Glutaral

Endpoint summary

Description of key information

For the oral route, three studies performed in compliance with OECD guidelines and following GLP are available. NOAELs determined in these three studies were similar and ranged from 14.6 to 23.0 mg/kg bw pure glutaraldehyde. The NOAEL after oral exposure was determined to be 15 mg/kg bw.

For the inhalation route, several studies indicate that glutaraldehyde affects primarily the respiratory tract. Systemic effects were also observed, but were rather mild and mostly at concentrations above the NOAEC for local effects. Based on all available data, the NOAECs for local and systemic effects were determined to be 0.25 and 0.5 mg/m3, respectively.

For the dermal route, a 13 week dermal rat study was conducted according to OECD TG 411. The NOAEL level for systemic toxicity was established at 150 mg/kg/day, the highest dose tested.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

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 oral 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. days);

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 day/week

Dose / conc.:

100 ppm

Remarks:

(nominal in drinking water)
- males: 6.1 mg/kg bw/day (test material) and 3 mg/kg bw/day active ingredient actual ingested
- females: 8.2 mg/kg bw/day (test material) and 4 mg/kg bw/day active ingredient actual ingested

Dose / conc.:

500 ppm

Remarks:

(nominal in drinking water)
- males: 29.9 mg/kg bw/day (test material) and 15 mg/kg bw/day active ingredient actual ingested
- females: 38.5 mg/kg bw/day (test material) and 19 mg/kg bw/day active ingredient actual ingested

Dose / conc.:

2 000 ppm

Remarks:

(nominal in drinking water)
- males: 106.9 mg/kg bw/day (test material) and 53 mg/kg bw/day active ingredient actual ingested
- females: 144.4 mg/kg bw/day (test material) and 72 mg/kg bw/day active ingredient 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 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; cyanmethemoglobin 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.

Sacrifice and pathology:

Organ weights:
The weight of following organs was determined: anesthetized animal, liver, kidneys, adrenals, testes/ovaries, epididymides, uterus, thymus, spleen, brain and heart.

Gross lesions:
All gross lesions were examined in all affected non-perfused animals per sex and group, for each test group (0, 100, 500 and 2000 ppm).

Histopathology:
Following organs and tissues were fixed with neutral buffered 4% formaldehyde for the purpose of (histo)pathological examination: all gross lesions, salivary glands, oesophagus, stomach, duodenum/jejunum/ileum, caecum/colon/rectum, liver, pancreas, brain, pituitary, sciatic nerve, spinal cord, eyes with optic nerve, adrenals, thyroid, parathyroid, trachea, lungs, pharynx, larynx, nose/nasal cavity, aorta, heart, bone marrow, lymph nodes, spleen, thymus, kidneys, urinary bladder, ureter, testes/ovaries, oviducts/uterus/vagina, epididymides/prostate/seminal vesicle, female mammary gland, skin, skeletal muscle, sternum with marrow, femur with knee joint and marrow, extraorbital lacrimal glands.

The following organ samples of all non-perfused animals per sex and group of the control-and the 2000 ppm group were processed for histological assessment (i.e. paraffin embedding, sectioning and 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, caecum/colon/rectum, urinary bladder, ureter, mesenteric and mandibular lymphnodes, female mammary gland, skeletal muscle, sciatic nerve, sternum with marrow, femur bone marrow, eyes with optic nerve, femur with knee joint all spinal cord.

Other examinations:

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. The data referring to the neurotoxicity part of the study are presented in chapter 7.9.1.

Statistics:

Statistical assessment of clinical data: Calculation of means and standard deviations;
Statistical assessment of food/water consumption, body weight, body weight change, food efficiency: Analysis of variance (ANOVA) followed by Dunnett´s test;
Statistical assessment of feces, rearing, grip strength (forelimbs, hindlimbs), landing foodsplay test, motor activity, hematological and clinical-chemical data (excepted differential blood count): 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 urinalysis data (excepted volume, color, turbidity and specific gravity): Fisher´s exact test;
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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 2000 ppm: slight impairment of body weight and body weight change in both sexes were reported.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 2000 ppm: slight impairment of food consumption in both sexes was reported.

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

effects observed, treatment-related

Description (incidence and severity):

At 100 ppm: impaired water consumption in females was reported;
At 500 ppm: impaired water consumption in females was reported;
At 2000 ppm: impaired water consumption in both sexes was reported.

Dose descriptor:

NOAEL

Effect level:

29.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effect observed at this tested dose

Remarks on result:

other: corresponding to 14.95 mg/kg bw/day of active ingredient

Dose descriptor:

NOAEL

Effect level:

38.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effect observed at this tested dose

Remarks on result:

other: corresponding to 19.25 mg/kg bw/day of active ingredient

Dose descriptor:

LOAEL

Effect level:

106.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 53 mg/kg bw/da of active ingredient

Dose descriptor:

LOAEL

Effect level:

144.4 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: corresponding to 72 mg/kg bw/da of active ingredient

Critical effects observed:

no

Analytical monitoring:
----------------------
Analyses confirmed the theoretical concentrations of test substance; means
were 94-106%.

The test substance intake was as follows:
-----------------------------------------
_______________________________________________________
Test group / Test Dose /     TS intake (mg/kg bw/day) 
                       /   Males      /   Females
-------------------------------------------------------
1          /    0 ppm  /     0        /    0 
-------------------------------------------------------
2          /  100 ppm  /     6.1      /    8.2
-------------------------------------------------------
3	   /  500 ppm  /    29.9      /    38.5
-------------------------------------------------------
4	   / 2000 ppm  /   106.9      /   144.4
_______________________________________________________

Conclusions:

The NOEL was 500 ppm in males, and below 100 ppm in females in this study. However, the impairment of water consumption observed at 100 ppm was assessed as being a palatability problem rather than an adverse effect. Therefore, the NOAEL with respect to the systemic toxicity was 500 ppm in both sexes. In terms of active ingredient, the respective NOAEL was ca. 15 and 20 mg/kg bw/day.
As no signs of neurotoxicity were detected, the NOEL for neurotoxicity was 2000 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study conduct was in principle similar to OECD TG 451, with the focus being on the non-neoplasic and neoplasic findings at necropsy; chronic toxicity was considered up to a certain point as only survival and body weight changes were considered.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 451

Principles of method if other than guideline:

The aim of the present study was to investigate the carcinogenic potential of glutaraldehyde when administered via inhalation over a period of two years to rats and mice (see 7.7); chronic toxicity also was considered up to a certain point.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Glutaraldehyde 25% aq. solution
- Supplier: Union Carbide Corporation (Specialty Chemicals Division, Charleston, WV)
- Physical state: liquid
- Analytical purity:
A purity of 91.2 to 92.9 % relative to the reference standard was reported for the test substance of batch No: IS-611699 (< 0.6% methanol)
A purity of 94.6 to 94.8 % relative to the reference standard was reported for the test substance of batch No: IS-678984 (< 0.3% methanol)
- Impurities (identity and concentrations): methanol < 0.6%
- Lot/batch No.: IS-611699 and IS-678984
- Stability under test conditions: the stability of the bulk material was monitored during the 2-years study by gas chromatography with flame ionization detection and by ultraviolet/visible spectroscopy. No degradation of the bulk chemical was detected.
- Storage condition of test material: To ensure stability, the bulk chemical used in present studies was stored under N2 headspace at ca. 0°C in 1-gallon amber glass bottles.

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: the mean weight at test initiation was ca. 26 g and 20 g for males and females, respectively
- Fasting period before study: no
- Housing: one animal per cage, in stainless-steel wire-bottom cages (Hazieton System, Inc., Aberdeen, MD)
- Diet (e.g. ad libitum): NIH-07 open formula pellet diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum except during exposure periods, changed weekly
- Water (e.g. ad libitum): Softened tap water via automatic watering system, available ad libitum except during exposure periods
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 12 - 18
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: No

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE
Glutaraldehyde (GA) vapour was generated with a rotary evaporation system with a hot-water operated at 44 °C and modified to included a heated stream of N2 metered into the flask; the GA and water vapors arising from the flask were carried through the generator by the N2. The temperature of the generator was sufficient to prevent condensation of the vapour during passage through the generator. Because of the evaporation rate of water (> than that of GA), ultrapure water was pumped into the evaporation flask throughout the generation period to maintain a constant volume in the flask. The vapour was conducted through a distribution manifold and was diluted with heated HEPA-and charcoal-filtered air.

TEST ATMOSPHERE
The flow into each exposure-chamber was controlled by means of vaccum pumps. Vapor flowed through separate metering valves for each chamber and was further diluted with filtered air to get the appropriate concentration. In order to maintain uniform exposure concentrations, chamber air circulation was increased by means of a recirculating system, which was added to each exposure chamber. The total active mixing volume of each chamber was 1.7 m3. A small particle detector was used to check that glutaraldehyde was present in the exposure chamber as vapour and not as aerosol, both in presence and absence of test animals. No particle counts above the minimum resolvable level of about 200 particles/cm3 were detected.

CONCENTRATION BUILD-UP AND DECAY
Build up and decay rates for the test concentrations in the chambers were determined in the presence of test animals. The time needed to reach 90% of the final stable concentration in the chamber was defined as T90, whereas the time needed for the exposure to decrease to 10% of the stable concentration was defined as T10. The theoritical value of T90 and T10 under 15-air changes/hour condition is ca. 12.5 minutes. During pre-start testing, T90-values ranged between 25 and 40 minutes in rat chambers whereas the T10 values were about 6 to 10 minutes. In mice chambers, the T90 values ranged between 18 and 31 minutes whereas the T10 values ranged between 9 and 11 minutes. During the studies, T90 was found to range between 9 and 24 minutes for rat chambers, and between 7 and 20 minutes for mice chambers; T10 was about 7 to 10 minutes for the rats and about 4 to 7 minutes for the mice. On the basis of these values, T90 was given a value of 25 minutes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Monitoring of the GA vapour concentrations in the distribution system was based on gas chromatography and showed that these concentrations were stable; for details see table below. Monitoring of the GA vapour concentrations in the exposure chambers was based on online gas chromatography, with the monitor being coupled to the exposure chamber via a computer-controlled 12-port steam select valve. Each chamber was samples approximately every 45 minutes.

