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Toxicological information

Carcinogenicity

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Administrative data

Description of key information

The oral administration of Glyoxal 40% via drinking water to Wistar rats over a period of either 12 or 24 months (OECD 453) resulted in changes with regard to body weight, clinical pathology parameters and pathology findings. Signs of systemic toxicity were determined at a target dose level of 75 mg/kg bw/d and above. The No Observed Adverse Effect Level (NOAEL) was considered to be 25 mg/kg bw/d in male and female Wistar rats for systemic effects. No substance related neoplastic lesions were observed either in males or females (BASF, 2012). Additionally, several published data which almost all have been validated and peer-reviewed (CICAD 57, 2004; The Toxicological Dossier of Glyoxal published by the EFfCI (Schilling 2004), BUA 187) exist, were considered as supporting studies. A carcinogenicity study on Glyoxal was conducted in the early eighties by the Bushy Run Research Center (Union Carbide, Pennsylvania, USA) and was entitled: “Evaluation of the dermal carcinogenicity of AEROTEXR Glyoxal 40 and European Glyoxal 40 in male C3H mice”. The data of this study were also considered as support.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
(1981)
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.4300 (Combined Chronic Toxicity / Carcinogenicity)
Version / remarks:
(1998)
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B61 (23.03.09); B61 (01.09.09); B62 (06.02.10); B61 (15.07.10); B61 (05.01.11)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, under N2
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain as described in the report: Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 42 +/- 1 days
- Housing: 5 animals per cage, in H-Temp (PSU) cages, floor area 610x435x215 mm (TECHNIPLAST, Germany)
- Diet: Ground Kliba mouse/rat maintenance diet “GLP”, meal (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: Drinking water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/ 12 h
- Any deviations will be documented

ANALYSIS OF FOOD, WATER, BEDDING
- The food used in the study will be assayed for chemical and microbial contaminants according to the Fed. Reg. Vol. 44, No. 91 of May. 09, 1979, p 27354 (EPA);
- The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants;
- The bedding (Type Lingocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany) is regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals). The values given in Lab Animal, Nov.–Dec. 1979, pp 24–33, will serve as the guideline for maximum tolerable contaminants
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
On the day of arrival the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. Prior to the first ophthalmological examinations, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20% of the mean weight of each sex. The list of randomization instructions was compiled with a computer. At the start of the administration period (study day 0) the male and female rats were 41-43 days old. The test substance was administered daily in the drinking water for about 12 (satellite groups) and 24 months (main groups). Control animals received drinking water only. At the end of the administration period the animals were sacrificed after a fasting period (withdrawal of food) of at least 16-20 hours.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany and/or at Competence Center Analytics, BASF SE, Ludwigshafen, Germany. The stability of the test substance in drinking water over a period of 4 days was proven prior to the study with the similar batch B62 (Date of production: 10 Oct 2008). At the start of the study, the concentration was demonstrated using 1 sample of all concentrations.
During the study, analyses of the test-substance preparations with respect to concentration control were conducted after about 3, 6, 9, 12, 15, 16, 18, 21 months as well as towards the end of the study. Concentration control analyses were performed with one sample per test group taken at the time points indicated above. The samples were taken out of randomly selected reserve water bottles being stored in the animal room. Thus, the stability of the test substance in drinking water was also proven under test conditions. Homogeneity was given because the test substance was completely miscible with water, in terms of a pure solution.
Duration of treatment / exposure:
12 months, satellite groups
24 months, main groups
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Main groups: fifty animals/sex and group
Satellite groups: ten animals/sex and group
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
MORTALITY AND CLINICAL OBSERVATIONS
A check for moribund or dead animals will be made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays; all animals will be checked daily for any abnormal clinical signs. Abnormalities and changes will be documented for each animal.

DETAILLED CLINICAL OBSERVATION
All animals will be subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0) and subsequently once a week (in the morning). For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed will be based on the current index of findings in Datatox F1 software and includes but is not limited to the following parameters listed:
abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, feces (appearance/consistency), urine, pupil size.

BODY WEIGHT
Body weight will be determined before the start of the administration period in order to randomize the animals. During the administration period the body weight will be determined on day 0 (start of administration period) and thereafter at weekly intervals until week 13. Thereafter, body weight measurement will be carried out at 4-week intervals and additionally at the end of the administration period. The difference between the body weight on the respective day of weighing and the body weight on day 0 will be calculated as body weight change.

