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 FG007.2.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Debra Milligan, Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

September 14, 2012
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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
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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 1218-04ε1. 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 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 Monoethanolamine

(mg/L)2

Measured 

Concentration 

of Monoethanolamine

(mg/L)

Measured 

Concentration 

of Monoethanolamine 

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.
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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.
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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.
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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.
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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 μmhos/cm.

A range-finding test was conducted to determine the concentration range for the definitive test. 

The nominal concentrations for the definitive test were: 15.6, 25.9, 43.2, 72.0, and 120 mg a.i./L. 

No solvent was utilized to solubilize the test material as Triazine is completely miscible in water.

Twenty rainbow trout were 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.
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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. 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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.
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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.
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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.
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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. 
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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.
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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 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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, rangefinding 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, 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 
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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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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. OD9211

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 μhos/cm, the pH ranged from 6.7-6.9, 

the temperature was 11-12 oC, the photoperiod was 16 hours light/8 hours dark, and the dissolved 

oxygen ranged from 83-94% of saturation. While being held, the fish were fed dry pellet food 

daily, ad libitum. Food was withheld for 48 hours prior to testing. The pretest mortality, 

observed for 48 hours prior to testing, was 0.2%. 

The dilution water consisted of soft water reconstituted from deionized water, using EPA 

recommended procedures, and had a hardness of 42 mg CaCO3/L and specific conductance of 

140 μhos/cm. The stock solution of Triadine 10 (15 mg/L) was prepared by adding distilled 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 rangefinding 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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 μhos/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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

25

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

Title: Acute Toxicity of Milidin X-2 to Bluegill (Lepomis macrochirus)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; Purity: not specified.

Remarks: Also referred to as Grotan and Milidin X-2.

METHOD

Method/guideline followed: Not specified

Test type: Acute Toxicity Test for Freshwater Fish, Bluegill (L. macrochirus), static

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 μmhos/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 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 μmhos/cm, and the photoperiod was 16 hours light/8 hours dark. 

Mortality rates observed after 96 hours at nominal concentrations of 22, 36, 60, and 100 mg/L 

were 10, 30, 90, and 100%, respectively. Sublethal effects were observed in all test groups, 

except the high-dose group where the effects 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. 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

27

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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

28

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

Title: Acute Toxicity of Triadine 10 to Channel Catfish (Ictalurus punctatus)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 63.6% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 6.4% sodium 

omadine, CAS No. not specified; 30% unidentified

Remarks: Formulation, also referred to as Triadine 10

METHOD

Method/guideline followed: Not specified

Test type: Acute Toxicity Test for Freshwater Fish, Channel Catfish (Ictalurus punctatus)

(static or flow-through not specified)

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. 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 μL 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 μmhos/cm. 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 μmhos/cm, respectively. At test termination, they were 32 mg/L, 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

32

96 mg/L, and 488 μmhos/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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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. 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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-Triazine1,3,5(2H,4H,6H)-Triethanol (77.5%) and water (22.5%); Supplier: Q2 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. 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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-

04ε1 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 105cells 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 104cells 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® 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® 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]
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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“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 (HPLCMS) 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 μl 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
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

38

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]
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 106cells/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 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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]
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

42

Study 2: Toxicity to Selenastrum capricornutum (Oldersma and Bowmer, 2002)

Title: Determination of the effect of KM200 on the growth of the freshwater green alga 

Selenastrum capricornutum (OECD 201 and EU C.3 Guidelines)

This study is: Adequate Not Adequate

The study is invalid because: (1) No 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.)1

Exposure duration: 70.5 hours

 

1Analytical monitoring was attempted by the study authors. However, due to inconsistent results from the chemical 

analyses, the measured concentrations of test substance were not reported by the study authors. The measurements 

were considered to be unreliable. See section on Study Reliability for more information.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

43

Concentrations tested: Nominal: 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L2; 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 105cells/mL was 

added to 200-mL flasks containing 100 mL of test solution, yielding an initial cell density of 8 x 

103cells/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 μmol/s/m2(measured with a Bottemanne Weather Instruments Photosynthetic Radiometer 

RA200 Q). 

The initial cell density was calculated from the measured density in the algal pre-culture used for 

the inoculation, divided by the appropriate dilution factor. Algal densities (cells/mL) and algal 

biovolume (μm3/mL) were determined after 23.5, 47, and 70.5 hours using an electronic particle 

counter (Coulter Multisizer IIe). Measured values were corrected for background values in the 

appropriate blanks. The mean measured values were used for calculation of effect levels.

The pH was measured at the start (in medium without algae) and after 70.5 hours in selected 

cultures. The morphology of the algae was examined visually with the aid of a microscope at the 

start and end of the test. The light intensity at two different culture positions was measured at 

the start of the test.

Samples for chemical 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 

 

2 The nominal concentrations were reported as 0, 3.2, 10, 18, 32, 56, 101, and 322 mg/L in section 2.5.3 and Annex 

D of the study report, but were reported as 0, 3.3, 10, 18, 33, 57, 102, and 326 mg/L in section 2.5.1 of the study 

report.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

44

was observed. The effect was considered significant if the average inhibition of the growth rate 

was higher than 5%. Statistical significance was determined with a one-tailed t-test (α = 5%).

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 μmol/s/m2, respectively. At the start of the test, the pH of each 

flask was found to be 8.4. At the end of the test, the pH range was 8.2-8.3 in the 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 104cells/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 106 μm3/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 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

45

appropriate method was not conducted;3(2) the composition of 10-30% of the test substance 

(described as 70-90% pure) is not defined, suggesting that the test substance may contain 

impurities; (3) it is not clear if the nominal concentrations have been adjusted to account for 

purity of the 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.

 

3Although the study report states that the test substance is unstable in water and, therefore, the test solutions were 

undefined mixtures of the test substance and hydrolysis products from the onset of testing, the U.S. EPA

Reregistration Eligibility Decision for Grotan (HHT) indicates that the hydrolysis half-life of CAS No. 4719-04-4 is 

50 and 302 days at pH 7 and 9, respectively. Therefore, the hydrolysis of the test substance is not expected to be 

rapid under the test conditions described for the algae study. 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

46

D-4. Acute Toxicity

Summary of endpoint: Adequate 

Study 1: Acute oral toxicity study in rats (Rush, 1989)

Title: Acute Oral Toxicity Study in Rats with Busan 1060

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 79.4% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 20.6 % 

unidentified

Remarks: Clear yellow liquid, referred to in the reviews as Busan 1060, HHT, hexahydro1,3,5-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.
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47

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).
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48

Study 2: Acute oral toxicity study in mice (Consultox Laboratories, 1973)

Title: Glokill 77: Acute Oral Toxicity

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 78.5% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 21.5% 

unidentified (Chen and Tao 2008) or 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. 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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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 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

51

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; 88110000067): 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]
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

52

Study 4: Acute sensory irritation and toxicity study in mice (Krystofiak, 1994)

Title: Evaluation of the Respiratory Effects from Components of a Metalworking Fluid in Mice

The study is: Adequate Not Adequate

TEST SUBSTANCE 

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

composition not indicated

Remarks: Amber liquid thicker than corn oil, referred to in the study as Triazine, CAS No. 

4719-04-4.

METHOD

Method/guideline followed: Unclear. The study in the TSCATS submission suggests that an 

ASTM method (ASTM 1984) was used but does not explicitly state what method was followed.

Test type: Acute sensory irritation from inhalation of airborne chemicals

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/m3(measured concentrations); the 

mass median aerodynamic diameter for the aerosol particles of triazine was 1.41 ± 2.33 μm.

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 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

53

from the test chamber onto glass fiber filters and the filters were weighed before and after 

sampling to determine test substance concentrations 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/m3. 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/m3.

Mice were noted to have extremely swollen heads after exposure to concentrations of ≥ 103 

mg/m3. Heads of one animal each at 103 and 1619 mg/m3 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/m3, 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/m3; animals were breathing through their mouths, gasping, and 

made noticeable clicking and “chirping” sounds. 

Endpoint value(s): RD50 = 137 mg/m3. Based on spontaneous deaths during the 7-day 

observation period, the LC50 was between 103 and 204 mg/m3

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/m3and 

sensory irritation even at the lowest concentration tested (55 mg/m3). The concentration that 

produced a 50% decrease in respiratory frequency (the RD50) was 137 mg/m3. Based on 

spontaneous deaths during the 7-day post-exposure observation period, the LC50 in male mice 

was between 103 and 204 mg/m3.
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54

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; 88950000037): 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.
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

55

Study 5: Acute dermal toxicity study in rabbits (Hughes et al., 1985)

Title: Acute dermal toxicity screen in rabbits of Bioban GK

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 79.96 % 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 20.04 % 

unidentified (Chen and Tao, 2008) or purity not indicated (Hughes, 1985)

Remarks: Yellow liquid with pungent odor, referred to in the reviews as HHT, hexahydro1,3,5-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. 
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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.
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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.
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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.
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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.
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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 
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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.
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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

Test type: Subacute repeated-dose toxicity

GLP compliant?: Not stated

Year study performed: ~1984

Test species: Sprague-Dawley rats

Number of animals/sex/dose: 5

Route of administration: Dermal

Vehicle: None. The test material was used undiluted as supplied.