Duration of treatment / exposure:

2 years (i.e. 104 weeks)

Frequency of treatment:

6 h/day, 5 days/week

Dose / conc.:

0.062 ppm

Remarks:

0.00025 mg/L; ppm value conversion in mg/L using a conversion factor of 0.0041 (Derelanko MJ, The Toxicologist's Pocket Handbook, Second Edition, Informa Healthcare USA, Inc., New York, 2008).

Dose / conc.:

0.125 ppm

Remarks:

0.0005 mg/L; ppm value conversion in mg/L using a conversion factor of 0.0041 (Derelanko MJ, The Toxicologist's Pocket Handbook, Second Edition, Informa Healthcare USA, Inc., New York, 2008).

Dose / conc.:

0.25 ppm

Remarks:

0.001 mg/L; ppm value conversion in mg/L using a conversion factor of 0.0041 (Derelanko MJ, The Toxicologist's Pocket Handbook, Second Edition, Informa Healthcare USA, Inc., New York, 2008).

No. of animals per sex per dose:

Fifty animals/sex/group

Control animals:

yes, sham-exposed

yes, historical

Observations and examinations performed and frequency:

All animals were observed twice daily for mortality. Clinical observations were recorded at test initiation and thereafter, every 4 weeks from week 5 to week 89, and every 2 weeks from week 93 until test ending. Body weights were recorded at test initiation and thereafter, every 4 weeks from week 5 to week 89, and every 2 weeks from week 93 until test ending.
No further parameters were considered.

Sacrifice and pathology:

- Complete necropsy as well as complete histopathology was conducted on all test animals.
- All organs and tissues were examined for gross pathology.
- No organ weights were assessed.
- All major tissues were fixed in 10% neutral buffered formalin and were processed and Hematoxylin/Eosin-stained for light microscopic examination. These tissues included: brain, pituitary, thyroid, parathyroid, thymus, oesophagus, gallblader, salivary glands, stomach, small and large intestines, liver, pancreas, pancreatic islets, kidneys, adrenals, spleen, heart, trachea, lungs, larynx, gonads, uterus, mammary gland, clitoral gland, prostate, preputial gland, urinary bladder, lymph nodes, bone marrow, skin and nose.
- All tumors as well as all potential target organs (larynx, lung, nose) were evaluated by a quality assessment pathologist.
- For the nose, 4 sections of the nasal passage were considered for examination.
- The kidneys of male mice were examined by the quality assessment pathologist for infarct or nephropathy.
- The livers of female mice were examined for eosinophilic foci.
- The thyroid glands of mice of both sexes were re-evaluated from hyperplasia.
- The (histo)pathological findings were submitted to the NTP Pathology Working Group chairperson for review.

Statistics:

- Survival data were statistically assessed by means of the product-limit procedure according to Kaplan and Meier (J. Am. Stat. Assoc. 53: 457-481, 1958), Cox´s method (J.R. Stat. Soc. B34: 187-220, 1972) and Tarone´s life table test (Biometrika 62: 679-682,1975);
- The incidence of neoplasms and non-neoplastic lesions was assessed using the Poly-k test according to Bailer and Portier (Biometrics 44: 417-431, 1988), Portier and Bailer (Fund. Appl. Toxicol. 12: 731-737, 1989), Piegorsch and Bailer (Statistics for Environmental Biology and Toxicology, Section 6.3.2, Chapman and Hall, London, 1997) and Bieler and Williams (Biometrics 49: 793-801, 1993);
- The body weight data were assessed using the parametric multiple comparison procedures according to Dunnett (J. Am. Stat. Assoc. 50: 1096-1121, 1955) and Williams (Biometrics 27: 103-117, 1971; Biometrics 28: 519-531, 1972); Mann-Whitney U test according to Hollander and Wolfe (Nonparametric Statistical Methods: 120-123, John Wiley and Sons, NY, 1973)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical symptoms were seen in mice, and survival in all test groups and for both sexes was similar to controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The male mice showed no treatment-related effects on body weight; for all concentrations and all intervals of time considered, the mean body weights as % of control ranged from 96 to 100%. The mean body weights of the female mice of the 0.250 ppm group were decreased compared to control; in fact, for the intervals of time from week 14 to week 52 and from week 53 to week 104, the mean body weights as % of control were about 91 and 93%.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant and/or biological relevant changes were mainly seen in the nose of the glutaraldehyde-treated mice.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The main non-neoplastic lesions in the nose are summarized in the table below. Non-neoplastic lesions were limited primarily to the most anterior region of the nasal cavity. The nasal lesions in the mice were qualitatively similar to those seen in rats. Squamous metaplasia of the respiratory epithelium was observed in both sexes of mice while female mice also had inflammation and hyaline degeneration of the respiratory epithelium.
Further effects were seen, which can be summarized as follows:
Main lesions in the thyroid gland of glutaraldehyde-treated mice:
An increased incidence in hyperplasia of the thyroid gland follicular cells, which was classified as minimal to mild, was reported for the females of the 0.250 ppm group (37 cases versus 26 in control). This effect however is a common spontaneous effect seen in aged mice. Furthermore, neither increased incidence in adenoma of the thyroid gland follicular cells in males and females, nor treatment-related effects in the thyroid gland of the males were seen. Therefore, the increased incidence in hyperplasia of the thyroid gland follicular cells seen in the 0.250 ppm females was considered as an incidental, not treatment-related finding.
Main lesions in the pituitary gland of glutaraldehyde-treated mice:
An increased incidence in hyperplasia of the pituitary gland (pars distalis), which was classified as minimal to mild, was reported for the females of the 0.250 ppm group (28 cases versus 19 in control). The females showed no increased incidence in adenoma of the pituitary gland, and the males were free of treatment-related effects affecting the pituitary gland. Therefore, the increased incidence in hyperplasia of the pituitary gland seen in the 0.250 ppm females was considered as an incidental, not treatment-related finding.
Main lesions in the liver of glutaraldehyde-treated mice:
The males of the 0.062 and the 0.250 ppm groups as well as the females of the 0.250 ppm group showed decreased incidences in hepatocellular adenoma when compared to control (males: 11 cases seen at 0.250 ppm versus 19 cases in control; females: 3 cases at 0.250 ppm versus 11 cases in control). In females, this effect was seen as secondary effect resulting from the decrease in body weight. In males, no such decrease in body weight was seen; therefore the decreased incidence in hepatocellular adenoma seen in males was not considered to be treatment-related.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No exposure-related neoplastic lesions were observed in the mice, neither in the males nor in the females.

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

0.062 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: 2.5E-4 mg/L

Remarks:

conversion in mg/L using a conversion factor of 0.0041 (Derelanko MJ, The Toxicologist's Pocket Handbook, Second Edition, Informa Healthcare USA, Inc., New York, 2008).

Dose descriptor:

LOAEC

Remarks:

Local

Effect level:

0.125 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: based on squamous metaplasia findings in the respiratory epithelium

Remarks on result:

other: 5.0E-4 mg/L

Dose descriptor:

NOAEC

Remarks:

Systemic

Effect level:

0.125 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed at this dose

Remarks on result:

other: 5.0E-4 mg/L

Dose descriptor:

LOAEC

Effect level:

0.25 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

not specified

Two-year inhalation study with mice, main non-neoplastic lesions in the nose:

Male mice
Lesions in the nose		0 ppm	0.062 ppm	0.125 ppm	0.250 ppm
Respiratory epithelium	Squamous metaplasia	2^a(1.0)^b	5 (1.0)	6 (1.2)	9* (1.1)
Turbinate	Necrosis	0	0	2 (2.0)	0
Female mice
Squamous epithelium	Inflammation	6 (1.2)	7 (1.3)	13 (1.4)	14* (1.4)
Respiratory epithelium	Squamous metaplasia	7 (1.1)	11 (1.0)	16* (1.3)	21** (1.5)
	Hyaline degeneration	16 (1.4)	35** (1.4)	32** (1.3)	30* (1.1)
Turbinate	Necrosis	0	3 (2.0)	1 (1.0)	4 (1.5)
*, p < 0.005; **, p < 0.001; a, Number of animals with lesions (Number of animals examined = 50, excepted for 0 ppb males: 48 and 62.5 ppb females: 49); b, Average severity grade of lesions (1 = minimal, 2 = mild, 3 = moderate, 4 = marked)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.5 mg/m³

Study duration:

chronic

Species:

mouse

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study conduct was in principle similar to OECD TG 451, with the focus being on the non-neoplasic and neoplasic findings at necropsy; chronic toxicity was considered up to a certain point as only survival and body weight changes were considered.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 451

Principles of method if other than guideline:

The aim of the present study was to investigate the carcinogenic potential of glutaraldehyde when administered via inhalation over a period of two years to rats and mice (see 7.7); chronic toxicity also was considered up to a certain point.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Glutaraldehyde 25% aq. solution
- Supplier: Union Carbide Corporation (Specialty Chemicals Division, Charleston, WV)
- Physical state: liquid
- Analytical purity:
A purity of 91.2 to 92.9 % relative to the reference standard was reported for the test substance of batch No: IS-611699 (< 0.6% methanol)
A purity of 94.6 to 94.8 % relative to the reference standard was reported for the test substance of batch No: IS-678984 (< 0.3% methanol)
- Impurities (identity and concentrations): methanol < 0.6%
- Lot/batch No.: IS-611699 and IS-678984
- Stability under test conditions: the stability of the bulk material was monitored during the 2-years study by gas chromatography with flame ionization detection and by ultraviolet/visible spectroscopy. No degradation of the bulk chemical was detected.
- Storage condition of test material: To ensure stability, the bulk chemical used in present studies was stored under N2 headspace at ca. 0°C in 1-gallon amber glass bottles.