FOOD CONSUMPTION
Food consumption will be determined weekly during the first 13 weeks and at 4-week intervals thereafter until test ending (as representative value over 3 or 4 days). Food consumption will be calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY
Food efficiency (group means) will be calculated based upon individual values for body weight and average food consumption for animal in each cage.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Drinking water consumption will be determined weekly during the first 13 weeks and at 4-week intervals thereafter until test ending (as representative value over 3 or 4 days). Water consumption will be calculated as mean water consumption in grams per animal and day. The test substance intake will be calculated for each animal on the basis of water consumption and body weight and will be given in mg/kg body weight/day.

OPHTHALMOSCOPIC EXAMINATION
At test initiation the eyes of all satellite animals will be examined using an ophthalmoscope after administration of a mydriatic. At the end of the administration period, the eyes of the satellite animals of the control and high dose will be examined. The eyes of the animals of the other satellite groups will be examined only if there is a striking discrepancy between the examined groups.

HAEMATOLOGY AND CLINICAL CHEMISTRY
For the purpose of haematology and clinical chemistry, blood samples will be taken from fasted animals by puncturing the retroorbital venous plexus under Isoflurane anesthesia. Blood sampling and examination will be carried out in a randomized sequence.
The haematological parameters considered will include leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, and prothrombin time.
The clinical chemical parameters considered will include alanine aminotransferase, aspartate aminotransferase,alkaline phosphatase, serum γ-glutamyl transferase, sodium, potassium, chloride, inorg. phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol and magnesium.

URINALYSIS
On the afternoon preceding the day fixed for urinalysis, the animals will be transferred individually into metabolism cages (no food or drinking water provided). On the following day, the samples will be examined in a randomized sequence. Following parameters will be considered: volume, color, turbidity, pH value, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity and microscopy of sediment.
Sacrifice and pathology:
The animals will be sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals will be necropsied and assessed by gross pathology. Animals which die intercurrently will be necropsied as soon as possible after their death and assessed by gross pathology.

ORGAN WEIGHTS
In addition to the weight of the anesthetized animals, the weights of following organs will be determined: liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart.

ORGAN/TISSUE FIXATION IN FORMALDEHYDE 4% SOL.:
In addition to all gross lesions, following organs and tissues will be fixed: salivary glands (mandibular and sublingual glands), esophagus, stomach (forestomach and glandular stomach), duodenum, jejunum and ileum, cecum, colon and rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and lumbar cords), eyes, adrenal glands, thyroid glands, parathyroid glands, trachea, lungs, pharynx, larynx, nose (nasal cavity), aorta, heart, bone marrow (femur), lymph nodes (mesenteric and axillary lymph nodes), spleen, thymus, kidneys, urinary bladder, testes, ovaries, oviducts, uterus and vagina, epididymides, prostate and seminal vesicle, female mammary gland, skin, skeletal muscle, sternum with marrow, femur with knee joint, and extraorbital lacrimal glands.

