SRC TR-12-073

      HPV2 TEST RULE DATA ADEQUACY REVIEW: SEPTEMBER 2012 UPDATE (FINAL)

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

                                 Prepared by:

     Jennifer Rayner, Teresa Manyin, Justine Von Runnen, Ashlee Aldridge,
                            and William L. Richards

                 Chemical, Biological and Environmental Center
                                   SRC, Inc.
                             7502 Round Pond Road
                           North Syracuse, NY 13212

                           Contract No. EP-W-09-027
                             Task FG<IP_ADDRESS>

                                 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

                               TABLE OF CONTENTS

A.  SOURCE MATERIAL USED FOR ASSESSMENT	1
B.  EVALUATING DATA ADEQUACY	1
C.  SUMMARY OF DATA ADEQUACY FOR TESTING 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.  SUPPLEMENTAL DATA (NON-SIDS ENDPOINTS)	88
E-1.  Genetic Toxicity  -  Other	88
E-2.  Skin Irritation	93
E-3.  Eye Irritation	99
E-4.  Skin Sensitization	103
F.  DATA ADEQUACY SUMMARY TABLE	107

A.  SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates an algal inhibition test study report, which was submitted to EPA on July 30, 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 herein as HPV2).  The C6 requirement consists of Toxicity to Plants (Algae) testing, following 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 previously 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 study 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 requirements.  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 page of this document (Section F).

C.  SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

The submitted algae study is summarized and evaluated in detail on pp. 35-41 of this report.  The following deficiencies in the algae study were noted:

(1) The study authors measured the concentrations of the primary degradation products (monoethanolamine and formaldehyde) 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 analytical testing scheme differs from the proposed methodology in the study plan submitted by SOCMA (the MEA Triazine Consortium) on January 6, 2012; this study plan indicated that verification of the test concentrations would be achieved by measurements of the parent 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 compound, the measured concentrations of the degradation products appear to suggest that complete degradation of CASRN 4719-04-4 was achieved quickly.  The measured concentrations of monoethanolamine and formaldehyde at the initiation of exposure (time = 0 hours) were similar 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 formaldehyde.  Tables 1 and 2 (below) show comparisons of the measured and nominal concentrations for monoethanolamine and formaldehyde, respectively.

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 monoethanolamine 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 formaldehyde.
[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.

Table 2.  Concentrations of formaldehyde in test medium.

Sample
Nominal Concentration of CASRN 4719-04-4 (mg a.i./L)[1]
Nominal Concentration of Formaldehyde
(mg/L)[2]
Measured Concentration of Formaldehyde
(mg/L)
Measured Concentration of Formaldehyde 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 formaldehyde 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 formaldehyde.
[3] Concentration of 4719-04-4 was calculated from the measured concentration of formaldehyde and assumes that each mole of CASRN 4719-04-4 produced 3 moles of formaldehyde.

(2) The study authors calculated the EC50 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 EC50 values is preferred.

At initiation of exposure (time = 0 hours), the measured concentrations of monoethanolamine and formaldehyde were 62-136% and 102-117% of nominal (expected) concentrations, respectively.  At the end of exposure (time = 96 hours), the measured concentrations of monoethanolamine and formaldehyde were 41-105% and 27-78% of nominal (expected) concentrations, respectively.  Thus, the measured concentrations of monoethanolamine and formaldehyde both demonstrated variations outside the range of +- 20% of nominal values during the exposure period, with concentrations of formaldehyde showing a higher degree of variability than those of monoethanolamine.  The variation in concentrations could have been the result of uptake by algae cells and/or reactions between monoethanolamine and formaldehyde to form other chemical compounds.

Nonetheless, the measured concentrations of monoethanolamine displayed a relatively low degree of variation (measured concentrations were 91-136% of nominal concentrations) at the nominal test concentrations of 3.2 and 10 mg a.i./L.  Since the 96-hour EC50 values presented for growth rate, yield, and biomass (4.2, 3.2, and 3.3 mg/L, respectively) 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 EC50 values (i.e., by using calculated concentrations of CASRN 4719-04-4 that correspond to measured concentrations of degradation products as shown in Tables 1 and 2, above).

(3) The complete composition of the test substance is not clearly stated in the submitted study report.  The "Certificate of Analysis" 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 only impurity were present in the test compound.  The "Certificate of Analysis" indicates that excess monoethanolamine (0.02 moles) 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 impurities other than water appear to be <1%, the test substance is acceptable.

Conclusions:  Despite the study deficiencies noted above, the algae toxicity data supplied on July 30, 2012 by the MEA Triazine Consortium were considered adequate to satisfy the Toxicity to Plants (Algae) C6 test requirement for CASRN 4719-04-4 in the HPV2 test rule.

There are no data gaps remaining for any SIDS endpoints.

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

GLP 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 immediately 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.

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) cycled at an average of 5.0 cycles per hour.  Organism loading was calculated to be 0.14 g/L/day, with a maximum loading at any point in time of 1.6 g/L.

Dilution water was municipal water, which was vigorously 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 moderately hard freshwater with a mean hardness of 52 mg CaCO3/L, mean alkalinity of 16 mg CaCO3/L, and a mean specific conductivity of 428 umhos/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 randomly distributed in the treatment and control chambers of the flow-through test system.  Survival of the trout was monitored daily and any dead fish were removed.  Any abnormalities in the behavior or physical appearance of rainbow trout were also noted.  Test solutions were gently 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 conductivity, 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.

