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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study according OECD 412 and GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzylalkohol
- Purity 99.9 %
- Appearance: clear colourless liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Crl:CD(SD) rats
- Source: Charles River Lab., Inc., Raleigh, NC, US
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: males 255-352 g, females 182-234 g; individual body weights at randomisation were within +/- 20 % of the mean for each sex.
- Housing: individually in stainless steel, wire-mesh cages; Animals were maintained in accordance with the "Guide for the care and use of laboratory animals" (National Researtch Council, 1996)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 22 days; animals were acclimated to restraint in nose-only tubes by increasing the restraint time over the acclimation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Aerosol particle size at 1000 mg/m³ air benzyl alcohol: MMAD: 3.3 µm/GSD 2.39

The aerosol particle size was not determined for the control group (filtered air) or for the 30, 100, or 300 mg/m³ benzyl alcohol exposure systems. In a previous method development and validation study it was determined that benzyl alcohol atmospheres at concentrations less than 100 mg/m³ did not contain aerosol particles. Additionally, due to the volatility of benzyl alcohol, and the sampling time required to obtain a measurable amount of test substance on impactor substrates, it was not possible to accurately assess the particle size of the 300 mg/m³ benzyl alcohol exposure atmosphere, although it was determined previously that aerosol particles were present at this concentration. It is not anticipated that the MMAD of aerosol particles in the 300 mg/m³ benzyl alcohol exposure atmosphere would have exceeded that observed for the 1000 mg/m³ benzyl alcohol exposure atmosphere.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Mode of exposure: Exposures were conducted using seven 7.9-L conventional nose-only exposure systems (designed and fabricated by WIL Research Laboratories, LLC) with synthetic rubber grommets in exposure ports to engage animal holding tubes.
- Generation of aerosols: A vapor/aerosol atmosphere was generated using a system which operated as follows. A syringe pump and appropriate size syringe were used to deliver test substance to an atomizer. The atomizer was comprised of a no. 2850 fluid cap and a no. 64 air cap. Using a Coilhose Pneumatics regulator, compressed air was supplied to the air port of the atomizer at a known, constant pressure to effect the atomization of the test substance.
The resulting vapor/aerosol atmosphere passed through a liquid trap prior to entering the exposure system. For Exposure Systems 2, 3, and 4, a siphon was placed in-line prior to the exposure system to reduce the concentration as needed. The siphon was controlled using a rotameter-type flowmeter. The approximate test substance delivery rates and syringe sizes are summarized in the following (Exposure System/ Syringe Size (mL)/ Test Substance Flow Rate (g/hour)): 2/ 3/ 0.1 to 1.1; 3/ 5/ 0.7 to 0.9; 4/ 10/ 4.2; 5/ 100/ 19 to 35.
- Conditioning of compressed air: Air supplied to the nose-only systems was provided from a dry compressed air source.
- Treatment of exhaust air: All test atmosphere exhaust passed through a Solberg filter prior to entering the facility exhaust system, which consisted of charcoal- and HEPA-filtration.

TEST ATMOSPHERE
- Samples taken from breathing zone: yes
- Brief description of analytical method used: Concentrations of benzyl alcohol in the exposure systems were determined at approximately 90-minute intervals using a gas chromatograph (GC). Samples were collected from the approximate animal-breathing zone of the exposure system using a series of 2 impingers containing isopropyl alcohol (IPA) as a trapping liquid.
- Particle size distribution: Aerosol particle size determinations were conducted for Exposure Systems 5, 6, and 7 using a 7-stage cascade impactor. Pre-weighed, 25-mm glass-fiber filters were used as the collection substrates. One sample was collected weekly at approximately 1.8 LPM
for 2, 300, and 60 minutes for Exposure Systems 5, 6, and 7, respectively. The filters were re-weighed and the particle size was calculated based on the impactor stage cut-offs.
The aerosol particle size was not determined for the control group (filtered air) or for the 30, 100, or 300 mg/m³ benzyl alcohol exposure systems. In a previous method development and validation study it was determined that benzyl alcohol atmospheres at concentrations less than 100 mg/m³ did not contain aerosol particles. Additionally, due to the volatility of benzyl alcohol, and the sampling time required to obtain a measurable
amount of test substance on impactor substrates, it was not possible to accurately assess the particle size of the 300 mg/m³ benzyl alcohol exposure atmosphere, although it was determined previously that aerosol particles were present at this concentration. It is not anticipated that the MMAD of aerosol particles in the 300 mg/m³ benzyl alcohol exposure atmosphere would have exceeded that observed for the 1000 mg/m³ benzyl alcohol exposure atmosphere.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): at 1000 mg/m³ MMAD 3.3 µm/GSD 2.39

