Glutaral

Neurotoxicity

Description of key information

The systemic toxicity and the neurotoxic potential of glutaraldehyde 50% were investigated in rats following repeated administration via drinking water over a period of 3 months (BASF, 2001). In fact, the study comprised a 90 day-repeated toxicity assessment according to OECD test guideline 408 and a neurotoxicity assessment using a neurotoxicity screening battery according to EPA test guideline OPPTS 870.6200. The study conduct followed GLP. No neurotoxic potential could be evidenced for glutaraldehyde. Moreover and because of the restricted bioavailability of glutaraldehyde, no exposure of the central and/or peripheral nervous system to glutaraldehyde has to be expected.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

Reference

Endpoint:

neurotoxicity: sub-chronic oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23.03.1999 - 08.11.2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study conducted in accordance with GLP; in fact, the study comprised a 90 day-repeated toxicity assessment according to OECD test guideline 408 and a neurotoxicity assessment using a neurotoxicity screening battery according to EPA test guideline OPPTS 870.6200. Due to an error in the protocol, no ophthalmological examinations were conducted prior starting the treatment; in fact, ophthalmological examinations only were performed at the end of the treatment period. However, as no treatment-related effects were seen, the validity of the study was not affected by this deviation.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 408 (1981)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.6200 (Neurotoxicity Screening Battery)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Physical state: homogeneous colourless-clear liquid
- Analytical purity: 50.5% (w/w); method: HNMR spectroscopy, analysis performed by BASF AG, the Analytical Department, Ludwigshafen/Rhein, Germany, Report 99L00151, 1999)
- Composition of test material, percentage of components: 50.5% and 49.5% water
- Lot/batch No.: 50-4402,
- Stability under test conditions: confirmed by reanalysis at the end of the experimental period; the reanalysis revealed 50.3% a.i. (w/w).
- Storage: Refrigerator (4°C -10 °C), under N2

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Boehringer Ingelheim Pharma KG
- Age at study initiation: 49 days
- Weight at study initiation:
Body weight for the males, group mean: 251.6 g (228.9 - 272) g
Body weight for the females, group mean: 169.4 g (152.8 – 187.5) g
- Housing: individual housing in type DK 111 stainless steel wire cages from Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding, supplied by SSNIFF, Soest, Germany).
- Diet (e.g. ad libitum): Kliba rats/mice/hamsters maintenance diet (meal, Provimi Kliba SA, Kaiseraugst, Switzerland) offered ad libitum except during motor activity measurements, functional observational batteries and during fasting periods.
- Water (e.g. ad libitum): drinking water water available ad libitum, except during motor activity measurements and functional observational batteries.
- Acclimatization: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): completely air-conditioned room
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance was administered as aqueous solution in the drinking water. The test substance was weighed in, depending on the dose group, then drinking water was filled up to the desired weight and mixed with a magnetic stirrer. The drinking water solutions were
prepared twice a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration control analyses of the test substance preparations were performed with samples of all concentrations drawn before the start, in the first week of administration as well as at end of the administration period. The latter samples were taken 4 days after the test substance preparation in order to cover the maximum of storage duration (the stability in drinking water over a period of 4 days at room temperature was guaranteed).

Duration of treatment / exposure:

90 day(s)

Frequency of treatment:

7 days/week

Dose / conc.:

100 ppm (nominal)

Remarks:

in water;
Males: 6.1 mg/kg bw/day of test material (ca. 3 mg a.i./kg bw/day) actual ingested
Females: 8.2 mg/kg bw/day of test material (ca. 4 mg a.i./kg bw/day) actual ingested

Dose / conc.:

500 ppm (nominal)

Remarks:

in water;
Males: 29.9 mg/kg bw/day of test material (ca. 15 mg a.i./kg bw/day) actual ingested
Females: 38.5 mg/kg bw/day of test material (ca. 19 mg a.i./kg bw/day) actual ingested

Dose / conc.:

2 000 ppm (nominal)

Remarks:

in water;
Males: 106.9 mg/kg bw/day of test material (ca. 53 mg a.i./kg bw/day) actual ingested
Females: 144.4 mg/kg bw/day of test material (ca. 4 mg a.i./kg bw/day) actual ingested

No. of animals per sex per dose:

Each dose group comprised 15 animals per sex.