HISTOPATHOLOGY:
Fixation will be followed by histotechnical processing and examination by light microscopy and assessment of findings. As a basic rule and in addition to all gross lesions, organs/tissues obtained from the control and high dose groups (main and satellite) will be examined in a comparative manner (salivary glands,esophagus, stomach, duodenum, jejunum and ileum, cecum, colon and rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord, eyes, adrenal glands, thyroid glands, parathyroid glands, trachea, lungs, pharynx, larynx, nose, aorta, heart, bone marrow lymph nodes, spleen, thymus, kidneys, urinary bladder, testes, ovaries, oviducts, uterus and vagina, epididymides, prostate and seminal vesicle, female mammary gland, skin).
Animals that die or are sacrificed in a moribund state will be processed histotechnically and assessed like control animals.
Statistics:
- Means and standard deviations will be calculated;
- TheDunnett test will be used for statistical analyses of body weight, body weight change, food consumption, water consumption and food efficiency;
- The KRUSKAL-WALLIS and WILCOXON test will be used for statistical analyses of blood parameters (except for differential blood count and reticulocytes) and of the weight of the anesthetized animals as well as the absolute and relative organ weights;
- The FISHER's exact test will be used for statistical analyses of urinalysis parameters (except for volume, color, turbidity and specific gravity)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
In the satellite groups no test substance-related clinical signs were observed for male and female Wistar rats. In the main groups abnormal clinical signs were equally distributed between control and test substance-treated animals or occurred incidentally in single animals.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The oral administration of Glyoxal 40% via the drinking water at dose levels of 0 mg/kg bw/d (test group 0), 25 (test group 1), 75 (test group 2) and 300 mg/kg bw/d (test group 3) to male and female Wistar rats over a period of either 12 (satellite groups) or 24 months (main groups) did not cause a higher incidence of treatment-related mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The effects on mean body weights and body weight change values observed in male and female animals of test groups 3S and 3 (300 mg/kg bw/d) were assessed as being related to the test compound administration. Differences observed at lower dose levels were not assessed as being of toxicological relevance.
Regarding pathology, the treatment with Glyoxal 40% caused decrease of terminal body weight in males of test group 3S (300 mg/kg bw/d) of the satellite group of -8%. Furthermore, males and females of the test group 3 (300 mg/kg bw/d) of the final sacrifice group showed also a decrease in terminal body weight of -11% and -12% respectively. These decreases in the terminal body weight were regarded to be a manifestation of a systemic toxic effect. It could not be addressed to a special organ but is regarded to be adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Relevant changes in water consumption were observed for the test groups 3S and 3 (300 mg/kg bw/d) most likely related to the taste of the test item.
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Vasodilatation in fundus oculi was observed only in 8 female animals of test group 3S (300 mg/kg bw/d) and in 6 female animals of test group 2S (75 mg/kg bw/d) at the end of the administration period of 12 months. This finding showed a clear dose-response relationship. Because a histopathological correlate was not observed, the vasodilation in fundus oculi was considered to be treatment-related, but assessed as non-adverse.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 3 months of compound administration, in male animals of test groups 2S and 3S (75 and 300 mg/kg bw/d) hemoglobin values were higher compared to controls. These alterations were not accompanied by any other changes of red blood cell parameters in these rats and the means were within the historical control range. After study months 6 and 12, in females of test group 2S (75 mg/kg bw/d) and at the later date also
in females of test group 3S (300 mg/kg bw/d) relative reticulocyte counts were lower compared to controls. After the 6th study month, reticulocyte counts were not changed dose-dependently. The values were not accompanied by any other alteration among red blood cell parameters. Therefore,
these alterations were regarded as not adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Regarding clinical pathology, a decreased ALT activity in male and female rats of test group 3 (300 mg/kg bw/d) was observed. Neither any other clinical pathology alteration indicating a microsomal enzyme induction nor any relevant liver weight increase was found in these dosed rats. Therefore, other reasons for an ALT activity decrease, including an effect on the pyridoxal 5’-phosphate levels, cannot be excluded (PSD guidance document, 2007). Lower cholesterol and globulin values in rats of both sexes of test group 3 (300 mg/kg bw/d) and, additionally, in males of test group 2 (75 mg/kg bw/d) were most probably due to a dysregulation of the liver cell metabolism or a decreased intestinal absorption of cholesterol combined with a lower synthesis of transport globulins.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The reason for the urinary excretion of higher keton body and urobilinogen levels in males could not be elucidated.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The terminal body weight was reduced in male and female animals of test group 3 (300 mg/kg bw/d). This was regarded to be related to treatment. The weight reduction of the male thyroid gland, female heart and liver in the same test group was regarded to be a consequence to the reduced terminal body weight. The increase of kidney and spleen weight in females of test group 1 (25 mg/kg bw/d) was regarded to be incidental due a missing dose-response relationship and no significant deviations in the relative organ weights. All other mean absolute weight parameters did not show significant differences when compared to the control group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Female animals of the final sacrifice group showed macroscopically an increase of erosions/ulcer in the glandular stomach (0/0/2/5) which was confirmed and even outnumbered by histopathology (0/1/6/9). The single erosion/ulcer in one animal of test group 1 (25 mg/kg bw/d) was regarded to be an incidental finding as erosion/ulcer are normally detected in a low incidence in the glandular stomach in long term studies. In former studies with Glyoxal 40% erosions/ulcer were already observed in the glandular stomach. Therefore, the erosions/ulcer in females of test group 2 (75 mg/kg bw/d) and test group 3 (300 mg/kg bw/d) were regarded to be substance related and adverse in nature.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The total numbers of primary, benign, malignant, systemic, and metastasized neoplasms were comparable between control and high-dose animals. They were biologically equally distributed over the control and treatment groups.
Key result
Dose descriptor:
NOAEL
Effect level:
> 300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
other: Effect type: carcinogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Effect type: systemic toxicity
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
GLP study according OECD TG 453

