Glyoxylic acid

Endpoint summary

Description of key information

In a repeated dose toxicity study according to OECD guideline 422 the NOAEL was 200 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Repeated dose toxicity of Glyoxylic Acid 50%

In a combined repeated dose toxicity study with the reproduction / developmental toxicity screening according to OECD TG 422, Glyoxylic acid 50% was administered to 5 Crj:CD (SD) rats per sex per dose by feed at dose levels of 0, 2000, 6000 or 18000 ppm for a period of 5 weeks (Myers, 2005). Bodyweight gains of males in the 18000 ppm and 6000 ppm groups were lower than in control. The change at 6000 ppm is regarded as a non adverse effect, because statistical significance was only observed temporarily at 18000 ppm, no such effect was recorded among females, bodyweight development was not impaired and weekly bodyweight gains were not statistically significantly changed. There was no effect on food consumption. The only possible treatment-related change evident concerning haematology was a slightly shorter activated partial thromboplastin time for males at 6000 ppm or 18000 ppm; the differences did not attain statistical significance. There were some changes in blood chemistry parameters for which an effect of treatment could not be discounted: a statistically significantly elevated level of cholesterol among males at 18000 ppm; a dose related reduction in the level of alkaline phosphatase among females, with differences attaining significance at 6000 ppm and 18000 ppm; slightly but not significantly lower levels of alanine amino-transferase and aspartate amino-transferase among females in the 6000 ppm and 18000 ppm groups. There were no effects of treatment on macroscopic pathology findings or absolute or bodyweight relative organ weights. There were no microscopic pathology changes in the tissues examined that were considered to be related to treatment. Based on the effects observed in this study a NOAEL of 6000 ppm (200 mg/kg/d) for males due to the statistically significantly reduced cumulative bodyweight gain among males at 18000 ppm and 18000 ppm (730 mg/kg bw/d) for females can be derived.

According to Council Regulation 1907/2006/EC, column 1, Annex IX a sub-chronic toxicity (90 days) study is the standard information required for the assessment of the sub-chronic toxicity of a substance manufactured in quantities of 1000 tonnes or more. This study is not available. However, in accordance to Council Regulation 1907/2006/EC, Annex XI, further testing on vertebrate animals is scientifically not justified. This is shown below in a weight of evidence approach.

Justification for the use of data on glyoxal for the assessment of the repeated dose toxicity of Glyoxylic Acid 50%

Glyoxal is produced endogenously and is commonly present in blood plasma of healthy subjects. The endogenous concentrations of glyoxal in human tissues and body fluids, as with other α-oxoaldehydes, are limited by the high catalytic efficiency of the glyoxalase system (Thornalley, 1995) as well as by the rapid reaction of glyoxal with proteins (Sady et al., 2000). Glyoxal reacts non-enzymatically with GSH with formation of a hemithioacetal, which is subsequently converted to S-glycolylglutathione by glyoxalase I. Glyoxalase II catalyses the hydrolysis of S-glycolylglutathione to glycolate, re-forming the GSH from the first reaction. Direct enzymatic transformation of glyoxal to glycolic acid is mediated by lactate dehydrogenase and/or aldehyde oxidase. The main path of the degradation of glycolic acid is to Glyoxylic Acid (mediated by lactic dehydrogenase or glycolic acid oxidase) (Corley et al., 2005). In addition, direct oxidation of glyoxal to Glyoxylic Acid occurs by enzymes such as 2 -ketoaldehydedehydrogenase. In vitro glyoxal is presumably metabolized to oxalic acid via glycolic and Glyoxylic Acid in the rat liver and by haemoglobin (Kun, 1952; Francoeur and Denstedt, 1954; Hills and Berry, 1967). In summary, the data given above demonstrate the central role of Glyoxylic Acid in the metabolism of glyoxal. Thus, whenever glyoxal has been tested in animals, Glyoxylic Acid has been tested as well. Therefore, the data on the repeated dose toxicity of glyoxal presented below can be used for the assessment of the repeated dose toxicity of Glyoxylic Acid 50%. Together with the OECD guideline 422 study (combined repeated dose toxicity study with a reproduction/developmental toxicity screening test) for Glyoxylic Acid 50% they represent sufficient weight of evidence to address the repeated dose toxicity potential of Glyoxylic Acid 50%. Therefore, an additional subchronic toxicity study with Glyoxylic Acid 50% is not needed.

