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/kg-bw/day

Statistical methods used:  Not stated

Test method/conditions remarks: Identity and stability of the test material were not determined by the investigating laboratory.  The test compound was held in contact with the skin by a porous gauze dressing covered with an Elastoplast elastic adhesive 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 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 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

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 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.

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.

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 ug/ 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.
Criteria for data evaluation were as follows:  (1) Mutagenic response:  a concentration-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; (2) Nonmutagenic response:  when no increase in the number of revertants was elicited by the chemical; and (3) Questionable response:  when there was an absence of a clear-cut concentration-related increase in revertants, when the concentration-related increases in the number of revertants were not reproducible, or when the response was of insufficient 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 his[-] pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn.
Positive control substances were tested concurrently.  Failure of the positive controls to induce mutation was considered reason to 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 ug/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.

REFERENCES

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

National Toxicology Program 1983.  Salmonella:  Study Summary for 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4.  Study ID 700221.
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 ug/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.
Criteria for data evaluation were as follows:  (1) Mutagenic response:  a concentration-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; (2) Nonmutagenic response:  when no increase in the number of revertants was elicited by the chemical; and (3) Questionable response:  when there was an absence of a clear-cut concentration-related increase in revertants, when the concentration-related increases in the number of revertants were not reproducible, or when the response was of insufficient 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 his[-] pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn.
Positive control substances were tested concurrently.  Failure of the positive controls to induce mutation was considered reason to 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-Triazine-1,3,5(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 ug/plate for strain TA100 and >= 333 ug/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.

REFERENCE

National Toxicology Program 2004.  Salmonella:  Study Summary for 1,3,5-Triazine-1,3,5(2H,4H,6H)-triethanol, CAS No. 4719-04-4.  Study ID A17569.
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 ug/ 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 concentration- response.  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 ug/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 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 ug/plate for TA1535 and TA100; 9-amino-acridine, 50 ug/plate for TA1537; 2-nitrofluorene, 0.5 ug/plate for TA1538 and TA98), but all to 2-aminoanthracene (2-AA, 2 ug/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 ug/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 ug/plate with or without S9 mix.

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

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.

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 ug/ 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.

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 ug/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 ug/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 ug/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.

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.
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.

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.
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.

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.

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.

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 hexahydro-1,3,5-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 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 14[th] ribs and retarded ossification of the vertebral thoracic centra that appeared to be dose-related.  However, since the increases were not statistically significant and the incidence of these abnormalities is highly variable in rats, the study authors and 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

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 per-litter 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

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:  Hexahydro-1,3,5-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 uL/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 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 uL/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 uL/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 uL/mL), the percent relative cytotoxicity was 1%; however, as the concentration was reduced, cytotoxicity increased to 52% at 1.0 uL/mL and 66% at 0.3 uL/mL.  Although the percent relative cytotoxicity at 0.1 uL/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 uL/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 uL/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 uL/mL.  The highest test material concentration scored for UDS (0.10 uL/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 uL/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 uL/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 uL/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.

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-uL/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.

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 ug/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 ug/mL), together with a constant concentration of tritiated thymidine (HTdr, 10 uCi/mL).  Other cultures were treated with 7, 12-dimethylebenz(a)anthracene (DMBA, 3 and 10 ug/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 stated that conventional criteria for assessing assay validity and genotoxic responses were followed.

RESULTS

The preliminary cytotoxicity test demonstrated that the test article produced concentration-related relative toxicities (based on LDH release) from >80% at 1.0 ug/mL down to 1% at 0.1 ug/mL.  Hence, the HDT selected for the UDS assay itself was 0.3 ug/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 ug/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 ug/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 ug/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 ug/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.
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-triazine-l,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-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.
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-triazine-l,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.

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).

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

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.

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.

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.
Study 2:  Eye irritation study in rabbits (Douds, 1995)

Title:  Not provided

TEST SUBSTANCE

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

Remarks:  Clear, light yellow viscous liquid, referred to in the review as Grotan, hexahydro-1,3,5-tris(2-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 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).

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-triazine-l,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.

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).

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:  Post-challenge scores are not presented here because they were not legible in Siglin et al. (1989).]