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Secondary quotation, scientifically acceptable data, peer-reviewed and reported in different acknowledged reviews (CICAD 57, The Toxicological Dossier on Glyoxal of the EFfCI, the BUA report 187)
Qualifier:
no guideline followed
Principles of method if other than guideline:
The present comparative study was designed to determine the dermal carcinogenic potential of AEROTEX glyoxal 40 and European glyoxal 40 in mice. No guideline was reported.
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BRRC sample number 41-400, identification number R9195-160 and BRRC sample number 41-429, identification number R9516-43

OTHER
- Impurities (identity and concentrations): 0.7% acid (calc. as acetic), 5.9% formaldehyde, 0.3% glycolaldehyde, 0.9% ethylene glycol (first batch)
- Composition of test material, percentage of components: 40.0% glyoxal in water, 0.7% acid (calc. as acetic), 5.9% formaldehyde, 0.3% glycolaldehyde, 0.9% ethylene glycol(first batch)
- Impurities (identity and concentrations): 0.1% acid (calc. as acetic), 0.7% formaldehyde, 0.2% glycolaldehyde, traces of ethylene glycol (second batch)
- Composition of test material, percentage of components: 40.4% glyoxal in water, 0.1% acid (calc. as acetic), 0.7% formaldehyde, 0.2% glycolaldehyde, traces of ethylene glycol (second batch)
Species:
mouse
Strain:
C3H
Sex:
male
Route of administration:
dermal
Details on exposure:
Treatment consisted of an application of 25 µL of diluted test substance to the clipped skin of the back of each animal. Negative control animals received similar treatments with deionized water alone.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
The animals were treated throughout their lifetime.
Frequency of treatment:
3 times per week (except holidays)
Post exposure period:
None
Dose / conc.:
63 mg/kg bw/day
Remarks:
calculated taking into account a density of 1.27g/cm3 and a mean body weight for the mice of 25 g
Dose / conc.:
158 mg/kg bw/day
Remarks:
calculated taking into account a density of 1.27g/cm3 and a mean body weight for the mice of 25 g
No. of animals per sex per dose:
The animals were randomized into 3 groups of 40 animals each
Control animals:
yes, concurrent vehicle
Details on study design:
Male C3H/HeJ mice were randomized into 3 groups of 40 animals each and were treated three times weekly (except holidays) throughout their lifetime with a 1:8 dilution of either AEROTEXR Glyoxal 40 or European Glyoxal 40 in deionized water. Treatment consisted of an application of  25 µL of diluted test substance to the clipped skin of the back of each  animal. Taking into account a density of 1.27g/cm3 and a mean body weight  for the mice of 25 g, the dosage was calculated to be about 158 mg/kg bw/day referring to the test substance containing ca. 40% glyoxal, and  ca. 63 mg/kg bw/day referring to the active ingredient as such (i.e.  Glyoxal). Negative control animals received similar treatments with  deionized water alone.
Positive control:
None
Observations and examinations performed and frequency:
The animals were observed for mortality and clinical symptoms, and were examined for skin lesions and skin tumors.
Sacrifice and pathology:
Necropsy was performed on all dead mice and on moribund mice,  which were sacrificed in extremis. Necropsy included careful examination of the skin and careful examination of the body cavities. The entire carcasses of all non-autolyzed mice that died after ca. 18 months of treatment were fixed in 10% neutral buffered formalin; sections were prepared and stained for the purpose of histopathological examination. The dorsal skin of all mice, with or 
without skin lesions, was also fixed in 10% neutral buffered formalin for further examinations.
Clinical signs:
not examined
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
The treatment with AEROTEX glyoxal 40 caused skin irritation resulting in inflammation and necrosis in some mice.
Mortality:
no mortality observed
Description (incidence):
In both treated groups (AEROTEX glyoxal 40 and European glyoxal 40), the mean survival time was statistically significantly greater than that of the deionized water control group (580 and 594 days, respectively, versus 488 days), indicating no treatment-related effect on survival. In the deionized water control group the mean survival time of 488 days. In all three groups, the last surviving mouse died after ca. 27 months of treatment.
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No skin neoplasms were found in both treated groups. In the deionized water control group, no tumors were found. No subcutaneous neoplasms were found in any mice treated with AEROTEX Glyoxal 40. In the European Glyoxal 40 treated mice, one case of infiltrate fibrosarcoma (left lateral rib cage and axilla, observed after 496 days of treatment) was reported, which however was without biological significance since this kind of tumor is known from historical control data to occur occasionally in the mouse strain used.
Key result
Sex:
male/female
Basis for effect level:
other: No treatment-related effects indicating carcinogenicity were observed.
Remarks on result:
other: Effect type: carcinogenicity
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
63 mg/kg bw/day
Study duration:
chronic
Species:
mouse