Exposure duration: 6 hours/day, 21 days

Doses/concentrations tested: 0 (sterile distilled water control), 100, 500, or 1000 mg/kgbw/day

Statistical methods used: Not stated

Test method/conditions remarks: Identity and stability of the test material were not determined 

by the investigating laboratory. The test compound was held in contact with the skin by a porous 

gauze dressing covered with an Elastoplast elastic adhesive bandage. At the end of each 6-hour 

exposure period the bandage and dressing were removed and the exposure site washed and dried. 

The test site of each animal was observed daily for local dermal irritation. Animals were 

observed daily for toxic signs. Animals that died during the study were subjected to a detailed 
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postmortem examination, and tissues were preserved for histopathological examination. Body 

weight was recorded immediately prior to the start of the study, and twice weekly thereafter. 

Food consumption was recorded weekly. Water consumption was monitored daily. Hematology 

and serum biochemical determinations were conducted on a separate group of 10 animals (5 

male, 5 female) from the same stock prior to start of the study, and on all test and control animals 

at termination of the study. Determinations were done for hemoglobin, erythrocytes, hematocrit, 

mean corpuscular volume, leukocytes (total and differential) and prothrombin time. Mean 

corpuscular hemoglobin and mean corpuscular hemoglobin concentration was calculated. Blood 

chemistry values were determined for blood urea nitrogen, total protein, albumin, A/G ratio, 

creatinine, total bilirubin, alkaline phosphatase, SGPT, SGOT, gamma glutamyltranspeptidase, 

glucose, calcium, potassium, sodium chloride, and phosphate. At termination of the study, all 

surviving animals were necropsied and terminal weights taken on liver, kidney, adrenals, and 

gonads. Histopathology was done on liver, kidney, lymph nodes, skin (treated and untreated) 

and organs showing lesions or change in size. Skin was examined histologically in all treatment 

groups, since compound-related changes were observed in the high-dose group. 

RESULTS

No animals died during the study. No toxic signs were reported except for local dermal 

irritation, which was dose-related and increased in severity with the duration of the study. All 

groups of treated males gained less weight than corresponding controls. Male weight gain in all 

groups was approximately 70% of the control male weight gain. The weights observed for all 

three dosed groups were within the range normally observed in the investigators’ laboratory, and 

therefore were not considered treatment related. Weight gain was comparable in control and 

treated females.

No difference in food consumption was reported between control and treated males, but during 

week 1, the mid and high-dose female food consumption was significantly decreased (p < 0.05) 

when compared to the controls. No intergroup differences in water consumption were reported.

Mean corpuscular volume was reduced in the low and high-dose males, and hemoglobin was 

reduced in the low and high-dose females, but the reductions were not considered biologically 

significant because they were within the normal range of values for Sprague-Dawley rats in the 

investigators’ laboratory.

The following statistically significant changes were noted in the treated animals when compared 

to the controls (p <0.05): increased protein in mid and high dose males and high dose females, 

increased sodium in all treated male groups, increased phosphate in mid and high dose females, 

increased AST in mid and high dose males, decreased A/G ratio in mid and high dose males and 

high dose females, and decreased chloride in mid and high dose females. These changes were 

considered biologically insignificant because the values observed were within the range observed 

in the animals used in the hematology and biochemical screens. Except for multifocal scab 

formation at treatment sites at all dose levels, no macroscopic abnormalities were reported. 

Absolute adrenal weight was significantly decreased in low and high-dose males and was 

reported to be due to two control animals with high adrenal weights. No difference was reported 

in relative organ weights. Treatment- and dose-related histopathological changes seen at treated 
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skin sites included epidermal ulceration, acanthosis, and dermal inflammatory infiltration with 

occasional necrosis and hemorrhage. No histological changes were found in other tissues. 

Endpoint value(s): LOAEL (systemic toxicity) > 1000 mg/kg-bw/day based on no effects at the 

highest dose tested; NOAEL = 1000 mg/kg-bw/day. LOAEL (application site irritation) = 100 

mg/kg-bw/day based on epidermal ulceration, acanthosis, and dermal inflammatory infiltration 

with occasional necrosis and hemorrhage at the lowest dose tested; NOAEL = Not established

Results remarks: Reproductive organs were not examined histologically in this study.

CONCLUSIONS

The LOAEL for systemic toxicity was > 1000 mg/kg-bw/day, based on no effects at the highest 

dose tested, and the NOAEL was 1000 mg/kg-bw/day. The LOAEL for irritation at the site of 

test compound application was 100 mg/kg-bw/day, the lowest dose tested, based on epidermal 

hyperplasia and ulceration; a NOAEL was not established.

STUDY RELIABILITY

[2] Reliable with restrictions. The 21-day duration of this study was shorter than the 28-day 

duration needed to adequately address the repeated-dose endpoint. Also, different values for 

percent active ingredient were reported in the reviews that formed the basis for this robust 

summary. In addition, a limited number of organs was preserved and examined histologically.

REFERENCE

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.

Thomas, M.; Brooks, P. (1984) Glokill 77: 21 Day Dermal Toxicity Study in the Rat: 

Experiment No. 508/8411. Unpublished study prepared by SafePharm Laboratories Ltd. 

EPA/OPP MRID 155989
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Study 3: Repeated-dose dermal toxicity study in rats (Hill and Newman, 1990b)

Title: 13 Week Dermal Toxicity Study in the Rat

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 78.5% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 21.5% 

unidentified

Remarks: Clear 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 82-3

Test type: Subchronic Dermal Toxicity 90-Day Study

GLP compliant?: Yes

Year study performed: 1990 (issue date)

Test species: Crl:CD(SD)BR rats weighing 197-234 g (females) and 230-276 g (males)

Number of animals/sex/dose: 10

Route of administration: dermal

Vehicle: pyrogen-free sterile water

Exposure duration: 5 days/week, 13 weeks

Doses/concentrations tested: 0 (vehicle control), 5, 50, or 250 mg/kg-bw/day

Statistical methods used: 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: During the exposure period, the application sites were 

covered with semi-occlusive bandages and animals were fitted with Elizabethan collars to 

minimize ingestion of the test chemical. The test site was not washed between doses. 

Hematology and clinical chemistry parameters were measured at study termination. Full 
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necropsy was performed on all animals. Complete histopathology was performed on all control 

and high dose animals. Normal and treated skin from all animals in all dose groups was 

examined histologically.

RESULTS

No deaths were reported during the study. The clinical signs observed during the study included 

periorbital, perianal, and cranial fur staining in all groups; yellow staining at the applications site 

in animals at 5 and 50 mg/kg-bw/day, and skin reactions at the application site in animals at 50 

and 250 mg/kg-bw/day. Occasional incidences of erythema were observed in female rats that 

received 5 mg/kg-bw/day.

In assessing the severity of the dermal lesions, erythema was scored as very slight to severe in 

animals at 50 and 250 mg/kg-bw/day. An increase in the numbers of animals affected and an 

increase in the severity of the reported reactions occurred at 250 mg/kg-bw/day. Moderate to 

very severe edema was present in animals at 250 mg/kg-bw/day. At 50 mg/kg-bw/day, only 

isolated incidences of edema were reported.

Statistically significant differences were found in blood chemistry values from individual 

animals. At 5 mg/kg-bw/day, affected parameters included one male with increased alanine 

aminotransferase, one female with increased phosphorous and one female with increased 

chloride. At 50 mg/kg-bw/day, blood chemistry parameters were increased in two animals. One 

female in this group had increased sodium and one male had increased chlorine levels. At 250 

mg/kg-bw/day, one female had elevated glucose levels and one male had elevated sodium levels.

Microscopically, there was an increase in the incidence of epidermal hyperplasia and ulceration 

of the treated area of the skin in animals treated with 50 or 250 mg/kg-bw/day. Eighty to 100 

percent of both the treated and control animals were found to have peribronchiolar lymphoid 

hyperplasia in the lungs. The significance of the lesions was not discussed.

Endpoint value(s): LOAEL (systemic toxicity) > 250 mg/kg-bw/day based on no effects at the 

highest dose tested; NOAEL = 250 mg/kg-bw/day. LOAEL (application site irritation) = 50 

mg/kg-bw/day based on epidermal hyperplasia and ulceration; NOAEL = 5 mg/kg-bw/day

Results remarks: Microscopic examinations were conducted on all tissues from control and 

high-dose animals, tissues from animals that died on study, gross lesions. No treatment-related 

histological effects were seen in the following reproductive organs: testes, epididymides, 

prostate, seminal vesicle, and ovaries.

CONCLUSIONS

The LOAEL for systemic toxicity was > 250 mg/kg-bw/day, based on no effects at the highest 

dose tested, and the NOAEL was 250 mg/kg-bw/day. The LOAEL for irritation at the site of test 

compound application was 50 mg/kg-bw/day based on epidermal hyperplasia and ulceration and 

the NOAEL was 5 mg/kg-bw/day.
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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 found in the lungs of both treated and control animals.

