Glutaral

Carcinogenicity

Description of key information

Valid studies have been conducted for the assessment of a carcinogenic potential of glutaraldehyde. A 2 year-oral study with rat treated via the drinking water was conducted according to the OECD TG 451 and followed GLP (BASF, 2003). A further 104 week study with Fischer 344 rats treated via the drinking water was recently published (Van Miller, 2002). No guideline was mentioned, and it was not specified whether the study followed GLP or not. However, the study design was well described and similar to the OECD TG 453, and the results were scientifically acceptable. An NTP technical report and a paper were published on the toxicology and carcinogenesis of glutaraldehyde administered in F344/N rats and B6C3F1 mice by inhalation (NTP, 1999; van Birgelen, 2000). The study conduct was in principle similar to OECD TG 451, with the focus being on the non-neoplasic and neoplasic findings at necropsy; survival and body weight changes also were considered. The (histo)pathological findings were submitted to the NTP Pathology Working Group chairperson for review. No carcinogenic potential could be evidenced from these oral and inhalative long-term animal studies conducted with glutaraldehyde.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test material (as cited in study report): Glutaraldehyde 50% aq. solution
- Physical state: homogeneous colourless-clear liquid
- Analytical purity: 50.5%
- Lot/batch No.: 50-4402
- Stability under test conditions: The stability of the test substance in drinking water over a period of 14 days at room temperature had been proven prior to starting the experiment. Further, the stability of the test substance was proven by reanalysis
- Storage conditions: 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: Elevage Janvier, Le Genest St Isle, France
- Age at study initiation: 42 +/- 1 days
- Weight at study initiation: mean body weight for the males, 211.3 g; mean body weight for the females, 166 g
- Housing: single, in type DK 11I stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2).
- Diet (e.g. ad libitum): basic maintenance diet for mice and rats, 9433 LL meal from Eberle Nafag AG, Gossau, Switzerland, ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: ca. 1 week
- Fasting: withdrawal of food for about 16 -20 hours prior sacrifice at test ending

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): fully air-conditioned room with central air-conditioning
- Photoperiod (hrs dark / hrs light): 12 hrs/2 hrs

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

- An amount of drinking water was weighed and the appropriate amount of test substance needed to get the wanted test concentration was added and mixed with a stirrer.
- The drinking test solutions were prepared twice a week.
- Control animals received drinking water without test substance.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The analytical investigations of the test substance preparations were carried out at the analytical chemistry laboratory of the department of Experimental Toxicology and Ecology of BASF AG;
- The stability of the test substance in drinking water over a period of 14 days at room temperature had been proven before study initiation. The analytical concentrations were within 90.5 to 109.3% of the nominal concentrations;
- As the test substance preparations were true aqueous solutions, no analyses for homogeneity were needed;
- During the study, analyses of the test substance preparations with respect to concentration control were conducted at test inititation, after about 3, 6, 9, 12, 15, 18 and 21 months, and towards the end of the study. The analyses from 3 months onwards were performed with samples taken at the end of
the time period for which the respective test substance preparations were used. The samples were taken out of randomly selected reserve drinking water bottles being stored in the animal room. Thus, the stability of the test substance in the drinking water was also proven under test conditions.

Duration of treatment / exposure:

2 years

Frequency of treatment:

daily

Post exposure period:

None

Dose / conc.:

100 ppm (nominal)

Remarks:

in water

Dose / conc.:

500 ppm (nominal)

Remarks:

in water

Dose / conc.:

2 000 ppm (nominal)

Remarks:

in water

No. of animals per sex per dose:

50 animals per sex and test group were used.

Control animals:

yes, concurrent no treatment

Details on study design:

- The test series comprised four groups of 100 rats (50/sex) each;
- Prior to the test inititation, the animals were allocated to the test groups according to weight.

Observations and examinations performed and frequency:

CLINICAL SYMPTOMS AND MORTALITY:
The animals were checked for symptoms of toxicity and mortality twice a day on working days, and once a day on Saturdays, Sundays and public holidays. A comprehensive clinical examination including palpation was conducted once a week.

BODY WEIGHT:
The rats were weighed prior test initiation, on day 0 (test start), once a week during the first 13 weeks of treatment and thereafter at 4 week-intervals until the end of the study; the last body weight assessment was done prior necropsy.