RESULTS

Endpoint value(s):  The 96-hour LC50 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 LC50 was 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 LC50 for rainbow trout was > 119 mg a.i./L, based on a lack of mortality and sublethal effects at the highest dose tested.

STUDY RELIABILITY

[1] Reliable without restrictions.  This study was classified as "core" by 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, Inc., Memphis, TN.  U.S. EPA MRID No. 431431-01.

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

GLP compliant?:  Yes

Year study was performed:  1994

Test species: Juvenile sheepshead 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) cycled 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 any point in time of 0.37 g/L.

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 randomly distributed in the treatment and control chambers of the flow-through test system.  Survival of the fish was monitored daily and any dead were removed.  Any abnormalities in the behavior or physical 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 hourly 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.

RESULTS

Endpoint value(s):  The 96-hour LC50 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 LC50 was greater than 118 mg a.i./L.  The NOEC was 118 mg a.i./L, based on lack of mortality and sublethal effects at this test concentration and all lower test concentrations.

CONCLUSIONS

The 96-hour LC50 for sheepshead minnows was greater than 118 mg a.i./L, based on a lack of mortality and sublethal effects at the highest concentration tested.

STUDY RELIABILITY

[1] Reliable without restrictions.  This study was classified as "core" by U.S. EPA's Office of Pesticide Programs.

REFERENCE

Ward, G.S. 1994. "Acute Toxicity to the Sheepshead Minnow, Cyprinodon variegatus, Under Flow-through Test Conditions".  Performing Laboratory: Toxikon Environmental Sciences, Jupiter, FL. Submitted by the Triazine Joint Venture, c/o Buckman Laboratories International,  Inc., Memphis, TN. U.S. EPA MRID No. 431431-02.

Study 3:  Acute toxicity test in 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)

GLP 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 LC50 was estimated using a binomial test.

RESULTS

Endpoint value(s):  The 96-hour LC50 value 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 LC50 value for bluegill was 77 mg/L and the confidence interval was 60 to 100 mg/L.

CONCLUSIONS

This study indicates that the test substance is slightly toxic to bluegill with an LC50 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 used, 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% saturation.  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 results may be used as part of a weight-of-evidence approach to describe 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, CT.  U.S. EPA MRID No. 00105191.

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

GLP 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 days 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 days prior to testing.  The holding and dilution water consisted 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 solubilize the test material.  Ten fish per test concentration were placed in each test vessel under static conditions.  The test vessels were lined with polyethylene bags.  There was one replicate per test concentration.  The fish were not fed during 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 oxygen and pH values were measured for the groups which experienced mortality and daily for the untreated control group.  The Litchfield and Wilcoxon tests were used to determine the LC50.

RESULTS

Endpoint value(s):  The 96-hr LC50 for rainbow trout was 74 mg/L.  The NOEC was 18 mg/L.

Results remarks: The untreated control had zero mortality.  The mortality 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 oxygen concentration ranged from 7.7 to 9.3 ppm (62 to 74%).  The control dissolved oxygen ranged from 6.6 to 8.0 ppm (53 to 64%).

The study authors reported a 96-hr LC50 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 mortality and sublethal effects.  An EPA OPP reviewer used EPA's TOXANAL program to verify the study author's values.  The LC50 determined by TOXANAL was 74 mg/L, with a 95% confidence interval of 64.2-83.4 mg/L.  The NOEC was not available through the TOXANAL program.

CONCLUSIONS

The 96-hr LC50 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 only 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: LC50 and 95% confidence limits for 24, 48, and 72 hours, range-finding study data, purity and physicochemical characteristics of test material, temperature of water during test period, water hardness and salinity, methods and data from water sample analysis verifying concentration and impurities, pretest mortality and disease treatment, description of acclimation and test facilities, 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 system and 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 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 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 Laboratories, Inc.  Submitted by the DeMille Chemical Corporation. U.S. EPA MRID No. 00073588.
Study 5:  Acute toxicity study in bluegill (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, Bluegill (Lepomis macrochirus), static

GLP 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 days 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 days prior to testing.  The holding and dilution water consisted 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, the nominal test concentrations were: 0 (control), 18, 32, 56, 78 and 100 mg/L.  No solvent was utilized to solubilize the test material.  Ten fish per test concentration were placed in each test vessel under static conditions.  The test vessels were lined with polyethylene bags.  There was one replicate per test concentration.  The fish were not fed during 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 oxygen and pH values were measured for the groups which experienced mortality and daily for the untreated control group.  The Litchfield and Wilcoxon tests were used to determine the LC50.

RESULTS

Endpoint value(s):  The 96-hr LC50 for bluegill was 40 mg/L.  The NOEC was 18 mg/L.