REFERENCE

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. (1990b) Triazine: 13 Week Dermal Toxicity Study in the Rat: Lab Project 

Number: LEF/4/89. Unpublished study prepared by Toxicol Laboratories, Ltd. EPA/OPP MRID 

41483002.
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D-6. Genetic Toxicity – Gene Mutation

Summary of endpoint: Adequate

Study 1: Mutagenicity study in Salmonella typhimurium (Mortelmans et al., 1986)

Title: Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4, purity not indicated

Remarks: Referred to in Table I of Mortelmans et al. (1986) as hexahydro-1,3,5-

tris(hydroxyethyl)-s-triazine.

METHOD

Method/guideline followed: Standard NTP Protocol for the Salmonella/E. coli Mutagenicity 

Test or Ames Test.

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Not indicated

Year study performed: 1983

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat and hamster livers)

Concentrations tested: 0 (water vehicle control) and at least five concentrations in the range 

0.1 to 333 μg/ plate

Statistical methods used: None

Test method/conditions remarks: In the standard protocol (preincubation) for conducting the 

Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium (or E. 

coli) plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37o C with the test 

chemical. Control cultures, with all the same ingredients except the test chemical, are also 

incubated. In addition, positive control cultures are prepared; these contain the particular 

bacterial tester strain under investigation, the various culture ingredients, and a known potent 

mutagen. After 20 minutes, agar is added to the cultures and the contents of the tubes are 

thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial 

culture medium. The plates are incubated, and bacterial colonies that do not require an excess of 
Final HPV2 Data Adequacy Review (September 2012) CASRN 4719-04-4

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supplemental histidine appear and grow. These colonies are comprised of bacteria that have 

undergone reverse mutation to restore function of the histidine-manufacturing gene. The number 

of colonies is usually counted after 2 days. All assays are repeated no less than one week after 

completion of the initial test.

Criteria for data evaluation were as follows: (1) Mutagenic response: a concentration-related, 

reproducible increase in the number of revertants over background, even if the increase was less 

than twofold; (2) Nonmutagenic response: when no increase in the number of revertants was 

elicited by the chemical; and (3) Questionable response: when there was an absence of a clearcut concentration-related increase in revertants, when the concentration-related increases in the 

number of revertants were not reproducible, or when the response was of insufficient magnitude 

to support a determination of mutagenicity.

In a preliminary concentration-setting experiment, 1,3,5-triazine-1,3,5(2H,4H,6H)-triethanol was 

tested with S. typhimurium strain TA100 in the presence and absence of the metabolic activation 

systems, over a wide concentration range with an upper limit of 10 mg/plate. The high 

concentration for further testing was selected to exhibit some degree of toxicity. One or more of 

the following was taken as evidence of toxicity: appearance of hispinpoint colonies, reduced 

numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. 

Positive control substances were tested concurrently. Failure of the positive controls to induce 

mutation was considered reason to discard the experiment.

RESULTS

In S. typhimurium strain TA100, 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol induced a 

concentration-related increase in revertants of up to 2-fold in the presence of metabolic 

activation from rat or hamster liver; no increase was observed in the absence of metabolic 

activation. The test substance was not mutagenic in strains TA98, TA1535, or TA1537 in the 

absence or presence of metabolic activation.

Genotoxicity result: Weakly positive

Cytotoxic concentration (if applicable): ≥ 100 μg/plate

Results remarks: Concurrent positive controls produced the expected mutagenic response.

CONCLUSIONS

1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol was mutagenic in S. typhimurium strain TA100 in the 

presence but not the absence of metabolic activation. The test substance was not mutagenic in S. 

typhimurium strains TA98, TA1535, or TA1537 with or without metabolic activation.

STUDY RELIABILITY

[1] Reliable without restrictions. The study followed the Standard NTP Protocol for the 

Salmonella/E. coli Mutagenicity Test or Ames Test.
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REFERENCES

Mortelmans, K. et al. 1986. Salmonella mutagenicity tests: II. Results from the testing of 270 

chemicals. Envir. Mutag. Vol. 8, Suppl. 7:1-119. 

National Toxicology Program 1983. Salmonella: Study Summary for 1,3,5-Triazine1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4. Study ID 700221.
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Study 2: Mutagenicity study in Salmonella typhimurium (NTP, 2004)

Title: Salmonella study for 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4, purity not indicated

Remarks: None

METHOD

Method/guideline followed: Standard NTP Protocol for the Salmonella/E. coli Mutagenicity 

Test or Ames Test.

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Not indicated

Year study performed: 2004

Test species: Salmonella typhimurium strains TA98 and TA100

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat and hamster livers)

Concentrations tested: 0 (water vehicle control) and at least five concentrations in the range 10 

to 500 μg/plate

Statistical methods used: None

Test method/conditions remarks: In the standard protocol (preincubation) for conducting the 

Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium (or E. 

coli) plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37o C with the test 

chemical. Control cultures, with all the same ingredients except the test chemical, are also 

incubated. In addition, positive control cultures are prepared; these contain the particular 

bacterial tester strain under investigation, the various culture ingredients, and a known potent 

mutagen. After 20 minutes, agar is added to the cultures and the contents of the tubes are 

thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial 

culture medium. The plates are incubated, and bacterial colonies that do not require an excess of 

supplemental histidine appear and grow. These colonies are comprised of bacteria that have 

undergone reverse mutation to restore function of the histidine-manufacturing gene. The number 

of colonies is usually counted after 2 days. All assays are repeated no less than one week after 

completion of the initial test.
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Criteria for data evaluation were as follows: (1) Mutagenic response: a concentration-related, 

reproducible increase in the number of revertants over background, even if the increase was less 

than twofold; (2) Nonmutagenic response: when no increase in the number of revertants was 

elicited by the chemical; and (3) Questionable response: when there was an absence of a clearcut concentration-related increase in revertants, when the concentration-related increases in the 

number of revertants were not reproducible, or when the response was of insufficient magnitude 

to support a determination of mutagenicity.

In a preliminary concentration-setting experiment, 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol 

was tested with S. typhimurium strain TA100 in the presence and absence of the metabolic 

activation systems, over a wide concentration range with an upper limit of 10 mg/plate. The high 

concentration for further testing was selected to exhibit some degree of toxicity. One or more of 

the following was taken as evidence of toxicity: appearance of hispinpoint colonies, reduced 

numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. 

Positive control substances were tested concurrently. Failure of the positive controls to induce 

mutation was considered reason to discard the experiment.

RESULTS 

In S. typhimurium strain TA100, 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol induced a 

concentration-related increase in revertants up to 4-fold in the absence of metabolic activation 

and up to 2-fold in the presence of metabolic activation from rat liver; no increase was observed 

with metabolic activation from hamster liver. In S. typhimurium strain TA98, 1,3,5-Triazine1,3,5(2H,4H,6H)-triethanol induced a concentration-related increase in revertants up to 2-fold in 

the presence of metabolic activation from hamster liver but no increase in the absence of 

activation or the presence of metabolic activation from rat liver.

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): ≥ 1000 μg/plate for strain TA100 and ≥ 333 μg/plate 

for strain TA98

Results remarks: Concurrent positive controls produced the expected mutagenic response.

CONCLUSIONS

1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol was mutagenic in S. typhimurium strains TA98 and 

TA100 in the presence of metabolic activation and was mutagenic in strain TA100 in the absence 

of metabolic activation. 

STUDY RELIABILITY

[1] Reliable without restrictions. The study followed the Standard NTP Protocol for the 

Salmonella/E. coli Mutagenicity Test or Ames Test.
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REFERENCE

National Toxicology Program 2004. Salmonella: Study Summary for 1,3,5-Triazine1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4. Study ID A17569.
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Study 3: Mutagenicity study in Salmonella typhimurium (Asquith, 1989a)

Title: Bacterial Reverse Mutation Assay: Triazine

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 78.5% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 21.5% not 

specified

Remarks: Clear yellow liquid, referred to in the review as Triazine and hexahydro-1,3,5-

tris(hydroxyethyl)-s-triazine.

METHOD

Method/guideline followed: OPPTS 870.5100

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1988

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538

Metabolic activation: With and without

Concentrations tested: 0 (distilled water control), 0.32, 1.6, 8, 40, or 200 μg/ plate

Statistical methods used: The number of revertant colonies per plate was counted 

electronically (Biotran III), treatment group means and standard deviations calculated, and 

resulting data statistically analyzed by ANOVA (F-statistic). If F was significant (p < 0.05) and 

concentration-responsiveness apparent, a correlation coefficient was calculated over the response 

range, and its significance read from standard (published) tables. It’s considered a substance 

positive if it produces a reproducibly statistically significant increase in the mean number of 

revertants that exceeds twice the concurrent solvent control, plus evidence for a concentrationresponse. Plates showing evidence of severe toxicity (markedly reduced or absent background 

bacterial lawn) were excluded from analysis, since these may yield significantly decreased 

revertant colony counts (of no relevance to mutagenic activity), or increased colonies from 

surviving non-revertant cells incorporating the histidine available because of (toxic) lysis (i.e., a 

false-positive result).

