats was positioned in a body plethy smograph, which was used to continuously monitor respiration pressure changes of the mouse. The head of each mouse extended through a dental latex dam, positioned with a tape collar. Respiratory frequency (f) was measured every

15 seconds. Tidal volume (VT) and f were plotted as a function of time for each mouse. Each experiment consisted of a 20 - minute control period to establish a baseline value for f, followed b y a 180 - minute exposure period. The mice were m onitored for 20 minutes after aerosol generation had ended to assess recovery . After removal from the test chamber, mice were observed for seven day s for signs of toxicity . RESUL TS Triazine produced severe sensory irritation at the start of exposure, with milder sensory irritation seen even at the lowest concentration of 55 mg/m 3 . Immediate decreases in respiratory frequency were noted at the start of exposure and a plateau of response was reached in 20 - 30 minutes. The plateau t yp ically lasted during the remainder of the exposure period. The exposure concentration that resulted in a 50% decrease in respiratory frequency

(the RD 50 ) was 137 mg/m 3

. Mice were noted to have extremely swollen heads after exposure to concentrations of ≥ 103 mg/m 3 . Heads o f one animal each at 103 and 1619 mg/m 3 were so swollen that rubber dams and tape restraints had to be cut to remove them from the exposure chamber; because this removal process caused hind leg paral ys is due to neck damage, these animals were euthanized on day 1 postexposure. Overall treatment - related deaths (including animals killed as noted in the preceding sentence) among the 4 mice/concentration were 1, 2, 2, 2, and 3 at concentrations of 55, 103, 204, 332, and 1619 mg/m 3 , respectivel y. All spontaneou s deaths occurred on day s 2 - 4 of the 7 - day

observation period. At 24 hours after exposure, up to 15% loss of body weight was seen in some mice at ≥ 103 mg/m 3 ; animals were breathing through their mouths, gasping, and made noticeable clicking and “chirping ” sounds. **Endpoint value(s):** RD 50 = 137 mg/m 3 . Based on spontaneous deaths during the 7 - day observation period, the LC 50 was between 103 and 204 mg/m 3 **Results remarks:** Triazine is a severe sensory irritant. CONCLUSIONS Acute exposure of mice to respirable aerosols of Triazine produced severe sensory irritation, signs of respiratory

distress, swollen heads, and deaths at concentrations ≥ 103 mg/m 3 and sensory irritation even at the lowest concentration tested (55 mg/m 3 ). The concentration that pr oduced a 50% decrease in respiratory frequency

(the RD 50 ) was 137 mg/m 3 . Based on spontaneous deaths during the 7 - day post - exposure observation period, the LC 50 in male mice was between 103 and 204 mg/m 3

.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

54 STUDY RELIABILITY [2] Reliable with restriction s. Information is needed on the composition and stability of the test substance. REFERENCE ASTM. (1984). American Society for Testing and Materials: Standard Guide for Estimating Sensory Irtritancy of Airborne Chemicals. ASTM Designation E 981 - 84. Philadelphia: ASTM, 1984. TSCATS submission (8EHQ - 1194 - 13247; 88950000037): General Motors Corporation, Environmental and Energy Staff, Chemical Hazard Assessment and Toxicology , November 4, 1994. Possible TSCA 8(e) Submission. Kry stofiak, S.P. (1994 ). Evaluation of the Respiratory Effects from Components of a Metalworking Fluid in Mice. Submitted to the Graduate Facult y of Environmental and Occupational Health, School of Public Health in partial fulfillment of the requirements for the degree of Mas ter of Science. University of Pittsburgh.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

55 **Study 5: Acute dermal toxicity study in rabbits (**  **Hughes et al., 1985) Title:**  Acute dermal toxicity screen in rabbits of Bioban GK **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 79.96 % 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 20.04 % unidentified (Chen and Tao, 2008) or purit y not indicated (Hughes, 1985) **Remarks:** Yellow liquid with pungent odor, referred to in the reviews as HHT, hexahy dro 1,3,5 - tris(2 - hydrox yeth yl) - s - triazine, dihydro - 5 - (2 - hydroxy eth yl ) - 1,3,5 - triazine - l,3(2H,4H) - dipropanol, and Bioban GK METHOD **Method/guideline followed:** OPPTS 870.1200 **Test type:** Acute Dermal -- Rabbit **GL P compliant?:**  Not indicated. **Year study performed:** 1985 (issue date) **Test species:** New Zealand White rabbits. 2.07 - 2.67 kg (males) and 2.27 - 2.59 kg (females) at study start. **Number of animals/sex/dose:** 5 **Route of administration:** Dermal **Vehicle:** None **Doses/concentrations tested:** 2000 mg/kg - bw undiluted Bioban GK **Statistical methods used:** Mean bod y weights (Days 0, 8, and 15) and body weight changes over the 15 - day

study period (and standard deviations) were calculated. **Test method/conditions remarks:** The exposure site (unabraded clipped flank skin) was occluded with rubber dental dam and exposure continued for 24 hours. Animals were weighed at study initiation (Day

0), Day 8, and at stud y termination (Day 15). At the end of the exposure period, the rub ber dams were removed and animals were observed for mortality , moribundity , gross s ys temic toxicity , and dermal irritation once on the day of treatment and twice dail y thereafter.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

56 RESUL TS No animals died on test. With the exception of one male, all animals appeared to be normal and gained weight during the entire study period. Gross necropsies were conducted on all animals at study

termination; the one abnormal - appearing male exhibited mottled lungs, a complete depletion of body fat stores, a green - p urple colored spleen, and a gall bladder distended with dark green fluid. Two other animals exhibited pale adrenal glands. None of these findings are considered to be elated to treatment. Dermal irritation, characterized b y edema, ery thema, necrosis, desqu amation, and coriaceousness, were seen at the site of application to the skin. **Endpoint value(s):** LD 50 >2000 mg/kg - bw **Results remarks:** None specified CONCLUSIONS The LD 50 was >2000 mg/kg - bw. The test substance was irritating to the skin. STUDY RELIABILITY [2] Reliable with restrictions. Information is needed on the composition and stability of the test substance. REFERENCES Chen, J.; Tao, J. (2008). Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the Is suance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Hughes, T.L. et al. (1985). Acute Dermal Toxicity Screen In Rabbits of Bioban GK. Study No. 85 - 0866 - 21. H ill Top Research, Inc. EPA/OPP MRID 155984 as cited in Locke (1995). Locke, R.K. (1995). H exahydro - 1,3,5 - tris(2 - hydroxyeth yl ) - s - triazine . Review of Toxicity Data. DP Barcode: D212071. Submission #: S481738. Document No. 011557.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

57 **Study 6: Acute**  **dermal toxicity study in rats ( Siglin et al., 1989a)**  **Title:** Acute Toxicity

Studies with Busan 1060

– Dermal Toxicity **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 78.5 % 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5 % unidentified. **Remarks:** Referred to in the study as Busan 1060 and hexahy dro - 1,3,5 - tris(2 - hydrox yeth yl ) - 5 - triazine METHOD **Method/guideline followed:** Not indicated **Test type:** Acute Dermal -- Rat **GL P compliant?:**  Not indicated **Year study performed:** 1989 **Test species:** Sprague - Dawley rat, 214 - 235 g **Number of animals/sex/dose:** 5 **Route of administration:** Dermal **Vehicle:** None **Doses/concentrations tested:** 2000 mg/kg - bw neat Busan 1060 **Statistical methods used:** None indicated **Test method/conditions remarks:**  Rats received a single occlusive dermal application of the test substance (neat) at a dose of 2000 mg/kg - bw. Animals were observed for clinical signs through day

15, body weights were determined on day s 1, 8, and 15, and gross n ecrops y was performed on day

15.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

58 RESUL TS The test substance produced no mortality , signs of toxicity , effects on bod y weight, or findings at necrops y. Following removal of the occlusive dressing, minimal erythema was seen at the application site but this was resolved b y day 5. **Endpoint value( s):**  LD 50 >2000 mg/kg - bw **Results remarks:** None specified CONCLUSIONS The LD 50 was >2000 mg/kg - bw. The test substance was minimally irritating to the skin. STUDY RELIABILITY [2] Reliable with restrictions. Information is needed on the composition and stability of the test substance. REFERENCE Siglin, J.C.; Rush, R.E.; Jenkins, P.K. et al. (1989a). Acute toxicity studies with Busan 1060. In: R.A. Parent (ed.), Acute Toxicity Data, J. Amer Coll. Toxicol., Part B, Vol. 1, No. 3, 1992, pp. 204 - 205.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

59 **D -**  **5. Repeated**  **- Dose Toxicity**  **Summary of endpoint:** Adequate **Study 1: Repeated** 

**- dose oral toxicity study in rats (**  **Hill and Newman, 1990a) Title:**  Triazine: 13 Week Oral (Gavage) Toxicity Study in the Rat **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% unidentified **Remarks:** Clear y ellow liquid, referred to in the review as hexahydro - 1,3,5 - tris(2 - hydrox yeth yl ) S triazine, Triazine, Grotan, and Bioban GK METHOD **Method/guideline followed:** OPP 82 - 1 **Test type:** Subchronic Oral Toxicity (Rodent and Non - Rodent): 90 - Day Study **GL P compliant?:**  Yes **Year study performed:** 1990 (issue date) **Test species:** Crl: CD(SD)BR rats **Number of animals/sex/dose:** 10 **Route of administration:** oral gavage **Vehicle:** py

rogen - free sterile water **Exposure duration:** 5 days/week, 13 weeks **Doses/concentrations tested:** 0 (vehicle control), 10, 50, 100, or 250 mg/kg - bw/day **Statistical methods used:** Analy sis of variance was used to evaluate body weights, hematology , and organ weight data. Where p = 0.05, pairwise T tests were used between control and treated groups. Blood chemistry parameters were anal yz ed using the Kruskal Wallis test. The Wilcoxon rank sum test was used to determine significant differences between tr eated and control groups at p = 0.05.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