Results remarks: The untreated control had zero mortality.  The mortality rates were 0, 20, 90, 100, 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 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 from 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 LC50 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 LC50 determined by TOXANAL 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 LC50 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 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 only measured daily for the control and if mortality occurred rather than daily 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 acceptable range (guideline specifies 6.0 to 8.0); (7) the following information was not provided: LC50 and 95% confidence limits for 24, 48, and 72 hours, range-finding study data, purity and physicochemical characteristics of test material, temperature of water during test period, water hardness and salinity, methods and data from water sample analysis verifying concentration and impurities, pretest mortality and disease treatment, description of acclimation and test facilities, 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 system and 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 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 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 Laboratories, Inc.  Submitted by the DeMille Chemical Corporation. U.S. EPA MRID No. 00073588.
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%; Impurity: ~6.4% sodium omadine, CAS No. not specified; >= 30% unidentified

Remarks:  Also referred to as Grotan and Triadine 10; Industrial Microbiostat, Lot No. OD-9211

METHOD

Method/guideline followed:  Not specified

Test type:  Acute toxicity test in for Freshwater Fish, Rainbow Trout (Oncorhynchus mykiss), static

GLP 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 days.  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 CaCO3/L, the alkalinity was 24-30 mg CaCO3/L, the specific conductance was 100 uhos/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 procedures, and had a hardness of 42 mg CaCO3/L and specific conductance of 140 uhos/cm.  The stock solution of Triadine 10 (15 mg/L) was prepared by adding distilled water to 7.5 grams of Triadine 10 in a 500-mL volumetric flask, until an appropriate volume had been reached.  The test vessels consisted of 19.6-L glass jars with 15 L of test solution.  No evidence of a range-finding study 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 each 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 analysis, 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 (by 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 by sodium omadine and Triadine 10 in this study, leaving about 30% of the compound as "unknown substance."

RESULTS

Endpoint value(s):  The 96-hr LC50 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.  Mortality occurred at nominal concentrations of 36 and 60 mg/L at rates of 40 and 80%, respectively.  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 LC50 for rainbow 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.

CONCLUSIONS

The 96-hr LC50 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 product, 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 two different sources and rainbow trout were not held in the test water dilution for a minimum of 7 days 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 replicates 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 provided: chemical analysis of dilution water, pretest mortality for the entire acclimation period, health of fish prior to test initiation, use and type of cover for test vessels, dissolved oxygen data at 72 hours, hourly temperatures in one replicate throughout the study, analysis 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" 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 results may be used as part of a weight-of-evidence approach to describe the acute toxicity to fish.

REFERENCE

Sousa, J.V., and LeBlanc, G.A. 1982a. Acute Toxicity of Triadine 10 to Rainbow Trout (Salmo gairdneri); Laboratory Report BW-82-2-1117. Performing Laboratory: EG&G, Bionomics, Aquatic Toxicology Laboratory, Wareham, MA. Submitted by the Olin Corporation, New Haven, CT.  U.S. EPA MRID Nos. 247712 and 00105190.
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

GLP compliant?:  No

Year study was performed:  1982

Test species:  Juvenile rainbow 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 days 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 CaCO3/L, alkalinity was 26-29 mg CaCO3/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 daily, 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 CaCO3/L and specific conductance of 140 uhos/cm, measured at the start of the definitive study.  The stock solution of Milidin X-2 (15 mg/L) was prepared by adding distilled water to 7.5 grams of Milidin X-2 in a 500-mL volumetric 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.

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 intervals.  A computer program (Stephan, C. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN) was used to perform the statistical analysis.  An EPA OPP reviewer used EPA's Toxanol program to verify the LC50 values using the moving average angle analysis and the probit method.

RESULTS

Endpoint value(s):  The 96-hr LC50 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 oxygen 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 and 100%, respectively.  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 LC50 of 71 mg/L, with a 95% confidence interval of 46-110 mg/L.  The 96-hr LC50 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 LC50 for rainbow trout was 71 mg/L.  The LOEC was 28 mg/L, based on sublethal effects, and the NOEC was 17 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions.  Deviations from OPPTS Guideline 850.1075 included the following: (1) purity and physicochemical characteristics of the test material were not provided; (2) test chemical concentrations were not monitored through the experiment (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 days 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 always fall within the recommended range for freshwater species of 6.0-8.0; and (6) the following information was not provided: chemical analysis 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 type 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, analysis 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" 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 results may be used as part of a weight-of-evidence approach to describe the acute toxicity to fish.

REFERENCE

Sousa, J.V. and LeBlanc, G.A. 1982b. Acute Toxicity of Milidin X-2 to Rainbow Trout (Salmo gairdneri); Laboratory Report BW-82-4-1145. Performing Laboratory: EG&G, Bionomics Aquatic Toxicology Laboratory, Wareham, MA. U.S. EPA MRID No. 248883.
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

GLP compliant?:  No

Year study was performed:  1982

Test species:  Juvenile bluegill, 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 days 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 CaCO3/L, the alkalinity was 26-29 mg CaCO3/L, the pH ranged from 6.8-7.0, the temperature was 20-21 ºC, the specific conductance was 105-120 umhos/cm, the photoperiod was16 hours light/8 hours dark, and the dissolved oxygen was100% of saturation at 20 ºC.  The fish were fed dry pellet food daily, ad libitum.  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 by adding distilled water to 7.5 grams of Milidin X-2 in a 500-mL volumetric flask.  No evidence of a range-finding study was provided.

Ten juvenile bluegill per replicate were distributed randomly into 19.6-L glass test jars containing 15 L of test solution within 30 minutes after the preparation of the test solution.  This 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.  Three 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 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.  A computer program (Stephan, C. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN) was used to perform the statistical analysis.  An EPA OPP reviewer used EPA's Toxanol program to verify the LC50 values using the moving average angle analysis and the probit method.