Test method/conditions remarks: Following a range-finding toxicity testing with TA98 

(only) at concentrations up to 5000 μg/plate, triplicate cultures of all five Ames strains were 

exposed to the test substance for 72 hours (extended from the customary 48 hours because the 
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test substance is bacteriostatic, thus slowing growth of revertant colonies) at each of five 

concentrations, both in the absence and presence of a mammalian metabolic activation system 

consisting of the liver microsomal fraction (S9) prepared from male Fischer 344 rats induced by 

Aroclor 1254 and containing 44 mg protein per mL, plus generating cofactors.

In addition to solvent controls (DW), bacterial strains were exposed to their selectively 

appropriate active mutagens in the absence of activation (Sodium azide, 1 μg/plate for TA1535 

and TA100; 9-amino-acridine, 50 μg/plate for TA1537; 2-nitrofluorene, 0.5 μg/plate for TA1538 

and TA98), but all to 2-aminoanthracene (2-AA, 2 μg/plate) under activation, thus serving as 

positive controls. The entire experiment was repeated.

RESULTS 

In the preliminary toxicity test, the test substance was toxic to both non-activated and activated 

TA98 cells at concentrations of 200 μg/plate and above, as revealed by reduced background lawn 

indicative of restricted growth. Hence, the five concentrations selected for the mutation assays 

were 200, 40, 8, 1.6, and 0.32 μg/plate with or without S9 mix.

In both experiments, the test substance consistently reduced background lawns at the HOT, 200 

μg/mL, in the absence of activation but with one exception did not induce significantly increased 

revertant colony counts. The exception was a significant F-value (p < 0.05) calculated for 

unactivated TA1537 cultures, derived from a wider-than-usual variation in mean numbers of 

revertants on test plates (3.3 to 7.7) compared to the concurrent mean control value (4.7). This 

was discounted by the investigator because there was neither a doubling of revertant rate nor 

evidence of a concentration-response relationship. 

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): ≥ 200 μg/plate

Results remarks: Positive controls were tested but responses to controls were not provided in 

the OPP review.

CONCLUSIONS

The test substance was not mutagenic in the Ames testing at levels up to levels of toxicity.

STUDY RELIABILITY

[1] Reliable without restriction.
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REFERENCES

Asquith, J. (1989a) Triazine Joint Venture: Bacterial Reverse Mutation Assay: Triazine: Toxicol

Study No. M/AMES/10658. Unpublished study prepared by Toxicol Laboratories Ltd. 

EPA/OPP MRID 41231702. As cited in an EPA OPP Data Evaluation Report by Irving Mauer, 

January 1990.

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.
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Study 4: Mutagenicity study in Salmonella typhimurium (Safe Pharm Laboratories, 1984)

Title: Ames Test (O.E.C.D.) Product Glokill 77.

The study is: Adequate Not Adequate

This study is not adequate because: 1) no results were provided for Salmonella typhimurium

strain TA1537; 2) results of assays in strains TA98 and TA1538 were inconclusive; and 3) the 

purity and composition of the test substance were not provided.

TEST SUBSTANCE 

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

and composition

Remarks: Referred to in the review as Glokill 77

METHOD

Method/guideline followed: Ames et al. and Gardner et al.

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Not indicated

Year study performed: 1984

Test species: Uncertain. The Materials and Methods section of the review of this study in 

Stewart (1986, pp. 34-37) indicates that Salmonella typhimurium strains TA98, TA100, TA1535, 

and TA1537 were tested. However, the results section provides results for strains TA98, TA100, 

TA1535, and TA1538 but not for TA1537.

Metabolic activation: With and without

Concentrations tested: 0 (distilled water control), 0.16, 0.8, 4, 20, or 100 μg/ plate

Statistical methods used: Not indicated.

Test method/conditions remarks: In the main study, each concentration was tested in 

triplicate, with and without metabolic activation. The test substance was considered mutagenic if 

it produced a dose-related and statistically significant increase in mutation rates (of at least twice 

the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence 

of the S-9 microsomal enzymes. Conversely, if the number of induced revertants compared to 

spontaneous revertants were less than twofold at all dose levels employed up to the limit of 

toxicity or solubility, the test compound was considered not to be mutagenic.
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RESULTS 

No significant increase in the number of revertant colonies was reported for TA100 and TA1535 

strains of Salmonella typhimurium; however, in strain TA98 a significant increase in the number 

of revertant colonies was reported in the assay with metabolic activation, and in strain

TA1538 an increase in the number of revertant colonies was reported both with and without 

metabolic activation.

In strain TA 98, the test compound caused an increase in the mutation frequency. In the first 

experiment, although the increase in mutation frequency was significantly increased over the 

spontaneous mutation frequency in untreated bacteria, the mean number of revertant colonies 

remained the same at all dosage levels in the study using metabolic activation. At the highest 

dosage level, 100 μg/plate, the test compound appeared to be cytotoxic. In experiment 2, the 

mean number of revertant colonies doubled, but the two highest dose levels had lower mutation 

frequencies than the two middle dose; thus the mutation frequency in strain TA 98 did not seem 

to be dose related.

In strain TA 1538, in experiment 1 without metabolic activation, the mutation frequency was 

significantly increased in a dose-dependent manner up to the high dose (100 μg/plate) which 

appeared to be cytotoxic. In the assay with metabolic activation, the increase in revertant 

colonies did not appear to be dose related. The highest dose level in this assay appeared to be 

cytotoxic. In the second experiment, the two highest dosage levels produced a small increase in 

the number of revertant colonies when the assay was performed without metabolic activation. 

When the assay was done with metabolic activation the increase in the number of revertant 

colonies was dose related.

In summary, the results of the assays in strains TA 98 and TA 1538 of Salmonella typhimurium

were not reproducible. Based on this, the results of the study seem to be inconclusive.

Genotoxicity result: Inconclusive

Cytotoxic concentration (if applicable): ≥ 100 μg/plate

Results remarks: Positive controls were tested and provided the expected responses. Test 

concentrations were chosen based upon the results of a preliminary cytotoxicity study.

CONCLUSIONS

The results of this Ames Salmonella reverse gene mutation assay were inconclusive.

STUDY RELIABILITY

[3] Not reliable. No results were provided for S. typhimurium strain TA1537. Results of assays 

in strains TA98 and TA1538 were inconclusive. The purity and composition of the test 

substance were not provided.
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REFERENCES

Safe Pharm Laboratories. (1984). Ames Test (O.E.C.D.) Product Glokill 77. Acc. No. 260195. 

September 11, 1984. As cited in Stewart (1986).

Stewart, J.E. (1986). Memorandum, Subject: Hexahydro-1.3.5-tris(2-hydroxyethy1)-s-triazine; 

Bioban GK; Glokill 77, EPA File Symbol 48301-RU. Document 005165, Caswell No. 481C, 

Project No. 902.
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D-7. Genetic Toxicity – Chromosomal Aberrations

Summary of endpoint: Adequate

Study 1: Micronucleus study in mice (Asquith, 1989b)

Title: Mouse Micronucleus Test: Triazine

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 78.5% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 21.5% not 

specified

Remarks: Clear yellow liquid, referred to in the review as Triazine and hexahydro-1,3,5-

tris(hydroxyethyl)-s-triazine.

METHOD

Method/guideline followed: OPPTS 870.5385

Test type: Mouse bone marrow chromosomal aberration test

GLP compliant?: Yes

Year study performed: 1988

Test species: CD-1 mice, 5/sex/dose

Metabolic activation: N/A

Doses tested: Single gavage dose of 0 (0.9% NaCl control), 200, 400, or 800 mg/kg

Statistical methods used: The micronuclei data were analyzed by the Mann—Whitney U—test, 

with probabilities for significant differences between test and control groups obtained from 

standard published tables.

Test method/conditions remarks: Following a range-finding study (at doses up to 2000 

mg/kg), test substance was administered to groups of CD-1 mouse by gavage in a single dose 

and the animals were sacrificed 24, 48, or 72 hours later. Control groups received 0.9% saline 

(solvent control), or cyclophosphamide (CPA, positive control); the latter were sacrificed only at 

24 hours. Femoral bone marrow harvested at scheduled sacrifice times was prepared for 

microscopic examination by conventional cytological techniques. At least 1000 polychromatic 

erythrocytes (PCE) per animal were scored for micronuclei (MN—PCE), and the ratio of PCE to 

normochromatic erythrocytes (NCE) also recorded for each animal. 
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RESULTS

In the range-finding test, deaths occurred at 1200 and 2000 mg/kg (respectively, 2 of 3 animals 

and 3 of 3 of each sex), but no mortalities at 800 mg/kg or below, providing an estimated LD50 of 

approximately 1000 mg/kg. Based upon the (OECD) convention of applying 80% of the LD50 as 

the maximum tolerated dose (MTD) for this type of study, the highest dose tested (HDT) for the 

main study was selected as 800 mg/kg, with two lower doses of 400 and 200 mg/kg.

