v>EPA United States Environmental Protection Agency Office of Chemical Safety and Pollution Prevention Final Risk Evaluation for n-Methylpyrrolidone Systematic Review Supplemental File: Data Quality Evaluation of Human Health Hazard Studies - Animal and In Vitro Studies CASRN: 872-50-4 December 2020 ------- EPA's Office of Pollution Prevention and Toxics (OPPT) developed data quality criteria for animal and in vitro studies, presented in the Application of Systematic Review in TSCA Risk Evaluations document (EPA Document #740-Pl-8001). This document presents data quality evaluation results for animal and in vitro studies evaluated for the NMP Risk Evaluation. ------- Table Listing Acute (<24 hr) 1 Animal toxicity evaluation results of Tatsuno et al., 2014 for an immunotoxicity study on immune outcomes 4 2 Animal toxicity evaluation results of Lashmar et al., 1989 for an irritation study, dermal 24-hour study on irritation outcomes 6 Short-term (1-30 days) 3 Animal toxicity evaluation results of Boenisch et al., 2012 for a 19-day immuno- toxicity (inhalation) study on hematological, immune, and respiratory outcomes . 9 4 Animal toxicity evaluation results of Lee et al., 1987 for a 4^week inhalation study on respiratory, hematological, and immune outcomes 12 5 Animal toxicity evaluation results of Malek et al., 1997 for a 28-day oral, rats and mice study on renal, hematology, and histopathology of various organs 14 6 Animal toxicity evaluation results of E.I. DuPont De Nemours Co. 1991 for a 4-week inhalation, mortality, and histopathology study on respiratory, endocrine, hematological, and immune outcomes 16 7 Animal toxicity evaluation results of Gopinathan et al., 2013 for a 5-day oral study on clinical chemistry/biochemical, renal, and hematological and immune outcomes 18 8 Animal toxicity evaluation results of N-Methylpyrrolidone Producers Group 1994 for a 4-week dietary study in mice on mortality, nutrition and metabolic/adult exposure body weight, renal, hepatic, hematological and immune, clinical chem- istry/biochemical, ADME/PBPK, and reproductive (male) outcomes 21 9 Animal toxicity evaluation results of N-Methylpyrrolidone Producers Group 1994 for 28-day diet study in rats on systemic and reproductive effects 24 Subchronic (30-90 days) 10 Animal toxicity evaluation results of Malley et al., 1999 for a 90-day oral rats and mice study on neurological/behavior, body weight, hepatic, and renal outcomes . 26 11 Animal toxicity evaluation results of BASF 1995 for a 90-day diet mouse-liver toxicity study on hepatic outcomes 28 12 Animal toxicity evaluation results of Becci et al., 1983 for a 13-week diet study in dogs on body weight, hematological and immune outcomes 30 Chronic (>90 days) 13 Animal toxicity evaluation results of Malley et al., 2001 for an oral cancer rats and mice study on cancer, hepatic, and renal outcomes 32 14 Animal toxicity evaluation results of N-Methylpyrrolidone Producers Group 1999 for 18-month oral cancer study in mice on cancer and hepatic outcomes 34 15 Animal toxicity evaluation results of E.I. Dupont De Nemours Co. 1982 for a 2-year inhalation study in rats on renal and cancer outcomes 36 16 Animal toxicity evaluation results of N-Methylpyrrolidone Producers Group 1997 for a 2-year cancer bioassay study 38 Genetic toxicity studies 17 In vitro evaluation results ofMortelmans et al., 1986 for bacterial reverse mutation 40 18 In vitro evaluation results of Wells et al., 1988 for Ames test 43 19 In vitro evaluation results of Wells et al., 1988 for Ames test with N-MeGABA . . 46 Page 1 of 110 ------- 20 Animal toxicity evaluation results of Engelhardt and Fleig 1993 for micronucleus and chromosomal aberrations assays 49 Developmental and Reproductive 21 Animal toxicity evaluation results of Lee et al., 1987 for an inhalation developmental study on growth (early life) and development outcomes 53 22 Animal toxicity evaluation results of Sitarek et al., 2008 for a reproductive-male study on reproductive outcomes 56 23 Animal toxicity evaluation results of Saillenfait et al., 2002 for an oral develop- mental rat and maternal effects study on growth (early life), development, and reproductive outcomes 58 24 Animal toxicity evaluation results of Saillenfait et al., 2003 for inhalation study on developmental and reproductive outcomes 60 25 Animal toxicity evaluation results of Exxon 1992 for a developmental toxicity study in rats on growth (early life) and development outcomes 62 26 Animal toxicity evaluation results of Solomon et al., 1995 for a 2-generation re- production/developmental study, inhalation study on reproductive, and growth (early life) and development outcomes 64 27 Animal toxicity evaluation results of Intl Specialty 1979 for dermal developmen- tal, offspring and maternal effects study on growth (early life) and development outcomes 67 28 Animal toxicity evaluation results of Exxon Biomedical 1991 for a multigenera- tion reproductive toxicity study on rats reproductive, growth (early life) and developmental outcomes 69 29 Animal toxicity evaluation results for NMP Producers Group 1999 for a 2-generation dietary reproductive toxicity study in Sprague Dawley rats 74 30 Animal toxicity evaluation results of NMP Producers Group 1999 for a 2-generation dietary reproductive toxicity study in Wistar rats 78 31 Animal toxicity evaluation results of E. I. du Pont de Nemours and Company 1990 for Reproductive and developmental inhalation toxicity study in rats on developmental outcomes 81 32 Animal toxicity evaluation results of E. I. du Pont de Nemours and Company 1990 for Reproductive and developmental inhalation toxicity study in rats on reproductive outcomes 84 33 Animal toxicity evaluation results of Ciba-Geigy 1987 for an oral developmental study in mice on growth (early life) and development outcomes 86 34 Animal toxicity evaluation results of Ciba-Geigy 1987 for an oral developmental rat study on growth (early life) and development outcomes 88 35 Animal toxicity evaluation results of GAF Chemicals Corp 1991 for developmental toxicity study in rabbits (GD 6-12) on growth (early life) and development outcomes 90 36 Animal toxicity evaluation results of Dupont 1979 for developmental range-finding study in rats on growth (early life) and development outcomes 92 37 Animal toxicity evaluation results of Sitarek et al., 2012 for single generation repro- ductive toxicity assessment in rats exposed orally study on reproductive outcomes 94 38 Animal toxicity evaluation results of Sitarek et al., 2012 for a single generation reproductive toxicity assessment in rats exposed orally study on hematological and immune, respiratory, endocrine, hepatic, renal, neurological/behavior, and thyroid outcomes 97 39 Animal toxicity evaluation results of Becci et al., 1982 for a dermal, developmental study in rats (rf and final-study report in 4214125) on growth (early life) and development outcomes 100 Page 2 of 110 ------- Mechanistic 40 In vitro evaluation results for Gjoksi et al., 2016 for an inhibition of bromodomain binding study 102 41 In vitro evaluation results for Gjoksi et al., 2015 for an inhibition of bromodomain binding study 105 42 In vitro evaluation results for Shortt et al., 2014 for an inhibition of bromodomain binding study 108 Page 3 of 110 ------- 1 Acute (<24 hr) Table 1: Animal toxicity evaluation results of Tatsuno et al., 2014 for an immunotoxicity study on immune outcomes Study Citation: Tatsuno, T; Miyazaki, K; Yamashiro, H (2014). Multiple solvent, N-methyl-2-pyrrolidone, acts as a novel adjuvant for enhancing cutaneous immune responses Bioscience, Biotechnology, and Biochemistry, 78(6), 954-959 Data Type: Immunotoxicity HERO ID: 3540753 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1 Test Substance Identity Medium X 2 4 Test substance identified by name, but more infor- mation may be in reference describing preparation of test tapes. Metric 2 Test Substance Source Low X 1 3 Source of test substance not identified. Metric 3 Test Substance Purity Low X 1 3 Purity not reported Domain 2: Test Design Metric 4 Negative and Vehicle Controls High X 2 2 Animals given a placebo were included. Metric 5 Positive Controls Not Rated NA NA Positive controls not required. Metric 6 Randomized Allocation Low X 1 3 Method of randomized allocation was not reported. Domain 3: Exposure Characterization Metric 7 Preparation and Storage of Test Substance Low X 1 3 Preparation not reported. Metric 8 Consistency of Exposure Administration High X 1 1 Exposures were administered consistently. Metric 9 Reporting of Doses/Concentrations Medium X 2 4 Percentages were reported. Metric 10: Exposure Frequency and Duration High X 1 1 Exposure frequencies were reported. Metric 11: Number of Exposure Groups and Dose Spac- Medium X 1 2 The number of groups and spacing were reported only. ing Metric 12: Exposure Route and Method High X 1 1 The route and method appear appropriate for the test substance and the purposes of the study. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium X 2 4 The source, species, strain, and age were reported. Health status and initial body weight were not re- ported. Metric 14: Adequacy and Consistency of Animal Hus- Low X 1 3 Husbandry conditions were not reported. bandry Conditions Metric 15: Number per Group High X 1 1 The number of animals per group were appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High X 2 2 Outcome assessment methodology was described. Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate. Continued on next page . . Page 4 of 110 ------- ... continued from previous page Study Citation: Tatsuno, T; Miyazaki, K; Yamashiro, H (2014). Multiple solvent, N-methyl-2-pyrrolidone, acts as a novel adjuvant for enhancing cutaneous immune responses Bioscience, Biotechnology, and Biochemistry, 78(6), 954-959 Data Type: Immunotoxicity HERO ID: 3540753 Domain Metric Rating^ MWF* Score Comments^ Metric 19: Blinding of Assessors Not Rated NA NA Blinding not required. Metric 20: Negative Control Response High x 1 1 Negative and placebo responses were reported and appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables were observed. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposure were re- ported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were appropriate. Metric 24: Reporting of Data High x 2 2 Outcome data were reported. Overall Quality Determination" High —~ Medium5 Extracted No MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = J]. (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. § Evaluator's explanation for rating change: "Additional information should be obtained from the reference." Page 5 of 110 ------- Table 2: Animal toxicity evaluation results of Lashmar et al., 1989 for an irritation study, dermal 24-hour study on irritation outcomes Study Citation: Lashmar, UT; Hadgraft, J; Thomas, N (1989). Topical application of penetration enhancers to the skin of nude mice: A histopathological study Journal of Pharmacy and Pharmacology, 41(2), 118-122 Data Type: irritation study, dermal 24-hour HERO ID: 3539872 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity Metric 2: Test Substance Source Metric 3: Test Substance Purity Medium X 2 4 Test substance identified by name; CASRN not re- ported. Medium X 1 2 The source manufacturer was reported, but batch/lot numbers were not; this omission is unlikely to have a substantial impact on results. Low X 1 3 Test substance reported to be "reagent grade," but no specific purity was reported. Domain 2: Test Design Metric 4: Negative and Vehicle Controls Metric 5: Positive Controls Metric 6: Randomized Allocation Medium x 2 Not Rated NA Low x 1 NA 3 A negative control group (untreated skin) was used; it is unclear if the untreated skin area was subjected to the same conditions as the exposed area. A 1% (w/w) neutralized carbomer gel was also tested as a control. The use of a positive control is not indicated by this study type. The study did not report how animals were allocated to study groups. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Metric 8: Consistency of Exposure Administration Metric 9: Reporting of Doses/Concentrations Metric 10: Exposure Frequency and Duration Metric 11: Number of Exposure Groups and Dose Spac- ing Metric 12: Exposure Route and Method Low X 1 3 Test substance preparation was briefly reported. There is a lack of reporting of the test substance storage. Medium X 1 2 Details of exposure protocol were limited; however, this deficiency is unlikely to have a substantial im- pact on the results. High X 2 2 Administered doses are reported. High X 1 1 Exposure duration (24- hours) was reported and ap- propriate for the study. Medium X 1 2 The number of exposure groups and the dose spacing were not justified by the study authors; however, the number of doses and spacing are adequate to show results relevant to the outcome of interest. Medium X 1 2 The exposure route and method are appropriate and reported with limited detail; however, this deficiency is unlikely to have a substantial impact on the re- sults. Continued on next page . .. Page 6 of 110 ------- .. . continued from previous page Study Citation: Lashmar, UT; Hadgraft, J; Thomas, N (1989). Topical application of penetration enhancers to the skin of nude mice: A histopathological study Journal of Pharmacy and Pharmacology, 41(2), 118-122 Data Type: irritation study, dermal 24-hour HERO ID: 3539872 Domain Metric Rating^ MWF* Score Comments^ Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium X 2 4 The test animal species, strain, sex, age, and start- ing body weight are reported, but there is no infor- mation on the health status of the mice reported. This omission in reporting is unlikely to have a sub- stantial impact on results. It is unclear if this species (nude mouse) is an adequate animal model for the outcome of interest. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Low X 1 3 Animal husbandry conditions were not sufficiently reported to determine whether husbandry was ade- quate. Metric 15: Number per Group High X 1 1 The number of animals per study group was reported (3) and appropriate for the study type. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Medium X 2 4 The outcome assessment methodology was partially addressed for the intended outcomes of interest re- ported Metric 17: Consistency of Outcome Assessment High X 1 1 The assessment protocol were reported and consis- tent across study groups and chemicals tested. Metric 18: Sampling Adequacy High X 1 1 Details regarding sampling for the outcomes of in- terest were reported and adequate. Metric 19: Blinding of Assessors Not Rated NA NA This metric is not applicable for initial histopathol- ogy review; therefore, is not rated for this study. Metric 20: Negative Control Response High X 1 1 The untreated control and 1% gel control response was adequate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Medium X 2 4 PVC and PVCD were used for occluded dermal ex- posure, it is unclear how this may have impacted the results. Metric 22: Health Outcomes Unrelated to Exposure Medium X 1 2 There is no reporting for attrition and/or health out- comes unrelated to exposure, though it is unlikely to have a substantial impact on results. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Medium X 1 2 No statistical analysis methods or statistical results were reported; however, calculation methods for ir- ritation scores were described. Continued on next page . . Page 7 of 110 ------- ... continued from previous page Study Citation: Lashmar, UT; Hadgraft, J; Thomas, N (1989). Topical application of penetration enhancers to the skin of nude mice: A histopathological study Journal of Pharmacy and Pharmacology, 41(2), 118-122 Data Type: irritation study, dermal 24-hour HERO ID: 3539872 Domain Metric Rating^ MWF* Score Comments^ Metric 24: Reporting of Data Medium X 2 4 Not all data was reported for the outcome. Only the model relative irritancy score for the representative tested animals. Irritation scores for individual ani- mals was not reported for any exposure group. Histopathological results are presented qualitatively as images of skin sections. Overall Quality Determination1" Medium 1.9 Extracted No MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 8 of 110 ------- 2 Short-term (1-30 days) Table 3: Animal toxicity evaluation results of Boenisch et al., 2012 for a 19-day immunotoxicity (inhalation) study on hematological, immune, and respiratory outcomes Study Citation: Boenisch, U; Boehme, A; Kohajda, T; Moegel, I; Schuetze, N; von Bergen, M; Simon, J; Lehmann, I; Polte, T (2012). Volatile organic compounds enhance allergic airway inflammation in an experimental mouse model PLoS ONE, 7(7), e39817 Data Type: 19-day immunotoxicity (inhalation) HERO ID: 2333837 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Metric 2: Test Substance Identity Test Substance Source Metric 3: Test Substance Purity High X 2 2 The test substance was identified definitively. Low X 1 3 The source and batch/lot number of the test sub- stance was not reported. Low X 1 3 Purity and grade of the test substance were not re- ported. Domain 2: Test Design Metric 4: Metric 5: Metric 6: Negative and Vehicle Controls Positive Controls Randomized Allocation High X 2 2 The study authors reported using a concurrent neg- ative control group. Not Rated NA NA Positive controls were not required. Low X 1 3 The study authors did not report how animals were allocated to study groups. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low x 1 Metric 8: Consistency of Exposure Administration Low x 1 Metric 9: Reporting of Doses/Concentrations Medium x 2 Metric 10: Exposure Frequency and Duration High x 1 Metric 11: Number of Exposure Groups and Dose Spac- High x 1 ing Metric 12: Exposure Route and Method Low x 1 3 The study authors did not describe the test sub- stance preparation or storage conditions. The re- porting deficiencies are likely to have a substantial impact on results. 3 Critical exposure details, including the methods for generating atmosphere in inhalation chambers, were not reported. 4 Mean concentrations were measured (as reported in supplemental file SI). 1 The exposure duration and frequency were reported and were suitable for the study type (the animals were exposed in a whole-body inhalation chamber from days 0-19 or days 17-19). 1 The number of exposure groups and spacing were reported and were relevant for the assessment. 3 The exposure route and method of exposure (whole body) were reported; however, there were reporting deficiencies in the chamber (e.g., number of changes per hour). Continued on next page .. Page 9 of 110 ------- .. . continued from previous page Study Citation: Boenisch, U; Boehme, A; Kohajda, T; Moegel, I; Schuetze, N; von Bergen, M; Simon, J; Lehmann, I; Polte, T (2012). Volatile organic compounds enhance allergic airway inflammation in an experimental mouse model PLoS ONE, 7(7), e39817 Data Type: 19-day immunotoxicity (inhalation) HERO ID: 2333837 Domain Metric Rating^ MWF* Score Comments^ Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium X 2 4 Ovalbumin-sensitized and non-sensitized mice were used. The body weight, and health status of mice at the start of the study were not reported. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions High X 1 1 All husbandry conditions were reported and were ad- equate and the same for the control and exposed populations. Metric 15: Number per Group Low X 1 3 The number per group was not clearly reported in the methods (other than stating that all animal ex- periments involved groups of 4-6 mice/cage). How- ever, the results appear to have been based on at least 9 animals per group (e.g., see Figures 4, 6A), although some results were based on only 4 animals (e.g., Fig 6B, 6C). Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High X 2 2 The outcome assessment methodology addressed or reported the intended outcomes of interest and was sensitive for the outcomes of interest. Metric 17: Consistency of Outcome Assessment High X 1 1 Details of the outcome assessment protocol were reported, and outcomes were assessed consistently across study groups. Metric 18: Sampling Adequacy High X 1 1 Sampling of the outcomes of interest were adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding is not required for objective outcomes. Metric 20: Negative Control Response High X 1 1 The negative control responses were reported and acceptable. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Low X 2 6 Respiratory rate measurement was not reported. This is may impact results since NMP is a poten- tial respiratory irritant. Metric 22: Health Outcomes Unrelated to Exposure Low X 1 3 Data on attrition and health outcomes unrelated to exposure were not reported for each study group and this deficiency may have a substantial impact on re- sults. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High X 1 1 Statistical methods were reported and were appro- priate for the data sets. Continued on next page . . Page 10 of 110 ------- ... continued from previous page Study Citation: Boenisch, U; Boehme, A; Kohajda, T; Moegel, I; Schuetze, N; von Bergen, M; Simon, J; Lehmann, I; Polte, T (2012). Volatile organic compounds enhance allergic airway inflammation in an experimental mouse model PLoS ONE, 7(7), e39817 Data Type: 19-day immunotoxicity (inhalation) HERO ID: 2333837 Domain Metric Rating^ MWF* Score Comments^ Metric 24: Reporting of Data High X 2 2 Data for exposure-related findings were presented for all outcomes by exposure group with quantal or continuous presentation. Negative findings were re- ported qualitatively and/or quantitatively. Overall Quality Determination1" Medium 1.8 Extracted No MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 11 of 110 ------- Table 4: Animal toxicity evaluation results of Lee et al., 1987 for a 4-week inhalation study on respiratory, hematological, and immune outcomes Study Citation: Lee, KP; Chromey, NC; Culik, R; Barnes, JR; Schneider, PW (1987). Toxicity of N-methyl-2-pyrrolidone (NMP): Teratogenic, subchronic, and two-year inhalation studies Fundamental and Applied Toxicology, 9(2), 222-235 Data Type: 4-wk inhalation HERO ID: 3539878 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Structure, nomenclature, physiochemical properties provided Metric 2: Test Substance Source Low x 1 3 Source not identified Metric 3: Test Substance Purity Medium x 1 2 Purity such that effects due to test substance. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Negative controls were included Metric 5: Positive Controls Not Rated NA NA Positive controls not required Metric 6: Randomized Allocation Low x 1 3 Method of allocation was not described Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Medium x 1 2 Preparation of test atmospheres was described, stor- age of the test material was not. Metric 8: Consistency of Exposure Administration High X 1 1 Exposures were administered consistently Metric 9: Reporting of Doses/Concentrations Low x 2 6 Aerosol particle size and MMAD were not reported. Metric 10: Exposure Frequency and Duration Medium x 1 2 Duration of exposure did not extend throughout organogenesis. Metric 11: Number of Exposure Groups and Dose Spac- Medium x 1 2 Rationale for dose selection was not provided Metric 12: ing Exposure Route and Method Low x 1 3 Route was appropriate, but insufficient detail was provided on the method (e.g., humidity, number of air changes per hour) Domain 4: Test Organism Metric 13: Test Animal Characteristics Low x 2 6 The source, species, strain, and sex were provided; age, and initial body weight, and health status were not reported. Metric 14: Adequacy and Consistency of Animal Hus- Low x 1 3 Husbandry was not adequately reported. bandry Conditions Metric 15: Number per Group High x 1 1 The number of animals exposed in each group was adequate Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Low x 2 6 Breathing rate and body temperature were not mea- sured. Continued on next page . .. Page 12 of 110 ------- . continued from previous page Study Citation: Lee, KP; Chromey, NC; Culik, R; Barnes, JR; Schneider, PW (1987). Toxicity of N-methyl-2-pyrrolidone (NMP): Teratogenic, subchronic, and two-year inhalation studies Fundamental and Applied Toxicology, 9(2), 222-235 Data Type: 4-wk inhalation HERO ID: 3539878 Domain Metric Rating^ MWF* Score Comments^ Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate for the outcomes Metric 19: Blinding of Assessors Not Rated NA NA Subjective outcomes were not assessed Metric 20: Negative Control Response High x 1 1 Negative controls responded appropriately Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Low x 2 6 Test animals showed signs of lethargy and irregular respiration. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 None were reported Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Medium x 1 2 Statistical methods were appropriate when applied, but all outcomes were not analyzed. Metric 24: Reporting of Data High x 2 2 Data were reported for outcomes Overall Quality Determination"1 Medium 2.0 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = J]. (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 13 of 110 ------- Table 5: Animal toxicity evaluation results of Malek et al., 1997 for a 28-day oral, rats and mice study on renal, hematology, and histopathology of various organs Study Citation: Malek, DE; Malley, LA; Slone, TW; Elliott, GS; Kennedy, GL; Mellert, W; Deckardt, K; Gembardt, C; Hildebrand, B; Murphy, SR; Bower, DB; Wright, GA (1997). Repeated dose toxicity study (28 days) in rats and mice with N-methylpyrrolidone (NMP) Drug and Chemical Toxicology, 20(1-2), 63-77 Data Type: 25-day oral, rats and mice HERO ID: 3539910 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Test substance identified by name and CASRN. Metric 2: Test Substance Source High x 1 1 The source was identified. Metric 3: Test Substance Purity High x 1 1 The reported purity was such that effects likely due to the test substance. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Concurrent negative control animals were included. Metric 5: Positive Controls Not Rated NA NA Positive controls not required. Metric 6: Randomized Allocation Medium x 1 2 Animals were allocated by computerized stratified randomization so no statistically significant differ- ences among body weights were observed. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low x 1 3 Limited preparation details were presented; how- ever, no information on NMP analytical concentra- tion, storage or stability were reported. Metric 8: Consistency of Exposure Administration High X 1 1 Exposures were administered consistently. Metric 9: Reporting of Doses/Concentrations High x 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration High x 1 1 Frequency and duration of exposure were reported. Metric 11: Number of Exposure Groups and Dose Spac- High x 1 1 The number of exposure groups and spacing were ing reported and justified. Metric 12: Exposure Route and Method High x 1 1 The exposure route and method were appropriate. Domain 4: Test Organism Metric 13: Test Animal Characteristics High x 2 2 The source, species, strain, sex, age, and initial body weight, and health status were reported. Metric 14: Adequacy and Consistency of Animal Hus- Medium x 1 2 All husbandry conditions except room air changes bandry Conditions were reported. Metric 15: Number per Group High x 1 1 The numbers of animals per group were appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 Outcome assessment methodology was appropriate. Metric 17: Consistency of Outcome Assessment High x 1 1 Outcomes were assessed consistently. Continued on next page . . Page 14 of 110 ------- .. . continued from previous page Study Citation: Data Type: HERO ID: Malek, DE; Malley, LA; Slone, TW; Elliott, GS; Kennedy, GL; Mellert, W; Deckardt, K; Gembardt, C; Hildebrand, B; Murphy, SR; Bower, DB; Wright, GA (1997). Repeated dose toxicity study (28 days) in rats and mice with N-methylpyrrolidone (NMP) Drug and Chemical Toxicology, 20(1-2), 63-77 25-day oral, rats and mice 3539910 Domain Metric Rating^ MWF* Score Comments^ Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding was not required. Metric 20: Negative Control Response High x 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables were reported. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health effects unrelated to exposure were re- ported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were reported and appropriate. Metric 24: Reporting of Data High x 2 2 Data were reported. Overall Quality Determination"1 High — -> High§ Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. This metric met the criteria for high confidence as expected for this type of study. § Evaluator's explanation for rating change: "Although no information on NMP analytical concentration, storage or stability were reported; the study protocol is valid.1 Page 15 of 110 ------- Table 6: Animal toxicity evaluation results of E.I. DuPont De Nemours Co. 1991 for a 4-week inhalation, mortality, and histopathol- ogy study on respiratory, endocrine, hematological, and immune outcomes Study Citation: Haskell Laboratories (1991). Initial submission: Four-week inhalation range-finding test on l-methyl-2-pyrrolidone (final report) with attachments and cover letter dated 112691 920000398 #88-920000398 Data Type: 4-wk inhalation, mortality, histopathology HERO ID: 3563360 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Test substance identified by name and CASRN. Metric 2: Test Substance Source Low x 1 3 Test substance submitted by identified persons. Metric 3: Test Substance Purity Low x 1 3 Purity was not reported. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Concurrent controls were used. Metric 5: Positive Controls Not Rated NA NA Positive controls were not required. Metric 6: Randomized Allocation Low x 1 3 Randomized allocation of animals was not reported. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Medium x 1 2 Methods and equipment used to generate the aerosol were reported; however, storage of the test material was not reported. Metric 8: Consistency of Exposure Administration High X 1 1 Exposures were administered consistently. Metric 9: Reporting of Doses/Concentrations Unacceptable x 2 8 Nominal and measured concentrations were re- ported, but particle size and MMAD were not re- ported. Metric 10: Exposure Frequency and Duration Medium x 1 2 Duration was reported, but frequency, in terms of days/week, was reported only as 21 six- hour expo- sures Metric 11: Number of Exposure Groups and Dose Spac- High x 1 1 The number of groups and spacing were reported ing and justified. Metric 12: Exposure Route and Method Low x 1 3 The number of air changes in the exposure chamber was not reported. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium x 2 4 The species, strain, sex, and initial body weight were reported. The age, health status, and source were not reported. Metric 14: Adequacy and Consistency of Animal Hus- Low x 1 3 Husbandry conditions were not reported. bandry Conditions Metric 15: Number per Group High x 1 1 The number of animals per group was appropriate. Domain 5: Outcome Assessment Continued on next page . .. Page 16 of 110 ------- . continued from previous page Study Citation: Haskell Laboratories (1991). Initial submission: Four-week inhalation range-finding test on l-methyl-2-pyrrolidone (final report) with attachments and cover letter dated 112691 920000398 #88-920000398 Data Type: 4-wk inhalation, mortality, histopathology HERO ID: 3563360 Domain Metric Rating^ MWF* Score Comments^ Metric 16: Outcome Assessment Methodology Low x 2 6 Breathing rates and body temperature were not measured to rule out reflex bradypnea from irri- tancy. Metric 17: Consistency of Outcome Assessment High x 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High x 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding was not required. Metric 20: Negative Control Response High x 1 1 Negative controls responded appropriately. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Low x 2 6 Test animals showed signs of lethargy and irregu- lar respiration which persisted until the end of each exposure. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposures were re- ported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Medium x 1 2 Statistical methods were described in the appendices and were appropriate. Analysis of histopathological results was not conducted. Metric 24: Reporting of Data High x 2 2 Data were reported. Overall Quality Determination1 Unacceptable*^ 2.0 Extracted No * Consistent with our Application of Systematic Review in TSCA Risk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4), EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score is presented solely to increase transparency. MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 17 of 110 ------- Table 7: Animal toxicity evaluation results of Gopinathan et al., 2013 for a 5-day oral study on clinical chemistry/biochemical, renal, and hematological and immune outcomes Study Citation: Data Type: HERO ID: Gopinathan, S; O'Neill, E; Rodriguez, LA; Champ, R; Phillips, M; Nouraldeen, Amr; Wendt, M; Wilson, AGE; Kramer, JA (2013). In vivo toxicology of excipients commonly employed in drug discovery in rats Journal of Pharmacological and Toxicological Methods, 68(2), 284-295 5-day oral 3037621 Domain Metric Ratingt MWF* Score Comments^ Domain 1: Test Substance Metric 1: Metric 2: Test Substance Identity Test Substance Source Metric 3: Test Substance Purity Medium X 2 4 The test substance was identified. Medium X 1 2 The source of the test substance was reported, but a batch/lot number was not reported. Low X 1 3 Test substance purity was not reported, but all sub- stances tested in the study were stated to have been of reagent or pharmacopeia grade. Domain 2: Test Design Metric 4: Metric 5: Metric 6: Negative and Vehicle Controls Positive Controls Randomized Allocation Low x 2 Not Rated NA Medium x 1 6 The study authors reported using a concurrent neg- ative control group; however, details were not fully reported on the negative control group (whether the negative control received the same preparation as used for the test substance (e.g., vehicle). NA Positive controls were not required. 2 The study reported methods of allocation of animals to study groups, but minor limitations were observed in that animals were randomly stratified by body weight. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low Metric 8: Consistency of Exposure Administration High Metric 9: Reporting of Doses/Concentrations High Metric 10: Exposure Frequency and Duration High Metric 11: Number of Exposure Groups and Dose Spac- High ing X 1 3 Preparation and storage of the test substance were not reported. X 1 1 Details of the exposure administration were re- ported, and exposures were administered consis- tently across study groups in a scientifically sound manner (e.g., dose volume was acceptable). X 2 2 Administered doses were reported without ambigu- ity. X 1 1 The exposure frequency and duration were reported and were appropriate for the study type and out- comes of interest (acute toxicity). X 1 1 The number of exposure groups and dose spacing was reported and was acceptable. Continued on next page ... Page 18 of 110 ------- . continued from previous page Study Citation: Data Type: HERO ID: Gopinathan, S; O'Neill, E; Rodriguez, LA; Champ, R; Phillips, M; Nouraldeen, Amr; Wendt, M; Wilson, AGE; Kramer, JA (2013). In vivo toxicology of excipients commonly employed in drug discovery in rats Journal of Pharmacological and Toxicological Methods, 68(2), 284-295 5-day oral 3037621 Domain Metric Rating^ MWF* Score Comments^ Metric 12: Exposure Route and Method High X 1 1 The exposure route and method were reported and were acceptable. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium Metric 14: Adequacy and Consistency of Animal Hus- Low bandry Conditions Metric 15: Number per Group High x 2 x 1 x 1 The test animal source, species, strain, and sex were reported; however, age, health status, and starting body weights were not reported. Most husbandry conditions (e.g., temperature, light cycle, housing) were reported and were adequate and similar for all groups; however, humidity levels were not reported. The number of animals per group (5 males/group) was reported, appropriate for the study type and outcome analysis, and consistent with studies of the same or similar type. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Metric 17: Consistency of Outcome Assessment Metric 18: Sampling Adequacy Metric 19: Blinding of Assessors Metric 20: Negative Control Response High High High x 2 x 1 x 1 Not Rated NA High x 1 NA 1 The outcome assessment methodology addressed or reported the intended outcomes of interest and was sensitive for the outcomes of interest (acute effects). Details of the outcome assessment methodology were reported, and outcomes were assessed consistently across study groups using the same protocol for all groups. Details regarding sampling for the outcomes of in- terest were reported and the study used adequate sampling for the outcomes of interest. Blinding was not required. The negative control responses were reported and acceptable. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and High x 2 Procedures Metric 22: Health Outcomes Unrelated to Exposure Low x 1 2 There were no reported differences among the study groups that would influence the outcome. 3 Data on attrition and health outcomes unrelated to exposure were not reported for each study group and this deficiency may have a substantial impact on re- sults. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were clearly described and were appropriate for the data sets. Continued on next page . .. Page 19 of 110 ------- .. . continued from previous page Study Citation: Data Type: HERO ID: Gopinathan, S; O'Neill, E; Rodriguez, LA; Champ, R; Phillips, M; Nouraldeen, Amr; Wendt, M; Wilson, AGE; Kramer, JA (2013). In vivo toxicology of excipients commonly employed in drug discovery in rats Journal of Pharmacological and Toxicological Methods, 68(2), 284-295 5-day oral 3037621 Domain Metric Rating^ MWF* Score Comments^ Metric 24: Reporting of Data Low X 2 6 Data for exposure-related outcomes were reported for most, but not all, outcomes by exposure group. For example, mottled kidneys were observed in all treated groups with a combined incidence of 8/15 rats (not observed in the control group). The in- cidence of mottled kidneys observed by dose group were not reported. Overall Quality Determination1" Medium — Medium5 4-r8 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. This metric met the criteria for high confidence as expected for this type of study. § Evaluator's explanation for rating change: "I would downgrade this paper to medium based on a relatively large number of reporting deficiencies (e.g., preparation and storage of the test substance; incomplete reporting of necropsy results)." Page 20 of 110 ------- Table 8: Animal toxicity evaluation results of N-Methylpyrrolidone Producers Group 1994 for a 4-week dietary study in mice on mortality, nutrition and metabolic/adult exposure body weight, renal, hepatic, hematological and immune, clinical chem- istry/biochemical, ADME/PBPK, and reproductive (male) outcomes Study Citation: N-Methylpyrrolidone Producers Group (1994). Repeated dose toxicity with N-Methylpyrrolidone in B6C3F1 mice Administration in the diet for 4 weeks (Range-finding study) Data Type: 4-wk dietary study HERO ID: 4214115 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 The test substance was identified definitely and CASRN reported. Metric 2: Test Substance Source High X 1 1 The source of the testing laboratory (industry spon- sored) test substance was reported, including man- ufacturer and batch/lot number. Metric 3: Test Substance Purity High X 1 1 The test substance purity was reported. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 Study authors reported using an appropriate con- current negative control group. Metric 5: Positive Controls Not Rated NA NA Not required for study type Metric 6: Randomized Allocation Medium X 1 2 Randomization list was drawn up by a computer based on body weight (laboratory data processing, Dept of Toxicology, BASF) Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High X 1 1 The test substance preparation and storage condi- tions were reported and appropriate for the test sub- stance. Metric 8: Consistency of Exposure Administration High X 1 1 Details of exposure administration were reported, and exposures were administered consistently across study groups. Metric 9: Reporting of Doses/Concentrations High X 2 2 The study authors reported the administered doses/concentrations, including the calculated infor- mation from ppm in diet to mg/kg-day in the diet for all the exposed groups without ambiguity. Metric 10: Exposure Frequency and Duration High X 1 1 The exposure frequency and duration of exposure were reported. Metric 11: Number of Exposure Groups and Dose Spac- High X 1 1 The number of exposure groups (n=4) and ing dose/concentration spacing were justified by study authors and considered adequate to address the pur- pose of the study. Metric 12: Exposure Route and Method High X 1 1 The route and method of exposure were reported and were suited to the test substance. Continued on next page . .. Page 21 of 110 ------- . continued from previous page Study Citation: N-Methylpyrrolidone Producers Group (1994). the diet for 4 weeks (Range-finding study) Data Type: 4-wk dietary study HERO ID: 4214115 Repeated dose toxicity with N-Methylpyrrolidone in B6C3F1 mice Administration in Domain Metric Rating^ MWF* Score Comments^ Domain 4: Test Organism Metric 13: Test Animal Characteristics High Metric 14: Adequacy and Consistency of Animal Hus- High bandry Conditions Metric 15: Number per Group Medium x 2 x 1 x 1 The test animal species, strain, sex, health status, age, and starting body weight were reported and the test animal was obtained from a commercial source or laboratory-maintained colony. The study authors reported all husbandry conditions and were adequate and the same for control and ex- posed populations, such that the only difference was exposure. The reported number of animals per study group was lower than the typical number used in studies of the same or similar type. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Metric 17: Consistency of Outcome Assessment Metric 18: Sampling Adequacy Metric 19: Blinding of Assessors Metric 20: Negative Control Response High X 2 2 The outcome assessment methodology addressed or reported the intended outcome(s) of interest and was sensitive for the outcomes(s) of interest. Medium X 1 2 Incomplete reporting of minor details of outcome as- sessment protocol execution, but these uncertainties or limitations are unlikely to have a substantial im- pact on results. High X 1 1 Details regarding sampling for the outcome(s) of in- terest were reported and the study used adequate sampling for the outcome(s) of interest. Low X 1 3 The study did not report whether assessors were blinded to treatment group for subjective outcomes, and this deficiency is likely to have a substantial im- pact on results. High X 1 1 The biological responses of the negative control group(s) were adequate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and High Procedures Metric 22: Health Outcomes Unrelated to Exposure High x 2 x 1 There were no reported differences among the study groups in initial body weight, food intake, or respi- ratory rate that could influence the outcome assess- ment. The authors did not report water intake, but it is not likely to have a significant impact on results. Details regarding animal attrition and health out- comes unrelated to exposure were reported for each study group. Domain 7: Data Presentation and Analysis Continued on next page Page 22 of 110 ------- ... continued from previous page Study Citation: N-Methylpyrrolidone Producers Group (1994). Repeated dose toxicity with N-Methylpyrrolidone in B6C3F1 mice Administration in the diet for 4 weeks (Range-finding study) Data Type: 4-wk dietary study HERO ID: 4214115 Domain Metric Rating^ MWF* Score Comments^ Metric 23: Statistical Methods Metric 24: Reporting of Data High High X 1 x 2 1 2 Statistical methods were clearly described and ap- propriate for dataset(s). Data for exposure-related findings were presented for all outcomes by exposure group. Overall Quality Determination"1' High 1.2 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 23 of 110 ------- Table 9: Animal toxicity evaluation results of N-Methylpyrrolidone Producers Group 1994 for 28-day diet study in rats on systemic and reproductive effects Study Citation: N-Methylpyrrolidone Producers Group (1994). Subchronic Oral Toxicity: 28-day Feeding study in Rats with N-Methylpyrrolidone (NMP) Data Type: 28-day diet rat-systemic and repro effects HERO ID: 42I4f24 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Test substance identified by name, synonyms, CASRN, form, and structure. Metric 2: Test Substance Source High x 1 1 The source was identified along with production date, and tank number. Metric 3: Test Substance Purity High x 1 1 Purity and composition are such that effects are likely due to the test substance. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Concurrent negative control animals were included. Metric 5: Positive Controls Not Rated NA NA Positive control animals not required. Metric 6: Randomized Allocation Medium x 1 2 Allocation wad by computerized stratified random- ization to prevent no statistically significant differ- ences among mean body weights by sex. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High x 1 1 Preparation and storage conditions were appropri- ate. Stability, homogeneity, and concentration anal- ysis were conducted and appropriate. Metric 8: Consistency of Exposure Administration High X 1 1 Diets were administered consistently. Metric 9: Reporting of Doses/Concentrations High x 2 2 Doses were reported without ambiguity. Metric 10: Exposure Frequency and Duration High x 1 1 Exposure frequency and duration were reported. Metric 11: Number of Exposure Groups and Dose Spac- ing High x 1 1 The number of exposure groups and dose spacing were based on the results of previous studies. Metric 12: Exposure Route and Method High x 1 1 The route and method were as specified in TPA TSCA testing consent order. Domain 4: Test Organism Metric 13: Test Animal Characteristics High x 2 2 The source, species, strain, sex, age, initial body weight, and health status were reported. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions High x 1 1 All husbandry conditions were reported and were ap- propriate. Metric 15: Number per Group High x 1 1 The number of animals per group was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 Outcome assessment methodology was reported. Continued on next page . . Page 24 of 110 ------- . continued from previous page Study Citation: N-Methylpyrrolidone Producers Group (1994). (NMP) Data Type: 28-day diet rat-systemic and repro effects HERO ID: 4214124 Subchronic Oral Toxicity: 28-day Feeding study in Rats with N-Methylpyrrolidone Domain Metric Rating^ MWF* Score Comments^ Metric 17: Consistency of Outcome Assessment High X 1 1 Outcome assessment was consistent across groups. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate for the outcomes of interest. Metric 19: Blinding of Assessors Not Rated NA NA Blinding not required for the outcomes. Metric 20: Negative Control Response High x 1 1 Negative controls responded appropriately. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and High x 2 2 A palatability study was conducted prior to the Procedures study which reported that the dietary levels was likely to be tolerated by the rats in a 28-day study. No other confounding variables in test design or pro- cedures were observed. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposure were re- ported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical analyses were described and appropriate. Metric 24: Reporting of Data High x 2 2 All outcomes were reported. Overall Quality Determination High 1.0 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. This metric met the criteria for high confidence as expected for this type of study. Page 25 of 110 ------- 3 Subchronic (30-90 days) Table 10: Animal toxicity evaluation results of Malley et al., 1999 for a 90-day oral rats and mice study on neurological/ behavior, body weight, hepatic, and renal outcomes Study Citation: Malley, LA; Kennedy, GL; Elliott, GS; Slone, TW; Mellert, W; Deckardt, K; Gembardt, C; Hildebrand, B; Parod, RJ; McCarthy, TJ; Griffiths, JC (1999). 90-day subchronic toxicity study in rats and mice fed N-methylpyrrolidone (NMP) including neurotoxicity evaluation in rats Drug and Chemical Toxicology, 22(3), 455-480 Data Type: 90-day oral rats and mice HERO ID: 3539912 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1 Test Substance Identity High x 2 2 Test substance identified by name and CASRN. Metric 2 Test Substance Source Medium x 1 2 The source was identified. Metric 3 Test Substance Purity High x 1 1 The reported purity was such that effects likely due to test substance. Domain 2: Test Design Metric 4 Negative and Vehicle Controls High x 2 2 Negative control animals were included. Metric 5 Positive Controls Not Rated NA NA Positive controls not required. Metric 6 Randomized Allocation Medium x 1 2 Animals allocated by computerized stratified ran- domization. Domain 3: Exposure Characterization Metric 7 Preparation and Storage of Test Substance High x 1 1 Limited preparation details were reported, but no storage information was presented. Stability of the test substance in the diet was established. Metric 8 Consistency of Exposure Administration Medium X 1 2 Exposures were administered consistently. Metric 9 Reporting of Doses/Concentrations High x 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration High x 1 1 Frequency and duration were reported. Metric 11: Number of Exposure Groups and Dose Spac- ing High x 1 1 The number of exposure groups and spacing were reported and justified. Metric 12: Exposure Route and Method High x 1 1 The route and method were reported. Domain 4: Test Organism Metric 13: Test Animal Characteristics High x 2 2 The source, species, strain, sex, age, initial body weight, and health status were reported. Metric 14: Adequacy and Consistency of Animal Hus- Medium x 1 2 All conditions except for room air changes were re- bandry Conditions ported. Metric 15: Number per Group High x 1 1 the number of animals per group was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 Outcome assessment methodology was reported. Continued on next page . . Page 26 of 110 ------- .. . continued from previous page Study Citation: Data Type: HERO ID: Malley, LA; Kennedy, GL; Elliott, GS; Slone, TW; Mellert, W; Deckardt, K; Gembardt, C; Hildebrand, B; Parod, RJ; McCarthy, TJ; Griffiths, JC (1999). 90-day subchronic toxicity study in rats and mice fed N-methylpyrrolidone (NMP) including neurotoxicity evaluation in rats Drug and Chemical Toxicology, 22(3), 455-480 90-day oral rats and mice 3539912 Domain Metric Rating^ MWF* Score Comments^ Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Medium X 1 2 Experimenters conducting the FOB evaluations were blind with respect to the exposure group of each an- Metric 20: Negative Control Response High X 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and High X 2 2 No confounding variables were reported. Procedures Metric 22: Health Outcomes Unrelated to Exposure High X 1 1 No health outcomes unrelated to exposure were ob- served. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High X 1 1 Statistical methods were appropriate. Metric 24: Reporting of Data High X 2 2 Data were reported. Overall Quality Determination1" High 1.2 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. This metric met the criteria for high confidence as expected for this type of study. Page 27 of 110 ------- Table 11: Animal toxicity evaluation results of BASF 1995 for a 90-day diet mo use-liver toxicity study on hepatic outcomes Study Citation: BASF (1995). N-methylpyrrolidone - subchronic oral toxicity study in b6c3fl mice by administration in the diet for 3 months, with cover letter dated 11/22/95 Data Type: 90-day diet mouse-liver toxicity HERO ID: 3585204 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 Test substance identified by name and CASRN Metric 2: Test Substance Source Medium X 1 2 Source not identified but production container was Metric 3: Test Substance Purity High X 1 1 Purity (99.8%) determined by gas chromatography and such that effects likely due to the test substance. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 Concurrent negative controls were used. Metric 5: Positive Controls Not Rated NA NA Positive controls not required. Metric 6: Randomized Allocation Medium X 1 2 Animals allocated by weight using a computerized randomization list. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High X 1 1 Preparation and storage details were provided. Sta- bility, homogeneity, and concentration tests were conducted. Metric 8: Consistency of Exposure Administration High X 1 1 Exposures were administered consistently. Metric 9: Reporting of Doses/Concentrations High X 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration High X 1 1 Frequency and duration of exposures were reported. Metric 11: Number of Exposure Groups and Dose Spac- High X 1 1 The number of groups and dose spacing were re- ing ported and justified. Metric 12: Exposure Route and Method High X 1 1 Route and method were appropriate. Domain 4: Test Organism Metric 13: Test Animal Characteristics High X 2 2 The source, species, strain, sex, initial body weight, and health status were reported. Metric 14: Adequacy and Consistency of Animal Hus- Medium X 1 2 All husbandry conditions except air changes were bandry Conditions reported. Metric 15: Number per Group High X 1 1 The number of animals per group was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High X 2 2 Outcome assessment methodology was appropriate. Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding not required. Continued on next page . . Page 28 of 110 ------- ... continued from previous page Study Citation: BASF (1995). N-methylpyrrolidone - subchronic oral toxicity study in b6c3fl mice by administration in the diet for 3 months, with cover letter dated 11/22/95 Data Type: 90-day diet mouse-liver toxicity HERO ID: 3585204 Domain Metric Rating^ MWF* Score Comments^ Metric 20: Negative Control Response High X 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and High x 2 2 No confounding variables were observed. Procedures Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposure were ob- served. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical analysis was conducted and appropriate. Metric 24: Reporting of Data High x 2 2 Data were presented. Overall Quality Determination"1' High 1.1 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 29 of 110 ------- Table 12: Animal toxicity evaluation results of Becci et al., 1983 for a 13-week diet study in dogs on body weight, hematological and immune outcomes Study Citation: Becci, PJ; Gephart, LA; Koschier, FJ; Johnson, WD; Burnette, LW (1983). Subchronic feeding study in beagle dogs of N- methylpyrrolidone Journal of Applied Toxicology, 3(2), 83-86 Data Type: 13-wk diet dogs HERO ID: 3539728 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Test substance identified by name and CASRN. Metric 2: Test Substance Source Low x 1 3 The test substance was obtained from the study sponsor (GAF Corp). Metric 3: Test Substance Purity High x 1 1 The purity of the test substance was reported (99.9%). Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Negative control animals were included. Metric 5: Positive Controls Not Rated NA NA Positive controls were not required. Metric 6: Randomized Allocation Low x 1 3 Allocation method was not reported. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low x 1 3 Preparation was reported, but storage and analysis was not. Metric 8: Consistency of Exposure Administration Medium X 1 2 Exposures were adjusted weekly according to body- weight in treated but not control animals. Metric 9: Reporting of Doses/Concentrations High x 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration Low x 1 3 The pattern of exposure is inadequate for assessing the outcome of interest(developmental toxicity). Metric 11: Number of Exposure Groups and Dose Spac- Low x 1 3 The number of groups and spacing were reported, ing but not justified. Metric 12: Exposure Route and Method Low x 1 3 It is unclear whether the test diet was prepared daily. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium x 2 4 The source, species, strain, and age were reported. Health status and initial body weight were not re- ported. Metric 14: Adequacy and Consistency of Animal Hus- Medium x 1 2 Humidity, housing, and room air changes were not bandry Conditions reported. Metric 15: Number per Group High x 1 1 The number of animals was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 Outcome assessment methodology was reported and appropriate. Continued on next page . . Page 30 of 110 ------- . continued from previous page Study Citation: Becci, PJ; Gephart, LA; Koschier, FJ; Johnson, WD; Burnette, LW (1983). methylpyrrolidone Journal of Applied Toxicology, 3(2), 83-86 Data Type: 13-wk diet dogs HERO ID: 3539728 Subchronic feeding study in beagle dogs of N- Domain Metric Rating^ MWF* Score Comments^ Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding was not required. Metric 20: Negative Control Response High x 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Low x 2 6 It is unclear why the test substance was diluted in corn oil or how this may impact toxicity. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposure were ob- served. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Medium x 1 2 Statistical methods were described with some omis- sions that may impact results. Metric 24: Reporting of Data High x 2 2 Data were reported. Overall Quality Determination Medium 1.7 Extracted No MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. This metric met the criteria for high confidence as expected for this type of study. Page 31 of 110 ------- 4 Chronic (>90 days) Table 13: Animal toxicity evaluation results of Malley et al., 2001 for an oral cancer rats and mice study on cancer, hepatic, and renal outcomes Study Citation: Malley, LA; Kennedy, GL; Elliott, GS; Slone, TW; Mellert, W; Deckardt, K; Kuttler, K; Hildebrand, B; Banton, MI; Parod, RJ; Griffiths, JC (2001). Chronic toxicity and oncogenicity of N-methylpyrrolidone (NMP) in rats and mice by dietary administration Drug and Chemical Toxicology, 24(4), 315-338 Data Type: Oral cancer rats and mice HERO ID: 3539913 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Test substance identified by name and CASRN. Metric 2: Test Substance Source Medium x 1 2 Source identified by name. Metric 3: Test Substance Purity High x 1 1 The reported purity was such that effects are likely due to the test substance. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Negative control animals were included. Metric 5: Positive Controls Not Rated NA NA Positive controls were not required. Metric 6: Randomized Allocation Medium x 1 2 allocation was by computerized stratified random- ization. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Medium x 1 2 Preparation details were limited, but stability, ho- mogeneity, and concentration analysis were con- ducted. Metric 8: Consistency of Exposure Administration High X 1 1 Exposures were administered consistently. Metric 9: Reporting of Doses/Concentrations High x 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration Medium x 1 2 Frequency and duration information were reported. Metric 11: Number of Exposure Groups and Dose Spac- ing High x 1 1 The number of groups and spacing were reported and justified. Metric 12: Exposure Route and Method High x 1 1 Route and method were appropriate. Domain 4: Test Organism Metric 13: Test Animal Characteristics High x 2 2 The source, species, strain, age, initial body weight, and health status were reported. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Medium x 1 2 All conditions were reported except for room air changes. Metric 15: Number per Group High x 1 1 The number of animals was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 Outcome assessment methodology was appropriate. Continued on next page . . Page 32 of 110 ------- .. . continued from previous page Study Citation: Data Type: HERO ID: Malley, LA; Kennedy, GL; Elliott, GS; Slone, TW; Mellert, W; Deckardt, K; Kuttler, K; Hildebrand, B; Banton, MI; Parod, RJ; Griffiths, JC (2001). Chronic toxicity and oncogenicity of N-methylpyrrolidone (NMP) in rats and mice by dietary administration Drug and Chemical Toxicology, 24(4), 315-338 Oral cancer rats and mice 3539913 Domain Metric Rating^ MWF* Score Comments^ Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were administered consistently. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding was not required. Metric 20: Negative Control Response High x 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables were reported. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposures were re- ported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were reported and appropriate. Metric 24: Reporting of Data High x 2 2 Data were reported. Overall Quality Determination High 1.2 Extracted No MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 33 of 110 ------- Table 14: Animal toxicity evaluation results of N-Methylpyrrolidone Producers Group 1999 for 18-month oral cancer study in mice on cancer and hepatic outcomes Study Citation: N-Methylpyrrolidone Producers Group (1999). N-methylpyrrolidone - carcinogenicity study in B6C3F1 mice, administration in the diet for 18 months, with cover letter dated 11/23/1999 Data Type: 18-month oral cancer mice HERO ID: 3566221 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 Test substance identified by name and CASRN. Metric 2: Test Substance Source Medium X 1 2 The source was not identified but the date of man- ufacture and container number were reported. Metric 3: Test Substance Purity High X 1 1 The purity was determined by gas chromatography and such that effects likely due to test substance. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 Negative controls were included. Metric 5: Positive Controls Not Rated NA NA Positive controls were not required. Metric 6: Randomized Allocation Medium X 1 2 Animals were distributed by weight using a comput- erized randomization list. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High X 1 1 Preparation methods, storage conditions, and re- sults from stability testing were reported and ap- propriate. Metric 8: Consistency of Exposure Administration High X 1 1 Exposures were administered consistently. Metric 9: Reporting of Doses/Concentrations High X 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration High X 1 1 Duration and frequency were reported. Metric 11: Number of Exposure Groups and Dose Spac- ing High X 1 1 The number of groups and spacing were reported and justified. Metric 12: Exposure Route and Method High X 1 1 The exposure route and method were appropriate. Domain 4: Test Organism Metric 13: Test Animal Characteristics High X 2 2 The source, species, strain, sex, , age, initial body weight, and health status were reported. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Medium X 1 2 All husbandry conditions except air changes were reported.. Metric 15: Number per Group High X 1 1 The number of animals per group was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High X 2 2 Outcome assessment methodology was appropriate. Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate. Continued on next page . . Page 34 of 110 ------- ... continued from previous page Study Citation: N-Methylpyrrolidone Producers Group (1999). N-methylpyrrolidone - carcinogenicity study in B6C3F1 mice, administration in the diet for 18 months, with cover letter dated 11/23/1999 Data Type: 18-month oral cancer mice HERO ID: 3566221 Domain Metric Rating^ MWF* Score Comments^ Metric 19: Blinding of Assessors Not Rated NA NA Blinding not required. Metric 20: Negative Control Response High x 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables were reported. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposure were ob- served. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were described in detail and suited to the data. Metric 24: Reporting of Data High x 2 2 Data for all outcomes were reported. Overall Quality Determination" High 1.1 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = J]. (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 35 of 110 ------- Table 15: Animal toxicity evaluation results of E.I. Dupont De Nemours Co. 1982 for a 2-year inhalation study in rats on renal and cancer outcomes Study Citation: DuPont (E. I. Dupont De Nemours and Company) (1982). 2-year inhalation study with n-methyl-2-pyrrolidone in rats (final) with cover letter dated 083090 Data Type: 2-year inhalation in rats HERO ID: 4214102 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity Medium X 2 4 Test substance identified by name. Metric 2: Test Substance Source Low X 1 3 Sponsor identified as source of test substance. Metric 3: Test Substance Purity Medium X 1 2 Purity such that effects likely due to the test sub- stance. Impurities were identified. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 Negative controls were used. Metric 5: Positive Controls Not Rated NA NA Positive controls were not required. Metric 6: Randomized Allocation Medium X 1 2 Selective allocation was used so that the mean body weights were equal across groups and sex. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Medium X 1 2 Generation of vapors were reported and appropriate; storage of test substance was not reported. Metric 8: Consistency of Exposure Administration Low X 1 3 Exposure periods were occasionally shorter than 6 hours or missed entirely because of mechanical prob- Metric 9: Reporting of Doses/Concentrations High X 2 2 Concentrations were presented without ambiguity. Metric 10: Exposure Frequency and Duration High X 1 1 Frequency and duration were reported and appro- priate. Metric 11: Number of Exposure Groups and Dose Spac- Medium X 1 2 The number of exposure groups and dose spacing ing were appropriate but not justified. Metric 12: Exposure Route and Method High X 1 1 Deficiencies in reporting of aerosol formation. Domain 4: Test Organism Metric 13: Test Animal Characteristics High X 2 2 The source, species, strain, age, health status, sex, and body weight at start of test were reported. Metric 14: Adequacy and Consistency of Animal Hus- High X 1 1 All husbandry conditions were reported. bandry Conditions Metric 15: Number per Group High X 1 1 The number of animals per group was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Low X 2 6 Breathing rate was not reported. Metric 17: Consistency of Outcome Assessment High X 1 1 Outcome assessments were consistent. Continued on next page Page 36 of 110 ------- . continued from previous page Study Citation: DuPont (E. I. Dupont De Nemours and Company) (1982). 2-year inhalation study with n-methyl-2-pyrrolidone in rats (final) with cover letter dated 083090 Data Type: 2-year inhalation in rats HERO ID: 4214102 Domain Metric Rating^ MWF* Score Comments^ Metric 18: Metric 19: Metric 20: Sampling Adequacy Blinding of Assessors Negative Control Response High Not Rated High X 1 NA x 1 1 NA 1 Sampling was adequate for the endpoints examined. Blinding was not required. Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Low Procedures Metric 22: Health Outcomes Unrelated to Exposure Low x 2 x 1 One male control rat was placed in the female group. Several rats in the low-exposure group escaped and mated. Several rats escaped and were never recaptured. Three female rats were impregnated by a male rat, gave birth, and were continued in the study. One low-exposure female was impregnated, gave birth, and was continued on the test. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Metric 24: Reporting of Data Low High x 1 x 2 3 2 Statistical methods were described. It does not ap- pear that incidence data were analyzed. Data were presented for all outcomes. Overall Quality Determination"1' Medium — -4- Medium^ Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. § Evaluator's explanation for rating change: "Paper downgraded due to uncertainties in the actual exposure regimen and deviations in the study protocol (e.g., pregnant/post- partum rats were allowed to continue in the study)." Page 37 of 110 ------- Table 16: Animal toxicity evaluation results of N-Methylpyrrolidone Producers Group 1997 for a 2-year cancer bioassay study Study Citation: N-Methylpyrrolidone Producers Group (1997). Final report, oncogenicity study with n-methylpyrrolidone (nmp) two-year feeding study in Sprague Dawley rats, with cover letter dated 5/22/1998 Data Type: 2 year cancer bioassay HERO ID: 4214107 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Identified by structure, nomenclature, CASRN. Metric 2: Test Substance Source High x 1 1 Source and production date provided Metric 3: Test Substance Purity High x 1 1 Purity such that effects due to test substance. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Concurrent negative controls were used Metric 5: Positive Controls Not Rated NA NA Positive control animals were not required Metric 6: Randomized Allocation Medium x 1 2 Computerized stratified randomization used to en- sure no body weight differences among the groups Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High x 1 1 Preparation and storage were appropriate based on stability analysis Metric 8: Consistency of Exposure Administration High X 1 1 Diets administered consistently across groups Metric 9: Reporting of Doses/Concentrations High x 2 2 Food intake and body weight were monitored in or- der to calculate daily intakes. Concentrations were analyzed in the diet Metric 10: Exposure Frequency and Duration Medium x 1 2 Diets were provided for "approximately 24 months" and were inferred to have been available ad libitum Metric 11: Number of Exposure Groups and Dose Spac- ing High x 1 1 The number of groups and spacing were based on previous repeated dose studies Metric 12: Exposure Route and Method High x 1 1 The route and methods were suitable for the test substance Domain 4: Test Organism Metric 13: Test Animal Characteristics High x 2 2 All characteristics were reported Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions High x 1 1 All conditions were reported Metric 15: Number per Group High x 1 1 The number of animals/group was adequate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 Outcomes methodology was reported and sensitive for outcome(s) of interest Metric 17: Consistency of Outcome Assessment High x 1 1 Outcomes were assessed consistently Metric 18: Sampling Adequacy High x 1 1 Sampling for outcomes was adequate Continued on next page . . Page 38 of 110 ------- ... continued from previous page Study Citation: N-Methylpyrrolidone Producers Group (1997). Final report, oncogenicity study with n-methylpyrrolidone (nmp) two-year feeding study in Sprague Dawley rats, with cover letter dated 5/22/1998 Data Type: 2 year cancer bioassay HERO ID: 4214107 Domain Metric Rating^ MWF* Score Comments^ Metric 19: Blinding of Assessors Medium X 1 2 Blinding was not reported for peer-review evaluation of histopathological findings, but this is not expected to impact on the results. Metric 20: Negative Control Response High X 1 1 The negative controls responded appropriately Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and High X 2 2 No confounding variables were reported Procedures Metric 22: Health Outcomes Unrelated to Exposure High X 1 1 No health outcomes unrelated to exposure were found. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High X 1 1 Statistical methods were appropriate for the out- Metric 24: Reporting of Data High X 2 2 All outcomes were reported Overall Quality Determination" High 1.1 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating, ft This metric met the criteria for high confidence as expected for this type of study. Page 39 of 110 ------- 5 Genetic toxicity studies Table 17: In vitro evaluation results of Mortelmans et al., 1986 for bacterial reverse mutation Study Citation: K. Mortelmans, S. Haworth, T. Lawlor, W. Speck, B. Tainer, E. Zeiger (1986). Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals Environmental Mutagenesis, 8(S7,S7), 1-119 Data Type: Bacterial reverse mutation for NMP HERO ID: 7315 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 Test substance was reported by name: N-methyl-2- pyrrolidone (NMP), CASRN 872-50-4 in table 1 and by structure in appendix 1 Metric 2: Test Substance Source High X 1 1 Test substance was obtained from Aldrich, Lot num- ber was not reported, however, the test substance is unlikely to vary in composition Metric 3: Test Substance Purity High X 1 1 Test substance purity reported in table 1: vendor purity-99% %, analyzed purity , blank testing lab -EGG Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Metric 5: Positive Controls High x 2 2 Metric 6: Assay Procedures High x 1 1 Metric 7: Standards for Tests Not Rated NA NA Domain 3: Exposure Characterization Metric 8: Preparation and Storage of Test Substance High X 1 1 Chemical preparation was reported in detail. Chem- ical was provided with stability and storage condi- tions, specific storage conditions for the test sub- stance were not reported; however, this is appropri- ate for the study design (single-dose administration). Metric 9: Consistency of Exposure Administration High x 1 1 Administration was consistent across study groups Metric 10: Reporting of Doses/Concentrations High X 2 2 Doses were reported in table 171 of appendix 2: 0, 100, 333, 1000, 3333, 10000 ug/plate Continued on next page . .. Concurrent solvent controls were tested with and without metabolic activation. Solvent controls in- clude water, DMSO and ethanol or acetone if not soluble in water or DMSO. The solvent used for the test substance was water reported in appendix 2 Positive controls were tested with and without metabolic activation: without metabolic activation: sodium azide for TA1535 and TA100, 4-nitro-o- phenylenediamine for TA98, and 9-aminoacridine for TA97 and TA1537; 2-aminoanthracene was used with all strains with hamster and rat liver metabolic activation systems. The assay procedure was well described Not applicable for this study type Page 40 of 110 ------- .. . continued from previous page Study Citation: K. Mortelmans, S. Haworth, T. Lawlor, W. Speck, B. Tainer, E. Zeiger (1986). Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals Environmental Mutagenesis, 8(S7,S7), 1-119 Data Type: Bacterial reverse mutation for NMP HERO ID: 7315 Domain Metric Rating^ MWF* Score Comments^ Metric 11: Number of Exposure Groups and Concentra- High x 2 2 Exposure duration was appropriate for the study tion Spacing type and was reported for each part of the procedure: 20 minute pre-incubation and 48 h plate incubation after exposure Metric 12: Exposure Route and Method High x 1 1 Number of doses was adequate for the study type. Dose spacing and upper limits were based on solu- bility and cytotoxicity Metric 13: Metabolic Activation High x 1 1 Testing was done in the presence and absence of S9 metabolic activation. Preparation of S9 was re- ported. Domain 4: Test Model Metric 14: Test Model High x 2 2 The identity and source of the S. typhimrium strains TA1535, TA1537, TA98, and TA100 were reported and appropriate. These strains are routinely used for the outcome of interest. Metric 15: Number per Group High x 1 1 Three plates per dose level were utilized. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 The outcome assessment was appropriate for the outcome of interest Metric 17: Consistency of Outcome Assessment High x 1 1 The outcome assessment was consistent in protocol and timing across all dose groups Metric 18: Sampling Adequacy Not Rated NA NA Not applicable for the study type Metric 19: Blinding of Assessors Not Rated NA NA Not applicable for the study type Domain 6: Confounding / Variable Control Metric 20: Confounding Variables in Test Design and High x 2 2 No confounding variables were reported. The num- Procedures ber or organisms was not reported but based on a citation it is assumed to be consistent across doses. Metric 21: Confounding Variables in Outcomes Unre- lated to Exposure High x 1 1 Data on confounding variables not related to expo- sure were not reported Domain 7: Data Presentation and Analysis Metric 22: Data Analysis High X 1 1 Statistical analysis was not conducted, however suf- ficient data were provided to allow for statistical testing. Metric 23: Data Interpretation High X 2 2 Evaluation criteria was cited previously and briefly described and were consistent with established prac- tice Metric 24: Cytotoxicity Data High X 1 1 Cytotoxicity testing was reported and used to deter- mine dose range for the test substance Continued on next page . .. Page 41 of 110 ------- . continued from previous page Study Citation: K. Mortelmans, S. Haworth, T. Lawlor, W. Speck, B. Tainer, E. Zeiger (1986). Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals Environmental Mutagenesis, 8(S7,S7), 1-119 Data Type: Bacterial reverse mutation for NMP HERO ID: 7315 Domain Metric Rating^ MWF* Score Comments^ Metric 25: Reporting of Data High X 2 2 Appendix 2 table 171 reports data as mean SEM for all dose groups. Table 1 includes summary +,-,eq Overall Quality Determination1" High 1.0 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = J]. (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 42 of 110 ------- Table 18: In vitro evaluation results of Wells et al., 1988 for Ames test Study Citation: D. A. Wells, H. F. Thomas, G. A. Digenis (1988). Mutagenicity and cyto-toxicity of n-methyl-2-pyrrolidinone and 4- (methylamino)butanoic acid in the salmonella microsome assay Journal of Applied Toxicology, 8(2,2), 135-139 Data Type: Ames for NMP HERO ID: 1459767 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 Test substance was identified as N-methyl-2- pyrrolidinone (NMP). Metric 2: Test Substance Source High X 1 1 Source of test substance was Aldrich Chemical Co (Milwaukee, WI). Metric 3: Test Substance Purity High X 1 1 Test substance is reported to be Gold Label grade. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 Sterile distilled water (vehicle) was used as a nega- tive control. Metric 5: Positive Controls Medium X 2 4 Positive controls were concurrently run. It was un- clear whether each positive control was appropri- ate for each strain according to current standards and guidelines. Positive control results were not re- ported. Metric 6: Assay Procedures Medium X 1 2 Details regarding the direct plate incorporation and pre-incubation methods were cited to other publica- tions, but the methodology described appeared ap- propriate and consistent with current standards and guidelines. Metric 7: Standards for Tests Not Rated NA NA Not applicable for this study. Domain 3: Exposure Characterization Metric 8: Preparation and Storage of Test Substance High X 1 1 Preparation of test substance was reported. Storage of the test substance was not reported, but this is appropriate given the study design (single-dose ad- ministration). Metric 9: Consistency of Exposure Administration High X 1 1 Exposures were consistently administered across study groups Metric 10: Reporting of Doses/Concentrations High X 2 2 Doses were reported without ambiguity. Metric 11: Number of Exposure Groups and Concentra- High X 2 2 Exposure duration was appropriate for the pre- tion Spacing incubation assay. The plate incorporation exposure duration was cited to another publication, but was considered to be appropriate given that the publica- tion cited was Maron and Ames 1983. Metric 12: Exposure Route and Method High X 1 1 Number of exposure groups was appropriate (six log- linear doses). Cytotoxicity was observed at highest dose. Continued on next page . .. Page 43 of 110 ------- . continued from previous page Study Citation: D. A. Wells, H. F. Thomas, G. A. Digenis (1988). Mutagenicity and cyto-toxicity of n-methyl-2-pyrrolidinone and 4- (methylamino)butanoic acid in the salmonella microsome assay Journal of Applied Toxicology, 8(2,2), 135-139 Data Type: Ames for NMP HERO ID: 1459767 Domain Metric Rating^ MWF* Score Comments^ Metric 13: Metabolic Activation High X 1 1 Exposure were performed in the presence and ab- sence of metabolic activation (S9). Domain 4: Test Model Metric 14: Test Model Medium x 2 4 Test strains were described; however, limited de- scriptive information were provided. Metric 15: Number per Group Medium x 1 2 The number of replicates per group is unclear. Fig- ure legends for both the direct plate incorporation assay (Figure 1) and the pre-incubation assay (Fig- ure 2) report n = 8 "obtained from pooled data from two independent experiments." In the methods de- scribing the direct plate incorporation test, it was reported that n = 4 plates/dose and "each experi- ment was repeated once." In the methods describing the pre-incubation test, it was implied but not ex- plicitly stated that n = 3. Despite this uncertainty, n = 3 replicates per dose level is considered adequate for the outcome of interest, so this deficiency i s not expected to have substantially affected results. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 The assessment methodology is appropriate for the outcome of interest. Metric 17: Consistency of Outcome Assessment Low x 1 3 It was reported that "revertant colonies were counted manually [...] or with a Biotran II auto- mated colony counter." It is not clear which treat- ment groups were assessed manually or automati- cally. This is considered to be a significant inconsis- tency that may have substantially impacted results. Metric 18: Sampling Adequacy Not Rated NA NA Not applicable for this study. Metric 19: Blinding of Assessors Not Rated NA NA Not applicable for thi Domain 6: Confounding / Variable Control Metric 20: Confounding Variables in Test Design and Low x 2 6 Information on potential confounding variables were Procedures not reported. Metric 21: Confounding Variables in Outcomes Unre- Medium x 1 2 Data on outcomes unrelated to exposure were not lated to Exposure reported. Domain 7: Data Presentation and Analysis Metric 22: Data Analysis High x 1 1 Data were analyzed by Bonferroni t-test of differ- ences between means of the treatment dose vs con- trol. P=0.05 Metric 23: Data Interpretation High x 2 2 Data interpretation was consistent with standards. Continued on next page . . Page 44 of 110 ------- . continued from previous page Study Citation: D. A. Wells, H. F. Thomas, G. A. Digenis (1988). Mutagenicity and cyto-toxicity of n-methyl-2-pyrrolidinone and 4- (methylamino)butanoic acid in the salmonella microsome assay Journal of Applied Toxicology, 8(2,2), 135-139 Data Type: Ames for NMP HERO ID: 1459767 Domain Metric Rating^ MWF* Score Comments^ Metric 24: Cytotoxicity Data Metric 25: Reporting of Data High Low X 1 x 2 1 6 Cytotoxic endpoints were described. Although the direct plate incorporation method was carried out for strains TA97, TA98, TA100, TA102, TA104, TA2638, UTH8413, and UTH8414, and the pre-incubation method was carried out for strains TA98 and TA104, only a small subset of these re- sults are reported quantitatively. It is unclear why the remainder of the results were not reported quan- titatively. Overall Quality Determination"1' High 1.5 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 45 of 110 ------- Table 19: In vitro evaluation results of Wells et al., 1988 for Ames test with N-MeGABA Study Citation: D. A. Wells, H. F. Thomas, G. A. Digenis (1988). Mutagenicity and cyto-toxicity of n-methyl-2-pyrrolidinone and 4- (methylamino)butanoic acid in the salmonella microsome assay Journal of Applied Toxicology, 8(2,2), 135-139 Data Type: Ames with N-MeGABA HERO ID: 1459767 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 Test substance was identified as 4- (methylamino)butanoic acid (N-MeGABA). Metric 2: Test Substance Source Low X 1 3 Test substance was synthesized by authors. Analyt- ical confirmation was not reported. Metric 3: Test Substance Purity Low X 1 3 Purity of test substance was not reported. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 Sterile distilled water (vehicle) was used as a nega- tive control. Metric 5: Positive Controls Medium X 2 4 Positive controls were concurrently run. It was un- clear whether each positive control was appropri- ate for each strain according to current standards and guidelines. Positive control results were not re- ported. Metric 6: Assay Procedures Medium X 1 2 Details regarding the direct plate incorporation and pre-incubation methods were cited to other publica- tions, but the methodology described appeared ap- propriate and consistent with current standards and guidelines. Metric 7: Standards for Tests Not Rated NA NA Not applicable for this study. Domain 3: Exposure Characterization Metric 8: Preparation and Storage of Test Substance High X 1 1 Preparation of test substance was reported. Storage of the test substance was not reported, but this is appropriate given the study design (single-dose ad- ministration). Metric 9: Consistency of Exposure Administration High X 1 1 Exposures were consistently administered across study groups Metric 10: Reporting of Doses/Concentrations High X 2 2 Doses were reported without ambiguity. Metric 11: Number of Exposure Groups and Concentra- High X 2 2 Exposure duration was appropriate for the pre- tion Spacing incubation assay. The plate incorporation exposure duration was cited to another publication, but was considered to be appropriate given that the publica- tion cited was Maron and Ames 1983. Metric 12: Exposure Route and Method High X 1 1 Number of exposure groups was appropriate (six log- linear doses). Cytotoxicity was observed at highest dose. This highest dose was selected based on solu- bility limits. Continued on next page . .. Page 46 of 110 ------- . continued from previous page Study Citation: D. A. Wells, H. F. Thomas, G. A. Digenis (1988). Mutagenicity and cyto-toxicity of n-methyl-2-pyrrolidinone and 4- (methylamino)butanoic acid in the salmonella microsome assay Journal of Applied Toxicology, 8(2,2), 135-139 Data Type: Ames with N-MeGABA HERO ID: 1459767 Domain Metric Rating^ MWF* Score Comments^ Metric 13: Metabolic Activation High X 1 1 Exposure were performed in the presence and ab- sence of metabolic activation (S9). Domain 4: Test Model Metric 14: Test Model Medium x 2 4 Test strains were described; however, limited de- scriptive information were provided. Metric 15: Number per Group Medium x 1 2 The number of replicates per group is unclear. Fig- ure legends for both the direct plate incorporation assay (Figure 1) and the pre-incubation assay (Fig- ure 2) report n = 8 "obtained from pooled data from two independent experiments". In the methods de- scribing the direct plate incorporation test, it was reported that n = 4 plates/dose and "each experi- ment was repeated once". In the methods describing the pre-incubation test, it was implied but not ex- plicitly stated that n = 3. Despite this uncertainty, n = 3 replicates per dose level is considered adequate for the outcome of interest, so this deficiency is not expected to have substantially affected results. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 The assessment methodology is appropriate for the outcome of interest. Metric 17: Consistency of Outcome Assessment Low x 1 3 It was reported that "revertant colonies were counted manually [...] or with a Biotran II auto- mated colony counter." It is not clear which treat- ment groups were assessed manually or automati- cally. This is considered to be a significant inconsis- tency that may have substantially impacted results. Metric 18: Sampling Adequacy Not Rated NA NA Not applicable for this study. Metric 19: Blinding of Assessors Not Rated NA NA Not applicable for this study. Domain 6: Confounding / Variable Control Metric 20: Confounding Variables in Test Design and Low x 2 6 Information on potential confounding variables were Procedures not reported. Metric 21: Confounding Variables in Outcomes Unre- Medium x 1 2 Data on outcomes unrelated to exposure were not lated to Exposure reported. Domain 7: Data Presentation and Analysis Metric 22: Data Analysis High x 1 1 Data were analyzed by Bonferroni t-test of differ- ences between means of the treatment dose vs con- trol. P=0.05 Metric 23: Data Interpretation High x 2 2 Data interpretation was consistent with standards. Continued on next page . . Page 47 of 110 ------- . continued from previous page Study Citation: D. A. Wells, H. F. Thomas, G. A. Digenis (1988). Mutagenicity and cyto-toxicity of n-methyl-2-pyrrolidinone and 4- (methylamino)butanoic acid in the salmonella microsome assay Journal of Applied Toxicology, 8(2,2), 135-139 Data Type: Ames with N-MeGABA HERO ID: 1459767 Domain Metric Rating^ MWF* Score Comments^ Metric 24: Cytotoxicity Data Metric 25: Reporting of Data High Low X 1 x 2 1 6 Cytotoxic endpoints were described. Although the direct plate incorporation method was carried out for strains TA97, TA98, TA100, TA102, TA104, TA2638, UTH8413, and UTH8414, and the pre-incubation method was carried out for strains TA98 and TA104, only a small subset of these re- sults are reported quantitatively. It is unclear why the remainder of the results were not reported quan- titatively. Overall Quality Determination"1' High 1.7 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 48 of 110 ------- Table 20: Animal toxicity evaluation results of Engelhardt and Fleig 1993 for micronucleus and chromosomal aberrations assays Study Citation: G. Engelhardt, H. Fleig (1993). l-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo Mutation Research, 298(3,3), 149-155 Data Type: Micronucleus and chromosomal aberrations assays for NMP HERO ID: 3539781 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 Test substance was clearly identified by name (1- methyl-2-pyrrolidinone; NMP). A CASRN was also provided (872-50-4). Metric 2: Test Substance Source High X 1 1 The source of the test substance (a manufacturer) was reported. Although a lot/batch number was not reported, the test substance is not expected to vary in composition. Metric 3: Test Substance Purity High X 1 1 The purity of the test substance was reported ( >99.8%). Any observed effects are likely due to the test substance itself. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 The study authors reported using an appropriate concurrent negative control groups (vehicle-only was clearly specified). Metric 5: Positive Controls High X 1 1 Positive controls were run concurrently. In both as- says, cyclophosphamide was used as a positive con- trol for clastogenic activity and vincristine sulfate was used as a positive control for spindle poison ef- fects. Benomyl was used in the micronucleus test only, Although fewer animals/group were used for these substances (2 to 3 animals/sex), significant positive responses were observed. Metric 6: Randomized Allocation High X 1 1 The study indicated that animals were randomly as- signed to test groups (separately according to sex). Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Medium X 1 2 The test substance preparation (i.e., dissolved in dis- tilled water) and storage conditions (i.e., kept at 4 to 6 degrees C under N2 conditions) were reported. Metric 8: Consistency of Exposure Administration High X 1 1 Exposures were administered consistently across study groups. Gavage volumes were consistent throughout (10 mL/kg). Metric 9: Reporting of Doses/Concentrations High X 2 2 Doses were reported without ambiguity (methods section and data tables). Metric 10: Exposure Frequency and Duration High X 1 1 The test substance was administered as a single dose via gavage; this treatment is adequate for the study types/outcomes of interest. Continued on next page . Page 49 of 110 ------- .. . continued from previous page Study Citation: G. Engelhardt, H. Fleig (1993). l-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo Mutation Research, 298(3,3), 149-155 Data Type: Micronucleus and chromosomal aberrations assays for NMP HERO ID: 3539781 Domain Metric Rating^ MWF* Score Comments^ Metric 11: Number of Exposure Groups and Dose Spac- Medium X 1 2 The number of dose groups was specified (3 plus neg- ing ative controls for the micronucleus assay and 2 plus negative controls for the chromosomal aberrations assay); the number of groups used in the chromo- somal assay was fewer than recommended by study type. The highest dose was justified by the authors as 80% of the LD50. Metric 12: Exposure Route and Method High X 1 1 The route and of exposure was reported (oral gav- age) and was suited to the test substance. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium X 2 4 Minor uncertainties in the reporting of test animal characteristics (health status, and age) are unlikely to have a substantial impact on results. The test an- imals were obtained from a commercial source. The test species used for the micronucleus assay (mice) was appropriate for the outcome of interest. Ham- sters were used for the chromosomal aberrations as- say; the use of this rodent species was not justified in the study report. Metric 14: Adequacy and Consistency of Animal Hus- Low X 1 3 Husbandry conditions were not sufficiently reported bandry Conditions to evaluate if husbandry was adequate and if differ- ences occurred between control and exposed popu- lations. Metric 15: Number per Group High X 1 1 The number of animals per study group was re- ported (i.e., 5/sex/time point) and appropriate for the study type/outcome analysis. Domain 5: Outcome Assessment Continued on next page . .. Page 50 of 110 ------- .. . continued from previous page Study Citation: G. Engelhardt, H. Fleig (1993). l-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo Mutation Research, 298(3,3), 149-155 Data Type: Micronucleus and chromosomal aberrations assays for NMP HERO ID: 3539781 Domain Metric Rating^ MWF* Score Comments^ Metric 16: Outcome Assessment Methodology Low X 2 6 The outcome assessment methodology was de- scribed; some details (bone marrow and slide prep for micronuclei and analysis of chromosomal aberra- tions) were cited to other publications. In the mi- cronucleus assay, outcomes were evaluated 16, 24, and 48 hours after dosing at the high-dose (only at 24 hours for the remaining two doses); at least two time points, no sooner than 24 hours and up to 48 hours, is recommended by guideline after a single treatment. With respect to chromosomal aberra- tions, the methods indicate that metaphases were analyzed 24 and 48 hours after dosing at the high- dose (only at 24 hours for the low-dose). However, the data table shows sacrifice intervals of 16 and 24 hours, and at least two time points (12 to 18 hours after dosing and up to 24 hours later) are recom- mended by the study type. Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently across study groups. Metric 18: Sampling Adequacy Medium X 1 2 Sampling for micronuclei was 1000 erythro- cytes/animal (500 recommended by study type); however, only 100 metaphases/animal were evalu- ated for chromosomal aberrations (200 metaphases recommended by study type). Metric 19: Blinding of Assessors Not Rated NA NA This metric was not applicable to the study type (blinding was not reported). Metric 20: Negative Control Response High X 1 1 The biological responses of the negative control groups were adequate (low incidences of micronu- clei/chromosomal aberrations). Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Low X 2 6 Information on potential confounding variables were Procedures not reported. Only the mean body weight of animals per assay was reported (29.9 g for mice and 25.9 g for hamsters). Metric 22: Health Outcomes Unrelated to Exposure Low X 1 3 Data on health outcomes unrelated to exposure were not reported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High X 1 1 Statistics were not reported in the methods; how- ever, Tables 2 and 3 indicate that data were analyzed using Fisher's exact test. For the micronuclei assay, data for individual animals was provided, allowing for independent analyses. Continued on next page . .. Page 51 of 110 ------- . continued from previous page Study Citation: G. Engelhardt, H. Fleig (1993). l-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo Mutation Research, 298(3,3), 149-155 Data Type: Micronucleus and chromosomal aberrations assays for NMP HERO ID: 3539781 Domain Metric Rating^ MWF* Score Comments^ Metric 24: Reporting of Data High X 2 2 All exposure-related data (by dose group, sex, and time point) were presented in Tables 2 and 3. Overall Quality Determination1" High 1.6 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = J]. (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 52 of 110 ------- 6 Developmental and Reproductive Table 21: Animal toxicity evaluation results of Lee et al., 1987 for an inhalation developmental study on growth (early life) and development outcomes Study Citation: Data Type: HERO ID: Lee, KP; Chromey, NC; Culik, R; Barnes, JR; Schneider, PW (1987). Toxicity of N-methyl-2-pyrrolidone (NMP): Teratogenic, subchronic, and two-year inhalation studies Fundamental and Applied Toxicology, 9(2), 222-235 3539878 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Chemical structure, established nomenclature, and physiochemical properties were reported Metric 2: Test Substance Source Medium x 1 2 The source was not reported. However, the concen- tration of exposures were measured by IR spectrom- etry and gas chromatography. Metric 3: Test Substance Purity Medium x 1 2 The compound was stated to be 100% pure without support for the statement. The concentration of ex- posures were measured by IR spectrometry and gas chromatography. Thus effects are likely due to the test substance. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 A concurrent control group was included Metric 5: Positive Controls Not Rated NA NA Positive controls not required Metric 6: Randomized Allocation Medium x 1 2 "Pregnant females were randomly assigned. How- ever, the article states, ""In the experiment for ter- atogenicity, the pregnant female rats were assigned at random into the three groups, but random distri- bution was not obtained. Five of twenty-five females in the 0.1 mg/liter exposure group were not impreg- nated. Two of twenty pregnant females had only four and one corpora lutea, respectively, and one implan- tation each. If these two females were removed from the group, the calculated mean value of parameters measuring the reproduction capability would be sim- ilar to those of the control group."" The NOAEC was 0.36 mg/L." Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High Metric 8: Consistency of Exposure Administration High Metric 9: Reporting of Doses/Concentrations Low X 1 1 Aerosol generation system was described and appro- priate. X 1 1 Exposures were consistent across groups X 2 6 The particle size distribution was not determined and there was uncertainty about whether analytical or nominal concentration was reported. Continued on next page Page 53 of 110 ------- .. . continued from previous page Study Citation: Lee, KP; Chromey, NC; Culik, R; Barnes, JR; Schneider, PW (1987). Toxicity of N-methyl-2-pyrrolidone (NMP): Teratogenic, subchronic, and two-year inhalation studies Fundamental and Applied Toxicology, 9(2), 222-235 Data Type: HERO ID: 3539878 Domain Metric Rating^ MWF* Score Comments^ Metric 10: Exposure Frequency and Duration High X 1 1 Exposed GD6-15 for 6 h/day Metric 11: Number of Exposure Groups and Dose Spac- ing Medium X 1 2 Rationale was not provided, but very little impact on results. Only 2 doses were used. Exposures were not high enough to cause toxicity in females or fetuses. Metric 12: Exposure Route and Method High X 1 1 Dynamic chambers were used Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium x 2 4 Age and overall health status were not reported Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Low x 1 3 Husbandry was not adequately reported. Metric 15: Number per Group High x 1 1 The number of animals/group was sufficient for out- come analysis Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 The outcomes of interest were addressed by the methodology Metric 17: Consistency of Outcome Assessment High x 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High x 1 1 Adequate sampling for the outcomes of interest was conducted Metric 19: Blinding of Assessors Not Rated NA NA Subjective outcomes were not assessed Metric 20: Negative Control Response High x 1 1 Negative controls responded appropriately Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Medium x 2 4 No confounding variables were reported. However, NMP is an irritant. Breathing rates and body tem- perature measurements were not made to rule our reflex bradypnea. Metric 22: Health Outcomes Unrelated to Exposure Medium x 1 2 The article states "In the experiment for teratogenic- ity, the pregnant female rats were assigned at ran- dom into the three groups, but random distribution was not obtained. Five of twenty-five females in the 0.1 mg/liter exposure group were not impregnated. Two of twenty pregnant females had only four and one corpora lutea, respectively, and one implanta- tion each. If these two females were removed from the group, the calculated mean value of parameters measuring the reproduction capability would be sim- ilar to those of the control group." The NOAEC was 0.36 mg/L. Domain 7: Data Presentation and Analysis Continued on next page . .. Page 54 of 110 ------- .. . continued from previous page Study Citation: Data Type: HERO ID: Lee, KP; Chromey, NC; Culik, R; Barnes, JR; Schneider, PW (1987). Toxicity of N-methyl-2-pyrrolidone (NMP): Teratogenic, subchronic, and two-year inhalation studies Fundamental and Applied Toxicology, 9(2), 222-235 3539878 Domain Metric Rating^ MWF* Score Comments^ Metric 23: Statistical Methods High X 1 1 The described statistical methods were appropriate Metric 24: Reporting of Data High x 2 2 Data were reported for outcomes Overall Quality Determination1" High 1.5 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. This metric met the criteria for high confidence as expected for this type of study. Page 55 of 110 ------- Table 22: Animal toxicity evaluation results of Sitarek et al., 2008 for a reproductive-male study on reproductive outcomes Study Citation: Sitarek, K; Stetkiewicz, J (2008). Assessment of reproductive toxicity and gonadotoxic potential of N-methyl-2-pyrrolidone in male rats International Journal of Occupational Medicine and Environmental Health, 21(1), 73-80 Data Type: Reproductive-males HERO ID: 3540734 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 The test substance was identified by name and CASRN. Metric 2: Test Substance Source Medium x 1 2 Source identified by name. Metric 3: Test Substance Purity Low x 1 3 Purity not reported. Analytical grade test substance used. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Concurrent negative control animals were included. Metric 5: Positive Controls Not Rated NA NA Positive control animals not required. Metric 6: Randomized Allocation Low x 1 3 Method of randomization for animal allocation was not reported. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low x 1 3 Limited preparation details were provided, and no storage information was provided. It is unclear whether dosing solutions were prepared daily. Since NMP degrades under aerobic conditions, this could have a substantial impact on results. Metric 8: Consistency of Exposure Administration Low X 1 3 It is unclear whether doses were administered con- sistently, as dosing volumes were not reported. Metric 9: Reporting of Doses/Concentrations High x 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration High x 1 1 Frequency and duration were reported. Metric 11: Number of Exposure Groups and Dose Spac- ing Medium x 1 2 No justification provided for the number of groups and dose spacing. Metric 12: Exposure Route and Method High x 1 1 The route and method were appropriate. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium x 2 4 The source, species, strain, sex, and age were re- ported. Initial body weight and health status were not reported. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Medium x 1 2 All husbandry conditions except air changes were reported. Metric 15: Number per Group High x 1 1 The number per group was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 Outcome assessment methodology was reported. Continued on next page . . Page 56 of 110 ------- . continued from previous page Study Citation: Sitarek, K; Stetkiewicz, J (2008). Assessment of reproductive toxicity and gonadotoxic potential of N-methyl-2-pyrrolidone in male rats International Journal of Occupational Medicine and Environmental Health, 21(1), 73-80 Data Type: Reproductive-males HERO ID: 3540734 Domain Metric Rating^ MWF* Score Comments^ Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding not required. Metric 20: Negative Control Response High x 1 1 Negative control responses were adequate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables were observed. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposure were re- ported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical analysis was described and appropriate. Metric 24: Reporting of Data Low x 2 6 Data were presented, but severity and incidence data for histopathological findings were not included. Overall Quality Determination High 1.6 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating, ft This metric met the criteria for high confidence as expected for this type of study. Page 57 of 110 ------- Table 23: Animal toxicity evaluation results of Saillenfait et al., 2002 for an oral developmental rat and maternal effects study on growth (early life), development, and reproductive outcomes Study Citation: Saillenfait, AM; Gallissot, F; Langonne, I; Sabate, JP (2002). Developmental toxicity of N-methyl-2-pyrrolidone administered orally to rats Food and Chemical Toxicology, 40(11), 1705-1712 Data Type: Oral developmental rat, maternal effects HERO ID: 3551103 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity Medium x 2 4 Test substance identified by name only. Metric 2: Test Substance Source Medium x 1 2 Source identified by name. Metric 3: Test Substance Purity High x 1 1 The reported purity was such that effects likely due to the test substance. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Concurrent negative controls were included. Metric 5: Positive Controls Not Rated NA NA Positive controls not required. Metric 6: Randomized Allocation Medium x 1 2 Stratified randomization was used so mean body weights were similar among groups on gestation day 0. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Medium x 1 2 limited details on preparation were reported. Fre- quency of preparation and storage were not re- ported. Metric 8: Consistency of Exposure Administration High X 1 1 Exposures were administered consistently. Metric 9: Reporting of Doses/Concentrations High x 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration High x 1 1 Frequency and duration were reported. Metric 11: Number of Exposure Groups and Dose Spac- ing High x 1 1 The number of groups and spacing were based on dose-range finding studies. Metric 12: Exposure Route and Method High x 1 1 The route and method were appropriate. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium x 2 4 The source, species, strain, sex, and initial body weight were reported. Health status and age were not reported. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Medium x 1 2 All husbandry conditions were reported except room air changes were reported. Metric 15: Number per Group High x 1 1 The number of dams per group was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 Outcome assessment methodology was appropriate. Metric 17: Consistency of Outcome Assessment High x 1 1 Outcomes were assessed consistently. Continued on next page . . Page 58 of 110 ------- ... continued from previous page Study Citation: Saillenfait, AM; Gallissot, F; Langonne, I; Sabate, JP (2002). Developmental toxicity of N-methyl-2-pyrrolidone administered orally to rats Food and Chemical Toxicology, 40(11), 1705-1712 Data Type: Oral developmental rat, maternal effects HERO ID: 3551103 Domain Metric Rating^ MWF* Score Comments^ Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding not applicable. Metric 20: Negative Control Response High x 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables were reported. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposure were ob- served. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical analysis was conducted and appropriate. Metric 24: Reporting of Data High x 2 2 Data were reported for all outcomes. Overall Quality Determination High 1.3 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating, ft This metric met the criteria for high confidence as expected for this type of study. Page 59 of 110 ------- Table 24: Animal toxicity evaluation results of Saillenfait et al., 2003 for inhalation study on developmental and reproductive outcomes Study Citation: Saillenfait, AM; Gallissot, F; Morel, G (2003). Developmental toxicity of N-methyl-2-pyrrolidone in rats following inhalation exposure Food and Chemical Toxicology, 41(4), 583-588 Data Type: HERO ID: 3551104 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1 Test Substance Identity High x 2 2 Nomenclature and CASRN were reported Metric 2 Test Substance Source High x 1 1 Source reported Metric 3 Test Substance Purity High x 1 1 Purity (99.5%) such that effects likely due to test substance Domain 2: Test Design Metric 4 Negative and Vehicle Controls High x 2 2 Concurrent negative control animals were included Metric 5 Positive Controls Not Rated NA NA Positive controls were not required Metric 6 Randomized Allocation High x 1 1 Females were randomly assigned to exposure groups Domain 3: Exposure Characterization Metric 7 Preparation and Storage of Test Substance Medium x 1 2 Method of generation and equipment used were re- ported; however, storage was not. Metric 8 Consistency of Exposure Administration High X 1 1 Exposures were administered consistently Metric 9 Reporting of Doses/Concentrations High x 2 2 Target and analytical concentrations were reported. The analytical method was reported and appropri- Metric 10: Exposure Frequency and Duration High x 1 1 Exposures occurred 6 hr/d on GD 6-20. Metric 11: Number of Exposure Groups and Dose Spac- Medium x 1 2 The purpose was to set a definitive NOAEL for ine developmental toxicity, and although not explicitly to stated, concentrations appeared to be based on a 2-gen toxicity study in which fetal weights were re- duced. The concentrations used were appropriate for the purposes of the study. Metric 12: Exposure Route and Method Low x 1 3 Methodological information provided, including an evaluation of aerosol formation (e.g., temperature, humidity). Exposures were conducted in dynamic chamber; however, aerosol particle size was not re- ported. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium x 2 4 Age, and health status were not reported. Metric 14: Adequacy and Consistency of Animal Hus- High x 1 1 Husbandry conditions were reported and appropri- bandry Conditions ate. Continued on next page . . Page 60 of 110 ------- .. . continued from previous page Study Citation: Data Type: HERO ID: Saillenfait, AM; Gallissot, F; Morel, G (2003). Developmental toxicity of N-methyl-2-pyrrolidone in rats following inhalation exposure Food and Chemical Toxicology, 41(4), 583-588 3551104 Domain Metric Rating^ MWF* Score Comments^ Metric 15: Number per Group High X 1 1 The number of females/group 25-26 was appropriate for the study type and outcomes. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Low X 2 6 Outcome assessment methodology for maternal and fetal evaluations addressed the outcomes of interest; however, breathing rate/body temperature was not measured. Metric 17: Consistency of Outcome Assessment High X 1 1 Outcome assessment was carried out consistently among groups Metric 18: Sampling Adequacy High X 1 1 Sampling details were reported and were adequate Metric 19: Blinding of Assessors Not Rated NA NA No subjective outcomes were assessed. Metric 20: Negative Control Response High X 1 1 Negative control responses were appropriate Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Low X 2 6 No confounding variables were reported. However, Procedures NMP is an irritant and no measures were made to as- sess changes in breathing rate or body temperature. The possibility of reflex bradypnea is not eliminated but the results of the study are consistent with oral and dermal study effects on fetal body weight. Metric 22: Health Outcomes Unrelated to Exposure High X 1 1 No health differences unrelated to exposure were re- ported Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High X 1 1 Statistical methods were described and appropriate for the dataset Metric 24: Reporting of Data High X 2 2 Data for all outcomes were reported. Overall Quality Determination High 1.5 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. This metric met the criteria for high confidence as expected for this type of study. Page 61 of 110 ------- Table 25: Animal toxicity evaluation results of Exxon 1992 for a developmental toxicity study in rats on growth (early life) and development outcomes Study Citation: ISP (1992). Initial submission: Developmental toxicity study in rats with n-methylpyrrolidone (draft report) with attachments and cover letter dated 041092 Data Type: Developmental toxicity study in rats HERO ID: 3563347 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity Medium X 2 4 Test substance identified by name. Characterization and analysis of the test article were reported to be documented by the sponsor, but no details were provided. Metric 2: Test Substance Source Medium X 1 2 The batch number was provided. The source was of the test substance was not provided but reported as sponsor. Metric 3: Test Substance Purity Medium X 1 2 The purity was assumed to be 100%, however no data was provided for support. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 A concurrent vehicle control was included. Metric 5: Positive Controls Not Rated NA NA Positive controls were not required. Metric 6: Randomized Allocation Medium X 1 2 Method of allocation was based on confirmed mat- ing. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High X 1 1 Test substance preparation and storage were re- ported. Stability and concentration were reported. Metric 8: Consistency of Exposure Administration High X 1 1 Doses were administered consistently. Metric 9: Reporting of Doses/Concentrations High X 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration High X 1 1 Duration and frequency were reported. Metric 11: Number of Exposure Groups and Dose Spac- Medium X 1 2 The number of groups and spacing were not justified ing but were sufficient to show results relevant to the outcome of interest. Metric 12: Exposure Route and Method High X 1 1 The exposure route and method were appropriate. Domain 4: Test Organism Metric 13: Test Animal Characteristics High X 2 2 The source, species, strain, sex, health status, and initial body weight were reported. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions High X 1 1 All husbandry conditions were reported. Metric 15: Number per Group High X 1 1 The number of animals per group was appropriate. Domain 5: Outcome Assessment Continued on next page . .. Page 62 of 110 ------- . continued from previous page Study Citation: ISP (1992). Initial submission: Developmental toxicity study in rats with n-methylpyrrolidone (draft report) with attachments and cover letter dated 041092 Data Type: Developmental toxicity study in rats HERO ID: 3563347 Domain Metric Rating^ MWF* Score Comments^ Metric 16: Outcome Assessment Methodology High x 2 2 Outcome assessment methodology was described and appropriate. Metric 17: Consistency of Outcome Assessment High x 1 1 Outcome assessment was consistent. Metric 18: Sampling Adequacy High x 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding not required in this assay. Metric 20: Negative Control Response High x 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Medium x 2 4 Distribution by mating confirmation resulted in 4 non-pregnant control animals, but there were a suf- ficient number of offspring available for comparison. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposure were ob- served. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were well described and appro- priate. Metric 24: Reporting of Data High x 2 2 Data were reported for all outcomes. Overall Quality Determination High 1.3 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 63 of 110 ------- Table 26: Animal toxicity evaluation results of Solomon et al., 1995 for a 2-generation reproduction/developmental study, inhalation study on reproductive, and growth (early life) and development outcomes Study Citation: Solomon, HM; Burgess, BA; Kennedy, GL, Jr; Staples, RE (1995). l-methyl-2-pyrrolidone (NMP): Reproductive and developmental toxicity study by inhalation in the rat Drug and Chemical Toxicology, 18(4), 271-293 Data Type: 2-generation reproduction/developmental study, inhalation HERO ID: 2761868 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Metric 2: Test Substance Identity Test Substance Source Metric 3: Test Substance Purity High Medium High X 2 2 Clearly identified X 1 2 Test substance source was reported. No lot or batch number was reported, but this omission of details is not likely to have a substantial impact on results. X 1 1 Purity reported (99.9%). Impurities were identified and present in quantities that would not influence the results. Domain 2: Test Design Metric 4: Metric 5: Metric 6: Negative and Vehicle Controls Positive Controls Randomized Allocation High X 2 2 A concurrent negative control was used and appears to be sham- exposed. Not Rated NA NA this metric is not rated/applicable for this study type. High X 1 1 Rats were distributed randomly into control and treated groups using a randomized block design based on body weight. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High Metric 8: Consistency of Exposure Administration High Metric 9: Reporting of Doses/Concentrations Medium Metric 10: Exposure Frequency and Duration High X 1 1 The test substance preparation, method, and equip- ment used to generate the vapor were reported and appropriate. X 1 1 Details of exposure administration were reported and administered consistently across study groups. X 2 4 Target and analytical concentrations were reported. The analytical high dose was reported as 116.4 ppm (target dose was 130 ppm). It was reported that technical restraints (condensation on the inside of the high dose chambers) prevented attainment of the target dose (130 ppm). X 1 1 The exposure frequency and duration of exposure were appropriately identified. Continued on next page .. Page 64 of 110 ------- . continued from previous page Study Citation: Solomon, HM; Burgess, BA; Kennedy, GL, Jr; Staples, RE (1995). l-methyl-2-pyrrolidone (NMP): Reproductive and developmental toxicity study by inhalation in the rat Drug and Chemical Toxicology, 18(4), 271-293 Data Type: 2-generation reproduction/developmental study, inhalation HERO ID: 2761868 Domain Metric Rating^ MWF* Score Comments^ Metric 11: Number of Exposure Groups and Dose Spac- Medium X 1 2 The number of exposure groups and concentrations ing were not justified by the study authors in the report. This is unlikely to impact results, as the number of exposure groups and spacing of the exposures were adequate to show results relevant to the outcome of interest. Metric 12: Exposure Route and Method Medium X 1 2 A dynamic whole-body chamber was used for vapors that may condense. The high-concentration target of 130 ppm was not attained due to condensation on the inside of the high dose chambers; the actual high-concentration exposure level was measured as 116.4 ppm. The number of air changes/hour was reported to be 12-15. Domain 4: Test Organism Metric 13: Test Animal Characteristics High Metric 14: Adequacy and Consistency of Animal Hus- High bandry Conditions Metric 15: Number per Group High x 2 x 1 x 1 The test animal species, strain, sex, age, and start- ing body weight were reported and obtained from a commercial source. The rats were weighed, and clin- ical signs were taken on all rats upon arrival and 2 times more before exposure. All husbandry conditions were reported, were ade- quate, and were the same for control and exposed groups. The number of animals per study group was reported and appropriate for the study type and outcome analysis. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Metric 17: Consistency of Outcome Assessment Metric 18: Sampling Adequacy Metric 19: Blinding of Assessors Metric 20: Negative Control Response High High High High High X 2 2 The outcome assessment methodology was reported and addressed the intended outcomes of interest. X 1 1 Details of the outcome assessment protocol were reported and outcomes were assessed consistently across study groups. X 1 1 Sampling was reported and adequate for the out- comes of interest. X 1 1 The study report reported that investigators assess- ing outcomes of the developmental were not aware of the exposure group to which any of the dams or offspring belonged to. X 1 1 The biological responses of the negative control groups were adequate. Domain 6: Confounding / Variable Control Continued on next page Page 65 of 110 ------- . continued from previous page Study Citation: Solomon, HM; Burgess, BA; Kennedy, GL, Jr; Staples, RE (1995). l-methyl-2-pyrrolidone (NMP): Reproductive and developmental toxicity study by inhalation in the rat Drug and Chemical Toxicology, 18(4), 271-293 Data Type: 2-generation reproduction/developmental study, inhalation HERO ID: 2761868 Domain Metric Rating^ MWF* Score Comments^ Metric 21: Confounding Variables in Test Design and Procedures Medium X 2 4 There were no reported differences among the study groups in initial body weight, but respiratory rates were not reported; this lack of reporting is not likely to have a significant impact on results. Metric 22: Health Outcomes Unrelated to Exposure High X 1 1 Details regarding animal attrition and health out- comes unrelated to exposure was reported. It was reported that 2 animals in the control group died during the reproduction phase of the study; one of these deaths was due to a handling injury. There were no differences among groups that would influ- ence the outcome assessment. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High X 1 1 Statistical methods were clearly described and ap- propriate. Metric 24: Reporting of Data High X 2 2 The data for all outcomes were reported by expo- sure group, sex, and generation and described ade- quately. Continuous data included means and the respective standard error. Overall Quality Determination^ High 1.2 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 66 of 110 ------- Table 27: Animal toxicity evaluation results of Intl Specialty 1979 for dermal developmental, offspring and maternal effects study on growth (early life) and development outcomes Study Citation: (1992). Initial submission: teratologic dose range-finding study w/ n-methylpyrrolidone in sprague dawley rats with cover letter dated 09/01/92 Data Type: Dermal developmental, offspring and maternal effects HERO ID: 3564297 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Metric 2: Metric 3: Test Substance Identity Test Substance Source Test Substance Purity Medium Low High XX X 2 1 1 4 3 1 Test substance identified by name. Sponsor (GAF Corp) was identified as the source of the test substance. Purity such that effects due to test substance. Domain 2: Test Design Metric 4: Metric 5: Metric 6: Negative and Vehicle Controls Positive Controls Randomized Allocation High High Medium XXX 2 1 1 2 1 2 Concurrent negative controls were used. Concurrent positive controls were used. Animals allocated by random number assignment sheet. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Metric 8: Consistency of Exposure Administration Metric 9: Reporting of Doses/Concentrations Metric 10: Exposure Frequency and Duration Metric 11: Number of Exposure Groups and Dose Spac- ing Metric 12: Exposure Route and Method Low Low High High High High XX XXX X 1 1 2 1 1 1 3 3 2 1 1 1 Preparation and storage were not reported. Test material was rubbed into the skin. Dosing vol- ume, diluent or dilution factor were not described. It is unclear whether doses were administered con- sistently. Doses were reported. Exposure duration and frequency were reported. The number of groups and dose spacing were re- ported and justified. The route and method of exposure were appropriate. Domain 4: Test Organism Metric 13: Metric 14: Metric 15: Test Animal Characteristics Adequacy and Consistency of Animal Hus- bandry Conditions Number per Group Medium Medium High X XX 2 1 1 4 2 1 The source, strain, sexual state, sex, and initial body weight were reported. Health status and specific nu- meric age were not reported. Temperature, lighting, and humidity were not re- ported. The number of animals per group was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Medium X 2 4 Treatment did not extend throughout the period of organogenesis. Continued on next page . Page 67 of 110 ------- . continued from previous page Study Citation: (1992). Initial submission: teratologic dose range-finding study w/ n-methylpyrrolidone in sprague dawley rats with cover letter dated 09/01/92 Data Type: Dermal developmental, offspring and maternal effects HERO ID: 3564297 Domain Metric Rating^ MWF* Score Comments^ Metric 17: Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently. Metric 18: Sampling Adequacy High X 1 1 Sampling was adequate for the outcomes of interest. Metric 19: Blinding of Assessors Not Rated NA NA Blinding was not required. Metric 20: Negative Control Response High x 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables in test design and proce- dures were noted. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposures were ob- served. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were described and appropriate. Metric 24: Reporting of Data High x 2 2 Data were presented for all outcomes. Overall Quality Determination"1 High 1.5 Extracted No MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = J]. (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 68 of 110 ------- Table 28: Animal toxicity evaluation results of Exxon Biomedical 1991 for a multigeneration reproductive toxicity study on rats reproductive, growth (early life) and developmental outcomes Study Citation: Exxon Biomedical (1991). Multigeneration Rat Reproduction Study with N-Methylpyrrolidone, Project Number 236535 Data Type: Multi-generation reproduction study in rats HERO ID: 3809420 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity Metric 2: Test Substance Source High Medium x 2 x 1 Metric 3: Test Substance Purity Medium x 1 The test substance was identified as N-methyl- pyrrolidone by name in the study title (CASRN not provided). The test substance is referred to as MRD- 89-365 throughout the study report. The study report indicates that the synthesis, fabri- cation, and/or derivation of the test substance were the responsibility of the sponsor (GAF Corpora- tion). The batch number is identified (I), but the manufacturer (if applicable) was not explicitly iden- tified. Although the materials and methods refer- ences a certificate of analysis, it was not included in the study report (i.e., not found in an appendix). The study report indicates that the test substance was assumed 100% pure for the purposes of dosing. Purity was not reported explicitly, but given other information (i.e., analyses of the test substance in feed mixtures), this was not expected to substan- tially impact the study results. Domain 2: Test Design Metric 4: Metric 5: Metric 6: Negative and Vehicle Controls Positive Controls Randomized Allocation High Not Rated High x 2 NA x 1 2 The study authors reported using an appropriate concurrent negative control group (conditions the same except for treatment). Controls were given the same rodent chow as treated animals (minus addi- tion of the test substance). NA Positive control not indicated by study type. 1 The study report indicates that rats (F0 generation) were selected using a computer-generated sorting program so that weight variations were within 20% of the group mean body weight. Rats were randomly allocated to groups by computer (with an attempt to nearly equalize initial body weights). Domain 3: Exposure Characterization Continued on next page .. Page 69 of 110 ------- . continued from previous page Study Citation: Exxon Biomedical (1991). Multigeneration Rat Reproduction Study with N-Methylpyrrolidone, Project Number 236535 Data Type: Multi-generation reproduction study in rats HERO ID: 3809420 Domain Metric Ratingt MWF* Score Comments^ Metric 7: Preparation and Storage of Test Substance High x 1 Metric 8: Consistency of Exposure Administration High Metric 9: Reporting of Doses/Concentrations Medium x 1 x 2 Metric 10: Exposure Frequency and Duration High Metric 11: Number of Exposure Groups and Dose Spac- High ing x 1 x 1 The test substance was thoroughly mixed into the diet; jars containing the feed were replaced at least weekly. Homogeneity, stability, and concentration analyses were performed. Homogeneity samples (top, middle, and bottom for the low- and high-dose groups only) were within 7% of the target values; stability analyses indicated the test substance was stable for at least one month. Details of exposure administration were reported and exposure were administered consistently across groups. All groups of rats were administered the test substance in the diet (offered ad libitum). Target doses were 0, 50, 160, and 500 mg/kg-day. Concentrations of the test substance in the diet were adjusted during pre-mating, and maintained con- stant thereafter (i.e., during mating, gestation, and lactation). Based on measured data, doses were generally within 20% of target levels during pre- mating and gestation. Although no test substance intake information was available during mating, the current guideline indicates that test substance in- take data are required except during cohabitation .The study notes that males were dosed at lower than target dose levels during this period (as dos- ing based on female body weight/food consumption data). Test substance intake was inconsistent during lactation. Doses were higher than target levels from PND 4 on, reaching 2 to 3x target levels by PND 21. In general, body weight and food consumption data were available to enable calculations. The exposure frequency and duration were reported and appropriate for the study type/outcomes of in- terest. Animals were dosed during pre-mating, mat- ing, gestation, lactation, and/or until weaning of the F2b litter. The target doses were based on data for a previously conducted dose-probe study. The high-dose was suf- ficient t o i nducet reatment-related e ffects ( i.e., se- lected doses enable evaluation of dose-response ef- fects). However, the target dose intervals were slightly more than 3-fold (current guideline suggests that for dietary studies, the dose interval should not be more than 3-fold). Continued on next page Page 70 of 110 ------- .. . continued from previous page Study Citation: Data Type: HERO ID: Exxon Biomedical (1991). Multigeneration Rat Reproduction Study with N-Methylpyrrolidone, Project Number 236535 Multi-generation reproduction study in rats 3809420 Domain Metric Rating^ MWF* Score Commentstt Metric 12: Exposure Route and Method High X 1 1 The route and method of exposure were reported and were suited to the test substance. Based on ho- mogeneity and stability analyses, the test substance was non-volatile in the diet. Domain 4: Test Organism Metric 13: Test Animal Characteristics High x 2 Metric 14: Adequacy and Consistency of Animal Hus- Medium bandry Conditions x 1 Metric 15: Number per Group High x 1 The test animal species, strain, sex, health status, age, and initial body weights were reported. Rats used in the study were obtained from a commercial source. The test species and strain were an appropri- ate model; the study report indicates that rats have historically been used for multi-generation reproduc- tion studies (rats are the preferred species based on OECD guideline). Husbandry conditions were reported and were gener- ally considered adequate. Some differences in condi- tions were noted (occasions in which room temper- ature and/or humidity were out of range), but all dose groups were presumably affected by these de- viations, and they are not expected to substantially impact the study results. The number of animals/group (30/sex/generation) was reported, and was appropriate for the study type (guideline recommendation of no fewer than 20 preg- nant females at or near parturition). Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Medium x 2 Metric 17: Consistency of Outcome Assessment High x 1 In general, the outcome assessment methodology ad- dressed/reported the intended outcomes of interest; reproductive/developmental parameters were evalu- ated over the course of two generations. However, several endpoints recommended by current guide- lines were not evaluated, including organ weights, estrous cycle parameters (normality), and sperm pa- rameters (motility and morphology) in parental an- imals. The timing/methods of outcome assessments were reported, and appeared to be consistent across treat- ment groups. Continued on next page Page 71 of 110 ------- . continued from previous page Study Citation: Exxon Biomedical (1991). Multigeneration Rat Reproduction Study with N-Methylpyrrolidone, Project Number 236535 Data Type: Multi-generation reproduction study in rats HERO ID: 3809420 Domain Metric Rating^ MWF* Score Comments^ Metric 18: Sampling Adequacy Medium X 1 2 In general, details regarding sampling were reported. However, it appears that mean fetal body weight data are based on n = number of fetuses (i.e., the litter was not the basis for analysis). Although histopathological examinations were performed for low- and high-dose groups only (which is acceptable as per the current guideline), additional dose groups were examined when histopathological findings were observed at the high dose (e.g., in the ovaries and uterus of female rats). Metric 19: Blinding of Assessors High X 1 1 The study report included a quality assurance state- ment with respect to histopathology examinations, indicating that evaluations were performed accord- ing to standard operating procedures, audited., and based on good laboratory practice. Other endpoints evaluated in the study were not subjective. Metric 20: Negative Control Response High X 1 1 The biological responses of the negative control group were adequate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and High X 2 2 There were no significant differences among study Procedures groups with respect to initial body weights or food consumption rates. Metric 22: Health Outcomes Unrelated to Exposure High X 1 1 There were no outcomes reported unrelated to ex- posure. There were only a few unscheduled deaths (which were considered incidental). Effects ob- served in histopathological examinations that were not treatment-related were considered spontaneous (not attributed to a cause like infection). Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Low X 1 3 The body of the study report indicates that mean body weights and food consumption were analyzed statistically for significant differences. However, the discussion references other "statistically significant effects" (e.g., on fertility and fecundity indices); these are not marked as statistically significant in the corresponding data tables. While sufficient data (n, mean, and measure of variance) are provided in most cases, some of these data would have to be ob- tained from data tables on individual animals. Ap- pendix AQ indicates that most data were not ana- lyzed statistically (including fertility indices). Continued on next page . .. Page 72 of 110 ------- . continued from previous page Study Citation: Exxon Biomedical (1991). Multigeneration Rat Reproduction Study with N-Methylpyrrolidone, Project Number 236535 Data Type: Multi-generation reproduction study in rats HERO ID: 3809420 Domain Metric Ratingt MWF* Score Comments^ Metric 24: Reporting of Data Medium x 2 Data for treatment-related findings were reported for most, but not all, outcomes appropriately. For example, reproductive indices were reported, but the numbers used for calculating these indices were not explicitly provided. However, these data could be pieced together by evaluating individual reproduc- tion data (available in appendices). Data for ges- tation length are also available based on individual data only. Similarly, necropsy data were not pre- sented in a summary table, but individual data were available. Overall Quality Determination1" High 1.4 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating, ft This metric met the criteria for high confidence as expected for this type of study. Page 73 of 110 ------- Table 29: Animal toxicity evaluation results for NMP Producers Group 1999 for a 2-generation dietary reproductive toxicity study in Sprague Dawley rats Study Citation: NMP Producers Group (1999). Two Generation Reproduction Toxicity Study with N-Methylpyrrolidone (NMP) in Sprague Dawley Rats - Administration in the Diet Data Type: 2-gen dietary repro tox study in SD rats HERO ID: 3809436 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity Metric 2: Test Substance Source Metric 3: Test Substance Purity High X 2 2 The test substance was identified as N- methylpyrrolidone (NMP); CAS 872-50-4 High X 1 1 The commercial source of the test substance was identified (BASF Aktiengesellschaft). The product number and batch/lot number were reported (Tank 3226-02-97) High X 1 1 The purity of the test substance was "considered 100%"; An analytical report was included in the appendix and the purity of the test material de- termined pretest, at 6 and 12 months was 97.3%, 98.5%,and 97.5% respectively. Domain 2: Test Design Metric 4: Metric 5: Negative and Vehicle Controls Positive Controls Metric 6: Randomized Allocation High X 2 2 An untreated diet control (basal diet only) Not Rated NA NA No positive control was used; however, is not neces- sary for this study type. High X 1 1 Parental Rats were randomly allocated into study group; a computerized randomization program was used and accounted for comparable body weight means. F1 parental generation were chosen at random using a random numbers table. Litters were culled to no more than ten pups using a computerized random- ization selection process. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High Metric 8: Consistency of Exposure Administration High x 1 x 1 The test substance preparation and storage condi- tions were reported and appropriate for the test sub- stance. Test diets were prepared every 6 days. Ho- mogeneity was analyzed. NMP is reported as stable in the diet for 32 days at room temperature at the tested concentrations. Details of exposure administration were reported and appear to be administered consistently across study groups. Continued on next page Page 74 of 110 ------- .. . continued from previous page Study Citation: NMP Producers Group (1999). Two Generation Reproduction Toxicity Study with N-Methylpyrrolidone (NMP) in Sprague Dawley Rats - Administration in the Diet Data Type: 2-gen dietary repro tox study in SD rats HERO ID: 3809436 Domain Metric Rating^ MWF* Score Comments^ Metric 9: Reporting of Doses/Concentrations High X 2 2 Parental PI and F1 Parental Administered dietary doses were reported without ambiguity. Dietary NMP concentrations were adjusted weekly based on mean body weight and food consumption measure- ments from the previous week. Metric 10: Exposure Frequency and Duration High X 1 1 Exposure frequency and duration were appropriate for this endpoint; continuously in the diet without restriction, 7 d/wk Metric 11: Number of Exposure Groups and Dose Spac- Medium X 1 2 The number of exposure groups and spacing were ing justified by the study authors (based on a previ- ously conducted 2-generation reproduction toxicity study). Initially, PI rats were treated with 0, 50, 160, or 500 mg/kg/day. After day 126, the high dose level was reduced to 350 mg/kg/day due to excessive pup mortality in Fla pups; thereafter, the admin- istered high dose level was 350 mg/kg/day for the remainder of the PI generation exposure. F1 rats were administered 0, 50, 160, and 350 mg/kg/day. The dose spacing was appropriate to evaluate the dose-response relationship Metric 12: Exposure Route and Method High X 1 1 The route or method of exposure was reported and suited to the test substance; administered orally via the diet. Domain 4: Test Organism Metric 13: Test Animal Characteristics High X 2 2 The test animal species, strain, sex, health status, age, and starting body weight were reported, and the test animal was obtained from a commercial source (Charles River Laboratories). The test species and strain were an appropriate animal model for the evaluation of the specific outcome(s) of interest (e.g., routinely used for similar study types and consistent with OECD TG 416). Metric 14: Adequacy and Consistency of Animal Hus- High X 1 1 All husbandry conditions were reported and ade- bandry Conditions quate. Conditions were the same for control and exposed population Metric 15: Number per Group High X 1 1 The number of animals per study group was reported (30 sex/dose in PI and F1 generations), appropriate for the study type and outcome analysis. Domain 5: Outcome Assessment Continued on next page . .. Page 75 of 110 ------- .. . continued from previous page Study Citation: NMP Producers Group (1999). Two Generation Reproduction Toxicity Study with N-Methylpyrrolidone (NMP) in Sprague Dawley Rats - Administration in the Diet Data Type: 2-gen dietary repro tox study in SD rats HERO ID: 3809436 Domain Metric Rating^ MWF* Score Comments^ Metric 16: Outcome Assessment Methodology High x 2 2 The outcome assessment methodologies were appro- priate for the endpoints of interest and were gener- ally consistent with OECD test guidelines for two- generation reproduction testing (OECD TG 416). Metric 17: Consistency of Outcome Assessment High x 1 1 Details of the outcome assessment protocol were re- ported and were assessed consistently across study groups. The rest period between Fla and Fib litters was shortened as compared to the rest period specified in the protocol and feed consumption was not mea- sured prior to the mating period for the Fib litter due to the time constraint. The shortening of the rest period is not likely to adversely affect the out- come assessment. Metric 18: Sampling Adequacy High x 1 1 Details regarding sampling for the outcomes of in- terest were reported and the study used adequate sampling for the outcomes of interest Metric 19: Blinding of Assessors Not Rated NA NA Not applicable for this study type Metric 20: Negative Control Response High x 1 1 The biological responses of the negative control group were reported and were adequate Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and High x 2 2 There were no differences among the study groups in Procedures initial body weight (individual wt of PI animals was not > 20% of the mean bd wt for each sex in each group). Food consumption was reported; palatabil- ity issues were not noted. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 Viability exams and clinical observations were con- ducted for all study groups; it was noted that there was a low incidence of sporadically occurring com- mon laboratory health issues in all treatment groups. It was noted if the cause of death was not treatment related. Mortality in 3 PI animals (across study groups) were considered not treatment-related; this incidence was low and is not likely to have an impact on results. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were clearly described and ap- propriate for datasets Continued on next page . . Page 76 of 110 ------- ... continued from previous page Study Citation: NMP Producers Group (1999). Two Generation Reproduction Toxicity Study with N-Methylpyrrolidone (NMP) in Sprague Dawley Rats - Administration in the Diet Data Type: 2-gen dietary repro tox study in SD rats HERO ID: 3809436 Domain Metric Rating^ MWF* Score Comments^ Metric 24: Reporting of Data High X 2 2 Data for exposure-related findings were presented for all dose groups and endpoints with quantal pre- sentation; summary and individual animal data were reported. Overall Quality Determination1" High 1.0 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 77 of 110 ------- Table 30: Animal toxicity evaluation results of NMP Producers Group 1999 for a 2-generation dietary reproductive toxicity study in Wistar rats Study Citation: NMP Producers Group (1999). Two Generation Reproduction Toxicity Study with N-Methylpyrrolidone (NMP) in Wistar Rats - Administration in the Diet Data Type: 2-gen dietary repro tox study in Wistar rats HERO ID: 3809437 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 The test substance was identified as N- methylpyrrolidone (NMP); CAS 872-50-4 Metric 2: Test Substance Source High x 1 1 The commercial source of the test substance was identified (BASF Aktiengesellschaft). The batch number was reported (Tank 32) Metric 3: Test Substance Purity High x 1 1 The purity of the test substance was reported (99.9%); analyzed using gas chromatography Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 An untreated diet control was used Metric 5: Positive Controls Not Rated NA NA No positive control was used; however, is not neces- sary for this study type. Metric 6: Randomized Allocation High x 1 1 F0 parental rats were randomly allocated into study groups; a computerized randomization program was used according to animal weights 2 days prior to the administration period. F1 parental rats were se- lected 'by lot'. Selection attempted to "take each litter into account" but if fewer than 25 litters were available for each dose group and gender, additional animals were taken from the available litters. Mat- ing partners were assigned randomly. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance High x 1 Metric 8: Consistency of Exposure Administration High x 1 The test substance preparation and storage condi- tions were reported and appropriate for the test sub- stance. Test diets were prepared at intervals that guaranteed the concentration of the test substance in the diet was stable through the feeding period. Ho- mogeneity was analyzed. NMP is reported as stable in the diet for 32 days at room temperature at the tested concentrations. Concentration control analy- sis was conducted. Details of exposure administration were reported and appear to be administered consistently across study groups. Continued on next page Page 78 of 110 ------- .. . continued from previous page Study Citation: NMP Producers Group (1999). Two Generation Reproduction Toxicity Study with N-Mythylpyrrolidone (NMP) in Wistar Rats Administration in the Diet Data Type: 2-gen dietary repro tox study in Wistar rats HERO ID: 3809437 Domain Metric Rating^ MWF* Score Comments^ Metric 9: Reporting of Doses/Concentrations High X 2 2 F0 and F1 administered dietary doses were reported without ambiguity. (0, 50, 160, 500/350 mg/kg bw/day). NMP dietary concentrations for each dose group and sex were adjusted weekly based on body weight and food consumption measurements from the previous week. The measured intakes of NMP correlated well with desired target doses. Metric 10: Exposure Frequency and Duration High X 1 1 Exposure frequency and duration were appropriate for this endpoint; continuously in the diet. Metric 11: Number of Exposure Groups and Dose Spac- Medium X 1 2 The number of exposure groups and spacing were ing justified by the study authors (based on a previ- ously conducted 2-generation reproduction toxicity study). Initially, F0 rats were treated with 0, 50, 160, or 500 mg/kg/day. The high dose level was re- duced to 350 mg/kg/day due to excessive pup mor- tality in Fla pups; thereafter, the administered high dose level was 350 mg/kg/day for the remainder of the study. The dose spacing was appropriate to evaluate the dose-response relationship. Metric 12: Exposure Route and Method High X 1 1 The route or method of exposure was reported and suited to the test substance; administered orally via the diet. Domain 4: Test Organism Metric 13: Test Animal Characteristics High X 2 2 The test animal species, strain, sex, health status, age, and starting body weight were reported. The test animal was obtained Dr. Karl Thomae, Biber- ach/ Riss, FRG. The test species and strain were an appropriate animal model for the evaluation of the specific outcomes of interest and routinely used for similar study types. Metric 14: Adequacy and Consistency of Animal Hus- High X 1 1 All husbandry conditions were reported and ade- bandry Conditions quate. Conditions were the same for control and exposed population. Metric 15: Number per Group High X 1 1 The number of animals per study group was reported (25 sex/dose in F0 and F1 generations) and appro- priate for the study type and outcome analysis. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High X 2 2 The outcome assessment methodologies were appro- priate for the endpoints of interest and generally consistent with OECD guidelines for two-generation reproduction testing (OECD TG 416). Continued on next page . Page 79 of 110 ------- . continued from previous page Study Citation: NMP Producers Group (1999). Two Generation Reproduction Toxicity Study with N-Mythylpyrrolidone (NMP) in Wistar Rats - Administration in the Diet Data Type: 2-gen dietary repro tox study in Wistar rats HERO ID: 3809437 Domain Metric Rating^ MWF* Score Comments^ Metric 17: Consistency of Outcome Assessment High X 1 1 Details of the outcome assessment protocol were reported and were generally assessed consistently across study groups. Metric 18: Sampling Adequacy High X 1 1 Details regarding sampling for the outcomes of in- terest were reported and the study used adequate sampling for the outcomes of interest Metric 19: Blinding of Assessors Not Rated NA NA Not applicable for this study type Metric 20: Negative Control Response High x 1 1 The biological responses of the negative control group were reported and were considered adequate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 There were no differences among the study groups in initial body weight. Food consumption was re- ported; palatability issues were not noted. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 Viability exams and clinical observations were con- ducted for all study groups. Details regarding health outcomes unrelated to exposure were reported for each study group. There were no differences among groups that could influence the outcome assessment. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were clearly described and ap- propriate for datasets Metric 24: Reporting of Data High x 2 2 Data for exposure-related findings were presented for all dose groups and endpoints with quantal pre- sentation; summary and individual animal data were reported. Overall Quality Determination1" High 1.0 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating, ft This metric met the criteria for high confidence as expected for this type of study. Page 80 of 110 ------- Table 31: Animal toxicity evaluation results of E. I. du Pont de Nemours and Company 1990 for Reproductive and developmental inhalation toxicity study in rats on developmental outcomes Study Citation: DuPont (E. I. du Pont de Nemours and Company) (1990). l-Methyl-2-pyrrolidinone (NMP): Reproductive and developmental toxicity in the rat. Unpublished data, 05 Oct 1990 Data Type: Reproductive and developmental toxicity study in rats exposed by inhalation (dev) HERO ID: 3833023 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Test substance identified by name, molecular weight, formula, and boiling point. Metric 2: Test Substance Source High x 1 1 Test substance source reported. Metric 3: Test Substance Purity High x 1 1 Test substance purity reported (99.867%) along with impurities and their concentrations Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Concurrent sham-treated controls were included. Metric 5: Positive Controls Not Rated NA NA Positive controls not typical for study type. Metric 6: Randomized Allocation Medium x 1 2 Animals allocated based on body weight using ran- domized block design. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low x 1 3 Exposure generation and measurement reported in Appendix B. Condensation of test material on cham- ber walls occurred in exposure group. Metric 8: Consistency of Exposure Administration Low X 1 3 Exposures not consistent due to test material con- densation in chambers of exposure group; there may have been oral and/or dermal exposure to conden- Metric 9: Reporting of Doses/Concentrations Medium x 2 4 Analytical chamber concentrations were reported in Appendix B. Target concentration not achieved due to condensation in chamber. Metric 10: Exposure Frequency and Duration High x 1 1 Exposure frequency and duration reported and Metric 11: Number of Exposure Groups and Dose Spac- Low ing x 1 suited to experiment. Only one exposure level was used for the teratogenic- ity study (130 ppm) and this exposure level resulted in condensation on chamber walls, with a potential for oral, dermal and inhalation exposure. Continued on next page Page 81 of 110 ------- .. . continued from previous page Study Citation: DuPont (E. I. du Pont de Nemours and Company) (1990). l-Methyl-2-pyrrolidinone (NMP): Reproductive and developmental toxicity in the rat. Unpublished data, 05 Oct 1990 Data Type: Reproductive and developmental toxicity study in rats exposed by inhalation (dev) HERO ID: 3833023 Domain Metric Rating^ MWF* Score Comments^ Metric 12: Exposure Route and Method Low X 1 3 Atmosphere generation was reported; however con- densation was observed. The ratio of vapor to aerosol is a function of the relative humidity and temperature. During the first 6 weeks of NMP ex- posure, the mean daily temperature exceeded 80 de- grees Fahrenheit on 29 days. This is expected to significantly impact results. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium X 2 4 Test species, sex, life stage and source were reported and use of the Crl:CD (SD) BR strain was justified. Health status was not reported. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Medium X 1 2 Animal husbandry conditions were reported. Sig- nificant variations in temperature was observed in chambers housing control versus treated animals. Metric 15: Number per Group High X 1 1 Number per group was reported and appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High X 2 2 Developmental outcome assessment methods were appropriate. Metric 17: Consistency of Outcome Assessment High X 1 1 There was no indication of inconsistency in outcome assessment; outcome assessments were adequately reported. Metric 18: Sampling Adequacy Medium X 1 2 Developmental study only included histopathologi- cal examinations of one dose group (130 ppm). Con- densation of the test material and porphyrin secre- tion was also observed at this concentration. Metric 19: Blinding of Assessors Medium X 1 2 Assessors were not blinded to treatment group for assessment of objective endpoints (narcosis, irrita- tion, performance). Metric 20: Negative Control Response High X 1 1 Control response was reported and appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Low X 2 6 Variations in temperature and humidity in the test chambers contributed to condensation of the test material. This is expected to influence the outcome. Metric 22: Health Outcomes Unrelated to Exposure Medium X 1 2 Two control dams died due to injury or other rea- sons; the remaining numbers per group were ade- quate. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High X 1 1 Statistical methods were reported and appropriate; litter served as the unit of statistical analysis. Continued on next page . Page 82 of 110 ------- . continued from previous page Study Citation: DuPont (E. I. du Pont de Nemours and Company) (1990). l-Methyl-2-pyrrolidinone (NMP): Reproductive and developmental toxicity in the rat. Unpublished data, 05 Oct 1990 Data Type: Reproductive and developmental toxicity study in rats exposed by inhalation (dev) HERO ID: 3833023 Domain Metric Rating^ MWF* Score Comments^ Metric 24: Reporting of Data Medium X 2 4 Litter parameters (e.g., live fetuses/litter) reported as means only without SD or SE. Overall Quality Determination1" Medium 1.8 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = J]. (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 83 of 110 ------- Table 32: Animal toxicity evaluation results of E. I. du Pont de Nemours and Company 1990 for Reproductive and developmental inhalation toxicity study in rats on reproductive outcomes Study Citation: DuPont (E. I. du Pont de Nemours and Company) (1990). l-Methyl-2-pyrrolidinone (NMP): Reproductive and developmental toxicity in the rat. Unpublished data, 05 Oct 1990 Data Type: Reproductive and developmental toxicity study in rats exposed by inhalation (repro) HERO ID: 3833023 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Test substance identified by name, molecular weight, formula, and boiling point. Metric 2: Test Substance Source High x 1 1 Test substance source reported . Metric 3: Test Substance Purity High x 1 1 Test substance purity reported (99.867%) along with impurities and their concentrations Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Concurrent sham-treated controls were included. Metric 5: Positive Controls Not Rated NA NA Positive controls not typical for study type. Metric 6: Randomized Allocation Medium x 1 2 Animals allocated based on body weight using ran- domized block design. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low x 1 3 Exposure generation and measurement reported in Appendix B. Condensation of test material on cham- ber walls occurred in high exposure group. Metric 8: Consistency of Exposure Administration Low X 1 3 Exposures not consistent due to test material con- densation in high exposure group. Metric 9: Reporting of Doses/Concentrations High x 2 2 Analytical chamber concentrations were reported in Appendix B. Metric 10: Exposure Frequency and Duration High x 1 1 Exposure frequency and duration reported and suited to experiment. Metric 11: Number of Exposure Groups and Dose Spac- ing Medium x 1 2 3 nonzero exposure concentrations were used; high- est exposure level resulted in condensation on cham- ber walls. Metric 12: Exposure Route and Method High x 1 1 Atmosphere generation was reported and appropri- ate. Domain 4: Test Organism Metric 13: Test Animal Characteristics High Metric 14: Adequacy and Consistency of Animal Hus- High bandry Conditions Metric 15: Number per Group High X 2 2 Test species, strain, sex, life stage, and source were reported and justified. X 1 1 Animal husbandry conditions were reported and ap- propriate. X 1 1 Number per group was reported and appropriate Continued on next page Page 84 of 110 ------- ... continued from previous page Study Citation: DuPont (E. I. du Pont de Nemours and Company) (1990). l-Methyl-2-pyrrolidinone (NMP): Reproductive and developmental toxicity in the rat. Unpublished data, 05 Oct 1990 Data Type: Reproductive and developmental toxicity study in rats exposed by inhalation (repro) HERO ID: 3833023 Domain Metric Rating^ MWF* Score Comments^ Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 Reproductive outcome assessment methods were re- ported and appropriate. Metric 17: Consistency of Outcome Assessment High x 1 1 There was no indication of inconsistency in outcome assessment; outcome assessments were adequately reported. Metric 18: Sampling Adequacy Medium x 1 2 Histopathology of testes and ovaries was not eval- uated except when grossly-observed effects or func- tional changes occurred. Metric 19: Blinding of Assessors Medium x 1 2 Investigators evaluating response to sound in P0 rats were not blinded to exposure status. Other out- comes were not subjective. Metric 20: Negative Control Response High x 1 1 Control response reported and appropriate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Medium x 2 4 Respiratory rate was not reported, but it is not clear that HBCD is an irritant. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 3 dams died due to injury or other reasons; 2 were controls. Remaining numbers per group were ade- quate. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were reported and appropriate. Metric 24: Reporting of Data Low x 2 6 Most data reported as means without SE or SD. Overall Quality Determination"1 High —~ Medium^ Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. § Evaluator's explanation for rating change: "Confounding by condensation on chamber walls at high exposure level; failure to report measures of variability for most data." Page 85 of 110 ------- Table 33: Animal toxicity evaluation results of Ciba-Geigy 1987 for an oral developmental study in mice on growth (early life) and development outcomes Study Citation: Ciba-Geigy Corp (1987). Letter from Ciba-Geigy Corporation to US EPA regarding information on the enclosed reports concerning n-methylpyrrolidone with attachments Data Type: Oral developmental mice HERO ID: 4214093 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 Test substance identified by name and structure. Metric 2: Test Substance Source Low X 1 3 The source was not identified. Metric 3: Test Substance Purity Low X 1 3 The purity was not reported; stated to be double distilled. Domain 2: Test Design Metric 4: Negative and Vehicle Controls Medium X 2 4 No information on the age/weight of negative (un- treated) control animals was provided. Metric 5: Positive Controls Not Rated NA NA Positive controls were not required. Metric 6: Randomized Allocation Low X 1 3 Method of randomization not reported. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low X 1 3 Information on storage/stability was not provided. Metric 8: Consistency of Exposure Administration High X 1 1 Exposures were administered consistently. Metric 9: Reporting of Doses/Concentrations Low X 2 6 Doses were reported as (mm3/kg); however, specific information needed to confirm dose calculations (pu- rity, body weight) was not provided. Metric 10: Exposure Frequency and Duration Low X 1 3 Frequency and duration were reported; however, the duration of exposure was inadequate based on OECD 422 standards. Metric 11: Number of Exposure Groups and Dose Spac- Low X 1 3 The number of exposure groups and dose spacing ing were inadequate based on OECD 422 standards. Metric 12: Exposure Route and Method Low X 1 3 Translated summaries provided for i.p., oral and in- halation exposures. Limited information provided for generation of in- halation concentrations is. Domain 4: Test Organism Metric 13: Test Animal Characteristics Low X 2 6 The source, species, strain, and sex were reported. Age, initial body weight, and health status were not reported. Metric 14: Adequacy and Consistency of Animal Hus- Medium X 1 2 Housing, feed, water, temperature, and humidity bandry Conditions were reported. Lighting and number of room air changes were not reported. Continued on next page . . Page 86 of 110 ------- ... continued from previous page Study Citation: Ciba-Geigy Corp (1987). Letter from Ciba-Geigy Corporation to US EPA regarding information on the enclosed reports concerning n-methylpyrrolidone with attachments Data Type: Oral developmental mice HERO ID: 4214093 Domain Metric Rating^ MWF* Score Comments^ Metric 15: Number per Group High X 1 1 The number of animals was adequate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Low x 2 6 Description of outcome assessment methodology is inadequate. Metric 17: Consistency of Outcome Assessment Low x 1 3 It is difficult to discern whether outcomes were as- sessed consistently based on translated text. Metric 18: Sampling Adequacy Medium x 1 2 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding not required. Metric 20: Negative Control Response Low x 1 3 Negative control responses were not appropriate. The total malformation rate in untreated controls (2.6%) exceeded the spontaneous value reported in historical controls (1.7%) Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables were reported. Metric 22: Health Outcomes Unrelated to Exposure Low x 1 3 No information provided regarding health outcomes unrelated to exposure were reported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Low x 1 3 Statistical analysis was not conducted, but data were available to conduct analysis. Metric 24: Reporting of Data Low x 2 6 Maternal body weights were not reported. Litter data were not presented. Overall Quality Determination Low 2.4 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 87 of 110 ------- Table 34: Animal toxicity evaluation results of Ciba-Geigy 1987 for an oral developmental rat study on growth (early life) and development outcomes Study Citation: Ciba-Geigy Corp (1987). Letter from Ciba-Geigy Corporation to US EPA regarding information on the enclosed reports concerning n-methylpyrrolidone with attachments Data Type: Oral developmental rats HERO ID: 4214093 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 Test substance identified by name and structure. Metric 2: Test Substance Source Low X 1 3 The source was not identified. Metric 3: Test Substance Purity Low X 1 3 The purity was not reported, but stated to be double distilled. Domain 2: Test Design Metric 4: Negative and Vehicle Controls Medium X 2 4 No information on the age/weight of negative (un- treated) control animals was provided. Metric 5: Positive Controls Not Rated NA NA Positive controls were not required. Metric 6: Randomized Allocation Low X 1 3 Method of randomization not reported. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Medium X 1 2 Information on storage/stability was not provided, but it is not expected to significantly impact results. Metric 8: Consistency of Exposure Administration High X 1 1 Metric 9: Reporting of Doses/Concentrations Medium X 2 4 Doses were reported as (mm3/kg); however, specific information needed to confirm dose calculations (pu- rity, body weight) was not provided. Metric 10: Exposure Frequency and Duration Low X 1 3 Frequency and duration were reported; however, the duration of exposure was inadequate based on OECD 422 standards. Metric 11: Number of Exposure Groups and Dose Spac- ing Low X 1 3 The number of exposure groups and dose spacing were reported and justified, but not considered ade- quate based on OECD 422 standards. Metric 12: Exposure Route and Method High X 1 1 The route and method were appropriate. Domain 4: Test Organism Metric 13: Test Animal Characteristics Low X 2 6 The source, species, strain, and sex were reported. Age, initial body weight, and health status were not reported. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Medium X 1 2 Housing, feed, water, temperature, and humidity were reported. Lighting and air changes were not reported. Metric 15: Number per Group High X 1 1 The number of animals was adequate. Domain 5: Outcome Assessment Continued on next page . .. Page 88 of 110 ------- . continued from previous page Study Citation: Ciba-Geigy Corp (1987). Letter from Ciba-Geigy Corporation to US EPA regarding information on the enclosed reports concerning n-methylpyrrolidone with attachments Data Type: Oral developmental rats HERO ID: 4214093 Domain Metric Rating^ MWF* Score Comments^ Metric 16: Outcome Assessment Methodology Low x 2 6 Outcome assessment methodology was described; however, it is not adequate based on OECD 422 standards. Metric 17: Consistency of Outcome Assessment Medium x 1 2 Outcomes were assessed consistently. Metric 18: Sampling Adequacy Medium x 1 2 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding not required. Metric 20: Negative Control Response Low x 1 3 Negative control responses were not appropriate. The total malformation rate in untreated controls (2.6%) exceeded the spon- taneous value reported in historical controls (1.7%) Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables were reported. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health outcomes unrelated to exposure were re- ported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Low x 1 3 Statistical analysis was not conducted, but data were available to conduct analysis. Metric 24: Reporting of Data Low x 2 6 Maternal body weights were not reported. Litter data were not presented. Overall Quality Determination1" Medium 2.2 Extracted No MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 89 of 110 ------- Table 35: Animal toxicity evaluation results of GAF Chemicals Corp 1991 for developmental toxicity study in rabbits (GD 6-12) on growth (early life) and development outcomes Study Citation: GAF (GAF Chemicals Corp) (1991). Letter from GAF Chem Corp to US EPA submitting preliminary results of n-methyl-2-pyrrolidone developmental toxicity study with attachments Data Type: developmental toxicity study in rabbits GD 6-12 HERO ID: 4214113 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High X 2 2 Identified by name and CAS number Metric 2: Test Substance Source Low X 1 3 Source and/or analytical verification not reported; impact on results uncertain Metric 3: Test Substance Purity Low X 1 3 Purity was not reported Domain 2: Test Design Metric 4: Negative and Vehicle Controls Medium X 2 4 Negative control was used, but vehicle was not re-ported Metric 5: Positive Controls Not Rated NA NA Not applicable for this study type. Metric 6: Randomized Allocation Low X 1 3 The study did not report how animals were allocated to study groups Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Unacceptable X 1 4 Test substance preparation and storage was not re-ported Metric 8: Consistency of Exposure Administration Unacceptable X 1 4 Method and vehicle were not reported Metric 9: Reporting of Doses/Concentrations Medium X 2 4 Method of administration not reported Metric 10: Exposure Frequency and Duration Unacceptable X 1 4 Duration and frequency not reported. Metric 11: Number of Exposure Groups and Dose Spac- ing Medium X 1 2 Sufficient number of exposure groups, but doses and spacing were not justified by the study authors; the number of groups and spacing were adequate to show dose-response. Metric 12: Exposure Route and Method Medium X 1 2 Method of administration reported. Domain 4: Test Organism Metric 13: Test Animal Characteristics Low X 2 6 The source, health status, age and starting body weight of the test animals were not reported Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Low X 1 3 animal husbandry conditions were not reported Metric 15: Number per Group Medium X 1 2 The number of animals in the study was reported and adequate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Unacceptable X 2 8 Limited information on outcome assessment re- ported. Continued on next page . .. Page 90 of 110 ------- . continued from previous page Study Citation: GAF (GAF Chemicals Corp) (1991). Letter from GAF Chem Corp to US EPA submitting preliminary results of n-methyl-2-pyrrolidone developmental toxicity study with attachments Data Type: developmental toxicity study in rabbits GD 6-12 HERO ID: 4214113 Domain Metric Rating^ MWF* Score Comments^ Metric 17: Consistency of Outcome Assessment Unacceptable X 1 4 Details regarding the execution of the study protocol for outcome assessment were not reported. Metric 18: Sampling Adequacy Low X 1 3 Details regarding sampling for outcomes of interest were not reported. Metric 19: Blinding of Assessors Not Rated NA NA Not applicable for this study Metric 20: Negative Control Response Medium x 1 2 Control responses reported without measure of vari- ability. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures Low x 2 6 Initial health status, body weight food/water intake not reported and could impact results. Metric 22: Health Outcomes Unrelated to Exposure Low x 1 3 Data on health outcomes unrelated to exposure for each study group was not reported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Unacceptable x 1 4 Statistical analysis not reported, and data required for independent analysis were not reported. Metric 24: Reporting of Data Low x 2 6 Statistical analysis not reported, and data required for independent analysis were not reported. Overall Quality Determination1 Unacceptable** 2.8 Extracted No * Consistent with our Application of Systematic Review in TSCA Risk Evaluations document, if a metric for a data source receives a score of Unacceptable (score = 4), EPA will determine the study to be unacceptable. In this case, one or more of the metrics were rated as unacceptable. As such, the study is considered unacceptable and the score is presented solely to increase transparency. MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating, ft This metric met the criteria for high confidence as expected for this type of study. Page 91 of 110 ------- Table 36: Animal toxicity evaluation results of Dupont 1979 for developmental range-finding study in rats on growth (early life) and development outcomes Study Citation: Dupont (E I Dupont De Nemours & Co) (1979). Initial submission: teratologic dose range-finding study w/ n-methylpyrrolidone in sprague dawley rats with cover letter dated 09/01/92 Data Type: Developmental range-finding: rats HERO ID: 4214130 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity Medium X 2 4 Test substance identified by name. Metric 2: Test Substance Source High X 1 1 The source and lot number were reported. Metric 3: Test Substance Purity High X 1 1 Test substance purity was reported Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 Concurrent negative controls were included. Metric 5: Positive Controls High X 1 1 Concurrent positive controls were used Metric 6: Randomized Allocation High X 1 1 Random number assignment sheet was used. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low X 1 3 Preparation and storage of test material was not pro- vided. However, it was reported that the positive control agent was prepared fresh daily. Metric 8: Consistency of Exposure Administration High X 1 1 Doses were administered consistently. Metric 9: Reporting of Doses/Concentrations High X 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration High X 1 1 Duration and frequency were reported. Metric 11: Number of Exposure Groups and Dose Spac- ing Medium X 1 2 The number of groups and dose spacing were re- ported but not justified. Metric 12: Exposure Route and Method Medium X 1 2 The exposure route was appropriate. This was a dermal exposure study. The method of "rubbing it in" was not described with regards to what was used and how it was done and what measures were taken to keep the test substance on. Domain 4: Test Organism Metric 13: Test Animal Characteristics High X 2 2 The source, species, strain, initial body weight, sex, and sexual state were reported. The specific age of the parents was not reported, but they were sexually mature. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions Medium X 1 2 Housing, feed and water were reported. Tempera- ture, humidity, lighting, and air changes were not reported. Metric 15: Number per Group High X 1 1 The number of animals was acceptable for a range- finding study. Continued on next page . .. Page 92 of 110 ------- ... continued from previous page Study Citation: Dupont (E I Dupont De Nemours & Co) (1979). Initial submission: teratologic dose range-finding study w/ n-methylpyrrolidone in sprague dawley rats with cover letter dated 09/01/92 Data Type: Developmental range-finding: rats HERO ID: 4214130 Domain Metric Rating^ MWF* Score Comments^ Domain 5: Outcome Assessment Metric 16 Outcome Assessment Methodology High X 2 2 Outcome assessment methodology was appropriate. Metric 17 Consistency of Outcome Assessment High X 1 1 Outcomes were assessed consistently. Metric 18 Sampling Adequacy High X 1 1 Sampling was adequate. Metric 19 Blinding of Assessors Not Rated NA NA Blinding was not required in this study. Metric 20 Negative Control Response High X 1 1 Negative control responses were appropriate. Domain 6: Confounding / Variable Control Metric 21 Confounding Variables in Test Design and Procedures High X 2 2 No confounding variables in test design and proce- dures were noted. Metric 22 Health Outcomes Unrelated to Exposure High X 1 1 No health outcomes unrelated to exposures were re- ported. Domain 7: Data Presentation and Analysis Metric 23 Statistical Methods High X 1 1 Statistical methods were reported and appropriate. Metric 24 Reporting of Data High X 2 2 Data presentation for all outcomes was adequate. Overall Quality Determination1" High 1.2 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating, ft This metric met the criteria for high confidence as expected for this type of study. Page 93 of 110 ------- Table 37: Animal toxicity evaluation results of Sitarek et al., 2012 for single generation reproductive toxicity assessment in rats exposed orally study on reproductive outcomes Study Citation: Sitarek, K; Stetkiewicz, J; Wasowicz, W (2012). Evaluation of reproductive disorders in female rats exposed to N-methyl-2-pyrrolidone Birth Defects Research, Part B: Developmental and Reproductive Toxicology, 95(3), 195-201 Data Type: single generation reproductive toxicity assessment in rats exposed orally HERO ID: 3043651 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 Test substance identified by name and CASRN Metric 2: Test Substance Source Medium x 1 2 Test substance obtained from commercial source but without certification or analytical verification of identity Metric 3: Test Substance Purity High x 1 1 Purity reported to be >98% Domain 2: Test Design Metric 4: Negative and Vehicle Controls Medium x 2 4 Negative controls were sham-treated with tap water; however, report did not specify whether water was the vehicle for NMP. Metric 5: Positive Controls Not Rated NA NA Positive controls not typical for this study type. Metric 6: Randomized Allocation Low x 1 3 Study did not report how animals were allocated to groups. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low x 1 3 Test material prep and storage were not reported. Metric 8: Consistency of Exposure Administration Medium X 1 2 Most exposure details were reported, but time of day of gavage administration was not reported. No inconsistencies in exposure administration were re- ported. Metric 9: Reporting of Doses/Concentrations Medium x 2 4 Gavage doses were reported in mg/kg bw; initial body weight was not reported. Metric 10: Exposure Frequency and Duration High x 1 1 Frequency and duration were reported and suited to the study time. Metric 11: Number of Exposure Groups and Dose Spac- High x 1 1 The number of exposure groups and concentrations ing were not justified by the study authors in the report. This is unlikely to impact results, as the number of exposure groups and spacing of the exposures were adequate to show results relevant to the outcome of interest. Continued on next page . .. Page 94 of 110 ------- .. . continued from previous page Study Citation: Sitarek, K; Stetkiewicz, J; Wasowicz, W (2012). Evaluation of reproductive disorders in female rats exposed to N-methyl-2-pyrrolidone Birth Defects Research, Part B: Developmental and Reproductive Toxicology, 95(3), 195-201 Data Type: single generation reproductive toxicity assessment in rats exposed orally HERO ID: 3043651 Domain Metric Rating^ MWF* Score Comments^ Metric 12: Exposure Route and Method Medium X 1 2 A dynamic whole-body chamber was used for vapors that may condense. The high- concentration target of 130 ppm was not attained due to condensation on the inside of the high dose chambers; the actual high-concentration exposure level was measured as 116.4 ppm. The number of air changes/hour was reported to be 12-15. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium X 2 4 The test animal species, strain, sex, life stage, and source (laboratory- maintained colony) were re- ported. Starting age and body weight were not re- ported. Test animal was appropriate to the out- come; however, only females were exposed, so effects through the male line could not be assessed. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions High X 1 1 All animal husbandry conditions were reported and appropriate. Metric 15: Number per Group High X 1 1 22 to 28 females/group tested. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High X 2 2 Metric 17: Consistency of Outcome Assessment Medium X 1 2 Females of the high dose group that were not preg- nant were sacrificed on day 25 post mating, while remaining females were sacrificed after 3 weeks of lactation (the latter had longer exposure durations). Metric 18: Sampling Adequacy High X 1 1 All animals were evaluated for all outcomes. Metric 19: Blinding of Assessors Not Rated NA NA Subjective outcomes not evaluated. Metric 20: Negative Control Response High X 1 1 Negative control responses were reported and ade- quate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High X 2 2 No potentially confounding factors were noted by the authors or apparent in the study. Metric 22: Health Outcomes Unrelated to Exposure Medium X 1 2 Details regarding animal attrition and health out- comes unrelated to exposure was reported. It was reported that 2 animals in the control group died during the reproduction phase of the study; one of these deaths was due to a handling injury. There were no differences among groups that would influ- ence the outcome assessment. Domain 7: Data Presentation and Analysis Continued on next page . . Page 95 of 110 ------- ... continued from previous page Study Citation: Sitarek, K; Stetkiewicz, J; Wasowicz, W (2012). Evaluation of reproductive disorders in female rats exposed to N-methyl-2-pyrrolidone Birth Defects Research, Part B: Developmental and Reproductive Toxicology, 95(3), 195-201 Data Type: single generation reproductive toxicity assessment in rats exposed orally HERO ID: 3043651 Domain Metric Rating^ MWF* Score Comments^ Metric 23: Statistical Methods Medium X 1 2 Statistical analysis was described and conducted; however, it is not clear that all offspring analyses considered the effect of litter size. Metric 24: Reporting of Data High x 2 2 Overall Quality Determination"1' High —~ Medium^ Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. § Evaluator's explanation for rating change: "The only metric that was unacceptable was test substance preparation and storage. The vehicle was not specified in the paper, but controls were given tap water, so water may have been the vehicle. According to the 2001 CICAD, NMP is very stable, and has low volatility from water. Coupled with the fact that the test material was given by gavage, the lack of information on preparation and storage is of limited concern." Page 96 of 110 ------- Table 38: Animal toxicity evaluation results of Sitarek et al., 2012 for a single generation reproductive toxicity assessment in rats exposed orally study on hematological and immune, respiratory, endocrine, hepatic, renal, neurological/behavior, and thyroid outcomes Study Citation: Sitarek, K; Stetkiewicz, J; Wasowicz, W (2012). Evaluation of reproductive disorders in female rats exposed to N-methyl-2-pyrrolidone Birth Defects Research, Part B: Developmental and Reproductive Toxicology, 95(3), 195-201 Data Type: single generation reproductive toxicity assessment in rats exposed orally HERO ID: 3043651 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Metric 2: Test Substance Identity Test Substance Source Metric 3: Test Substance Purity High Medium High X 2 2 Test substance identified by name and CASRN X 1 2 Test substance obtained from commercial source but without certification or analytical verification of identity X 1 1 Purity reported to be >98% Domain 2: Test Design Metric 4: Negative and Vehicle Controls Metric 5: Positive Controls Metric 6: Randomized Allocation Medium X 2 4 Negative controls were sham-treated with tap water; however, report did not specify whether water was the vehicle for NMP. Not Rated NA NA Positive controls not typical for this study type. Low X 1 3 Study did not report how animals were allocated to groups. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low Metric 8: Consistency of Exposure Administration Medium Metric 9: Reporting of Doses/Concentrations Medium Metric 10: Exposure Frequency and Duration High Metric 11: Number of Exposure Groups and Dose Spac- High ing Metric 12: Exposure Route and Method Medium X 1 3 Test material prep and storage were not reported. X 1 2 Most exposure details were reported, but time of day of gavage administration was not reported. No inconsistencies in exposure administration were re- ported. X 2 4 Gavage doses were reported in mg/kg bw; initial body weight was not reported. X 1 1 Frequency and duration were reported and suited to the study time. X 1 1 Three nonzero doses ranging —7-fold were selected based on fractions of the LD50. Effects were seen at all doses, so it is not clear that the low dose was low enough. X 1 2 Study does not report whether compound adminis- tered neat or in a vehicle. Controls were given tap water, so it is possible that water was the vehicle. Domain 4: Test Organism Continued on next page Page 97 of 110 ------- .. . continued from previous page Study Citation: Sitarek, K; Stetkiewicz, J; Wasowicz, W (2012). Evaluation of reproductive disorders in female rats exposed to N-methyl-2-pyrrolidone Birth Defects Research, Part B: Developmental and Reproductive Toxicology, 95(3), 195-201 Data Type: single generation reproductive toxicity assessment in rats exposed orally HERO ID: 3043651 Domain Metric Rating^ MWF* Score Comments^ Metric 13: Test Animal Characteristics Medium x 2 4 The test animal species, strain, sex, life stage, and source (laboratory- maintained colony) were re- ported. Starting age and body weight were not re- ported. Test animal was appropriate to the out- come; however, only females were exposed, so effects through the male line could not be assessed. Metric 14: Adequacy and Consistency of Animal Hus- bandry Conditions High x 1 1 All animal husbandry conditions were reported and appropriate. Metric 15: Number per Group High x 1 1 22 to 28 females/group tested. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Low x 2 6 Endpoints relevant to these outcomes were limited to organ weights, histopathology, and hematocrit. Metric 17: Consistency of Outcome Assessment Medium x 1 2 Females of the high dose group that were not preg- nant were sacrificed on day 25 post mating, while remaining females were sacrificed after 3 weeks of lactation (the latter had longer exposure durations). Metric 18: Sampling Adequacy High x 1 1 All animals were evaluated for all outcomes. Metric 19: Blinding of Assessors Not Rated NA NA Subjective outcomes not evaluated. Metric 20: Negative Control Response High x 1 1 Negative control responses were reported and ade- quate. Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No potentially confounding factors were noted by the authors or apparent in the study. Metric 22: Health Outcomes Unrelated to Exposure Medium x 1 2 There were 2 deaths of nonpregnant females at the high dose, and pneumonia was diagnosed, suggesting possible gavage errors, but the authors did not draw a conclusion with respect to cause of death. This attrition was reported and unlikely to impact the results. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods Medium x 1 2 Statistical analysis was described and conducted; however, it is not clear that all offspring analyses considered the effect of litter size. Metric 24: Reporting of Data Low x 2 6 Histopathology data were reported qualitatively. Overall Quality Determination • Medium — —~ Low^® Extracted Yes Continued on next page . .. Page 98 of 110 ------- ... continued from previous page Study Citation: Sitarek, K; Stetkiewicz, J; Wasowicz, W (2012). Evaluation of reproductive disorders in female rats exposed to N-methyl-2-pyrrolidone Birth Defects Research, Part B: Developmental and Reproductive Toxicology, 95(3), 195-201 Data Type: single generation reproductive toxicity assessment in rats exposed orally HERO ID: 3043651 Domain Metric Rating^ MWF* Score Comments^ * MWF = Metric Weighting Factor t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. + The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. if any metric is unacceptable Overall rating = J]. (Metric Score; x MWF;) / J] . MWFj (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. § Evaluator's explanation for rating change: "The only metric that was unacceptable was test substance preparation and storage. The vehicle was not specified in the paper, but controls were given tap water, so water may have been the vehicle. According to the 2001 CICAD, NMP is very stable, and has low volatility from water. Coupled with the fact that the test material was given by gavage, the lack of information on preparation and storage is of limited concern." Page 99 of 110 ------- Table 39: Animal toxicity evaluation results of Becci et al., 1982 for a dermal, developmental study in rats (rf and final-study report in 4214125) on growth (early life) and development outcomes Study Citation: Becci, PJ; Knickerbocker, MJ; Reagan, EL; Parent, RA; Burnette, LW (1982). Teratogenicity study of N-methylpyrrolidone after dermal application to Sprague-Dawley rats Fundamental and Applied Toxicology, 2(2), 73-76 Data Type: Dermal developmental rats (rf and final-study report in 4214125) HERO ID: 3539729 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity Medium x 2 4 Test substance was identified by name. Metric 2: Test Substance Source Low x 1 3 The sponsor (GAF Corp) was identified as the source. Metric 3: Test Substance Purity Low X 1 3 Concurrent positive controls were used; however, a similar incidence was observed for incomplete ossifi- cation between negative and positive controls. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High X 2 2 Concurrent negative controls were used in both stud- Metric 5: Positive Controls Low X 1 3 Concurrent positive controls were used; however, a similar incidence was observed for incomplete ossifi- cation between negative and positive controls. Metric 6: Randomized Allocation Low X 1 3 Method of allocation not reported. Domain 3: Exposure Characterization Metric 7: Preparation and Storage of Test Substance Low X 1 3 Authors note that the positive control solutions were prepared fresh daily, but preparation and storage of the test material was not reported. Metric 8: Consistency of Exposure Administration Low X 1 3 Test material was rubbed into skin and the test site remained uncovered. It is unclear whether doses were administered consistently. Metric 9: Reporting of Doses/Concentrations High X 2 2 Doses were reported. Metric 10: Exposure Frequency and Duration High X 1 1 Frequency and duration were adequate. Metric 11: Number of Exposure Groups and Dose Spac- Low X 1 3 Exposure groups (n) was adequate; however, doses ing and spacing were not adequately justified (i.e., no ef- lliO fects observed at 500 mg/kg in range finding study). Metric 12: Exposure Route and Method High X 1 1 Route and method were reported. Domain 4: Test Organism Metric 13: Test Animal Characteristics Medium X 2 4 The source, species, strain, and sex were reported. Age, initial body weight, and health status were not reported. Metric 14: Adequacy and Consistency of Animal Hus- Medium X 1 2 Housing, temperature, and lighting were reported. bandry Conditions Humidity and air changes were not reported. Continued on next page . .. Page 100 of 110 ------- .. . continued from previous page Study Citation: Becci, PJ; Knickerbocker, MJ; Reagan, EL; Parent, RA; Burnette, LW (1982). Teratogenicity study of N-methylpyrrolidone after dermal application to Sprague-Dawley rats Fundamental and Applied Toxicology, 2(2), 73-76 Data Type: Dermal developmental rats (rf and final-study report in 4214125) HERO ID: 3539729 Domain Metric Rating^ MWF* Score Comments^ Metric 15: Number per Group High X 1 1 The number of animals was appropriate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology Medium x 2 4 Treatment through organogenesis (GD 16 in rats) is recommended for teratology assessments. Metric 17: Consistency of Outcome Assessment Low x 1 3 Based on examination of urine, only 1 animal treated at 75 mg/kg showed evidence of exposure. Metric 18: Sampling Adequacy High x 1 1 Sampling was adequate. Metric 19: Blinding of Assessors Not Rated NA NA Blinding was not required. Metric 20: Negative Control Response Low x 1 3 A similar incidence was observed for incomplete os- sification between negative and positive controls Domain 6: Confounding / Variable Control Metric 21: Confounding Variables in Test Design and Procedures High x 2 2 No confounding variables were reported. Metric 22: Health Outcomes Unrelated to Exposure High x 1 1 No health effects unrelated to exposure were re- ported. Domain 7: Data Presentation and Analysis Metric 23: Statistical Methods High x 1 1 Statistical methods were described and appropriate. Metric 24: Reporting of Data High x 2 2 Data were reported for all outcomes. Overall Quality Determination Medium 1.8 Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. This metric met the criteria for high confidence as expected for this type of study. Page 101 of 110 ------- 7 Mechanistic Table 40: In vitro evaluation results for Gjoksi et al., 2016 for an inhibition of bromodomain binding study Study Citation: Gjoksi, B.,Ghayor, C.,Bhattacharya, I.,Zenobi-Wong, M.,Weber, F. E. (2016). The bromodomain inhibitor N-methyl pyrrolidone reduced fat accumulation in an ovariectomized rat model Clinical Epigenetics, 8 42 Data Type: Inhibition of bromodomain binding (AlphaScreen assay) HERO ID: 3539796 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 The test substance was identified by established nomenclature. Metric 2: Test Substance Source Low x 1 3 The source of the test substance was not reported. Metric 3: Test Substance Purity Low x 1 3 Purity was not reported. Domain 2: Test Design Metric 4: Negative and Vehicle Controls Low x 2 6 Negative control groups were shown in Figure 5. The study indicated that the assay was performed in the absence of the compound (defined as 100% activity). The assay was also performed in the absence if the bromodomain/BET ligand (defined as 0% activity). Metric 5: Positive Controls Not Rated NA NA The assay test kits may have contained reference compounds, but no data for reference compounds was reported. Reference compound (JQ1) data was provided in an earlier publication by the same au- thors (Gjoksi et al., 2015; HERO ID 3539797) Metric 6: Assay Procedures Medium x 1 2 Methods were partially described and were cited to a standard AphaScreening assay method from BPS Bioscience (San Diego, CA). Metric 7: Standards for Tests Not Rated NA NA Not applicable to the study type. Domain 3: Exposure Characterization Metric 8: Preparation and Storage of Test Substance Not Rated NA Metric 9: Consistency of Exposure Administration Medium x 1 Metric 10: Reporting of Doses/Concentrations High x 2 Metric 11: Number of Exposure Groups and Concentra- Not Rated NA tion Spacing NA Preparation and storage of NMP was not described; however, this information was likely standardized and provided in the AlphaScreen assay instructions (cited to BPS Bioscience, San Diego, CA). 2 Exposure details were not reported; however, the Al- phaScreen assay is conducted in a microplate for- mat which suggests consistent administration across groups. 2 Concentrations were reported in Fig 5. NA Assay duration was not reported; however, this in- formation was likely standardized and provided in the AlphaScreen assay instructions (cited to BPS Bioscience, San Diego, CA). Continued on next page .. Page 102 of 110 ------- .. . continued from previous page Study Citation: Gjoksi, B.,Ghayor, C.,Bhattacharya, I.,Zenobi-Wong, M.,Weber, F. E. (2016). The bromodomain inhibitor N-methyl pyrrolidone reduced fat accumulation in an ovariectomized rat model Clinical Epigenetics, 8 42 Data Type: Inhibition of bromodomain binding (AlphaScreen assay) HERO ID: 3539796 Domain Metric Rating^ MWF* Score Comments^ Metric 12: Exposure Route and Method High X 1 1 9 concentrations were used; spacing across 5 log units. Not applicable to the study design. Metric 13: Metabolic Activation Not Rated NA NA Domain 4: Test Model Metric 14: Test Model Medium x 2 4 The test model was reported along with limited de- scriptive information (recombinant bromodomains and bromodomain ligands or recombinant BET bromodomains and BET ligands). Metric 15: Number per Group Medium x 1 2 Number of replicates was not reported; however, er- ror bars in Figure 5 suggest more than one replicate. An earlier publication by the same authors (Gjoksi et al., 2015; HERO ID 3539797) indicated that binding experiments were performed in duplicate. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 The outcome assessment method reported and was sensitive for the outcome of interest. Metric 17: Metric 18: Metric 19: Consistency of Outcome Assessment Sampling Adequacy Blinding of Assessors High Not Rated Not Rated x 1 NA NA 1 NA NA Outcomes were assessed consistently across groups (microplate format). Not applicable to the study design. Automated measurements only (fluorescence inten- sity). Domain 6: Confounding / Variable Control Metric 20: Confounding Variables in Test Design and Procedures High x 2 2 There were no differences reported among study groups (assay test kit). Metric 21: Confounding Variables in Outcomes Unre- lated to Exposure Low x 1 3 Data on outcome differences unrelated to exposure were not reported (e.g., altered fluorescence by test substance). Domain 7: Data Presentation and Analysis Metric 22: Data Analysis High x 1 1 Statistical methods and calculation of IC50s were well described and appropriate. Metric 23: Data Interpretation Not Rated NA NA Scoring and/or evaluation criteria do not apply to the study design. Metric 24: Cytotoxicity Data Not Rated NA NA A cell-free test system was used. Metric 25: Reporting of Data High x 2 2 Data for exposure-related findings were presented for all outcomes by exposure group. Overall Quality Determination^ High 1.7 Continued on next page . .. Page 103 of 110 ------- ... continued from previous page Study Citation: Gjoksi, B.,Ghayor, C.,Bhattacharya, I.,Zenobi-Wong, M.,Weber, F. E. (2016). The bromodomain inhibitor N-methyl pyrrolidone reduced fat accumulation in an ovariectomized rat model Clinical Epigenetics, 8 42 Data Type: Inhibition of bromodomain binding (AlphaScreen assay) HERO ID: 3539796 Domain Metric Rating^ MWF* Score Comments^ Extracted Yes MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; x MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 104 of 110 ------- Table 41: In vitro evaluation results for Gjoksi et al., 2015 for an inhibition of bromodomain binding study Study Citation: Gjoksi, B.,Ghayor, C.,Siegenthaler, B.,Ruangsawasdi, N.,Zenobi-Wong, M.,Weber, F. E. (2015). The epigenetically active small chem- ical N-methyl pyrrolidone (NMP) prevents estrogen depletion induced osteoporosis Bone, 78 114-121 Data Type: Inhibition of bromodomain binding (AlphaScreen assay) HERO ID: 3539797 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 The test substance was identified by established nomenclature. Metric 2: Test Substance Source Low x 1 3 The source of the test substance was not reported. Metric 3: Test Substance Purity Low x 1 3 Purity was not reported. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 Negative control groups were shown in Figure 1. The study indicated that the assay was performed in the absence of the compound (defined as 100% activity). The assay was also performed in the absence if the bromodomain/BET ligand (defined as 0% activity). Metric 5: Positive Controls High x 2 2 The pan-BET inhibitor JQ1 was used as a positive control. Metric 6: Assay Procedures Medium x 1 2 Methods were partially described and were cited to a standard AphaScreening assay method from BPS Bioscience (San Diego, CA). Metric 7: Standards for Tests Not Rated NA NA Not applicable to the study type. Domain 3: Exposure Characterization Metric 8: Preparation and Storage of Test Substance Metric 9: Consistency of Exposure Administration Metric 10: Reporting of Doses/Concentrations Metric 11: Number of Exposure Groups and Concentra- tion Spacing Metric 12: Exposure Route and Method Metric 13: Metabolic Activation Not Rated NA NA Medium High Not Rated High Not Rated x 1 x 2 NA x 1 NA 2 NA NA Preparation and storage of NMP was not described; however, this information was likely standardized and provided in the AlphaScreen assay instructions (cited to BPS Bioscience, San Diego, CA). Exposure details were not reported; however, the Al- phaScreen assay is conducted in a microplate for- mat which suggests consistent administration across groups. Concentrations were reported in Figure IB. Assay duration was not reported; however, this in- formation was likely standardized and provided in the AlphaScreen assay instructions (cited to BPS Bioscience, San Diego, CA). 9 concentrations were used; spacing across 5 log units. Not applicable to the study design. Domain 4: Test Model Continued on next page Page 105 of 110 ------- .. . continued from previous page Study Citation: Gjoksi, B.,Ghayor, C.,Siegenthaler, B.,Ruangsawasdi, N.,Zenobi-Wong, M.,Weber, F. E. (2015). The epigenetically active small chem- ical N-methyl pyrrolidone (NMP) prevents estrogen depletion induced osteoporosis Bone, 78 114-121 Data Type: Inhibition of bromodomain binding (AlphaScreen assay) HERO ID: 3539797 Domain Metric Rating^ MWF* Score Comments^ Metric 14: Test Model Medium x 2 4 The test model was reported along with limited de- scriptive information (recombinant bromodomains and bromodomain ligands or recombinant BET bromodomains and BET ligands). Metric 15: Number per Group High x 1 1 Binding experiments were performed in duplicate. According to the guide for this assay type, Al- phaScreen assays are typified by very low variability between replicate wells. Running samples in dupli- cate is typically sufficient (the assay can even be run in singlicate). Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 The outcome assessment method reported and was sensitive for the outcome of interest. Metric 17: Consistency of Outcome Assessment High x 1 1 Outcomes were assessed consistently across groups (microplate format). Metric 18: Sampling Adequacy Not Rated NA NA Not applicable to the study design. Metric 19: Blinding of Assessors Not Rated NA NA Automated measurements only (fluorescence inten- sity). Domain 6: Confounding / Variable Control Metric 20: Confounding Variables Procedures in Test Design and High x 2 2 There were no differences reported among study groups (assay test kit). Metric 21: Confounding Variables lated to Exposure in Outcomes Unre- Low x 1 3 Data on outcome differences unrelated to exposure were not reported (e.g., altered fluorescence by test substance). Domain 7: Data Presentation and Analysis Metric 22: Data Analysis High x 1 1 Statistical methods and calculation of IC50s were well described and appropriate. Metric 23: Data Interpretation Not Rated NA NA Scoring and/or evaluation criteria do not apply to the study design. Metric 24: Cytotoxicity Data Not Rated NA NA A cell-free test system was used. Metric 25: Reporting of Data High x 2 2 Data for exposure-related findings were presented for all outcomes by exposure group. Overall Quality Determination" High 1.4 Extracted Yes Continued on next page . .. Page 106 of 110 ------- ... continued from previous page Study Citation: Gjoksi, B.,Ghayor, C.,Siegenthaler, B.,Ruangsawasdi, N.,Zenobi-Wong, M.,Weber, F. E. (2015). The epigenetically active small chem- ical N-methyl pyrrolidone (NMP) prevents estrogen depletion induced osteoporosis Bone, 78 114-121 Data Type: Inhibition of bromodomain binding (AlphaScreen assay) HERO ID: 3539797 Domain Metric Rating^ MWF* Score Comments^ * MWF = Metric Weighting Factor t High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. + The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. if any metric is unacceptable Overall rating = J]. (Metric Score; x MWF;) / J] . MWFj (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. ^ This metric met the criteria for high confidence as expected for this type of study. Page 107 of 110 ------- Table 42: In vitro evaluation results for Shortt et al., 2014 for an inhibition of bromodomain binding study Study Citation: Shortt, J., Hsu, A. K., Martin, B. P., Doggett, K., Matthews, G. M., Doyle, M. A., Ellul, J., Jockel, T. E., Andrews, D. M., Hogg, S. J., Reitsma, A., Faulkner, D., Bergsagel, P. L., Chesi, M., Heath, J. K., Denny, W. A., Thompson, P. E., Neeson, P. J., Ritchie, D. S., Mcarthur, G. A., Johnstone, R. W. (2014). The drug vehicle and solvent N-methylpyrrolidone is an immunomodulator and antimyeloma compound 7(4,4), 1009-1019 Data Type: Inhibition of bromodomain binding (BromoMax screening assay) HERO ID: 3540731 Domain Metric Rating^ MWF* Score Comments^ Domain 1: Test Substance Metric 1: Test Substance Identity High x 2 2 The test substance was identified by establish nomenclature. Metric 2: Test Substance Source High x 1 1 Commercial source was identified. Metric 3: Test Substance Purity Low x 1 3 Purity was not reported. Domain 2: Test Design Metric 4: Negative and Vehicle Controls High x 2 2 IC50 values were reported for DMSO (see Figure 3A). The affinity of NMP for bromodomain proteins was expressed as the percent of control (Table S3). Metric 5: Positive Controls High x 2 2 Data were reported for JQ1 (a pan-BET inhibitor). Metric 6: Assay Procedures Low x 1 3 Assay procedures were not well described. Bromod- omain competition assays were BROMOscan (Dis- coverex). Metric 7: Standards for Tests Not Rated NA NA Not applicable to the study type. Domain 3: Exposure Characterization Metric 8: Preparation and Storage of Test Substance Not Rated NA NA Preparation and storage of NMP was not described; however, this information was likely standardized and provided in the Bromoscan assay instructions (cited to Discoverex). Metric 9: Consistency of Exposure Administration Medium x 1 2 Exposure details were not reported; however, the BROMOscan assay format suggests consistent ad- ministration across groups. Metric 10: Reporting of Doses/Concentrations High x 2 2 IC50 was reported (Fig 3A); also 25mM concentra- tion of NMP was reported in Table S3. Metric 11: Number of Exposure Groups and Concentra- Not Rated NA NA Assay duration was not reported; however, this in- tion Spacing formation was likely standardized and provided in the BROMOoscan assay instructions. Metric 12: Exposure Route and Method Not Rated NA NA Multiple exposure groups must have been used to generate the IC50 values; however, the concentration were not indicated. The BromoMax screening as- say (Table S3) used a single (presumably optimized) concentration of NMP. Metric 13: Metabolic Activation Not Rated NA NA Not applicable to the study design. Continued on next page . .. Page 108 of 110 ------- . continued from previous page Study Citation: Shortt, J., Hsu, A. K., Martin, B. P., Doggett, K., Matthews, G. M., Doyle, M. A., Ellul, J., Jockel, T. E., Andrews, D. M., Hogg, S. J., Reitsma, A., Faulkner, D., Bergsagel, P. L., Chesi, M., Heath, J. K., Denny, W. A., Thompson, P. E., Neeson, P. J., Ritchie, D. S., Mcarthur, G. A., Johnstone, R. W. (2014). The drug vehicle and solvent N-methylpyrrolidone is an immunomodulator and antimyeloma compound 7(4,4), 1009-1019 Data Type: Inhibition of bromodomain binding (BromoMax screening assay) HERO ID: 3540731 Domain Metric Rating^ MWF* Score Comments^ Domain 4: Test Model Metric 14: Test Model Low x 2 6 The test model was reported, but no additional de- tails were provided. Metric 15: Number per Group Not Rated NA NA The number of replicates was not indicated, but may have been indicated in the BROMOscan assay in- structions. Domain 5: Outcome Assessment Metric 16: Outcome Assessment Methodology High x 2 2 The outcome assessment method reported and was sensitive for the outcome of interest (qPCR). Metric 17: Consistency of Outcome Assessment High x 1 1 Outcomes were asessed consistently across groups. Metric 18: Sampling Adequacy Not Rated NA NA Not applicable to the study design. Metric 19: Blinding of Assessors Not Rated NA NA Automated measurements only (qPCR). Domain 6: Confounding / Variable Control Metric 20: Confounding Variables in Test Design and Procedures High x 2 2 There were no differences reported among study groups (assay test kit). Metric 21: Confounding Variables in Outcomes Unre- lated to Exposure High x 1 1 Differences among the study replicates or groups in test model unrelated to exposure were not antici- pated (qPCR readout). Domain 7: Data Presentation and Analysis Metric 22: Data Analysis Low x 1 3 Statistical analysis was not described. Metric 23: Data Interpretation Not Rated NA NA Scoring and/or evaluation criteria do not apply to the study design. Metric 24: Cytotoxicity Data Not Rated NA NA A cell-free test system was used. Metric 25: Reporting of Data Low x 2 6 Data for exposure-related findings were not shown for each study group (IC50 values only in Figure 3A). Overall Quality Determination High 1.7 Extracted Yes Continued on next page . .. Page 109 of 110 ------- .. . continued from previous page Study Citation: Data Type: HERO ID: Shortt, J., Hsu, A. K., Martin, B. P., Doggett, K., Matthews, G. M., Doyle, M. A., Ellul, J., Jockel, T. E., Andrews, D. M., Hogg, S. J., Reitsma, A., Faulkner, D., Bergsagel, P. L., Chesi, M., Heath, J. K., Denny, W. A., Thompson, P. E., Neeson, P. J., Ritchie, D. S., Mcarthur, G. A., Johnstone, R. W. (2014). The drug vehicle and solvent N-methylpyrrolidone is an immunomodulator and antimyeloma compound 7(4,4), 1009-1019 Inhibition of bromodomain binding (BromoMax screening assay) 3540731 Domain Metric Ratingt MWF* Score Comments^ MWF = Metric Weighting Factor High = 1; Medium = 2; Low = 3; Unacceptable = 4; N/A has no value. The overall rating is calculated as necessary. EPA may not always provide a comment for a metric that has been categorized as High. Overall rating = ]T\ (Metric Score; X MWF;) / J] . MWFj if any metric is unacceptable (round to the nearest tenth) otherwise where High: > 1 to < 1.7; Medium: > 1.7 to < 2.3; Low: > 2.3 to < 3.0. If the reviewer determines that the overall rating needs adjustment, the original rating is crossed out and an arrow points to the new rating. This metric met the criteria for high confidence as expected for this type of study. Page 110 of 110 ------- |