RESULTS

Endpoint value(s):  The 96-hr LC50 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 CaCO3/L.  The alkalinity was 31 mg CaCO3/L, specific conductance was 140 umhos/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 were 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 96-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 LC50 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 LC50 was 39 mg/L (95% C.I. = 31-50 mg/L) using the moving average method and the 96-hr LC50 was 40 mg/L (95% C.I. = 32-50 mg/L) using the probit method.

CONCLUSIONS

The 96-hr LC50 for bluegill was 39 mg/L.  The NOEC was < 13 mg/L, based on sublethal effects at the lowest dose tested.

STUDY RELIABILITY

[2] Reliable with restrictions.  Deviations from OPPTS Guideline 850.1075 included the following: (1) purity and physiochemical 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 days 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: physical condition and signs of disease in the fish, chemical analysis of dilution water, pretest mortality rates for the entire 14-day acclimation period; use and type of cover for test vessels, dissolved oxygen concentration at 72 hours, range-finding test data, explicit statement of the number of replicates for each concentration (assumed three replicates based on n = 30), hourly temperatures in one replicate throughout the study, analysis of the stability of the test chemical, mortality at 6 hours, graph of the concentration-mortality curve, and quality assurance and GLP compliance statements.  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 results may be used as part of a weight-of-evidence approach to describe the acute toxicity to fish.

REFERENCE

Sousa, J.V., and LeBlanc, G.A. 1982c. Acute Toxicity of Milidin X-2 to Bluegill (Lepomis macrochirus); Laboratory Report BW-82-4-1144. Performing Laboratory: EG&G, Bionomics Aquatic Toxicology Laboratory, Wareham, MA. U.S. EPA MRID No. 248883.

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)

GLP 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 LC50 value.

RESULTS

Endpoint value(s):  The 96-hr LC50 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, respectively.  Using the binomial probability method, the LC50 was 36 mg/L, with a 95% confidence interval of 28 to 46 mg/L.

CONCLUSIONS

The 96-hr LC50 for channel catfish was 36 mg/L, estimated by binomial probability.  The test substance was found to 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" 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 results may be used as part of a weight-of-evidence approach to describe the acute toxicity to fish.

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 MRID No. 00105189.

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 Toxicity Test

GLP 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./L and a dilution water control.  The definitive test was conducted under flow-through conditions in a modified proportional diluter system constructed of glass, silicone adhesive, and silicone tubing.  A total volume of 53.9 uL of test 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 moderately hard fresh water, with a mean hardness of 113 mg/L as CaCO3, mean alkalinity of 32 mg/L, and a mean specific conductivity of 658 umhos/cm.

A test solution of ~ 220 mL was delivered to each test chamber during every cycle: 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-mL volume of test solution; the diluter cycled at an average rate of 2.9 cycles/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 and 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 analyzed using HPLC.

The median effective concentrations (EC50) and associated 95% confidence intervals (C.I.) were calculated using a computer program (moving average angle, probit, logit, and nonlinear interpolation).

RESULTS

Endpoint value(s):  The 48 hour EC50 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 mg a.i./L test concentration.

Results remarks:  The diluter functioned properly throughout the test.  The mean measured concentrations ranged from 7.49 to 95.2 mg a.i./L and 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, respectively.  The slope of the concentration response curve could not be determined using the binomial probabi1ity method.  The study author determined the 48-hour EC50 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 EC50 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 study author, which was 19.7 mg a.i./L.

Initial alkalinity, hardness, and conductivity of the dilution water as measured in the control were 32 mg/L, 130 mg/L, and 828 umhos/cm, respectively.  At test termination, they were 32 mg/L, 96 mg/L, and 488 umhos/cm, respectively.  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 concentrations.

CONCLUSIONS

The 48 hour EC50 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./L test concentration.

STUDY RELIABILITY

[1] Reliable without restrictions.  The study was scientifically sound and generally in accordance with SEP and ASTM guidelines.  This study was classified as "core" by 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.
Study 2:  Acute toxicity study in 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)

GLP 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 formulated 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 analysis.  An EPA OPP reviewer used EPA's Toxanol program to verify the EC50 value using the moving average angle analysis and the probit method.

RESULTS

Endpoint value(s): The 48-hour EC50 value for Daphnia magna was 5.2mg/L.

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%, respectively.

The study authors reported a 48-hour EC50 of 5.2 mg/L.  Using Toxanol, the 48-hour EC50, estimated by the moving average angle method was 5.4 mg/L, with a 95% confidence interval of 3.6-10.79.4 mg/L.  Using the probit method, the 48-hour EC50 was 5.2 mg/L, with a 95% confidence interval of 3.5-9.5 mg/L.

CONCLUSIONS

The 48-hour EC50 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" 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 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 Water 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.