60 **Test method/conditions remarks:** Bod y weights and food consumption were recorded weekl y. Hematology

and clinical chemistry

parameters were measured at study termination. Full necrops y was performed on all animals. Complete histopathology was performed on all control and high dose animals. The stability of the test material was confirmed by analy sis on the day

of preparation of the test material and on day s 1, 7, 8, 14, and 29 of the study . RESUL TS One male at 50 mg/kg - bw/day and seven females (one each at 0, 50, and 100 mg/kg - bw/day and four at 250 mg/kg - bw/day) were found dead during the study period. With the exception of one female from the high dose group, the decedents had gross changes in the lung and/or thoracic cavity . The lungs in these animals did not collapse upon opening the thoracic cavity and adhesions or fluid was present in the area. Inflammatory changes and vascular congestions were present in the thoracic viscera. In addition to the thoracic lesions, all decedents from the high dose group showed a white discoloration and prominence of the limiting ridge of the stomach. There were significant differences in the bod y weight gain and food consumption between treated and control rats. With regar d to organ weights, absolute adrenal weights were increased for females at 250 mg/kg - bw/day ; this increase was not significant. In addition, an increase in splenic weight was observed in all treated males. The sponsor attributes this difference to unusua lly low weights in control animals. Grossly , a white discoloration and/or prominence of the limiting ridge of the stomach were present in 10/10 high dose males which were sacrificed at the end of the study . This was also present in the remaining high dos e females which survived the duration of the study . In animals receiving 100 mg/kg/day, the change occurred in 3/10 males and 1/10 females. A treatment - related distribution of lesions was in the stomach and to a lesser degree in the liver. In the stomac h, erosion of the glandular mucosa was observed in 4/10 males and 2/10 females at 250 mg/kg - bw/day . This finding was also present in one male at 100 mg/kg. Ly mphocy tic infiltration was reported in rats at 100 mg/kg - bw/day (1/10 males and 2/10 females) an d at 250 mg/kg - bw/day

(4/10 males and 6/10 females). Epithelial hy perplasia was reported in three animals at 250 mg/kg - bw/day (1/10 males and 2/10 females) and in the controls (1/10 females), but there was no statistical significance for this finding. In the liver, the incidence of hepatocy te vacuolation was increased in high - dose male rats when compared to other groups. On the other hand, margination of hepatocy te cy toplasm was more pronounced in animals at 0 and 10 mg/kg - bw/day than in rats at 50, 100, or 250 mg/kg - bw/day . A high frequency

of peribronchiolar l ymphoid hyperplasia was reported for all groups. The incidence of this finding ranged from 70 to 100 percent on both sexes. While this finding is not believed to be treatment - related, the spon sor has not addressed its significance. A statistically

significant reduction in hematocrit was found in all treated males. Additionally , males at 100 mg/kg - bw/day demonstrated a statistically - significant decrease in hemoglobin**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

61 concentration. Although these findings were statistically significant, they

were within the expected laboratory

range. Significant differences in blood chemistry values included reduced bilirubin in all treated groups, reduced total protein in both sexes of rats at 250 mg/kg, and a statistically significant increase in sodium concentration for all treated males and for fem ales at 50, 100, or 250 mg/kg - bw/day . The bilirubin and total protein values were within the normal range and were not considered to be biologically

significant. Urinaly sis was unremarkable and no treatment - related ocular lesions were present. **Endpoin t value(s):**  LOAEL = 100 mg/kg - bw/day based on gastric lesions (erosion of the mucosa and l ymphocy

tic infiltration) in males and females; NOAEL = 50 mg/kg - bw/day **Results remarks:** Microscopic examinations were conducted on all tissues from control and high - dose animals, tissues from animals that died on study , gross lesions. No treatment - related histological effects were seen in the following reproductive organs: testes, epididymides, prostate, seminal vesicle, ovaries, uterus, and vagina. CONCLUSIONS T he LOAEL and NOAEL were 100 and 50 mg/kg - bw/day , respectivel y. STUDY RELIABILITY [2] Reliable with restrictions. While not expected to affect the outcome of this study , the sponsor should address the significance of the high incidence of peribronchiolar ly mphoid hy perplasia as it related to the overall health of the test animals. For animals that died during the study , gross necrops y and microscopic examinations revealed that test substance had entered the lungs in 6 of the 7 decedents; aspiration pneumonia likely resulted from failure to correctl y administer the test compound. REFERENCES Chen, J.; Tao, J. (2008) Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Hill, R.; Newman, A. (1990a) Triazine: 13 Week Oral (Gavage) Toxicity Study in the Rat: Lab Project Number: LEF/3/90: L EF/3/89. Unpublished study prepared by

Toxicol. Laboratories, Ltd. EPA/OPP MRI D 41483001.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

62 **Study 2: Repeated** 

**- dose de** **rmal toxicity study in rats (**  **Thomas and Brooks, 1984) Title:**  Glokill 77: 21 Day Dermal Toxicity

Study in the Rat **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 94.8% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 5.2 % unidentified (Thomas and Brooks 1984) or 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% unidentified (Chen and Tao 2008) **Remarks:** Clear viscous liquid, referred to in the reviews as Glokill 77 and hexahydro - 1,3,5 - tris(2 - hydroxy eth yl ) S triazine METHOD **Method/guideline followed:**  Not indicated **Test type:** Subacute repeated - dose toxicity **GL P compliant?:**  Not stated **Year study performed:** ~1984 **Test species:** Sprague - Dawley rats **Number of animals/sex/dose:** 5 **Route of administration:** Dermal **Vehicle:** None. The test material was used undiluted as supplied. **Exposure duration:** 6 hours/day, 21 day s **Doses/concentrations tested:** 0 (sterile distilled water control), 100, 500, or 1000 mg/kg bw/day **Statistical methods used:** Not stated **Test method/conditions remarks:** Identity and stability of the test material were not determined by the investigating laboratory . The test compound was held in contact with the skin by a porous gauze dressing covered with an Elastoplast elastic adhesive ba ndage. At the end of each 6 - hour exposure period the bandage and dressing were removed and the exposure site washed and dried. The test site of each animal was observed daily for local dermal irritation. Animals were observed dail y for toxic signs. Ani mals that died during the study were subjected to a detailed**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

63 postmortem examination, and tissues were preserved for histopathological examination. Body weight was recorded immediately prior to the start of the study , and twice weekl y thereafter. Food cons umption was recorded weekl y. Water consumption was monitored daily . Hematology and serum biochemical determinations were conducted on a separate group of 10 animals (5 male, 5 female) from the same stock prior to start of the study , and on all test and c ontrol animals at termination of the study. Determinations were done for hemoglobin, erythrocytes, hematocrit, mean corpuscular volume, leukocy tes (total and differential) and prothrombin time. Mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration was calculated. Blood chemistry values were determined for blood urea nitrogen, total protein, albumin, A/G ratio, creatinine, total bilirubin, alkaline phosphatase, SGPT, SGOT, gamma glutamyltranspeptidase, glucose, calcium, potassium, sodi um chloride, and phosphate. At termination of the study , all surviving animals were necropsied and terminal weights taken on liver, kidney , adrenals, and gonads. Histopathology

was done on liver, kidney , ly mph nodes, skin (treated and untreated) and organs showing lesions or change in size. Skin was examined histologically in all treatment groups, since compound - related changes were observed in the high - dose group. RESUL TS No animals died during the study . No toxic signs were reported except for local dermal irritation, which was dose - related and increased in severit y with the duration of the study. All groups of treated males gained less weight than corresponding controls. Male weight gain in all groups was approximately 70% of the control male weight gain. The weights observed for all three dosed groups were within the range normally observed in the investigators’ laboratory , and therefore were not considered treatment related. Weight gain was comparable in control and treated females. No di fference in food consumption was reported between control and treated males, but during week 1, the mid and high - dose female food consumption was significantl y decreased (p < 0.05) when compared to the controls. No intergroup differences in water consumpt ion were reported. Mean corpuscular volume was reduced in the low and high - dose males, and hemoglobin was reduced in the low and high - dose females, but the reductions were not considered biologicall y significant because they

were within the normal range of values for Sprague - Dawley rats in the investigators’ laboratory . The following statistically significant changes were noted in the treated animals when compared to the controls (p <0.05): increased protein in mid and high dose males and high dose females , increased sodium in all treated male groups, increased phosphate in mid and high dose females, increased AST in mid and high dose males, decreased A/G ratio in mid and high dose males and high dose females, and decreased chloride in mid and high dose fem ales. These changes were considered biologicall y i nsignificant because the values observed were within the range observed in the animals used in the hematology and biochemical screens. Except for multifocal scab formation at treatment sites at all dose l evels, no macroscopic abnormalities were reported. Absolute adrenal weight was significantly decreased in low and high - dose males and was reported to be due to two control animals with high adrenal weights. No difference was reported in relative organ wei ghts. Treatment - and dose - related histopathological changes seen at treated**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

64 skin sites included epidermal ulceration, acanthosis, and dermal inflammatory infiltration with occasional necrosis and hemorrhage. No histological changes were found in other tis sues. **Endpoint value(s):** LOAEL (s ys temic toxicity) > 1000 mg/kg - bw/day based on no effects at the highest dose tested; NOAEL= 1000 mg/kg - bw/day . LOAEL (application site irritation) = 100 mg/kg - bw/day

based on epidermal ulceration, acanthosis, and derma l inflammatory infiltration with occasional necrosis and hemorrhage at the lowest dose tested ; NOAEL = Not established **Results remarks:** Reproductive organs were not examined histologically in this study . CONCLUSIONS The LOAEL for s ys temic toxicity was > 1000 mg/kg - bw/day , based on no effects at the highest dose tested, and the NOAELwas 1000 mg/kg - bw/day . The LOAEL for irritation at the site of test compound application was 100 mg/kg - bw/day, the lowest dose tested, based on epidermal hy perplasia and ulc eration; a NOAEL was not established. STUDY RELIABILITY [2] Reliable with restrictions. The 21 - day duration of this study

was shorter than the 28 - day duration needed to adequately address the repeated - dose endpoint. Also, different values for percent active ingredient were reported in the reviews that formed the basis for this robust summary . In addition, a limited number of organs was preserved and examined histologicall y. REFERENCE Chen, J.; Tao, J. (2008) Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - T riazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Thomas, M.; Brooks, P. (1984) Glokill 77: 21 Day Dermal Tox icity Study in the Rat: Experiment No. 508/8411. Unpublished study prepared by

