Methacrylonitrile

Endpoint summary

Description of key information

In accordance with Regulation (EC) 1907/2006, the toxicokinetic behaviour of the substance has been assessed to the extent that can be derived from the relevant available information.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Additional information

METHODOLOGY

Unlabelled test material and two C-14 radio-labelled versions of the test substance have been used in toxicokinetic studies: 2 -methyl-2¹⁴C-propenenitrile and a dual radio-labelled version [2 -methyl-¹⁴C]-2¹⁴C-propenenitrile. The toxicokinetics of the radio-labelled test substance was studied in rats and mice and the unlabelled test substance in rats, mice and Mongolian gerbils.

Rats were orally dosed with radio-labelled test substance at dose levels up to 200 mg/kg and up to 116 mg/kg for an intravenous dose. Mice were orally dosed with radio-labelled test substance up to 17 mg/kg and gerbils up to 3.8 mg/kg with unlabelled test substance. Following dosing with radio-labelled test substance, glass animal metabolism cages were employed to separate and collect expired air, urine and faeces for up to 72 hours post dose and tissues and organs were collected for analysis at various time points and at the termination of the experiment. Incomplete mass balances from these types of studies will be obtained unless there is effective trapping of radioactivity from the expired air, as the test substance itself is relatively volatile and some of its metabolites are also volatile (for example, carbon dioxide and acetone).

Characterisation of isolated metabolites was by HPLC, co-chromatography with synthesised substances and¹H NMR. In addition to the quantitation of the test substance in blood and tissues in some experiments, cyanide levels were also determined. Quantitative whole-body autoradiography (QWBA) was performed for tissue distribution studies and the autoradiographs developed for 30 days by exposure to X-ray film. The quantitation of the autoradiographs was determined using an autoscanning image analyser.

ABSORPTION

As the test substance is a low molecular weight organic molecule which is water soluble (approximately 29 g/L), a liquid at room temperature, relatively volatile (vapour pressure of 86.3 hPa at 25 degrees Centigrade) and has a Log Kow of 0.76, it has the potential to be readily absorbed following oral, dermal and inhalation exposure. Approximately 90 % of an oral dose of radio-labelled test substance was recovered in the urine and expired air (carbon dioxide and volatiles) within 24 hours of dosing to both rats and mice(Ghanayem, Sanchez et al. 1994) confirming the exceedingly high oral absorption and very rapid excretion of the test substance. No specific toxicokinetic studies on the dermal and inhalation rates of absorption of the test substance are reported, however if a comparison is made of the acute toxicity data in the rat by oral (LD50 64 mg/kg) and dermal (LD50 256 mg/kg) and inhalation (LC50 1092 mg/m³) routes then it can be seen that the test substance must also be absorbed to a significant degree by these routes of administration, albeit to a lower extent than by the oral route.

DISTRIBUTION AND EXCRETION

A comparison of the excretion of the radio-labelled test substance in rats and mice is presented in the following tables:

Excretion of radioactivity following dosing of radio-labelled test substance to rats and mice after 24 hours (results expressed as percentage of dose)

Study	Ahmed et al (1996)	Burka et al (1994)	Cavazos et al (1989)	Ghanayem et al (1994)
Dose level, species and (route)	14.5 mg/kg, rats, (iv)	58 mg/kg, rats, (oral)	100 mg/kg, rats, (oral)	1.15 mg/kg, rats, (oral)	11.5 mg/kg, rats, (oral)	1.15 mg/kg, mice, (oral)	11.5 mg/kg, mice, (oral)
Urine	13.4	21.8	32.0	20.0	22.2	43.5	54.1
CO2	36.2	39.2	2.1	61.7	54.9	48.4	32.5
Volatiles	2.8	30.7	-	8.8	12.0	2.1	1.2
Faeces	6.2	4.9	8.5	2.2	3.2	6.9	4.5
Tissues	-	4.0	-	-	7.7	-	2.0
Total recovered	58.6	100.6	42.6	92.7	100	100.9	94.3

 

Excretion of radioactivity following oral dosing of radio-labelled test substance to rats after 72 hours, results expressed as percentage of dose (Ghanayem et al, 1992).

