4-methylmorpholine 4-oxide, monohydrate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-04-11 to 2021-01-18

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

hepatic clearance 

metabolism

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of the study was to investigate the metabolism of the test item NMMO in primary hepatocytes from rat and human by liquid chromatography-mass spectrometry. In a first pilot study (assay 1), 3 different test item concentrations (i.e. 30, 100 and 300 μM) were tested to select an appropriate condition for the main study part. However, NMMO was highly stable during 120 min of incubation with hepatocytes from both species and no difference was observed for the different test item concentrations. Therefore, the test concentration was further recuded to 0.3 and 3 μM in a second pilot study (assay 2). Also with this conditions, the test item was poorly metabolized with rat and human hepatocytes and the sponsor decided to cancel any further testing.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Identity: 4-methylmorpholine 4-oxide, monohydrate, CAS 7529-22-8 (NMMO)
- Lot/batch number of test material: 12.01.2018
- Purity: 50% aqueous solution

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Working solutions of test item were diluted from the stock solutions in water, to obtain working solutions of 100-fold higher strength than the final incubation concentrations and the respective final organic solvent content. In a second step, working solutions were further diluted in incubation medium to reach 10-fold concentrated starting solutions.

Radiolabelling:

no

Species:

other: rat and human

Route of administration:

application in vitro

Vehicle:

water

Duration and frequency of treatment / exposure:

120 min

Dose / conc.:

300 other: µM

Dose / conc.:

100 other: µM

Dose / conc.:

30 other: µM

Dose / conc.:

3 other: µM

Dose / conc.:

0.3 other: µM

Positive control reference chemical:

7-Ethoxycoumarin (Sigma-Aldrich, E1379, Lot: MKBN6201V)

Details on study design:

- Preparation of test solutions:
The concentration of the test item stock solution in water was 4880.75 mM, while the reference item stock solution contained 10 mM 7-ethoxycoumarin in acetonitrile. The concentration of the working solutions of the test item NMMO was 30, 10, 3, 0.3 and 0.03 mM in water, while the reference item working solution was 0.5 mM 7-ethoxycoumarin in ACN. By default, working solutions of test and reference items were diluted from the stock solutions in an appropriate solvent, to obtain working solutions of 100-fold higher strength than the final incubation concentrations and the respective final organic solvent content. In a second step, working solutions were further diluted in incubation medium to reach 10-fold concentrated starting solutions.

- Preparation of calibration standards of test and reference items:
By default, working solutions of the test and reference items were prepared for each calibration level by appropriate dilution of the corresponding stock solution with the corresponding solvent by serial dilution. Calibration solutions were processed for ACN/MeOH precipitation and quantitative bioanalysis.
Calibration standards were prepared by mixing 196 μl of incubation medium (WME supplemented with 25 mM HEPES and 2 mM L-glutamine) and the corresponding working solution (4 μl). The final standard solutions of the reference item contained 1% ACN. The standard solutions of the test item contained no organic solvent.

- Sample preparation (ACN precipitation):
As internal standards (ISTDs), compounds were chosen from the Pharmacelsus pool known to be suitable for ACN precipitation. Injection volumes of all measurements were 2 µl for NMMO samples and 0.5 µl (orbitrap) and 15 µl (triple quadrupole) for 7-ethoxycoumarin samples, respectively.
Metabolic stability samples from hepatocyte incubations were stopped by addition of 200 μl ACN/MeOH containing the internal standards (1 μM Diazepam, 1 μM Griseofulvin and 10 μM Diclofenac) to 200 μl sample or calibration standards, respectively. Samples were shaken vigorously (10 seconds) and spun down (4,800 x g, room temperature, 5 minutes). The resulting supernatant was transferred to auto sampler vials and subsequently subjected to LC-MS analysis.

- Liquid chromatography – mass spectrometry (LC-MS):
For quantitative analysis of test and reference items LC-MS systems Surveyor MS Plus HPLC (Thermo Electron) HPLC system connected to a TSQ Quantum Discovery Max (Thermo Electron) triple quadrupole mass spectrometer equipped with an electrospray (ESI) or APCI interface (Thermo Fisher Scientific, USA); connected to a PC running the standard software Xcalibur 2.0.7.
LC-HRMS: Accela U-HPLC pump and an Accela Open auto sampler (Thermo Fisher Scientific, USA) connected to an Q-Exactive mass spectrometer (Orbitrap); data handling with the standard software Chromeleon 7.2 SR5 MVf.