In the micronucleus assay itself, two high-dose (800 mg/kg) animals (one of each sex) died 

before their scheduled sacrifice at 48 hours. No cytotoxicity, as measured by reduction in 

PCE/NEC ratios, was found in any treated group. Random variation around control values for 

numbers of micronuclei was found at all sacrifice times and in both sexes of test animals. A 

singular statistically significant increase in micronuclei over concurrent control was calculated 

for the 400 mg/kg male group sacrificed at 72 hours (0.14 vs. 0.02). This increase was 

considered to be of no biological significance since it fell within solvent control ranges at other

sampling times (notably 24 hours when optimally most of the micronuclei would be visualized).

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): N/A

Results remarks: Positive controls were tested and produced the expected responses.

CONCLUSIONS

The test substance did not induce micronuclei in bone marrow cells of mice treated orally up to a 

nominal dose (800 mg/kg) considered a MTD by OECD (80% of the LD50).

STUDY RELIABILITY

[2] Reliable with restrictions. Although the EPA’s limit dose in the absence of any toxicity 

(1000 mg/kg) was not assayed (represented by the LD50 calculated in the range-finding study), 

testing up to 800 mg/kg was sufficiently high to validate the negative result obtained.

REFERENCES

Asquith, J. (1989b) Triazine Joint Venture: Mouse Micronucleus Test: Triazine: Toxicol Study 

No. M/MMN/10659. Unpublished study prepared by Toxicol Laboratories Ltd. EPA/OPP 

MRID 41231701. As cited in an EPA OPP Data Evaluation Report by Irving Mauer, January 

1990.

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.
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Study 2: Micronucleus study in mice (Richardson, 1983)

Title: Evaluation of some formaldehyde-release compounds and other biocides in the mouse 

micronucleus test

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 75% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 25% not 

specified

Remarks: Referred to in the study report as Glokill 77 (75% pure, CA Reg. No. 56996-62-4).

METHOD

Method/guideline followed: Salamone, M.F. et al. (1981). Mutagenic activity of 41 

compounds in the in vivo micronucleus assay. Progr. Mutation Res. 1:686-697.

Test type: Mouse bone marrow micronucleus test

GLP compliant?: Not indicated

Year study performed: 1983 (publication date)

Test species: Male C57Bl/6J mice, 6-8 weeks old, 8-10/sampling time

Metabolic activation: N/A

Doses tested: Two consecutive daily doses by intraperitoneal injection of 0 (vehicle not 

indicated) or 176 mg/kg-bw. The latter dose represented 80% of the intraperitoneal LD50/7 days

(based on deaths occurring when animals were observed for 7 days following administration of a 

single i.p. dose). Mice were also dosed at 50% of the intraperitoneal LD50/7 days, but the 

micronucleus data were not evaluated for these animals.

Statistical methods used: The micronuclei results in treated animals were analyzed for 

significant differences from controls using a one-sided Student’s t-test.

Test method/conditions remarks: For the study in which micronuclei were evaluated, mice 

were given two consecutive daily doses, 24 hours apart, using dose levels equivalent to 80% of 

the LD50/7 days. Animals were killed at 24 or 48 hours after the second dose. Femurs were 

removed, bone marrow smears were applied to microscope slides, and cells were stained in 

Leishman’s stain. Slides were coded and scored blind. Five hundred polychromatic erythrocytes 

(PCEs) were examined from each animal and the number containing micronuclei was scored. 

The ratio of PCEs to mature erythrocytes (ME) was also determined as a measure of cytotoxicity. 
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RESULTS

Genotoxicity result: Negative

Cytotoxic dose: >176 mg/kg-bw, the highest dose tested, based on absence of effect on the 

PCE: ME ratio

Results remarks: A positive control, cyclophosphamide, was tested and produced the expected, 

significant increase in micronucleus frequency. 

CONCLUSIONS

The test substance did not induce micronuclei in bone marrow erythrocytes of mice treated by 

two consecutive daily intraperitoneal injections (dose = 80% of the LD50/7days), 24 hours apart, 

followed by evaluation of micronucleus frequency at 24 or 48 hours after the second dose.

STUDY RELIABILITY

[2] Reliable with restriction. The study did not present evidence that the test substance 

produced signs of toxicity during the main study. The study did not indicate the vehicle used for 

the test substance (if any) or if control animals were injected i.p. with the vehicle. OPPTS 

870.5395 (Mammalian Erythrocyte Micronucleus Test) recommends that at least 2000 immature 

erythrocytes per animal should be scored for the incidence of micronucleated immature 

erythrocytes, whereas the current study evaluated micronucleus frequency in only 500 PCE per 

animal.

REFERENCE

Richardson, C.R. (1983). Evaluation of some formaldehyde-release compounds and other 

biocides in the mouse micronucleus test. Mutat. Res. 124:241-246.
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D-9. Reproductive Toxicity 

Summary of endpoint: Adequate

Although no reproductive toxicity studies were available, the reproductive endpoint has been 

acceptably addressed on the basis that no effects on developmental endpoints were seen in an 

adequate oral developmental toxicity study in rats and no treatment-related effects on the 

histology of reproductive organs of either sex were seen in adequate 90-day oral and dermal 

toxicity studies in rats. 
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D-10. Developmental Toxicity 

Summary of endpoint: Adequate

Study 1: Developmental toxicity study in rats (Ridgway, 1989)

Title: Triazine Rat Teratology Study

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 78.5% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 21.5% 

unidentified

Remarks: Clear yellow fluid, referred to in the review as Grotan, Triazine, and hexahydro1,3,5-tris(2-hydroxyethyl)s- triazine

METHOD

Method/guideline followed: OPP 83-3

Test type: Developmental Toxicity

GLP compliant?: Yes

Year study performed: 1989 (issue date)

Test species: OFA-SD(IOPS-Caw) rats, 200-219 grams

Number of animals/sex/dose: 24 timed-pregnant females

Route of administration: Oral gavage

Exposure duration: Daily on gestation days 6-15

Doses/concentrations tested: 0 (vehicle control), 250, 500, or 750 mg/kg-bw/day

Vehicle: Deionized distilled water 

Statistical methods used: The significance level was set at p ≤ 0.05. ANOVA was followed by 

Student’s t-test for analysis of maternal body weight, body weight gain, and food consumption, 

number of corpora lutea, number of live fetuses, implantation sites, and fetal body weights. The

Kruskal-Wallis test was used for analysis of % pre- and post-implantation losses, fetal sex ratios, 

fetal malformations, and fetal variations. The percentage of fetus in each litter that showed each 
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category of malformation and/or variation was calculated, group means were calculated from 

litter means, and the results were compared using the Kruskal-Wallis test.

Test method/conditions remarks: Females were time mated with males of the same strain and 

source. One male was caged with two females. The day on which the vaginal plug was seen, 

taken as evidence of mating, was designated day 0 of gestation. Pregnant females were then 

randomly assigned to dose groups. Analysis of dosing solution for concentration (times of 

analysis not stated) indicated that mean measured concentrations were 99.2-105% of target 

values and that the compound was stable in solution for 28 days.

RESULTS

One dam at 750 mg/kg-bw/day was sacrificed on GD 14. Prior to sacrifice on that day the 

animal exhibited labored breathing, rales, emaciation, piloerection, red staining around the 

mouth and nose, and pale extremities. All other animals survived the study.

Post dosing salivation was seen in females at 750 mg/kg-bw/day. In dams at 250 and 500 

mg/kg-bw/day, rales, labored breathing, wheezing, and tachypnea were observed occasionally in 

toward the end of the dosing period. No other clinical signs were reported.

At 750 mg/kg-bw/day, maternal body weight gain (p<0.01) and food consumption (p<0.001) 

were significantly lower during the dosing period than in controls. Stomach lesions, 

characterized by ulceration and/or scarring of the mucosa, were observed in 14 of 20 females at 

750 mg/kg-bw/day, including the dam sacrificed on day 14. No gross abnormalities were 

reported in the other dose groups.

The data did not demonstrate any differences between the control and treated dams with respect 

to pregnancy rates, number of corpora lutea, implantation sites, number of live fetuses, or early 

and late resorptions. There were no abortions and no premature deliveries. In addition, those 

treatments did not produce evidence of developmental toxicity as measured by mean fetal weight 

or external or visceral, abnormalities. There were increased per-fetus and per-litter incidences of 

vestigial 14th ribs and retarded ossification of the vertebral thoracic centra that appeared to be 

dose-related. However, since the increases were not statistically significant and the incidence of 

these abnormalities is highly variable in rats, the study authors and OPP reviewer did not 

consider them to be treatment-related.

Endpoint value(s): LOAEL (maternal toxicity) = 750 mg/kg-bw/day based on decreased 

maternal body weight gain and ulcerations and/or scarring of the stomach mucosa; NOAEL = 

500 mg/kg-bw/day. LOAEL (developmental toxicity) > 750 mg/kg-bw/day based on no effects

at highest dose tested; LOAEL = 750 mg/kg-bw/day.