Control animals:

yes, concurrent no treatment

Details on study design:

The test substance was administered as aqueous solution in the drinking water. 0, 100, 500 and 2000 ppm; the animals were treated daily, 7 days per week, over a period of 90 days. Each dose group comprised 15 animals per sex and was subdivided into 3 sections as follows:
- Section A comprised the first 5 animals of each sex and group;
- Section B comprised the further 5 animals of each sex and group;
- Section C comprised the remaining 5 animals of each sex and group.
The animals of section A and B were subjected to the functional observational batteries (FOB) and the motor activity assessment (MA); The animals of section A were sacrificed at the end of the experimental period and were then perfusion-fixed for neuropathological examination. The animals of section B and C were subjected to urinalysis and ophthalmological examination, and after sacrifice, they were subjected to necropsy and blood examination.

Observations and clinical examinations performed and frequency:

- The rats were checked twice daily for mortality from Mondays to Fridays, and once daily on Saturdays, Sundays and public holidays.
- The rats were checked twice daily for clinical signs of toxicity from Mondays to Fridays, and once daily on Saturdays, Sundays and public holidays; general clinical examinations were furthermore carried out daily.
- The body weight of the rats was determined prior to the first neurofunctional test for randomized distribution of the animals in the test groups. This parameter was again determined at test initiation (day 0) and thereafter once a week. A further body weight measurement was done on the days when functional observational batteries (FOB) were conducted. The body weight parameter was expressed as body weight change.
- The food consumption (as grams/animals/day) of the rats was determined once a week during the experiment.
- The food efficiency was calculated on the basis of the body weight and the food consumption on days where both parameters were determined simultaneously.
- Water consumption of the rats was determined once a week over a period of 4 days, and was calculated as mean water consumption in grams per animal per day.
- The mean daily intake of test substance (group mean) was calculated on the basis of water consumption.
- Referring to the ophthalmological examinations, due to an error in the protocol, no such examinations were conducted prior starting the treatment but only at the end of the treatment period. However, as no treatment-related effects were seen, the validity of the study was not affected by this deviation. The eye examination was done in the control and the high-dose group.
- Referring to haematology, following parameters were considered: leukocyte count (WBC; peroxidase method), erythrocyte count (RBC; flow cytometric laserlight scattering), hemoglobin (HGB; cyanmethehoglobin method), hematocrit (HCT; calculated on the basis of MCV and RBC), mean corpuscular volume (MCV; RBC/PLT method), mean corpuscular hemoglobin (MCH; calculated on the basis of hemoglobin/ erythrocytes), mean corpuscular hemoglobin concentration (MCHC; calculated on the basis of hemoglobin/hematocrit), platelet count (PLT; flow cytometric laserlight scattering), differential blood count (cytochemistry coupled with flow cytometry). Clotting analysis was performed and the prothrombin time (Hepato Quick´s Test; HQT) was determined (citrated blood with calcium thromboplastin method).
- Referring to clinical chemistry, following parameters were considered: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-gamma-glutamyl transferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulina, triglycerides, cholesterol and magnesium.
- Referring to urinalysis, following parameters were considered: volume, colour, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity and sediment.

Specific biochemical examinations:

None

Neurobehavioural examinations performed and frequency:

FUNCTIONAL OBSERVATIONAL BATTERY
The animals were subjected to functional observational batteries (FOB) as well as to motor activity measurements. Five animals per sex and group were examined for neuropathology. FOBs were conducted on all animals of the section A and B at defined time points (see study design). The FOB consisted of following 4 steps:

(1) passive observation of the animals without disturbing them:
The animals were observed for posture, tremor, convulsions, abnormal movements, impairment of gait, general state.

(2) open field observations in a standard arena following removal of the rats from their home cage:
Attention was given to behaviour out of the cage, fur, skin, posture, salivation, respiration, activity/arousal level, tremor, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (number of pellets produced within 2 minutes, appearance and consistence), urine (volume and color, 2 min.) and number of rearings within 2 minutes.

(3) sensorimotor tests and (4) reflex tests:
These tests included a series of responses, such as the approach response, the touch response, the visual placing response, the startle response and the righting response. Furthermore the animals were checked for their pupillary, winking and pinna reflexes. The grip strength of the fore- and hind limbs was tested as well as the pain perception (tail pinch). The animals were furthermore observed for vocalisation, olfaction and catalepsy, and they were subjected to the landing foot-splay test.