The pump flow rate was set to 600 μl/min and the analytes were separated as follows: 7-Ethoxycoumarin using a Kinetex Phenyl-Hexyl analytical column, 2.6 μm, 50x2.1 mm (Phenomenex, Germany) or Poroshell 120, 2.7 μm, 100x3.0 mm (Agilent, Germany), and NMMO using a Poroshell EC-C18, 2.7 μm, 100x3 mm (Agilent, Germany) with a corresponding pre-column using the gradients as presented in Table 1 in Section "Any other information on materials and methods incl. tables".
For measurements of 7-Ethoxycoumarin applying the triple quadrupole technology, full scan mass spectra were acquired in the positive mode using syringe pump infusion to identify the protonated quasimolecular ions [M+H]+. Auto-tuning was carried out for maximising ion abundance followed by the identification of characteristic fragment ions using a generic parameter set: ESI ion-transfer-capillary temperature 350°C, capillary voltage 3.8 kV, collision gas 0.8 mbar argon, sheath gas, ion sweep gas and auxiliary gas pressure were 40, 2 and 10 arbitrary units. Ions with the highest S/N ratio were used to quantify the item in the selected reaction monitoring mode (SRM) and as qualifier, respectively.
For measurements of 7-Ethoxycoumarin and NMMO applying the OrbitrapTM technology with accurate mass (Q-Exactive), as MS tune file, a generic tune file was used and as a lock mass for internal mass calibration the [M+H]+ ion of the Diisooctyl phthalate (m/z 391.28429), which is ubiquitously present in the solvent system, was used. Full MS-SIM (m/z ranges are given in Table 2 in Section "Any other information on materials and methods incl. tables") analysis was applied with the mass resolution of the Orbitrap set as given in Table 2. Further analyser settings were as follows: max. trap injection time 250 ms, sheath gas 40, aux gas 10, sweep gas 2, capillary voltage 4 kV for the positive and 2.8 kV for the negative mode, capillary temperature 350°C, H-ESI heater temperature 350°C.

QUALITY CONTROL
- Bioanalysis:
For quantification the ISTD method was applied and the system calibrated using an appropriate regression as mathematical model. To improve the accuracy at low concentrations, a suitable weighting was applied. The limit of quantification (LOQ) was defined as the lowest standard concentration used for the corresponding calibration curve.
- Biological assays:
In order to demonstrate adequate enzyme activity of the cryopreserved hepatocytes during the metabolic stability assay, the positive control substrate 7-ethoxycoumarin (5 μM) was incubated for 0, 60 and 120 min and the depletion of the marker compound was monitored. Test items were classified based on their intrinsic clearance (Clint) expressed as μl/min/10E6 cells. High stability is assumed if the calculated Clint was determined to be ≤ 3.5 or 5.0 μl/min/106 cells with human or rat hepatocytes, respectively. Low metabolic stability is characterized by Clint values of ≥ 19.0 or 27.5 μl/min/10E6 cells with human or rat hepatocytes, respectively.

Details on dosing and sampling:

- Test concentration selection:
The selected test concentrations were 0.3, 3, 30, 100 and 300 μM NMMO in all test item incubations (in absence of any organic solvent) and 5 μM with a solvent content of 1% ACN in reference item incubations.
Working solutions were prepared as described above. Primary hepatocytes from rats (pooled, male) and humans (5-donor-pool) were thawed according to the instructions of the manufacturer. The incubation samples were composed of 0.2 x 106 cells/well in 225 μl incubation medium and 25 μl test item solution (3, 30, 300, 1000 or 3000 μM in incubation medium), resulting in a final concentration of 0.3, 3, 30, 100 and 300 μM without any organic solvent. 200 μl samples were taken from the suspension cultures after 0, 30, 60, 90 and 120 minutes of incubation and processed for LC-MS analysis, applying protein precipitation using 1 volume of ACN/MeOH (1:1, v/v). After centrifugation the supernatant was analyzed by LC-MS.