SafePharm Laboratories Ltd. EPA/OPP MRID 155989**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

65 **Study 3: Repeated** 

**- dose dermal toxicity study in rats (**  **Hill and Newman, 1990b) Title:**  13 Week Dermal Toxicity Study in the Rat **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% unidentified **Remarks:** Clear y ellow liquid, referred to in the review as hexahydro - 1,3,5 - tris(2 - hydrox yeth yl ) S - triazine, Triazine, Grotan, and Bioban GK METHOD **Method/guideline followed:**  OPP 82 - 3 **Test type:** Subchronic Dermal Toxicity 90 - Day Study **GL P compliant?:**  Yes **Year study performed:** 1990 (issue date) **Test species:** Crl:CD(SD)BR rats weighing 197 - 234 g (females) and 230 - 276 g (males) **Number of animals/sex/dose:** 10 **Route of administration:** dermal **Vehicle:** py

rogen - free sterile water **Exposure duration:** 5 days/week, 13 weeks **Doses/concentrations tested:** 0 (vehicle control), 5, 50, or 250 mg/kg - bw/day **Statistical methods used:** Analy sis of variance was used to evalu ate body weights, hematology , and organ weight data. Where p = 0.05, pairwise T tests were used between control and treated groups. Blood chemistry parameters were anal yz ed using the Kruskal Wallis test. The Wilcoxon rank sum test was used to determine significant differences between treated and control groups at p = 0.05. **Test method/conditions remarks:** During the exposure period, the application sites were covered with semi - occlusive bandages and animals were fitted with Elizabethan collars to minimi ze ingestion of the test chemical. The test site was not washed between doses. Hematology

and clinical chemistry

parameters were measured at study termination. Full**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

66 necrops y was performed on all animals. Complete histopathology was performed on all control and high dose animals. Normal and treated skin from all animals in all dose groups was examined histologically . RESUL TS No deaths were reported during the stud y. The clinical signs observed during the stud y included periorbital, perianal, and cran ial fur staining in all groups; y ellow staining at the applications site in animals at 5 and 50 mg/kg - bw/day , and skin reactions at the application site in animals at 50 and 250 mg/kg - bw/day . Occasional incidences of ery thema were observed in female rats that received 5 mg/kg - bw/day. In assessing the severity

of the dermal lesions, erythema was scored as very slight to severe in animals at 50 and 250 mg/kg - bw/day . An increase in the numbers of animals affected and an increase in the severity

of the reported reactions occurred at 250 mg/kg - bw/day . Moderate to very severe edema was present in animals at 250 mg/kg - bw/day . At 50 mg/kg - bw/day , only isolated incidences of edema were reported. Statistically

significant differences were found in blood chemistry values from individual animals. At 5 mg/kg - bw/day , affected parameters included one male with increased alanine aminotransferase, one female with increased phosphorous and one female with increased chloride. At 50 mg/kg - bw/day , blood chemistry parameters were increased in two animals. One female in this group had increased sodium and one male had increased chlorine levels. At 250 mg/kg - bw/day , one female had elevated glucose levels and one male had elevated sodium levels. Microscopicall y, the re was an increase in the incidence of epidermal hy perplasia and ulceration of the treated area of the skin in animals treated with 50 or 250 mg/kg - bw/day . Eight y to 100 percent of both the treated and control animals were found to have peribronchiolar ly mphoid hy perplasia in the lungs. The significance of the lesions was not discussed. **Endpoint value(s):** LOAEL (s ys temic toxicity) > 250 mg/kg - bw/day based on no effects at the highest dose tested; NOAEL= 250 mg/kg - bw/day . LOAEL (application site irritati on) = 50 mg/kg - bw/day

based on epidermal hy

perplasia and ulceration; NOAEL = 5 mg/kg - bw/day **Results remarks:** Microscopic examinations were conducted on all tissues from control and high - dose animals, tissues from animals that died on study , gross lesions. No treatment - related histological effects were seen in the following reproductive organs: testes, epididymides, prostate, seminal vesicle, and ovaries. CONCLUSIONS The LOAEL for s ys temic toxicity was > 250 mg/kg - bw/day , based on no effects at the high est dose tested, and the NOAELwas 250 mg/kg - bw/day . The LOAEL for irritation at the site of test compound application was 50 mg/kg - bw/day based on epidermal h yperplasia and ulceration and the NOAEL was 5 mg/kg - bw/day .**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

67 STUDY RELIABILITY [2] Reliable wi th restrictions. While not expected to affect the outcome of this study , the sponsor should address the significance of the high incidence of peribronchiolar ly mphoid hy perplasia found in the lungs of both treated and control animals. REFERENCE Chen, J.; Tao, J. (2008) Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Hill, R.; Newman, A. (1990b) Triazine: 13 Week Dermal Toxicity Study in the Rat: L ab Project Number: LEF/4/89. Unpublished study prepared by

Toxicol Laboratories, Ltd. EPA/OPP MRID 41483002.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

68 **D -**  **6. Genetic Toxicity**  **– Gene Mutation**  **Summary of endpoint:**  Adequate **Study 1: Mutagenicity study in Salmonella typhimurium (**  **Mortelmans et al., 1986) Title:**  Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4, purity not indicated **Remarks:** Referred to in Table I of Mortelmans et al. (1986) as hexahydro - 1,3,5 - tris(hydrox ye thyl) - s - triazine. METHOD **Method/guideline followed:** Standard NTP Protocol for the Salmonella/E. coli Mutagenicity Test or Ames Test. **Test type:** Ames Salmonella reverse gene mutation assay **GL P compliant?:**  Not indicated **Year study performed:** 1983 **Test species:** Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 **Metabolic activation:** With and without (tested separately with S - 9 metabolic activation from rat and hamster livers) **Concentrations tested:** 0 (water vehicle control) and at least five concentrations in the range 0.1 to 333 μg/ plate **Statistical methods used:** None **Test method/conditions remarks:**  In the standard protocol (preincubation) for conducting the Ames assay , a test tube containing a suspension of one strain of Salmonella typhimurium (or E. coli ) plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37o C with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughl y mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

69 supplemental histidine appear and grow. These colonies are comprised of bacteria that have undergone reverse mut ation to restore function of the histidine - manufacturing gene. The number of colonies is usually

counted after 2 day s. All assay s are repeated no less than one week after completion of the initial test. Criteria for data evaluation were as follows: (1) Mutagenic response: a concentration - related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; (2) Nonmutagenic response: when no increase in the number of revertants was elicited by

the chemical ; and (3) Questionable response: when there was an absence of a clear cut concentration - related increase in revertants, when the concentration - related increases in the number of revertants were not reproducible, or when the response was of insufficient ma gnitude to support a determination of mutagenicity . In a preliminary

concentration - setting experiment, 1,3,5 - triazine - 1,3,5(2H,4H,6H) - triethanol was tested with S. typhimurium strain TA100 in the presence and absence of the metabolic activation sy stems, ov er a wide concentration range with an upper limit of 10 mg/plate. The high concentration for further testing was selected to exhibit some degree of toxicity . One or more of the following was taken as evidence of toxicity : appearance of his pinpoint col onies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Positive control substances were tested concurrently . Failure of the positive controls to induce mutation was considered reason to discard the experiment. RESUL TS In S. typhimurium strain TA100, 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol induced a concentration - related increase in revertants of up to 2 - fold in the presence of metabolic activation from rat or hamster liver; no increase was observed in th e absence of metabolic activation. The test substance was not mutagenic in strains TA98, TA1535, or TA1537 in the absence or presence of metabolic activation. **Genotoxicity result:** Weakly positive **Cytotoxic concentration (if applicable):** ≥ 100 μg/plate **Results remarks:** Concurrent positive controls produced the expected mutagenic response. CONCLUSIONS 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol was mutagenic in S. typhimurium strain TA100 in the presence but not the absence of metabolic activation. The test substance was not mutagenic in S. typhimurium strains TA98, TA1535, or TA1537 with or without metabolic activation. STUDY RELIABILITY [1] Reliable without restrictions. The study followed the Standard NTP Protocol for the Salmonella /

E. coli Mutagenicity

Test or Ames Test.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

70 REFERENCES Mortelmans, K. et al. 1986. Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Envir. Mutag. Vol. 8, Suppl. 7:1 - 119. National Toxicology

Program 1983. Salmonella: Study Summary

for 1,3,5 - Triazine 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4. Study I D 700221.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

71 **Study 2 :**  **Mutagenicity study in Salmonella typhimurium (NTP, 2004) Title:**  Salmonella study

for 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4 **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4, purity not indicated **Remarks:** None METHOD **Method/guideline followed:** Standard NTP Protocol for the Salmonella / E. coli Mutagenicity Test or Ames Test. **Test type:** Ames Salmonella reverse gene mutation assay **GL P compliant?:**  Not indicated **Year study performed:** 2004 **Test species:** Salmonella typhimurium strains TA98 and TA100 **Metabolic activation:** With and without (tested separately with S - 9 metabolic activation from rat and hamster livers) **Concentrations tested:** 0 (water vehicle control) and at least five concentrations in the range 10 to 500 μg/plate **Statistical methods used:** None **Test method/conditions remarks:** In the standard protocol (preincubation) for conducting the Ames assay , a test tube containing a suspension of one strain of Salmonella typhim urium (or E. coli ) plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37o C with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughl y mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow. These colonies are comprised of bacteria that have unde rgone reverse mutation to restore function of the histidine - manufacturing gene. The number of colonies is usually

counted after 2 day s. All assay s are repeated no less than one week after completion of the initial test.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