Results remarks: None specified
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CONCLUSIONS

The LOAEL for maternal toxicity was 750 mg/kg-bw/day based on decreased body weight gain 

and ulcerations and/or scarring of the stomach mucosa and the NOAEL was 500 mg/kg-bw/day. 

The LOAEL for developmental toxicity was > 750 mg/kg-bw/day based on no effects at the 

highest dose tested; the NOAEL was 750 mg/kg-bw/day.

STUDY RELIABILITY

[2] Reliable with restrictions. There was no indication from the OPP reviews that the study 

provided and analyzed certain developmental toxicity data (e.g., fetal weight, sex ratio) on a perlitter basis. 

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.

Ridgway, P. (1989) Triazine Rat Teratology Study: Reference No. LEF/8/89. Unpublished study 

prepared by Toxicol Laboratories Ltd. EPA/OPP MRID 41161801
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E. SUPPLEMENTAL DATA (NON-SIDS ENDPOINTS)

E-1. Genetic Toxicity – Other 

Study 1: Unscheduled DNA synthesis study in primary rat hepatocytes (Curren, 1988)

Title: Unscheduled DNA Synthesis in Rat Primary Hepatocytes: Test Article: Hexahydro1,3,5-tris(2-hydroxyethyl)-s-triazine

TEST SUBSTANCE 

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

and composition not provided

 

Remarks: Viscous yellow liquid, referred to in the review as hexahydro-1,3,5-

tris(hydroxyethyl)-s-triazine, Grotan, and Bioban GK.

METHOD

Method/guideline followed: OPPTS 870.5550

Test type: Primary rat hepatocyte unscheduled DNA systhesis assay

GLP compliant?: Not indicated

Year study performed: 1988 (date report was issued)

Test species: Primary hepatocytes from male F344 rats

Metabolic activation: Without

Concentrations tested: Based on the results of a preliminary cytotoxicity study, nine 

concentrations ranging from 0.0001 to 1.0 μL/mL were tested.

Statistical methods used: Not indicated. 

Test method/conditions remarks: For the assay to be considered valid, the following criteria 

must be satisfied: (1) the proportion of cells in repair in the solvent control group must be <15% 

and the net nuclear grain count of the solvent control group must be < 1.0; and (2) the positive 

control compound must induce a significant increase in the net nuclear grain count (≥ 5 

grains/nucleus over the negative control.

The assay was considered positive if the test material induced a concentration-related increase in 

mean net nuclear grains and one or more of the doses had an increase in the mean net nuclear 

grain count that was ≥ 5 grains/nucleus over the negative control. In the absence of a 
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concentration-related effect, a compound that showed nuclear grain counts that were ≥ 5 

grains/nucleus over two successive concentrations was also considered positive. 

RESULTS

In the preliminary cytotoxicity assay, ten concentrations (0.0003 to 10 μL/mL) of the test 

material were examined. The study author stated that the pH of the stock solution was adjusted 

to 7.4 with 1 N HC1 prior to dilution. However, there was a basic shift in the pH of cultures 

treated with the two highest assayed levels (3.0 and 10 μL/mL) at initiation and termination of 

treatment. The study author further stated that the lactate dehydrogenase (LDH) value could not 

be obtained for the highest test concentration, presumably because of test material interference 

with the LDH determination. Cytotoxicity, as indicated by increased leakage of LDH into the 

culture medium, did not proceed in a conventional concentration-related manner. At the highest 

assayed concentration for which the LDH level could be determined (3.0 μL/mL), the percent 

relative cytotoxicity was 1%; however, as the concentration was reduced, cytotoxicity increased 

to 52% at 1.0 μL/mL and 66% at 0.3 μL/mL. Although the percent relative cytotoxicity at 0.1 

μL/mL was 8%, the microscopic evaluation of the hepatocyte cultures revealed a cytotoxic 

effect. Below this level, the test material was not cytotoxic.

Two UDS and parallel cytotoxicity assays were conducted with nine concentrations of the test 

material (0.0001 to 1.0 μL/mL). Because of unspecified technical difficulties, the first assay was 

terminated.

In the repeat assay, the two highest test concentrations (0.3 and 1.0 μL/mL) induced marked 

cytotoxicity (85%) and were, therefore, not scored for UDS activity. The five levels selected for 

the evaluation of UDS induction were 0.1, 0.03, 0.01, 0.003, and 0.001 μL/mL. The highest test 

material concentration scored for UDS (0.10 μL/mL) induced a marked increase in the net 

nuclear grain count compared to the solvent control. Similarly, the percentage of cells with ≥5 

net nuclear grains was markedly higher at this level compared to the solvent control value. 

However, the evidence for a genotoxic effect was confined to the 0.10 μL/mL level. The mean 

net nuclear grain counts and percentage of cells with ≥5 net nuclear grains for hepatocytes 

exposed to 0.001, 0.003, 0.01, or 0.03 μL/mL of the test material, although higher than the 

solvent control, did not indicate a positive or a concentration-related response. The study author 

concluded that hexahydro-l,3,5-tris(2- hydroxyethyl)-s-triazine induced an equivocal response in 

this test system.

Genotoxicity result: Equivocal

Cytotoxic concentration (if applicable): 0.1 μL/mL

Results remarks: Responses to the positive control were acceptable.

CONCLUSIONS

The test material is classified as presumptively genotoxic for induction of unscheduled DNA 

synthesis in this test system.
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STUDY RELIABILITY

[2] Reliable with restrictions. In the absence of a concentration-related effect, confirmation of a 

“significant” increase at a single concentration is necessary to conclude a positive response in the 

UDS assay. However, the positive response was not concentration-related, was limited to the 

0.l0-μL/mL level, and the results were not confirmed. In addition, the study author failed to 

provide information on test material purity, the batch number of the assayed sample, and 

analytical data to verify actual concentrations used in the assay. 

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.

Curren, R. (1988) Unscheduled DNA Synthesis in Rat Primary Hepatocytes: Hexahydro-1,3,5-

tris(2-hydroxyethyl)-s-triazine: Study No. T8102.380. Unpublished study prepared by 

Microbiological Associates, Inc. EPA/OPP MRID 41262301.
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Study 2: Unscheduled DNA synthesis study in primary rat hepatocytes (San and Raabe, 1993)

Title: Unscheduled DNA synthesis in rat primary hepatocytes

TEST SUBSTANCE 

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

and composition not provided

 

Remarks: Straw-colored liquid, referred to in the review as hexahydro-1,3,5-tris(hydroxyethyl)-

s-triazine and Grotan.

METHOD

Method/guideline followed: OPPTS 870.5550

Test type: Primary rat hepatocyte unscheduled DNA systhesis assay

GLP compliant?: Yes

Year study performed: 1993 (date report was issued)

Test species: Primary hepatocytes from male F344 rats

Metabolic activation: Without

Concentrations tested: Based on the results of a preliminary cytotoxicity study, nine 

concentrations ranging from 0.01 to 0.3 μg/mL were tested.

Statistical methods used: Not indicated. 

Test method/conditions remarks: Following preliminary cytotoxicity testing (by lactate 

dehydrogenase, LDH, release determinations), triplicate coverslip cultures (per does) of rat 

hepatocytes were exposed to hexahydro-l, 3, 5-tris(2-hydroxyethyl)-s-triazine for 18-20 hours to 

a series of nine concentrations of test article (ranging from 0.01 to 0.30 μg/mL), together with a 

constant concentration of tritiated thymidine (HTdr, 10 μCi/mL). Other cultures were treated 

with 7, 12-dimethylebenz(a)anthracene (DMBA, 3 and 10 μg/mL), to serve as positive controls. 

Fifty cells per slide were read; net nuclear counts (NNC) were calculated (crude nuclear count 

less mean of three cytoplasmic counts) and were averaged for each treatment. In addition, the 

percentage of cells in repair was recorded for each test concentration. Means, standard 

deviations and percent survival were computed. The grain count results represent unscheduled 

DNA synthesis

A test result was considered positive if the NNC in test substance-treated cells was at least five 

counts over the solvent control. Criteria for assay validity were not presented. The study author 
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stated that conventional criteria for assessing assay validity and genotoxic responses were 

followed.

RESULTS

The preliminary cytotoxicity test demonstrated that the test article produced concentrationrelated relative toxicities (based on LDH release) from >80% at 1.0 μg/mL down to 1% at 0.1 

μg/mL. Hence, the HDT selected for the UDS assay itself was 0.3 μg/mL (at which related 

toxicity was 79%).

For the UDS assay, examination of the fixed and stained cells which had been treated at 

concentrations of 0.15, 0.2, 0.25, or 0.3 μg/mL revealed that these could not be evaluated for 

UDS “because of excessive cytotoxicity”, resulting in relative cell survivals less than 35%. 

Although none of the remaining (lower) test article concentrations (0.01 to 0.1 μg/mL) caused an 

increase in mean NNC, according to the authors’ criteria established for declaring a positive 

result, there was a concentration-related increase trend in both mean NNC, as well as percentage 

of cells showing DNA repair. 

