glyoxal … %; ethandial … %

Endpoint summary

Description of key information

Oral:

The oral toxicity of Glyoxal (40 % aq. sol.) was assessed in rats following repeated administration via drinking water over 28 days (CIT 2619 TSR, OECD TG 407). The LOAEL was 300 mg/kg bw/day and the NOAEL was 100 mg/kg bw/day, referring to the active ingredient. Two 90 days oral studies (drinking water) were conducted according to the OECD TG 408. The NOAEL of the first study with rats (BASF 50S0496/01233) was 1000 ppm, corresponding to 72 mg/kg bw/day in males and 92.6 mg/kg bw/day in females. The second one was conducted with mice (BASF 51S0496/01234), yielding a NOAEL of 1000 ppm, corresponding to 160 and 212 mg/kg bw/day for males and females. The NOAEL value of 72 mg/kg bw/day obtained from the rat study was retained as general NOAEL for the oral repeated dose toxicity. 

Dermal:

Referring to the dermal route of exposure, data from a preliminary test conducted for the purpose of dosage estimation for a main dermal carcinogenicity study are available and were considered as weight of evidence (BRRC 45-508). The study resulted in a NOAEL for systemic toxicity of ca. 125 mg/kg bw/day (highest dose tested) and a NOAEL for local effects (skin lesions) of ca. 63 mg/kg bw/day.

Inhalation:

The subacute inhalation toxicity was conducted in rats of both sexes exposed to Glyoxal aerosol (6 hrs/day, 5 days/week) over 29 days (Hoechst 94.1056, OECD TG 412). This study resulted in a NOAEC of 0.4 mg/m3 air. Local effects were observed in the larynx. Thus, the upper respiratory tract appears to be the main target for Glyoxal under repeated inhalation conditions. As support, the publication of Gamer and coworkers (Gamer et al., 2008) investigating laryngeal epithelial alteration related to inhalation also was considered.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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:

other: OPPTS 870.4300; Combined Chronic Toxicity/Carcinogenicity (August 1998)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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
- Stability under test conditions: stable

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
- Weight at study initiation: no data
- Fasting period before study: no data
- 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
- Acclimation period: planned, duration not specified

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 oral 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 (S)
24 months, main groups

Frequency of treatment:

Daily

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 were calculated;
- The Dunnett test was used for statistical analyses of body weight, body weight change, food consumption, water consumption and food efficiency;
- The KRUSKAL-WALLIS and WILCOXON test was 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 was 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):

With regard to clinical observations, 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

Description (incidence and severity):

In the satellite group (12 month period of treatment) no changes in food consumption were observed. In the main groups food consumption was increased on several time points but no clear dose-response relationship was observed and the findings were assessed as incidental.

Food efficiency:

no effects observed

Description (incidence and severity):

No test substance-related effects on food efficiency were obtained.

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 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 increases were 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. 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. The higher urine volume in males of test group 2 (75 mg/kg bw/d) after the 6th study month were regarded as incidental and not treatment-related.

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:

ca. 25 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP study according OECD TG 453

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

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 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(1981)

Deviations:

yes

Remarks:

; the test period was 29 days instead of 28 as recommended by the guideline. This however did not affect the validity of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(31 July 1992)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 94073/70

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: guaranteed by the re-analysis

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: About 5 to 6 weeks old at beginning of the prestudy period
- Weight at study initiation:
Initial body weight range for the males: 193 – 206 g
Initial body weight range for the females: 171– 192 g
- Fasting: during exposure and at the period in which the animals were kept in diuresis cages (both, feed and water)
- Housing: five animals/cage, Makrolon cages Type 4 with soft wood granulate
- Diet (e.g. ad libitum): Altromin 1324 rat diet (Altromin GmbH, Lage/Lippe), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: ca. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): fully-air conditioned rooms
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Mass median aerodynamic diameter, standard deviation and other parameters of the particle size distribution were calculated using linear regression (Probit values versus the logarithm of the particle size). The measurements were carried out once for each concentration.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE
A defined amount of test material was injected into the nozzle at a constant speed by means of a continuous infusion apparatus. The primary aerosol was formed in a separator and was diluted instantaneously with moisturised air in order to prevent changes in the concentration of the aerosol particles. Smaller aerosol particles (secondary aerosol) passed through a rising tube into the exposure chamber. The total amount of test atmosphere generated was 1100 litres per hour.
A suction device at the bottom of the exposure chamber drew off the test atmosphere through a gas cleaning equipment at a rate of 1100 litres/hour.

