2-hydroxy-2-methylpropionitrile

Endpoint summary

Description of key information

Reliable data on repeated dose toxicity are available only for the inhalation route of exposure. Acute lethality appears to be the critical adverse health effect. Beside lethality and signs of irritation (red nasal discharge, clear nasal discharge, perioral wetness, encrustations) no substance related toxic effects were seen. Allowing for a very steep dose response curve, an NOAEL of 61.2 ppm 2-hydroxy-2-methylproanenitril (216 mg/m³ or 66 mg CN-/m, the highest concentration tested, can be defined from a reliable 14 week inhalation study in rats. Concentrations slightly overrun this NOAEL are already acute lethal.

Key value for chemical safety assessment

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Dose descriptor:

NOAEC

216 mg/m³

Additional information

Reliable data on repeated dose toxicity are available from a 4 week and a 14 week inhalation study on rats. Further on supporting information on repeated dose toxicity can be drawn from two reliable inhalation studies on fertility in which female or male rats were exposed to 2-hydroxy-2-methylproanenitril for 21 days or 10 weeks prior to mating, respectively and an oral teratogenicity study in rats, in which 2-hydroxy-2-methylproanenitril doses up to and including the maternal toxic effect level were used. All these studies have been quoted as well in an assessment report of the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC report, JACC No. 53, Volume I, 2007; in the following cited as ECETOC) as in a report on AEGLs (Acute Exposure Guideline Levels) established by AEGL-Committee (US-NAC, Acetone Cyanohydrin, Interim Acute Exposure Guideline Levels (AEGLs), Interim final draft, 2005; in the following cited as AEGL). In these reports 2-hydroxy-2-methylproanenitril is designated as acetone cyanohydrin (ACH).

Repeated dose toxicity: inhalation

Subacute

“Sprague-Dawley rats were exposed (whole-body) to ACH vapours at concentrations of up to 59.9 ppm (measured) for 4 weeks (28 days). The test atmospheres were generated by bubbling nitrogen gas through liquid ACH into an airflow (1,643 l/min) for low exposure (9.2 ppm) or by nebulising pressurised ACH for medium (29.9 ppm) and high (59.9 ppm) exposures, both into an airflow of 1,727 l/min. The air concentrations were monitored by infra-red spectrophotometry of samples taken (4 x/d) at a port in the centre of the exposure chamber door. Clinical observations were made daily prior to, between 2 and 5 hours during, and after exposure. Observations during exposure included irritation of the eyes and/or nose and breathing difficulties in all animals at 29.9 and 59.9 ppm. In rats at 59.9 ppm, hypoactivity was observed. Soon after commencement of exposure, 4 males at 59.9 ppm showed symptoms of anoxia such as respiratory distress, tremors or convulsions, foam around the mouth and prostrate posture. Three of the 10 high-dose males subsequently died. An additional observation (pre- and post exposure and weekly) was chromo-rhinorrhoea that appeared more frequently in rats at 59.9 ppm and less at 29.9 ppm, but also on single occasions in controls and at 9.2 ppm. The mean body weight (recorded weekly) of the males at 59.9 ppm was lower than that of controls, but the difference was not statistically significant. The body-weight development of the other exposure groups did not differ from that of the controls. At necropsy, haematological examination revealed statistically significant decreases in red blood cell counts, haemoglobin and mean corpuscular haemoglobin concentrations in females at 59.9 ppm compared to controls, but the levels were still in the range of historical control data. Females at 59.9 ppm also revealed a significant increase in blood urea nitrogen (BUN) and a decrease in lactate dehydrogenase (LDH1). Total serum protein levels were statistically significantly decreased in males at 29.9 and 59.9 ppm. In the males at 29.9 ppm, a significant decrease in lactate dehydrogenase levels was also noted. All these changes were within the biological variation of this rat strain and were not considered treatment-related by the authors. Serum thyroid hormone (T3 and T4) levels were not significantly different from controls except for the 29.9 ppm males that showed elevated T3 levels. Increases in serum thiocyanate and urinary thiocyanate excretion were observed in all test groups. The increases in serum thiocyanate reached statistical significance in rats of both sexes at 9.2 and 29.9 ppm and the males at 29.9 ppm, while the increase in urinary thiocyanate excretion was significant in animals at 29.9 and 59.9 ppm. The terminal body weights were reduced in males at 59.9 ppm only, but the reduction was not statistically significant. No changes in absolute organ weights were observed, but there were increased relative liver weights in males at 29.9 and 59.9 ppm (statistically significant only at 29.9 ppm), but these changes were within normal biological variation and a result of the decreased terminal body weights. There were no exposure related gross or microscopic pathological changes of the following organs: adrenals, bone and bone marrow (femur), optic nerves, testes, heart, intestines and duodenum, kidney, liver, lung, nasal turbinates, spleen, skeletal muscle, thyroid, trachea at 59.9 ppm compared to controls. The NOAEL of the study was reported to be 9.2 ppm based on the clinical observations. The LOEL was 29.9 ppm (Monsanto, 1981d). The LOEL is based on the clinical observation of local irritation of the eyes, nose and respiratory tract. Test substance related systemic effects were not observed at 29.9 ppm. Thus the NOAEL for systemic effects would be 29.9 ppm in this study. During the first day of exposures the chamber concentrations were reported to fluctuate considerably. At the four measurement times during day one a level of 60 ppm was exceeded 3 times with a maximum concentration of 63.5 ppm. In all other measurements of the high-dose group at the following days the levels stayed mostly below 60 ppm and only occasionally went to a maximum 61.5 ppm. Deaths that occurred following exposure on day 1 were obviously due to acute toxicity and reflect the steep dose response for cyanide and ACH and the difficulty in designing repeated dose studies without exposing the animals to acutely lethal doses. A meaningful systemic NOAEL for repeated dose toxicity can therefore not be derived from this study” (quotation from ECETOC).