Genotoxicity result: Equivocal

Cytotoxic concentration (if applicable): 0.1 μg/mL

Results remarks: Positive controls produced an acceptable response.

CONCLUSIONS

The assay is considered equivocally positive based on slight, but concentration-related, increases 

in both mean NNC and percentage of cells in repair at the highest three usable (i.e., non-toxic) 

concentrations of 0.05, 0.07, and 0.10 μg/mL. However, results at these concentrations did not 

reach the criterion for significance proposed by the investigators, namely, increase of ≥ 5 counts 

over control. 

STUDY RELIABILITY

[2] Reliable with restrictions. The study author did not provide information on test material 

purity, the batch number of the assayed sample, and analytical data to verify actual 

concentrations used in the assay. 

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.

San, R.; Raabe, H. (1993) Unscheduled DNA Synthesis in Rat Primary Hepatocytes: Final 

Report: Lab Project Number: TC836.380: SPGT380. Unpublished study prepared by 

Microbiological Associates, Inc. EPA/OPP MRID 43020001.
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E-2. Skin Irritation

Study 1: Dermal irritation study in rabbits (Guest and Collier, 1985)

Title: Primary Dermal Irritation Test: Determination of the Degree of Primary Cutaneous 

Irritation Caused by Glokill 77 in the Rabbit.

TEST SUBSTANCE 

Identity: 79.96% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 20.04% 

unidentified (Chen and Tao 2008) or composition not indicated (Guest and Collier 1985)

Remarks: Clear colorless viscous liquid, referred to in the reviews as Glokill 77, HHT, 

hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, and dihydro-5-(2-hydroxyethyl)-1,3,5-triazinel,3(2H,4H)-dipropanol

METHOD

Method/guideline followed: OPPTS 870.2500

Test type: Primary Dermal Irritation -- Rabbit

GLP compliant?: Not stated

Year study performed: 1985 (issue date)

Test species: New Zealand White rabbits, 12-16 weeks of age

Number of animals/sex/dose: 12, sex not indicated

Route of administration: Dermal

Vehicle: None

Doses/concentrations tested: 0.5 mL, undiluted

Statistical methods used: None

Test method/conditions remarks: Glokill 77 (undiluted; 0.5 ml) was applied to clipped back 

skin of six young adult (12-16 weeks of age; no sex data given) New Zealand White rabbits. The 

test sites were occluded with a patch of surgical gauze and elasticized corsets (Tubigrip), and 

exposure continued for 4 hours. Test sites were swabbed with cotton wool soaked in distilled 

water, and skin irritation was scored using a Draize method at 1, 24, 48, and 72 hours after 

treatment. The test substance produced minimal erythema in three rabbits and minimal edema in 

one rabbit at one hour after dosing. However, these effects had disappeared at 24 hours post-
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dosing, and no other effects were observed throughout the 72-hour study period. The calculated 

Primary Cutaneous Irritation Index was reported to be 0.0. 

RESULTS

Endpoint value(s): Not irritating. Primary Irritation Index was 0.0. No erythema or edema was 

seen from 24 to 72 hours. 

Results remarks: None specified

CONCLUSIONS

The test substance was not irritating to rabbit skin.

STUDY RELIABILITY

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

substance. 

REFERENCE

Chen, J.; Tao, J. (2008). 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.

Guest, R.L.; Collier, T.A. (1985). Primary Dermal Irritation Test: Determination of the Degree 

of Primary Cutaneous Irritation Caused by Glokill 77 in the Rabbit. Study Nos. 317/8505. 

Safepharm Laboratories Limited. EPA/OPP MRID 155986, as cited in Locke (1995).

Locke, R.K. (1995). Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine. Review of Toxicity Data. 

DP Barcode: D212071. Submission #: S481738. Document No. 011557.
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Study 2: Dermal irritation study in rabbits (Thomas, 1974)

Title: Glokill 77: Draize Skin Tests.

TEST SUBSTANCE 

Identity: 79.96% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 20.04% 

unidentified (Chen and Tao 2008) or composition not indicated (Thomas 1974)

Remarks: Clear viscous liquid, referred to in the reviews as Glokill 77 (Batch No. K249), HHT, 

hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, and dihydro-5-(2-hydroxyethyl)-1,3,5-triazinel,3(2H,4H)-dipropanol.

METHOD

Method/guideline followed: OPPTS 870.2500

Test type: Primary Dermal Irritation -- Rabbit

GLP compliant?: Not stated

Year study performed: 1974 (issue date)

Test species: New Zealand White rabbits [Note: According Table 3 of Chen and Tao (2008), 

the Guinea pig was the test species]

Number of animals/sex/dose: 4, sex not indicated

Route of administration: Dermal

Vehicle: Distilled water 

Doses/concentrations tested: 0.5 mg/site or 5 mg/site from application of 0.5 mL of a 0.1 or 1% 

solution, respectively.

Statistical methods used: None

Test method/conditions remarks: Test material was applied to clipped and depilated intact 

abraded back skin of four New Zealand White rabbits for each concentration of dosing solution. 

The treatment sites were occluded with sterile gauze and exposure continued for 24 hours. 

After 24 hours of exposure, the patches were removed and the skin reactions on both the abraded 

and unabraded skin scored by a Draize scoring system. Additional scores were recorded at 72 

hours post-treatment. 
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RESULTS

The 0.1% concentration was non-irritating to intact skin and, on abraded skin, produced only 

mild erythema in one animal at 24 hours post-treatment, which was absent at 72 hours after 

treatment. A concentration of 1.0% on intact skin produced only mild erythema in one animal at 

24 hours after treatment, which was absent at 72 hours post-treatment; on abraded skin, mild 

erythema was evident at 24 hours post-treatment for all four animals, but this condition persisted 

only for one animal at 72 hours post-treatment. Primary Irritation Indices were 0.06 for the 0.1% 

solution and 0.44 for the 1.0% solution. 

Endpoint value(s): Mildly irritating. Primary Irritation Indices were 0.06 for the 0.1% solution 

and 0.44 for the 1.0% solution. 

Results remarks: None specified

CONCLUSIONS

The test substance was mildly irritating to rabbit skin.

STUDY RELIABILITY

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

substance. 

REFERENCES

Chen, J.; Tao, J. (2008). 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.

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.

Thomas, M.B. (1974). Glokill 77: Draize Skin Tests. Study Nos. CL74:74:1024. Consultox 

Laboratories Ltd. EPA/OPP MRID 155987, as cited in Locke (1995).
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Study 3: Dermal irritation study in rabbits (Siglin et al., 1989b)

Title: Acute Skin Irritation/Sensitization Studies with Busan 1060

TEST SUBSTANCE 

Identity: 78.5% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 21.5% 

unidentified

Remarks: Referred to in the study report as Busan 1060, HHT, hexahydro-1,3,5-tris(2-

hydroxyethyl)-s-triazine, and 1,3,6-triazine, 1,3,5(2H,4H, 6H)-triethanol.

METHOD

Method/guideline followed: Not indicated.

Test type: Primary Dermal Irritation -- Rabbit

GLP compliant?: Not stated

Year study performed: 1989

Test species: New Zealand White rabbit

Number of animals/sex/dose: 6, sex not indicated

Route of administration: Dermal

Vehicle: None 

Doses/concentrations tested: 0.5 mg/site or 5 mg/site from application of 0.5 mL of a 0.1 or 1% 

solution, respectively.

Statistical methods used: None

Test method/conditions remarks: Animals received a single dermal application of Busan 1060 

(neat) as a 4-hour semi-occluded exposure. Dermal reactions were evaluated by the Draize 

scoring system at 1, 24, 48, and 72 hours after treatment.

RESULTS

Very slight erythema and edema were observed at test sites of 3/6 animals at 1 hour after 

treatment. Irritation resolved completely by 24 hours post-treatment.

Endpoint value(s): Mildly irritating. The Primary Irritation Index was 0.2/8 
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Results remarks: None specified

CONCLUSIONS

The test substance was mildly irritating to rabbit skin.

STUDY RELIABILITY

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

substance, the study method, and the sex of animals on test. 

REFERENCE

Siglin, J.C.; Rush, R.E.; Jenkins, P.K. et al. (1989b). Acute skin irritation/sensitization studies 

with Busan 1060. In: R.A. Parent (ed.), Acute Toxicity Data, J. Amer Coll. Toxicol., Part B, 

Vol. 1, No. 3, 1992, pp. 203-204.
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E-3. Eye Irritation

Study 1: Eye irritation study in rabbits (Collier and Axelrad, 1984)

Title: OECD Eye Irritation Test: Determination of the Degree of Ocular Irritation Caused by 

Glokill 77 in the Rabbit

TEST SUBSTANCE 

Identity: 79.96% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 20.04% 

unidentified (Chen and Tao 2008) or composition not indicated (Collier and Axelrad, 1984).

Remarks: Clear Liquid, referred to in reviews as Glokill 77, HHT, hexahydro-1,3,5-tris(2-

hydroxyethyl)-s-triazine, and dihydro-5-(2-hydroxyethyl)-1,3,5-triazine-l,3(2H,4H)-dipropanol.

