Registration Dossier

Administrative data

Description of key information

In a 13 week dose-finding study male and female rats received daily up to 800 mg/kg bw benzyl alcohol. The NOAEL was considered to be 400 mg/kg bw/daybased on signs of neurotoxicity, reduced body weight development and histopathological effects mainly in the brain at next higher dose (800 mg/kg bw/day)(NTP 1989). This NOAEL was confirmed in a 2 year study (carcinogenicity study).
In a 13 week dose-finding study male and female mice received daily up to 800 mg/kg bw benzyl alcohol. The NOEL was considered to be 200 mg/kg bw/day. At 400 mg/kg a slight decreased body weight gain was reported and at 800 mg/kg additionally staggering after dosing during the first and second weeks of the studies was observed. No compound-related histopathological effects were found in the mice study, indicating adaptive response to the compound and no adverse effect at 400 or 800 mg/kg. This NOEL was confirmed in a 2 year study (carcinogenicity study).
In a subacute inhalation toxicity study according to OECD TG 412, male and female rats were exposed nose-only to benzyl alcohol at mean analytical exposure concentrations up to 1072 mg/m³ air for 6 hour/day, 5 day/week for 4 weeks. The NOAEL was concluded to be 1072 mg/m³ based based on the fact that the exposure was tolerated without adverse effects up to and including this test substance concentration (Roper 2010).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study on carcinogenicity (supervised by NTP); no data on hematology, clinical chemistry or urinalysis
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 451 (Carcinogenicity Studies)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: F344/N rats
- Source: Charles River Breeding Laboratories (Portage, MI)
- Age at study initiation: 8-9 weeks
- Weight at study initiation (mean): males: 211-213 g; females: 145-150 g
- Housing: 5 per cage
- Diet ad libitum
- Water ad libitum
- Acclimation period: 26 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9-31 (66-88 °F)
- Humidity (%): 20-80
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Administration volume: 5 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
extraction of benzyl alcohol with methanol followed by gas chromatographic analysis
Duration of treatment / exposure:
103 weeks
Frequency of treatment:
once daily, 5 days/week
Remarks:
Doses / Concentrations:
200, and 400 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
50
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: For dose selection results from previously conducted 2-week and 3-month studies were used. "Because of reductions in relative weight gain, deaths, and lesions of the brain, thymus, skeletal muscle, and kidney, doses selected for rats for the 2-year studies were 200 and 400 mg/kg benzyl alcohol, administered in corn oil by gavage, 5 days per week."
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, all animals
- Time schedule: observed twice daily; clinical signs were recorded at least once per month.

BODY WEIGHT: Yes, all animals
- Time schedule for examinations: Body weights were recorded initially, once per week for the first 12 weeks of the studies and once per month thereafter.

CLINICAL PATHOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, necropsies were performed on all animals

HISTOPATHOLOGY: Yes, complete histopathology was performed on all female rats and on vehicle control and on high dose male rats, on male rats that died before month 22 and male rats with gross lesions.
The following tissues were examined: adrenal glands, brain, cecum, clitoral or preputial gland, colon, costochondral junction, duodenum, esophagus, eyes, gross lesions and tissue masses with regional lymph nodes, heart, ileum, jejunum, kidneys, larynx, liver, lungs and bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, oral cavity, pancreas, parathyroids, pharynx, pituitary gland, rectum, salivary glands, sciatic nerve, scrotal sac/tunica vaginalis/seminal vesicles/ prostate/epididymis/testes or ovaries/uterus, skin, spinal cord, spleen, sternebrae or vertebrae or femur including marrow, thigh muscle, thymus, thyroid gland, trachea, urinary bladder, and Zymbal gland; pituitary gland and testis examined for low dose male rats.
Other examinations:
no further data
Statistics:
According to Kaplan and Meier, method of Cox (1972) and Tarone's (1975) life table test, indicential tumor analysis, Fisher's exact/Cochrane-Armitage trend analysis
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHTS AND CLINICAL SIGNS
Mean body weights of dosed and vehicle control male and female rats were generally comparable throughout the studies. No compound-related clinical signs were observed.

SURVIVAL
Survival in male rats was not affected by treatment (final survival rates: vehicle control 28/50, low dose: 27/50, high dose 24/50) but reduced survival of dosed female rats by half (final survival rates: vehicle control 36/50; low dose 18/50; high dose 17/50). Many of the early deaths were considered to be related to the gavage procedure.