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.
F.  DATA ADEQUACY SUMMARY TABLE

                                   Endpoint
                          Study Information Available
                                  Acceptable
                                   Data Gap
Aquatic Effects
                            Acute Toxicity to Fish
                                      Yes
                                      Yes
                                      No
                    Acute Toxicity to Aquatic Invertebrates
                                      Yes
                                      Yes
                                      No
                          Toxicity to Aquatic Plants
                                      Yes
                                      Yes
                                      No
Health Effects
                                Acute Toxicity
                                      Yes
                                      Yes
                                      No
                            Repeated-Dose Toxicity
                                      Yes
                                      Yes
                                      No
                                 Gene Mutation
                                      Yes
                                      Yes
                                      No
                            Chromosomal Aberrations
                                      Yes
                                      Yes
                                      No
                            Reproductive Toxicity
                                      No
                                      --
                                     No[1]
                            Developmental Toxicity
                                      Yes
                                      Yes
                                      No

[1]The reproductive endpoint has been acceptably addressed on the basis that no effects on developmental endpoints were seen in an adequate oral developmental toxicity study in rats and no treatment-related effects on the histology of reproductive organs of either sex were seen in adequate 90-day oral and dermal toxicity studies in rats.


 ADVANCE \y63

	UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

	REGION III

1650 Arch Street

	Philadelphia, Pennsylvania  19103-2029

January 13, 2015

The Honorable Dana Aunkst

Acting Secretary, Pennsylvania Department of Environmental Protection

Rachel Carson State Office Building

P.O. Box 2063

Harrisburg, PA  17105-2063

Dear Acting Secretary Aunkst:

On December 4, 2014, the U.S. Environmental Protection Agency (EPA)
received a revision to the Pennsylvania State Implementation Plan (SIP)
from the Pennsylvania Department of Environmental Protection.  The SIP
revision requests redesignation from nonattainment to attainment, and
approval of the maintenance plan of the Johnstown 1997 annual and the
2006 24-hour PM2.5 area.  EPA has determined that the submittal is
administratively and technically complete, and is reviewing it to
prepare Notice of Proposed Rulemaking.

If members of your staff have any questions, they may direct them to
Rose Quinto, Office of Air Program Planning, at<PHONE_NUMBER>.  She is
the principal contact for this rulemaking.

	     Sincerely,

							/s/

David L. Arnold, Acting Director

                          			            Air Protection Division

cc:  Joyce E. Epps, Director, PADEP Bureau of Air Quality

	Customer Service Hotline: 1-800-438-2474


Page 1 of 8 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D. C. 20460 APR 14, 2006 MEMORANDUM: OFFICE OF PREVENTION, PESTICIDES AND TOXIC SUBSTANCES SUBJECT: Initial Ethical Review of Carbofuran Human Dermal Study FROM: John M. Carley TO: John Liccione, HED REF: Arnold, J. D. ( 1977) Carbamate ( Carbofuran) Human Dermal Study. Unpublished study prepared by Quincy Research Center. 36 p. ( MRID 92827; pp. 106 141 in CDL Accession 241303.)

I have performed an initial review of available information concerning the referenced document. This review characterizes the ethical conduct of the research in terms of both current ethical standards and ethical standards prevailing when the study was performed. The review applies the " Summary Framework for Ethical Assessment Using Seven Criteria of Emanuel et al." developed by the EPA Science Policy Committee's Human Studies Work Group. The completed " framework" is attached. This framework was derived from the work of Emanuel, et al. ( 2000), which summarizes seven general principles for ethical treatment of human subjects in scientific research. The Emanuel article was primarily directed at those who consider proposals for new medical research and decide which are worthy of funding or approval. These are very different decisions from those we in EPA must make when we determine whether we can ethically consider already completed human studies.

The Emanuel article reflects current standards for ethical research prevailing in the U. S. This study was conducted in the U. S. in late 1976, but cites no standard of ethical research conduct. I have applied FIFRA § 12( a)( 2)( P) and assumed the Declaration of Helsinki ( 1975) to have prevailed when the research was conducted.

A. Summary Assessment of Ethical Conduct of the Research Page 2 of 8 Here is a summary of my observations about the study under the seven headings used in the Emanuel framework. Supporting details are in the attachment.

1. Value of the Research to Society: This research has never been published, suggesting its purpose was not mainly to advance generalizable knowledge. Its purpose is characterized differently for different audiences. It is clear from the protocol that the scientific purpose was to define a LOAEL; it was described to volunteers in terms of a " safe dosage", perhaps to make them more likely to consent to participate. The study may provide some information on the toxicity of carbofuran to humans that could help to inform EPA's assessment of human health risks.

2. Scientific Validity of the Research: I defer to others for a full review of the scientific validity of this study. If it were determined not to have scientific validity, it would also not be ethically acceptable.