72 Criteria for data evaluation were as follows: (1) Mutagenic response: a concentration - related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; (2) Nonmutagenic response: when no increase in the number of revertants was elicite d by

the chemical; and (3) Questionable response: when there was an absence of a clear cut concentration - related increase in revertants, when the concentration - related increases in the number of revertants were not reproducible, or when the response was o f insufficient magnitude to support a determination of mutagenicity . In a preliminary

concentration - setting experiment, 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol was tested with S. typhimurium strain TA100 in the presence and absence of the metabolic activ ation sy stems, over a wide concentration range with an upper limit of 10 mg/plate. The high concentration for further testing was selected to exhibit some degree of toxicity . One or more of the following was taken as evidence of toxicity : appearance of his pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Positive control substances were tested concurrently . Failure of the positive controls to induce mutation was considered reason to disc ard the experiment. RESUL TS In S. typhimurium strain TA100, 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol induced a concentration - related increase in revertants up to 4 - fold in the absence of metabolic activation and up to 2 - fold in the presence of metaboli c activation from rat liver; no increase was observed with metabolic activation from hamster liver. In S. typhimurium strain TA98, 1,3,5 - Triazine 1,3,5(2H,4H,6H) - triethanol induced a concentration - related increase in revertants up to 2 - fold in the presenc e of metabolic activation from hamster liver but no increase in the absence of activation or the presence of metabolic activation from rat liver. **Genotoxicity result:** Positive **Cytotoxic concentration (if applicable):** ≥ 1000 μg/plate for strain TA100 and ≥ 333 μg/plate for strain TA98 **Results remarks:** Concurrent positive controls produced the expected mutagenic response. CONCLUSIONS 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol was mutagenic in S. typhimurium strains TA98 and TA100 in the presence of metabolic activation and was mutagenic in strain TA100 in the absence of metabolic activation. STUDY RELIABILITY [1] Reliable without restrictions. The study followed the Standard NTP Protocol for the Salmon ella /

E. coli Mutagenicity

Test or Ames Test.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

73 REFERENCE National Toxicology

Program 2004. Salmonella: Study Summary

for 1,3,5 - Triazine 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4. Study I D A17569.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

74 **Study 3 :**  **Mutagenicity study in Salmonella typhimurium (**  **Asquith, 1989a) Title:**  Bacterial Reverse Mutation Assay : Triazine **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% not specified **Remarks:** Clear y ellow liquid, referred to in the review as Triazine and hexahydro - 1,3,5 - tris(hydrox ye thyl) - s - triazine. METHOD **Method/guideline followed:** OPPTS 870.5100 **Test type:** Ames Salmonella reverse gene mutation assay **GL P complian**  **t?:** Yes **Year study performed:** 1988 **Test species:** Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 **Metabolic activation:** With and without **Concentrations tested:** 0 (distilled water control), 0.32, 1.6, 8, 40, or 200 μg/ plate **Statistical methods used:** The number of revertant colonies per plate was counted electronically

(Biotran III), treatment group means and standard deviations calculated, and resulting data statistically anal yz ed b y ANOVA (F - statistic). If F was significant (p < 0.05) and concentration - responsiveness apparent, a correlation coefficient was calculated over the response range, and its significance read from standard (published) tables. It’s considered a substance positive if it produces a reproducibl y statis tically significant increase in the mean number of revertants that exceeds twice the concurrent solvent control, plus evidence for a concentration response. Plates showing evidence of severe toxicity (markedl y reduced or absent background bacterial lawn) were excluded from analy sis, since these may yield significantly

decreased revertant colon y counts (of no relevance to mutagenic activity), or increased colonies from surviving non - revertant cells incorporating the histidine available because of (toxic) l ys is (i.e., a false - positive result). **Test method/conditions remarks:** Following a range - finding toxicity testing with TA98 (only) at concentrations up to 5000 μg/plate, triplicate cultures of all five Ames strains were exposed to the test substance for 72 hours (extended from the customary 48 hours because the**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

75 test substance is bacteriostatic, thus slowing growth of revertant colonies) at each of five concentrations, both in the absence and presence of a mammalian metabolic activation sy stem consisting of the liver microsomal fraction (S9) prepared from male Fischer 344 rats induced b y Aroclor 1254 and containing 44 mg protein per mL, plus generating cofactors. In addition to solvent controls (DW), bacterial strains were exposed to their selectivel y approp riate active mutagens in the absence of activation (Sodium azide, 1 μg/plate for TA1535 and TA100; 9 - amino - acridine, 50 μg/plate for TA1537; 2 - nitrofluorene, 0.5 μg/plate for TA1538 and TA98), but all to 2 - aminoanthracene (2 - AA, 2 μg/plate) under activatio n, thus serving as positive controls. The entire experiment was repeated. RESUL TS In the preliminary

toxicity test, the test substance was toxic to both non - activated and activated TA98 cells at concentrations of 200 μg/plate and above, as revealed b y reduced background lawn indicative of restricted growth. Hence, the five concentrations selected for the mutation assays were 200, 40, 8, 1.6, and 0.32 μg/plate with or without S9 mix. In both experiments, the test substance consistently reduced backgro und lawns at the HOT, 200 μg/mL , in the absence of activation but with one exception did not induce significantl y increased revertant colon y counts. The exception was a significant F - value (p < 0.05) calculated for unactivated TA1537 cultures, derived fro m a wider - than - usual variation in mean numbers of revertants on test plates (3.3 to 7.7) compared to the concurrent mean control value (4.7). This was discounted b y the investigator because there was neither a doubling of revertant rate nor evidence of a concentration - response relationship. **Genotoxicity result:** Negative **Cytotoxic concentration (if applicable):** ≥ 200 μg/plate **Results remarks:** Positive controls were tested but responses to controls were not provided in the OPP review. CONCLUSIONS The test substance was not mutagenic in the Ames testing at levels up to levels of toxicity . STUDY RELIABILITY [1] Reliable without restriction.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

76 REFERENCES Asquith, J. (1989a) Triazine Joint Venture: Bacterial Reverse Mutation Assay : Triazine: Toxicol Study No. M/AMES/10658. Unpublished study prepared b y Toxicol L aboratories Ltd. EPA/OPP MRID 41231702. As cited in an EPA OPP Data Evaluation Report by Irving Mauer, January 1990. Chen, J.; Tao, J. (2008) Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazin e: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

77 **Study 4 :**  **Mutagenicity study in Salmonella typhimurium ( Safe**  **Pharm Laboratories, 1984) Title:**  Ames Test (O.E.C.D.) Product Glokill 77. **The study is:** Adequate Not Adequate This study

is not adequate because: 1) no results were provided for Salmonella typhimurium strain TA1537; 2) results of assay s in strains TA98 and TA1538 were inconclusive; and 3) the purity and composition of the test substance were not provided. TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4 of unspecified purity and composition **Remarks:** Referred to in the review as Glokill 77 METHOD **Method/guideline followed:** Ames et al. and Gardner et al. **Test type:** Ames Salmonella reverse gene mutation assay **GL P compliant?:**  Not indicated **Year study performed:** 1984 **Test species:** Uncertain. The Materials and Methods section of the review of this study in Stewart (1986, pp. 34 - 37) indicates that Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 were tested. However, the results section provides results for strains TA98, TA100, TA1535, and TA 1538 but not for TA1537. **Metabolic activation:** With and without **Concentrations tested:** 0 (distilled water control), 0.16, 0.8, 4, 20, or 100 μg/ plate **Statistical methods used:** Not indicated. **Test method/conditions remarks:** In the main study , each concentration was tested in triplicate, with and without metabolic activation. The test substance was cons idered mutagenic if it produced a dose - related and statistically significant increase in mutation rates (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S - 9 microsomal enzy mes. Conversel y, if the number of induced revertants compared to spontaneous revertants were less than twofold at all dose levels employ ed up to the limit of toxicity or solubility , the test compound was considered not to be mutagenic.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

78 RESUL TS No significant increase in the number of revertant colonies was reported for TA100 and TA1535 strains of Salmonella typhimurium ; however, in strain TA98 a significant increase in the number of revertant colonies was reported in the assay with metabolic activation, and in strain TA1538 an increase in the number of revertant colonies was reported both with and without metabolic activation. In strain TA 98, the test compound caused an increase in the mutation frequency . In the first experiment, although the increase in mutation frequency was significantly

increased over the spontaneous mutation frequency in untreated bacteria, the mean numbe r of revertant colonies remained the same at all dosage levels in the study using metabolic activation. At the highest dosage level, 100 μ g/plate, the test compound appeared to be cy totoxic. In experiment 2, the mean number of revertant colonies doubled, but the two highest dose levels had lower mutation frequencies than the two middle dose; thus the mutation frequency in strain TA 98 did not seem to be dose related. In strain TA 1538, in experiment 1 without metabolic activation, the mutation frequency was significantl y increased in a dose - dependent manner up to the high dose (100 μ g/plate) which appeared to be cy

totoxic. In the assay

with metabolic activation, the increase in revertant colonies did not appear to be dose related. The highest dose level in this assay appeared to be cy totoxic. In the second experiment, the two highest dosage levels produced a small increase in the number of revertant colonies when the assay was performed without metabolic activation. When the assay

was done with metabolic activation the increase in the number of revertant colonies was dose related. In summary , the results of the assay s in strains TA 98 and TA 1538 of Salmonella typhimurium were not reproducible. Based on this, the results of the study seem to be inconclusi ve. **Genotoxicity result:** In conclusive **Cytotoxic concentration (if applicable):** ≥ 100 μg/plate **Results remarks:** Positive controls were tested and provided the expected responses. Test concentrations were chosen based upon the results of a preliminary cy totoxicity study . CONCLUSIONS The results of this Ames Salmonella reverse gene mutation assay were inconclusiv e. STUDY RELIABILITY [3] Not reliable. No results were provided for S. typhimurium strain TA1537. Results of assay s in strains TA98 and TA1538 were inconclusive. The purity and composition of the test substance were not provided.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

79 REFERENCES Safe Pharm Laboratories. (1984). Ames Test (O.E.C.D.) Product Glokill 77. Acc. No. 260195. September 11, 1984. As cited in Stewart (1986). Stewart, J.E. (1986). Memorandum, Subject: Hexahydro - 1.3.5 - tris(2 - hydroxyeth y1 ) - s - triazine; Bioban GK; Glokill 7 7, EPA File Sy mbol 48301 - RU. Document 005165, Caswell No. 481C, Project No. 902.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