FOOD CONSUMPTION:
Food consumption was determined once a week over a period of 7 days during the first 13 weeks of treatment and thereafter at 4-week intervals until the end of the study. Food consumption was expressed as grams per animal and day.

FOOD EFFICIENCY:
The food efficiency (as group means) was calculated on the basis of the body weight and the food consumption using the appropriate formula.

WATER CONSUMPTION AND COMPOUND INTAKE:
Water consumption was determined once a week over a period of 7 days during the first 13 weeks of treatment and thereafter at 4-week intervals until the end of the study. Water consumption was expressed as grams per animal and day. The mean daily intake of test substance as group means was calculated using the appropriate formula.

OPHTHALMOSCOPIC EXAMINATION:
None.

HAEMATOLOGY:
Blood samples were collected from all animals at necropsy; differential blood smears were prepared. The blood smears of the control and the highest test group (2000 ppm) were evaluated. Blood smears of all animals, which were killed in extremis during the administration period, also were evaluated, independently from the test group. Leukocyte differential count as well as white and red cell morphology (light microscopical examination) were considered.

CLINICAL CHEMISTRY:
Not considered

URINALYSIS:
Not considered

Sacrifice and pathology:

Animals that died during the administration period were subjected to necropsy as soon as possible following death; the survivors were sacrificed at test ending.

ORGAN WEIGHT:
Prior sacrifice, the whole body of the anesthetized animals was weighed. The following organs were collected from all sacrificed animals for weighing: liver, kidneys, adrenals, testes, epididymes, uterus, ovaries, spleen, brain, heart.

GROSS PATHOLOGY:
All animals were subjected to gross pathological examination.

HISTOPATHOLOGY:
In addition to all gross lesions found, the following organs and tissues were fixed in 4% formaldehyde for more detailed examination: the target organs, brain, pituitary, thyroid, parathyroid, thymus, oesophagus, salivary glands, stomach, small and large intestines, liver, pancreas, kidneys, adrenals, spleen, heart, trachea, lungs, pharynx, larynx, nasal cavity, aorta, gonads, uterus, female mammary gland, prostate, urinary bladder, lymph nodes, bone, bone marrow, femur with knee joint, eyes, epididymes, sciatic nerve, spinal cord, seminal vesicles, skeletal muscle, skin.
All gross lesions were subjected to light microscopical examination.
Following organs and tissues were examined in the control and the high dose (i.e. 2000 ppm) sacrificed animals: brain, cecum, cervical cord, colon, duodenum, epididymides, esophagus, eyes, mammary glands, heart, ileum, jejunum, mandibular gland, mandibular lymphnode, mesenteric lymph nodes, ovaries, oviduct, uterus, vagina, pituitary, thyroid, parathyroid, thymus, pancreas, adrenals, spleen, nasal cavity, aorta, prostate, urinary bladder, bone marrow, femur with knee joint, lumbar cord, rectum, sternum with marrow, sublingual gland, thoracic cord, sciatic nerve, seminal vesicles, skeletal muscle, skin.
Following organs and tissues were examined in the control and all treated groups (100, 500 and 2000 ppm) sacrificed animals: forestomach, glandular stomach, kidneys, larynx, liver, lungs, pharynx, testes, trachea.
Animals that died intercurrently were assessed like control animals.

Statistics:

The statistical evaluation of the clinical parameters (food consumption, water consumption, body weight, body weight change, food efficiency) was based on the calculation of the means and standard deviation.
For the assessment of the statistical significance of the differences observed versus control, Dunnett´s test was applied (Dunnett CW, JASA, Vol. 50: 1096-1121, 1955; Dunnett CW, Biometrics, Vol. 20: 482-491, 1964).
The haematological parameters were assessed by calculation of the means and standard deviations.
For terminal body weight and absolute and relative organ weights, means and standard deviation were calculated.
Further statistical assessments were based on the Kruskal-Wallis test and if necessary, on the Wilcoxon test (Hettmannsperger TP, Statistical inference based on ranks, John Wiley & Sons, New York, 132-140, 1984; International Mathematical and Statistical Libraries, Inc., 2500 Park West Tower One, Houston, Texas 77042-3020, USA, nakl-1 – nakl-3; Miller RG, Simultaneous statistical inference, Springer-Verlag, New York Inc., 165-167, 1981; Nijenhuis A & Wilf SW, Combinatorial Algorithms, Academic Press, New York, 32-33, 1978).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 2000 ppm respiratory sounds brought in association with metaplastic changes in larynx and/or trachea as well as increased incidence in reddish-discolored urine were reported.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 2000 ppm, 2 males and 9 females died from asphyxia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Compared to control the 2000 ppm rats showed statistically significantly decreased body weights (BW) and BW changes (BWC). For the males the maximum BW reduction of 9.6% was seen on day 14, and the BWC reached –39% on day 7; for the females, the max. BW reduction of 11.1% was seen on day 595 and BWC reached -39.6% on day 7. For the 500 ppm females, BW and BWC also were significantly decreased.
The mean terminal body weight of the 2000 ppm rats was significantly decreased (6.1 and 10% below control respectively for males and females).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was significantly reduced for the 2000 and 500 ppm groups. The maximum reduction was 18.1% and 7.4% below control for the 2000 and the 500 ppm males respectively, and 15.2% and 8.9% below control for the 2000 and the 500 ppm females.