EXPOSURE CHAMBER
The rats were placed individually in cylindrical plastic tubes and exposed to defined aerosol concentrations. The plastic tubes leading into the exposure chamber were arranged such as only the noses of the animals were inside the chamber. The exposure chamber itself consisted of a stainless-steel and glass cylinder with a volume of 80 litres, standing in a vent pipe with a volume of approx. 4 m3. The chambers were shown to yield a uniform distribution of aerosols in the different breathing zones of the animals.

PARTICLE SIZE DETERMINATION
Determination of the particle size distribution was performed with an Anderson 7 stage cascade impactor (U.S.A.). The test atmosphere was impacted at each stage onto steel discs which were weighed before and after sampling. Sampling was performed at a volume rate of 9.5 l/minute, resulting in a flow velocity of 1.25 m/s. Total volume sampled was 2850 litres in the 0.4 mg/m3 air group, 2708 litres in the 2.0 mg/m3 air group and 1140 litres in the 10.0 mg/m3 air group, respectively.
The aerodynamic diameters were measured in the following ranges:
< 0.6 micrometer
0.6 – 0.8 µm
0.8 – 1.5 µm
1.5 – 3.0 µm
3.0 – 4.8 µm
4.8 – 7.0 µm
7.0 – 10.3 µm

MONITORING OF CO, CO2, O2, TEMPERATURE AND HUMIDITY
During the exposures CO, CO2, O2 in all exposure chambers were measured continuously by means of air-monitoring equipment manufactured by Hartmann & Braun.
Likewise, atmospheric humidity and temperature in the exposed groups were checked continuously by alternatively measuring the individual chambers. Measurement of temperature was performed via the CMR-transformer TEU 320 and humidity was checked by means of the
transmitter HMT12 manufactured by VAISALA.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- All samplings were performed by replacing a lid of the chamber (otherwise closed) with the sampling equipment and thus represent the conditions of the breathing zone of the animals.
- The concentration of Glyoxal 40 in the high concentration group was measured gravimetrically at intervals of approximately 30 minutes. For this purpose, the test atmosphere was drawn off by vacuum (through a rotary gas counter, a glass fibre filter and a membrane filter with a pore width of 0 .65 μm). The extraction rate was 3 litres/minute, resulting in a flow rate of 1 .25 m/sec. The filters were weighed before and after sampling by means of an electronic balance (Mettler AE 163, Mettler GmbH, Gießen).
- In the intermediate concentration group, Glyoxal 40 was monitored continuously via an aerosol photometer whereas in the low dose group, no monitoring was possible due to the low mass.
- Analytical examinations for determination of the amount of test material present in the exposure chambers were done. Therefore, 31 litres of the test atmosphere were conducted through 3 washing flasks filled with deionised water and then through a rotary gas counter. The test material was isolated by HPLC and determined spectrophotometrically .

Duration of treatment / exposure:

29 days

Frequency of treatment:

6 hours/day, 5 days /week, total of 20 exposures

Dose / conc.:

0.4 mg/m³ air

Dose / conc.:

2 mg/m³ air

Dose / conc.:

10 mg/m³ air

No. of animals per sex per dose:

Five animals per sex and group were exposed

Control animals:

yes, sham-exposed

Details on study design:

- The test concentrations in the present study were selected on the basis of a preliminary, range-findings study.
- At the beginning of the acclimatisation period, the 40 animals were randomised and assigned to the different groups.
- Each animal received 20 exposures of 6 hours each.

