Methacrylamide

Neurotoxicity

Description of key information

Methacrylamide can cause neurotoxic effects after single and repeated exposure.

Additional information

Acute effects (for related systemic effects, see Endpoint Study Summary “Acute Toxicity”)

Methacrylamide caused neurotoxic effects in rats after single oral exposure to < 2000 mg/kg bw.

 

Effects after repeated dosage (for related systemic effects, see respective Endpoint Study Summaries “Repeated Dose Toxicity” or “Reproductive Toxicity”)

Oral

A 12 month repeated dose toxicity study in male rats and male mice given methacrylamide in drinking water (200, 400, 800 and 1200 ppm corresponding to ca. 10, 20, 40 and 60 mg/kg for rats, and ca. 33.4, 67, 133 and 200 mg/kg/day for mice) was conducted (Aratani 1993). The NOAELs for the 12 month repeated dose study were considered to be ca. 20 mg/kg/day for rats (400 ppm, based on reduction in the rotarod performance, distension of the urinary bladder, shrinkage and loss of myelinated fibers of sciatic nerve, atrophy of gastrocnemius muscle, symptoms of peripheral neuropathy including decrease in grip strength and abnormal gait) and ca. 33.4 mg/kg/day for mice (200 ppm; based on reduction in the rotarod performance, symptoms of peripheral neuropathy including decrease in grip strength and abnormal gait, atrophy of gastrocnemius muscle, distension of the urinary bladder,  and shrinkage and loss of myelinated fibers of sciatic nerve).

In a 28 day repeated dose study in rats according to OECD TG 407 by gavage at the dose levels of 0, 30, 100 and 300 mg/kg/day (MHLW 1999), some clinical and functional changes (decrease in muscle tone, ataxia and decrease in grip strength) were found at 300 mg/kg/day. Males at 100 mg/kg/day and higher and females at 30 mg/kg/day and higher showed a decrease in locomotor activity. These functional changes were observed continuously throughout the recovery period. Histopathological examination revealed a degeneration of the sciatic nerve fibers and axonal swelling in the cerebellar peduncle at 300 mg/kg/day of both sexes. Systemic NOAELs were considered to be 30 mg/kg/day for males and less than 30 mg/kg/day for females. 

In a reproductive developmental toxicity screening study according to OECD 421 (MHLW 2001), methacrylamide was administered to Spraque-Dawley (Crj: CD) rats (male/female) in purified water by gavage at dose levels of 0, 12.5, 50, and 200 mg/kg bw/day. Adverse effects of systemic toxicity as mortality, neurotoxicity, changed behaviour were observed at 200 mg/kg/day. No effects on fertility or other signs of toxicity to reproduction were observed below the range of general/maternal toxicity.

In a developmental toxicity study according to OECD TG 426 (CRL 2021), Methacrylamide was given by gavage to Wistar Han rats from Day 6 post-coitum up to and including lactation Day 20 in doses of 15, 50 and 150 mg/kg/day to determine the potential functional and/or morphological effects of the developing nervous system of the offspring that may arise from exposure in utero and during lactation/ early life.

For the F0-generation, grip strength was evaluated as neurotoxicity parameter in the respective dose range finding study.

For the F1-generation, the following neurotoxicity parameters and end points were evaluated in this study: functional observations including acoustic startle response, learning and memory test (Biel maze), motor activity and grip strength, gross necropsy findings, brain weights and histopathologic examinations of central and peripheral nervous tissues (including brain morphometry).

In the F0-generation, in the presence of adverse clinical signs (piloerection, hunched posture, uncoordinated movements and abnormal gait of the hindlegs/ forelegs), reduced body weight and lower food intake, a markedly reduced grip strength was recorded at 150 mg/kg/day on PND 20 (0.52x and 0.40x of control for fore- and hindlimb grip strength, respectively).

In the F1 animals, lower mean body weights were observed at 150 mg/kg/day throughout the study, while the effects strength decreased over time (0.77x of controls for males and females combined on PND 21 vs. 0.89x and 0.93x of controls for males and females, respectively, at the end of the study period). In the F1-generation, treatment-related neurotoxicity effects consisted of lower grip strength on PND 20, 35 and 60, reduced startle response on PND 25 and 60 and reduced motor activity on PND 13 at 150 mg/kg/day. At 50 mg/kg/day, treatment related effects were confined to a slightly lower startle response on PND 25. In detail, at 150 mg/kg/day a lower mean forelimb grip strength was recorded on PND 20 and 35 for both sexes (0.55x and 0.73x of control for males and females, respectively on PND 20 and 0.82x and 0.88x of control for males and females, respectively on PND 35). Males at 150 mg/kg/day additionally showed a lower mean hindlimb grip strength on PND 20 and 35 (0.76x and 0.90x of control, respectively). On PND 60, only mean hindlimb grip strength of males at 150 mg/kg/day appeared marginally lower (0.90x of control), while the effect on forelimb grip strength had recovered. The effect on grip strength was most pronounced in males. Since grip strength largely recovered to control values towards completion of the study period, this was considered not to represent an adverse effect. Fore- and hindlimb grip strength at 15 and 50 mg/kg/day was considered not affected by treatment with the test item on PND 20,35 and 60.