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was significantly reduced at 2000 ppm (males: max. 29.2% below control on day 728; females: max. 38.5% below control on day 595) and at 500 ppm (males: max. 14% below control on day 35; females: max. 24.3% below control on day 595 and 623). At 100 ppm, water consumption was significantly reduced for females only (max. 21.4% below control on day 707).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean terminal body weight of the 2000 ppm rats was significantly decreased (6.1 and 10% below control respectively for males and females). Consequently, liver, hear and brain showed decreased organ weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence of erosions/ulcer in the mucosa of the glandular stomach was reported for the females of the 2000 ppm group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Squamous epithelial metaplasia in the larynx was reported for the 2000 ppm group (18/50 males and 30/50 females, versus 0 cases in control). Squamous metaplasia in trachea also was seen at 2000 ppm (4/50 males and 11/50 females, versus 0 cases in control). Metaplasia was nearly always accompanied by accumulation of keratin detritus in the laryngeal and/or tracheal lumen. In five 2000 ppm females with laryngeal/tracheal metaplasia, inflammation in the tracheal lumen with/without purulent contents was seen. One 2000 ppm male with squamous metaplasia in larynx and trachea also showed squamous metaplasia of the bronchial epithelium. Some 2000 ppm animals with squamous laryngeal/tracheal metaplasia also displayed foreign body granuloma in their lungs. Purulent inflammation in the nasal cavity was seen in 6 females and 3 males of the 2000 ppm group. Furthermore, an increased combined incidence of grossly and/or histopathologically confirmed erosions/ulcer of the glandular stomach was reported for the 2000 ppm group (males: 20 cases versus 13 in control; females: 29 cases versus 18 in control). At 500 ppm diffuse a squamous metaplasia of the epithelium of the Iarynx was observed in 1 male rat.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Neoplastic findings were spontaneous in origin and showed no treatment-relationship.

Dose descriptor:

LOAEL

Effect level:

500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed at this dose

Remarks on result:

other: test material: 7 and 10 mg/kg bw/day for male and female active ingredient: 3.5 and 5 mg/kg bw/day for male and female

Mean daily test substance intake (mg/kg bw/day):

Concentration in the drinking water	Mean daily test substance intake (mg/kg bw/day)
	Males	Females
100 ppm	6.1	10.5
500 ppm	31.9	48.5
2000 ppm	120.7	176.4

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No guideline was mentioned and it was not specified in the publication whether the study followed GLP or not. However, the study design was well described and similar to guideline, and the results were scientifically acceptable.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Principles of method if other than guideline:

Method: other: no data

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report): Glutaraldehyde 50% aq. solution
- Supplier: Union Carbide Corporation (Bound Brook, NJ)
- Physical state: liquid
- Analytical purity: 50.0 - 51.3% (w/w)
- Stability under test conditions: The stability of the 50 and 1000 ppm solutions of glutaraldehyde (GA) was confirmed by analysis directly after preparation and after 7, 14 and 21 days of storage at room temperature. GA was found to be stable for at least 21 days.