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms (Germantown, NY)
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: the mean weight at test initiation was ca. 26 g and 20 g for males and females, respectively
- Fasting period before study: no
- Housing: one animal per cage, in stainless-steel wire-bottom cages (Hazieton System, Inc., Aberdeen, MD)
- Diet (e.g. ad libitum): NIH-07 open formula pellet diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum except during exposure periods, changed weekly
- Water (e.g. ad libitum): Softened tap water via automatic watering system, available ad libitum except during exposure periods
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 12 - 18
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: No

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE
Glutaraldehyde (GA) vapour was generated with a rotary evaporation system with a hot-water operated at 44 °C and modified to included a heated stream of N2 metered into the flask; the GA and water vapors arising from the flask were carried through the generator by the N2. The temperature of the generator was sufficient to prevent condensation of the vapour during passage through the generator. Because of the evaporation rate of water (> than that of GA), ultrapure water was pumped into the evaporation flask throughout the generation period to maintain a constant volume in the flask. The vapour was conducted through a distribution manifold and was diluted with heated HEPA-and charcoal-filtered air.

TEST ATMOSPHERE
The flow into each exposure-chamber was controlled by means of vaccum pumps. Vapor flowed through separate metering valves for each chamber and was further diluted with filtered air to get the appropriate concentration. In order to maintain uniform exposure concentrations, chamber air circulation was increased by means of a recirculating system, which was added to each exposure chamber. The total active mixing volume of each chamber was 1.7 m3. A small particle detector was used to check that glutaraldehyde was present in the exposure chamber as vapour and not as aerosol, both in presence and absence of test animals. No particle counts above the minimum resolvable level of about 200 particles/cm3 were detected.

CONCENTRATION BUILD-UP AND DECAY
Build up and decay rates for the test concentrations in the chambers were determined in the presence of test animals. The time needed to reach 90% of the final stable concentration in the chamber was defined as T90, whereas the time needed for the exposure to decrease to 10% of the stable concentration was defined as T10. The theoritical value of T90 and T10 under 15-air changes/hour condition is ca. 12.5 minutes. During pre-start testing, T90-values ranged between 25 and 40 minutes in rat chambers whereas the T10 values were about 6 to 10 minutes. In mice chambers, the T90 values ranged between 18 and 31 minutes whereas the T10 values ranged between 9 and 11 minutes. During the studies, T90 was found to range between 9 and 24 minutes for rat chambers, and between 7 and 20 minutes for mice chambers; T10 was about 7 to 10 minutes for the rats and about 4 to 7 minutes for the mice. On the basis of these values, T90 was given a value of 25 minutes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Monitoring of the GA vapour concentrations in the distribution system was based on gas chromatography and showed that these concentrations were stable; for details see table below. Monitoring of the GA vapour concentrations in the exposure chambers was based on online gas chromatography, with the monitor being coupled to the exposure chamber via a computer-controlled 12-port steam select valve. Each chamber was samples approximately every 45 minutes.

Duration of treatment / exposure:

2 years (i.e. 104 weeks)

Frequency of treatment:

6 h/day, 5 days/week

Dose / conc.:

0.062 ppm

Remarks:

0.00025 mg/L; ppm value conversion in mg/L using a conversion factor of 0.0041 (Derelanko MJ, The Toxicologist's Pocket Handbook, Second Edition, Informa Healthcare USA, Inc., New York, 2008).

Dose / conc.:

0.125 ppm

Remarks:

0.0005 mg/L; ppm value conversion in mg/L using a conversion factor of 0.0041 (Derelanko MJ, The Toxicologist's Pocket Handbook, Second Edition, Informa Healthcare USA, Inc., New York, 2008).

Dose / conc.:

0.25 ppm

Remarks:

0.001 mg/L; ppm value conversion in mg/L using a conversion factor of 0.0041 (Derelanko MJ, The Toxicologist's Pocket Handbook, Second Edition, Informa Healthcare USA, Inc., New York, 2008).

No. of animals per sex per dose:

Fifty animals/sex/group

Control animals:

yes, sham-exposed

yes, historical

Observations and examinations performed and frequency:

All animals were observed twice daily for mortality. Clinical observations were recorded at test initiation and thereafter, every 4 weeks from week 5 to week 89, and every 2 weeks from week 93 until test ending. Body weights were recorded at test initiation and thereafter, every 4 weeks from week 5 to week 89, and every 2 weeks from week 93 until test ending.
No further parameters were considered.

Sacrifice and pathology:

- Complete necropsy as well as complete histopathology was conducted on all test animals.
- All organs and tissues were examined for gross pathology.
- No organ weights were assessed.
- All major tissues were fixed in 10% neutral buffered formalin and were processed and Hematoxylin/Eosin-stained for light microscopic examination. These tissues included: brain, pituitary, thyroid, parathyroid, thymus, oesophagus, gallblader, salivary glands, stomach, small and large intestines, liver, pancreas, pancreatic islets, kidneys, adrenals, spleen, heart, trachea, lungs, larynx, gonads, uterus, mammary gland, clitoral gland, prostate, preputial gland, urinary bladder, lymph nodes, bone marrow, skin and nose.
- All tumors as well as all potential target organs (larynx, lung, nose) were evaluated by a quality assessment pathologist.
- For the nose, 4 sections of the nasal passage were considered for examination.
- The kidneys of male mice were examined by the quality assessment pathologist for infarct or nephropathy.
- The livers of female mice were examined for eosinophilic foci.
- The thyroid glands of mice of both sexes were re-evaluated from hyperplasia.
- The (histo)pathological findings were submitted to the NTP Pathology Working Group chairperson for review.

Statistics:

- Survival data were statistically assessed by means of the product-limit procedure according to Kaplan and Meier (J. Am. Stat. Assoc. 53: 457-481, 1958), Cox´s method (J.R. Stat. Soc. B34: 187-220, 1972) and Tarone´s life table test (Biometrika 62: 679-682,1975);
- The incidence of neoplasms and non-neoplastic lesions was assessed using the Poly-k test according to Bailer and Portier (Biometrics 44: 417-431, 1988), Portier and Bailer (Fund. Appl. Toxicol. 12: 731-737, 1989), Piegorsch and Bailer (Statistics for Environmental Biology and Toxicology, Section 6.3.2, Chapman and Hall, London, 1997) and Bieler and Williams (Biometrics 49: 793-801, 1993);
- The body weight data were assessed using the parametric multiple comparison procedures according to Dunnett (J. Am. Stat. Assoc. 50: 1096-1121, 1955) and Williams (Biometrics 27: 103-117, 1971; Biometrics 28: 519-531, 1972); Mann-Whitney U test according to Hollander and Wolfe (Nonparametric Statistical Methods: 120-123, John Wiley and Sons, NY, 1973)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical symptoms were seen in mice, and survival in all test groups and for both sexes was similar to controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The male mice showed no treatment-related effects on body weight; for all concentrations and all intervals of time considered, the mean body weights as % of control ranged from 96 to 100%. The mean body weights of the female mice of the 0.250 ppm group were decreased compared to control; in fact, for the intervals of time from week 14 to week 52 and from week 53 to week 104, the mean body weights as % of control were about 91 and 93%.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant and/or biological relevant changes were mainly seen in the nose of the glutaraldehyde-treated mice.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The main non-neoplastic lesions in the nose are summarized in the table below. Non-neoplastic lesions were limited primarily to the most anterior region of the nasal cavity. The nasal lesions in the mice were qualitatively similar to those seen in rats. Squamous metaplasia of the respiratory epithelium was observed in both sexes of mice while female mice also had inflammation and hyaline degeneration of the respiratory epithelium.
Further effects were seen, which can be summarized as follows:
Main lesions in the thyroid gland of glutaraldehyde-treated mice:
An increased incidence in hyperplasia of the thyroid gland follicular cells, which was classified as minimal to mild, was reported for the females of the 0.250 ppm group (37 cases versus 26 in control). This effect however is a common spontaneous effect seen in aged mice. Furthermore, neither increased incidence in adenoma of the thyroid gland follicular cells in males and females, nor treatment-related effects in the thyroid gland of the males were seen. Therefore, the increased incidence in hyperplasia of the thyroid gland follicular cells seen in the 0.250 ppm females was considered as an incidental, not treatment-related finding.
Main lesions in the pituitary gland of glutaraldehyde-treated mice:
An increased incidence in hyperplasia of the pituitary gland (pars distalis), which was classified as minimal to mild, was reported for the females of the 0.250 ppm group (28 cases versus 19 in control). The females showed no increased incidence in adenoma of the pituitary gland, and the males were free of treatment-related effects affecting the pituitary gland. Therefore, the increased incidence in hyperplasia of the pituitary gland seen in the 0.250 ppm females was considered as an incidental, not treatment-related finding.
Main lesions in the liver of glutaraldehyde-treated mice:
The males of the 0.062 and the 0.250 ppm groups as well as the females of the 0.250 ppm group showed decreased incidences in hepatocellular adenoma when compared to control (males: 11 cases seen at 0.250 ppm versus 19 cases in control; females: 3 cases at 0.250 ppm versus 11 cases in control). In females, this effect was seen as secondary effect resulting from the decrease in body weight. In males, no such decrease in body weight was seen; therefore the decreased incidence in hepatocellular adenoma seen in males was not considered to be treatment-related.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No exposure-related neoplastic lesions were observed in the mice, neither in the males nor in the females.

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

0.062 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: 2.5E-4 mg/L

Remarks:

conversion in mg/L using a conversion factor of 0.0041 (Derelanko MJ, The Toxicologist's Pocket Handbook, Second Edition, Informa Healthcare USA, Inc., New York, 2008).

