Glycerol

Endpoint summary

Description of key information

In the best available dietary study, groups of 22 rats (Long-Evans)/sex/treatment received 5, 10 and 20% glycerol (natural or synthetic) in their diet (males 2000, 4000 and 8000 mg/kg bw; females 2500, 5000 and 10000 mg/kg bw) for 2 years. Although the results were not described in detail, based on this limited dietary study it can be concluded that no adverse effects were observed at up to 10,000 mg/kg bw.

 

The effect of glycerine following administration for 90 days in a subchronic toxicity study was examined.  Rats fed 5 or 20% glycerine in the diet for 90 days gained weight at a faster rate than control animals. There were no adverse treatment related effects noted in male or female rats fed 5% glycerine in the diet.  In the male rats which received 20 percent glycerine, there was an increase in the final liver/body weight ratio and upon microscopic examination generalized cloudy swelling and hypertrophy of the parenchymal cells was observed. The only effect in the female rats on this level was some generalized cloudy selling upon microscopic examination of the liver.  A 5% glycerol in the diet corresponded to 4580 and 6450 mg/kg/day for male and female rats, respectively, after 4 weeks and a 20% glycerol in the diet corresponded to 18,750 and 25,800 mg/kg/day for male and female rats, respectively, after 4 weeks.

 

A number of other studies have been incorporated in the dossier. These studies are considered less reliable indicators of the systemic effects of glycerol following repeated administration, mainly because of limited toxicity assessments and/or deficient experimental design. The effects they do report are consistent with those observed in the key studies and as such they may contribute to the overall assessment of toxicity of glycerol.

 

 

The effects following repeated dermal application of glycerin was examined. There were no effects noted in rabbits dosed 8 hours/day, 5 days/week for 45 weeks with dose levels as high as 4.0 ml/kg.  Using a density of 1.2611 g/cm3 at 20 °C, a dose of 4.0 ml/kg corresponds to 5040 mg/kg/day.

 

The subchronic toxicity of glycerol was examined following aerosol exposure. The NOAEC for local and systemic toxicity was 622 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was conducted prior to GLP and test guidelines, but sufficient data is available for interpretation of results

Qualifier:

no guideline available

Principles of method if other than guideline:

Test material was administered in the diet for up to 2 years. Study design followed intent of OECD 452. The number of animals/dose level used was less than required and minimal histopathology was conducted.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Long-Evans

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS
- Age: not indicated
- Weight at study initiation: 96-109 g (males), 92-108 g (females)
- Number of animals: 22/sex/treatment, 26/sex for controls
- Source: Institute of Experimental Biology of University of California

Upon arrival at the laboratory, each rat was assigned to an individual numbered cage. Distribution among the experimental groups was accomplished by reference to a standard random number table.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

ADMINISTRATION / EXPOSURE
- Exposure period: 2 year (1 year for the high dose group)
- Route of administration: oral in diet. Diet consisted of a standard dog-food meal with which the glycerin was thoroughly mixed. Feed was prepared once a week and stored in stoneware crocks. The crocks were weighed and refilled weekly, and the unconsumed feed was discarded.
- Doses: 5, 10 and 20% in diet; males 2000, 4000 and 8000 mg/kg bw, females 2500, 5000 and 10000 mg/kg bw
- Water was allowed freely.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not applicable.

Duration of treatment / exposure:

Continuous

Frequency of treatment:

Daily for up to two years

Remarks:

Doses / Concentrations:
5, 10 and 20% in diet; males 2000, 4000 and 8000 mg/kg bw, females 2500, 5000 and 10000 mg/kg bw
Basis:
nominal in diet

No. of animals per sex per dose:

22/sex/treatment, 26/sex for controls

Control animals:

yes, concurrent no treatment

Details on study design:

No additional information available.

Positive control:

No data.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: daily in cage and weekly examination outside the home cage
- Mortality: daily
- Body weight: weekly
- Food consumption: weekly
- Haematology: erythrocyte and leucocyte count and haemoglobin after 3, 6, 12, 18 and 24 months
- Urinalysis: albumin, glucose, casts and red and white blood cells after 3, 6, 12, 18 and 24 months (24-48 hr urine collection)

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Organ weights: liver, kidneys, heart, spleen and lungs
- Macroscopic: no details provided
- Microscopic: liver, spleen, adrenals, kidney, small intestine, gonads and urinary bladder

Other examinations:

glycogen and lipid content of the liver of surviving rats at 0 and 20% glycerol.

