Glutaral

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: EPA OPP 83-5

Deviations:

yes

Remarks:

This exception is that verified copies of data collected on heat-sensitive paper and of some data printed on calculator tape were retained and the original data were discarded.

GLP compliance:

yes

Specific details on test material used for the study:

- Purity: approx. 50-51% as measured by titration
Assumed to be stable under typical storage conditions.

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: approximately 155 g (males) and 110 g (females)
- Fasting period before study: not reported
- Housing: The animals were housed 2 per cage for approximately 3 weeks in stainless steel, wire mesh cages (22.5 x 15.5 x 18.0 cm high). Following this 3-week acclimation period, the animals were individually housed for the remainder of the study. Approximately 11 months into the study, all male animals were moved to slightly larger cages (30.5 x 15.5 x 18.0). Male animals were housed in this cage size until the end of the study.
- Diet (e.g. ad libitum): ground, certified Rodent Chow® #5002, ad libitum
- Water (e.g. ad libitum): tap water or filtered tap water, ad libitum
- Acclimation period: approximately 3 weeks

DETAILS OF FOOD AND WATER QUALITY: Water analyses were provided by the supplier, the N US Corporation, Materials Engineering and Testing Co., and Lancaster Laboratories, Inc. at regular intervals. Analyses for chemical composition and possible contaminants of each feed lot were performed by the Ralston Purina Co. or Purina Mills, Inc.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9-25°C (66-77°F)
- Humidity (%): 40-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hour photoperiod (approximately 0500 to 1700 hours for the light phase)

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: In all cases a concentrated premix was prepared by addition of the appropriate amount of the test substance to water to yield a concentration of 10000 ppm. The dosing solutions were prepared by dilution of the appropriate amount of the premix.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose solutions were analyzed by gas chromatography. Homogeneity and stability in the drinking water were also verified. Solution concentrations were verified for all dose levels for the first 4 weeks of study prior to administration to the animals. Thereafter, at least one sample from each preparation was analyzed every 4 weeks along with a control sample.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

Continuous in the drinking water

Dose / conc.:

50 ppm

Remarks:

equivalent to 3.6 mg/kg/day (males) and 5.5 mg/kg/day (females)

Dose / conc.:

250 ppm

Remarks:

equivalent to 17.1 mg/kg/day (males) and 25.0 mg/kg/day (females)

Dose / conc.:

1 000 ppm

Remarks:

equivalent to 63.9 mg/kg/day (males) and 85.9 mg/kg/day (females)

No. of animals per sex per dose:

100

Control animals:

yes, concurrent vehicle

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes - Time schedule: Twice daily - Cage side observations: Mortality

DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: once weekly, with palpitations for masses beginning on the 27th week

BODY WEIGHT: Yes - Time schedule for examinations: prior to study start, and weekly thereafter until termination

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly for the first 13 weeks, and every other week thereafter

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes - Time schedule for examinations: weekly for the first 13 weeks, and every other week thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes - Time schedule for examinations: prior to study start, at weeks 52, 78, and 104, and prior to termi nation - Dose groups that were examined: all animals except those chosen for clinical pathology evaluations

HAEMATOLOGY: Yes - Time schedule for collection of blood: weeks 13, 26, 52, 78, and 104 - Anaesthetic used for blood collection: methoxyflurane - Animals fasted: Yes - How many animals: 10/sex/group - Parameters checked: Haematocrit, haemoglobin, mean corpuscular volume (MCV), mean corpus cular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), erythrocyte count, leukocyte count, differential leukocyte count, platelet count

CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: weeks 12, 25, 51, 77, and 103 - Animals fasted: Yes - How many animals: all - Parameters checked: Glucose (fasting), urea nitrogen, creatinine, total protein, albumin, globulin ( calculated), total bilirubin, direct bilirubin, indirect bilirubin, calcium, phosphorus, sodium, potassium, chloride, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALK), glutamate dehydrogenase (GLDH)

URINALYSIS: Yes - Time schedule for collection of urine: weeks 10, 25, 51, and 103 - Metabolism cages used for collection of urine: Yes - Animals fasted: No - Parameters checked: Urine osmolality, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, total volume, colour and turbidity, microscopic constituents

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: On the day of sacrifice, body weights were obtained to allow expression of relative organ weights. A complete necropsy was performed on all animals. The liver, kidneys, brai n, heart, adrenals, ovaries (females), spleen, and testes (male) were weighed. The following tissue s were collected and retained in 10% neutral buffered formalin: gross lesions, lungs with mainstem bronchi, nasopharyngeal tissue, brain (cerebral cortex, cerebellar cortex, medulla/pons), pituitary, thyroid – parathyroid complex, thymic region, trachea, heart, sternum (including marrow), salivary gland, liver, spleen, kidneys, adrenals, pancreas, testes – males, prostate – males, seminal vesicles – males, ovaries – females, vagina – females, uterus (corpus and cervix) – females, aorta, skin, oesoph agus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, representative lymph nodes (mesenteric and submandibular), mammary gland – females, thigh musculature, periph eral nerve (sciatic), eyes, femur (including articular surface), spinal cord (cervical, midthoracic, and lumbar), Zymbal’s gland, exortibal lacrimal gland.