Dose descriptor:

LOAEC

Remarks:

Local

Effect level:

0.125 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: based on squamous metaplasia findings in the respiratory epithelium

Remarks on result:

other: 5.0E-4 mg/L

Dose descriptor:

NOAEC

Remarks:

Systemic

Effect level:

0.125 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse systemic effects observed at this dose

Remarks on result:

other: 5.0E-4 mg/L

Dose descriptor:

LOAEC

Effect level:

0.25 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Critical effects observed:

not specified

Two-year inhalation study with mice, main non-neoplastic lesions in the nose:

Male mice
Lesions in the nose		0 ppm	0.062 ppm	0.125 ppm	0.250 ppm
Respiratory epithelium	Squamous metaplasia	2^a(1.0)^b	5 (1.0)	6 (1.2)	9* (1.1)
Turbinate	Necrosis	0	0	2 (2.0)	0
Female mice
Squamous epithelium	Inflammation	6 (1.2)	7 (1.3)	13 (1.4)	14* (1.4)
Respiratory epithelium	Squamous metaplasia	7 (1.1)	11 (1.0)	16* (1.3)	21** (1.5)
	Hyaline degeneration	16 (1.4)	35** (1.4)	32** (1.3)	30* (1.1)
Turbinate	Necrosis	0	3 (2.0)	1 (1.0)	4 (1.5)
*, p < 0.005; **, p < 0.001; a, Number of animals with lesions (Number of animals examined = 50, excepted for 0 ppb males: 48 and 62.5 ppb females: 49); b, Average severity grade of lesions (1 = minimal, 2 = mild, 3 = moderate, 4 = marked)

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.25 mg/m³

Study duration:

chronic

Species:

mouse

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1999 - May 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)

Version / remarks:

(1998)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Glutaraldehyde 50% aq. sol.
- Supplier: sponsor
- Physical state: colorless clear liquid
- Analytical purity: 50.3%
- Lot/batch No.: 50-4402
- Stability under test conditions: the stability of the test substance was guaranteed until February 2000 by the sponsor
- Storage condition of test material: at + 4 °C, under nitrogen

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Saint-Aubin-lès-Elbeuf, France (Breeder)
- Age at study initiation: about 9 weeks old
- Weight at study initiation: mean body weight of the males, 340 g (320 g- 362 g); mean body weight of the females, 221 g (207 g – 243 g)
- Fasting period: at least 14 hours fasting prior sacrifice at test ending
- Housing: individually in suspended wire-mesh cages, with metallic tray, containing autoclaved sawdust placed under each cage.
- Diet (e.g. ad libitum): A04 C pelleted maintenance diet
- Water (e.g. ad libitum): filtered tap water (0.22 micron filter)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Type of coverage:

semiocclusive

Vehicle:

water

Details on exposure:

- The route of administration was dermal;
- Protectol GDA was diluted with water to give concentrations of 5, 10 and 15% test concentrations;
- The applied test volume was 2 ml/kg bw;
- The test solution was applied to the clipped dorsal skin of each animal;
- The site of application was about 10% of the total body surface;
- The application site was covered with a gauze held in place with a semi-occlusive dressing for 6 hours;
- Following removal of the dressing, residual test substance on the skin was removed using a gauze pad moistened with water;
- The control animals were treated similarly as above, but with the vehicle only (i.e. water).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of a sample taken from each dosage form (including the control) prepared for use in weeks 1, 4, 8 and 13 was determined by means of UV spectrophotometry at 233 nm; the concentration of Protectol GDA was determnined from a calibration curve obtained by linear regression analysis of absorbance against concentration of Protectol GDA in standard solutions (external standard calibration).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

The animals were treated 5 days a week over a period of 13 weeks, implying 67 days of test substance-administration.

Dose / conc.:

50 mg/kg bw/day

Remarks:

(active ingredient); calculated on the basis of an application volume of 2 ml/kg bw;
5 % test material, 2.5% (active ingredient)

Dose / conc.:

100 mg/kg bw/day

Remarks:

(active ingredient); calculated on the basis of an application volume of 2 ml/kg bw;
10 % test material, 5% (active ingredient)

Dose / conc.:

150 mg/kg bw/day

Remarks:

(active ingredient); calculated on the basis of an application volume of 2 ml/kg bw;
15 % test material, 7.5% (active ingredient)

No. of animals per sex per dose:

Ten/sex/group

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

MORTALITY, CLINICAL SYMPTOMS OF TOXICITY, SKIN REACTION:
Starting from the day prior test initiation, the animals were checked at least once daily for clinical signs of toxicity (including mortality) and signs of skin irritation. In addition to these standard examinations, the animals also were subjected once a week to a more detailed examination of following parameters: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, autonomic activity, changes in gait and posture, reaction to handling, presence of clonic or tonic movements, and stereotypical or bizarre behaviour.

BODY WEIGHT:
The body weight of each animal was recorded prior allocation of the animals to each test group, on the first day of treatment and thereafter once a week over the whole treatment period.

FOOD CONSUMPTION:
Food consumption for each animal was recorded once a week over a 7-day period, from test initiation until test ending.

FOOD EFFICIENCY:
Not considered

WATER CONSUMPTION:
Not considered

OPHTHALMOSCOPIC EXAMINATION:
All animals were subjected to ophthalmologic examinations prior test initiation. At test ending, the eyes of the control animals and of the animals of the high-dose group were examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY:
Blood samples were collected via orbital sinus puncture from all surviving animals at the end of the experimental period (week 13). Prior to blood sampling, the animals were fasted overnight. The blood samples served for the evaluation of the haematological and clinical-chemical parameters.
Following haematological parameters were considered: erythrocytes, hemoglobin, mean cell volume, packed cell volume, mean cell hemoglobin concentration, mean cell hemoglobin, thrombocytes, leucocytes, differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes, monocytes), and prothrombin time.
Following clinical-chemical parameters were considered: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total protein, albumin, albumin/globulin ratio, cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase.

URINALYSIS:
Not considered

NEUROBEHAVIOURAL EXAMINATION:
See clinicals symptoms

Sacrifice and pathology:

At the end of the treatment period the animals were fasted for at least 14 hours and were then sacrificed for necropsy. Animals that died during the experiment also were subjected to necropsy.

ORGAN WEIGHT:
The animals were weighed a last time prior sacrifice, and after sacrifice, following organs were taken for weighing: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus. Both, the absolute and the relative organ weights were determined.

GROSS PATHOLOGY:
Following sacrifice, all animals were subjected to a complete macroscopic examination, including the external surfaces, all orifices, the cranial cavity, the brain surface, the surface of the spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues, and the neck with its associated organs and tissues.

HISTOPATHOLOGY:
Following organs/tissues were preserved for the purpose of histopathology: all gross lesions, adrenals, aorta, brain (cerebellar and cerebral cortex, medulla/pons), caecum, colon, duodenum, epididymes, eyes with Harderian glands, femoral bone with articulation, heart, ileum, jejunum, kidneys, larynx, liver, lung with bronchi, lymph nodes, mammary glands, nose, oesophagus, optic nerve, ovaries, pancreas, pharynx, pituitary gland, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids and parathyroids, tongue, trachea, urinary bladder uterus and vagina.
Following organs/tissues were embedded in paraffin wax, sectioned and stained with hematoxylin-eosin for microscopical examination:all organs/tissues listed above for all sacrificed animals of the control group and of the high-dose group (excepted for the femoral bone, the skeletal muscle, the tongue and the vagina), all organs/tissues listed above for all animals that died or had to be killed prematurely during the experiment, and all gross lesions.

Statistics:

A series of tests were used for statistical assessment of the findings, including among other Kolmogorov-Lilliefors´ test, Fisher´s test, Bartlett´s test and Student´s test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Poor condition including pallor of eyes and/or extremities, piloerection, round back, tremors, staggering gait, half-closed eyes and/or dyspnea, was seen in week 11 or 13 in 4 animals (1 male, 3 females) of the 100 mg/kg bw group. Soft feces were seen in one male of the 50 mg/kg bw group, cutaneous lesions on the abdomen were reported for a further male of this group (from week 12). Exophthalmus, and hair loss on one forelimb were respectively reported for one male (from week 11) and one female (from week 10) of the 150 mg/kg bw group. The low incidence of clinical signs and the absence of a dose-response relationship indicated that the clinical effects were not treatment-related.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Signs of local irritation, including scabs, desquamation and very slight or well-defined erythema, were noted at the application site of almost all treated animals.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the control group, one male rat died on day 44 from acute rhinitis. A female was killed in extremis on day 46 for ethical reasons.
In the 50 mg/kg bw group, one male was found dead on day 86; no clinical signs had been seen prior to death. The low incidence of mortality, the absence of a dose-relationship and the occurrence of mortality in the control group indicated that mortality was not treatment-related.
In the 100 mg/kg bw group, one male was found dead on day 80; prior to death the animal showed poor clinical condition.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For the males, no significant differences between treated and control groups were seen. In fact, individual differences in body weight were observed in all control and treated groups, which were not considered to be treatment-related.
For the females, the mean final body weight was slightly decreased compared to control in both the 100 and the 150 mg/kg bw/day groups. In fact, the mean body weight of females given 150 mg/kg bw/day was slightly lower than that of control animals. The observed difference was statistically significant on weeks 2 and 5 and decreased thereafter. Except for week 13, when a statistically significant lower mean body weight was recorded, the mean body weight of females given 100 mg/kg bw/day was similar to that of controls. The mean body weight of females given 50 mglkglday was similar to that of controls. Thus, the slight effect seen here were not considered to be treatment-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption for both sexes was similar for all groups including control.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were seen. In fact, ophthalmologic examinations prior test initiation revealed findings such as variation in corneal thickness and vacuolization of the cornea, which mainly were seen in the control group. On week 13, ophthalmological examination revealed hyphemia, exophthalmia and luxation of the lens in one male of the 150 mg/kg bw/day group.

Haematological findings:

no effects observed

Description (incidence and severity):

In males of the 50 mg/kg bw/day group, slightly increased mean cell volume and mean cell haemoglobin as well as slightly decreased erythrocyte count and mean cell haemoglobin concentration were reported.
In males of the 100 and 150 mg/kg bw/day groups and in females of the 50 mg/kg bw/day group, increased eosinophil count was reported.
In females of the 150 mg/kg bw/day group, increased thrombocyte count was reported.
All these effects however were slight, without any dose-response relationship and had values in the range of historical control data. They were therefore considered to be of no biological/ toxicological relevance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In males and females of the 100 and 150 mg/kg bw/day groups, lower glucose levels were reported.
In females of the 100 and 150 mg/kg bw/day groups, lower calcium levels were reported.
These effects were slight, without any dose-relationship and without any pathological significance. They therefore were considered to be of no biological/ toxicological relevance.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Differences in organ weights were minimal and without any dose-relationship or correspondence with microscopical findings.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Yellowish coloration of the skin was seen in control and test animals. Due to lack of corresponding microscopic findings this was regarded as being of no toxicological importance. Scabs were seen in all treated animals; they were associated with microscopic findings and were considered to be treatment-related. Changes in liver (yellowish or greyish areas, colored foci, changes in size or consistency) were seen both in control and treated animals (similar incidence and degree of severity). These changes were therefore not considered to related to the treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Slight to moderate acanthosis together with the presence of inflammatory cells in the dermis was reported for the skin of treated animals. Furthermore, ulceration and scabs also were reported for the treated animals. The incidence and severity of these skin findings did not differ notably between the individual treated groups. These skin findings were considered to be treatment-related.
Lesions in the liver included coagulative hepatocellular necrosis, interlobular fibroplasia, tension lipidosis and macrophages with yellow pigment contents. These findings were in accordance with the macroscopical findings in the liver and were found with similar incidence and severity in both, treated and control animals. The effects affecting the liver possibly were related to the wearing of bandage and slight compression at the abdomen.