Observations and examinations performed and frequency:

CLINICAL SYMPTOMS, MORTALITY. NEUROLOGICAL DISTURBANCES:
The animals were observed for behaviour and state of health twice a day on working days (before and after exposure), once a day on Saturdays, Sundays and public holidays. During the 6 hours of exposure, the animals were subjected to continuous observation.
Moreover, the animals were examined weekly for neurological disturbances, damage to the oral mucosa and impairment of dental growth.

BODY WEIGHT
The animals were weighed twice weekly throughout the study.

FOOD CONSUMPTION
Food consumption was measured twice a week throughout the study. Food consumption data were expressed as g/100 g bw/day.

WATER CONSUMPTION
Water consumption was measured once a week over a period of 16 hours. Water consumption data were expressed as ml/animal/day.

OPHTHALMOSCOPIC EXAMINATION
The animals were examined weekly for opacity of the refracting media of the eyes.

HAEMATOLOGY AND CLINICAL CHEMISTRY
At the end of the treatment period, blood samples were collected from all non-fasted animals. These samples were used for haematological and clinical-chemical examinations.
Following haematological parameters were considered:
Haematocrit, haemoglobin (HB), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular haemoglobin (MCH), erythrocyte count, white cell count (WBC), differential white cell count, coagulation time, thromboplastin time (PT), activated partial thromboplastin time (APTT) , mean corpuscular volume (MCV).
Reticulocyte count and Heinz bodies were scored in the animals of the control group and the high concentration group only
Following clinical chemical parameters were considered:
Sodium, potassium, glucose, chloride, calcium, inorganic phosphorus, blood urea, uric acid, creatinine, total cholesterol, triglycerides, total lipids, total proteins, albumin, total bilirubin, alkaline phosphatase (AP), alanine aminotransferase (ALAT/GPT), aspartate aminotransferase (ASAT/GOT), Gamma-glutamyltransferase (GGT).

URINALYSIS
Urine was sampled from the animals for a 16 hour period (overnight), from day 25 to 26. Following parameters were considered:
Appearance, color, volume, specific gravity, pH, haemoglobin, protein, glucose, ketones, bilirubin, nitrites.
Sediment was scored in the animals of the control group and the high concentration group only.

Sacrifice and pathology:

The animals were sacrificed at the end of the 29 day exposure period for the purpose of necropsy. Body weights were recorded before exsanguination.

ORGAN WEIGHING
Following organs were weighed: heart, lung, liver, kidneys, spleen, epididymes, adrenals and gonads. Both, the absolute and the relative organ weights were considered.

GROSS PATHOLOGY AND HISTOPATHOLOGY:
Following sacrifice, all superficial tissues (skin, eyes, teeth, oral mucosa) and natural orifices were examined as were the internal organs before and after removal. Any abnormality in size or appearance was noted.
Samples from following organs/tissues as well as gross lesions were collected and fixed in an appropriate fixative (probably 10% buffered formalin) for further histopathological examinations:
Heart, adrenals, liver, kidneys, stomach, trachea, nasal cavity, lung, nasopharynx, larynx, epididymes, spleen, ovaries and testes.

Statistics:

The following parameters were compared statistically with the control group values at the level of significance p = 0.05:
Body weights at the designated measurement times
Haematological data
Clinical chemical data
Urine data
Absolute and relative organ weights

The statistical assessment of findings almost was based on the one-way analysis of variance with sequentially rejective multiple comparisons,
The evaluation was performed by Pharma Research and Development Informatics with the aid of a program package for the evaluation of toxicological studies.