3. Subject Selection: Subjects were 18 healthy adult men. Restriction to men was consistent with the stated intent to explore occupational exposure patterns. Based on documentation in MRID 92829, the pool from which subjects were drawn consisted mainly of unemployed semi skilled workers.

4. Risk Benefit Ratio: Potential symptoms were accurately listed in the information for volunteers, but risks were not minimized by the study design, which committed to dose escalation until toxic signs were observed. Volunteers were also told plainly that they would not benefit from participating. How the potential societal benefit of improved safety for production workers was weighed against the risks to subjects  either by the investigator or by the review committee  was not reported.

5. Independent Ethical Review: The protocol and related materials, including both the procedures and the information associated with informed consent, were reported to have been approved by the Community Review Committee, Inc., of Kansas City, MO. The critical changes to the protocol to raise the doses in phase 2 were made after ethics approval was obtained, and without consulting the committee.

6. Informed Consent: Both the information for subjects and the consent form itself were remarkably clear and complete for research conducted in this period. Subjects were given no indication, however, that the design of the research required dose escalation until frank toxic signs were elicited.

7. Respect for Potential and Enrolled Subjects: Subjects' privacy was not compromised. They were free to withdraw at any time.

B. Compliance with Ethical Standard Prevailing when the Research Was Conducted No standard of ethical research conduct is cited, either by the reviewing ethics committee or by the authors. The research was conducted in the U. S. after 1972, so FIFRA § 12( a)( 2)( P) Page 3 of 8 applies. In addition, as clinical research this falls within the scope of the Declaration of Helsinki. I have applied both standards in assessing the conduct of this study.

  FIFRA Sec. 12( a)( 2)( P) states: " In general, [ i] t shall be unlawful for any person . . . to use any pesticide in tests on human beings unless such human beings ( i) are fully informed of the nature and purposes of the test and of any physical and mental health consequences which are reasonably foreseeable therefrom, and ( ii) freely volunteer to participate in the test."

Although the information provided to subjects was extraordinarily complete for research from this period, subjects were not told that the design of the study required dose escalation until frank toxic effects were elicited. Instead they were told that " The purpose of the testing . . . is to find out the maximum safe dosage when applied to the skin . . . . The nature of the test compound . . . is such that we do not expect serious complications from its use." This falls short of the requirement that they be " fully informed of . . . any physical . . . consequences which are reasonably foreseeable."

  Basic Principle # 5 of the Declaration of Helsinki ( 1975) reads " Every biomedical research project involving human subjects should be preceded by careful assessment of predictable risks in comparison to foreseeable benefits to the subject or to others. Concern for the interests of the subject must always prevail over the interest of science and society."

Societal benefits are not explicitly weighed against the risks to subjects. Comparable benefit could probably have been achieved through better methods of measuring ChE inhibition, without proceeding to elicitation of frank toxicity requiring administration of an antidote. The decisions to raise dose levels in phase 2 does not show that the interests of the subjects prevailed over other interests.

  Basic Principle # 12 of the Declaration of Helsinki ( 1975) reads " The research protocol should always contain a statement of the ethical considerations involved and should indicate that the principles enunciated in the present Declaration are complied with."

No discussion of substantive ethical considerations appears in the protocol.

C. Standards for Judging Ethical Acceptability On February 6, 2006, EPA published a final rule, " Protections for Subjects in Human Research," effective on April 7, 2006. Section 26.1704 of that regulation provides in pertinent part:

EPA shall not rely on data from any research initiated before [ effective date of the final rule] if there is clear and convincing evidence that the conduct of the research Page 4 of 8 was fundamentally unethical ( e. g., the research was intended to seriously harm participants or failed to obtain informed consent), or was significantly deficient relative to the ethical standards prevailing at the time the research was conducted.

In addition, section 26.1703 of the final rule provides in pertinent part:

EPA shall not rely on data from any research involving intentional exposure of any human subject who is a pregnant woman ( and therefore her fetus) or child.

I have applied the standards in sections 26.1704 and 26.1703 in arriving at the conclusions below.

D. Conclusion All subjects were adult males. Section 26.1703 therefore does not prohibit reliance on this study.

Although there are some gaps in the documentation of the ethical conduct of this study, it is extraordinarily well documented for research from this period. There is no clear evidence that the research was intended to harm participants, or that it was fundamentally unethical in other ways. Deficient documentation does not itself constitute evidence that the ethical conduct of this study was deficient relative to standards prevailing when it was conducted.

