Outcome of assessment of 3-Nitrooxypropanol “3-NOP” as a feed additive for all ruminants for milk production and reproduction
Outcome of assessment of 3-Nitrooxypropanol “3-NOP” - Appendix 1 List of toxicological studies
Table 8 studies [resented in support of Section III: Safety for 3-NOP.
Tolerance and residue studies
| Study | Year | OECD | Animals | Doses tested |
|---|---|---|---|---|
| Pilot tolerance study, 90 days | 2018 | N/A | 16 (4 x4 groups) dairy cows | 0, 1.6, 8, 16g 3-NOP/cow/day = 100, 500 and 1000mg/kg feed DM |
| Pivotal tolerance study, 56 days | 109 | N/A | 80 (20 x 4 groups) dairy cows | 0, 80, 100, 200 mg 3-NOP/kg DM |
| Milk analysis for NOPA from University of Reading efficacy study | 2019 | N/A | 5 dairy cows | Milk samples from 5 cows receiving 3-NOP at approx 60 mg/kg, during 3 days in week 1, 6 and 15. |
ADME
| Study | Year | OECD | Animals | Doses tested |
|---|---|---|---|---|
| Stability of 3-NOP under Different Conditions | 2015 | N/A | N/A | 202 µmol/L |
| Stability of 3-NOP under Different Conditions II – Plasma Protein Binding and Chemical Oxidation | 2017 | N/A | Wistar rat plasma | 34 µmol/L |
| Plasma Protein Binding of 14C-NOPA | 2019 | N/A | Wistar rat plasma | 31.3 µmol/L and 6.26 µmol/L at 37c for up to 24 hours |
| Stability of 3-NOP under Different Conditions III – In-vitro Incubations Leading to the Major Metabolite NOPA | 2017 | N/A | Rat (Wistar and Sprague Dawley), Dog (Beagle) and Human Liver Function | 34 to 36 µmol/L |
| Metabolite Profiles and Kinetics of 3-NOP after In-vitro Incubation | 2014 | N/A | Cow rumen fluid | 2.2 and 23 mg/L at 38 degrees for 24 hours |
| Metabolite Profiles of 3-NOP after In-vitro Incubation | 2016 | N/A | Sheep, Goat and Cow Rumen Fluid | 1 mg/L at 39 degrees for 16 hours |
| ADME tissue distribution and plasma kinetics | 2013 | 417 | Wistar rats | 505 mg/kg bw |
| ADME in the Rat Following Single and Multiple Oral Administration | 2018 | N/A | 4M/4F Wistar rats | 2 exps each with 50 and 500 mg/kg bw (exps in total). 50 given as a single dose and as a 50 x 5 daily doses. 500 just as single doses. |
| ADE with volatiles | 2015 | 417 | Wistar rats | 506 mg/kg bw |
| Metabolites in plasma, liver and GIT | 2014 | 417 | Wistar rats | 505 mg/kg bw |
| Nitrate/ nitrite in plasma | 2014 | 417 | Wistar rats | 100 and 500 mg/kg bw |
| 3-NOP in lactating goats | 2015 | 503 | 2 goats | 7 daily doses of 4.34 and 3.28 mg/kg bw being equiv to 112, 102 mg / kg DM (feed) |
| ADME in Dairy Cattle Following Multiple Oral Administration | 2018 | N/A | 4 dairy cows | Every 12 hours for 7 days at dose level of 3.6 mg / kg bw / d (1.8 g / animal / d) being equiv to 150-160 mg / kg DM (feed) |
| ADME in Dairy Cattle Following Multiple Oral Administration (part 2) |
2021 | N/A | 10 dairy cows | Every 12 hours for 5 days at dose level of 3.6 mg / kg bw / d (2.1 g/animal/day) being approximately equivalent to 150 mg/kg dry feed |
| NOPA and nitrate analysis of plasma | 2016 | N/A | 4 Beef cattle and 4 controls | 29 days of 3 mg/kg bw (2g / animal) being equiv to 284 mg/kg (feed) |