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The substance is already listed in Annex VI of Regulation (EC) No 1272/2008 but not classified for carcinogenicity. Owing to the available experimental test data, there is no need for classification of the test item with respect to carcinogenicity according to Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

Additional information

Oral

The oral administration of Glyoxal 40% via drinking water to Wistar rats over a period of either 12 or 24 months (OECD TG 453) resulted in changes with regard to body weight, clinical pathology parameters and pathology findings. Signs of systemic toxicity were determined at a target dose level of 75 mg/kg bw/day and above. There were no substance-related organ weight changes, gross lesions, and non-neoplastic findings in male and female Wistar rats after 12 and 24 months of treatment with 25 mg/kg bw, respectively. Especially no substance related neoplastic lesions were observed either in males or females after 12 and 24 months of treatment with Glyoxal 40% respectively. All further pathological findings recorded were considered to be incidental in nature and not related to treatment. The No Observed Adverse Effect Level (NOAEL) was considered to be 25 mg/kg bw/day in male and female Wistar rats (24 mg/kg bw/day was achieved in both sexes) for systemic effects (BASF SE 80S0496/01236, 2012).

Validated and peer-reviewed data (CICAD 57, 2004; The Toxicological Dossier on Glyoxal published by the EFfCI (Schilling, 2004)), had already mentioned in the chapter on repeated dose exposure (Ueno 1991a). The study comprised two experimental series defined as Phase I and Phase II. In Phase I, five male Sprague-Dawley rats per group were treated with Glyoxal (98.7% purity) in drinking-water at concentrations of 2000, 4000, or 6000 mg/L active ingredient for periods of 30, 60, or 90 days. The animals received drinking water and were fed basal diet ad libitum. Since mean body weight gain and food consumption were found to decrease proportionately to Glyoxal dosage in this first experiment, the Phase II experiment was undertaken. The Phase II test series consisted of a 6000 mg/L Glyoxal test group (as above), a control group receiving diet ad libitum, and an additional control group where diet was no more offered ad libitum but was limited to an amount similar to that consumed by the animals of the treated group; the treatment period was 90 and 180 days. For each experiment, the study design included observations of clinical signs, body weights, major organ weights (liver, kidneys, spleen, heart, testes, brain), serum clinical chemistry, and biochemical examinations of glyoxalase activity and extent of lipid peroxidation (content of GSH and 2-thiobarbituric acid-reactive substances) in liver, kidneys, and erythrocytes. In the Phase II experiment examinations further comprised gross pathological and histopathological examinations of liver, kidneys, spleen, stomach, thymus, and mesenteric lymph nodes. After exposure of the Sprague-Dawley rats to dosages of 6000 mg Glyoxal/L of drinking water for up to 180 days, there were no neoplastic changes found upon gross and histopathological examination of liver, kidneys, spleen, stomach, thymus, and mesenteric lymph nodes.oral repeated dose study was performed and published by Ueno H et al. (1991a) has already mentioned in the chapter on repeated dose exposure. The study comprised two experimental series defined as Phase I and Phase II. In Phase I, five male Sprague-Dawley rats per group were treated with Glyoxal (98.7% purity) in drinking-water at concentrations of 2000, 4000, or 6000 mg/L active ingredient for periods of 30, 60, or 90 days. The animals received drinking water and were fed basal diet ad libitum. Since mean body weight gain and food consumption were found to decrease proportionately to Glyoxal dosage in this first experiment, the Phase II experiment was undertaken. The Phase II test series consisted of a 6000 mg/L Glyoxal test group (as above), a control group receiving diet ad libitum, and an additional control group where diet was no more offered ad libitum but was limited to an amount similar to that consumed by the animals of the treated group; the treatment period was 90 and 180 days. For each experiment, the study design included observations of clinical signs, body weights, major organ weights (liver, kidneys, spleen, heart, testes, brain), serum clinical chemistry, and biochemical examinations of glyoxalase activity and extent of lipid peroxidation (content of GSH and 2-thiobarbituric acid-reactive substances) in liver, kidneys, and erythrocytes. In the Phase II experiment examinations further comprised gross pathological and histopathological examinations of liver, kidneys, spleen, stomach, thymus, and mesenteric lymph nodes. After exposure of the Sprague-Dawley rats to dosages of 6000 mg Glyoxal/L of drinking water for up to 180 days, there were no neoplastic changes found upon gross and histopathological examination of liver, kidneys, spleen, stomach, thymus, and mesenteric lymph nodes.

