Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: inhalation
Remarks:
other: subacute and subchronic
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Concise report, no details on results, non GLP, however MMAO information and range of parameters included.

Data source

Reference
Reference Type:
publication
Title:
2-week and 13-week inhalation studies of aerosolised glycerol in rats.
Author:
Renne R.A. et al
Year:
1992
Bibliographic source:
Inhalation Toxicology 1992, 4: 95-111

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
not applicable
GLP compliance:
not specified
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Glycerol, (Superol glycerin, USP)
purity > 99.8%
Source: Proctor & Gamble Company, Cincinnati, Ohio

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Raleigh, N.C.
- Age at study initiation: no data
- Weight at study initiation: no data (weight gains reported in the article)
- Fasting period before study: not applicable
- Housing: individual cage compartments in Hazelton chambers
- Diet: Purina certified pellet chow 5002 ad libitum
- Water: ad libitum
- Acclimation period: 3 week quarantine (incl. health screening)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 +/- 7
- Humidity (%): 71 +/- 6
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: 2-week study:
Target concentration 1.0 mg glycerol/ l air -- MMAD = 1.46; GSD = 1.75
Target concentration 2.0 mg glycerol/ l air -- MMAD = 1.45; GSD = 2.11
Target concentration 4.0 mg glycerol/ l air -- MMAD = 1.36; GSD = 1.91

Target concentration vs. mean exposure concentration determined gravimetrically (mg/l)
Target concentration 1.0 -- mean exposure concentration = 1.00
Target concentration 2.0 -- mean exposure concentration = 1.93
Target concentration 4.0 -- mean exposure concentration = 3.91


13 week study:
Target concentration 0.033 mg glycerol/ l air -- MMAD = 1.09; GSD = 1.90
Target concentration 0.165 mg glycerol/ l air -- MMAD = 1.49; GSD = 1.69
Target concentration 0.660 mg glycerol/ l air -- MMAD = 1.61; GSD = 1.75

Target concentration vs. mean exposure concentration determined gravimetrically (mg/l)
Target concentration 0.033 -- mean exposure concentration = 0.033
Target concentration 0.165 -- mean exposure concentration = 0.167
Target concentration 0.660 -- mean exposure concentration = 0.660
Details on inhalation exposure:
2-week study:
Apparatus: a viscous - liquid aerosol generator designed in Battelle's Pacific Northwest Laboratory.
Exposure took place in Cannon flow-past chambers. Before the start of exposures uniformly distributed target aerosol was documented using a real-time aerosol monitor (model RAM-1). Temporal and spatial variations in glycerol concentrations were characterized.
During exposures, aerosol concentrations were measured hourly for each exposure group.
Aerodynamic particle size and associated geometric standard deviation were determined weekly.


13-week study:
The study followed the experimental procedures similar to the 2-week study. The control of temporal homogenity was verified in seven 6-h mock exposures to each concentration prior to the beginning of animal exposures. Six samples of each concentration were acquired with a RAM-1 (Real Time Aerosol Monitor.). Uniformity to aerosol concentrations at different breathing ports of the nose-only exposure system was determined by 20 RAM-1 samples acquired at each concentration. Exposure atmosphere was measured daily.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
RAM (Real Time Aerosol Monitor) determined aerosol mass concentrations derived from glass-fiber filter.
Duration of treatment / exposure:
2 weeks or 13 weeks
Frequency of treatment:
2 week study: 5 days/week (weekends excluded)
13-week study: 5 days/week for 13 consecutive weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2 week study: 1.0, 2.0, 4.0 mg glycerol/l of filtered air
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
13-week study: 0.033; 0.165 or 0.660 glycerol/l of filtered air
Basis:
other: target concentrations
No. of animals per sex per dose:
2-week study: 10 rats/sex/exposure group
13-week study: 15 rats/sex/exposure group
Control animals:
yes, sham-exposed
Details on study design:
2-week study:
10 rats/sex/exposure group received 10 nose-only exposures of 6h/day over period of 14 days (weekends excluded) to target aerosol concentration of 1.0, 2.0, 4.0 mg glycerol/l of filtered air. An identical group of rats served as control and was exposed to filtered room air.