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest tested dose.

Critical effects observed:

not specified

Analytical monitoring revealed a good agreement between the nominal and measured concentrations of the test substance in all dosage forms. In fact the deviation of the measured from the nominal values did not exceed 6%. 

Summary of the mean body weight data:

Body weight	Dose level (mg/kg bw/day)
	0	50	100	150
Males
Initial body weight (g/animal)	341	341	339	341
Final body weight (g/animal)	491	500	501	513
Body weight gain (g/animal; week 1 to13)	150	159	162	172
Body weight gain (%)	44	47	48	50
Females
Initial body weight (g/animal)	224	223	219	217
Final body weight (g/animal)	305	302	293*	291
Body weight gain (g/animal; week 1 to 13)	81	79	74	74
Body weight gain (%)	36	35	34	34
*, p<0.05

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Oral repeated dose toxicity

In a 90-day oral toxicity study performed in accordance with OECD Guideline 408 and following GLP, 15 male and 15 female Wistar rats were exposed to 50% glutaraldehyde in drinking water at concentrations of 0, 100, 500 and 2,000 ppm for 3 months (BASF, 2001). Food and water consumption was determined once a week. Body weight was determined once a week and on the days when functional observational batteries were performed. A check of the general state of health was made at least daily. Furthermore, the animals were thoroughly examined and palpated once a week. Functional observational batteries and motor activity measurements were carried out in 10 animals per sex and group on days -7, 22, 50 and 85. Ophthalmological and clinicochemical, haematological examinations and urinalyses were carried out towards the end of the administration period. Five animals per sex and dose were fixed by in situ perfusion and subjected to neuropathological examinations. The remaining animals were subjected to gross-pathological assessment, followed by histopathological examinations. In the 2,000 ppm dose group slight impairment of food consumption in both sexes, impairment of water consumption in both sexes and slight impairment of body weight and body weight change in both sexes, (the values of body weight change being 4.5% and 18.8% [males and females] below control on day 91 was observed. In the 500 ppm dose group and 100 ppm dose group, impairment of water consumption in females was observed. The impairment of water consumption was assessed as being a palatability problem, but not an adverse effect. The no observed adverse effect level (NOAEL) with respect to systemic toxicity was therefore 500 ppm in both sexes (29.9 and 38.5 mg/kg bw/day in males and females, respectively for 50% glutaraldehyde and 15.0 and 19.3 mg/kg bw/day in males and females, respectively for pure glutaraldehyde). No signs of neurotoxicity were detected. The no observed effect level for neurotoxicity was therefore 2,000 ppm (106.9 and 144.4 mg/kg bw/day in males and females, respectively for 50% glutaraldehyde and 53.5 and 72.2 mg/kg bw/day in males and females, respectively for pure glutaraldehyde.

In a 12 -month toxicity study performed in accordance with OECD Guideline 452 and following GLP, 20 male and 20 female Wistar rats were exposed to 50% Glutaraldehyde in drinking water at concentrations of 0, 100, 500, and 2,000 ppm for 12 months. (BASF, 2002). Food consumption, water consumption and body weight were determined once a week during the first 13 weeks, thereafter at 4-week intervals. The animals were examined for signs of toxicity or mortality at least once a day. Moreover, detailed clinical examinations in an open field (including palpation) were conducted prior to the start of the administration period and weekly thereafter. Ophthalmological observations were carried out prior to the start and towards the end of dosing. Urinalysis, clinicochemical and haematological examinations were carried out 3, 6 and 12 months after start of the administration period. At the end of the administration period the animals were subjected to full gross-pathological assessment, followed by detailed histopathological examinations. The administration of 2,000 ppm of 50% Glutaraldehyde resulted in respiratory sounds, decreased food consumption, followed by an impairment of body weight. Moreover, erosion/ulcer in the glandular stomach, a slight increase of clear cell foci in the liver of male animals as well as a slight diffuse squamous metaplasia in the epithelium of the larynx in one male rat were obtained in pathology. The reduced water consumption in both sexes of the high dose group (2,000 ppm) as well as in male animals of the mid dose group (500 ppm) was most probably caused by the physical properties (bad taste) of the test compound rather than presenting an adverse effect. Therefore the no observed adverse effect level (NOAEL) under the conditions of this study was 500 ppm in both sexes (30.5 and 46.0 mg/kg bw/day in males and females, respectively for 50% glutaraldehyde and 15.3 and 23.0 mg/kg bw/day in males and females, respectively for pure glutaraldehyde).

In a 12 -month toxicity study performed in accordance with OECD Guideline 452 and following GLP, 5 male and 5 female purebred Beagle dog were exposed to 50% Glutaraldehyde in drinking water at concentrations of 0, 20, 100, and 500 ppm for 12 months. (BASF, 2001). Water consumption of the animals was determined once a week (over a period of four days each); food consumption of the animals was determined daily and their body weight once a week. The animals were examined at least once each working day for any signs of toxicity and a check tor any moribund or dead animals was made twice a day (Mondays to Fridays) and once a day (Saturdays, Sundays and on public holidays). Clinical chemistry and haematological examinations as well as urinalyses were carried out once before the beginning of test substance administration and after approx. 3, 6 and 12 months of test substance administration. Ophthalmological examinations were carried out 14 days before the beginning of the administration period and on study day 360. All animals were subjected to gross-pathological assessment, followed by histopathological examinations. As a consequence of treatment reduced drinking water consumption in both sexes occurred at a dose of 500 ppm. Furthermore slight erythema in the in the inguinal region was recorded in one male and one female of this dose. At 100 ppm slight erythema in the inguinal region was recorded in one female. Due to the localization of the erythema, it was attributed to incidental skin contact with the test substance preparation by licking. This unintentional test substance contact with the skin caused a local inflammatory response. Regarding clinical pathology, the oral administration of 500 ppm of the test compound caused reduced excretion of urine with increased specific gravity in the males, only. No treatment-related changes were observed in the animals given 20 ppm and 100 ppm of the test compound. Regarding pathology, there were no macroscopical or histopathological findings that could be related to the test substance administered. as the impairment of water consumption was assessed as being a palatability problem, rather than an adverse effect. The no observed adverse effect level (NOAEL) was therefore 500 ppm in both sexes (29.1 and 33.2 mg/kg bw/day in males and females, respectively for 50% glutaraldehyde and 14.6 and 16.6 mg/kg bw/day in males and females, respectively for pure glutaraldehyde).

In a study by the Dow Chemical Company (1994), Fischer 344 rats (100/sex/group) were dosed with glutaraldehyde at 0, 50, 250 or 1000 ppm in drinking water for 7 days/week for 104 weeks. Body weight, weight gains and food consumption were generally decreased throughout the study for 250 and 1000 ppm animals, compared to control values, while for water consumption the dose-related effect was also apparent at 50 ppm. At necropsy (at 52, 78 and 104 weeks), the only statistically significant changes in organ weights were for the kidney. Changes seen in urinary parameters and kidney weights were likely related the decreased water consumption rather than to a direct toxic action of glutaraldehyde. There were no significant, treatment related effects on haematology or clinical chemistry. Gross evidence of gastric irritation was present in many 250 and 1000 ppm animals and included thickening of the stomach wall and ulceration of the mucosa, with mucosal hyperplasia in males and females at 104 weeks at 1000 ppm. The main finding of the study was a statistically significant increase in the number of large granular lymphocyte leukemia (LGLL) observed in the liver and spleen of females only. The main cause of death during the study was LGLL. No other significant oncogenic effects were observed. Although the increase in incidence in female in treatment groups was statistically significant when compared the control value, the toxicological significance of these effect is uncertain. LGLL is a commonly occurring spontaneous neoplasm in Fisher 344 rats, with an incidence in control female rats varied 6-52%; in this study it was 24 % (low control value). Finally, decreased water consumption throughout the study can have some effect on this condition. A pathology peer review and pathology working group was formed to confirm the incidence and stage involvement of LGL leukemia found in Report 91U0012 to render an opinion on the biological significance of the findings. The Working group concluded that the finding of increased LGL leukemia in one sex, one species, and one strain is not toxicologically relevant to human risk assessment, even when the increase was noted as statistically significant.

In a study by Miller (2002), performed similar to OECD 453 and following GLP, 100 male and 100 female Fischer 344 rats were exposed to glutaraldehyde in drinking water at concentrations of 50, 250, and 1000 ppm. Main effects were observed in the 250 and the 1000 ppm groups and included reduction in body weight and body weight gain, reduction in food consumption, and reduction in water consumption, increased statistically significant incidence of nucleated erythrocytes and of large monocytes, decreases in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamate dehydrogenase (GD), dose-related decrease in urine volume accompanied by a dose-related increase in osmolality, changes in absolute and relative kidney weight, gastric irritation, increase in bone marrow hyperplasia, and increased incidence of renal tubular pigmentation. With regard to the reduced water consumption, similar as in other studies, glutaraldehyde offered to the animals via the drinking water resulted in a decreased water consumption, which probably was related to the bad taste, smell and/or irritancy of the test substance. Therefore the reduction in water consumption was considered to be of no toxicological relevance. As consequence of the reduced water intake, a decrease in urine production coupled with increased osmolality and decreased pH was reported. These effects were indicative of renal physiological adaptive changes resulting from the decreased water intake, and were in accordance with the findings referring to the absolute and relative changes in kidney weight. Effects affecting the haematological and clinical-chemical parameters were marginal and of no toxicological relevance. In fact, the main haematological finding seen at the end of the test period and which consisted of the appearance of nucleated erythrocytes and large monocytes in all treated groups (statistically significant for the males of the 250 and the 1000 ppm groups) was related to the incidence of large granular lymphocytic leukaemia (LGLL) in the spleen. Both, bone marrow hyperplasia and renal tubular pigmentation were put into relation with the occurrence/incidence of large granular lymphocytic leukaemia (LGLL), and were considered by the authors of the study as being secondary to a low grade haemolytic anaemia in animals with LGLL. Due to the background and variable incidence of LGLL in the Fischer 344 rat, the finding of a statistical significance only for female rats and because, there was no clear dose-response relationship, the biological significance of the LGLL findings is unclear. Based on the available information, the NOAEL was determined to be 50 ppm (4 and 6 mg/kg bw/day in males and females, respectively for glutaraldehyde).