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related symptoms of toxicity were seen. In fact, blood-colored encrusted nose was the only reported symptom and was observed in all groups including the control group.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain were not impaired by the repeated treatment with the test substance aerosol.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption remained unaffected by the treatment throughout the study and was comparable in all groups. The amount of food consumed ranged between 8.96 - 9.13 and 8.53 - 8.91 mg/100 g bw/day for males and females, respectively, for all groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Water consumption remained unaffected by the treatment throughout the study and was comparable in all groups. The amount of water consumed ranged between 29.76 - 33.28 and 24.96 - 27.40 mL/animal/day for males and females, respectively, for all groups.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No opacity of the refracting media of the eyes was seen.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed. In fact, Haematological examinations revealed statistically significant decreases in erythrocyte counts in females of the high concentration group. However, these changes were observed in one sex only and the values were within the physiological range of rats. Furthermore, there were no signs indicative for anaemia observable by other parameters and by histopathological examination. Therefore, the findings were not considered to be treatment-related. All other parameters were inconspicuous.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed. In fact, statistically significant decreases in potassium and inorganic phosphate levels were reported for the females of the high concentration group; the changes however were slight and could not be evidenced in males. These findings were therefore rather incidental than treatment-related.
Furthermore, following statistically significant changes were reported: decreases in GOT values in males of the mid concentration group, increases in bilirubin levels in females of the mid concentration group, and increases in creatinine values in females of the low concentration. As no dose-relationship was evident for these findings, they were not considered to be treatment-related. All other parameters were inconspicuous.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed. In fact urine volume was increased to a statistically significant degree in males of the low concentration group; additionally, pH-values were statistically significantly decreased in males of all concentration groups. As there was no dose-dependency, the findings were not considered to be treatment-related. All other parameters were inconspicuous.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Statistical evaluation of absolute and relative organ weights did not reveal any difference between the control and treatment groups.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Description (incidence and severity):

No neurological disturbances, opacity of the refracting media of the eyes, impairment of dental growth or changes in the oral mucosa were observed.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examinations revealed minimal squamous metaplasia of the cuboidal epithelium of the epiglottis in the larynx of all animals from the high concentration group, as well as in most animals of the mid concentration group. In addition, some animals showed submucosal infiltration with lymphoid cells. In one female of the high concentration group, focal epithelial necrosis with moderate inflammation was seen in the same area. In the animals from the low concentration group, no comparable alterations were observed.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

0.4 mg/m³ air

Sex:

male/female

Basis for effect level:

other: (measured: 0.0006 mg/L); based on histopathological findings in the upper respiratory tract.

Key result

Dose descriptor:

LOAEC

Effect level:

2 mg/m³ air

Sex:

male/female

Basis for effect level:

other: (measured: 0.0023 mg/L); based on histopathological findings in the upper respiratory tract.

Key result

Dose descriptor:

NOAEC

Remarks:

(systemic toxicity)

Effect level:

10 mg/m³ air

Sex:

male/female

Basis for effect level:

other: (measured: 0.0089 mg/L)

Critical effects observed:

not specified

Results of the particle size distribution analysis:

The results of the analysis indicate that the aerosol was well-respirable to the test animals( 99.7% of particles < 3 µm); for details see table below.

Test group	Particle size
	3 µm	1 µm	MMAD (µm)	GSD
Group 2 (0.4 mg/m^3)	99.7%	55.1%	0.95	1.52
Group 3 (2.0 mg/m^3)	99.7%	67.7%	0.80	1.63
Group 4 (10.0 mg/m^3)	96.6%	39.0%	1.16	1.68
MMAD = Mass Median Aerodynamic Diameter; GSD: Geometric Standard Deviation

MONITORED PARAMETERS IN THE EXPOSURE CHAMBERS:

All measured parameters (O2, CO, CO2, temperature and humidity) were inconspicuous and in the expected range

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.4 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study according OECD TG 412

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

The present data are from a preliminary test conducted for the purpose of dosage estimation for a main dermal carcinogenicity study (see 7.7). The data are available as secondary peer-reviewed quotation, reported in following acknowledged reviews, the CICAD 57 (2004) and the Toxicological Dossier of Glyoxal of the EFfCI (2004).