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)-Triethanol (CAS No. 4719-04-4): Algal Inhibition Test

This study is:	 Adequate		 Not Adequate

TEST SUBSTANCE

Identity:  1,3,5-triazine-1,3,5(2H,4H,6H)-triethanol, CASRN 4719-04-4

Remarks:  Physical state: yellow slightly viscous liquid; Composition: 1,3,5-Triazine-1,3,5(2H,4H,6H)-Triethanol (77.5%) and water (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 toxicity test to freshwater algae

GLP compliant?: Yes

Year study was performed:  2012

Test species:  Freshwater green algae (Pseudokirchneriella subcapitata strain CCAP 278/4, formerly Selenastrum capricornutum); culture supplied by the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

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 (nominal): 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.  The 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 CO2 and to counteract the increase in pH due to algal growth in an enclosed system." [Vryenhoef 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 ml) of each of the stock solutions was separately inoculated with algal suspension (37 ml) 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 by chemical analysis at 0 and 96 hours..." [Vryenhoef and Mullee 2012, p. 10]

"Six [250 mL glass conical] flasks each completely filled with test preparation were used for the control and three flasks each completely 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 ml.  Inoculation of 1800 ml of test medium with 37 ml of this algal suspension gave an initial nominal cell density of 1 x 10[4] cells per ml and had no significant dilution effect on the final test concentration." [Vryenhoef and Mullee 2012, p. 10]

"The flasks were sealed with ground glass stoppers and incubated (INFORS Multitron(R) Version 2 incubator) at 24 +- 1°C under continuous illumination (intensity approximately 7000 lux) provided by 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 Coulter(R) Multisizer Particle Counter" [Vryenhoef 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 hourly using the incubators' internal pt-100 temperature probe and Multicom Software Package." [Vryenhoef and Mullee 2012, p. 11]

"A re-growth test was performed after 96 hours to determine the algicidal or algistatic effect of the test item.  Aliquots were removed from each replicate control and test culture (0.25 ml and 0.50 ml respectively) and the replicates pooled.  Fresh sterile culture medium (100 ml) was added to each to ensure that the test concentration was reduced to below the inhibiting level.

The flasks were plugged with polyurethane 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." [Vryenhoef and Mullee 2012, p. 11]

"Samples were taken from the control (replicates R1-R6 pooled) and each test group (replicates R1-R3 pooled) at 0 and 96 hours for quantitative analysis.

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 additional replicates were taken for unopened vessel analysis at 96 hours." [Vryenhoef and Mullee 2012, p. 11]

"[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 item was known to degrade immediately in water to form ethanolamine (0.84 mg/l ethanolamine ≡ to 1 mg ai/l) and formaldehyde (0.41 mg/l formaldehyde ≡ to 1 mg ai/l).  Therefore analysis of the test preparations was [performed] for the 2 degradation products only.  The ethanolamine concentration in the test samples was determined by high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) using an external standard.  The formaldehyde concentration in the test samples was determined by derivatisation with 2,4-dinitrophenylhydrazine phosphoric acid solution with high performance liquid chromatography (HPLC) using an external standard.  The ethanolamine and the formaldehyde 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-dinitrophenylhydrazine phosphoric acid solution* [* Fluka  -  0.2M derivatising agent] and 50 ul of pentafluoropropionic acid.  The test sample was then shaken to mix and allowed to stand for 15 minutes before analysis for both ethanolamine and formaldehyde." [Vryenhoef and Mullee 2012, p. 40]

Statistical Analysis: "One way analysis of variance incorporating Bartlett's test for homogeneity 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 any statistically significant differences between the test and control group.  All statistical analyses were performed using the SAS computer software package..." [Vryenhoef and Mullee 2012, pp. 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 using the Xlfit software package (IDBS).  ECx values were then determined from the equation for the fitted line.  Where appropriate 95% confidence limits for the EC50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949)." [Vryenhoef 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 concentration of the control cultures must increase by a factor of at least 16 after 72 hours and by a factor of at least 100 after 96 hours; 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 exceed 7%; and the coefficient of variation of the cell density values in replicate control cultures must not exceed 20% after 72 and 96 hours).

A positive control study was conducted using zinc chloride as the reference item.

RESULTS

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." [Vryenhoef 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 EC50 (growth): 5.2 mg ai/L*
96-h EC50 (growth): 4.2 mg ai/L; 95% confidence limits 3.9  -  4.6 mg ai/L
96-h NOEC (growth): 1.0 mg ai/L
72-h EC50 (yield): 3.5 mg ai/L; 95% confidence limits 3.2  -  3.8 mg ai/L
96-h EC50 (yield): 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 EC50 (biomass): 3.6 mg ai/L; 95% confidence limits 3.3  -  3.9 mg ai/L
96-h EC50 (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

"*It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits." [Vryenhoef 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]

Measurements of degradation products:  Ethanolamine (HPLC-MS) concentrations were 0.519, 11.0, and 88.6 mg/L at nominal concentrations of 1.0, 10, and 100 mg ai/L, respectively, 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, respectively, at 96 hours.  Formaldehyde (HPLC) concentrations were 0.498, 3.95, and 44.2 mg/L at nominal concentrations of 1.0, 10, and 100 mg ai/L, respectively, 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, respectively, at 96 hours.  "Only the test concentrations spanning the range of concentrations to be employed in the definitive test were analysed." [Vryenhoef and Mullee 2012, p. 16]

Definitive Test:
Growth, Yield, and Biomass Integral Data:  After 96 hours of exposure, the mean cell density was 1.29, 1.43, 0.639, 0.00930, 0.00731, and 0.0114 x 10[6] cells/mL at nominal concentrations of 0, 1.0, 3.2, 10, 32, and 100 mg ai/L, respectively.  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, respectively.  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, respectively.  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, respectively.