METHOD

Method/guideline followed: OPPTS 870.2400

Test type: Primary Eye Irritation -- Rabbit

GLP compliant?: Not stated

Year study performed: 1984 (issue date)

Test species: New Zealand White rabbit, sex not indicated, 12-16 weeks old

Number of animals/sex/dose: 6 

Route of administration: Ocular

Vehicle: None

Doses/concentrations tested: 0.1 mL

Statistical methods used: None

Test method/conditions remarks: None specified

RESULTS

Glokill 77 (undiluted, 0.1 mL) was instilled into the right eyes and irritation was scored by the 

Draize method at 1, 24, 48, and 72 hours after treatment.
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Endpoint value(s): The test substance was severely irritating (corrosive) to the eyes of all 6 

rabbits. Effects at 72 hours included iritis, beefy-red coloration of the conjunctivae accompanied 

by considerable swelling, copious discharge from the eye, and areas of complete corneal opacity.

Results remarks: None specified

CONCLUSIONS

The test substance was severely irritating (corrosive) to rabbit eyes.

STUDY RELIABILITY

[2] Valid with restrictions. Information is needed on test substance composition and stability.

REFERENCES

Chen, J.; Tao, J. (2008). 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.

Collier, T.A.; Axelrad, J.C. (1984) OECD Eye Irritation Test: Determination of the Degree of 

Ocular Irritation Caused by Glokill 77 in the Rabbit. EPA/OPP MRIDs 41675206; 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.
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Study 2: Eye irritation study in rabbits (Douds, 1995)

Title: Not provided

TEST SUBSTANCE 

Identity: 78.5% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 21.5%

unidentified inert ingredients.

Remarks: Clear, light yellow viscous liquid, referred to in the review as Grotan, hexahydro1,3,5-tris(2-hydroxyethyl)-s-triazine, and BD-024-3A. 

METHOD

Method/guideline followed: OPP 81-4; OPPTS 870.2400

Test type: Primary Eye Irritation -- Rabbit

GLP compliant?: Not stated

Year study performed: 1995 (report date)

Test species: New Zealand White rabbit, 2.832-3.363 g

Number of animals/sex/dose: 3 

Route of administration: Ocular

Vehicle: None

Doses/concentrations tested: 0.5 mL

Statistical methods used: Not indicated

Test method/conditions remarks: None specified 

RESULTS

The test substance (undiluted, 0.5 mL) was instilled into the conjunctival sac of “six pre 

examined” eyes and irritation was scored by the Draize method at 1, 24, 48, and 72 hours and on 

days 7, 10, 14, and 21, 2 after treatment.

Endpoint value(s): The test substance was severely irritating (corrosive) to the eyes of all 6 

rabbits, with corneal opacity, unspecified effects on the iris, and redness, chemosis, and 

discharge of conjunctivae in 6/6 animals at 72 hours. The reviewer also noted sloughing of the 

corneal epithelium, vascularization of the cornea, raised areas on the cornea, blanching of the 
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conjunctivae, and hair loss around the eye (incidences of these effects were not provided in the 

review). 

Results remarks: None specified

CONCLUSIONS

The test substance was severely irritating (corrosive) to rabbit eyes.

STUDY RELIABILITY

[2] Valid with restrictions. Information is needed on test substance composition and stability. 

The stated volume of test material instilled into the eye (0.5 mL) exceeded the volume 

recommended by OPPTS Guideline 870.2400 (0.1 mL). The OPP reviewer questioned the 

Quality Assurance statement provided by the testing laboratory; the reviewer noted that the only 

inspected in-life portion of the study was indicated to be dose preparation, whereas it is “difficult 

to understand how the dose needed any preparation” because the study “used the liquid material 

as received.”

REFERENCE

Douds, D.A. (1995). Study title not provided. Springborn Report No. 3157-167, January 6, 

1995. EPA/OPP MRID 435173-01. Data Review for Acute Eye Irritation Testing (§81-4).
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E-4. Skin Sensitization

Study 1: Dermal sensitization study in guinea pigs (Thomas, 1974)

Title: Glokill 77: Guinea Pig Sensitization Test

TEST SUBSTANCE

Identity: 79.96% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 20.04% 

unidentified (Chen and Tao 2008) or composition not indicated (Thomas 1974)

Remarks: Clear viscous liquid, referred to in the reviews as Glokill 77 (Batch No. K249), HHT, 

hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, and dihydro-5-(2-hydroxyethyl)-1,3,5-triazinel,3(2H,4H)-dipropanol.

METHOD

Method/guideline followed: OPPTS 870.2600

Test type: Dermal Sensitization – Guinea Pigs

GLP (Y/N): Not stated

Species/strain: Hartley Guinea pigs, 300-400 g

Sex (male/female/both): Male

Number of animals/sex/dose: 6

Dose/concentration: Induction: 0.1 mL of 0.1% Glokill 77 in distilled water was injected 

intradermally every other day, using a different injection site for each treatment, for a total of 9 

injections. Injection sites were scored for erythema and edema by the Draize method at 24 hours 

after each injection. 

Challenge Period: Two weeks after final induction injection.

Challenge dose: 0.1 mL of 0.1% Glokill 77 in distilled water, injected intradermally 

Control: The Draize scores resulting from the challenge dose were compared with those obtained 

during the induction phase to determine if sensitization had be induced.

Test condition: Not stated

RESULTS

All animals survived to study termination. Slight erythema and/or edema were noted in all 

animals after each of the induction injections. No greater edema and/or erythema was noted

in any animal following the challenge injection than had been observed in the same animal 

following the induction injections.
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CONCLUSIONS

The test substance was mildly irritating but not a sensitizer to guinea pig 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.

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.

Thomas, M.B. (1974). Glokill 77: Guinea Pig Sensitization Test. Study Nos. CL74:74:1024. 

Consultox Laboratories Ltd. EPA/OPP MRID 155987, as cited in Locke (1995).
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Study 2: Dermal sensitization study in guinea pigs (Siglin et al., 1989b)

Title: Acute Skin Irritation/Sensitization Studies with Busan 1060

TEST SUBSTANCE

Identity: 78.5% 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4; 21.5% 

unidentified

Remarks: Referred to in the study report as Busan 1060, HHT, hexahydro-1,3,5-tris(2-

hydroxyethyl)-s-triazine, and 1,3,6-triazine, 1,3,5(2H,4H, 6H)-triethanol.

METHOD

Method/guideline followed: Buehler, E.V.; Griffith, F. (1975). Experimental skin sensitization 

in the guinea pig and man. In: Animal Models in Dermatology, Maibach, H. (ed.), pp. 56-66.

Test type: Delayed contact sensitization (modified Buehler design)

GLP (Y/N): Not stated

Species/strain: Hartley guinea pigs, 301-428 g

Sex (male/female/both): Both

Number of animals/sex/dose: 5

Dose/concentration: Induction: 0.4 mL topical occlusive application of 10% w/v Busan 1060 in 

distilled water once/week for 3 consecutive weeks. 

Challenge Period: Following a resting period of 2 weeks, the 10 induced animals received the 

first challenge application; 7 days later, the animals were rechallenged.

Challenge dose: 5% Busan 1060 in distilled water (volume not indicated)

Control: Naïve irritation control groups received 5% Busan 1060 at week 2 (10 animals) or week 

3 (a new group of 10 animals). 

Test condition: Not stated

RESULTS

Similar dermal irritation scores were seen in control and induced animals at 24 hours after the 

initial challenge. By 48 and 72 hours after the initial challenge, the response in induced animals 

“generally appeared more severe.” Following re-challenge, elevated irritation scores were seen in 

induced animals at 24 hours (7/10 animals) and 48 hours (3/10 animals) whereas minimal 

responses were seen in the naïve re-challenge control animals at both times. [NOTE: Postchallenge scores are not presented here because they were not legible in Siglin et al. (1989).]
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CONCLUSIONS

The test substance was considered to be a mild delayed contact sensitizer to guinea pig skin. 

STUDY RELIABILITY

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

substance. The irritation scoring system was not characterized and irritation scores were not 

legible in the cited study.

REFERENCE

Siglin, J.C.; Rush, R.E.; Jenkins, P.K. et al. (1989b). Acute skin irritation/sensitization studies 

with Busan 1060. In: R.A. Parent (ed.), Acute Toxicity Data, J. Amer Coll. Toxicol., Part B, 

Vol. 1, No. 3, 1992, pp. 204.
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F. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Aquatic 

Invertebrates

Yes Yes No

Toxicity to Aquatic Plants Yes Yes No

Health Effects

Acute Toxicity Yes Yes No

Repeated-Dose Toxicity Yes Yes No

Gene Mutation Yes Yes No

Chromosomal Aberrations Yes Yes No

Reproductive Toxicity No -- No1

Developmental Toxicity Yes Yes No

1The reproductive endpoint has been acceptably addressed on the basis that no effects on developmental endpoints 

were seen in an adequate oral developmental toxicity study in rats and no treatment-related effects on the histology 

of reproductive organs of either sex were seen in adequate 90-day oral and dermal toxicity studies in rats.