glyoxal … %; ethandial … %

Neurotoxicity

Administrative data

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 408 (1998)

Principles of method if other than guideline:

The study was an oral 90-day repeated dose toxicity study conducted according to the OECD TG 408 (1998). The study included neurobehavioural examination and a functional observational battery (FOB).

GLP compliance:

yes (incl. QA statement)

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B62

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (RT), under N2
- Stability under test conditions: the stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer until 10 April 2009

Test animals

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: the mean body weight of males at day 0 was about 190 g; the mean body weight of females at day 0 was about 140 g
- Fasting period before study: no
- Housing: 5 animals per cage, in H-Temp (PSU) cages, floor area 610x435x215 mm (TECHNIPLAST, Germany); for measurement of motor activity, Polycarbonate cages with a floor area of ca. 800 cm2 (EHRET, Emmendingen, Germany)
- Diet (e.g. ad libitum): Ground Kliba mouse/rat maintenance diet “GLP”, meal (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): Drinking water, ad libitum
- Acclimation period: ca. 10 days

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

ANALYSIS OF FOOD, WATER, BEDDING
- The food used in the study was 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 serves 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, serve as the guideline for maximum tolerable contaminants.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

The stability of the test item in drinking water over a period of 4 days was proven prior to the study in a comparable batch (BASF Project No. 08L00341).
For each concentration, the test item was weighed out, and then corresponding amounts of drinking water, depending on dose group, were added to obtain the desired concentrations. Mixing was carried out for about 5 minutes at a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test-item preparations were carried out as a separate study at GKA Competence Center Analytics, BASF SE under the responsibility of the Study Director of this test facility.
Concentration control analyses of the test item preparations were performed in samples of all concentrations at the start and at the end of the administration period.
Homogeneity was given because the test item was completely miscible with water, in terms of a pure solution.

Duration of treatment / exposure:

3 months (i.e. 90 days)

Frequency of treatment:

Daily, continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

200 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

5 000 ppm (nominal)

No. of animals per sex per dose:

The test system comprised 4 test groups; each test group consisted of 20 animals (i.e. 10 animals/sex).

Control animals:

yes, concurrent no treatment

Examinations

Observations and clinical examinations performed and frequency:

MORTALITY AND CLINICAL OBSERVATIONS
Check for moribund and dead animals was made and the animals were examined daily for evident signs of toxicity. For details, see entry in chapter 7.5.1.

DETAILLED CLINICAL OBSERVATION
Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. For details, see entry in chapter 7.5.1.

BODY WEIGHT
Body weight was determined before the start of the administration period, at test initiation, weekly thereafter. For details, see entry in chapter 7.5.1.

FOOD CONSUMPTION
Group food consumption was determined weekly for each cage. For details, see entry in chapter 7.5.1.

WATER CONSUMPTION AND COMPOUND INTAKE
Group water consumption was determined weekly for each cage and the mean daily intake of test item as group means was calculated. For details, see entry in chapter 7.5.1.

OPHTHALMOSCOPIC EXAMINATION
The eyes of all animals were examined prior test initiation and at test ending. For details, see entry in chapter 7.5.1.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS
Blood was taken from the retro orbital venous plexus of the fasted animals in the morning; these samples were used for haematological and clinical-chemical examinations. For details on haematological and clinical chemical parameters, see entry in chapter 7.5.1.
Urine was sampled for urinalysis from the individual animals overnight. For details on urine parameters, see entry in chapter 7.5.1.

Specific biochemical examinations:

No neurotoxicity-relevant biochemical parameters (e.g. cholinesterase activity) were examined.

Neurobehavioural examinations performed and frequency:

NEUROBEHAVIOURAL EXAMINATION, FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
The functional observational battery was carried out in all animals once at the end of the administration period (day 84 to 89). The FOB was performed in all animals at the end of the administration period starting at about 10:00 h. It started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

HOME CAGE OBSERVATION:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behaviour of the animals. Following parameters were considered: posture, tremor, convulsions, abnormal movements, impairment of gait, other findings.