NONNEOPLASTIC AND NEOPLASTIC LESIONS
Cited from NTP report: "No apparent compound-related non-neoplastic responses were observed. Dose-related negative trends in the incidences of anterior pituitary gland neoplasms were seen in female rats (vehicle control, 29/50; low dose, 17/47; high dose, 9/49)"....., which is an incidence of 58, 36, and 18 %, respectively. Historical incidence in NTP studies: 692/1654 (42 %).
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: based on the fact that body weight gains in male and female mice were not affected and compound-related clinical signs were not observed at this dose level; no apparent compound-related non-neoplastic findings at pathology/histopathology
Critical effects observed:
not specified

Cited from abstact of NTP report: "No apparent compound-related non-neoplastic responses were observed."

Executive summary:

In a study equivalent to OECD TG 451 (supervised by NTP) male and female F344/N rats received daily by gavage 0, 200 and 400 mg/kg bw/day benzyl alcohol diluted in corn oil for 104 weeks (5 days/week). No effect on body weight gain and and no compound-related clinical signs were observed throughout the study. Survival was reduce only for female rats, but in many cases deaths were attributed the gavage procedure. Gross necropsy and histopathology revealed no apparent compound-related non-neoplastic responses. Thus, 400 mg/kg can be considered a NOAEL from this study (NTP TR 343, 1989).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes
Limit test:
no
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
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
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
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
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.
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)
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 072 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
No effect observed even at the highest dose concentration tested.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
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
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes
Limit test:
no
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
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
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
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
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.
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)
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 072 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
No effect observed even at the highest dose concentration tested.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral route:

There are subchronic and chronic studies available which were conducted within the US NTP programme (NTP 1989). They do not fully comply with the current respective guidelines due to the lack of clinical chemistry data, hematology data, and urinalysis data. Instead, clinical signs, body weight development and extensive gross and histopathology examinations were performed. The studies were assessed as of high quality, since they were peer-reviewed by a panel nominated by the US NTP.

In the 13 week dose-finding study male and female rats received daily 0, 50, 100, 200, 400, 800 mg/kg bw/day benzyl alcohol by gavage. At 800 mg/kg clinical signs of neurotoxicity including staggering, laboured breathing, and lethargy were reported; additionally reduced body weight development and histopathological changes mainly in the brain. The NOAEL was concluded therefore 400 mg/kg bw/day (NTP 1989).

The respective 13 week dose-finding study in male and female mice revealed no compound-related adverse effects up to the highest dose of 800 mg/kg. Only staggering after dosing during the first and second weeks and reduced body weight development were reported. No compound-related histopathologic effects were observed. The NOEL was considered 200 mg/kg bw/day (NTP 1989).

In the consecutive chronic studies (studies on carcinogenicity; NTP 1989), rats and mice received by gavage 0, 200, 400 mg/kg bw/day and 0, 100, 200 mg/kg bw/day, respectively. The exposure was well tolerated and the NOAELs of the subchronic studies were confirmed: NOAEL rats 400 mg/kg bw/day; NOEL mice 200 mg/kg bw/day (highest dose tested). This provides evidence that for benzyl alcohol toxicity is not depending on the duration of exposure.

(For the assessment of carcinogenic effects see the chapter on carcinogenicity.)

Dermal route

No studies available.

Inhalation route

There is no subchronic study with inhalation exposure available.

In a subacute inhalation toxicity study according to OECD TG 412 male and female rats were nose-only exposed to concentrations of up to 1072 mg/m³ aerosolised benzyl alcohol. The exposure regimen was 6 hours/day, 5 days/week for 4 weeks (minimum of 20 exposures). The aerosol was of good respirability for the rats.

In this study exposure was tolerated without adverse effects up to the highest concentration of 1072 mg/m³, leading to a NOAEC of 1072 mg/m³.

Moreover, the low inhalation toxicity indicated that the NOAEC obtained is not more sensitive than the NOAEL derived from the repeated oral toxicity studies. (Roper 2010)


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The rat study with the more relevant NOAEL was selected from the reliable studies; the respective mouse study revealed no compound-related histopathological effect at any dose, and no adverse effect up to the highest dose.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Only one reliable repeated inhalation study available for the substance.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
comprehensive study

Justification for classification or non-classification

According to legal classification (Directive 67/548/EEC and Regulation (EC) No. 1272/2008) benzyl alcohol is not classified for repeated dose toxicity.

Based on the available data and according to criteria of Regulation (EC) No. 1272/2008, Annex I, classification for repeated dose toxicity would not be justified.