Statistics:

Chi-sqare test, student t-test, ANOVA (Fisher) were used as appropriate.

Clinical signs:

not specified

Mortality:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slightly increased (significant) in males at 5 and 10% natural glycerin

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality and time to death: not indicated
- Clinical signs: not reported
- Body weight gain: no statistically significant differences between treated and control animals
- Food consumption: slightly increased (significant) in males at 5 and 10% natural glycerin
- Haematology: no treatment related effects
- Urinalysis: albumin: no significant treatment related effects (92% incidence in females at 20% natural glycerin compared to 54-64% in controls); glucose, casts, red and white blood cells: no treatment related effects
- Organ weights: incidental increases and decreases were reported without apparent relationship to treatment
- Gross pathology: no lesions related to treatment.
- Histopathology: Incidental bronchiectasis, pneumonia, pulmonary abcesses, taenia infestation of the liver, hydronephosis and pyelonephritis (total 27 rats were affected).
- Other: liver glycogen and lipid did not significantly differ between 0 and 20% glycerin (liver glycogen natural glycerin 4.2-4.3% and synthetic glycerin 3.7-4.2%)

Dose descriptor:

NOAEL

Effect level:

> 8 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Effect level:

> 10 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

No additional information available.

Conclusions:

The NOAEL was 8000-10,000 mg/kg bw based on the absence of treatment related effects in high dose animals.

Executive summary:

The chronic toxicity of glycerin was examined in rats fed the test material in the diet at concentrations of 5, 10 and 20% for up to two years. The NOAEL was 8000-10,000 mg/kg bw based on the absence of treatment related effects in high dose animals.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between July and August 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: The experimental period was 28 days, in which food
and water were given ad libitum. Glycerol was supplied by gavage at specific doses for each group

- Short description of test conditions:

- Parameters analysed / observed:
# The feed and leftovers were weighed and recorded to determine the individual feed intake.
# The animals were weighed at the beginning and at the end of the experiment
to determine the average daily weight gain (final weight – initial weight/28 days).
# The volumes of water intake and urine were measured daily with the
aid of tubes, to assess the effects of glycerol on the water balance
# Levels of glucose, triacylglycerol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), high-density lipoprotein (HDLc) and low-/very low-density lipoprotein (LDLc + VLDLc) fractions in plasma were quantified
# the abdominal cavity of the animals was surgically opened to expose the internal organs.
# In sequence, the pancreas, kidneys and liver were collected for the determination of the relative weight (organ weight/body weight of the animal
#Liver fragments were removed, immersed in isopentane and immediately cryopreserved in liquid nitrogen. Cryostate sections were stained with haematoxylin and eosin to characterise the structural integrity.
# The other organs (pancreas and kidneys) were collected and fixed in 10% formalin for 24 h and subsequently stored in 70% ethanol until histological
processing. All samples were subjected to routine techniques for paraffin embedding. Sections of 5 lm thickness were obtained and stained with haematoxylin– eosin. Digital images were obtained using a system
for capturing and analysing images.

GLP compliance:

not specified

Remarks:

no information provided in publication

Specific details on test material used for the study:

pure glycerol (glycerine with double distilled 99.85% of glycerol – ALMAD, Aracatuba, SP, Brazil)
fatty acids and esters, 0.02%;
heavy metals, <5 ppm;
density, 1.2604 g/ml;
pH 6.09;
water
content, 0.20%;
glycerol content, 99.85%.