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)
- Age at study initiation: at test initiation, the males and females were about 48 days old (ca. 27 days old at arrival, 3 weeks of acclimatisation prior test starting).
- Weight at study initiation: the body weights of the males at test initiation ranged between 131.03 and 185.23 g ; for the females, the body weights ranged between 98.48 and 124.36 g .
- Housing: single housing in stainless steel, wire-mesh cages
- Diet (e.g. ad libitum): Certified Rodent Chow # 5002 (Purina Mills, Inc.), ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: three weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 19 - 25
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12 hrs/ 12 hrs

PRELIMINARY HEALTH CHECK
A representative number of animals was subjected to a preliminary health screening including necropsy, histology of various tissues, hematology, serum chemistry, serum viral antibodies and examination for fecal parasites.

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

- The selection of the test concentrations was based on the results of a preliminary subchronic (90-day) study;
- A premix of 10000 ppm GA in water was prepared and served for the preparation of the dosing solutions by dilution; the dosing solutions were prepared weekly.
- Control animals received drinking water without test substance.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The concentrations were verified for all solutions for the first 4 weeks prior test starting; thereafter, at least one sample from each test solution was verified for test substance concentration every 4 weeks.
- The homogeneity of the solutions was tested and confirmed by removing 3 samples from the top, the middle and the bottom of each test solution.
- GA in aqueous solutions was analysed by means of gas chromatography (GC, equipped with a flame ionization detector).

Duration of treatment / exposure:

104 weeks (interim sacrifice at week 52 and 78)

Frequency of treatment:

daily

Post exposure period:

None

Dose / conc.:

50 ppm

Remarks:

males: 4 mg/kg bw/day
females: 6 mg/kg bw/day
actual ingested

Dose / conc.:

250 ppm

Remarks:

males: 17 mg/kg bw/day
females: 25 mg/kg bw/day
actual ingested

Dose / conc.:

1 000 ppm

Remarks:

males: 64 mg/kg bw/day
females: 86 mg/kg bw/day
actual ingested

No. of animals per sex per dose:

100 animals per sex and test group.

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

MORTALITY AND CLINICAL SYMPTOMS:
The rats were checked for mortality at least twice a day; they were examined for clinical symptoms of toxicity at least twice a day; additional detailed clinical examinations were conducted once a week. Palpation for masses was started from week 27 of treatment.

BODY WEIGHT:
The rats were weighed prior test initiation, weekly during the test period, and immediately prior sacrifice.

FOOD CONSUMPTION:
Food consumption was determined weekly until week 13 of treatment; thereafter food consumption was determined on alternate weeks.

WATER CONSUMPTION AND COMPOUND INTAKE:
Water consumption was determined weekly until week 13 of treatment; thereafter water consumption was determined on alternate weeks. The mean daily intake of test substance as group means was determined on the basis of water consumption.

OPHTHALMOSCOPIC EXAMINATION:
The eyes of the animals were examined prior test starting and thereafter, after week 52, 78 and 104 of treatment.

HAEMATOLOGY:
For haematology and clinical chemistry , blood samples were collected from 20 fasted animals per sex and test group; they were taken from the retro-orbital venous plexus on following weeks of treatment: 2, 12, 26, 52, 78 and 104.
Following haematological parameters were considered: haematocrit (HCT), haemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), erythrocyte count (RBC), leukocyte count (WBC), platelet count (PLT), differential leukocyte count.

CLINICAL CHEMISTRY:
Following blood chemistry parameters were considered: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum gamma glutamyl transferase, creatine kinase, lactate deshydrogenase, sorbitol deshydrogenase, glutatamate deshydrogenase, sodium, potassium, chloride, calcium, urea nitrogen, creatinine, glucose, total bilirubin, total protein, albumin, globulins.

URINALYSIS:
For urine sampling, ten animals per sex and group were placed in metabolism cages and urine was collected over 24 hours. Urine sampling was conducted on week 12, 25, 51, 77 and 103 of treatment.
Urinalysis consisted of following parameters: volume, color, microscopic elements, osmolality, pH, protein, glucose, ketones, urobilinogen, bilirubin and blood.

Sacrifice and pathology:

Ten rats per sex and group were sacrificed for the purpose of necropsy after 52 and after 78 weeks of treatment; the remaining animals were sacrificed at test ending, i.e. after 104 weeks of treatment.

ORGAN WEIGHT:
Liver, kidneys, brain, heart, adrenals and testes were removed for weighing.

GROSS PATHOLOGY:
Following necropy, the animals were examined for gross pathology.