Qualifier:

no guideline followed

Principles of method if other than guideline:

The present study was a preliminary test conducted for the purpose of dosage estimation for a main dermal carcinogenicity study reported in chapter 7.7.

GLP compliance:

no

Remarks:

GLP was not compulsory at the time the study was conducted

Limit test:

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)
- Impurities (identity and concentrations): 0.1% acid (calc. as acetic), 0.7% formaldehyde, 0.2% glycolaldehyde, traces of ethylene glycol (second batch)

Species:

mouse

Strain:

C3H

Sex:

male

Type of coverage:

not specified

Vehicle:

water

Details on exposure:

-Each substance was tested as 1:4 and 1:8 dilutions in distilled water. 
-Taking into account a density of 1.27g/cm3 and a mean body weight for the mice of 25 g, the dosage for the 1:4 dilution was calculated to be about 317 mg/kg bw/day referring to the test substance containing ca. 40% glyoxal, and ca. 125 mg/kg bw/day referring to the  active ingredient as such. 
- For the 1:8 dilution, the dosage was about 158 mg/kg bw/day referring to the test substance containing ca. 40% glyoxal, i.e. ca. 63 mg/kg bw/day referring to the active ingredient.

Duration of treatment / exposure:

2 week

Frequency of treatment:

each animal received one application/day, and a total of 10 applications.

Dose / conc.:

63 mg/kg bw/day

Remarks:

referring to the active ingredient; 1:4 dilution of each test substance in distilled water

Dose / conc.:

125 mg/kg bw/day

Remarks:

referring to the active ingredient; 1:8 dilution of each test substance in distilled water

No. of animals per sex per dose:

Five animals per group were used

Control animals:

yes, concurrent no treatment

Details on study design:

Male C3H /HeJ mice were randomized into 5 groups of 5 animals each and received a series of 10 applications of diluted test substance onto the clipped skin of the back throughout a period of 2 weeks. The application  volume was 25 µL. No further details were provided. Negative control animals received similar treatment with distilled water alone.

Observations and examinations performed and frequency:

- The animals were observed and examined for mortality, health state and skin lesions. 
- Body weight was recorded at  test initiation and thereafter,following the first, third, fourth, sixth, eighth, ninth and tenth application; a semi-automatic weighing system was used. Body weight changes were assessed statistically.

Sacrifice and pathology:

After the last application, all animals were sacrificed for the purpose of necropsy.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Both substances tested as 1:4 dilutions in water resulted in open lesions of the skin; no such lesions were seen in the animals treated with the 1:8 dilutions of both test substances.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 63 mg/kg bw/day

Sex:

male

Basis for effect level:

other: referring to skin lesions

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 125 mg/kg bw/day

Sex:

male

Basis for effect level:

other: referring to systemic toxicity

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

dose finding study

Additional information

Oral

The oral administration of Glyoxal 40% via the drinking water at dose level of 0 mg/kg bw/day (test group 0), 25 (test group 1), 75 (test group 2) and 300 mg/kg bw/day (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. With regard to clinical observations, 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/day) were assessed as being related to the test compound administration. Differences observed at lower dose levels were not assessed as being of toxicological relevance. Relevant changes in water consumption were observed for these test groups most likely related to the taste of the test item. No changes in food consumption were obtained. Vasodila in fundus oculi was observed only in 8 female animals of test group 3S (300 mg/kg bw/day) and in 6 female animals of test group 2S (75 mg/kg bw/day) 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. Regarding clinical pathology, a decreased ALT activity in male and female rats of test group 3 (300 mg/kg bw/day) was observed. Neither any other clinical pathology alteration indicating a microsomal enzyme induction nor any relevant liver weight increase were 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/day) and, additionally, in males of test group 2 (75 mg/kg bw/day) 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. The reason for the urinary excretion of higher keton body and urobilinogen levels in males could not be elucidated. Regarding pathology, the treatment with Glyoxal 40% caused decrease of terminal body weight in males of test group 3S (300 mg/kg bw/day) of the satellite group of -8%. Furthermore, males and females of the test group 3 (300 mg/kg bw/day) 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. 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/day) 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/day) and test group 3 (300 mg/kg bw/day) were regarded to be substance related and adverse in nature. Besides the above mentioned adverse findings, there were no other substance-related organ weight changes, gross lesions, and non-neoplastic findings in male and female Wistar rats after 12 and 24 months of treatment 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 [BASF, 2012].