| NOPA and nitrate analysis of plasma | 2016 | N/A | 28 beef cattle per dosing group | 0,100,200 mg/kg feed for 238 days |
Toxicity
| Study | Year | OECD | Animals | Doses tested |
|---|---|---|---|---|
| In-vitro Ames Microsuspension Test | 2010 | 471 | N/A | 0, 1.6, 5, 15.8, 50, 158, 500 μg / plate, with and without S9 mix |
| In-vitro Salmonella typhimurium and Escherichia coli reverse mutation assay | 2014 | 471 | N/A | 52, 164, 512, 1600 and 5000 μg/plate, with and without S9 mix |
| In-vitro Salmonella typhimurium and Escherichia coli reverse mutation assay II | 2015 | 471 | N/A | 52, 164, 512, 1600, 5000 µg/plate (experiment I), 492, 878, 1568, 2800, 5000 µg/plate (experiment II) with and without S9 mix |
| Screening in-vitro Micronucleus Test in Chinese Hamster V79 Cells | 2010 | 487 | N/A | 0, 310.8, 621.6, 1243.2 μg/mL (without S9-mix), 0, 77.7, 155.4, 310.8 μg/mL (with S9-mix) |
| In-Vitro V79 Micronucleus Assay | 2020 | 487 | N/A | 0, 300, 480, 540, 570, 600 μg/mL (with S9-mix) |
| In-vitro Micronucleus assay in cultured peripheral human lymphocytes | 2014 | 487 | N/A | 164, 512, 1211 μg/mL, with and without S9 mix |
| In-vitro mammalian cell gene mutation test (Mouse lymphoma assay) | 2015 | 476 | N/A | 0, 0.55, 1.7, 5.4, 17, 52, 164, 512 and 1211 μg/mL, with and without S9 mix |
| Cell transformation (SHE) assay | 2013 | N/A (followed OECD draft proposal) | N/A | 0, 500, 1000, 1500, 2000, 2250, 2500 μg/mL |
| In-Vitro TK6 Micronucleus Assay | 2021 | 487 | N/A | 0, 750, 1000, 1220 µg/ml with and without S9 mix |
| Salmonella typhimurium and Escherichia coli reverse mutation assay (NOPA) | 2020 | 471 | N/A | NOPA: 0, 3,10,33,100, 333, 1000, 2500 and 5000 μg/plate with and without S9 mix |
| Micronucleus Test in Human Lymphocytes In vitro (NOPA) | 2020 | 487 | N/A | NOPA: 10.4,18.2,31.8,55.7,97.5,171,299,525,915,1372 µg/ml with and without S9 mix |
| Acute Oral Toxicity Test | 2014 | 423 | Wistar rats | 300 - 2000 mg/kg bw |
| Assessment of acute inhalation toxicity | 2017 | 436 | Wistar rats | 1 and 5 mg/L |
| Micronucleus test in bone marrow cells of the mouse (screening) | 2011 | 474 | NMRI Male mice (intraperitoneal) | 0, 250, 500, 1000 mg/kg bw |
| Micronucleus test in bone marrow cells of the rat | 2014 | 474 | Wistar rats |
0, 375, 750, 1500 mg/kg bw |
| 10-day dose range finding study | 2012 | N/A | Wistar rats (n= 3 per group per sex) | 0, 100, 300, 1000 mg/kg bw |
| Combined 28-day repeated dose toxicity study and reproduction / developmental toxicity screening test | 2013 | 422, 407 | Wistar rats | 0, 10, 20, 100, 500 mg/kg bw |
| 90-day oral gavage toxicity study | 2015 | 408 | Wistar rats | 0, 50, 100, 300 mg/kg bw |
| Dose range finding study and the maximum Tolerated Dose (MTD study) | 2014 | N/A | Beagle dogs, n = 2 (1 xM, 1x F) DRF, n = 2 per sex per dose MTD | 25, 125 and 500 mg/kg bw (DRF) 0,30,100,200 (MTD study) mg/kg bw |