"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 "No Observed Effect Concentration" (NOEC) based on [growth rate, yield and biomass integral] was 1.0 mg ai/l.  Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on [growth rate, yield, and biomass integral] was 3.2 mg ai/l." [Vryenhoef and Mullee 2012, pp. 18-19]

Validation Criteria:
"[T]he cell concentration of the control cultures increased by 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 hours) 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 the cell density values in replicate control cultures was 14% after 72 hours and 7% after 96 hours..." [Vryenhoef and Mullee 2007, p. 17]

Observations on Cultures:
"All test and control cultures were inspected microscopically at 96 hours.  There were no abnormalities detected in any of the control or test cultures." [Vryenhoef and Mullee 2012, p. 19]

Observations on test material solubility:
"At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 96-Hour test period all control and 1.0 mg ai/l test cultures were observed to 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 extremely pale green dispersions whilst the 32 and 100 mg ai/l test cultures were observed to be clear colorless solutions." [Vryenhoef and Mullee 2007, pp. 19  -  20]

Physico-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 hours. 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 CO2 from the gaseous phase to the aqueous phase and hence CO2 required for photosynthesis 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 culture 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 concentration in the control cultures exceeded the validation criterion given in the Test Guidelines." [Vryenhoef and Mullee 2012, p. 20]

Verification of test concentrations:
Measured ethanolamine (HPLC-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, respectively, 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, respectively, at 96 hours.  Measured formaldehyde (HPLC) concentrations 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, respectively, 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, respectively, at 96 hours.

At initiation of exposure (time = 0 hours), the measured concentrations of monoethanolamine and formaldehyde were 62-136% and 102-117% of nominal (expected) concentrations, respectively, assuming complete degradation of each mole of 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 formaldehyde were 41-105% and 27-78% of nominal (expected) concentrations, respectively.  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, respectively, at 96 hours.  Measured formaldehyde 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, respectively, at 96 hours.  "These results were similar to the results obtained from the test samples which had been opened on a daily 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." [Vryenhoef and Mullee 2012, p 21]

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 formaldehyde and ethanolamine were found, and... these 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" [Vryenhoef and Mullee 2012, p. 50]

Re-Growth Experiment:
"A re-growth test was performed which showed the test item to be algistatic in effect." [Vryenhoef and Mullee 2012, p. 21]

Positive Control:
"The results from the positive control with zinc chloride were within the normal ranges for this reference item."  [Vryenhoef and Mullee 2012, 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:..." [Vryenhoef and Mullee 2007, p. 21]

The 72-hr EC50 values were 5.2 mg ai/L for growth rate, 3.5 mg ai/L for yield, and 3.6 mg ai/L for biomass integral.  The 96-hr EC50 values were 4.2 mg ai/L for growth rate, 3.2 mg ai/L for yield, and 3.3 mg ai/L for biomass integral.  The 96-hour NOEC and LOEC were 1.0 and 3.2 mg ai/L, respectively, for growth rate, yield, and biomass integral.

STUDY RELIABILITY

[2] Reliable with restrictions.  The following study deficiencies were noted: (1) The study authors measured the concentrations of the primary degradation products (monoethanolamine and formaldehyde) 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 EC50 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 formaldehyde from solution during the algae test is not clearly explained; and (5) 95% confidence limits were not calculated for the 72-hour ErC50 value because the data generated did not fit the models available for the calculation of confidence limits.

REFERENCE

Vryenhoef, H., and Mullee, D.M. 2012. 1,3,5-Triazine-1,3,5(2H,4H,6H)-Triethanol (CAS No. 4719-04-4): Algal Inhibtion Test; Project No. 41103115. Performing Laboratory: Harlan Laboratories Ltd., Shardlow, Derbyshire, UK. Sponsor: Society of Chemical Manufacturers & Affiliates, Washington, DC, USA.

Text in quotes is taken directly from Vryenhoef and Mullee, 2012.
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 analytical measures of the test substance were obtained during testing.  An appropriate method for performing analytical measurements of the test substance was not used.  The test needs to be redone using an appropriate method.  (2) The purity 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 is not clear that the reported nominal concentrations take into account the purity of the test substance.

TEST SUBSTANCE

Identity:  1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4

Remarks:  Purity: 70-90%; trade name: KM200; clear liquid; Batch no. L11381

METHOD

Method/guideline followed:  OECD Guideline 201 and EU C.3 Guidelines

Test type:  Static toxicity test to freshwater algae

GLP compliant?:  Yes

Year study was performed:  2002

Test species:  Green algae (Pseudokirchneriella subcapitata, formerly Selenastrum capricornutum); culture supplied by CCAP, The Freshwater Biological Association, Cumbria, England.

Analytical monitoring:  No (Analytical monitoring was attempted, but results were not reported.)

Exposure duration:  70.5 hours

Concentrations tested:  Nominal: 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L; 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 NaHCO3/L and had a hardness of 24.3 mg CaCO3/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 mL of an algal suspension containing 8 x 10[5] cells/mL was added to 200-mL flasks 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 only and a single set of flasks containing test substance without 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 umol/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 (um3/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 analysis 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 analysis was based on an HPLC method using a fluorescence detector and an external standard.  The test substance was fluorogenically labeled with a mixed o-phtalaldehyde/mercaptoethanol reagent before HPLC analysis.

The EC values with respect to growth rate (ErC 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 (EbC values) were calculated by the method provided in OECD Guideline 201.  The values were calculated by linear interpolation of a plot of the percentage reduction in growth (IA) against the log concentration of the test substance.  The NOEC was determined as the highest concentration at which no statistically significant inhibition 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%).