Subchronic toxicity of glyoxal

The data presented below were taken from the OECD SIDS publication on glyoxal.

Glyoxal (40 %) was administered with the diet to 10 Harlan-Wistar rat/group in a 90-day study. Based on the food consumption, doses of 32.7, 63.2, 132 and 253 mg glyoxal (based on the pure ingredient)/kg bw/day were hereby reached for the male animals and doses of 32, 63.2, 127 and 271 mg glyoxal body weight/day for the female animals. Data on the stability of glyoxal in the diet are missing. No substance-related deaths occurred. An effect on the food consumption could not be observed. For the male animals of the highest dose group, a significant retardation of the body weight gain was noted only during the first 2 weeks of the study. During the subsequent study period, however, the body weight was in the range of the controls. Moreover, a significant increase of the relative liver and kidney weight was determined for the male animals in the highest dose group. The organ weight changes in both intermediate dose groups was not significant. Substance related macroscopic or histo-pathological organ changes (13 different organs) were not observed in any dose groups. The authors derived a no effect level of ca. 125 mg/kg bw/day (active ingredient). Haematological and clinical-chemical examinations were not performed (Union Carbide Company, 1966).

In a 28 -day study on 6 rats each per dose and control group as well as sex (strain Crl CD(SD)BR), glyoxal (40 % aqueous solution) was administered in doses of 100, 300 and 1000 mg/kg bw/d via the drinking water according to OECD guideline 407. The concentrations in drinking water were geared weekly to the body weight and the drinking water consumption. No deaths occurred during the test period. The body weight gain was not retarded in the low dose group, only slightly retarded in the intermediate dose group and was significantly retarded in the high dose group. The body weight retardation coincided with a decreased food consumption. A dose dependent decrease of the water consumption was noted for the male animals from the lowest dose group and for the female animals from the intermediate dose group. A slight increase of the erythrocyte count in the male rats of the high dose groups was evaluated as secondary effect of the reduced water consumption. The effect on the various organ weights in the high dose group was attributed to the reduced body weight. Moreover, in none of the dose groups a substance-related effect on haematological and biochemical parameters and of the urinary status was seen. During the final autopsy, substance related macroscopic or histopathological organ changes were not observed in any dose group. Based on these findings and specially on the dose related decrease of the water, food consumption and body weight, a no toxic effect level of 100 mg 40 % glyoxal/kg bw/d was established (Société Française Hoechst, 1987).

The subchronic toxicity of glyoxal (purity 98.7 %) was studied on male Sprague-Dawley rats with oral administration via the drinking water at concentrations of 0, 2000, 4000 or 6000 mg/L for 30, 60 or 90 days (corresponding to 0, 188, 407 or 451 (30-d subgroup); 0, 135, 239 or 344 (60-d supgroup) and 0, 107, 234 or 315 (90-d subgroup) mg/kg bw/d). 5 animals were used per group. Clinical-chemical studies were performed at the end of the administration period. In addition, the activities of glyoxalase I and II, the glutathione content and the formation of 2-thiobarbituric acid reactive substances were measured in the liver, kidney and erythrocytes. The liver, kidneys, spleen, heart, testes and brain were weighed and the liver, kidneys, spleen, stomach, thymus and mesenteric lymph nodes examined histopathologically. For the 90-day exposure period, a dose-dependent and, in the intermediate and high dose group, significant decrease of the food-and water consumption as well as a corresponding body weight retardation were observed. In the low dose group, only the water consumption was significantly reduced. A dose-dependent decrease of the absolute weight of the examined organs, excluding the weights of the testes and brain, was seen in the animals of all dose groups and exposure periods. In the upper dose group, the relative kidney weights after 90 days exceeded those of the controls. In the intermediate and high dose groups, the clinical-chemical examination showed decreased activities of alanine and aspartate aminotransferase as well as lactate dehydrogenase and reduced albumin and total protein values. In the lowest dose group, a decreased alanine aminotransferase activity and a reduced total protein value were determined. Only after a 30-day exposure a significant increase of the activity of glyoxalase I and II was measured in the liver and in the erythrocytes in the animals of the intermediate and high dose groups as well as the glyoxalase I activity in the kidneys in the animals of the high dose group. In contrast, no substance related effect on the enzymatic activity of glyoxalase I and II was detectable for the longer exposure periods. Neither the glutathione level nor the synthesis of 2-thiobarbituric acid-active substances were affected in the liver, kidney or erythrocytes. Substance-related macroscopic or histopathological organ changes were no found. According to the authors, a no observed adverse effect level could not be determined due to the reduced serum protein levels in the lowest dose group (lowest observed effect level 107 mg/kg bw/d) (Ueno et al., 1991).