VEHICLE
No vehicle used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For benzyl alcohol, a mixed aerosol and vapor exposure atmosphere may have been present. Therefore, the exposure concentration was reported as total test substance (i.e., aerosol plus vapor).
Concentrations of benzyl alcohol in the exposure systems were determined at approximately 90-minute intervals using a gas chromatograph (GC). Additional samples were collected as needed. Samples were collected from the approximate animal-breathing zone of the exposure system using a series of 2 impingers containing isopropyl alcohol (IPA) as a trapping liquid. Test atmosphere samples were pulled through the impinger sampling train. This sampling method was used to collect the test atmosphere vapor, as well as aerosol (if present). Following sample collection, the liquid in the impingers was pooled, mixed using a laboratory vortex, and manually injected into a calibrated, validated GC. Impinger samples for the presence of benzyl alcohol were not collected or analyzed for the control group. Exposure of the control group in a separate room from the benzyl alcohol exposures eliminated the possibility of exposure of the control group to benzyl alcohol.
Duration of treatment / exposure:
4 weeks (a minimum of 20 exposures/animal)
Frequency of treatment:
6 h/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30, 100, 300, and 1000 mg/m³
Basis:
other: target conc.
Remarks:
Doses / Concentrations:
41, 102, 290, 1072 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: All benzyl alcohol exposure concentrations were selected by the Sponsor based on known toxicity information.
- Objective of the study: The objective of this study was to evaluate the potential toxic effects of aerosolized benzyl alcohol. Due to known toxicity information (metabolism of benzyl alcohol to benzoic acid) and for a comparative evaluation in addition to a control group exposed to filtered air and treatment groups exposed to 4 concentrations of benzyl alcohol, 2 additional groups were exposed to benzoic acid.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
All animals were observed twice daily for mortality and moribundity, once in the morning and once in the afternoon, except on the day of scheduled necropsy. Clinical examinations were performed prior to exposure, at the approximate mid-point during the exposure period (beginning on study day 2; third exposure), 0 to 1 hour post-exposure (designated as 1 hour post-exposure for report presentation purposes), and once daily on non-exposure days.

DETAILED CLINICAL OBSERVATIONS: Yes, all animals
Detailed physical examinations were conducted on all animals at least once during the pretest period, at the time of randomization and group assignment, and weekly during the exposure phase (including prior to scheduled necropsy).

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes, all animals
- Time shedule: weekly
Food intake was calculated as g/animal/day for the corresponding body weight intervals.

OPHTHALMOSCOPIC EXAMINATION: Yes, all animals
- Time schedule for examinations: during pretest (study week -2) and prior to the scheduled necropsy (study week 3)

HAEMATOLOGY: Yes, all animals
- Time schedule for collection of blood: on the days of the scheduled necropsies (study week 3)
- Animals fasted: Yes, animals were fasted overnight prior to blood collection
- Parameters: Total leukocyte count (White Cells), Erythrocyte count (Red Cells), Hemoglobin, Hematocrit, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (Platelet), Prothrombin time (ProTime), Activated partial thromboplastin time (APTT), Reticulocyte count (Percent and asolute), Red cell distribution width (Red CellWidth), Hemoglobin distribution width (HGB Width), Differential leukocyte count (Percent and absolute: -Neutrophil -Lymphocyte -Monocyte -Eosinophil -Basophil -Large unstained cell), Platelet estimate, Red cell morphology (RBC Morphology)

CLINICAL CHEMISTRY: Yes, all animals
- Time schedule for collection of blood: on the days of the scheduled necropsies (study week 3)
- Animals fasted: Yes, animals were fasted overnight prior to blood collection
- Parameters: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (AlkalinePhos'tse), Alanine aminotransferase (Alanine Transfer), Aspartate aminotransferase (AspartatTransfer), Gamma glutamyltransferase (GlutamylTransfer), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Sorbitol dehydrogenase (Sorbitol 'Genase)

URINALYSIS: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals
The necropsies included an examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
ORGAN WEIGHTS: The following organs were weighed from all animals at the scheduled necropsy:
Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Lungs (prior to inflation with fixative), Ovaries and oviducts, Spleen, Testes, Thymus, Uterus with cervix. Paired organs were weighed together.