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 202.7 +/- 29.98
- Diet: comercial diet at libitum
- Water: ad libitum
- Acclimation period: 7 days


DETAILS OF FOOD AND WATER QUALITY:
The basal diet consisted of a commercial feed for rats
(Nuvilab; Nuvital Feeding, Curitiba, PR, Brazil),
containing by weight: 19% protein, 56% carbohydrate,
3.5% fat, 4.5% cellulose, 5.0% vitamins and
minerals, 12% humidity and an energy content of
17.03 kJ/g.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Photoperiod (hrs dark / hrs light): 12/12
- housing in individual metabolic cages

Route of administration:

oral: gavage

Vehicle:

water

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Dose / conc.:

1 600 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

The volumes of water intake and urine were measured daily

Sacrifice and pathology:

On the 28th day, the animals were fasted for 8 h and were killed by exsanguination by cardiac puncture under general anaesthesia
pancreas, kidneys and liver were collected for the determination of the relative weight
(organ weight/body weight)
Liver fragments were removed, immersed in isopentane and immediately cryopreserved in liquid
nitrogen. The sections were stained with haematoxylin and eosin to characterise the structural integrity.
The other organs (pancreas and kidneys) were collected and fixed in 10% formalin for 24 h and subsequently stored in 70% ethanol until histological processing.

Other examinations:

The feed and leftovers were weighed and recorded to determine the individual feed intake.

Statistics:

Data were subjected to analysis of variance and then multivariate regression.
For the variables, triglycerides, AST, VLDLc + LDLc, percentage of carcass fat, weight gain and average daily urine volume, the data were processed by square root transformation. All statistical
analyses were performed using the statistical program SISVAR (version 5.3).

Clinical signs:

not examined

Mortality:

not specified

Body weight and weight changes:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

The differential count for lymphocytes and neutrophils did not change between the experimental groups.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Plasma glucose and total cholesterol levels demonstrated a significant increase in values up to a dose of 800 mg/kg, followed by a decrease at 1,600 mg/kg bw per day.

In terms of high-density lipoprotein levels, there was an increase up to a dose of 800 mg/kg bw per day, followed by a decrease at 1,600 mg/kg bw per day, but levels were still within the normal range for the species.

For very low-density lipoprotein and low density lipoprotein levels, there was a decrease with increasing glycerol doses, which remained within the standards.

The liver enzymes AST and ALT showed a decrease trend up to the dose of 800 mg/kg bw per day, with an increase at 1,600 mg/kg bw per day.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No lesions were observed in any tissue (pancreas, kidney, liver) from animals receiving different levels of glycerol.

Dose descriptor:

NOAEL

Effect level:

> 1 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

The NOAEL was higher than 1600 mg/kg bw based on the absence of treatment related effects in high dose animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to guideline/s and GLP but the report contains sufficient data for interpretation of study results

Principles of method if other than guideline:

Study design appears to follow intent of OECD 413 but publication does not indicate that OECD 413 was followed.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were obtained from Charles River Breeding Laboratories. Animals were subjected to a 3-week quarantine, health screening five rats of each sex for viral and Mycoplasma infection at the beginning and toward the end of quarantine, physical examinations, body weight measurements, acclimation to the restraining tubes of the nose only exposure system, randomized assignment to exposure groups and individual identification.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: MMAD <2.0 um (respirable)

Details on inhalation exposure:

A viscous-liquid aerosol generator was used to generate aerosol.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES: target concentration and homogeneity of aerosol
- Method: sampling with aerosol monitor and gravimetric and GC analyses
- Sampling times: actual concentration 2 samples per exposure chamber; homogeneity and uniformity in mock exposure before start of the experiment (6 samples) and during animal exposure (20 samples/concentration)

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week for 13 weeks

Remarks:

Doses / Concentrations:
33, 165 and 660 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

15/sex/dose level

Control animals:

yes, concurrent no treatment

Details on study design:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Mortality/clinical signs: twice daily
- Body weight: weekly
- Food consumption: weekly
- Haematology: not specified (complete blood count included)
- Biochemistry: blood urea nitrogen, creatinine, glucose, protein, albumin, albumin/globulin, ASAT, ALAT, LDH, gamma glutamyl transferase, cholesterol, triglycerides and phospholipids

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Organ weights: lungs, liver, kidneys, brain and heart
- Macroscopic: not specified (complete necropsy).
- Microscopic: total of 40 tissues of high dose and control animals; lungs trachea and anterior nasal cavity were stained with hematoxylin and eosin and duplicate slides with Alcian blue/periodic acid Schiff (Goblet cell changes)