80 **D -**  **7. Genetic Toxicity**  **– Chromosomal Aberrations**  **Summary of endpoint:** Adequate **Study 1: Micronucleus study in mice (**  **Asquith, 1989b) Title:**  Mouse Micronucleus Test: Triazine **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% not specified **Remarks:** Clear y ellow liquid, referred to in the review as Triazine and hexahydro - 1,3,5 - tris(hydrox ye thyl) - s - triazine. METHOD **Method/guideline followed:** OPPTS 870.5385 **Test type:** Mouse bone marrow chromosomal aberration test **GL P compliant?:**  Yes **Year study performed:** 1988 **Test species:** CD - 1 mice, 5/sex/dose **Metabolic activation:** N/A **Doses tested:** Single gavage dose of 0 (0.9% NaCl control), 200, 400, or 800 mg/kg **Statistical methods used:** The micronuclei data were anal yz ed b y the Mann

—Whitney

U

—test, with probabilities for significant differences between test and control groups obtained from standard published tables. **Test method/conditions remarks:** Following a range - finding study (at doses up to 2000 mg/kg), test substance was administered to groups of CD - 1 mouse b y gavage in a single dose and the animals were sacrificed 24, 48, or 72 hours later. Control groups received 0.9% saline (solvent control), or cycl ophosphamide (CPA, positive control); the latter were sacrificed onl y at 24 hours. Femoral bone marrow harvested at scheduled sacrifice times was prepared for microscopic examination by conventional cy

tological techniques. At least 1000 polychromatic ery throcy tes (PCE) per animal were scored for micronuclei (MN

—PCE), and the ratio of PCE to normochromatic ery

throcy tes (NCE) also recorded for each animal.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

81 RESUL TS In the range - finding test, deaths occurred at 1200 and 2000 mg/kg (respectively , 2 of 3 an imals and 3 of 3 of each sex), but no mortalities at 800 mg/kg or below, providing an estimated LD 50 of approximately

1000 mg/kg. Based upon the (OECD) convention of appl ying 80% of the LD 50 as the maximum tolerated dose (MTD) for this ty pe of study , the highest dose tested (HDT) for the main study

was selected as 800 mg/kg, with two lower doses of 400 and 200 mg/kg. In the micronucleus assay itself, two high - dose (800 mg/kg) animals (one of each sex) died before their scheduled sacrifice at 48 hours. No cy totoxicity , as measured by reduction in PCE/NEC ratios, was found in any treated group. Random variation around control values for numbers of micronuclei was found at all sacrifice times and in both sexes of test animals. A singular statisticall y sign ificant increase in micronuclei over concurrent control was calculated for the 400 mg/kg male group sacrificed at 72 hours (0.14 vs. 0.02). This increase was considered to be of no biological significance since it fell within solvent control ranges at oth er sampling times (notabl y 24 hours when optimally most of the micronuclei would be visualized). **Genotoxicity result:** Negative **Cytotoxic concentration (if applicable):** N/A **Results remarks:** Positive controls were tested and produced the expected responses. CONCLUSIONS The test substance did not induce micronuclei in bone marrow cells of mice treated orally up to a nominal dose (800 mg/kg) considered a MTD b y OECD (80% of the LD 50 ). STUDY RELIABILITY [2] Reliable with restrictions. Although the EPA’s limit dose in the absence of an y toxicity (1000 mg/kg) was not assay ed (represented by the LD 50 calculated in the range - finding study), testing up to 800 mg/kg was sufficientl y high to vali date the negative result obtained. REFERENCES Asquith, J. (1989b) Triazine Joint Venture: Mouse Micronucleus Test: Triazine: Toxicol Study No. M/MMN/10659. Unpublished study prepared by

Toxicol Laboratories Ltd. EPA/OPP MRID 41231701. As cited in an EP A OPP Data Evaluation Report by Irving Mauer, January 1990. Chen, J.; Tao, J. (2008) Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

82 **Study 2: Micronucleus study in mice (Richardson, 1983)**  **Title:** Evaluation of some formaldeh yd e - release compounds and other biocides in the mouse micronucleus test **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 75% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 25% not specified **Remarks:** Referred to in the study report as Glokill 77 (75% pure, CA Reg. No. 56996 - 62 - 4). METHOD **Method/guideline followed:** Salamone, M.F. et al. (1981). Mutagenic activity of 41 compounds in the in vivo micronucleus assay . Progr. Mutation Res. 1:686 - 697. **Test type:** Mouse bone marrow micronucleus test **GL P compliant?:**  Not indicated **Year study performed:** 1983 (publication date) **Test species:** Male C57Bl/6J mice, 6 - 8 weeks old, 8 - 10/sampling time **Metabolic activation:** N/A **Doses tested:** Two consecutive dail y doses b y intraperitoneal injection of 0 (vehicle not indicated) or 176 mg/kg - bw. The latter dose represented 80% of the intraperitoneal LD 50/7 days (based on deaths occurring when animals wer e observed for 7 day s following administration of a single i.p. dose). Mice were also dosed at 50% of the intraperitoneal LD 50/7 days , but the micronucleus data were not evaluated for these animals. **Statistical methods used:** The micronuclei results in treated animals were anal yz ed for significant differences from controls using a one - sided Student’s t - test. **Test method/conditions remarks:** For the stud y in which micronuclei were evaluated, mice were given two consecutive daily doses, 24 hours apart, usi ng dose levels equivalent to 80% of the LD 50/7 days . Animals were killed at 24 or 48 hours after the second dose. Femurs were removed, bone marrow smears were applied to microscope slides, and cells were stained in Leishman’s stain. Slides were coded an d scored blind. Five hundred pol yc hromatic ery throcy tes (PCEs) were examined from each animal and the number containing micronuclei was scored. The ratio of PCEs to mature ery throcy tes (ME) was also determined as a measure of cy totoxicity .**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

83 RESUL TS **Genotoxicity result:** Negative **Cytotoxic dose:** >176 mg/kg - bw, the highest dose tested, based on absence of effect on the PCE: ME ratio **Results remarks:** A positive control, cy clophosphamide, was tested and produced the expected, significant increase in micronucleus frequency . CONCLUSIONS The test substance did not induce micronuclei in bone marrow ery throcy tes of mice treated by two consecutive dail y intraperitoneal injections (dose = 80% of the LD 50/7days) , 24 hours apart, followed b y evaluation of micronucleus frequency at 24 or 48 hours after the second dose. STUDY RELIABILITY [2] Reliable with restriction. The study did not present evidence that the test substance produced signs of toxicity during the main study . The study did not indicate the vehicle used for the test substance (if an y) or if control animals were injected i.p. with the vehicle. OPPTS 870.5395 (Mammalian Ery throcy te Micronuc leus Test) recommends that at least 2000 immature erythrocy tes per animal should be scored for the incidence of micronucleated immature erythrocy tes, whereas the current study evaluated micronucleus frequency in only 500 PCE per animal. REFERENCE Richardson, C.R. (1983). Evaluation of some formaldehyde - release compounds and other biocides in the mouse micronucleus test. Mutat. Res. 124:241 - 246.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

84 **D -**  **9. Reproductive Toxicity Summary of endpoint:**  Adequate Although no reproductive toxicity studies were available, the reproductive endpoint has been acceptabl y addressed on the basis that no effects on developmental endpoints were seen in an adequate oral developmental toxicity study in rats and no treatment - related effects on the histology of reproductive organs of either sex were seen in adequate 90 - day oral and dermal toxicity studies in rats.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

85 **D -**  **10. Developmental Toxicity Summary of endpoint:**  Adequate **Study 1: Developmental toxicity study in rats (**  **Ridgway, 1989) Title:**  Triazine Rat Teratology Study **The study is:** Adequate Not Adequate TEST SUBSTANCE **Identity:** 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% unidentified **Remarks:** Clear y ellow fluid, referred to in the review as Grotan, Triazine, and hexahydro 1,3,5 - tris(2 - hydrox yeth yl )s - triazine METHOD **Method/guideline followed:** OPP 83 - 3 **Test type:** Developmental Toxicity **GL P compliant?:**  Yes **Year study performed:**  1989 (issue date) **Test species:** OFA - SD(IOPS - Caw) rats, 200 - 219 grams **Number of animals/sex/dose:** 24 timed - pregnant females **Route of administration:** Oral gavage **Exposure duration:** Daily on gestation day s 6 - 15 **Doses/concentrations tested:**  0 (vehicle control), 250, 500, or 750 mg/kg - bw/day **Vehicle:** Deionized distilled water S **tatistical methods used:** The significance level was set at p ≤ 0.05. ANOVA was followed by Student’s t - test for anal ysis of maternal bod y weight, bod y weight gain, and food consumption, number of corpora lutea, number of live fetuses, implantation sites, and fetal body weights. The Kruskal - Wallis test was used for analy sis of % pre - and post - implantation losses, fetal sex ratios, fetal malformations, and fetal variations. The percentage of fetus in each litter that showed each**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

86 category of malformation and/or variation was calculate d, group means were calculated from litter means, and the results were compared using the Kruskal - Wallis test. **Test method/conditions remarks:** Females were time mated with males of the same strain and source. One male was caged with two females. The day on which the vaginal plug was seen, taken as evidence of mating, was designated day 0 of gestation. Pregnant females were then randomly assigned to dose groups. Anal ys is of dosing solution for concentration (times of anal ys is not stated) indicated that mean measured concentrations were 99.2 - 105% of target values and that the compound was stable in solution for 28 day s. RESUL TS One dam at 750 mg/kg - bw/day was sacrificed on GD 14. Prior to sacrifice on that day the animal exhibited labored breathing, ra les, emaciation, piloerection, red staining around the mouth and nose, and pale extremities. All other animals survived the study . Post dosing salivation was seen in females at 750 mg/kg - bw/day . In dams at 250 and 500 mg/kg - bw/day , rales, labored breath ing, wheezing, and tachy pnea were observed occasionall y in toward the end of the dosing period. No other clinical signs were reported. At 750 mg/kg - bw/day , maternal body weight gain (p<0.01) and food consumption (p<0.001) were significantly

lower during the dosing period than in controls. Stomach lesions, characterized b y ulceration and/or scarring of the mucosa, were observed in 14 of 20 females at 750 mg/kg - bw/day , including the dam sacrificed on day 14. No gross abnormalities were reported in the oth er dose groups. The data did not demonstrate an y differences between the control and treated dams with respect to pregnancy

rates, number of corpora lutea, implantation sites, number of live fetuses, or earl y and late resorptions. There were no abortions and no premature deliveries. In addition, those treatments did not produce evidence of developmental toxicity as measured by

mean fetal weight or external or visceral, abnormalities. There were increased per - fetus and per - litter incidences of vestigial 14 th ribs and retarded ossification of the vertebral thoracic centra that appeared to be dose - related. However, since the increases were not statistically significant and the incidence of these abnormalities is highly variable in rats, the study authors and O PP reviewer did not consider them to be treatment - related. **Endpoint value(s):** LOAEL (maternal toxicity ) = 750 mg/kg - bw/day

based on decreased maternal bod y weight gain and ulcerations and/or scarring of the stomach mucosa; NOAEL = 500 mg/kg - bw/day . LOAEL (developmental toxicity) > 750 mg/kg - bw/day based on no effects at highest dose tested; LOAEL = 750 mg/kg - bw/day . **Results remarks:** None specified**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