| 14-day oral gavage toxicity study | 2016 | N/A | Beagle dogs 2 x M and 2 x F per dose | 0, 150, 300 mg/kg bw (300 given as a split dose of 150 x 2, each 6 hours apart) |
| 3-months oral gavage toxicity study | 2016 | 409 | Beagle dogs | 0, 10, 30, 100, 300 mg/kg bw |
| 1 year oral gavage toxicity study | 2016 | 452 | Wistar rats |
Males: 0, 25, 50, 100, 300 mg/kg bw Female: 0, 50, 100, 600 mg/kg bw |
| 2-year carcinogenicity study | 2019 | 451 | Wistar rats | Males: 0, 25, 50, 100 mg/kg bw Female: 0, 50, 100, 300 mg/kg bw |
| 6-day DRF in mice | 2018 | 451 and 417 | CbyB6F1 hybrid mouse | 0, 124, 372, 742, 1224 mg/kg bw |
| 28-day study in mice | 2019 | 451 | CbyB6F1 hybrid mouse | 0, 100, 300, 700 mg/kg bw |
| NOPA In-Vivo 14-Day Dose Range Finder Assay in Rats | 2021 | N/A | Fischer rats | NOPA: 0, 112, 335, 558 and 892 mg/kg bw/d (n=6, male), 0, 335, 670 and 1000 mg/kg bw/day (n=6, female) |
| NOPA In-Vivo Mutation Assay at the cII Locus and In-Vivo Micronucleus Assay in Male and Female Big Blue® Transgenic F344 Rats | 2021 | 488, 474 | Fischer rats | NOPA: 0, 150, 300 and 600 mg/kg/day (n=6_ male), 0, 250, 500 and 1000 mg/kg/day (n=6_female) |
Reprotoxicity
| Study | Year | OECD | Animals | Doses tested |
|---|---|---|---|---|
| 28-day oral gavage mechanistic study | 2014 | Based on 407 | Wistar rats | 0, 100, 300, 500 mg / kg bw |
| Prenatal developmental toxicity study | 2015 | 414 | Wistar rats | 0, 100, 300, 1000 mg/kg bw |
| Prenatal developmental toxicity study | 2016 | 414 | NZW Rabbits | 0, 50, 150, 450 mg/kg bw |
| Two-generation reproduction study | 2016 | 416 | Wistar rats | 0, 25, 50, 100 (Male and Female), extra satellite group of females dosed at 600 mg/kg bw |
| 6-10-day preliminary mechanistic study | 2017 | N/A | Wistar rats (n=9 across the two dosing levels) | 800 and 1000 mg/kg bw |
| Dose range finding (mechanistic) | 2018 | N/A | Wistar rats (n=1 per dosing group) | 3-NOP: 1000 mg/kg bw (Oral) NOPA (metabolite): 75,250,600 mg/kg bw (IV) HPA (metabolite): 75, 250, 400 mg/kg bw (IV) HPA: 75,250,350 mg/kg bw (SC) |
| Influence of metabolites on testicular toxicity in male rats, 10-day study | 2018 | N/A | Wistar rats (n=5 per dosing group) | 3-NOP: 800 mg/kg bw (Oral) NOPA: 425 mg/kg bw (IV) HPA: 350 then 250 (day 3 onwards) mg/kg bw (IV) HPA: 350 mg/kg bw (SC) |
| Single dose transcriptomics study | 2017 | N/A | Wistar rats (n=8 per dosing group) | 0, 100, 1000 mg/kg bw |
| Benchmark-Dose-Modelling | 2019 | N/A | N/A | N/A |
| In-vitro Steroidogenesis | 2015 | N/A | Human adrenal cells | 0, 0.00001, 0.001, 0.01, 0.1, 1, 10 mM (3-NOP, NOPA and HPA) |
| Ex-vivo model testicular toxicity evaluation (3-NOP, NOPA, HPA, inorganic nitrate) | 2015 | N/A | Sprague Dawley rat | 0, 0.002, 0.02, 0.5, 2 mM (all compounds) |
| Ex-vivo model testicular toxicity evaluation of NOPA | 2016 | N/A | Sprague Dawley rat | 0, 0.02, 0.5, 2 mM (NOPA) |
| In-vitro / ex-vivo species comparison study using NOPA | 2019 | N/A | Testicular tissue from Wistar rats, Beagle dog, and Cynomolgus monkey (n=34 tissue samples for each species) | 0,1,20,500,1200,2500 µM (NOPA) |