RESULTS

Endpoint value(s):  Based on nominal concentrations reported: ErC50 = 62 mg/L; EbC50 = 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 umol/s/m[2], 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 absence 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 by the study 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/mL at nominal concentrations of 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L, respectively.  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] um[3]/mL at nominal concentrations of 3.2, 10, 18, 32, 56, 101, and 322 mg/L, respectively.  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 ErC50 was found to be 62 mg/L, with a 95% confidence interval of 55 to 70 mg/L.  The ErC10 and ErC90 were 31 and 120 mg/L, respectively.  The EbC50 was found to be 33 mg/L, with a 95% confidence interval of 32 to 56 mg/L.  The EbC10 and EbC90 were 21 and 79 mg/L, respectively.  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 EC50 was found to be 62 mg/L based 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 analytical measures of the test substance were obtained during testing.  The analytical method attempted
for measuring test substance concentrations during the toxicity test was not appropriate because it relied on rapid hydrolysis of the test substance and, therefore, analytical testing using an
appropriate method was not conducted; (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 test substance, such that the concentrations of the test substance reported in the study may be overestimated by as much as 30%; hence, the EC50s, NOECs, and LOECs may be overestimated by as much as 30%; and (4) the definitive test used only two replicates per 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.

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 yellow liquid, referred to in the reviews as Busan 1060, HHT, hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, and dihydro-5-(2-hydroxyethyl)-1,3,5-triazine-l,3(2H,4H)-
dipropanol

METHOD

Method/guideline followed: OPPTS 870.1100

Test type: Acute Oral -- Rat

GLP compliant?: Not indicated

Year study performed: 1989 (issue year)

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 analysis. LD50 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.

Test method/conditions remarks: Different doses were tested in males and females because a rangefinding study was thought to indicate sex-related differences in lethality.

RESULTS

All deaths occurred by Day 2 after dosing. Incidences of mortality 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 production, 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 necropsy findings for animals found dead included: dark red adrenal glands, reddened mucosa of the digestive tract, dark red lymph nodes, and colored fluid/mucoid contents in the digestive tract.

Endpoint value(s): LD50 (male rats) = 1250 (840-1858) mg/kg-bw; LD50 (female rats) = 763 (701-832) mg/kg-bw

Results remarks: None specified

CONCLUSIONS

The LD50 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). Hexahydro-1,3,5-tris(2-hydroxyethyl)-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 MRID 41675206 as cited in Locke (1995).

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 purity unstated (Consultox Laboratories 1973).

Remarks:  None

METHOD

Method/guideline followed:  OPPTS 870.1100

Test type: Acute oral -- mouse

GLP 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, tested 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 days.

RESULTS

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): LD50 = 0.75, 1, 1.5, 1.75, or 2 mL/kg.

Results remarks: None specified

CONCLUSIONS

The oral (gavage) LD50 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 daily for signs of toxicity, surviving animals were not examined and necropsied, the LD50 was not provided for each sex, the method used to calculate the LD50 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-Hydroxyethyl)-s-Triazine:  Toxicology Disciplinary Chapter for the Issuance of the Reregistration Eligibility Decision (RED) Document.  Case No.:  3074.  PC Code:  083301.  DP Barcode:  346237.  February 4, 2008.

Consultox 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-hydroxyethy1)-s-triazine;
Bioban GK; Glokill 77, EPA File Symbol 48301-RU.  Document 005165, Caswell No. 481C, Project No. 902.

Study 3:  Acute inhalation toxicity study in rats (BASF Corporation, 2010)

Title: Acute Inhalation Study in Wistar Rats (RccHan:WIST(SPF)) with hexahydro-1,3,5-tris(2-hydroxyethyl)-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

GLP 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-only

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 days.

RESULTS

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 days after exposure.  At 0.26 mg/L, 1 of 5 males and 2 of 5 females died during exposure or by study day 2.  Clinical signs of toxicity included gasping, respiration sound, abdominal respiration, nose discharge, red encrusted 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 necropsy.  Histological examination, performed only at 1.07 mg/L, showed moderate to severe diffuse erosion and ulceration of the nasal cavity and larynx with moderate to severe diffuse erosion and ulceration.

Endpoint value(s): LC50 = 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 LC50 (both sexes combined) was 0.371 mg/L.  The LC50 values for males and females were 0.4 and 0.338 mg/L, respectively.

STUDY RELIABILITY

 [2] Reliable with restrictions.  Information is needed on the composition and stability of the test substance.

REFERENCE

TSCATS submission (8EHQ-10-18183;<PHONE_NUMBER>7): BASF Corporation, November 18, 2010.  Notice in Accordance with Section 8(e):  Results of an Acute Inhalation Study in Wistar Rats with hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine (CAS No. 4719-04-4) as Liquid Aerosol.  [This is an abstract; the full study was not available]
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

GLP 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-only

Vehicle: Air

Doses/concentrations tested:  55, 103, 204, 332, or 1619 mg/m[3] (measured concentrations); the mass median aerodynamic diameter for the aerosol particles of triazine was 1.41 +- 2.33 um.

Statistical methods 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.  Least-squares regression analysis was then conducted to establish concentration-response relationships (i.e., testing that the slope of the line from regression analysis 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 (RD50) 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 from the test chamber onto glass fiber filters and the filters were weighed before and after sampling to determine test substance concentrations gravimetrically.  For each concentration of aerosolized test substance, each of 4 male rats was positioned in a body plethysmograph, 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 by a 180-minute exposure period.  The mice were monitored for 20 minutes after aerosol generation had ended to assess recovery.  After removal from the test chamber, mice were observed for seven days for signs of toxicity.