87 CONCLUSIONS The LOAEL for maternal toxicity was 750 mg/kg - bw/day

based on decreased body weight gain and ulcerations and/or scarring of the stomach mucosa and the NOAEL was 500 mg/kg - bw/day . The LOAEL for developmental toxicity was > 750 mg/kg - bw/day based on no effects at the highest dose tested; the NOAEL was 750 mg/kg - bw/day . STUDY RELIABILITY [2] Reli able with restrictions. There was no indication from the OPP reviews that the study provided and analyzed certain developmental toxicity data (e.g., fetal weight, sex ratio) on a per litter basis. REFERENCES Chen, J.; Tao, J. (2008) Hexahy dro - 1,3,5 - tr is(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Ridgway , P. (1989) Triazine Rat Terat ology Study: Reference No. LEF/8/89. Unpublished study prepared b y Toxicol L aboratories Ltd. EPA/OPP MRID 41161801**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

88 **E. SUPPLEMENTAL DATA (NON -**  **SIDS ENDPOINTS) E**  **- 1**  **. Genetic Toxicity**  **– Other**  **Study 1: Unsched** 

**uled DNA synthesis study in primary rat hepatocytes (**  **Curren, 1988) Title:**  Unscheduled DNA Sy nthesis in Rat Primary

Hepatocy

tes: Test Article: Hexahydro 1,3,5 - tris(2 - hydrox yeth yl ) - s - triazine TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4, test substance purit y and composition not provided **Remarks:** Viscous ye llow liquid, referred to in the review as hexah ydro - 1,3,5 - tris(hydrox ye thyl) - s - triazine, Grotan, and Bioban GK. METHOD **Method/guideline followed:** OPPTS 870.5550 **Test type:** Primary

rat hepatocy

te unscheduled DNA sy sthesis assay **GL P compliant?:**  Not indicated **Year study performed:** 1988 (date report was issued) **Test species:** Primary

hepatocy tes from male F344 rats **Metabolic activation:** Without **Concentrations tested** : Based on the results of a preliminary cy totoxicity study , nine concentrations ranging from 0.0001 to 1.0 μL/mL were tested. **Statistical methods used** : Not indicated. **Test method/conditions remarks:** For the assay to be considered valid, the following criteria must be satisfied: (1) the proportion of cells in repair in the solvent control group must be <15% and the net nuclear grain count of the solvent control group must be < 1.0; and (2) the positive control compound must induce a significant increase in the net nuclear grain count ( ≥ 5 grains/nucleus over the negative control. The assay was considered positive if the test material induced a concentration - related increase in mean net nuclear grains and one or more of the doses had an increase in the mean net nucle ar grain count that was ≥ 5 grains/nucleus over the negative control. In the absence of a**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

89 concentration - related effect, a compound that showed nuclear grain counts that were ≥ 5 grains/nucleus over two successive concentrations was also considered positiv e. RESUL TS In the preliminary

cy totoxicity assay , ten concentrations (0.0003 to 10 μL/mL ) of the test material were examined. The study author stated that the pH of the stock solution was adjusted to 7.4 with 1 N HC1 prior to dilution. However, there was a basic shift in the pH of cultures treated with the two highest assay ed levels (3.0 and 10 μL/mL) at initiation and termination of treatment. The stud y author further stated that the lactate deh ydrogenase (LDH) value could not be obtained for the hi ghest test concentration, presumably because of test material interference with the LDH determination. Cy totoxicity , as indicated by increased leakage of LDH into the culture medium, did not proceed in a conventional concentration - related manner. At the h ighest assay ed concentration for which the LDH level could be determined (3.0 μL/mL), the percent relative cy

totoxicity was 1%; however, as the concentration was reduced, cytotoxicity increased to 52% at 1.0 μL/mL and 66% at 0.3 μL/mL. Although the percent relative cy totoxicity at 0.1 μL/mLwas 8%, the microscopic evaluation of the hepatocy te cultures revealed a cy

totoxic effect. Below this level, the test material was not cy totoxic. Two UDS and parallel cytotoxicity assay s were conducted with nine conce ntrations of the test material (0.0001 to 1.0 μL/mL). Because of unspecified technical difficulties, the first assay was terminated. In the repeat assay , the two highest test concentrations (0.3 and 1.0 μL/mL) induced marked cy totoxicity (85%) and were, therefore, not scored for UDS activit y. The five levels selected for the evaluation of UDS induction were 0.1, 0.03, 0.01, 0.003, and 0.001 μL/mL . The highest test material concentration scored for UDS (0.10 μL/mL ) induced a marked increase in the net nu clear grain count compared to the solvent control. Similarly , the percentage of cells with ≥5 net nuclear grains was markedl y higher at this level compared to the solvent control value. However, the evidence for a genotoxic effect was confined to the 0.1 0 μL/mLlevel. The mean net nuclear grain counts and percentage of cells with ≥5 net nuclear grains for hepatocy tes exposed to 0.001, 0.003, 0.01, or 0.03 μL/mL of the test material, although higher than the solvent control, did not indicate a positive or a concentration - related response. The study author concluded that hexah ydro - l,3,5 - tris(2 - hydrox yethyl) - s - triazine induced an equivocal response in this test sy stem. **Genotoxicity result:** Equivocal **Cytotoxic concentration (if applicable):** 0.1 μL/mL **Re sults remarks:**  Responses to the positive control were acceptable. CONCLUSIONS The test material is classified as presumptivel y genotoxic for induction of unscheduled DNA sy nthesis in this test sy stem.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

90 STUDY RELIABILITY [2] Reliable with restrictions. In the absence of a concentration - related effect, confirmation of a “significant” increase at a single concentration is necessary to conclude a positive response in the UDS assay . However, the positive response was not co ncentration - related, was limited to the 0.l0 - μL/mLlevel, and the results were not confirmed. In addition, the study author failed to provide information on test material purit y, the batch number of the assayed sample, and anal yt ical data to verify

actual concentrations used in the assay . REFERENCES Chen, J.; Tao, J. (2008) Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Curren, R. (1988) Unscheduled DNA S ynthesis in Rat Primary Hepatocytes: Hexahydro - 1,3,5 - tris(2 - hydroxy eth yl ) - s - triazine: Study No. T8102.380. Unpublished study prepared by Microbiological Associates, Inc. EPA/OPP MRID 41262301.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

91 **Study 2: Unscheduled DNA synthesis study in primary rat hepatocytes (**  **San and Raabe, 1993) Title:**  Unscheduled DNA sy nthesis in rat primary

hepatocy tes TEST SUBSTANCE **Identity:** 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4, test substance purit y and composition not provided **Remarks:** Straw - colored liquid, referred to in the review as hexahydro - 1,3,5 - tris(hydroxy eth yl ) - s - triazine and Grotan. METHOD **Method/guideline followed:** OPPTS 870.5550 **Test type:** Primary

rat hepatocy

te unscheduled DNA sy sthesis assay **GL P compliant?:**  Yes **Year study performed:** 1993 (date report was issued) **Test species:** Primary

hepatocy tes from male F344 rats **Metabolic activation:**  Without **Concentrations tested** : Based on the results of a preliminary cy totoxicity study , nine concentrations ranging from 0.01 to 0.3 μg/mLwere tested. **Statistical methods used** : Not indicated. **Test method/conditions remarks:** Following p reliminary cytotoxicity

testing (by

lactate dehydrogenase, LDH, release determinations), triplicate coverslip cultures (per does) of rat hepatocy tes were exposed to hexahydro - l, 3, 5 - tris(2 - hydrox yeth yl ) - s - triazine for 18 - 20 hours to a series of nine conce ntrations of test article (ranging from 0.01 to 0.30 μg/mL ), together with a constant concentration of tritiated thy midine (HTdr, 10 μCi/mL ). Other cultures were treated with 7, 12 - dimethylebenz(a)anthracene (DMBA, 3 and 10 μg/mL), to serve as positive co ntrols. Fifty cells per slide were read; net nuclear counts (NNC) were calculated (crude nuclear count less mean of three cy

toplasmic counts) and were averaged for each treatment. In addition, the percentage of cells in repair was recorded for each tes t concentration. Means, standard deviations and percent survival were computed. The grain count results represent unscheduled DNA s ynthesis A test result was considered positive if the NNC in test substance - treated cells was at least five counts over th e solvent control. Criteria for assay validity were not presented. The stud y author**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

92 stated that conventional criteria for assessing assay validity and genotoxic responses were followed. RESUL TS The preliminary

cy totoxicity test demonstrated that the te st article produced concentration related relative toxicities (based on LDH release) from >80% at 1.0 μg/mLdown to 1% at 0.1 μg/mL . Hence, the HDT selected for the UDS assay itself was 0.3 μg/mL(at which related toxicity was 79%). For the UDS assay , ex amination of the fixed and stained cells which had been treated at concentrations of 0.15, 0.2, 0.25, or 0.3 μg/mL revealed that these could not be evaluated for UDS “because of excessive cy totoxicity”, resulting in relative cell survivals less than 35%. Although none of the remaining (lower) test article concentrations (0.01 to 0.1 μg/mL) caused an increase in mean NNC, according to the authors’ criteria established for declaring a positive result, there was a concentration - related increase trend in both mean NNC, as well as percentage of cells showing DNA repair. **Genotoxicity result:** Equivocal **Cytotoxic concentration (if applicable):** 0.1 μg/mL **Results remarks:** Positive controls produced an acceptable response. CONCLUSIONS The assay is considered equivocally positive based on slight, but concentration - related, increases in both mean NNC and percentage of cells in repair at the highest three usable (i.e., non - toxic) concentrations of 0.05, 0.07, and 0.10 μg/mL. However, resul ts at these concentrations did not reach the criterion for significance proposed b y the investigators, namel y, increase of ≥ 5 counts over control. STUDY RELIABILITY [2] Reliable with restrictions. The study author did not provide information on test material purity , the batch number of the assay ed sample, and anal yt ical data to verify actual concentrations used in the assay . REFERENCES Chen, J.; Tao, J. (2008) Hexahydro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. San, R.; Raabe, H. (1993) Unscheduled DNA S yn thesis in Rat Primary Hepatocy

tes: Final Report: L ab Project Number: TC836.380: SPGT380. Unpublished study prepared b y Microbiological Associates, Inc. EPA/OPP MRID 43020001.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