HISTOPATHOLOGY:
A complete set of standard tissues was collected and fixed in 10% buffered formalin for further histopathological examination.

Other examinations:

Particular attention was given to the occurrence and incidence of tumors.

Statistics:

- The statistical assessment of quantitative continuous variables was based on Levene´s test for equality of variance, analysis of variance (ANOVA) and t tests;
- The statistical evaluation of non-parametric data (e.g. haematological and clinical-chemical parameters) was based on the one-way analysis using the Kruskal-Wallis test followed by the Mann-Whitney U-test;
- Incidence data were compared using Fisher´s Exact test and incidental fatal tumor analysis according to Haseman JK (Statistical issues in the design, analysis and interpretation of animal carcinogenicity studies. Environ Health Perspect. 58:385-392, 1984). Time to tumor analyses were based on Mantel-Cox, Tarone-Ware and Breslow trend tests (Dixon WJ, BMDP Statistical Software. University of California Press, Berkeley, California, 1990). Additional statistical assessments considered the dose-response trend for the incidence of large granular lymphocytic leukaemia (LGLL) severity grade in spleen in order to determine if increased severity of leukaemia was associated with higher dosages. The severity of LGLL in spleen was determined by evaluating the degree of alteration of the normal spleen morphology. All tumor incidence data were used for a single overall statistical test for the presence of any carcinogenic effect found in the study.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

With regard to neoplastic findings, an increased incidence of large granular lymphocytic leukaemia (LGLL) was reported as only statistically/biologically relevant finding. LGLL was seen in males and females, in all groups including the control. LGLL which was diagnosed in spleen and liver mainly occurred in animals that died and in animals sacrificed after 104 weeks. The statistical assessment of the LGLL findings revealed that the incidence of LGLL in treated male rats was not significantly increased. In contrast, the treated females showed a significantly increased incidence of LGLL; this was particularly true for those females that were sacrificed at test ending, i.e. after 104 weeks of treatment. Analysis for dose-response trend for the severity of LLGL in the spleen revealed an increased severity in females at the higher dosages (53% in spleen and 54% in liver versus respectively 20% and 23% in untreated females); no such observation were made for the males. Time to tumor analysis revealed a significant difference to control for the females only, which mainly was due to the findings reported for the 104 weeks sacrifice time point. The single overall statistical test for the presence of any carcinogenic effect found in the study revealed a statistically significant trend for total tumor incidence in the females, but not in the males; the combination of male and female data resulted in a statistical significance for total tumor response of the entire population. Particular attention was given to the spleen, which is considered to be the most reliable and sensitive indicator for the occurrence of LGLL as LGLL originates from there (Losco PE and Ward JM, The early stage of large granular lymphocyte leukaemia in the F344 rat. Vet. Pathol. 21(3): 286-291, 1984); 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 was related to the incidence of large granular lymphocytic leukemia (LGLL) in the spleen. Furthermore, both, bone marrow hyperplasia and renal tubular pigmentation were put into relation with the occurrence/incidence of large granular lymphocytic leukemia (LGLL) and were considered by the authors of the study as being secondary to a low grade hemolytic anemia in animals with LGLL.
LGLL is known to be a tumor with high incidence in untreated Fischer 344 rats, which further is the most common cause of spontaneous death for this strain. Hermansky et al. (Hermansky SJ, Longhorn KA, Ballantyne B, Large granular lymphocytes leukemia in Fischer 344 (F344) rats: summary of incidence from several laboratories. J. Am. Coll. Toxicol. 12: 115-116, 1992) reviewed the LGLL incidence F344 rats in various laboratories and reported an overall incidence in untreated animals of 29% (34% for males, 24% for females), however with variations between laboratories.
Considering the findings of present study, it is rather unclear whether the increased occurrence of LGLL in treated groups and especially in treated females after 104 weeks of treatment was due to glutaraldehyde or was an artifact. Arguments rejecting the relationship between finding and glutaraldehyde treatment are the following: LGLL was seen in all groups including control; the incidence of LGLL in the 1000 ppm group was high compared to control, but no clear dose-response relationship was evident; LGLL mainly affected the treated females whereas the incidence in treated males was within control range. The authors suggested that the statistical significance of the findings in treated animals might have result from the lower incidence seen in control animals, which again might have been related to the known biological variability of LGLL incidence in untreated F344 rats.
A further possibility considered by the authors was that the increased incidence of LGLL in females following chronic treatment with GA might have result from the influence of the chronic GA supply on factors influencing the expression of LGLL in F344 rats.

