3-hydroxypropiononitrile

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Toxicological evaluation and pharmacokinetic profile of beta-hydroxypropionitrile in rats.

Author:

Sauerhoff MW, Braun WH, Ramsey JC

Year:

1976

Bibliographic source:

J. Toxicol. Environ. Health 2(1): 31-44

Materials and methods

Objective of study:

other: Pharmacokinetic and Metabolism Studies: Rates and routes of absorption and clearance/excretion of C14 activity (from plasma, in selected tissues,in expired air (as HCN)).

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

3 male and 3 female Sprague-Dawley rats were treated orally (feeding needle) with [14C]ethylene cyanohydrin ( nitrile carbon labeling) to provide for each animal a single dose of 20 mg/kg (ethylene cyanhydrin/bodyweight) and 19 µCi/kg [14C] activity, in order to characterize the concentration of [14C] activity in plasma as a function of time, e.g. to determine a pharmakokinetic profile of the test substance.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-hydroxypropiononitrile

EC Number:

203-704-8

EC Name:

3-hydroxypropiononitrile

Cas Number:

109-78-4

Molecular formula:

C3H5NO

IUPAC Name:

3-hydroxypropanenitrile

Constituent 2

Reference substance name:

Ethylene cyanohydrin

IUPAC Name:

Ethylene cyanohydrin

Details on test material:

- Name of test material (as cited in study report): beta-Hydroxypropionitrile (beta-HPN)
- Substance type: aliphatic nitrile
- Physical state: colourlesss liquid
- Analytical purity: > 99% (by NMR)
- Impurities (identity and concentrations): ethylene glycol 0.2 ±0.1 wt%; acrylonitrile 0.3 ±0.2 wt%; formic acid 0.3 ±0.2 wt%; acrylamide < 0.01 wt%;
beta-Hydroxypropionamide 0.1 ±0.1 wt%; alpha-Hydroxypropionotrile 0.12 ±0.3 wt%;
- Purity test date: no data
- Lot/batch No.: no data
- Radiochemical purity (if radiolabelling): > 99.5%( by thin-layer chromatography)
- Specific activity (if radiolabelling): 0.94 µCi/mg
- Locations of the label (if radiolabelling): nitrile carbon of beta-HPN/ethylene cyanhydrin
- Expiration date of radiochemical substance (if radiolabelling): no data
- Stability under test conditions: stable in water under test conditions
- Storage condition of test material: urine, feces and trapping solutions for CO2 and HCN were stored at -2°C

Radiolabelling:

yes

Remarks:

[14C]beta-Hydroxypropionitrile/[14C] Ethylene cyanhydrin was labeled in the nitrile carbon; the specific activity was 0.94 µCi/mg. A radiopurity of 99,5% [14C] Ethylene cyanhydrin was established by thin-layer chromatography .

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Spartan Research Animals Inc., Haslett, Michigan
- Age at study initiation: 6-7 wk old
- Weight at study initiation: approximately 200g
- Fasting period before study: 8 hours before (and 1 hour after administration of the test substance)
- Housing: individual housing
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days prior to dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ±1°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hour light-dark cycle

IN-LIFE DATES: no data

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An aliquot [14C]ethylene cyanhydrin in ethanol was evaporated in dryness and the crystals were redisolved in distilled water. Subsequently an
aqueous solution of [14C]ethylene cyanhydrin was prepared such that the concentration of [14C]ethylene cyanhydrin and [14C]activity were
6.76 mg/ml and 6.33 µCi/ml respectively. Using a syringe fitted with a needle, approximately 3.0 ml/kg bw of this solution was administered orally to provide doses of 20 mg/kg bw [14C]ethylene cyanhydrin and µCi/kg bw [14C]activity.

DIET PREPARATION
no data

VEHICLE
- Concentration in vehicle: 6.76 mg/ml concentration of [14C]ethylene cyanhydrin in aqueous solution
- Amount of vehicle (if gavage): 3.0 ml/kg bw aqueous solution of [14C]ethylene cyanhydrin administered orally per animal

Duration and frequency of treatment / exposure:

one single dose was administered to each animal

Doses / concentrations

Remarks:

Doses / Concentrations:
20 mg/kg bw [14C]ethylene cyanhydrin (µCi/kg bw [14C]activity) per animal

No. of animals per sex per dose / concentration:

3 male and 3 female animals per dose

Control animals:

yes, concurrent no treatment

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: no data
- Rationale for animal assignment (if not random): random

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces, blood (plasma, serum), tissues (muscle, fat, skin, spleen liver, kidney, caracass), cage washes, air
leaving the cage, bubbled throgh trapping solutions, to get expired HCN and CO2.
Time and frequency of sampling: urine, feces and trapping solutions for CO2 and HCN were collected at 8hr intervals; blood samples were collected
from a cut section of tail 1, 2, 4,8, 12, 20, 28, 36, 44, 52, 60, 68, 76, 80, 92, 104 and 116 hours after administration of the single dose; the sampling
of other tissues was done after decapitation of the animals (120 hours after administration of the single dose).