Daily administration of Glyoxal (40% in drinking water) to rats for over a period of 28 days at dose levels of 100, 300 and 1000 mg/kg bw/day (CIT 2619 TSR; 1987) resulted in a dose-related decrease in water consumption, decrease in food consumption and in a decrease in body weight gain at the mid and high dose levels. In the 1000 mg/kg bw/day group, body weight gain was statistically significantly reduced for both, males and females when compared to controls. In the 300 mg/kg bw/day group, body weight gain was reduced for both, males and females when compared to controls; the differences from control however were not statistically significant. At dose levels of 300 and 1000 mg/kg bw and as a consequence of the decreased water uptake, changes in haematological and clinical-chemical parameters were also seen, which consisted of a slight statistically significant increase in red blood cell count for the males of the high dose group, and a statistically significant increase in inorganic phosphorus in males of the mid and high dose groups and in females of the high dose group. Urinalysis findings which consisted of decrease in the urinary volume in males (statistically significant) and females (not statistically significant) from the mid and high dose groups and increase in specific gravity also were seen as resulting from the decrease in water consumption. Necropsy revealed no treatment-related abnormalities. For almost all findings, male animals were more sensitive than the females. The LOAEL was 300 mg/kg bw/day and the NOAEL was 100 mg/kg bw/day [CIT, 1987]. The administration of Glyoxal (40% in drinking water) to male and female Wistar rats for 90 days caused signs of general systemic toxicity at the highest tested dose level of 5000 ppm (BASF 50S0496/01233; 2009). The treatment-related effects consisted of impaired body weight and several changed parameters in clinical pathology. No test-item related findings were observed in mid dose (1000 ppm) and low dose (200 ppm) groups of either sex. Body weight was significantly decreased in both sexes. The clinical-chemical/enzymatic changes consisted of decreased alanine aminotransferase (ALT) values in rats of both sexes, increased urea values in rats of both sexes, decreased globulin and total protein values in females, increased inorganic phosphate levels in males, increased incidences of higher urine ketone body and urobilinogen levels in males, and increased incidences of higher urine protein levels in females. All other considered parameters were unaffected or showed incidental changes which were not treatment-related. The FOB and Motor Activity Measurements were inconspicuous. Thus, the no observed adverse effect level (NOAEL) was 1000 ppm in male and female Wistar rats, corresponding to 72 mg/kg bw/day in males and 92.6 mg/kg bw/day in females [BASF, 2009]. The administration of Glyoxal (40% in drinking water) to male and female C57BL mice for 90 days (BASF 51S0496/01234; 2009) resulted in treatment-related effects at a dose level of 5000 ppm, which corresponded to a daily intake of 684 and 933 mg/kg bw/day for males and females. The treatment-related effects included decreased albumin, globulin and total protein as well as cholesterol levels in males, and reduced urea and magnesium levels in females. A treatment-related decrease in water consumption was reported for males and females of the high dose group (5000 ppm); however the finding appears to be related to a palatability problem rather than reflecting a real toxicologically relevant adverse substance-related effect. No treatment-related effects were reported for the low and mid-dose levels (200 and 1000 ppm). Therefore, under the conditions of the present study, the no observed adverse effect level (NOAEL) was 1000 ppm in male and femaleC57BL/6 J Rj mice (160 and 212 mg/kg bw/day for males and females) [BASF, 2009]. The results reported above are supported by a series of validated and peer-reviewed data on repeated oral exposure studies (Ueno et al., 1991).