HISTOPATHOLOGY: All tissues to be examined microscopically were paraffin embedded, sectioned at approximately 5 microns, and stained with haematoxylin and eosin. Lesions, when appropriate, were graded as to severity into 5 categories (minimal, mild, moderate, marked, or severe). The saved tissues were examined microscopically for all rats in the control and high dose groups and for all rats from the remaining groups which died or were sacrificed moribund during the study. In addition, the lungs, liver, kindeys, spleen, and all gross lesions were examined histologically from all animals in the other dosage groups.

Statistics:

The data for quantitative continuous variables were intercompared for the three treatment groups and the control group by use of Levene's test for equality of variances, analysis of variance (ANOVA), and t-tests. The t-tests were used when the f value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated a heterogeneous variance, all groups were compared by an ANOVA for unequal variances, followed by a separate variance t-test for pairwise comparisons if needed. Non-parametric data was statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U-test when appropriate. Incidence data were compared using the Fisher's exact test. Additional analyses included a dose-response trend for the incidence of LGL luekemia to determine if increased severity of the leukemia was related to higher doses. All tumour incidence data was used to conduct a single overall statistical test for the presence of any carcinogenic effect. For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadic findings of urine soiling, emaciation, laboured respiration, pallor, and yellow cutis was observed, but the relevance of the observations was unclear due to the lack of a dose-response relationship.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In male or female rats, there were no differences in the incidence of mortality or longevity of treated animals compared to the controls that were considered to be related to treatment. A statistically significant decrease in the mean survival time in the 50 ppm dose group of female rats was not considered to be related to treatment due to the lack of a dose-response relationship.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight and weight gains were generally decreased throughout the study for 250 and 1000 ppm animals, compared to control values .

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was generally decreased throughout the study for 250 and 1000 ppm animals, compared to control values.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was generally decreased throughout the study for 50, 250, and 1000 ppm an imals, compared to control values.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant, treatment-related effects in any treated group.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant, treatment-related effects in any treated group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant, treatment-related effects in any treated group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased urine volume and increased urine osmolality were noted in males and females at 250 and 1000 ppm, and are considered to be a compensatory effect associated with decreased water cons umption. There were sporadic changes in urine pH. Urinary protein and bilirubin concentrations were increased in high-dose males and females at weeks 12 and 25, respectively. These changes are also considered secondary to decreased water consumption.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At necropsy (at 52, 78 and 104 weeks), the only statistically significant changes in organ weights w ere for the kidney. Changes seen in urinary parameters and kidney weights were likely related the d ecreased water consumption rather than to a direct toxic action of glutaraldehyde.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross evidence of gastric irritation was present in many 250 and 1000 ppm animals. Gross lesions of the stomach included multifocal colour change, nodules, thickening of the wall, and ulceration of the gastric mucosa.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings included oedema, gastritis, keratinized cysts (1000 ppm dose group of males at 104 weeks only), and hyperplasia of the squamous epithelium.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The main finding of the study was a statistically significant increase in the number of large granular lymphocytis 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.

Dose descriptor:

NOEL

Effect level:

< 50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

water consumption and compound intake

Critical effects observed:

yes

Organ:

liver

spleen

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

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. All slides containing sections of spleen, liver, and lung were examined by the reviewing pathologist, and the quality of the slides was considered to be of good quality and prepared according to the study protocol. The working group confirmed an increased incidence of LGL leukemia in all groups compared to controls, but the responses were not proportional to the dose despite the 20-fold increase in doses between low and high dose levels. Furthermore, the percentage of female rats with Stage 3 leukemia was greatest in the control group, while stage 1 and 2 leukemia were most frequent in the high dose group. When a weight-of-evidence approach was applied, the working group concluded that the observed increase in LGL leukemia in female F344 rats had an uncertain relationship to ingestion of glutaraldehyde in drinking water. There was no known mechanism for the increased incidence in female rats, and there was no comparable finding in male rats. There was no evidence of systemic, acute, subchronic, or chronic toxicity involving the hematopoetic system.In vivobone marrow cytogenicity studies were reported to be negative in both sexes, nor did the NTP report similar findings following a 2-year inhalation study. A chronic drinking water study conducted by BASF at the same dose levels in the Wistar rat (a strain with a low spontaneous frequency of background LGL leukemia) also could not repeat the findings. The Working group therefore 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.