In studies by the Bushy Run Research Center, rats and dogs were exposed to 50% glutaraldehyde for 2 and 13 weeks. In one two-week range finding study, 10 male and 10 female Fischer 344 rats were exposed 10, 100, 1000 ppm 50% glutaraldehyde (BRRC, 1985). Male rats exposed to 1000 ppm exhibited decreased water and food consumption each day of the study. Females in the high dose group had decreased water consumption all 14 days of the study and a slightly reduced body weight gain at day 14 of the study. No clinical signs of toxicity, alterations in clinical chemistry or haematology measurements, changes in absolute or relative organ weights or histologic changes resulting from glutaraldehyde in the drinking water were evident. Based on the results of this study, sub-chronic testing of glutaraldehyde should be conducted using a concentration not greater than 1000 ppm. In the other two-week range finding study, performed under GLP, Beagle dogs (2/sex/group) were administered glutaraldehyde in the drinking water or by gavage (BRRC, 1990). Glutaraldehyde in drinking water was administered at 0, 100 and 250 ppm. Oral gavage dosing of glutaraldehyde appeared unfeasible because of resultant vomiting when the test material was administered at a concentration of 1 % in dose volumes ranging between 0.5 and 10.0 ml/kg and at concentrations of 0.25% and 0.5% in a dose volume of 10.0 ml/kg. The 4 dogs (2 of each sex) given 250 ppm glutaraldehyde in drinking water appeared to have depressed water consumption and possible mild dehydration occurred in one of the females as evidenced by apparent increases in red blood cell counts, haemoglobin, haematocrit, and serum sodium and chloride concentrations. No other treatment-related findings as determined by clinical signs of toxicity, body weight, food consumption, clinical chemistry measurements, urinalysis, or necropsy findings were observed in male or female dogs treated with 100 or 250 ppm glutaraldehyde in the drinking water for 14 days. The results of this study indicated that gavage dosing of glutaraldehyde was not a feasible route of administration for repeated subchronic administration, and that based on the reduced water consumption and possible mild dehydration in at least one animal, 250 ppm is the maximum drinking water concentration that can be used for subchronic toxicity testing of glutaraldehyde in dogs.

In the follow-up 90 -day study, 20 male and 20 female Fischer 344 rats were exposed 10, 100, 1000 ppm 50% glutaraldehyde in drinking water (BRRC, 1985). The study was performed similar to OECD 408 and following GLP and for the control, 250 and 1000 ppm group an additional 10 animals per sex were treated for 13 weeks and this treatment period was followed by a recovery period of 4 weeks. There were no treatment-related effects on the incidence of mortality, clinical signs or ophthalmologic findings, haematology or clinical chemistry measurements, gross necropsy findings or histopathologic findings. Dose-related decreased water consumption in the 250 and 1000 ppm groups was attributed to aversion to the glutaraldehyde solutions. Decreased food consumption and body weights in the high dose group were related to the decreased water consumption. Alterations in urine parameters, including increased specific gravity and decreased total volume, in males and females from the 250 and 1000 ppm groups were also considered sequelae to the decreased water consumption. A statistically significant increase in urea nitrogen was observed in females from the intermediate and high dose groups at six weeks, but this change was not observed at the 13-week evaluation. A dose-related increase in absolute and relative kidney weights in females and kidney weights relative to final body weight in males was observed after 90 days of exposure to glutaraldehyde at 250 and 1000 ppm. All parameters were normal following a four-week recovery period. Based on the limited biological response and the rapid recovery, 1000 ppm glutaraldehyde was considered a minimally toxic dose, and 250 ppm was a dose which may have produced a marginal physiological response. The NOAEL was determined to be 50 ppm (5 and 7 mg/kg bw/day in males and females, respectively for glutaraldehyde).

In the other follow-up 90 -day study (BRRC, 1990) performed under GLP, glutaraldehyde was administered to dogs in drinking water for at least 13 weeks. There were 32 dogs assigned at random to 4 groups (4/sex/group). The animals received 0, 50, 150, or 250 ppm glutaraldehyde daily in the drinking water. Measurements or observations for clinical signs of toxicity, ophthalmic changes, water consumption, food consumption, body weights, haematology, clinical chemistry, urinalysis, necropsy, and histology were made. For males and females, there were intermittent occurrences of fluid-like and food-like vomitus. This was considered to be related to the acute irritant properties of the test material on the gastric mucosa. This irritancy and some moderate aversion to the test solutions due to the presence of the glutaraldehyde presumably resulted in the reduced water consumption in the males from the mid and high dose group and females from the high dose group as well as in the non-biologically significant changes in body weight gain observed in all groups of females. An increase in kidney weight relative to body weight for females from the high dose group was observed. This was considered to be of minimal biological significance based on the findings that urine analyses were unaltered and there was neither biochemical nor microscopic evidence for renal injury. Absolute left and right ovary weights, and left and right ovary to-body weight percentages were higher than controls in the females given 250 ppm. However, the biological significance of this finding is unclear, but is presumably related to the stage of the oestrus cycle and was not considered a response to treatment with glutaraldehyde. Therefore the no observed adverse effect level (NOAEL) under the conditions of this study was 250 ppm for both sexes (14.1 and 15.1 mg/kg bw/day in males and females pure glutaraldehyde).

Ballantyne (1986) performed a 4 -day and 7 day toxicity study. In the 4 -day study, male Wistar rats were exposed to glutaraldehyde in drinking water. Animals ingested 180, 440, and 640 mg/kg bw glutaraldehyde. No mortality occurred, body weight, food and water consumption and urine production were significantly depressed in all treated groups. Organ weighing revealed that the absolute liver weight as well as the relative kidney weight was significantly depressed in the 1000 and 2000 ppm treated groups. In the 7 -day study, male Wistar rats were exposed to glutaraldehyde in feed. Animals ingested 0, 100, 480, 1020, and 1630 mg /kg bw/day glutaraldehyde. Neither mortality nor clinical signs of toxicity were observed. Body weight was statistically significantly depressed at the highest tested dose. Food consumption was reduced at the highest tested dose. In the highest tested dose, a decrease in absolute liver and kidney weights was reported. In the 1020 mg/kg bw/day group, the relative liver weight for the males was statistically significantly increased compared to control; however, as the difference was minimal, the effect seen at 1000 mg/kg bw/day was considered to be incidental. Therefore, the NOAEL was determined to be 1020 mg/kg bw/day.

An in vivo study of laboratory rats was performed to determine the effect of glutaraldehyde exposure on the esophagus (Isserow 1998). Ten laboratory rats were divided into five groups that received daily gavage of the esophagus with either 3.2% glutaraldehyde solution (treated rats) or 1 mL of normal saline (control rats) for varying durations (1, 2, 3, and 4 days). After the rats were killed (10 hrs after last treatment), histologic sections from the esophagus were reviewed in a blind fashion. Histologic examination revealed greater neutrophilic infiltration in the submucosa of the gastroesophageal junction in three of four glutaraldehyde-treated rats compared with controls. Treated rats also had evidence of segmental esophageal vasculitis not seen in any of the controls. Both controls and treated rats had areas of myositis and myonecrosis within the esophagus. It was concluded by the authors that glutaraldehyde has a toxic effect on the rat esophagus.

Inhalation repeated dose toxicity

Rat

In a 13 -week study by the NTP, male and female Fischer 344 rats were exposed to 0, 0.0625, 0.125, 0.25, 0.5 and 1 ppm by whole body exposure, 5 days a week, 6 hours per day (NTP, 1993). Clinical signs of toxicity were seen during the first 5 weeks of treatment at 1 ppm, including dyspnoea, ruffled fur and emaciation. The final body weight of the 1000 ppm males were as ca. 90% of control and the final body weights of the 0.5 and 1 ppm females were ca. 93 -96% of control. At the end of the 13-week experiment, no biologically relevant changes in haematology and clinical chemistry were seen; effects were sporadic and not treatment-related. Considering sperm morphology and vaginal cytology, the treated rats of both sexes inconspicuous. Necropsy revealed some statistically significant increases in relative weights of some organs, which however were small and without any toxicological significance. Referring to the findings in the nasal passages of glutaraldehyde-treated rats, a small number of rats exposed to 1 ppm died before the 6-week time point; these deaths were attributed to glutaraldehyde exposure-associated occlusion of the external nares. Necropsy of the treated rats revealed lesions, which mainly were confined to the anterior third of the nose and were present in both sexes. In contrast, no histopathological evidence of treatment-related effects was observed in the trachea, the central airways or the lungs; minimal changes were seen in the larynx. The nasal lesions affected the respiratory mucosa (nasal cavity and tips of the turbinates) and the olfactory epithelium (dorsal meatus); hyperplasia, squamous metaplasia, olfactory degeneration, squamous exfoliation (accumulation of keratin, cell debris and bacteria in the lumen of the nasal vestibule) and focal erosions were reported. Neutrophilic inflammation was seen in most 1 ppm rats. The local NOAEC was determined to be 0.125 ppm (equivalent to 0.0005 mg/L glutaraldehyde). The NOAEC for systemic effects was determined to be 1 ppm (equivalent to 0.004 mg/L)