Three rats/sex of control and high dose group were killed during week 10, lung lobes were excised and 2 samples/rat were examined by transmission electron microscopy for abnormalities associated with the Clara cells. The same procedure was followed for 3 rats/sex of all groups during terminal necropsy

ANALYSES: target concentration and homogeneity of aerosol
- Method: sampling with aerosol monitor and gravimetric and GC analyses
- Sampling times: actual concentration 2 samples per exposure chamber; homogeneity and uniformity in mock exposure before start of the experiment (6 samples) and during animal exposure (20 samples/concentration)

Positive control:

No data

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Mortality/clinical signs: twice daily
- Body weight: weekly
- Food consumption: weekly
- Haematology: not specified (complete blood count included)
- Biochemistry: blood urea nitrogen, creatinine, glucose, protein, albumin, albumin/globulin, ASAT, ALAT, LDH, gamma glutamyl transferase, cholesterol, triglycerides and phospholipids

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Organ weights: lungs, liver, kidneys, brain and heart
- Macroscopic: not specified (complete necropsy)
- Microscopic: total of 40 tissues of high dose and control animals; lungs trachea and anterior nasal cavity were stained with hematoxylin and eosin and duplicate slides with Alcian blue/periodic acid Schiff (Goblet cell changes)

Three rats/sex of control and high dose group were killed during week 10, lung lobes were excised and 2 samples/rat were examined by transmission electron microscopy for abnormalities associated with the Clara cells. The same procedure was followed for 3 rats/sex of all groups during terminal necropsy

Other examinations:

No additional information available.

Statistics:

STATISTICAL METHODS: ANOVA, least significant difference

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 specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

triglycerides decreased in males at 33 (34%) and 167 mg/m3 (22%) only.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

minimal squamous metaplasia of the epiglottis in 2/25, 1/19, 4/20 and 10/21 rats at 0, 33, 167 and 662 mg/m3; 1 male at 662 mg/m3 showed mild squamous metaplasia. No differences in morphology of the Clara cells in control and high dose rats

Histopathological findings: neoplastic:

not specified

Details on results:

ANALYTICAL ANALYSES:
- Actual dose level: 33, 167 and 662 mg/m3 (100-101% of target)
- Homogeneity (uniformity): relative standard deviation <1% of mean value

- Hematology: No differences between the exposed rats in any group of either sex when compared to their respective control group.
- Clinical chemistry: triglycerides decreased in males at 33 (34%) and 167 mg/m3 (22%) only.
- Gross lesions: few gross lessions observed with no evidence of changes attributed to exposure to glycerol.
- Histopathology: minimal squamous metaplasia of the epiglottis in 2/25, 1/19, 4/20 and 10/21 rats at 0, 33, 167 and 662 mg/m3; 1 male at 662 mg/m3 showed mild squamous metaplasia.
No differences in morphology of the Clara cells in control and high dose rats

STATISTICAL RESULTS: all effects mentioned showed statistical significance (squamous metaplasia only significant at high concentration)

Dose descriptor:

NOAEC

Effect level:

662 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEC

Effect level:

167 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on local irritant effects on the upper respiratory tract.

Critical effects observed:

no

No additional information available.

Conclusions:

Because the metaplasia of the squamous epithelium of the larynx seen in rats at 662 mg/m3 with glycerol aerosol was only minimal to slight, is not interpreted as adverse.

The NOEC of 165 is mg/m3 for local toxicity was based on local irritant effects on the upper respiratory tract.
The NOAEC for local and systemic toxicity is 662 mg/m3.

Executive summary:

The subchronic toxicity of glycerol was examined following aerosol exposure.

Because the metaplasia of the squamous epithelium of the larynx seen in rats at 662 mg/m3 with glycerol aerosol was only minimal to slight, is not interpreted as adverse.

The NOEC of 165 for local toxicity is mg/m3 was based on local irritant effects on the upper respiratory tract.
The NOAEC for local and systemic toxicity is 662 mg/m3.