Conclusions:

No clear NOEL was established for this study due to increased incidence of LGLL in all dose groups of female rats.

Executive summary:

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. Observations for mortality were made twice daily. Detailed clinical observations were performed once weekly, with palpitations for masses beginning on the 27th week. Observations for overt clinical signs were made on all other days. Body weights were collected prior to study start, and weekly thereafter until termination. Food and water consumption were measured weekly for the first 13 weeks, and every other week thereafter. Eyes were examined by indirect ophthalmoscopy prior to study start, at weeks 52, 78, and 104, and prior to termination for all animals except those chosen for clinical pathology evaluations. Clinical pathology evaluations were conducted on weeks 12, 25, 51, 77, and 103. Animals were fasted and bled for haematology and clinical chemistry measurements. Urine was evaluated for osmolality, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, total volume, color and turbidity, and microscopic constituents at weeks 10, 25, 51, and 103.Rats (10/sex/dose) were sacrificed at week 52 and 78, and all remaining animals at 104 weeks. A complete necropsy was performed on all animals.

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. All slides containing sections of spleen, liver, and lung were examined by the reviewing pathologist, and the quality of the slides was considered to be of good quality and prepared according to the study protocol. The working group confirmed an increased incidence of LGL leukemia in all groups compared to controls, but the responses were not proportional to the dose despite the 20-fold increase in doses between low and high dose levels. Furthermore, the percentage of female rats with Stage 3 leukemia was greatest in the control group, while stage 1 and 2 leukemia were most frequent in the high dose group. When a weight-of-evidence approach was applied, the working group concluded that the observed increase in LGL leukemia in female F344 rats had an uncertain relationship to ingestion of glutaraldehyde in drinking water. There was no known mechanism for the increased incidence in female rats, and there was no comparable finding in male rats. There was no evidence of systemic, acute, subchronic, or chronic toxicity involving the hematopoetic system.In vivobone marrow cytogenicity studies were reported to be negative in both sexes, nor did the NTP report similar findings following a 2-year inhalation study. A chronic drinking water study conducted by BASF at the same dose levels in the Wistar rat (a strain with a low spontaneous frequency of background LGL leukemia) also could not repeat the findings. The Working group therefore 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA OPP 83-5

Deviations:

yes

Remarks:

This exception is that verified copies of data collected on heat-sensitive paper and some data printed on calculator tape were retained and the original data were discarded.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Glutaral

EC Number:

203-856-5

EC Name:

Glutaral

Cas Number:

111-30-8

Molecular formula:

C5H8O2

IUPAC Name:

glutaraldehyde

Test material form:

liquid

Specific details on test material used for the study:

- Purity: approx. 50-51% as measured by titration
Assumed to be stable under typical storage conditions.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY, USA
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: approximately 155 g (males) and 110 g (females)
- Fasting period before study: not reported
- Housing: The animals were housed 2 per cage for approximately 3 weeks in stainless steel, wire mesh cages (22.5 x 15.5 x 18.0 cm high). Following this 3-week acclimation period, the animals were individually housed for the remainder of the study. Approximately 11 months into the study, all male animals were moved to slightly larger cages (30.5 x 15.5 x 18.0). Male animals were housed in this cage size until the end of the study.
- Diet (e.g. ad libitum): ground, certified Rodent Chow® #5002, ad libitum
- Water (e.g. ad libitum): tap water or filtered tap water, ad libitum
- Acclimation period: approximately 3 weeks

DETAILS OF FOOD AND WATER QUALITY: Water analyses were provided by the supplier, the NUS Corporation, Materials Engineering and Testing Co., and Lancaster Laboratories, Inc. at regular intervals. Analyses for chemical composition and possible contaminants of each feed lot were performed by the Ralston Purina Co. or Purina Mills, Inc.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9-25°C (66-77°F)
- Humidity (%): 40-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hour photoperiod (approximately 0500 to 1700 hours for the light phase)

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: In all cases a concentrated premix was prepared by addition of the appropriate amount of the test substance to water to yield a concentration of 10000 ppm. The dosing solutions were prepared by dilution of the appropriate amount of the premix.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose solutions were analyzed by gas chromatography. Homogeneity and stability in the drinking water were also verified. Solution concentrations were verified for all dose levels for the first 4 weeks of study prior to administration to the animals. Thereafter, at least one sample from each preparation was analyzed every 4 weeks along with a control sample.