In the 2 -year study by the NTP, male and female Fischer 344 rats were exposed to 0, 0.25, 0.5 and 0.75 ppm by whole body exposure, 5 days a week, 6 hours per day (NTP, 1999). Some female rats of the 0.75 ppm group were thin to emaciate and were sacrificed in extremis. The survival of female rats treated with 0.50 and 0.75 ppm was statistically significantly decreased compared to controls; survival of the treated males was similar to control. The mean body weights of all treated males and of the females of the 0.50 and 0.75 ppm groups were below control values; this effect was considered to be treatment-related. Statistically significant and/or biological relevant changes were mainly seen in the nose of the glutaraldehyde-treated rats. No treatment-related neoplastic lesions were observed. The non-neoplastic lesions were limited primarily to the most anterior region of the nasal cavity. Hyperplasia and inflammation of the squamous epithelium; hyperplasia, goblet cell hyperplasia, inflammation, and squamous metaplasia of the respiratory epithelium, and hyaline degeneration of the olfactory epithelium were observed. No local NOAEC could be derived; the local LOAEC was determined to be 0.25 ppm (equivalent to 0.001 mg/L glutaraldehyde). The NOAEC for systemic effects was determined to be 0.25 ppm (equivalent to 0.001 mg/L)

In a 9 -day repeated dose toxicity study by Ballantyne et al. (1986), male and female Fischer 344 rats were whole body exposed to 0, 0.2, 0.63, and 2.1 ppm glutaraldehyde vapour (Analytical concentration: 0.00083, 0.0026 and 0.0087 mg/L), 6 hours per day. Exposure to glutaraldehyde resulted in mortalities (9/10 males and 7/10 females) at the highest tested concentration. At the two lower concentrations, sensory irritation, reduction in feed consumption and in body weight gain and clinical symptoms including nasal discharge, lacrimation and salivation, were observed. In addition, for the males of the 0.63 ppm group, significant difference in absolute and relative liver, lung, kidney and testes weight. For the 0.63 ppm females, a significant difference in relative liver and lung weight (27%) were reported. Thus the LOAEC was 0.2 ppm, corresponding to 0.00083 mg/l air. 

In a 11-day repeated dose toxicity study by Ballantyne et al. (1986), male and female Fischer 344 rats were whole body exposed to 0, 0.3, 1.1, and 3.1 ppm glutaraldehyde vapour, 6 hours per day, 5 days per week. The results of this study were in accordance with the findings of the preliminary nine-day inhalation study on rats. According to the author, the effects referring to body weight, food consumption, water consumption and organ weight were probably due to the sensory irritant and related stress effects of the treatment. The haematological and chemical-clinical findings probably reflected the marked disturbances due to reduced food and water intake, and a possible early liver damage. The inflammatory changes in the nasal mucosa were compatible with the irritant potential of glutaraldehyde. No peripheral neurotoxic effects could be evidenced.

In a 14 -week repeated dose toxicity study by Ballantyne et al. (1986), male and female Fischer 344 rats were whole body exposed to 0, 0.02, 0.05, 0.2 ppm glutaraldehyde vapour, 6 hours per day, 5 days per week. No deaths occurred throughout the exposure period. Signs of irritation, consisting of perinasal discharge and encrustation, were seen in males and females at 0.05 and 0.2 ppm. Males exposed to 0.2 and 0.05 ppm glutaraldehyde showed a reduced body weight over the whole study. At 0.2 ppm, increased creatinine phosphokinase activity was reported for both sexes. At 0.05 ppm, increased hydroxybutyrate dehydrogenase and lactate dehydrogenase activities were reported for both sexes. Necropsy revealed no treatment-related abnormalities. The results of the present subchronic inhalation study confirmed the known sensory irritating potential of glutaraldehyde at low concentrations, but there was no evidence of respiratory tract pathology at the concentrations of vapour generated (i.e. 0.02, 0.05 and 0.2 ppm). In conclusion, based on the reduced body weight and signs of irritation, the NOAEC was determined to be 0.02 ppm (0.00008 mg/L).

Rat: Respiratory effect-specific studies

In a 28-day repeated dose toxicity study by Halatek et al. (2003), male Wistar rats were whole body exposed to 0, 0.025, or 0.1 ppm glutaraldehyde vapour (0.0001 and 0.0004 mg/L), 6 hours per day, 5 days per week. At the end of the exposure period of 28 days, groups of rats were sacrificed following a post-exposure period of 24 h, 48 h or 7 days. The lungs of the sacrificed animals were subjected to bronchoalveolar lavage. Serum CC16 protein was determined. Furthermore, total protein concentration in the BALF, glutathione S-transferase in postmitochondrial supernatant (GST), and hyaluronic acid (HA) in the BALF were determined. Pieces of lung tissue were prepared for ultrastructural examination by means of electron microscopy. After 4 weeks of exposure to 0.1 ppm glutaraldehyde and a post-exposure period of 24 hours, lung weight was significantly decreased. Furthermore, lower levels of CC16 increased levels of HA and lower levels of GST activity were reported. At 0.025 ppm and after 48 h post exposure, the same pattern was observed for CC16 and HA, however the changes were not significant when compared to control. Total protein in BALF was inconspicuous, indicating that the vascular and epithelial permeability at all post-exposure time points were unchanged. The ultrastructural examination of lung tissue revealed pronounced morphological changes in rats treated with 0.1 ppm after a post-exposure period of 24 hours; in fact, numerous vacuoles and dilated spaces in epithelial cells in bronchioles were reported, which were indicative of a destructive effect of glutaraldehyde on the cellular membrane. After 48 h post-exposure and at 0.1 ppm, lipid vacuoles were seen in the Clara cells of the bronchial epithelium, and in endothelial cells of the alveolar capillaries; these vacuoles were attributed to disturbed lipid metabolism. Many foci of collagen fibres were observed after 7 days post-exposure.

The acute, sub-acute, and sub-chronic responses to glutaraldehyde in the respiratory tract of rats were characterized using histopathology and epithelial cell labeling index (ULLI) as end points in the publication by Gross (1994). Therefore, male and female F344 rats were whole-body exposed for 1 day, 4 days, 6 weeks, or 13 weeks to 0, 0.625, 0.125, 0.25, 0.5, or 1.0 ppm glutaraldehyde using a recycling inhalation chamber. Exposure to the higher concentrations of the test substance resulted in clinical observations such as bloating and/or gasping. During week 3 of the study, 2 male and 3 female rats from the 1.0 ppm exposure group died. These deaths were attributed to glutaraldehyde exposure-associated occlusion of the external nares. No macroscopic abnormalities were found at necropsy. Treatment-induced lesions, including epithelial erosions, inflammation, and squamous metaplasia, were confined to the anterior third of the nose and were present in both sexes. No histopathological evidence of glutaraldehyde-induced responses was observed in the trachea, central airways, or lungs, while the larynx showed minimal changes. There were clear increases in ULLI in association with acute and subacute cytotoxic responses, with similar concentration-response relationships. Neutrophilic infiltration of the squamous epithelium of the nasal vestibule, became progressively more severe with increasing exposure time and was associated with increased ULLI.

Mouse

In a 13 -week study by the NTP, male and female B6C3F1 mice were exposed to 0, 0.0625, 0.125, 0.25, 0.5 and 1 ppm by whole body exposure, 5 days a week, 6 hours per day (NTP, 1993). Mortality was observed in mice from 0.5 ppm upwards (20% in 0.5 ppm-females, 100% at 1 ppm). Clinical signs of toxicity were seen at 0.5 ppm (dyspnoea during the first weeks of treatment) and 1 ppm, mainly including dyspnoea, emaciation and abnormal posture. For male mice of the 0.125, 0.25 and 0.5 ppm groups, a concentration-related reduction in body weight gain was reported. This was also true for the females of the 0.25 and 0.5 ppm groups. Sperm morphology in treated male mice were was inconspicuous. The female mice of the 250 and 500 ppb groups displayed longer oestrus and diestrus phases and shorter metestrus and proestrus phases when compared to control. At necropsy, increases in relative weights of some organs were reported, which however were of no toxicological significance. Gross pathological changes were reported for mice that died during the experiment or were sacrificed in extremis. The more common findings included dilation of the stomach and intestine (probably related to dyspnoea and subsequent air swallowing), and paler and smaller spleen (probably due to lymphoid depletion). Histopathological lesions mainly affected the nasal passages and turbinates of mice. No local NOAEC could be derived; the local LOAEC was determined to be 0.0625 ppm (equivalent to 0.00025 mg/L glutaraldehyde). The NOAEC for systemic effects was determined to be 0.125 ppm (equivalent to 0.0005 mg/L)

In the 2 -year study by the NTP, male and female B6C3F1 mice were exposed to 0, 0.0625, 0.125, and 0.25 ppm by whole body exposure, 5 days a week, 6 hours per day (NTP, 1999). No treatment-related clinical symptoms were seen in mice, and survival in all test groups and for both sexes was similar to controls. The male mice showed no treatment-related effects on body weight. The mean body weights of the female mice of the 0.250 ppm group were decreased compared to control. Statistically significant and/or biological relevant changes were mainly seen in the nose of the glutaraldehyde-treated mice. No exposure-related neoplastic lesions were observed in the mice, neither in the males nor in the females. Non-neoplastic lesions were limited primarily to the most anterior region of the nasal cavity. The nasal lesions in the mice were qualitatively similar to those seen in rats. Squamous metaplasia of the respiratory epithelium was observed in both sexes of mice while female mice also had inflammation and hyaline degeneration of the respiratory epithelium. The local NOAEC was determined to be 0.0625 ppm (equivalent to 0.00025 mg/L glutaraldehyde). The NOAEC for systemic effects was determined to be 0.125 ppm (equivalent to 0.0005 mg/L).