13-week study:
15 rats/sex/exposure group received nose only exposure 6h/day; 5 days/week for 13 consecutive weeks to glycerol aerosols. Target aerosol concentrations were 0.033; 0.165 or 0.660 mg/l. The study followed the experimental procedures similar to the 2-week study.
Positive control:
Not required

Examinations

Observations and examinations performed and frequency:
2-week study:
The animals were observed twice daily for signs of toxicity, morbidity and mortality and weighted at 2 or 3 day intervals. Diet consumption was measured weekly during one 17-h non-exposure period.

13-week study:
Body weights were measured weekly. The other parameters measured as above.
Sacrifice and pathology:
2-week study:
Immediately prior to terminal necropsy, the animals were anesthetized with 70% CO2 in air. Blood samples were obtained from the retro-orbital plexus for hematologic evaluations (complete blood count, serum chemistry incl. blood urea nitrogen (BUN), creatinine, glucose, protein, albumin, albumin/globulin ratios, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), gamma glutamyl transferase (GGT), cholesterol, triglicerydes, phospholipides.
All rats underwent a complete necropsy. After anasthesis, rats were ensanguined via the branchial artery. Terminal necropsy was performed within 24h of the final exposure.
The entire respiratory tract, associated lymph nodes and the liver, kidneys, heart and gross lesions observed at necropsy were fixed in 10% neutral formaline buffer. The lungs, liver, kidneys, brain, heart of all animals were weighted. After weighing, the lungs were perfused with NBF via the trachea at 25 cm hydrostatic pressure. Four transverse sections of nasal cavity, transverse sections of larynxs, transverse and longitudinal sections of trachea, coronal sections of lungs, mainstem bronchi and associated lumph nodes, and sections of thymic lymph nodes from all rads in all groups were examined microscopically. Duplicate slides of lungs, trachea, and anterior nasal cavity from all groups were examined microscopically to evaluate goblet cell changes. H&E sections of liver, kidneys and heart were examined microscopically from all rats exposed to 4 mg glycerol/l air and from all sham-exposed control rats.

13-week study:
Tissue collection and necropsy was more extensive than in the 2-week study and included in addition to the tissues collected in the 2-week study, a complete set of 40 tissues from each rat.

Other examinations:
none
Statistics:
Body weight, body weight gain, diet consumption, organ:body weight and organ:brain weight ratios, and clinical pathology data were analysed statistically for each sex by exposure group (ANOVA).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see also below
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
data not reported
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
2-WEEK study:
CLINICAL SIGNS AND MORTALITY
No effects. Two rats died when confined in the restraining tubes during exposure but these deaths were not treatment-related.

BODY WEIGHT AND WEIGHT GAIN
Mean weight gain was decreased for all glycerol-exposed groups, compared with sham controls of the same sex. These decreases were statistically significant for all groups of exposed female rats with an inverse relationship between exposure concentrations and body weight gain.
The authors argue that the decreases in body weight are rather considered to be related to the combined stress of confinement in the restraining tubes and exposure to the glycerol aerosol than a specific toxic effect of the inhaled glycerol.

FOOD CONSUMPTION
No effects.

HAEMATOLOGY
No Effects

CLINICAL CHEMISTRY
Sporadic statistical differences were observed in comparing hematology data from various groups but there were no differences or trends to suggest an effect related to glycerol exposure. The only consistent significant effect noted was a reduction on the mean blood glucose values from all female groups exposed to glycerol. The decreases in these glucose concentrations were modest but these differences between exposure groups were statistically significant.

NEUROBEHAVIOUR
No effects

ORGAN WEIGHTS
No effects.