Overall statistically significant increased incidence of LGLL:
__________________________________________________

Test group  0 ppm    50 ppm     250 ppm   1000 ppm
--------------------------------------------------
Males       43%      51%        40%        46%
Females     24%      41%        41%        53%
__________________________________________________

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study conduct was in principle similar to OECD TG 451, with the focus being on the non-neoplasic and neoplasic findings at necropsy; survival and body weight changes also were considered. The (histo)pathological findings were submitted to the NTP Pathology Working Group chairperson for review.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

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; chronic toxicity also was considered up to a certain point.

GLP compliance:

yes

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 (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on 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 (nominal)

Remarks:

0.00025 mg/L; other: 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 (nominal)

Remarks:

0.0005 mg/L; other: 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.:

250 ppm (nominal)

Remarks:

0.001 mg/L; other: 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.

Mortality:

no mortality observed

Description (incidence):

Survival in all test groups and for both sexes was similar to controls.

Body weight and weight changes:

no effects observed

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%.

Histopathological findings: non-neoplastic:

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.
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.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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

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)

Reference 2

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study conduct was in principle similar to OECD TG 451, with the focus being on the non-neoplasic and neoplasic findings at necropsy; survival and body weight changes also were considered. The (histo)pathological findings were submitted to the NTP Pathology Working Group chairperson for review.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

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; chronic toxicity also was considered up to a certain point.

GLP compliance:

yes

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:

rat

Strain:

Fischer 344

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. 150 g and 112 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 (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on 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.:

0.25 ppm (nominal)

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).

Dose / conc.:

0.5 ppm (nominal)

Remarks:

0.002 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.:

0.75 ppm (nominal)

Remarks:

0.003 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 92 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 92 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, 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.
- Tooth degeneration also was evaluated in rats.
- Brain of rats was examined in case of hydrocephalus or hemorrhage.
- 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)

Mortality:

mortality observed, treatment-related

Description (incidence):

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. For details, see table below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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. For details, see table below.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Main lesions in the kidney of glutaraldehyde-treated rats:
Nephropathy is a common spontaneous change seen in almost all male rats surviving to 2 years. In the present case, a decrease in the severity of the nephropathy was observed with increasing test concentration. This effect also was seen as a consequence of the decrease in body weight observed for the treated rats (i.e. secondary effect).

Histopathological findings: non-neoplastic:

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 rats.
The main non-neoplastic lesions in the nose of the glutaraldehyde-treated rats are summarized in a table presented below. In fact, 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.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related neoplastic lesions were observed.
One male of the 0.25 ppm group, one male of the 0.50 ppm group, two males of the 0.75 ppm group and one female of the 0.50 ppm group showed alveolar/bronchiolar adenomas; one 0.75 ppm male further displayed a carcinoma. These effects however were within the historical control range for inhalation studies and were not related to the treatment with glutaraldehyde.

Main lesions in the lung of glutaraldehyde-treated rats:
In females, an increased incidence of histiocyte infiltration at 0.75 ppm and of interstitial fibrosis at 0.50 and 0.750 ppm was reported; in males, the incidence of histiocyte infiltration was decreased at 0.50 ppm and the incidence of fibrosis was increased at 0.50 ppm. These effects however, were not considered directly related to the treatment and were of little biological relevance.

Main lesions in the thyroid of glutaraldehyde-treated rats:
In two females of the 0.75 ppm group, the occurrence of thyroid gland follicular cell adenoma was over the historical control range for inhalation studies. As neither hyperplasia nor other treatment-related effects were seen, the two cases of follicular cell adenomas were not related to the treatment with glutaraldehyde.

Main lesions in the mammary gland of glutaraldehyde-treated rats:
Single and multiple fibroadenomas occurred in all groups with a decreasing incidence observed from the 0 ppm (24 cases) to the 0.75 ppm group (10 cases). The incidence of fibroadenoma or carcinoma (combined) in the females of the 0.75 ppm group also was significantly decreased compared to the control group (11 cases versus 26 cases). Moreover, the incidences of fibroadenomas or fibroadenoma /carcinoma were below the historical control range for inhalation studies. The decrease in fibroadenomas or fibroadenoma /carcinoma was seen as a consequence of the decrease in body weight and was therefore not seen as a direct effect due to glutaraldehyde.