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, blood (plasma, serum), tissues (muscle, fat, skin, spleen liver, kidney, caracass)
- From how many animals: (samples pooled or not): pooled
- Method type(s) for identification (e.g. GC-FID, GC-MS, HPLC-DAD, HPLC-MS-MS, HPLC-UV, Liquid scintillation counting, NMR, TLC): TLC
- Limits of detection and quantification: no data

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): hydrolization; the major component was hydrolyzable, one minor component was not
hydrolyzable

Sampling:
 - Feces and urine traps were immersed in dry ice baths. Room air was drawn through the Roth-type cages at 500 cc/min and air leaving  the cages
continuously bubbled through trapping solutions:    0.02M Ag2SO4 in 0.1 N H2SO4 to trap expired HCN and  5 M 2-aminoethanol in 
2-methoxyethanol to trap expired CO2.
   Urine, feces and trapping solutions for CO2 and HCN were collected at 8hr intervals and stored at -2°C.  
- Samples of excreta collected during the acclimation period were used to determine background radioactivity.
 - Samples of blood were collected in heparinized capillary tubes from acut section of tail 1, 2, 4,8, 12, 20, 28, 36, 44, 52, 60, 68, 76, 80, 92, 104 and
116 hours after administration of the single dose.     
The capillary tubes were sealed with clay, centrifuged and broken at the plasma-packed cell interface.The plasma was transferred to tared 
scintillations vials and counted for C14 activity.
 - Tissues and caracass:  All rats were killed by decapitation 120 hr after dosing. 
   The caracasses were skinned and and all tissues were collected and frozen until assayed.

Sample preparation: 
Samples were prepared four counting as follows:
1. Urine: 250 µl urine was added to 750µl H2O and 15ml Aquasol.
2. Plasma: After determining the weight, 3.5ml H2O and 11.5 ml Aquasol were added to the scintillation vials.
3. CO2 trapping solution: 5ml trapping solution from each collection period was added to 5ml fresh trapping solution 
    and 10 ml of a scintillation cocktail composed of 120ml CONCIFLUOR; 220 ml DOWANOL and 660ml toluene.
4. Tissues and feces were prepared as 50% aqueous homogenates. 300mg of the homogenates were oxidized by combustion in a Beckman Biological Material Oxidizer. 
The [14]CO2 generated by combustion was bubbled through  and trapped in 10ml 5 M 2amino-ethanol in 2-methoxyethanol. 
10milliliters of scintillation vials, followed by addition of 11.5 ml Aquasol. a uniform dispersion of particles in the gel was obtained on shaking.
6. HCN trapping solution: 1.5 ml trapping solution was added to 15 ml Aquasol.
7. Separatipon of urinary metabolites: 1 ml samples of urine and 1 ml concentrated hydrochlorid acid were heated for 4hr at 60°C. 
Aliquots of the hydrolyzate urine were spotted on a Brinkman-F-254 silika gel thin-layer plate and developed in a solvent system consisting of 
chloroforme: acetone: glacial acetic acid (75: 20: 10). 
[C14]thiocyanate in urine was determined according to the method of Boxer and Richards (1951).
8. Radioactivity determination: All [14C] activity was quantitated with the Nuclear Chicago Mark.II liquid scintillation system using external standard 
channell ratios to make routine corrections for counting efficiency.

Statistics:

Rate constant for the slow phase of elimination of C14 activity from plasma and C14 excretion in urine and CO2 were obtained by linear regression
and analysis. To obtain the rate constants for the rapid elimination phasesand the absorption of C14 activity the feathering technique was employed
(Wagner and Metzler, 1967).

Results and discussion

Preliminary studies:

no data

Toxicokinetic / pharmacokinetic studies

Details on absorption:

Absorption of [14C]activity via absorption of [C14] Ethylene cyanohydrin was a first order process with a rate constant of 1/hr corresponding to a
half-life (t1/2) of 0.69 hr. Peak plasma levels were attained at 4hr.(-> See: attached Document: Kinetics_14C_activity).

Details on distribution in tissues:

Ethylene cyanohydrin is extensively, if not completely absorbed from the gastrointestinaltract with peak plasma levels of [14C] activity attained
4 hr following administration of the dose. If one assumes the plasma volume of the rat to be 40ml/kg bw, then 6.7% of the dose is distributed in
the plasma at 4hr. The apparent volume of distribuition of Ethylene cyanohydrin is 333 ml/kg. This indicates, that Ethylene cyanohydrin may be
distribuited to the extracellular compartment.