Positive control incubations were performed using 7-ethoxycoumarin as substrate. The incubation samples were composed of 0.2 x 106 cells/well in 225 μl incubation medium and 25 μl reference item solution (50 μM in incubation medium), resulting in a final concentration of 5 μM in presence of 1% ACN. Metabolic turnover rates were measured at 0, 60 and 120 minutes of incubation. Aliquotes were taken from the incubations for sample preparation and analysis. Hepatocyte enzyme activity was assessed in terms of 7-ethoxycoumarin turnover, i.e. loss of 7-ethoxycoumarin.
Negative controls were performed to observe non-metabolic degradation processes; i.e. test item concentrations remaining stable over the investigated time suggests that a decrease of the parent compound is mainly due to metabolism. Negative control incubations were performed in line with all experiments using incubation medium with test and reference item in absence of hepatocytes. Samples were taken from the incubations at 0 and 120 minutes and processed as described above.

Statistics:

The amount of compound in the samples was expressed as percentage of remaining compound compared to time point zero (=100%). These percentages were plotted against the corresponding time points. In vitro intrinsic clearance (CLint) and half-life (t1/2) estimates were determined using the rate of precursor disappearance and following formula, based on the well-stirred liver model.

Equation 1: t1/2= ln2/-k
t1/2 = half life [min]
k = slope from the linear regression of log [test compound] versus time plot [1/min]

Equation 2: CL int=(-k) * V * fu
CLint = in vitro intrinsic clearance [μl/min/106 cells]
t1/2 = half life [min]
k = slope from the linear regression of log [test compound] versus time plot [1/min]
V = ratio of incubation volume and cell number
fu = unbound fraction in the blood

As fu is not known for the tested compound, the calculation was performed with fu =1.
CLint was used to calculate in vivo intrinsic clearance (CLint in vivo) on the basis of Equation 3. Scaling parameters are given in Table 3 in Section "Any other information on materials and methods incl. tables".

Equation 3: CL int in vivo = CLint* w liver *cd
CLint in vivo = in vivo intrinsic clearance [ml/min/kg]
CLint = in vitro intrinsic clearance [ml/min/106 cells]
w liver = liver weight [g/kg]
cd = liver cell density [10E6 hepatocytes / g liver]

Hepatic clearance (CLhep) was calculated as follows:

Equation 4: CLhep= CLint in vivo*Q/CLint in vivo+Q
CLhep= hepatic clearance [ml/min/kg]
CLint in vivo = in vivo intrinsic clearance [ml/min/kg]
Q = blood flow [ml/min/kg]

Type:

metabolism

Results:

NMMO was highly stable during 120 min of incubation with pimary hepatocytes from rat and human, resulting in 98.9 to 108.4% or 102.6 to 112.8% remaining parental compound for rat and human hepatocytes, respectively.

Type:

metabolism

Results:

Positive control 7-ethoxycoumarin showed half-lives of 13.3 and 19.3 min, (CLint in vitro 65.2 and 44.9 μl/min/10E6cells) in human hepatocytes and half-lives of 81.4 and 79.9 minutes (CLint in vitro 10.7 and 10.9 μl/min/10E6 cells) in rat hepatocytes.

Table 4: Stability of test item (0.3, 3, 30, 100 and 300 μM) with cryopreserved primary rat hepatocytes and William’s Medium E (n=1)

Time [± 1 min]	Rat hepatocytes (male), % remaining compound
	0.3 μM	3 μM	30 μM	100 μM	300 μM
0	100.0	100.0	100.0	100.0	100.0
30	109.7	102.8	106.8	108.8	105.0
60	110.0	96.4	103.4	105.0	103.2
90	104.3	106.7	110.7	110.5	108.1
120	108.4	104.4	106.3	108.1	98.9

Table 5: Stability of test item (0.3, 3, 30, 100 and 300 μM) with cryopreserved primary human hepatocytes and William’s Medium E (n=1)

Time [± 1 min]	Human hepatocytes (5-donors, mixed gender), % remaining compound
	0.3 μM	3 μM	30 μM	100 μM	300 μM
0	100.0	100.0	100.0	100.0	100.0
30	104.2	93.6	108.8	106.2	100.0
60	101.7	95.7	112.6	105.7	108.0
90	107.2	96.2	108.2	115.0	103.3
120	105.9	102.6	110.9	112.8	109.4

Conclusions:

The test item 4-methylmorpholine 4-oxide, monohydrate (NMMO) was tested for its metabolic stability at different concentrations (i.e. 0.3, 3, 30, 100 and 300 μM) with primary hepatocytes from rat and human. Independent on the test concentration, NMMO was highly stable within 120 min of incubation, resulting in 98.9 to 108.4% or 102.6 to 112.8% remaining parental compound for rat and human hepatocytes, respectively.