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

Continuous in the drinking water

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 ppm

Remarks:

equivalent to 3.6 mg/kg/day (males) and 5.5 mg/kg/day (females)

Doses / concentrations 2

Dose / conc.:

250 ppm

Remarks:

equivalent to 17.1 mg/kg/day (males) and 25.0 mg/kg/day (females)

Doses / concentrations 3

Dose / conc.:

1 000 ppm

Remarks:

equivalent to 63.9 mg/kg/day (males) and 85.9 mg/kg/day (females)

No. of animals per sex per dose:

100

Control animals:

yes, concurrent vehicle

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations: Mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly, with palpitations for masses beginning on the 27th week

BODY WEIGHT: Yes
- Time schedule for examinations: prior to study start, and weekly thereafter until termination

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly for the first 13 weeks, and every other week thereafter

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly for the first 13 weeks, and every other week thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to study start, at weeks 52, 78, and 104, and prior to termination
- Dose groups that were examined: all animals except those chosen for clinical pathology evaluations

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 13, 26, 52, 78, and 104
- Anaesthetic used for blood collection: methoxyflurane
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters checked: Haematocrit, haemoglobin, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), erythrocyte count, leukocyte count, differential leukocyte count, platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 12, 25, 51, 77, and 103
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Glucose (fasting), urea nitrogen, creatinine, total protein, albumin, globulin (calculated), total bilirubin, direct bilirubin, indirect bilirubin, calcium, phosphorus, sodium, potassium, chloride, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALK), glutamate dehydrogenase (GLDH)

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 10, 25, 51, and 103
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: Urine osmolality, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, total volume, colour and turbidity, microscopic constituents

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: On the day of sacrifice, body weights were obtained to allow expression of relative organ weights. A complete necropsy was performed on all animals. The liver, kidneys, brain, heart, adrenals, ovaries (females), spleen, and testes (male) were weighed. The following tissues were collected and retained in 10% neutral buffered formalin: gross lesions, lungs with mainstem bronchi, nasopharyngeal tissue, brain (cerebral cortex, cerebellar cortex, medulla/pons), pituitary, thyroid – parathyroid complex, thymic region, trachea, heart, sternum (including marrow), salivary gland, liver, spleen, kidneys, adrenals, pancreas, testes – males, prostate – males, seminal vesicles – males, ovaries – females, vagina – females, uterus (corpus and cervix) – females, aorta, skin, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, representative lymph nodes (mesenteric and submandibular), mammary gland – females, thigh musculature, peripheral nerve (sciatic), eyes, femur (including articular surface), spinal cord (cervical, midthoracic, and lumbar), Zymbal’s gland, exortibal lacrimal gland.

HISTOPATHOLOGY: All tissues to be examined microscopically were paraffin embedded, sectioned at approximately 5 microns, and stained with haematoxylin and eosin. Lesions, when appropriate, were graded as to severity into 5 categories (minimal, mild, moderate, marked, or severe). The saved tissues were examined microscopically for all rats in the control and high dose groups and for all rats from the remaining groups which died or were sacrificed moribund during the study. In addition, the lungs, liver, kindeys, spleen, and all gross lesions were examined histologically from all animals in the other dosage groups.

Statistics:

The data for quantitative continuous variables were intercompared for the three treatment groups and the control group by use of Levene's test for equality of variances, analysis of variance (ANOVA), and t-tests. The t-tests were used when the f value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated a heterogeneous variance, all groups were compared by an ANOVA for unequal variances, followed by a separate variance t-test for pairwise comparisons if needed. Non-parametric data was statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U-test when appropriate. Incidence data were compared using the Fisher's exact test. Additional analyses included a dose-response trend for the incidence of LGL luekemia to determine if increased severity of the leukemia was related to higher doses. All tumour incidence data was used to conduct a single overall statistical test for the presence of any carcinogenic effect. For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Sporadic findings of urine soiling, emaciation, laboured respiration, pallor, and yellow cutis was observed, but the relevance of the observations was unclear due to the lack of a dose-response relationship.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In male or female rats, there were no differences in the incidence of mortality or longevity of treated animals compared to the controls that were considered to be related to treatment. A statistically significant decrease
in the mean survival time in the 50 ppm dose group of female rats was not considered to be related to treatment due to the lack of a dose-response relationship.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight and weight gains were generally decreased throughout the study for 250 and 1000 ppm animals, compared to control values