At 150 mg/kg/day, startle response measurements showed a lower mean average and maximum response amplitude for males and females on PND 25 (overall mean response for average response amplitude was 0.62x and 0.70x of control for males and females, respectively; overall mean response for maximum response amplitude was 0.68x and 0.62x of control for males and females, respectively). On PND 60, average and maximum response amplitude remained lower for males at 150 mg/kg/day (overall mean response for average and maximum response amplitude was 0.77x and 0.78x of control, respectively). For females at 150 mg/kg/day, only maximum response amplitude on PND 60 appeared marginally lower than controls (overall mean response for maximum response amplitude was 0.85x of control). These data indicated that the startle response only partially recovered during the study period.

At 150 mg/kg/day, lower mean motor activity (both for total movements and ambulations) was recorded for females on PND 13 (0.64x and 0.47x of control for total movements and ambulations, respectively). This effect on motor activity appeared reversible since on PND 17, 25 and 60, motor activity was considered not affected by treatment with the test item.

No direct test item-related effects on fresh and fixed brain weight were recorded.

No test item-related changes were noted in any of the other F1-generation

(neuro-)developmental parameters investigated in this study (i.e. time to acquire righting reflex, arena observations, hearing and pupillary reflex, rectal temperature, learning and memory, macroscopy, gross brain dimensions, brain morphometry, and peripheral and central nervous tissue histopathology).

Thus, essentially reversible effects were recorded for motor activity and grip strength at 150 mg/kg/day, based on which these effects were considered not adverse. Reduced startle response at 150 mg/kg/day was not completely reversible during the study period. While there were no supportive histopathological correlates and means remained within historical control ranges, the magnitude and non-reversible nature of the reduced startle response was considered to represent an adverse effect.

In conclusion, based on the results of this Developmental Neurotoxicity Study, the following No Observed Adverse Effect Levels (NOAEL) of Methacrylamide were established:

Maternal NOAEL:   50 mg/kg/day (based on clinical signs, weight loss or reduced weight gain and reduced food intake at 150 mg/kg/day)

Developmental NOAEL:     50 mg/kg/day (based on non-complete recovery and magnitude of reduced startle response at 150 mg/kg/day)

In a two-generation reproduction study (according to modified reproductive assessment continuous breeding protocol (RACB)) Methacrylamide was administered to Swiss CD-1 mice (male/female) in drinking water at dose levels of 0, 24, 80 and 240 ppm (corresponding to F0: 4.5, 15.4, 49 mg/kg bw/day; F1: 6.8, 23.8, 71.3 mg/kg bw/day for males and 8 - 69 mg/kg bw/day for females; “Task 4” of the RACB program).

In the respective 4 week dose range finding study (“Task 1”), reduced grip strength was observed in F0 females at 720 ppm Methacrylamide in drinking water (corresponding to 248-552 mg/kg bw/day; unintended high exposure due to increased water consumption), but not at 540 ppm (dose in mg/kg bw/d unclear).

It is acknowledged that in the Developmental Neurotoxicity study, the effects on startle response and grip strength were observed together with a reduced pup body weight in rats. The same is basically true for the temporary grip strength effects seen in the RACB study in mice. Here, published data reveal a strong correlation between body weight (and as consequence, muscle mass) of the test animals and grip strength (Maurissen JP et al: Factors affecting grip strength testing; Neurotoxicol Teratol. 2003 Sept-Oct; 25 (5): 543-53). Also the startle response appears to depend upon the body weight (Fodor A et al.: The prepulse inhibition deficit appearance is largely independent on the circadian cycle, body weight, and the gender of vasopressin deficient Brattleboro rat; Endocrine Regulations, Vol. 50, 16–23, 2016). While these publications describe the correlation in rats, mice are expected to show comparable but not identical patterns.

In conclusion, taking into account the bias caused by diminished body weights of the dosed animals versus control animals it is questionable whether the effects on startle response and grip strength are a direct neurotoxic effect or alternatively are an indirect consequence of the reduced body weight, at least partially.

 

Inhalation

In a subchronic inhalation toxicity study acc. OECD TG 413, exposure to methacrylamide dust did not cause any evidence for neurotoxicity (RCC 2008).  Therefore, the no-observed-adverse-effect-level for neurotoxic effects after inhalation was set at the highest dose level administered, 62.5 mg/m³.

Justification for classification or non-classification

According to CLP, Methacrylamide is classified with STOT-SE Category 2, H302, for its neurotoxicological potential. Furthermore, based on results on studies after repeated oral dosing in rats and mice, Methacrylamide is classified with STOT RE Category 2, H373: May cause damage to organs, Affected organs: nervous system.