Mouse: Respiratory effect-specific studies

For assessment of respiratory tract injury, in a sub-acute repeated dose toxicity study by Zissu et al. (1994), male Swiss mice were whole body exposed to 0.3, 1.0, and 2.6 ppm (4 day exposure) and 0.3 and 1.0 ppm (9 and 14 day exposure). The animals were observed for clinical symptoms of toxicity. At necropsy, the lungs of the mice examined. The mice exposed to 1.0 and 2.6 ppm suffered from respiratory difficulties. 4/10 mice of the 2.6 ppm group died, that is why the testing with this concentration was stopped after 5 days. The mice exposed to 1.0 ppm glutaraldehyde during 14 days showed nervous running around indicative of increased excitation; they further suffered from abdominal swelling and a bad general state of health, and the fur was rough. The mice treated with 0.3 ppm glutaraldehyde were inconspicuous compared to controls. A decrease in body weight of 20% was reported for the mice of the 1.0 ppm group. Lesions affecting the nasal cavity were seen in all treated mice; the earliest lesions were observed after 4 days of exposure to 0.3 ppm. These lesions mainly affected the respiratory epithelium of the septum, the naso- and the maxilloturbinates. The epithelium covering the lateral wall also was concerned but at a lesser extent; the olfactory epithelium was not altered. An increase in severity of the lesions was evident between 0.3 and 1.0 ppm whereas for 1.0 and 2.6 ppm, the degree of severity of the lesions was the same. The severity of the lesions showed no exposure time-dependency. In the mice subjected to a 14 day-exposure to 1.0 ppm test substance followed by a resting period of 1 or 2 weeks, no amelioration of the lesions in terms of recovery was evident; in contrast, following a resting period of 4 weeks, a significant decline in severity of the lesions became evident. No exposure-related histological abnormalities were detected in the trachea and lungs. Both, control and treated mice, showed erythrocytes within the alveolar parenchyma of the lung.

In addition Zissu et al. (1998) also performed a chronic repeated dose toxicity study. Male and female B6C3F1 mice were whole body exposed to 0.1 ppm glutaraldehyde vapour, 6 hours per day, 5 days per week for 52 or 78 weeks. No treatment-related mortalities were observed. The treated animals showed no nasal discharge, no swelling of the nose/adjacent tissues, no discomfort dyspnoea and no nose pruritus. A statistically significant decrease in body weight gain was reported for the treated females compared to controls. In contrast, the treated males had an increased body weight gain compared to the concurrent control males. Treatment-related changes were found in the nasal vestibule of female mice that survived the experiment. No treatment-related changes such were seen in the trachea and the lungs. Few neoplastic changes were reported, but these findings were incidental and not treatment-related. The results of present study are in accordance with those of further repeated dose inhalation studies, all demonstrating and confirming that chronic exposure the glutaraldehyde vapours at concentrations close to the current threshold limit values induces lesions at the more anterior part of the nasal passages in rats and mice, with an anterior-posterior gradient being evident. The lesions likely are due to an irritation mechanism.

The acute, sub-acute, and sub-chronic responses to glutaraldehyde in the respiratory tract of rats were characterized using histopathology and epithelial cell labeling index (ULLI) as end points in the publication by Gross (1994). Therefore, male and female B6C3F1 mice were whole-body exposed for 1 day, 4 days, 6 weeks, or 13 weeks to 0, 0.625, 0.125, 0.25, 0.5, or 1.0 ppm glutaraldehyde using a recycling inhalation chamber. A total of 10 male and 10 female mice from the 0.1 ppm group died or were found moribund over the course of the study. One 0.5 ppm exposed female died during the study (week 8). Mice died due to substance induced occlusion of the external nares. No macroscopic abnormalities were found at necropsy, except at the 4 day time point, when a small number of mice that had been exposed to the highest concentrations of the test substance exhibited distension of the stomach and intestines with gas. Similar as with the exposed rats, treatment-induced lesions, including epithelial erosions, inflammation, and squamous metaplasia, were confined to the anterior third of the nose and were present in both sexes and species. No histopathological evidence of glutaraldehyde-induced responses was observed in the trachea, central airways, or lungs, while the larynx showed minimal changes. There were clear increases in ULLI in association with acute and subacute cytotoxic responses, with similar concentration-response relationships. The latter responses were generally most severe at the higher glutaraldehyde exposure concentrations, however, in female mice they were present at all concentrations of glutaraldehyde studied.

Dermal repeated dose toxicity

In a subchronic dermal toxicity study performed in accordance with OECD Guideline 411 and following GLP, 10 male and 10 female Sprague-Dawley rats per dose were exposed under semi-occlusive conditions 5 days per week, 6 hours per day for 13 weeks to the test substance (Centre International de Toxicologie, 2000). The test substance was applied at concentrations of 0, 5, 10 and 15% (w/w) (i.e. 0, 2.5, 5 and 7.5% glutaraldehyde) to groups of 10 males and 10 females Sprague-Dawley rats. The administration volume was 2 ml/kg body weight. The achieved dose-levels of glutaraldehyde were 0, 50, 100 and 150 mg/kg bw/day. The animals were checked daily for mortality, morbidity and clinical signs; in addition, a detailed clinical observation was performed on all animals of each group, once weekly. Body weight was recorded before allocation of the animals into groups, on the first day of treatment, and then once a week during die treatment period. Food consumption was recorded once a week during the treatment period. Ophthalmological examinations were performed on all animals before the beginning of the treatment period and on the animals of the control and high-dose groups in week 13. Haematological and blood biochemical investigations were performed on all surviving animals at the end of die treatment period. On completion of the treatment period, all animals were submitted to a complete macroscopic post-mortem examination and selected organs were weighed. Macroscopic lesions and specified tissues were preserved. Microscopic examination was performed on specified tissues/lesions of specified groups. Repeated cutaneous application of the test substance induced only dose-related signs of local irritation. There were no signs of systemic toxicity which were formally attributed to treatment with the test substance. Consequently, under the experimental conditions, the No Observed Adverse Effect Level for systemic toxicity was established at 150 mg/kg/day.

In a study by the Dow Chemical Company (1994), male and female F344 rats were dermally exposed to glutaraldehyde. Groups of 15 male and 15 female rats were assigned to each of 4 dose groups (0, 50, 100, 150 mg/kg/day). Five of the animals for each dose group/sex were maintained an additional 4 weeks post-dosing as a recovery group. Dosing solution was added directly to the skin, and the entire test site was covered with gauze and bandaging tape. The dosing site was occluded for 6 hours per day. There were no signs of systemic toxicity or treatment-related mortality in any dose group. Local effects were noted at the dermal test site. Male body weights were depressed 5.6% from the controls, but trended towards recovery during the additional 4-week observation phase. Body weight changes in females were seen, but not considered biologically significant due to the small magnitude of the changes. Food consumption in the high dose group animals was also decreased relative to controls. There were no clinical chemistry or haematology findings for males. Female platelet counts were increased for all exposure groups (nearly resolved by the end of recovery phase). Reticulocyte counts were elevated in a dose-related manner (resolved by end of recovery phase). Urea nitrogen was elevated in mid and high dose males, and was considered associated with skin irritation (resolved at the end of recovery phase). All organ weight findings were considered spurious and not attributed to direct treatment. Significant histopathological findings were noted at the dermal test site. All gross skin lesions were graded as minimal to moderate, and were confined to superficial layers of the skin. The severity of the skin lesions generally increased with increasing concentration of the dosing solution. Glutaraldehyde did not elicit any significant systemic toxicity in rats when dosed via the dermal route for 28 days. Findings were limited to gross and histopathological findings at the dermal test site, and effects secondary to repeated irritation. The LOEL is 50 mg/kg/day based on dermal effects at the test site.

In a study by the Bushy Run Research Center (1981), male C3H mice were exposed to glutaraldehyde in three experiment. 50µL of test solution was applied to the skin under open conditions, 5 times per week for 2 week. The test material was not removed from the skin by washing. In the first experiment, 100, 50 and 10% test solution was applied (50, 25, and 5% glutaraldehyde). In the second experiment, 1, 0.5, and 0.1% dilutions (0.5, 0.25, and 0.05% glutaraldehyde) and in the third experiment, 5% dilution (2.5% glutaraldehyde) was applied. Animals were checked for mortality, clinical signs, and body weight. Necropsy was performed and skin and body cavities of the mice were carefully examined and dorsal skin and gross lesions were fixed in buffered formalin for further examinations. In the first experiment all animals receiving 50 and 100% of test material lost weight and died after 4 to 9 applications, indicating that cumulative toxicity is possible by repeated dermal contact with the test material. No consistent features were seen an necropsy of these animals. Mice receiving 10 % glutaraldehyde had decreased body weights after 4 and 6 doses, but not thereafter. In the two other experiments, no evidence of short-term cumulative toxicity was observed. The NOAEL was therefore determined to be 5% test material (2.5% glutaraldehyde).

In a study by Stonehill et al. (1963), rabbits were exposed daily for 6 weeks to 0.5mL of 2% glutaraldehyde solution under open conditions. After 7 days a yellowish discoloration of skin and hair was seen after the first application, which gradually became more intense turning to golden brown over the whole period of treatment. Spotty, mild questionable (because of skin discoloration) erythema was reported, as well as slight to moderate, reversible rash. Questionable scarring also was seen, which cleared despite of further applications. After 42 days excepted for the discoloration reported above, no further intensification of the effects described above was seen. The discoloration persisted for 35 days following the last application.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Three WoE studies were identified. These were all performed in compliance with OECD guidelines and following GLP. NOAELs determined in these three studies were similar and ranged from 14.6 to 23.0 mg/kg bw pure glutaraldehyde. The lowest NOAEL in rat was observed in a 90-day study.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Based on all available data, most studies indicate that the NOAEC for systemic effects after repeated inhalation exposure will be between 0.5 and 1 mg/m3. The lowest NOAEC of 0.5mg/m3 was observed in a 2-year mouse study.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Based on all available data, most studies indicate that the NOAEC for local effects after repeated inhalation exposure will be between 0.25 and 0.5 mg/m3. The lowest NOAEC of 0.25 mg/m3 was observed in a 2-year mouse study.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The study performed in accordance with OECD guideline and following GLP has been selected as key. This was also the study with the longest exposure period.

Justification for classification or non-classification

Systemic toxicity of glutaraldehyde under repeated oral exposure conditions is not expected, possible effects rather would be related to the irritating potential of this substance. No systemic toxicity of glutaraldehyde under repeated dermal exposure conditions is expected. Under repeated inhalation exposure conditions, the upper respiratory tract was identified as target for the toxicity of glutaraldehyde vapours. Therefore, according to Annex VI of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and based on the available data, glutaraldehyde does not have to be classified.