From the documentation available, I have identified some deficiencies relative to the standards of FIFRA § 12( a)( 2)( P) and the 1975 Declaration of Helsinki. These deficiencies do not, in my judgment, amount to " clear and convincing evidence" that this study was " fundamentally unethical." This review, however, does not take a position on either the persuasiveness of the evidence or the overall significance of the identified deficiencies relative to the prevailing ethical standards. This decision is deferred pending review of the research by the Human Studies Review Board as required by EPA regulation before EPA takes an action relying on this study.

Attachment Cited reference:

Emanuel, E.; Wender, D.; Grady, C. ( 2000) What Makes Clinical Research Ethical? JAMA 283: 2701 2711. Page 5 of 8 Framework for Ethical Assessment Using Seven Criteria of Emanuel et al. 1 April 14, 2006]

Arnold, JD ( 1977) Carbamate ( Carbofuran) Human Dermal Study. Unpublished study prepared by Quincy Research Center. 36 p. MRID 92827; pp. 106 141 in CDL Accession 241303.

1. Value: This research has never been published, suggesting its purpose was not mainly to advance generalizable knowledge. Its purpose is characterized differently for different audiences. It is clear from the protocol that the scientific purpose was to define a LOAEL; it was described to volunteers in terms of a " safe dosage", perhaps to make them more likely to consent to participate. The study may provide some information on the toxicity of carbofuran to humans that could help to inform EPA's assessment of human health risks.

a. What was the stated purpose of the research? " To determine the threshold toxicity level in normal male volunteers to single and multiple dermal doses of carbamate ( carbofuran) under normal and elevated temperatures and/ or humidities." ( p. 108)

" To find out the maximum safe dosage when applied to the skin for human beings. . . ." ( p. 120)

b. Does it evaluate a diagnostic or therapeutic intervention that could lead to improvements in health or well being? No c. Does it test a hypothesis that can generate important knowledge about structure or function of human biological systems? No d. Will society benefit from the knowledge gained from this research? Will its results be disseminated? It has never been published. Subjects were told " We do not expect you to derive any benefit from taking the test compound; however it is essential to determine the levels of this test compound that are safe for the people making it as well as the people exposed to it during its use." ( p. 120)

e. What government, organization, company and/ or institution( s) funded the research? FMC 2. Scientific Validity: I defer to others for a full review of the scientific validity of this study. If it were determined not to have scientific validity, it would also not be ethically acceptable.

a. Did the research have a clear scientific objective? See item 1( a) above. The scientific objective was to establish a human dermal LOAEL.

b. Was the research designed using accepted principles, methods, and reliable practices? I defer to the science reviewer. Page 6 of 8 c. In what way were human subjects intentionally dosed in this research, and what endpoints were identified or measured? Testing took place in three phases. In phase 1, subjects entered the clinic the evening before dosing. After eating a standard breakfast, two subjects at a time entered a controlled environment with high temperature and humidity. They were pre tested for ChE and neurovegetative signs immediately before administration of the dose, and repeatedly from 15 minutes to 6 h. post dose. For four hours post dose subjects alternated 5 minutes on an exercise bicycle with 15 minutes of rest, all the time in the chamber with elevated temperature and humidity. After a first pass with only the vehicle as a control, the initial dose was 0.5 mg/ kg; after evaluation of symptoms and data at each dose level, doses escalated by doubling. Escalation was discontinued at 2.0 mg/ kg because of frank toxic signs in both subjects.

In phase 2 subjects similarly were dosed after breakfast and then alternated exercise and rest for four hours, but at lower temperature and humidity. In this phase dosing began at 2.0 mg/ kg ( although the protocol originally specified it should begin at half the LOAEL from phase 1, or 1.0 mg/ kg) and doubled each time to 4.0 mg/ kg, then to 8.0 mg/ kg, and then the planned dose of 16 mg/ kg was skipped in favor of 32 mg/ kg.

In phase 3 two additional subjects received a dose of 1.0 mg/ kg under high temperature and humidity each day for three consecutive days.