Dose level, strain and vehicle	1.15 mg/kg, F344, water	11.5 mg/kg, F344, water	115 mg/kg, F344, water	115 mg/kg, SD, water	115 mg/kg, SD, oil
Urine	23.5	28.2	20.8	22.1	29.7
CO2	70.5	63.8	25.8	30.4	37.7
Volatiles	9.6	12.0	42.5	35.0	38.7
Faeces	3.6	4.5	1.4	2.2	2.1
Tissues	-	2.0	1.7	1.9	2.5
Total recovered	107.2	110.5	92.2	91.6	110.7

The total recovery of radioactivity at 48 hours from rats following intravenous dosing at 14.5 mg/kg (Ahmed et al, 1996) was only 65.2 % and the authors gave no explanation for the very low recovery. The tissue concentrations at 48 hours were generally comparable to the blood concentrations of radioactivity with only the liver and intestinal mucosa having higher levels, even so these levels would not likely account for the missing dose. One likely explanation for an incomplete recovery when using a test substance (and its metabolites) that has some volatility is incomplete trapping of expired radioactivity. As such, the reliability of this study is considered poor.

Another study also showed a low recovery of radioactivity after oral dosing of a dual¹⁴C-radio-labelled test substance at 100 mg/kg to rats (Cavazos et al, 1989) and these data were also reported in another publication (Farooqui et al, 1990). At 48 hours after dosing a total of 59.3 % of dose was recovered with 42.5 % in the urine, 14.4 % in the faeces and only 2.4 % in expired air. The authors concluded that the low recovery was due to 40 % of dose remaining in the body, either bound to macromolecules or in the form of unexcretable conjugates; however no data was presented to substantiate this claim. As such, the reliability of this study is also considered poor.

Conclusions on the absorption and excretion of dose following oral dosing of the radiolabelled test substance to rats from the remaining studies (all apparently from the same group of workers) can be summarised as follows:

- Rapid excretion of more than 90 % of dose within 24 hours.

- At low doses (1.15 or 11.5 mg/kg), the majority of dose was excreted in expired air, mostly as carbon dioxide.

- At a higher dose (115 mg/kg), whilst the total amount of radioactivity recovered in expired air remained similar to the lower doses, the proportion of carbon dioxide decreased and the proportion of other volatiles (including unchanged parent test substance) increased. This would mean that there is an apparent saturation of a route of metabolism of the test substance at higher doses.

-  Even at the high dose (115 mg/kg), there was very little radioactivity remaining in the tissues at 72 hours after dosing (approximately 2 %). As such, no evidence of bioaccumulation of the test substance was seen.

- A difference in excretion and toxicity of the test substance was seen between different strains of rat (F344 and SD) and also when oil was used instead of water for the dose vehicle.

The absorption of dose following oral administration to mice was similar to rats however the relative routes of excretion were different. Mice excreted a larger proportion of dose in urine (which increased with increasing dose) and a lower proportion in expired air (which decreased with increasing dose). The proportion of dose exhaled as volatiles other than carbon dioxide was also much less than in the rat. The relative proportions of urinary metabolites were also very different between the rat and mouse.

Blood toxicokinetics of the test substance (unlabelled) was investigated following either single intravenous or oral doses to male F344 rats(Demby et al, 1993). The blood concentration of the test substance versus time profiles was biphasic and showed that the Vss and terminal (β) half-life were independent of dose. The longest β half-life was 41 minutes (116 mg/kg, iv). The α half-life (approximately 2 minutes) indicated that distribution of the test substance into tissues was very rapid. The short terminal half-life indicates that systemic exposure to the test substance following intravenous dosing was very limited (< 5 hour) and suggests a minimal bioaccumulation potential for test substance following multiple exposures. Clearance of the test substance was faster at the lower dose than the higher doses which again suggests that there was a saturation of first-pass metabolism or elimination, or both, at high doses. Following oral dosing, the absorption of the test substance was slow and erratic and suggested that absorption controlled the rate and extent of systemic exposure to the test substance. Comparison of excretion following oral and intravenous dosing revealed that a higher proportion of dose was metabolised via the oral route. This is clearly related to a first pass effect of orally administered test substance and also means that other routes of administration (dermal and inhalation) may lead to less metabolism than by oral administration.

The tissue distribution following an intravenous dose of 14.5 mg/kg radio-labelled test substance was studied in male F344 rats using quantitative whole-body autoradiography(Ahmed et al, 1996). Levels of radioactivity were determined at 5 minutes, 8, 24 and 48 hours after dosing. The dose was rapidly absorbed (the C_maxin many tissues was at 5 minutes), widely distributed and levels fell quickly in most tissues (except thymus) after 8 hours. At 48 hours after dosing only the lacrimal gland, thymus and liver had radioactivity levels of just above 100 nCi/mg tissue (equivalent to 0.29 μg/mg tissue) which was approximately 10-fold greater than blood radioactivity. Following a single oral dose of radiolabelled test substance at 58 mg/kg to F344 rats no tissues analysed had greater than 5-fold more radioactivity than was in blood(Burka et al, 1994).