The following tissues and organs were collected and placed in 10% neutral-buffered formalin, except otherwise stated:
Adrenals (2), Aorta, Bone with marrow (femur with joint, Sternum), Brain (Cerebrum (2 levels), Cerebellum with pons/medulla), Cervix, Epididymides (2; fixed in Bouin’s solution), Eyes with optic nerves (2; fixed in Davidson’s solution), Exorbital lacrimal glands (2), Gastrointestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum), Harderian glands (2), Heart Kidneys (2), Larynx, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by constant pressure inflation with fixative), Lymph nodes (Axillary (2), Mediastinal and bronchial (if visible), Mesenteric and mandibular), Mammary gland (females only; A corresponding section of skin was collected from the same anatomic area for males), Nasal cavity with turbinates (the entire head was removed and preserved. Following decalcification, 6 cross-sections of the nasal cavities were prepared for microscopic examination in accordance with the method described by Morgan, 1991) Ovaries (2) with oviducts (Examined if in plane of section and in all cases when a gross lesion of the organ was present), Pancreas, Peripheral nerve (sciatic), Peyer's patches, Pharynx, Pituitary, Prostate, Salivary glands [mandibular (2)], Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin Spinal cord (cervical, thoracic, lumbar), Spleen, Testes (2; fixed in Bouin’s solution), Thymus, Thyroid gland with parathyroids (2; Examined if in plane of section and in all cases when a gross lesion of the organ was present), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

HISTOPATHOLOGY: Yes
Microscopic examination was performed on all tissues from all animals at least in the control and 1000 mg/m³ benzyl alcohol group.
Other examinations:
no further data
Statistics:
Parametric one-way analysis of variance (ANOVA), Dunnett's test, Kruskal-Wallis nonparametric ANOVA, Dunn's test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no test substance-related deaths. There were no test substance-related clinical observations.

BODY WEIGHT AND WEIGHT GAIN
Body weights were unaffected by test substance administration.

FOOD CONSUMPTION
Food consumption was unaffected by test substance administration.

OPHTHALMOSCOPIC EXAMINATION
No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups.

HAEMATOLOGY
Hematology parameters were unaffected by test substance administration.

CLINICAL CHEMISTRY
Serum chemistry parameters were unaffected by test substance administration.

ORGAN WEIGHT
There were no statistically significant rest substance related alterations in final body weight or organ weights. However, non-statistically significant lower mean final body weights in the males exposed to either 300 or 1000 mg/m³ benzyl alcohol resulted in non-adverse statistically significant higher mean epididymides weights relative to final body weights but not relative to brain weights.
male - final mean body weight (g) at doses 30, 100, 300, and 1000 mg/m³: 376 - 383 -363 - 358 g versus 383 g of controls; final relative epididymides weight (g) at doses 30, 100, 300, and 1000 mg/m³: 0.307 - 0.304 - 0.314 (p: 0.05) - 0.322 (p: 0.01) versus 0.279 of controls.

GROSS PATHOLOGY
There were no test substance-related macroscopic findings at the scheduled necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test substance-related microscopic findings.

Effect levels

Dose descriptor:
NOAEC
Effect level:
1 072 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: based on the fact that the exposure was tolerated without adverse effects up to and including the highest test concentration (1072 mg/m³)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

In a study according to OECD TG 412 aerosolized benzyl alcohol was administered via nose-only inhalation for 6 hours per day on a 5-day/week basis for a period of 4 weeks (a minimum of 20 exposures/animal) to 4 groups (Groups 2-5) of Sprague-Dawley rats. Target exposure concentrations were 30, 100, 300, and 1000 mg/m³ for Groups 2, 3, 4, and 5, respectively. A concurrent control group (Group 1) was exposed to filtered air on a comparable regimen. Each group consisted of 10 animals/sex. All animals were euthanized on the day following the last exposure.

Based on the results of this study, repeated inhalation exposure of male and female rats to benzyl alcohol at mean analytical concentrations of 41, 102, 290 and 1072 mg/m³ was well-tolerated with no adverse effects at any exposure concentration. The

no-observed-adverse-effect concentration (NOAEC) was considered to be 1072 mg/m³.