In another experiment of this research group with approximately the same study design as described above, the male Sprague-Dawley rats (5 to 7 animals/group) obtained glyoxal (100%) in a concentration of 6000 mg/l for 90 or 180 days in the drinking water. In addition to one control group fed the diet ad libitum, a control group was led which obtained the same amount of diet as the treated group (pair-fed control group). The daily substance intake in the 90 day group corresponded to that in the study described above. In the 180-day group, it was 298 mg/kg bw/d. The body weight retardation after administration of glyoxal for 180 days was greater than that in the pair-fed control group. With the exception of those of the brain and testes, the absolute weights of the weighed organs were below those of the controls. The relative weights of the liver, kidneys and heart were increased compared to the pair-fed control group. Slightly reduced activities of alanine and aspartate aminotransferase as well as lactacte dehydrogenase were determined after 180 days. The total protein content in the serum was significantly below that of both control groups. After 180 days, haemorrhage and polyps in the forestomach were observed macroscopically in 2 of the treated animals which, however, were assessed by the authors not to be treatment-related. A slight swelling of the papillary epithelial cells in the kidneys as well as a papillary interstitial oedema and congestion of the lymph nodes in this area were observed after 90 and 180 days in 4 animals of the glyoxal group. Electron microscopic examinations of the liver and kidneys showed no findings (Ueno et al., 1991).

10 male and female Fischer-344 rats each obtained 0, 1000, 2000, 4000, 8000 and 16000 mg glyoxal/l drinking water for ca. 90 days (no data on drinking water consumption). Depending on the concentration, there was a reduced food and water intake and the body weight gain was correspondingly retarded. All male and female rats of the high concentration group had to be killed on the 12th day in a cachectic state. Other symptoms were not observed. Haematological and clinical-chemical examinations were not performed. The histopathological examination showed minor findings in all groups such as haemorrhages in the mesenteric lymph nodes (male and female rats) and mild to moderate hyperplasia of the mandibular lymph nodes (male rats) as well as atrophic and degenerative changes of the submandibular salivary glands and slight to minimum changes in the kidneys (male rats) in the 8000 and 16000 mg/l-groups. Moreover, hypospermia in the epididymis with atypical cells and slight degenerative changes of the germ epithelium in the testes occurred in the male animals in the highest concentration group. For the female rats of the high dose group, atrophy of the thymus was observed. These histopathological findings were possibly not substance-related, but rather were attributed to cachexia of the animals caused by the reduced water intake (NTP, 1991). A no effect level cannot be derived, because the feed and drinking water intake was reduced down to the lowest tested concentration of 1000 mg/l. It is unclear whether the reduced consumption of drinking water and food indicates toxic effects or more likely a palatability effect.

In the same way, groups of 10 male and female B6C3F1 mice were given glyoxal in the drinking water at doses of 0, 1000, 2000, 4000, 8000 and 16000 mg/l (no data on drinking water consumption). No deaths occurred, but a concentration-dependent reduction of the food and water intake resulted here, too. Consequently, the body weight gain was delayed and the organ weights reduced. Other symptoms did not appear. Haematological and clinical-chemical tests were not conducted. In the male mice of all dose groups histological changes in the submanidibular salivary glands (secretion depletion) were observed which were possibly evaluated to be substance-related, whereby the authors interpret the toxicological relevance to be unclear (NTP, 1991). A no effect level cannot be derived, because the food and drinking water intake was reduced down to the lowest tested concentration of 1000 mg/l. It is unclear whether the reduced consumption of drinking water and food indicates toxic effects or more likely a palatability effect.

In another 90 -day study, glyoxal was administered in the diet to 3 Beagle dogs each per dose and control group (no data on sex). The doses were 31, 65 and 115 mg (based on pure glyoxal/kg bw/d. All the animals survived. No substance-related effect on the body weight as well as on the relative or absolute weight of the liver and kidney was ascertained. A substance-related effect on haematological or clinical-chemical parameters of the blood (haematocrit, erythrocyte and leukocyte count, haemoglobin and urea nitrogen levels, alkaline phosphatase activity, bromosulphthalein retention) did not occur in any dose group. In addition, no subtance-related macroscopic or histopathological organ changes were observed (18 different organs). The authors derived a no effect level of ca. 115 mg/kg bw/d (Union Carbide Company, 1966).