Groups of rats that had also been used for excretion and metabolite identification work were also used for quantitative tissue distribution by the oxidising of samples and radio-analysis(Ghanayem et al, 1992). Male F344 rats received a single oral dose of radio-labelled test substance at 1.15, 11.5 or 115 mg/kg and were sacrificed for tissue collection at 8, 24 or 72 hours. The concentrations of radioactivity were dose dependent and were particularly high in the metabolising/excretory organs (liver, kidney, and urinary bladder), intestines, adrenals and thymus. The proportion of dose that remained in the tissues of rats 72 hours after dosing was less than 3 % of the administered dose with none of the tissues having a radioactivity concentration of more than 4-fold that of blood.

Concentrations of radioactivity in tissues of males F334 rats following oral administration of radiolabelled test substance, values are expressed as μg equiv/g (Ghanayem et al, 1992)

 

Tissues	11.5 mg/kg			115 mg/kg
	8 hour	24 hour	72 hour	24 hour	72 hour
Blood	3.5 ± 0.1	1.9 ± 0.1	1.1 ± 0.2	18.1 ± 2.3	7.8 ± 0.5
Liver	12.4 ± 1.1	6.1 ± 0.8	2.2 ± 0.1	34.6 ± 1.9	15.6 ± 0.8
Kidney	7.8 ± 0.1	3.8 ± 0.0	2.3 ± 0.1	33.7 ± 7.9	8.9 ± 0.4
Adrenals	8.6 ± 0.7	7.0 ± 0.5	4.3 ± 0.4	24.0 ± 3.6	27.5 ± 2.1
Thymus	5.1 ± 0.3	4.0 ± 0.8	3.3 ± 0.4	16.6 ± 4.6	11.2 ± 1.3
Urinary bladder	8.6 ± 1.5	2.1 ± 0.2	2.2 ± 0.3	16.4 ± 3.7	9.4 ± 2.7
Spleen	4.5 ± 0.4	3.3 ± 0.0	1.8 ± 0.1	12.7 ± 0.7	8.1 ± 0.2
Lung	5.5 ± 0.4	2.8 ± 0.1	1.5 ± 0.0	13.0 ± 0.3	8.4 ± 2.6
Brain	1.8 ± 0.2	0.8 ± 0.1	0.6 ± 0.1	10.1 ± 0.7	3.4 ± 0.3
Glandular stomach	5.6 ± 0.4	2.4 ± 0.1	2.0 ± 0.3	14.0 ± 0.2	8.1 ± 1.0
Forestomach	4.6 ± 0.2	3.0 ± 0.1	1.7 ± 0.3	17.2 ± 1.1	6.9 ± 2.2
Small intestine	9.5 ± 0.6	6.0 ± 0.5	1.8 ± 0.1	36.2 ± 4.7	8.2 ± 0.4
Large intestine	9.9 ± 1.00	4.2 ± 0.5	1.5 ± 0.4	20.4 ± 2.9	6.3 ± 0.4
Zymbal gland	11.9 ± 0.4	4.4 ± 0.3	1.3 ± 0.2	10.2 ± 1.7	8.2 ± 1.2

METABOLISM

 

In order to investigate the relationship between toxicity and metabolism of the test substance, the blood and tissue cyanide concentrations were measured in rats, mice and Mongolian gerbils following oral dosing of the test substance at the reported LD₅₀values or fractions thereof (Farooqui et al, 1992). The signs of toxicity (reported to be cyanide related) were seen earlier in mice and gerbils than rats which also correlated with earlier C_maxvalues of cyanide in blood for each species. The authors concluded that the toxicity signs were a function of the metabolism of the test substance to cyanide and that mice and gerbils were able to metabolise the test substance faster to cyanide or to detoxify it slower than rats. The formation of cyanide was also dose dependent in all three species.