Subchronic

Sprague-Dawley rats were exposed to ACH vapours at concentrations of up to 61.2 ppm (measured) for 14 weeks (90 days). Pressurised ACH was nebulised into an airflow (1,699 l/min) to generate low (10.5 ppm), medium (32.3 ppm) and high (61.2 ppm) exposures. The air concentrations were monitored by infra-red spectrophotometry of samples taken (4 x/d) at a port in the centre of the exposure chamber door. Even distribution of the test material in the chamber was verified at regular intervals. No deaths occurred during the course of the study. Clinical observations were made daily prior to, between 2 and 5 hours during, and after exposure. Nasal discharge, salivation, integument and swaying movements were noted before and after exposure. These effects were not considered to be treatment-related by the authors since they occurred with a similar incidence in the control group or only at single times in individual animals, and were not related to concentration. No statistically significant decrease in mean body weight (recorded weekly) was observed in the exposed animals compared to controls. Following necropsy, the slight changes seen in haematological parameters, in particular red blood cell count, were not statistically significant and within the background variation of the Sprague-Dawley rat strain used. In females exposed to 32.3 and 61.2 ppm ACH, a statistically significant decrease in blood glucose levels was observed. In these females, there were also slight (statistically significant) decreases in globulin levels compared to controls. In females at 10.5 ppm and 32.3 ppm, total serum protein levels were decreased. The observed changes in the clinical chemical parameters were within the historical background ranges and not considered treatment-related by the authors. Increases in urinary thiocyanate levels compared to control animals were observed in a dose-related manner, but these only reached statistical significance in rats at 32.3 and 61.2 ppm. A statistically significant elevation of serum thiocyanate levels was only observed in female rats at 10.5 and 32.3 ppm. The serum levels fluctuated considerably and were not clearly dose related. (This gives rise to some doubt about the appropriateness of the analytical method used for the determination of the serum SCN^- concentrations.) No changes in serum T3 or T4 levels or in serum protein fractions were observed in the treated animals compared to controls. There were no treatment-related changes in body weight or organ weights. Neither were there macroscopic or microscopic changes of the following organs in rats at 61.2 ppm compared to controls: adrenals, bone and bone marrow (femur), optic nerves, testes, heart, intestines and duodenum, kidney, liver, lung, nasal turbinates, spleen, skeletal muscle, thyroid and trachea. The following additional organs were also unchanged: oesophagus, eyes, ovaries, epididymis, mesenteric lymph nodes, mammary glands, pancreas, pituitary, prostate, salivary gland, sciatic nerves, skin, spinal cord, stomach, thymus, trachea, parathyroid and urinary bladder. The NOAEL was 61.2 ppm ACH (216 mg/m³ or 66 mg CN^-/m³), the highest concentration tested (Monsanto, 1984). This corresponds to approximately 10.4 mg CN^-/kgbw/d. Contrary to the 28-day study (Monsanto, 1981d), this 90-day study did not reveal any significant local irritating effect of the test substance when compared to controls. This could be due to the fact that a different batch with perhaps another spectrum of impurities (e.g. acid stabiliser content) was used, but no firm conclusions can be drawn from the report. With regard to systemic toxicity the fluctuations of the test substance concentrations were better controlled in the 90-day study compared to the 28-day study and did not lead to inhalation of acutely lethal doses by the test animals” (quotation from ECETOC).