Executive summary:

In a non-guideline metabolic stability study, the test item 4-methylmorpholine 4-oxide, monohydrate (NMMO), was incubated with primary hepatocytes from male rats and primary human hepatocytes from 5 mixed gender donors in vitro at concentrations of 0.3, 3, 30, 100 and 300 μM. Samples were taken after 0, 30, 60, 90 and 120 min and analysed by liquid chromatography-mass spectrometry (LC-MS).

Independent of the test concentration, NMMO was highly stable within 120 min of incubation, resulting in 98.9 to 108.4% or 102.6 to 112.8% remaining parental compound for rat and human hepatocytes, respectively. Due to the high stabililty, clearance and half-life calculation was not applicable. Based on these results, the hepatocyte-specific metabolism of NMMO is predicted to be low, inter-species differences between rat and human were not observed.

Incubation of hepatocytes from all test species with the positive control 7-ethoxycoumarin confirmed the high metabolic activity of the primary cell model and were in accordance with historical laboratory control data.

This metabolism study in rat and human hepatocytes is classified acceptable for the analysis of metabolic stability of NMMO.

Reference 2

Endpoint:

basic toxicokinetics, other

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Objective of study:

toxicokinetics

Qualifier:

no guideline available

Principles of method if other than guideline:

Human oral equivalence doses (OEDs) were calculated based on data from liver metabolism in rat and human primary hepatocytes. Clearance rates determined from the metabolism study were used to support the In vitro to in vivo extrapolation (IVIVE) model and determination of OEDs. This approach assumes oral bioavailability and minimal plasma binding. Thus, steady state concentrations are primarily determined by glomerular filtration and hepatic metabolism.

Species:

other: rat and human

Dose / conc.:

3 other: µM

Remarks:

media concentrations from in vitro assays

Dose / conc.:

30 other: µM

Remarks:

media concentrations from in vitro assays

Dose / conc.:

300 other: µM

Remarks:

media concentrations from in vitro assays

Dose / conc.:

1 200 other: µM

Remarks:

media concentrations from in vitro assays

Dose / conc.:

4 800 other: µM

Remarks:

media concentrations from in vitro assays

Details on study design:

Experimental method for metabolism study with NMMO is described in section 7.1.1 (Basic toxicokinetics) of the dossier (Przibilla J & Mayer M., 2021, study report).

Estimation of Oral equivalent Doses Using IVIVE:
The equivalent daily oral dose (OED) required to achieve a steady-state concentration in the blood (CSS) equal to the concentration in an in vitro assay a was estimated from the in vivo clearance (Yoon et al 2012):

Css = DR * BW / (CLr + CLh)

where DR = the daily dose (mg/kg/d)
BW = body weight (kg)
CLr = renal clearance (L/hr)
CLh = hepatic clearance (L/hr)

In the case of NMMO, the available evidence indicates that:
1. Hepatic clearance in both rat and human is insignificant. No metabolism was detected over 2 hours for concentrations ranging from 30 to 0.3 uM (Przibilla J. & Mayer M., 2021).
2. NMMO is highly water soluble and has a very low vapor pressure (1.35E-008 mmHg: Chemspider), so exhalation would not be a significant clearance factor.
3. Based on read-across from similar compounds, clearance is primarily by glomerular filtration (GFR) and the fraction unbound in the blood (fu) is on the order of 1.

Therefore, we can estimate the Oral Equivalent Dose (OED, mg/kg/d) for an in vitro assay concentration (Cmedia) using the following formula (Yoon et al. 2012):
OED = Cmedia / Css= Cmedia *GFR / (0.042*BW)

Based on the formulas provided under "Details on study design" in Materials and methods section, for a 0.25 kg rat, the GFR is about 1.31 mL/min (Davies & Morris), or 1.89 L/d, so a concentration of 300uM (35.1 mg/L) would be equivalent to a dose of 35.1*1.89/0.25 = 265 mg/kg/day

 

For a human adult, the typical GFR is around 120 ml/min (Davies & Shock 1950), or 173 L/d, so a concentration of 300 uM (35.1 mg/L) would be equivalent to a dose of 35.1*173/70 = 87 mg/kg/day.

