glyoxal … %; ethandial … %

Repeated dose toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

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.

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Glyoxal

EC Number:

203-474-9

EC Name:

Glyoxal

Cas Number:

107-22-2

Molecular formula:

C2H2O2

IUPAC Name:

oxalaldehyde

Test material form:

liquid

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

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.4 mg/m³ air

Doses / concentrations 2

Dose / conc.:

2 mg/m³ air

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

1

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.

2

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.

3

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)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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