Further validated and scientifically acceptable data referring to the tumour initiation and/or promotion potential of Glyoxal in mice and rats have been peer-reviewed and published in CICAD 57 (2004), in the Toxicological Dossier of Glyoxal of the EFfCI (2004), in the Opinion on Glyoxal published by the The Scientific Committee on Consumer Products (SCCP) of the European Commission (2005), and in the BUA Report 187 (1998). The potential tumour-initiating effect of Glyoxal at dilutions of 37 to 43% on the skin was examined in groups of 20 female CD-1 mice each, which were about 7 weeks old at test begin (Miyakawa et al., 1991). The mice received two applications/week of 0.1 mL test material onto the shaven skin of the back for a period of 5 weeks, defined as initiation phase. After a post - exposure of 1 week, the animals received applications of 12-O-tetradecanoylphorbol- 13-acetate (TPA) as a promoter for 47 weeks. 7,12-dimethylbenzo(a)-anthracene (DMBA) was used as positive control, and dimethylsulfoxide (DMSO) was used as negative control. The total initiation dose of Glyoxal per mouse was 500 μmol, corresponding to 30 mg/mouse). All animals survived the entire 53-week test period. In the Glyoxal-treated group, 2 of 20 mice showed papilloma. No tumours were found in the DMSO group. In the positive control group (DMBA/TPA), all 20 mice had a total of 134 skin tumours (99 cases of papilloma and 31 cases of squamous cell carcinomas). Thus, Glyoxal was not shown to be a tumour-initiator in the present study.

The potential tumour-promoting effect of Glyoxal in the stomach was examined in groups of 7 week old male Wistar rats in a 2-stage model assay (Takahashi et al. 1989). In the initiation phase, 2 groups of 30 animals each received N-methyl- N’-nitro-N-nitrosoguanidine (MNNG) in drinking water at a concentration of 100 mg/L, for a period of 8 weeks; the animals simultaneously received diet containing 10% sodium chloride. From week 8 to week 40, the first group received drinking water containing 0.5% Glyoxal whereas the second group received drinking water as such, i.e. without any additive. A third group of 10 animals received a diet and drinking water without additives in the first 8 weeks and then received drinking water with 0.5% Glyoxal from the 8th to the 40th week of the experiment. In the group of rats pre-treated with MNNG and NaCl, a statistically significant incidence of adenocarcinomas in the glandular stomach of ca. 43% (12 of 28 animals affected; for more details, see table below) was observed, with the tumours being localized mostly within the pylorus region; a hyperplasia of the mucosa was also found there. In the group of animals which had been pre-treated with MNNG and NaCl but not treated with Glyoxal from week 8 to week 40, 5 tumours were reported, in 5/30 animals, corresponding to 17%. In the third group of animals, no tumours were seen. The findings of this study indicated that Glyoxal possessed a local tumour promoting potential on the glandular stomach; these findings are in accordance with the in vivo mutagenic/genotoxic effect of Glyoxal seen in the pyloric mucosa of the stomach.