Because the clearance of NMMO is expected to be linear, OEDs can be calculated for other in vitro concentrations via linear extrapolation of the above values.

The OEDs associated with the in vitro media concentrations in the experimental studies are shown in Table 1:

Table 1: Oral Equivalent Doses (mg/kg/d)

In vitro:	3 uM	30 uM	300 uM	1200 uM	4800 uM
Rat	2.65	26.5	265	1060	4240
Human	0.87	8.7	87	348	1392

Conclusions:

Oral equivalence doses (OEDs) were calculated based on data from liver metabolism in rat and human primary hepatocytes. Based on this approach, an in vitro concentration of 300 µM results in an oral equivalent dose of 267 mg/kg bw/day for rats and 86 mg/kg bw/day in humans.

Executive summary:

An in vitro to in vivo extrapolation (IVIVE) approach for 4-methylmorpholine 4-oxide (NMMO) was performed based on data from liver metabolism studies in rat and human primary hepatocytes (Przibilla J. & Mayer M., 2021, section 7.1.1 of the NMMO dossier).

Oral equivalence doses (OEDs) were calculated based on data from liver metabolism studies in rat and human primary hepatocytes. Clearance rates determined from the metabolism study were used to support the In vitro to in vivo extrapolation (IVIVE) model and determination of OEDs. This approach assumes oral bioavailability and minimal plasma binding. Thus, steady state concentrations are primarily determined by glomerular filtration and hepatic metabolism.

Based on a body weight of 0.25 kg for rats and a GFR of 1.31 mL/min, or 1.89 L/d, a concentration of 300uM (35.1 mg/L) would be equivalent to an OED of 265 mg/kg/day for rats. 

For a human adult, the typical GFR is around 120 ml/min, or 173 L/d, so a concentration of 300 uM (35.1 mg/L) would be equivalent to a dose of = 87 mg/kg/day in humans.

Because the clearance of NMMO is expected to be linear, OEDs can be calculated for other in vitro concentrations via linear extrapolation of the above values.

Description of key information

For NMMO, no study is available for the determination of toxicokinetics and distribution. Therefore, a qualitative assessment is performed on the basis of the physico-chemical properties of the substance.

In addition, data on metabolism of NMMO was obtained from incubation experiments with rat and human primary hepatocytes. Independent of the test concentration, NMMO was highly stable within 120 min of incubation, resulting in 98.9 to 108.4% or 102.6 to 112.8% remaining parental compound for rat and human hepatocytes, respectively.

Based on this data, an in vitro to in vivo extrapolation was performed, in which concentrations typically used in cell culture experiments were converted to human oral equivalent doses (OEDs). A concentration of 300uM (35.1 mg/L) would therefore be equivalent to an OED of 87 mg/kg/day for humans. Because clearance of NMMO is expected to be linear, OEDs can be calculated for other in vitro concentrations via linear extrapolation of the above values.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

Morpholine, 4-methyl-, 4-oxide (or n-methylmorpholine oxide; CAS 7529-22-8; hereafter named NMMO) is a solid substance but it is always marketed as an aqueous solution (mostly as a 50% solution). NMMO is a high water soluble compound (> 100 g/L) with a low log Kow (-1.2) and a moderate vapour pressure (141 Pa). Its pKa is 5.14. NMMO surface tension is 68.9 mN/m at test concentration of 1g/L at 20°C and therefore the substance is not considered to be surface active. The substance was proven not to be skin irritant nor a skin sensitiser.

Besides an in vitro metabolic stability study, no further toxicokinetic data (animal or human studies) are available on this substance. The information present in this document is mostly based on physico-chemical parameters which allow a qualitative assessment of the toxicokinetic behaviour of NMMO rather than a quantitative assessment.

Absorption

Oral/Gastrointestinal absorption

Following its high water solubility, NMMO will readily dissolve into the gastrointestinal (GI) fluids and subsequently pass through aqueous pores or be carried through the epithelial barrier by the bulk passage of water.

Based on the moderate log Kow, absorption by passive diffusion will be favoured. Passive diffusion is also considered not to be significantly hindered as its molecular weight is <200 (117 g/mole).