Details on excretion:

Percent of [14C]activity eliminated in urine, feces and expired air 120 hr after administration of a single dose of 20 mg/kg
[14C]Etylene cyanohydrin: 53.2% (including urinary SCN) in urine; 7.39% in feces and in expired air 0.44% as HCN and 25.6% as CO2.
Excretion is expressed as a percent of the totale dose of [14C]activity found during successive time intervals. (->see table 1, in the field:" remarks on results")!
Excretion of [C14]activity in both urine and CO2 seems to be biphasic. The rate constant and half-life values for the alpha and beta phase of CO2
and urinary [C14]excretion are summerized in table2 (-> see table2, in the field: " remarks on results").
The net elimination of [C14]HCN excreted in expired air appears to follow first-order kinetics. The log of percent of dose of [14C]HCN elemination
is linear to time over the first 40 hr following administration of the dose. The rate constant of elimination is 0.113 1/hr (95% confidence limits :
±0.022), corresponding to a t1/2 of 6.13 hr. Net elimination of [14C]SCN in urine reached peak levels at 8 and 16hr time interval.
Because of the scatter in the data, rate constants and half-lifes (t1/2) were not calculated (-> see attached document: " Percentual [C14]activity in
excretion products").
The major component in urine was hydrolyzable to a compound when chromatographed using the TLC methods described that exhibited the same
Rf as Etylencyanhydrin itself. One minor component found in unhydrolyzed urine exhibited the same Rf as Ethylene cyanohydrin itself.
The percent of the total dose of [C14]activity excreted as Ethylene cyanohydrin conjugate and Ethylene cyanohydrin (probably) itself during
successive 8hrs intervals (following the single oral dose of 20mg/kg bw [C14]Ethylene cyanohydrin) is shown by the attached document :
"ECH_Metaboliten_Diagram".

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

The main constituent containing [14C ]activity in urine over the 40-120 hr intervals was SCN. Thiocyanate(SCN) excretion persisted long after
CN(-)/HCN in the expired air was undetectable. In addition to urinary thiocyanate (SCN) two further components in urine were found to contain
[14C]activity. The major component in urine was hydrolyzable to a compound, when chromatographed using TLC methods described, that
exhibited the same Rf as Ethylene cyanohydrin. One minor component found in unhydrolyzed urine exhibited the same Rf as the Ethylen
cyanhydrin itself.
The percent of the total dose of [C14]activity excreted as Ethylene cyanohydrin conjugate and Ethylene cyanohydrin (probably) itself during
successive 8hrs intervals (following the single oral dose of 20mg/kg bw [C14]Ethylene cyanohydrin) is shown by the attached document:
"ECH_Metaboliten_Diagram".

Any other information on results incl. tables

Table1: Percent of [14C ] activity eliminated in urine, feces and expired air during successive time intervals following 

a single oral dose  of  20 mg/kg [14C]Ethylene cyanohydrin to 3 male/3 female rats.

Excretion -  Urine    Faces    CO2    HCN (expired air)    Totale  interval (hr)  mean  +/- SD  mean   +/- SD  mean   +/- SD  mean   +/- SD    0-8  18.3 3.12   -  -  1.95 0.91   0.21 0.11  20.4   8-16  22.9  9.34 2.46  1.56  8.46  5.04  0.15   0.05  33.9  16-24 7.53  2.35  1.69  1.09  10.7  3.40  0.05   0.02 19.9   24-32  1.30  0.38  0.64  0.48  2.20  0.85  0.02   0.01  4.16  32-40  0.89  0.31 1.00   0.46  0.69  0.12  0.01  0.01  2.69  40-48  0.56  0.17 0.48  0.29  0.30  0.03     1.34  48-56  0.20  0.03  -  -  0.27  0.03      0.47  56-64  0.30  0.08  -  -  0.22  0.04      0.53  64-72  0.32  0.05  0.76  0.89 0.16  0.02      1.23  72-80  0.18  0.04  -  -  0.14  0.02      0.32  80-88  0.11  0.03  -  -  0.14  0.03      0.25  88-96  0.21  0.08  0.22  0.06  0.12 0.03      0.55  96-104  0.11  0.03  -  -  0.10  0.027      0.21  104-112  0.13  0.02  -  -  0.09  0.02      0.22  112-120  0.18  0.05  0.14  0.03  0.08  0.02      0.40    5.32    7.39    25.4        86.7

Table2: Rates of Clearance of 14C activity from plasma, urine, and CO2  in rats following oral administration of 20mg/kg               [14C]Ethylene cyanohydrine.

Sample  k (1/hr);  elimination rate constant  t 1/2 (hr); elimination half-life value plasma  alpha 0.157 (+/- 0.036);   beta   0.013 (+/- 0.0036) 4.41; 53.3 urine  alpha 0.174 (+/- 0.0025);  beta   0.019 (+/- 0.0031) 3.98; 36.5  CO2  alpha 0.209 (+/- 0.0031);  beta   0.018 (+/- 0.0023) 3.32; 38.5

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results
The total recovery of [C14]activity of 95.6±5.8% (mean± SD) allows the interpretation, that Ethylenen cyanohydrin does not accumulate in the body of the test animal, so there should be no hazard of bioaccumulation for human organism.
Since the production of thiocyanate exceeded strongly the production of free cyanide via methabolic pathway - according to the test result net
cyanide production was 80µg/48hr following administration of 20mg/kg bw Ethylene cyanohydrin - the hazard by methabolic transformation of
Ethylene cyanohydrin to cyanide has to be judged as negligable for the organism of the test animals in relation to the administered dose of Ethylene
cyanohydrin. Therefore the increase in cyanide production does not appear to be toxicologically significant.