For testing the subacute inhalation toxicity according to OECD guideline no. 412, groups of 5 male and female Wistar rats each (average initial weight 193 and 171 g, respectively) inhaled nominal concentrations of 0, 0.4, 2.0 and 10 mg glyoxal (40 % aqueous solution)/m³ as an aerosol for 6 hours daily, 5 times per week over a period of 29 days (nose only, a total of 20 exposures). The analytically controlled concentrations amounted to 0.6 (± 0.2), 2.3 (± 0.8) and 8.9 (± 1.9) mg/m3, and the mean aerodynamic mass diameter was 0.8 to 1.2 μm with a mean geometric standard deviation of 1.5 to 1.7. With regard to the systemic toxicity, the no observed effect level was given at > 10 mg/m3 (Hoechst, 1995).

In conclusion, by oral route the key study is the study conducted by Société Française Hoechst (1987) according to OECD guideline 407 on rats. A no effect level of 100 mg/kg bw/d was established for glyoxal 40% (i.e. 40 mg/kg bw/d related to the active ingredient). This value is supported by the studies by Ueno (1991a, b) on rats which obtained a LOEL of 107 mg/kg bw/d related to pure glyoxal, as well as by the study from Union Carbide (1966) on dogs which derived a NOEL of 115 mg/kg bw/d related to pure glyoxal. In a subacute inhalation study on rats for 29 days, a NOEL of > 10 mg/m3 was derived for the systemic toxicity (40% glyoxal). No dermal repeated toxicity studies have been performed.

Conclusion

The dataset for the repeated dose toxicity of glyoxal together with the OECD TG 422 study on Glyoxylic Acid 50% provide sufficient weight of evidence for the assessment of the repeated dose toxicity of Glyoxylic Acid 50%. There was no specific target organ toxicity at regulatroy relevant dose levels. Reduced body weight or body weight gain was the most sensitive or among the most sensitive adverse effects. In general, the NOAEL did not decrease with increasing exposure time. Thus, the NOAEL of 200 mg Glyoxylic Acid 50%/kg bw/d derived from the OECD TG 422 study represents a valid and reliable value for the assessment of the repeated dose toxicity of Glyoxylic Acid 50%. A sub-chronic toxicity study with Glyoxylic Acid 50% is scientifically not justified and can be omitted according to Council Regulation 1907/2006/EC, Annex XI.

References

Corley RA et al. (2005). Development of a physiologically based pharmacokinetic model for ethylene glycol and its major metabolite, glycolic acid, in rats and humans. Toxicological Sciences 85(1): 476 -490.

Francoeur M and Denstedt OF (1954). Metabolism of mammalian erythrocytes. VI. Heme as a catalyst for the oxidation of glyoxal in the erythrocyte. Can. J. Biochem. Physiol. 32: 655-662.

Hills PR and Berry RJ (1967). Cytotoxicity of Carbohydrates Heavily Irradiated in Solution. Nature 215: 309.

Hoechst AG (1995). Unpublished report No 94.1056

Kun E (1952). A study on the metabolism of glyoxal in vitro. J. Biol. Chem. 194: 603 -611.

OECD Screening Information DataSet (SIDS) High Production Volume Chemicals. Glyoxal –Cas No. 107-22-2. Jan-2003.

Sady C et al. (2000). Maillard reactions by α-oxoaldehydes: detection of glyoxal-modified proteins. Biochimica et Biophysica Acta 1481: 255–264.

Société Française Hoechst (1987). Unpublished report by CIT No. 2619 TSR (HOE 87.1678).

Thornalley PJ (1995). Advances in glyoxalase research. Glyoxalase expression in malignancy, anti-proliferative effects of methylglyoxal, glyoxalase I inhibitor diesters and S-D-lactoylglutathione, and methylglyoxal-modified protein binding and endocytosis by advanced glycation endproduct receptor. Critical Reviews in Oncology/Hematology 20(1–2): 99–128.

Ueno H et al. (1991). Subchronic oral toxicity of glyoxal via drinking water in rats. Fund. Appl. Toxicol. 16: 763-772.

Union Carbide Company (1966). Unpublished report No. 29-1 (HOE 87.0733).

Justification for classification or non-classification

Based on the available data Glyoxylic Acid 50% is not subject to classification and labeling for repeated dose toxicity according to Directive 67/548/EEC or Regulation 1272/2008/EC.