GROSS PATHOLOGY
No effects

HISTOPATHOLOGY:
Very few gross lesions were observed at necropsy, with no evidence of gross lesions attributable to glycerol exposure. Minimal to mild squamous metaplasia of the epithelium lining the base of the epiglottis was observed in most rats exposed to all three concentrations of glycerol. In the sham control, the epithelium in the center of the base of the epiglottis was composed of a mixture of slightly flattened, rounded, cuboidal and ciliated columnar epithelial cells two to four cells deep. Squamous metaplasia was diagnosed when the center of the base of the epiglottis contained epithelium characterised by a surface layer of more rounded cells with slightly darker nuclei. The increased incidence of laryngeal squamous metaplasia was statistically significant in all groups exposed to glycerol. There was a low incidence of microscopic lesions in other organs with no evidence of a relationship to glycerol exposure.




13-WEEK study:
CLINICAL SIGNS AND MORTALITY
No effects.

BODY WEIGHT AND WEIGHT GAIN
Some differences seen without biological significance (no details available).

FOOD CONSUMPTION
Some differences seen without biological significance (no details available).

HAEMATOLOGY
No Effects

CLINICAL CHEMISTRY
In the serum chemistry assays, the only statistically significant difference between the exposed and control groups was the increased triglyceride values in the low and medium exposure groups of males (134% and 123% of controls). These values seem to be related to exposure concentrations. Although the toxicological significance remains unclear in absence of details of related parameters an inverse relationship between exposure concentration and triglyceride values was noted.



NEUROBEHAVIOUR
No effects

ORGAN WEIGHTS
No effects.

GROSS PATHOLOGY
No effects.

HISTOPATHOLOGY:
Few gross lesions were observed with no evidence of changes attributable to glycerol exposure. Minimal squamous metaplasia of epithelium lining the base of epiglottis was observed in 10 of the 21 rats from the group exposed to 0.662 mg/l. One rat from this group had mild squamous metaplasia. This incidence was statistically significant and was considered to be exposure related. No glycerol-related effects were observed at the lower concentrations.
Al low incidence of other microscopic lesions was observed in tissues examined from the sham-exposed control and glycerol-exposed groups with no evidence of an effet of glycerol exposure.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
0.165 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
>= 0.66 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In a 2-week inhalation study in rats (0, 1, 2 or 4 mg/L), a decrease in mean body weight gain was noted for all exposure groups; decreases were statistically significant in females with an inverse relationship between exposure concentrations and body weight gain. Furthermore, a decreased in blood glucose values was in females (inverse dose response). No further changes were noted in food consumption, haematology, clinical biochemistry, organ weights and macroscopy. At histopathology, minimal to mild squamous metaplasia of the epithelium lining of the base of the epiglottis in larynx was observed in most rats of all concentration groups. The increase incidence of this finding was statistically significant at all dose groups.

The toxicological significance of the inverse dose response on mean body weight gain and blood glucose levels in female rats remains unclear. In absence of further data and since the reductions in body weight gain were significant (42-62% of controls), these findings are considered toxicologically relevant and therefore the NOAEC for systemic effects is < 1 mg/L. Based on the observed increased in laryngeal squamous metaplasia, the LOAEC for local effects is < 1 mg/L.

In a 13-week inhalation study in rats (0, 0.033, 0.165 or 0.66 mg/L) no effects on mortality, clinical signs, body weight and food consumption, hematology, organ weights and gross pathology were reported. In clinical biochemistry, a statistically significant increase in triglyceride values in the low and medium male exposure groups (134 and 123% of controls) were observed; an inverse relation between exposure and triglyceride values. In females, slightly lower values were measured at the low and medium exposure groups (83 and 93% of controls). No further changes were reported for clinical biochemistry.

At histopathology, minimal squamous metaplasia of epithelium lining of the base of the epiglottis was observed half of the animals of the high dose group, one high dose animal showed mild laryngeal squamous metaplasia.
The toxicological relevance of the observed changes in triglycerides at the low and medium dose groups is unclear. However, since no corroborative findings were reported in the liver (clinical biochemistry, liver weight and histopathology) and no effect on body weight was reported, this finding is not considered adverse. Therefore the NOAEC for systemic effects is ≥ 0.66 mg/L. Based on the laryngeal squamous metaplasia at the high dose group, the NOAEC for local effects is 0.165 mg/L.