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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.

Last updated: 31 March 2023
Last updated: 31 March 2023

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)