A short-term liver carcinogenicity screening assay was undertaken by Hasegawa and Ito (1992). A group of 12 male, 6 weeks old F344 rats received a single intraperitoneal injection of 200 mg diethylnitrosamine/kg bw as initiator. After a period of 2 weeks, the animals received 5000 ppm Glyoxal in the diet, corresponding to a dosage of 333 mg/kg bw/d day, over a period of 6 weeks. The animals were subjected to a 2/3 hepatectomy after a feeding period of one week. The animals of a control group received the initiator (i.p. injection of diethylnitrosamine) but were not treated with Glyoxal. At the end of the study, the liver was evaluated for GST-P-(placental glutathione-S-transferase) positive foci as indicator for a carcinogenic potential. The results revealed no Glyoxal-related increase of the number of foci, but rather a significant reduction was reported. Thus, no promoting potential of Glyoxal in the liver of rat could be evidenced in this study.

In vitro cell transformation assays with Glyoxal using an embryonic mouse cell-line (C3H/10T½CL8 cells) were negative (Thilagar A 1980, also cited in CICAD 57 and in the Toxicological Dossier of Glyoxal of the EFfCI; American Cyanamid Co 029 -626 -292 -8; 1988).

 

Dermal

Referring to the dermal route, a carcinogenicity study on Glyoxal was conducted in the early eighties by the Bushy Run Research Center (Union Carbide, Pennsylvania, USA, 1982) and was entitled: “Evaluation of the dermal carcinogenicity of AEROTEX Glyoxal 40 and European Glyoxal 40 in male C3H mice”. The data of this study were peer reviewed and published in different acknowledged reviews including CICAD 57 (2004), the Toxicological Dossier on Glyoxal of the EFfCI (Schilling K, 2004), and the BUA Report 187 (1998). Male C3H/HeJ mice randomized into 3 groups of 40 animals each were treated three times weekly (except holidays) throughout their lifetime with a 1:8 aqueous dilution of Glyoxal 40%. In fact, Glyoxal 40% aqueous solution was obtained from two different suppliers and the two test substances were comparatively tested; we refer to them as US Glyoxal 40 and EU Glyoxal 40. Treatment consisted of an application of 25 µL of diluted test substance to the clipped skin of the back of each animal. Taking into account a density of 1.27g/cm3 and a mean body weight for the mice of 25 g, the dosage was calculated to be about 158 mg/kg bw/day referring to the test substance containing ca. 40% Glyoxal, and ca. 63 mg/kg bw/day referring to the active ingredient. Negative control animals were treated with deionized water. The animals were observed for mortality and examined for neoplastic and non-neoplastic skin findings. In addition to examination of the skin, necropsy also included examination of the body cavities. The entire carcasses of all non-autolyzed mice that died after ca. 18 months of treatment were fixed in 10% neutral buffered formalin; sections were prepared and stained for the purpose of histopathological examination. The dorsal skin of all mice, with or without skin lesions, was also fixed in 10% neutral buffered formalin for further examinations. Survival was not affected by the long-term treatment with Glyoxal; in fact, the mean survival time was statistically significantly greater in both treated groups than in the control (580 and 594 days, respectively, versus 488 days). Neither skin neoplasms nor subcutaneous neoplasms were found in the control and the two treated groups. In fact, the only treatment-related finding reported was skin irritation resulting in inflammation and necrosis in some mice treated with US Glyoxal 40. One case of infiltrative fibrosarcoma (left lateral rib cage and axilla, observed after 496 days of treatment) was reported for the EU Glyoxal 40 group; the finding however was without biological significance since this kind of tumour is known from historical control data to occur occasionally in the mouse strain used. This long-term dermal carcinogenicity study had some significant limitations. In fact, the study (1) has been designated as screening study because observations were limited to one gender, (2) was based on a MTD assessment from a ten days dose range finding study, (3) did not comply with current test guideline requirements with respect to the number of animals per dose group and the dose levels, and (4) did not consider systemic toxicity. Despite of these deficiencies, the study is regarded as sufficient to support a conclusion of the lack of carcinogenic potential of Glyoxal by the dermal exposure route. No carcinogenic potential could be evidenced when repeatedly applied to the skin of mice throughout lifetime at a dose level of ca. 63 mg/kg bw/day of active ingredient.