93 **E -**  **2 .**  **Skin Irritation Study 1:**  **Dermal irritation study in rabbits (**  **Guest and Collier, 1985) Title:**  Primary

Dermal Irritation Test: Determination of the Degree of Primary Cutaneous Irritation Caused b y Glokill 77 in the Rabbit. TEST SUBSTANCE **Identity:** 79.96% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 20.04% unidentified (Chen and Tao 2008) or composition not indicated (Guest and Collier 1985) **Remarks:** Clear colorless viscous liquid, referred to in the reviews as Glokill 77, HHT, hexahydro - 1,3,5 - tris(2 - hydroxy ethyl) - s - triazine, and dihydro - 5 - (2 - hydroxyethyl) - 1,3,5 - triazine l,3(2H,4H) - dipropanol METHOD **Method/guideline followed:** OPPTS 870.2500 **Test type:** Primary

Dermal Irritation -- Rabbit **GL P compliant?:**  Not stated **Year study performed:** 1985 (issue date) **Test species:**  New Zealand White rabbits, 12 - 16 weeks of age **Number of animals/sex/dose:** 12, sex not indicated **Route of administration:** Dermal **Vehicle:** None **Doses/concentrations tested:** 0.5 mL , undiluted **Statistical methods used:** None **Test method/conditions remarks:**  Glokill 77 (undiluted; 0.5 ml) was applied to clipped back skin of six young adult (12 - 16 weeks of age; no sex data given) New Zealand White rabbits. The test sites were occluded with a patch of surgical gauze and elasticized cor sets (Tubigrip), and exposure continued for 4 hours. Test sites were swabbed with cotton wool soaked in distilled water, and skin irritation was scored using a Draize method at 1 **,** 24, 48, and 72 hours after treatment. The test substance produced minimal ery thema in three rabbits and minimal edema in one rabbit at one hour after dosing. However, these effects had disappeared at 24 hours post -**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

94 dosing, and no other effects were observed throughout the 72 - hour study period. The calculated Primary Cutaneous Ir

ritation Index was reported to be 0.0. RESUL TS **Endpoint value(s):** Not irritating. Primary Irritation Index was 0.0. No ery thema or edema was seen from 24 to 72 hours. **Results remarks:** None specified CONCLUSIONS The test substance was not irritating to rabbit skin. STUDY RELIABILITY [2] Valid with restrictions. Information is needed on the composition and stability of the test substance. REFERENCE Chen, J.; Tao, J. (2008). Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Guest, R.L .; Collier, T.A. (1985). Primary Dermal Irritation Test: Determination of the Degree of Primary

Cutaneous Irritation Caused by Glokill 77 in the Rabbit. Study Nos. 317/8505. Safepharm Laboratories Limited. EPA/OPP MRID 155986, as cited in Locke (1995). Locke, R.K. (1995). H exahydro - 1,3,5 - tris(2 - hydroxyeth yl ) - s - triazine . Review of Toxicity Data. DP Barcode: D212071. Submission #: S481738. Document No. 011557.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

95 **Study 2: Dermal irritation study in rabbits (**  **Thomas, 1974) Title:**  Glokill 77: Draize Skin Tests. TEST SUBSTANCE **Identity:** 79.96% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 20.04% unidentified (Chen and Tao 2008) or composition not indicated (Thomas 1974) **Remarks:** Clear viscous liquid, referred to in the reviews as Glokill 77 (Batch No. K249), HHT, hexahydro - 1,3, 5 - tris(2 - hydroxy ethyl) - s - triazine, and dihydro - 5 - (2 - hydroxyethyl) - 1,3,5 - triazine l,3(2H,4H) - dipropanol. METHOD **Method/guideline followed:** OPPTS 870.2500 **Test type:** Primary

Dermal Irritation -- Rabbit **GL P compliant?:**  Not stated **Year study performed:** 1974 (issue date) **Test species:** New Zealand White rabbits [Note: According Table 3 of Chen and Tao (2008), the Guinea pig was the test species] **Number of animals/sex/dose:** 4, sex not indicated **Route of administration:** Dermal **Vehicle:** Distilled water **Doses/concentrations tested:** 0.5 mg/site or 5 mg/site from application of 0.5 mLof a 0.1 or 1% solution, respectively . **Statistical methods used:** None **Test method/conditions remarks:** Test material was applied to clipped and depilated intact abraded back skin of four New Zealand White rabbits for each concentration of dosing solution. The treatment sites were occluded with sterile gauze and exposure continued for 24 hours. After 24 hours of exposure , the patches were removed and the skin reactions on both the abraded and unabraded skin scored by a Draize scoring s ys tem. Additional scores were recorded at 72 hours post - treatment.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

96 RESUL TS The 0.1% concentration was non - irritating to intact skin and, on abraded skin, produced onl y mild ery thema in one animal at 24 hours post - treatment, which was absent at 72 hours after treatment. A concentration of 1.0% on intact skin produced onl y mild erythema in one animal at 24 hours after treatment, which was ab sent at 72 hours post - treatment; on abraded skin, mild erythema was evident at 24 hours post - treatment for all four animals, but this condition persisted only for one animal at 72 hours post - treatment. Primary Irritation Indices were 0.06 for the 0.1% solution and 0.44 for the 1.0% solution. **Endpoint value(s):** Mildly irritating. Primary

Irritation Indices were 0.06 for the 0.1% solution and 0.44 for the 1.0% solution. **Results remarks:** None specified CONCLUSIONS The test substance was mildly irritating to rabbit skin. STUDY RELIABILITY [2] Valid with restrictions. Information is needed on the composition and stability of the test substance. REFERENCES Chen, J.; Tao, J. (2008). Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicolog y Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Locke, R.K. (1995). H exahydro - 1,3,5 - tris(2 - hydroxyeth yl ) - s - triazine . Review of Toxicity Data. DP Barcode: D212071. Submission #: S481738. Document No. 011557. Thomas, M.B. (1974). Glokill 77: Draize Skin Tests. Study Nos. CL74:74:1024. Consultox Laboratories Ltd. EPA/OPP MRID 155987, as cited in L ocke (1995).**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

97 **Study 3 :**  **Dermal irritation study in rabbits ( Siglin et al., 1989b)**  **Title:** Acute Skin Irritation/Sensitization Studies with Busan 1060 TEST SUBSTANCE **Identity:** 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% unidentified **Remarks:** Referred to in the study report as Busan 1060, HHT, hexahy dro - 1,3,5 - tris(2 - hydrox ye thyl) - s - triazine, and 1,3,6 - triazine, 1,3,5(2H,4H, 6H) - triethanol. METHOD **Method/guideline followed:** Not indicated. **Test type:** Primary

Dermal Irritation -- Rabbit **GL P compliant?:**  Not stated **Year study performed:** 1989 **Test species:** New Zealand White rabbit **Number of animals/sex/dose:** 6, sex not indicated **Route of administration:** Dermal **Vehicle:** None **Doses/concentrations tested:** 0.5 mg/site or 5 mg/site from application of 0.5 mLof a 0.1 or 1% solution, respectively . **Statistical methods used:** None **Test method/conditions remarks:** Animals received a single dermal application of Busan 1060 (neat) as a 4 - hour semi - occluded exposure. Dermal reactions wer e evaluated by the Draize scoring s ys tem at 1, 24, 48, and 72 hours after treatment. RESUL TS Very slight ery

thema and edema were observed at test sites of 3/6 animals at 1 hour after treatment. Irritation resolved completel y b y 24 hours post - treatment. **Endpoint value(s):** Mildly irritating. The Primary

Irritation Index was 0.2/8**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

98 **Results remarks:** None specified CONCLUSIONS The test substance was mildly irritating to rabbit skin. STUDY RELIABILITY [2] Valid with restrictions. Information is needed on the composition and stability of the test substance, the stud y method, and the sex of animals on test. REFERENCE Siglin, J.C.; Rush, R.E.; Jenkins, P.K. et al. (1989b). Acute skin irritation/sensi tization studies with Busan 1060. In: R.A. Parent (ed.), Acute Toxicity Data, J. Amer Coll. Toxicol., Part B, Vol. 1, No. 3, 1992, pp. 203 - 204.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

99 **E -**  **3 .**  **Eye Irritation Study 1:**  **Eye irritation study in rabbits ( Collier and Axelrad, 1984)**  **Title:** OECD Ey e Irritation Test: Determination of the Degree of Ocular Irritation Caused by Glokill 77 in the Rabbit TEST SUBSTANCE **Identity:** 79.96% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 20.04% unidentified (Chen and Tao 2008) or composition not indicated (Collier and Axelrad, 1984). **Remarks:** Clear Liquid, referred to in reviews as Glokill 77, HHT, hexahydro - 1,3,5 - tris(2 - hydrox ye thyl) - s - triazine, and dihydro - 5 - (2 - hydroxy ethyl) - 1,3,5 - triazine - l,3(2H,4H) - dipropanol. METHOD **Method/guideline followed:** OPPTS 870.2400 **Test type:** Primary

Ey e Irritation -- Rabbit **GL P compliant?:**  Not stated **Year study performed:** 1984 (issue date) **Test species:** New Zealand White rabbit, sex not indicated, 12 - 16 weeks old **Number of animals/sex/dose:**  6 **Route of administration:** Ocular **Vehicle:** None **Doses/concentrations tested:** 0.1 mL **Statistical methods used:** None **Test method/conditions remarks:** None specified RESUL TS Glokill 77 (undiluted, 0.1 mL ) was instilled into the right ey es and irritation was scored b y the Draize method at 1, 24, 48, and 72 hours after treatment.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