It is generally thought that ionised substances do not readily diffuse across biological membranes. The intestine is where absorption after oral administration normally takes place. The pKa of NMMO suggests that this substance will be predominantly in its ionised form in the GI tract and hence diffusion can be hampered in some extent.

In an acute oral toxicity study (Mallory, 1981) NMMO was tested via gavage at 7000, 8000, 9000 and 10000 mg/kg male/female Sprague-Dawley rats. Necropsy of animals dosed at 8000, 9000 and 10000 mg/kg and sacrificed during the study, revealed distended fluid-filled stomachs and intestines, mottled lungs, darkened thymuses and darkened lymph nodes. Terminal necropsy revealed no visible lesions in any of the remaining rats. The acute oral LD50 for male and female rats was determined to be 9200 mg/kg,indicating absorption of the test substance.

In a combined repeated dose/reprotox-developmental screening study performed with NMMO (as 50% aqueous solution), males and female rats were exposed to 0,10, 100 and 1000 mg/kg bw/d via oral gavage according to OECD 422 (Martell, 2013). Treatment related effects at the high dose level were observed for body weight, body weight gain and food consumption of male and female rats. Under the experimental conditions of the study, the NOAEL of NMMO (as 50% aqueous solution) was 100 mg/kg bw/ d for males and females. The NOAEL for embryo-fetal toxicity was 100 mg/kg bw/day. These results support the assumption for oral absorption of NMMO.

The oral absorption factor is set to 50%, based on the anticipated hampered diffusion of NMMO as an ionized substance. The results of the toxicity studies do not provide reasons to deviate from this proposed value. 

Respiratory absorption

Given the vapour pressure of 141 Pa, NMMO is a low volatile substance and the availability for inhalation as a vapour is limited.

Once in the respiratory tract, NMMO would deposit on the walls of the airways. Deposited substances may be absorbed directly from the respiratory tract or, through the action of clearance mechanisms, may be transported out of the respiratory tract and swallowed. In that last case the substance needs to be considered as contributing to the oral/GI absorption rather than to the inhalation rate.

Although absorption directly across the respiratory tract epithelium by passive diffusion is favoured in view of the moderate log Kow value, NMMO is a highly water-soluble substance and, as suggested by its pKa value, predominantly in its ionised form at physiological pH. Based on this ionization, diffusion can be hampered in some extent.

Based on the above considerations, the inhalatory absorption factor is set to 100%, as a worst case assumption.

Dermal absorption

In view of its high water solubility and moderate log Kow, penetration into the lipid-rich stratum corneum and hence dermal absorption through deeper epidermis layers might be limited although its physical form (liquid) favours dermal absorption.

In an acute dermal toxicity study (Auletta, 1981), New Zealand White male/female rabbits were acutely exposed to 8000 mg/kg bw of NMMO. All animals survived the 4-day post-dose period. The LD50 value of the substance is greater than 8000 mg/kg.

The substance was proved not to be irritant to the skin and not to be a skin sensitiser.

Generally, default values of 10% and 100% are used for dermal absorption, based on molecular weight and log Kow value (ECHA guidance on IR&CSA, R.7c). The dermal absorption factor is therefore set to 100% (default), based on a molecular weight < 500 and a log Kow in the range of -1 to 4. However, it is also generally acknowledged that dermal absorption will not be higher compared to oral absorption; as a result, the dermal absorption factor for NMMO is set to 50%. The results of the available toxicity studies using the dermal route do not provide reasons to deviate from this proposed value.

Distribution

The high water solubility, moderate log Kow and low molecular weight predict that the substance will distribute widely through the body.

 

Accumulation

In view of the moderate log Kow and the high water solubility, NMMO will not easily accumulate in the body (lung, adipose tissue, stratum corneum).

Metabolism

Once absorbed, NMMO is expected to be metabollically stable, as observed from in vitro metabolism studies with rat and human primary hepatocytes. Independent of the test concentration, NMMO was highly stable within 120 min of incubation. Based on these results, the hepatocyte-specific metabolism of NMMO is predicted to be low and inter-species differences between rat and human were not observed.

 

Excretion

Given the high water solubility and low molecular weight, NMMO will be mainly excreted via the urine. Considering the high metabolic stability observed from in vitro studies with primary hepatocytes from rats and humans, a significant contribution of hepatic clearance to the overall excretion process is not expected.