 

Percent of dose recovered as cyanide following oral dosing of the test substance at 0.5 LD50 (Farooqui et al, 1992)

 

Species	Dose (mg/kg)	Liver	Kidneys	Brain	Blood
	(0.5 LD50)	Cyanide (% dose)/g tissue	Cyanide (% dose)/g tissue	Cyanide (% dose)/g tissue	Cyanide (% dose)/mL blood, (nmoles/mL blood)
Rat	100	0.05	0.02	0.01	0.09 (338)
Mouse	17	1.00	0.45	0.24	2.42 (238)
Mongolian gerbil	3.8	3.97	1.84	1.57	5.31 (59)

 

The identity of the metabolites in expired air and urine from rats and mice dosed with radiolabelled test substance were characterised by HPLC, co-chromatography and NMR(Ghanayem et al, 1992; Ghanayem, 1994). Carbon dioxide, unchanged test substance and acetone were identified as the major metabolites in expired air. The test substance/acetone ratio was directly proportional to dose and decreased with a function of time. A minimum of 4 metabolites were detected in the urine of both rats and mice after a high dose of test substance, although the ratios of these metabolites varied significantly. Three major metabolites were isolated and identified as: an isomer of deoxyuridine, and two mercapturic acids: N-acetyl-S-(2-cyanopropyl)-L-cysteine (NACPC) and N-acetyl-S-(2-hydroxypropyl)-L-cysteine (NAHPC). The proposed metabolic pathway for for the test substance in rats and mice(Ghanayem et al, 1994) is attached.

A more recent study looked at the route of metabolism of the test substance and related substances, in particular on an enzymatic basis, but also measuring blood cyanide levels(El Hadri et al, 2005). A schematic representation of the metabolic pathways of the unsaturated aliphatic nitriles metabolism that may lead to cyanide release is attached.

Results from El-Hadri et al (2005) plus early work on the metabolism of the test substance (Day et al, 1988; Ghanayem et al, 1992; Burka et al, 1994; Ghanayem et al, 1994; Ghanayem et al, 1999) lead to the following major conclusions on the metabolism of the test material:

- The metabolism of the test substance is dose dependent, species and strain dependent, and sex dependent.

- Direct glutathione (GSH) conjugation of the test substance is considered primarily a detoxification pathway.

- The “detoxification” metabolic pathway of the test substance becomes saturated as the dose increases leading to increased cyanide production and subsequent toxicity.

- Cytochrome P-450 mediated oxidation forming an epoxide intermediate (1-cyano-1-methyloxirane) is considered to be a bioactivation pathway.

- Although CYP2E1 plays a major role in the epoxide formation and subsequent release of cyanide, other enzymes (for example, epoxide hydrolases) also contribute to this mechanism (rats).

-Subsequent metabolism of the epoxide, including conjugation with GSH, is considered to lead to cyanide release.

EXTRAPOLATION TO HUMAN

No human data on the toxicokinetics of the test substance have been identified; however there is one report that describes exposure of humans to test substance vapour (Pozzani et al, 1968). The first group (8 to 9) was exposed to test substance vapour in a set sequence of 24, 14, zero, 7, 14, 24, 7, 2, zero and 2 ppm. The group inhaled the same concentrations twice with an interval of at least 45 minutes between exposure periods. The second group was exposed to 2 ppm (9 subjects) or 14 ppm (7 subjects) for 10 minutes. Irritation to the nose, throat and eyes were reported in a minority of subjects.

From rodent studies it is known that the mechanism of acute toxicity of the test substance is via the generation of excess cyanide during metabolism and elimination. The saturation of the detoxification pathway and subsequent increase in the proportion of the reactive epoxide intermediate leading to increased cyanide production is a process that is dose, species (strain) and sex dependent. A significant part of the rate limiting step appears to be the availability of glutathione. It can be reasonably expected that humans will also metabolise the test substance in a similar manner to rodents in the sense that they will have the ability to safely detoxify and excrete low levels of the test substance. In the same manner, humans are also likely to have a threshold whereby there is a metabolic shift from detoxification to bioactivation and subsequent acute toxicity. It is also likely that this threshold will be lower in individuals with impaired liver functions and there may even be sub-populations with greater genetic susceptibility. 

Without direct human data, for example well designedin vitro metabolism studies, it is impossible to extrapolate an exposure level for the threshold in humans. Hence, conservative risk assessment methods have been applied in accordance with ECHA Guidance on information requirements and chemical safety assessment Chapter R.8: Characterisation of dose [concentration]-response for human health and (ECETOC Guidance on assessment factors to derive a DNEL: Technical Report 110, ISSN-0773-8072-110 (2010, in press)).