RESULTS

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 typically lasted during the remainder of the exposure period.  The exposure concentration that resulted in a 50% decrease in respiratory frequency (the RD50) was 137 mg/m[3].

Mice were noted to have extremely swollen heads after exposure to concentrations of >= 103 mg/m[3].  Heads of 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 paralysis 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], respectively.  All spontaneous deaths occurred on days 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): RD50 = 137 mg/m[3].  Based on spontaneous deaths during the 7-day observation period, the LC50 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 produced a 50% decrease in respiratory frequency (the RD50) was 137 mg/m[3].  Based on spontaneous deaths during the 7-day post-exposure observation period, the LC50 in male mice was between 103 and 204 mg/m[3].

STUDY RELIABILITY

 [2] Reliable with restrictions.  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;<PHONE_NUMBER>7): General Motors Corporation, Environmental and Energy Staff, Chemical Hazard Assessment and Toxicology, November 4, 1994.  Possible TSCA 8(e) Submission.  Krystofiak, S.P. (1994).  Evaluation of the Respiratory Effects from Components of a Metalworking Fluid in Mice.  Submitted to the Graduate Faculty of Environmental and Occupational Health, School of Public Health in partial fulfillment of the requirements for the degree of Master of Science.  University of Pittsburgh.

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 purity not indicated (Hughes, 1985)

Remarks:  Yellow liquid with pungent odor, referred to in the reviews as HHT, hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, dihydro-5-(2-hydroxyethyl)-1,3,5-triazine-l,3(2H,4H)-
dipropanol, and Bioban GK

METHOD

Method/guideline followed: OPPTS 870.1200

Test type: Acute Dermal -- Rabbit

GLP 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 body 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 study termination (Day 15). At the end of the exposure period, the rubber dams were removed and animals were observed for mortality, moribundity, gross systemic toxicity, and dermal irritation once on the day of treatment and twice daily thereafter.

RESULTS

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-purple 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 by edema, erythema, necrosis, desquamation, and coriaceousness, were seen at the site of application to the skin.

Endpoint value(s): LD50 >2000 mg/kg-bw

Results remarks: None specified

CONCLUSIONS

The LD50 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). Hexahydro-1,3,5-tris(2-Hydroxyethyl)-s-Triazine:  Toxicology Disciplinary Chapter for the Issuance 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.  Hill Top Research, Inc.  EPA/OPP MRID 155984 as cited in Locke (1995).

Locke, R.K.  (1995).  Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine.  Review of Toxicity Data.  DP Barcode:  D212071.  Submission #:  S481738.  Document No. 011557.

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 hexahydro-1,3,5-tris(2-hydroxyethyl)-5-triazine

METHOD

Method/guideline followed: Not indicated

Test type: Acute Dermal -- Rat

GLP 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 days 1, 8, and 15, and gross necropsy was performed on day 15.

RESULTS

The test substance produced no mortality, signs of toxicity, effects on body weight, or findings at necropsy.  Following removal of the occlusive dressing, minimal erythema was seen at the application site but this was resolved by day 5.

Endpoint value(s): LD50 >2000 mg/kg-bw

Results remarks: None specified

CONCLUSIONS

The LD50 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.

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 yellow liquid, referred to in the review as hexahydro-1,3,5-tris(2-hydroxyethyl) 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

GLP 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: pyrogen-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:  Analysis 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 analyzed 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:  Body weights and food consumption were recorded weekly.  Hematology and clinical chemistry parameters were measured at study termination.  Full necropsy 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 analysis on the day of preparation of the test material and on days 1, 7, 8, 14, and 29 of the study.

RESULTS

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 body weight gain and food consumption between treated and control rats.  With regard 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 unusually 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 dose 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 stomach, 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.  Lymphocytic infiltration was reported in rats at 100 mg/kg-bw/day (1/10 males and 2/10 females) and at 250 mg/kg-bw/day (4/10 males and 6/10 females).  Epithelial hyperplasia 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 hepatocyte vacuolation was increased in high-dose male rats when compared to other groups.  On the other hand, margination of hepatocyte cytoplasm 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 lymphoid 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 sponsor 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 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 females 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.

Urinalysis was unremarkable and no treatment-related ocular lesions were present.

Endpoint value(s): LOAEL = 100 mg/kg-bw/day based on gastric lesions (erosion of the mucosa and lymphocytic 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

The LOAEL and NOAEL were 100 and 50 mg/kg-bw/day, respectively.

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 lymphoid hyperplasia as it related to the overall health of the test animals. For animals that died during the study, gross necropsy and microscopic examinations revealed that test substance had entered the lungs in 6 of the 7 decedents; aspiration pneumonia likely resulted from failure to correctly administer the test compound.

REFERENCES

Chen, J.; Tao, J.  (2008) Hexahydro-1,3,5-tris(2-Hydroxyethyl)-s-Triazine:  Toxicology Disciplinary Chapter for the Issuance 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: LEF/3/89. Unpublished study prepared by Toxicol. Laboratories, Ltd. EPA/OPP MRID 41483001.

Study 2:  Repeated-dose dermal 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-hydroxyethyl) S triazine

METHOD

Method/guideline followed:  Not indicated