Main lesions in the pituitary gland of glutaraldehyde-treated rats:
Adenoma occurred in all groups with a decreasing incidence from 0 to 0.75ppm; this was associated to the decrease in body weight of the treated animals.

Two-year inhalation study with rats, survival data:

Test animals	Test group (ppm)	Survival at study ending (2 years)
		Males	Females
Rats (Ninitial= 50/sex)	0	12/50 (24%; p = 0.032)	26/50 (52%; p < 0.001)
	0.25	14/50 (28%; p = 0.395)	31/50 (62%; p = 0.454)
	0.50	9/50 (18%; p = 0.788)	15/50 (30%; p = 0.023)
	0.75*	6/50 (12%; p = 0.094)	14/50 (28%; p = 0.008)
*, 8 male and 5 female rats of the 750 ppb group were removed from the study between week 13 and 21 because of breathing problems likely related to nasal lesions.

 

Two-year inhalation study with rats, mean body weight data (MBW), summary:

Male rats
Weeks	0	0.25 ppm				0.50 ppm				0.75 ppm
	MBW (g)	MBW (g)		MBW as % of control		MBW (g)		MBW as % of control		MBW (g)		MBW as % of control
1 - 13	263	258		98%		255		97%		248		94%
14 - 52	428	412		96%		412		96%		394		92%
53 - 104	478	456		95%		449		94%		428		90%
Female rats
Weeks	0		0.25 ppm				0.50 ppm				0.75 ppm
	MBW (g)		MBW (g)		MBW as % of control		MBW (g)		MBW as % of control		MBW (g)	MBW as % of control
1 - 13	156		156		100%		152		97%		148	95%
14 - 52	236		231		98%		219		93%		211	89%
53 - 104	335		325		97%		300		90%		278	83%

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

Male rats
Lesions in the nose		0 ppm	0.25 ppm	0.50 ppm	0.75 ppm
Squamous epithelium	Hyperplasia	3^a(2.0)^b	11* (1.6)	39** (2.2)	48** (2.9)
	Inflammation	6 (2.0)	17* (1.5)	41** (2.7)	49** (3.6)
Respiratory epithelium	Hyperplasia	6 (2.0)	5 (2.0)	17** (1.9)	35** (1.9)
	Inflammation	17 (2.1)	10* (1.5)	25 (2.4)	43** (3.2)
	Squamous metaplasia	1 (2.0)	2 (1.5)	11** (2.0)	24** (2.2)
	Goblet cell hyperplasia	1 (1.0)	0	6 (1.8)	6* 81.2)
Olfactory epithelium	Hyaline degeneration	4 (1.0)	8 (1.3)	9 (1.1)	14** (1.1)
Female rats
Squamous epithelium	Hyperplasia	3 (1.3)	15** (1.7)	29** (2.0)		45** (2.7)
	Inflammation	6 (2.5)	26** (1.5)	42** (2.1)		48** (3.2)
Respiratory epithelium	Hyperplasia	1 (3.0)	6 (1.7)	15** (1.9)		29** (1.9)
	Inflammation	5 (2.2)	9 (1.7)	26** (2.1)		42** (2.5)
	Squamous metaplasia	1 (2.0)	1 (3.0)	11** (1.6)		16** (2.3)
	Goblet cell hyperplasia	1 (2.0)	3 (1.3)	5 (1.4)		8** (1.6)
Olfactory epithelium	Hyaline degeneration	4 (1.0)	5 (1.0)	12* (1.1)		15** (1.1)
*, p < 0.005; **, p < 0.001; a, Number of animals with lesions (Number of animals examined = 50); b, Average severity grade of lesions (1 = minimal, 2 = mild, 3 = moderate, 4 = marked)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No carcinogenic potential was evident from oral and inhalative long-term animal studies conducted with glutaraldehyde. A carcinogenic effect resulting from long-term dermal exposure to glutaraldehyde is not expected, as severe skin reactions come to the fore. Therefore, no classification is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.