OPEN FIELD OBSERVATION
The animals were transferred into a standard arena (50 x 50 cm with sides of 25 cm high) and were observed for at least 2 minutes. Following parameters were examined: behaviour when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements, impairment of gait, activity/arousal level, faeces (number of faecal pellets/appearance/consistency) within two minutes, urine (appearance/quantity) within two minutes, number of rearing within two minutes.

SENSORY MOTOR TESTS/REFLEX TESTS
The animals were removed from the open field and subjected to following sensorimotor or reflex tests:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behaviour during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hind limbs
13. landing foot-splay test
14. other findings

MEASUREMENT OF MOTOR ACTIVITY (MA)
Motor activity measurements were carried out in all animals at the end of the administration period, on the same day as FOB was performed. The examinations were performed using the TSE Labmaster System (TSE Systems GmbH, Bad Homburg, Germany). For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. 18 beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out starting at 14:00 h. On account of the measuring variant "staggered", the starting time varied according to the time needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements.

Sacrifice and (histo)pathology:

Prior sacrifice, the anesthetized animals were weighed. They were sacrificed by decapitation under anaesthesia (isoflurane). Following exsanguination, the animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
Following organs were weighed: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus and thyroid glands. Both, the absolute and the relative organ weights were considered.

ORGAN/TISSUE FIXATION IN FORMALDEHYDE 4% SOLUTION
Following sacrifice, all organs were examined before and after removal. Any abnormality was noted.
Samples from following organs/tissues as well as gross lesions were collected and fixed in neutral 4% buffered formaldehyde for further histopathological examinations: all gross lesions, salivary glands (mandibular and sublingual glands), oesophagus, 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 and 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, gonads, oviducts, uterus and vagina, epididymides, prostate and seminal vesicle, female mammary gland, skin, skeletal muscle, sternum with marrow, femur with knee joint, extra orbital lacrimal glands.

HISTOPATHOLOGY
The following organ samples of all animals per sex and group of the control-and the 5000 ppm group were processed for histological assessment (i.e. paraffin embedding, sectioning, haematoxylin and eosin staining):
Brain, pituitary, thyroid, parathyroid, thymus, trachea, lungs, pharynx, larynx, nasal cavity, aorta, heart, salivary glands, liver, spleen, kidneys, adrenals, pancreas, testes/ovaries, oviducts/uterus/vagina, epididymides/prostate/seminal vesicle, skin, oesophagus, stomach, duodenum/jejunum/ileum, cecum/colon/rectum, urinary bladder, mesenteric and axillary lymph nodes, female mammary gland, sciatic nerve, femur bone marrow, eyes, all spinal cord.
Gross lesions seen in all groups, also were processed for histopathology.
The haematoxylin-eosin stained slides were examined by light microscopy and assessed. A correlation between gross lesions and histopathological findings was performed.

Statistics:

The tests used for the statistical assessment of the results for the different parameters considered can be summarized as follows:
- Body weight, body weight change: Comparison of each group with control group using DUNNETT's test (two-sided) for the hypothesis of equal means; - Faeces, rearing, grip strength forelimbs, grip strength hind limbs, footsplay test, motor activity, clinical pathology parameters, urine volume, urine specific gravity, weight parameters at necropsy: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- Urinalysis, remaining parameters (except colour): Pairwise comparison of each dose group with the control group using FISHER's exact test for the hypothesis of equal proportions.

Results and discussion

Results of examinations

Details on results:

For details on systemic toxicity findings, see entry in chapter 7.5.1.
Referring to neurotoxicity, all considered parameters (home cage observations, open field observations, sensorimotor tests/reflexes) were inconspicuous. No test item-related or significantly deviations were noted for motor activity measurement.
In fact, deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

5 000 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: The FOB and Motor Activity Measurements were inconspicuous.

Any other information on results incl. tables

The approximate, mean daily intake of test material in mg/kg bw/day over the entire study period was calculated to be as follows:

Test dose group	Test material concentration in the drinking water	Mean daily intake of test material (mg/kg bw/day)
		Males	Females
low	200 ppm	15.0	18.3
mid	1000 ppm	72.0	92.6
high	5000 ppm	290.0	351.7