Subjects remained in the clinic for 24 hours post dose. Measures included RBC and plasma ChEI, pulse, blood pressure, pupil size, eye accommodation, Fukuda step test, and ECG.

d. Did the research design have sufficient power to definitively test the objective? I defer to the science reviewer e. To what purpose is the study used, or proposed for use, in the Agency? To inform the WOE for reassessment of carbofuran 3. Fair Subject Selection: Subjects were 18 healthy adult men. Restriction to men was consistent with the stated intent to explore occupational exposure patterns. Based on documentation in MRID 92829, the pool from which subjects were drawn consisted mainly of unemployed semi skilled workers.

a. Were the groups and individuals recruited and enrolled determined solely on the basis of the scientific goals of the study? Eighteen healthy adult men participated in the research. Limitation to adult men was consistent with the stated intent to explore occupational exposure patterns. Exclusion factors included organic disease, use of medications before or during the study, or use of alcohol during the study. The protocol states " special attention will be paid to the use of tobacco", but does not indicate that it was controlled.

b. Were any susceptible groups used in the study, such as children, prisoners, infirm, or impoverished? Did the burden of participation fall disproportionately on a particular group? The pool from which volunteers were drawn was described in these terms: "[ V] olunteers were 19 to 58 year old men with 60% between 20 and 40 and a median age of 34. Seventeen percent were black, 80% were white, and 3% were from other ethnic groups. Fifty two percent had completed the twelfth grade, and 16 percent had attended college. Sixty percent were semi skilled workers and 8% were steadily employed." [ MRID 92829, p. 85]

4. Favorable Risk Benefit Ratio: Potential symptoms were accurately listed in the information for volunteers, but risks were not minimized by the study design, which committed to dose escalation until toxic signs were observed. Volunteers were also told plainly that they would not benefit from participating. How the potential societal benefit of improved safety for production workers was weighed against the risks to subjects  either by the investigator or by the review committee  was not reported. Page 7 of 8 a. How were the risks to individual subjects minimized? Research was conducted in a clinic under medical supervision. Subjects were told what kinds of symptoms they might experience, and encouraged to report any complaints. Atropine was available in the clinic, and was administered intravenously to the two subjects who received the 2 mg/ kg dose at high temperature and humidity. A study design with a more robust baseline and better methods for analyzing ChE inhibition could probably have generated similar knowledge without exposing the subjects to doses producing frank toxic signs requiring administration of an antidote. The decision to change the protocol for phase 2 to raise the starting dose level and the subsequent decision to quadruple the NOAEL level of 8 mg/ kg did not show a commitment to risk minimization.

b. If the research presents no health related benefits to individual subjects, what are the societal benefits in terms of knowledge from the study, and do these justify the excess risk to individual subjects? The results of this research may have been used to improve safety of Furadan production workers.

c. What compensation was paid to the participants in the study? Not reported 5. Independent Review: The protocol and related materials, including both the procedures and the information associated with informed consent, were reported to have been approved by the Community Review Committee, Inc., of Kansas City, MO. The critical changes to the protocol to raise the doses in phase 2 were made after ethics approval was obtained, and without consulting the committee.

a. Was the research asserted to have been overseen by an ethics review body? Yes, the protocol and informed consent materials were reportedly reviewed and approved by the Community Review Committee, Inc., of Kansas City, MO.

b. Was the research subject to independent review by individuals unaffiliated with the clinical research? Yes.

c. Was the research conducted in compliance with the Common Rule? It pre dates the Common Rule d. Does/ did the research institution ( or any institution participating in the research) hold a Federal Wide Assurance or Multi Project Assurance during the period of the study? n/ a e. Was the research conducted in compliance with another standard? What standard? No ethical standard was cited. FIFRA § 12( a)( 2)( P) applies, as does the Declaration of Helsinki ( 1975).

6. Informed Consent: Both the information for subjects and the consent form itself were remarkably clear and complete for research conducted in this period. Subjects were given no indication, however, that the design of the research required dose escalation until frank toxic signs were elicited.

a. Does the research assert that informed consent was obtained from participants? Yes b. How and under what circumstances was informed consent obtained? The " Volunteer agreement and informed consent" was read aloud, verbatim, and then the subjects were asked to sign the consent form.

7. Respect for Potential and Enrolled Subjects: Subjects' privacy was not compromised. They were free to withdraw at any time. Page 8 of 8 a. Was information about individual subjects managed so as to ensure their privacy? " The collection and submission of the medical information from this study will be accomplished with strict adherence to professional standards of confidentiality."

b. Were subjects free to withdraw from the research without penalty? " As a volunteer, I understand that I am free to withdraw and discontinue my participation at any time upon my request. Both participation in the study as well as possible withdrawal are at my own free will without coercion, duress, or intimidation of any sort."

1 Emanuel, E; Wender, D; Grady, C ( 2000) What Makes Clinical Research Ethical? JAMA 283: 2701 2711.