Two 90-day feeding studies with Wistar rats and with Beagle dogs receiving Glyoxal (40%) in the diet have been conducted (Mellon Institute 29-1). The peer reviewed data were also reported in CICAD 57 (2004) and in the Toxicological Dossier of Glyoxal of the EFfCI, (2004). In the rat study, 10 animals/sex/group were exposed to the following dosages referring to 100% a.i.: 32, 63, 125, and 250 mg/kg bw/day. It was reported that the male animals of the high-dose group showed a reversible significant retardation of body weight gain during the first 2 weeks of exposure without a concomitant reduction of food intake. Significant increases of liver and kidney weights were observed in the high-dose group; no other organs were weighed. No relevant macroscopic or histopathological changes were observed in thoracic and abdominal organs. Haematological and biochemical parameters were not analysed. From these investigations, a NOAEL of 125 mg/kg bw/day of active ingredient was estimated for the rat. In the dog study, 3 animals per group were exposed to the same preparation of Glyoxal by feeding dosages of 31, 65, or 115 mg/kg bw/day (dosages corresponding to 100% Glyoxal). Up to the high dosage, no substance-related changes of body weight, food consumption, liver or kidney weights, or haematological or serum clinical chemistry parameters and no macroscopic or histopathological changes were observed in thoracic and abdominal organs (pancreas not examined). Thus, the NOEL for 90-day feeding of Glyoxal to dogs was >115 mg/kg bw/day (dosage corresponding to 100% Glyoxal) [Mellon Institute, 1966].

In a further study, Fischer-344 rats were exposed daily to drinking water containing either 0, 1, 2, 4, 8, or 16 mg/mL of glyoxal for approximately 90 days. Decreased dose-related body and organ weights, decreased food and water consumptions, and moribundity of the animals in the 16 mg/mL dose group on Day 12 were observed. Possible chemical-related histopathological changes observed were minimal hemorrhages of the mesenteric lymph nodes (males and females) and mild to moderate hyperplasia of the mandibular lymph nodes (males). In the male rats of the 8 and 16 mg/mL dose groups, moderate salivary gland atrophy and mild to minimal kidney changes (i.e., swelling and vacuolization of the papillary duct epithelium, with renal tubular dilitation) were also observed. Hypospermia and atypical cells (males) and thymic atropy (females) were seen in the animals of the 16 mg/mL dose group. The above histopathological changes were considered equivocal, and may represent changes secondary to the cachectic state of the animals due to decreased water consumption.The absence of definitive histological or chemical-related clinical observations makes dose setting for the chronic study uncertain.Water intake appears to be the only criteria for establishing dose levels. Based on the data in this study, it would appear that female Fischer-344 rats show few ill effects when water consumption is decreased by as much as 46%. Male Fischer-344 rats, however, appear to be more sensitive than the females with a weight gain of only 90% of control values when water consumption is decreased by 40%. Recommended chronic study dose levels for the female Fischer-344 rats are 1.0, 2.0 and 4.0 mg/mL and for the male Fischer-344 rats are 0.5, 1.0 and 2.0 mg/mL. These dose levels should result in the "maximum tolerated dose" being tested, while producing dose-response relationships for carcinogenicity interpretation. Long-term effects of a decreased water consumption in the range of 35-45% are unknown (Southern Research Institute, 1992).