Additional information

Oral

The findings of the 2 year-oral study in rat revealed no carcinogenic potential of glutaraldehyde when administered to male and female Wistar rats in the drinking water over a period of 24 months (BASF, 2003). The non-neoplastic findings confirmed that particularly the upper respiratory tract (nasal cavity, larynx, trachea) is a target for the toxicity of glutaraldehyde; furthermore, at 2000 ppm, lesions also were seen within the lungs and the bronchi.

In a 104 week study by Van Miller (2002) conducted with Fischer 344 rats of both sex, which were repeatedly treated with glutaraldehyde 50% via drinking water (50, 250 and 1000 ppm), a significant and dose-related increase in the incidence and severity of large granular lymphocytic leukaemia (LGLL) in spleen was reported for the females only; following percentages of incidence were reported for the females: 24% in the control, 41% in the 50 and the 250 ppm group respectively, and 53% in the 1000 ppm group. LGLL is a tumour with a high incidence in Fischer 344 rats and is the most common cause of spontaneous death for this strain. Due to (1) the background and variable incidence of LGLL in the Fischer 344 rat, (2) the finding of a statistical significance only for female rats, and (3) because, there was no clear dose-response relationship (see 50 and 2500 ppm groups), the biological significance of the LGLL findings is unclear. The authors suggested that the observed significance was a statistical artefact due to the low incidence of LGLL in the female control animals as a result of biological variability within the study. They also considered the possibility that the chronic dosage of GA in the drinking water resulted in a modification of one or more of the factors responsible for the expression of this common and spontaneously occurring neoplasm in the Fischer 344 rat.

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.

Inhalation

No neoplastic lesions were observed in rats and mice after inhalation exposure to glutaraldehyde over 2 years; however, exposure to glutaraldehyde resulted in considerable non-neoplastic lesions in the noses of both species (NTP, 1999; van Birgelen, 2000). As both formaldehyde and glutaraldehyde result in similar acute and sub-chronic histopathological lesions in the nasal epithelium of rats, but only long-term treatment with formaldehyde induces tumours, the RNA transcription pattern underlying aldehyde-induced lesions resulting from formaldehyde and glutaraldehyde treatments was examined, as a possible marker for the different cancer outcomes (Hester, 2005). The study delivered insight into the potential specific (cyto)toxicity-mechanisms of glutaraldehyde and formaldehyde, leading in the case of glutaraldehyde to an increased apoptosis in comparison to formaldehyde, and thus to the lack of carcinogenicity of this aldehyde. These data suggest that although both aldehydes induced similar short-term cellular phenotypes, gene expression could distinguish glutaraldehyde from formaldehyde in accordance with their different carcinogenic potential.

Zissu et al (1998, supporting) 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 were seen in the trachea and the lungs. Few neoplastic changes were reported, but these findings were incidental and not treatment-related. The lesions likely are due to an irritation mechanism. No substance related carcinogenic effects were observed.

Dermal

Because of the unlikeliness of long-term dermal exposure of humans to glutaraldehyde, no chronic dermal carcinogenicity study with glutaraldehyde was conducted. In fact, the repeated application of glutaraldehyde over a period of 13 weeks was shown to induce no systemic toxicity (BASF, 2000); this was related to the limited percutaneous absorption and the low systemic bioavailability of glutaraldehyde and/or metabolites following dermal application; in fact, the main reported effects referred to skin reactions which were related to the irritant potential of the substance and showed increasing incidence and severity during time elapsed. Therefore severe skin reactions come to the fore in case of repeated dermal exposure to glutaraldehyde, and because of the growing incidence and severity of these reactions, a 2-year dermal exposure study with animals would be of doubtful relevance and ethical acceptance.

Carcinogen Human data:

In an extended follow-up study using death certificates of male workers working in a glutaraldehyde producing unit, standardized mortality ratios (SMRs) were calculated for three cumulative exposure categories of glutaraldehyde (Collins et al 2006). The mortality rates were compared to the mortality rates of the U.S. population. In this study no increasing trend with increasing exposure was observed for any cause of death. No leukaemia deaths were observed among exposed workers. No cancer of the nasal cavity and sinus or of the nasopharynx was observed among the glutaraldehyde-exposed workers. In an epidemiological study from the Union Carbide Corporation (1993), the mortality was examined of 186 men assigned to glutaraldehyde production from its start-up in 1959 through 1978, at a chemical plant in West Virginia and compared to US white males and 29000 chemical workers. Results showed no excess deaths due to all causes or total malignancies.