For details on the supporting inhalation studies with focus on reproductive toxicity see Chapter 7.8. However, these studies came to comparable results, as discussed in a valuing summary given in AEGL: “Four studies exposed rats repeatedly to acetone cyanohydrin at about 10, 30 and 60 ppm for 6 h/d, 5 d/wk for a total of 4 wk (Monsanto 1986a; using groups of 10 male and 10 female rats), 10 wk (Monsanto 1982b; using groups of 15 male rats) and 14 wk (Monsanto 1986b; using groups of 15 male and 15 female rats) or for 6 h/d for 21 d (Monsanto 1982c; using groups of 15 female rats). Death was observed at 60 ppm after the first exposure in three animals of the Monsanto (1986a) study, but not in subsequent exposures or in the other studies conducted under similar protocols. Preceding death, respiratory distress, prostration, convulsions, and tremors were obvious. In all studies, exposure to 60 and 30 ppm caused signs of irritation (red nasal discharge, clear nasal discharge, perioral wetness, encrustations) during the first and subsequent weeks of exposure. At 10 ppm, red nasal discharge was not observed in one study (Monsanto 1986a); its incidence was not increased compared with the concurrent control group in two studies (Monsanto 1982b, 1982c), but it was increased compared with the control group in the fourth study (Monsanto 1986b). No other signs of intoxication were reported in these four studies”

Further on, the discrepancy in mortality at the 60 ppm exposure are discussed in AEGL: “Monsanto (1986a) exposed groups of 10 female and 10 male Sprague-Dawley rats to acetone cyanohydrin at 0, 10, 30 or 60 ppm for 6 h/d, 5 d/wk for 20 exposure d (28 d in total). Concentrations in the exposure chamber were calculated by dividing the net amount of acetone cyanohydrin delivered to the chamber per unit time by the airflow per unit time and, in addition, measured by a Miran infrared analyzer (using the C-N triple bond frequency, which detects both acetone cyanohydrin and hydrogen cyanide) four times daily. For the total exposure period, mean analytic concentrations (±SD) were determined as 9.2 ± 0.9, 29.9 ± 1.2 and 59.6 ± 1.4 ppm, respectively. In the highest exposure group respiratory distress and tremors or convulsions or both, foaming at the mouth, and prostration were observed in four males following the first exposure. Of these four animals, three died. No deaths occurred in the 29.9-ppm group In three other studies conducted under similar protocols, no deaths were observed at 60 ppm for 6 h/d (Monsanto 1982b, 1982c, 1986b) . The authors suggested that the differences between the 28-d study and the 14-wk study (Monsanto 1986b) were possibly due to the very steep dose-response for acetone cyanohydrin or to the normal variation in experimental animals of the same strain. Evaluation of the nominal and analytic concentrations revealed that the animals in the 60-ppm group may, indeed, have been exposed to a slightly higher concentration during the second half of the first day: the nominal concentration of 64.8 ppm for the first day was the highest of all days (mean for the other 19 exposure days was 60.4 ± 1.8 ppm), likewise, the last two analytic concentrations measured during the first day (55.5, 60.5, 63.5, and 63.5 ppm; mean 60.8 ± 3.8) were greater than those measured on all subsequent exposure days (the highest individual value for exposure days 2-20 was 61.5 ppm; mean for exposure days 2-20 was 59.5 ± 1.4 ppm).”

In an acute inhalation study, 2/6 rats exposed for 4 hours to 62,5 ppm died (Smythe et al., 1962, see Chapter 7.2.2).

Repeated dose toxicity: oral

“In a teratogenicity study, pregnant Sprague-Dawley rats (25/group) were dosed on days 6 to 15 of gestation with 0, 1, 3 or 10 mg ACH/kg bw (corresponding to 0, 0.31, 0.92 and 3.06 mg CN^-/kg bw). Dose selection was made on the basis of marked maternal toxicity observed at higher doses observed in a range-finding study (Monsanto, 1983a). No deaths were observed during the exposure period. Maternal toxicity was evident by slight reductions in body-weight gain in mid and high exposure groups” (quotation from ECETOC).”

Conclusion

In summary, reliable data on repeated dose toxicity are available only for the inhalation route of exposure. Acute lethality appears to be the critical adverse health effect. Beside lethality and signs of irritation (red nasal discharge, clear nasal discharge, perioral wetness, encrustations) no substance related toxic effects were seen. Allowing for a very steep dose response curve, an NOAEL of 61.2 ppm 2-hydroxy-2-methylproanenitril (216 mg/m³ or 66 mg CN^-/m, the highest concentration tested, can be defined from a reliable 14 week inhalation study in rats. Concentrations slightly overrun this NOAEL are already acute lethal.

Justification for classification or non-classification

Reliable data on repeated dose toxicity are available from a 4 week and a 14 week inhalation study on rats. Acute lethality appears to be the critical adverse health effect of 2-hydroxy-2-methylproanenitril. There is no evidence on a specific target organ toxicity.

Thus 2-hydroxy-2-methylpropionitrile does not comply with the classification requirements regarding specific target organ toxicity — repeated exposure outlined in regulation (EC) 1272/2008 or the former Directive on classification and labelling 67/548/EWG.