100 **Endpoint value(s):** The test substance was severely irritating (corrosive) to the ey es of all 6 rabbits. Effects at 72 hours included iritis, beefy - red color ation of the conjunctivae accompanied by considerable swelling, copious discharge from the ey e, and areas of complete corneal opacit y. **Results remarks:** None specified CONCLUSIONS The test substance was severel y irritating (corrosive) to rabbit eyes. STUDY RELIABILITY [2] Valid with restrictions. Information is needed on test substance composition and stability . REFERENCES Chen, J.; Tao, J. (2008). Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Collier, T.A.; Axelrad, J.C. (1984) OECD Ey e Irritation Test: Determination of the Degree of Ocular Irritation Caus ed by Glokill 77 in the Rabbit. EPA/OPP MRIDs 41675206; 155984. As cited in L ocke (1995). Locke, R.K. (1995). H exahydro - 1,3,5 - tris(2 - hydroxyeth yl ) - s - triazine . Review of Toxicity Data. DP Barcode: D212071. Submission #: S481738. Document No. 011557.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

101 **Study 2: Eye irritation study in rabbits (**  **Douds, 1995) Title:**  Not provided TEST SUBSTANCE **Identity:** 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% unidentified inert ingredients. **Remarks:** Clear, light yellow viscous liquid, referred to in the review as Grotan, hexahydro 1,3,5 - tris(2 - hydrox yeth yl ) - s - triazine , and BD - 024 - 3A. METHOD **Method/guideline followed:** OPP 81 - 4; OPPTS 870.2400 **Test type:** Primary

Ey e Irritation -- Rabbit **GL P compliant?:**  Not stated **Year study performed:** 1995 (report date) **Test species:** New Zealand White rabbit, 2.832 - 3.363 g **Number of animals/sex/dose:** 3 **Route of administration:** Ocular **Vehicle:** None **Doses/concentrations tested:** 0.5 mL **Statistical methods used:** Not indicated **Test method/conditions remarks:** None specified RESUL TS The test substance (undiluted, 0.5 mL ) was instilled into the conjunctival sac of “six pre examined” ey es and irritation was scored b y the Draize method at 1, 24, 48, and 72 hours and on day s 7, 10, 14, and 21, 2 after treatment. **Endpoint value(s):** The test substance was severely irritating (corrosive) to the ey es of all 6 rabbits, with corneal opacity , unspecified effects on the iris, and redness, chemosis, and discharge of conjunctivae in 6/6 animals at 72 hours. The reviewer also noted sloughing of the corneal epithelium, vascularization of the cornea, raised areas on the cornea, blanching of the**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

102 conjunctivae, and hair loss around the ey e (incidences of these effects were not provided in the r eview). **Results remarks:** None specified CONCLUSIONS The test substance was severel y irritating (corrosive) to rabbit eyes. STUDY RELIABILITY [2] Valid with restrictions. Information is needed on test substance composition and stability . The stated volume of test material instilled into the ey e (0.5 mL) exceeded the volume recommended b y OPPTS Guideline 870.2400 (0.1 mL ). The OPP reviewer questioned the Quality Assurance statement provided by the testing laboratory ; the reviewer noted that the onl y inspected in - life portion of the study was indicated to be dose preparation, whereas it is “difficult to understand how the dose needed an y preparation” because the stud y “used the liquid material as received.” REFERENCE Douds, D.A. (1995). Study title not provided. Springborn Report No. 3157 - 167, January 6, 1995. EPA/OPP MRI D 435173 - 01. Data Review for Acute Ey e Irritation Testing (§81 - 4).**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

103 **E -**  **4 .**  **Skin Sensitization Study 1:**  **Dermal sensitization study in guinea pigs ( Thomas, 1974)**  **Title:** Glokill 77: Guinea Pig Sensitization Test TEST SUBSTANCE **Identity:** 79.96% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 20.04% unidentified (Chen and Tao 2008) or composition not indicated (Thomas 1974) **Remarks:** Clear viscous liquid, referred to in the reviews as Glokill 77 (Batch No. K249), HHT, hexahydro - 1,3,5 - tris(2 - hydroxy ethyl) - s - triazine, and dihydro - 5 - (2 - hydroxyethyl) - 1,3,5 - triazine l,3(2H,4H) - dipropanol. METHOD **Method/guideline followed:** OPPTS 870.2600 **Test type:** Dermal Sensitization

– Guinea Pigs **GL P (Y/N):**  Not stated **Species/strain:** Hartley Guinea pigs, 300 - 400 g **Sex (male/female/both):** Male **Number of animals/sex/dose:** 6 **Dose/concentration:** Induction: 0.1 mLof 0.1% Glokill 77 in distilled water was inject ed intradermall y every other day, using a different injection site for each treatment, for a total of 9 injections. Injection sites were scored for ery thema and edema by

the Draize method at 24 hours after each injection. Challenge Period: Two weeks af ter final induction injection. Challenge dose: 0.1 mLof 0.1% Glokill 77 in distilled water, injected intradermally Control: The Draize scores resulting from the challenge dose were compared with those obtained during the induction phase to determine if s ensitization had be induced. **Test condition:** Not stated RESUL TS All animals survived to study termination. Slight ery

thema and/or edema were noted in all animals after each of the induction injections. No greater edema and/or erythema was noted in any animal following the challenge injection than had been observed in the same animal following the induction injections.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

104 CONCLUSIONS The test substance was mildly irritating but not a sensitizer to guinea pig skin. STUDY RELIABILITY [2] Reliable with restrictions. In formation is needed on the composition and stability of the test substance. REFERENCES Chen, J.; Tao, J. (2008). Hexahy dro - 1,3,5 - tris(2 - Hydroxy eth yl ) - s - Triazine: Toxicology Disciplinary

Chapter for the I ssuance of the Reregistration Eligibility Decision (RED) Document. Case No.: 3074. PC Code: 083301. DP Barcode: 346237. February 4, 2008. Locke, R.K. (1995). H exahydro - 1,3,5 - tris(2 - hydroxyeth yl ) - s - triazine . Review of Toxicity Data. DP Barcode: D212071. Submission #: S481738. Document No. 011557. Thomas, M.B. (1974). Glokill 77: Guinea Pig Sensitization Test. Study Nos. CL 74:74:1024. Consultox Laboratories Ltd. EPA/OPP MRI D 155987, as cited in Locke (1995).**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

105 **Study 2: Dermal sensitization study**  **in guinea pigs (Siglin et al., 1989b) Title:**  Acute Skin Irritation/Sensitization Studies with Busan 1060 TEST SUBSTANCE **Identity:** 78.5% 1,3,5 - Triazine - 1,3,5(2H,4H,6H) - triethanol, CAS No. 4719 - 04 - 4; 21.5% unidentified **Remarks:** Referred to in the study report as Busan 1060, HHT, hexahy dro - 1,3,5 - tris(2 - hydrox ye thyl) - s - triazine, and 1,3,6 - triazine, 1,3,5(2H,4H, 6H) - triethanol. METHOD **Method/guideline followed:** Buehler, E.V.; Griffith, F. (1975). Experimental skin sensitization in the guinea pig and man. In: Animal Models in Dermatology , Maibach, H. (ed.), pp. 56 - 66. **Test type:** Delay ed contact sensitization (modified Buehler design) **GL P (Y/N):**  Not stated **Species/strain:** Hartley

guinea pigs, 301 - 428 g **Sex (male/female/both):** Both **Number of animals/sex/dose:** 5 **Dose/concentration:** Induction: 0.4 mLtopical occlusive application of 10% w/v Busan 1060 in distilled water once/week for 3 consecutive weeks. Challenge Period: Following a resting period of 2 weeks, the 10 induced animals received the first challenge application; 7 day s later, the animals were rechallenged. Challenge dose: 5% Busan 1060 in distilled water (volume not indicated) Control: Naïve irritation control groups received 5% Busan 1060 at week 2 (10 animals) or week 3 (a new group of 10 animals). **Test condition:** Not stated RESUL TS Similar dermal irritation scores were seen in control and induced animals at 24 hours after the initial challenge. By

48 and 72 hours after the initial challenge, the response in induced animals “generally

appeared more severe.” Following re - challenge, elevated irritation scores were seen in induced animals at 24 hours (7/10 animals) and 48 hours (3/10 anim als) whereas minimal responses were seen in the naïve re - challenge control animals at both times. [NOTE: Post challenge scores are not presented here because they were not legible in Siglin et al. (1989).]**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

106 CONCLUSIONS The test substance was considered to be a mild delay ed contact sensitizer to guinea pig skin. STUDY RELIABILITY [2] Reliable with restrictions. In formation is needed on the composition and stability of the test substance. The irritation scoring s ys tem was not characterized and irritation scores were not legible in the cited study . REFERENCE Siglin, J.C.; Rush, R.E.; Jenkins, P.K. et al. (1989b). Acute skin irritation/sensitization studies with Busan 1060. In: R.A. Parent (ed.), Acute Toxicity Data, J. Amer Coll. Toxicol., Part B, Vol. 1, No. 3, 1992, pp. 204.**Final HPV**  **2 Data Adequacy Review**  **(September 2012) CAS**  **RN 4719 -**  **04 -**  **4**

107 **F. DATA ADEQUACY SUMMARY TABLE** 

**Endpoint Study Information**  **Available Acceptable**  **Data Gap Aquatic Effects**  Acute Toxicity

to Fish Yes Yes No Acute Toxicity

to Aquatic Invertebrates Yes Yes No Toxicity to Aquatic Plants Yes Yes No **Health Effects** Acute Toxicity Yes Yes No Repeated - Dose Toxicity Yes Yes No Gene Mutation Yes Yes No Chromosomal Aberrations Yes Yes No Reproductive Toxicity No -- No 1 Developmental Toxicity Yes Yes No 1 The reproductive endpoint has been acceptably addressed on the basis that no effects on developmental endpoints were seen in an adequate oral developmental toxicity study in rats and no treatment - related effects on the histology of reproductive organs of e ither sex were seen in adequate 90 - day oral and dermal toxicity studies in rats.