In another study, B6C3F1 mice were exposed daily to drinking water containing 0, 1, 2, 4, 8, or 16 mg/mL of glyoxal for approximately 90 days. The salient features observed were decreased body and selected organ weights, decreased feed and water consumptions, and possible chemical-related salivary gland changes in the male mice of all dose groups. It was felt that the decreased water consumptions were due to unsatisfactory palatability of the dosed water, subsequently leading to lower daily doses and decreased feed consumptions.With only salivary gland changes occurring in the male mice at all dose levels, the absence of definitive histopathological or chemical-related clinical observations makes dose setting for the chronic study uncertain. Body weight and water intake appear to be the only criteria for establishing dose levels. Based on the data in this study, female B6C3F1 mice show few ill effects when water consumption is decreased by as much as 27%. Male B6C3F1 mice appear to be more sensitive than the females, with a weight gain of only 88% of control values when water consumption is decreased 26%. It is felt that the salivary gland changes noted in the male mice would not be life-threatening and would not affect the mortality outcome in a chronic study. Recommended chronic study dose levels for the female B6C3F1 mice are 1, 2, and 4 mg/mL and for the male B6C3F1 mice are 0.5, 1, and 2 mg/mL. These dose levels should result in the "maximum tolerated dose" being tested, while producing dose-response relationships for carcinogenicity interpretation. Long-term effects of decreased water consumption in the range of 12-27% are unknown (Southern Research Institute, 1992).

Dermal

Data from a preliminary test conducted for the purpose of dosage estimation for a main dermal carcinogenicity study are available and were considered as weight of evidence (BRRC 45-508). A NOAEL for systemic toxicity of ca. 125 mg/kg bw/day and a NOAEL for local effects (skin lesions) of ca. 63 mg/kg bw/day could be established. A long-term dermal carcinogenicity study conducted with mice (BRRC, 1992) was peer-reviewed and reported in the Concise International Chemical Assessment Document of the International Program of Chemical Safety of the WHO, IPCS (CICAD 57, 2004), and in the Toxicological Dossier of Glyoxal of the European Federation for Cosmetic Ingredients, (EFfCI, 2004). In this study, mice were treated over their entire life span with either US or EU Glyoxal 40 (1:8 dilution), i.e. ca. 158 mg/kg bw/day Glyoxal 40% and ca. 63 mg/kg bw/day a.i. The treatment with US Glyoxal 40 caused skin irritation resulting in inflammation and necrosis in some mice; no such effect was seen with EU Glyoxal 40. The study however was found to have significant limitations. Despite of these deficiencies, the study is regarded as sufficient, however, to support a conclusion of the lack of carcinogenic potential by the dermal exposure route. But restriction of dermal contact is expected due to the sensitising properties of the test substance.

Inhalation

A systemic toxicity of Glyoxal aerosol after repeated inhalation exposure could not be evidenced in a subacute, 29 days study conducted with rats subjected to test concentrations up to 0.01 mg a.i./L air (Hoechst 94.1056, Hoechst 94.0246, range-finder). In fact, local effects were observed in the larynx, that consisted of minimal squamous metaplasia of the epiglottal epithelium with minimal submucosal lymphoid cell infiltration; the NO(A)EC was 0.0004 mg/L air. The reported effects are indicative of an adaptive change of the epithelium because of repeated irritation. Thus, the upper respiratory tract appears to be the main target for Glyoxal under repeated inhalation conditions, and it can be assumed that systemic toxicity only plays a secondary role.

No long-term inhalation studies are available. However, the short-term repeated dose inhalation study gives a good idea of the main target of Glyoxal aerosol in case of repeated inhalative exposure and clearly showed that repeated inhalation exposure to Glyoxal aerosol implies the risk of local effects in the larynx.

A risk from repeated inhalation exposure could take place in situations where Glyoxal is released as an aerosol. Such an exposure situation might occur under conditions where products containing Glyoxal are applied by spray; however, the production of an aerosol would then depend on the dispersion energy used for spraying. Since handling with care and with respect to the official Annex I classification for Glyoxal is expected, an inhalation risk can be ruled out.                                                    

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 repeated dose toxicity.

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No 1272/2008. Repeated inhalation exposure to the aerosol of the test item implies the risk of local effects in the larynx. Therefore, classification for STOT SE Cat.3 (H335 ”May cause respiratory irritation”) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776, is warranted.