LOCOMOTOR ACTIVITY
The animals were subjected to motor activity measurements.
Motor activity assessment was conducted in parallel to FOB on all animals of the A and B sections. The measurements were conducted in polycarbonate cages with wire covers from Ehret (Emmendingen, Germany) and a floor area of about 800 cm2, in the dark. Measurements were based on a Multi-Varimex-system with 4 infrared beams per cage. The number of beam interrupts was assessed over 12 intervals of 5 minutes each (total: 60 minutes).

Sacrifice and (histo)pathology:

GROSS PATHOLOGY
The animals of the section A were sacrificed by perfusion fixation (4% buffered formaldehyde solution).
The animals were subjected to gross pathology; thereafter a series of organs/tissues was removed including the brain, which was first weighed (without olfactory bulb) prior any further preparation.
Brain, eyes with optic nerve, spinal cord (cervical and lumbar) one hind limb and all gross lesions were sampled and preserved in fixative.

HISTOPATHOLOGY
A series of tissue samples was processed for histopathology by plastic embedding; for details, see table 1 below.
A series of tissue samples was processed for histopathology by paraffin embedding; for details, see table 2 below.

Statistics:

Statistical assessment of grip strength (forelimbs, hindlimbs), landing foodsplay test, motor activity: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided) followed when necessary by the Mann-Whitney U-test (two-sided);
Statistical assessment of organ weight data: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided), followed when necessary by the Wilcoxon test.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observational batteries (FOB):
Only incidental effects were seen, which clearly were not treatment-related as they either were equally distributed between treated and control animals or occurred in single animals only.

Motor activity assessment (MA):
Only incidental, not treatment-related deviations from controls were observed. These sometimes statistically significant deviations occurred isolated and/or showed no dose-response relationship.

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no statistical significant changes in brain weight (see table below). Neither gross lesions nor treatment-related histological abnormalities were found.

Dose descriptor:

NOAEL

Remarks:

neurotoxicity

Effect level:

2 000 ppm (nominal)

Sex:

male/female

Basis for effect level:

other: No tretatment-related neurotoxicity observed up to the highest tested dose.

Remarks on result:

other:

Remarks:

Males: 106.9 mg/kg bw/day of test material (ca. 53 mg a.i./kg bw/day) , Females: 144.4 mg/kg bw/day of test material (ca. 4 mg a.i./kg bw/day)

The absolute brain weights:

Males (N=5/group)
Dose		Control	100 ppm	500 ppm	2000 ppm
Brain weight (g)	Mean	2.144	2.124	2.06	2.148
	SD	0.102	0.064	0.097	0.101
*, p<0.05; **, p<0.01

Females (N=5/group)
Brain weight (g)	Mean	1.924	1.856	1.906	1.87
	SD	0.086	0.066	0.066	0.107
*, p<0.05; **, p<0.01

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the 90 day rat study, the neurotoxic potential of glutaraldehyde (GA 50%) was investigated in addition to the systemic toxicity following repeated dosing. For the neurotoxicity part of the study, Wistar rats of both sexes were distributed into test groups. Each of these group which comprised 15 animals per sex was further subdivided into 3 sections of 5 animals/sex each (section A, B and C). The test substance was administered as aqueous solution in the drinking water. 0, 100, 500 and 2000 ppm; the animals were treated daily, 7 days per week, over a period of 90 days. For assessment of the neurotoxic potential of glutaraldehyde, the animals of section A and B were subjected to a functional observational battery (FOB) and to motor activity assessment (MA); the animals of section A were sacrificed at the end of the experimental period and were then perfusion-fixed for neuropathological examination. Neither the FOB nor the MA assessment revealed treatment-related effects, and the neuropathological examination was inconspicuous.

This result was in agreement with Spencer et al. (1978). In this study the neurotoxic effect of glutaraldehyde was determined at concentrations of 0.05, 0.1 and 0.25% in drinking water over a period of 11 weeks. Each test group comprised 3 rats. In this study no symptoms of toxicity were seen and body weight gain was inconspicuous. In addition, histopathological examination of the tissue samples removed from the nervous system of the treated animals revealed no abnormalities.

Justification for classification or non-classification

No neurotoxic potential could be evidenced for glutaraldehyde. Moreover and because of the restricted bioavailability of glutaraldehyde, no exposure of the central and/or peripheral nervous system to glutaraldehyde has to be expected. This no classification is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.