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was generally decreased throughout the study for 250 and 1000 ppm animals, compared to control values.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was generally decreased throughout the study for 50, 250, and 1000 ppm animals, compared to control values.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant, treatment-related effects in any treated group.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant, treatment-related effects in any treated group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant, treatment-related effects in any treated group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased urine volume and increased urine osmolality were noted in males and females at 250 and 1000 ppm, and are considered to be a compensatory effect associated with decreased water consumption. There were sporadic changes in urine pH. Urinary protein and bilirubin concentrations were increased in high-dose males and females at weeks 12 and 25, respectively. These changes are also considered secondary to decreased water consumption.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross evidence of gastric irritation was present in many 250 and 1000 ppm animals. Gross lesions of the stomach included multifocal colour change, nodules, thickening of the wall, and ulceration of the gastric mucosa.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings included oedema, gastritis, keratinized cysts (1000 ppm dose group of males at 104 weeks only), and hyperplasia of the squamous epithelium.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The main finding of the study was a statistically significant increase in the number of large granular lymphocytis 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.

Effect levels

Dose descriptor:

NOEL

Effect level:

< 50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

50 ppm

System:

other: hepatobiliary and haematopoietic

Organ:

liver

spleen

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

Any other information on results incl. tables

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. All slides containing sections of spleen, liver, and lung were examined by the reviewing pathologist, and the quality of the slides was considered to be of good quality and prepared according to the study protocol. The working group confirmed an increased incidence of LGL leukemia in all groups compared to controls, but the responses were not proportional to the dose despite the 20-fold increase in doses between low and high dose levels. Furthermore, the percentage of female rats with Stage 3 leukemia was greatest in the control group, while stage 1 and 2 leukemia were most frequent in the high dose group. When a weight-of-evidence approach was applied, the working group concluded that the observed increase in LGL leukemia in female F344 rats had an uncertain relationship to ingestion of glutaraldehyde in drinking water. There was no known mechanism for the increased incidence in female rats, and there was no comparable finding in male rats. There was no evidence of systemic, acute, subchronic, or chronic toxicity involving the hematopoetic system.In vivobone marrow cytogenicity studies were reported to be negative in both sexes, nor did the NTP report similar findings following a 2-year inhalation study. A chronic drinking water study conducted by BASF at the same dose levels in the Wistar rat (a strain with a low spontaneous frequency of background LGL leukemia) also could not repeat the findings. The Working group therefore 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.

Applicant's summary and conclusion

Conclusions:

No clear NOEL was established for this study due to the decreased water consumption in all dose groups of male and female rats and increased incidence of LGLL in all dose groups of female rats.

Executive summary:

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. Observations for mortality were made twice daily. Detailed clinical observations were performed once weekly, with palpitations for masses beginning on the 27th week. Observations for overt clinical signs were made on all other days. Body weights were collected prior to study start, and weekly thereafter until termination. Food and water consumption were measured weekly for the first 13 weeks, and every other week thereafter. Eyes were examined by indirect ophthalmoscopy prior to study start, at weeks 52, 78, and 104, and prior to termination for all animals except those chosen for clinical pathology evaluations. Clinical pathology evaluations were conducted on weeks 12, 25, 51, 77, and 103. Animals were fasted and bled for haematology and clinical chemistry measurements. Urine was evaluated for osmolality, pH, protein, glucose, ketones, bilirubin, blood, urobilinogen, total volume, color and turbidity, and microscopic constituents at weeks 10, 25, 51, and 103. Rats (10/sex/dose) were sacrificed at week 52 and 78, and all remaining animals at 104 weeks. A complete necropsy was performed on all animals.

 

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. All slides containing sections of spleen, liver, and lung were examined by the reviewing pathologist, and the quality of the slides was considered to be of good quality and prepared according to the study protocol. The working group confirmed an increased incidence of LGL leukemia in all groups compared to controls, but the responses were not proportional to the dose despite the 20-fold increase in doses between low and high dose levels. Furthermore, the percentage of female rats with Stage 3 leukemia was greatest in the control group, while stage 1 and 2 leukemia were most frequent in the high dose group. When a weight-of-evidence approach was applied, the working group concluded that the observed increase in LGL leukemia in female F344 rats had an uncertain relationship to ingestion of glutaraldehyde in drinking water. There was no known mechanism for the increased incidence in female rats, and there was no comparable finding in male rats. There was no evidence of systemic, acute, subchronic, or chronic toxicity involving the hematopoetic system.In vivobone marrow cytogenicity studies were reported to be negative in both sexes, nor did the NTP report similar findings following a 2-year inhalation study. A chronic drinking water study conducted by BASF at the same dose levels in the Wistar rat (a strain with a low spontaneous frequency of background LGL leukemia) also could not repeat the findings. The Working group therefore 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.