Endpoint conclusion

Dose descriptor:

NOAEC

622 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information meets the information requirements under REACH

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

repeated dose toxicity: inhalation, other

Type of information:

other: published expert opinion

Adequacy of study:

other information

Conclusions:

As the severity of the squamous metaplasia observed in the 13-week study with glycerol was minimal to slight, the effect is not regarded as adverse

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to guideline/s and GLP but the report contains sufficient data for interpretation of study results

Principles of method if other than guideline:

Study design appears to follow intent of OECD 413 but publication does not indicate that OECD 413 was followed.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were obtained from Charles River Breeding Laboratories. Animals were subjected to a 3-week quarantine, health screening five rats of each sex for viral and Mycoplasma infection at the beginning and toward the end of quarantine, physical examinations, body weight measurements, acclimation to the restraining tubes of the nose only exposure system, randomized assignment to exposure groups and individual identification.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: MMAD <2.0 um (respirable)

Details on inhalation exposure:

A viscous-liquid aerosol generator was used to generate aerosol.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES: target concentration and homogeneity of aerosol
- Method: sampling with aerosol monitor and gravimetric and GC analyses
- Sampling times: actual concentration 2 samples per exposure chamber; homogeneity and uniformity in mock exposure before start of the experiment (6 samples) and during animal exposure (20 samples/concentration)

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week for 13 weeks

Remarks:

Doses / Concentrations:
33, 165 and 660 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

15/sex/dose level

Control animals:

yes, concurrent no treatment

Details on study design:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Mortality/clinical signs: twice daily
- Body weight: weekly
- Food consumption: weekly
- Haematology: not specified (complete blood count included)
- Biochemistry: blood urea nitrogen, creatinine, glucose, protein, albumin, albumin/globulin, ASAT, ALAT, LDH, gamma glutamyl transferase, cholesterol, triglycerides and phospholipids

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Organ weights: lungs, liver, kidneys, brain and heart
- Macroscopic: not specified (complete necropsy).
- Microscopic: total of 40 tissues of high dose and control animals; lungs trachea and anterior nasal cavity were stained with hematoxylin and eosin and duplicate slides with Alcian blue/periodic acid Schiff (Goblet cell changes)

Three rats/sex of control and high dose group were killed during week 10, lung lobes were excised and 2 samples/rat were examined by transmission electron microscopy for abnormalities associated with the Clara cells. The same procedure was followed for 3 rats/sex of all groups during terminal necropsy

ANALYSES: target concentration and homogeneity of aerosol
- Method: sampling with aerosol monitor and gravimetric and GC analyses
- Sampling times: actual concentration 2 samples per exposure chamber; homogeneity and uniformity in mock exposure before start of the experiment (6 samples) and during animal exposure (20 samples/concentration)

Positive control:

No data

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Mortality/clinical signs: twice daily
- Body weight: weekly
- Food consumption: weekly
- Haematology: not specified (complete blood count included)
- Biochemistry: blood urea nitrogen, creatinine, glucose, protein, albumin, albumin/globulin, ASAT, ALAT, LDH, gamma glutamyl transferase, cholesterol, triglycerides and phospholipids

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Organ weights: lungs, liver, kidneys, brain and heart
- Macroscopic: not specified (complete necropsy)
- Microscopic: total of 40 tissues of high dose and control animals; lungs trachea and anterior nasal cavity were stained with hematoxylin and eosin and duplicate slides with Alcian blue/periodic acid Schiff (Goblet cell changes)

Three rats/sex of control and high dose group were killed during week 10, lung lobes were excised and 2 samples/rat were examined by transmission electron microscopy for abnormalities associated with the Clara cells. The same procedure was followed for 3 rats/sex of all groups during terminal necropsy

Other examinations:

No additional information available.

Statistics:

STATISTICAL METHODS: ANOVA, least significant difference

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 specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

triglycerides decreased in males at 33 (34%) and 167 mg/m3 (22%) only.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

minimal squamous metaplasia of the epiglottis in 2/25, 1/19, 4/20 and 10/21 rats at 0, 33, 167 and 662 mg/m3; 1 male at 662 mg/m3 showed mild squamous metaplasia. No differences in morphology of the Clara cells in control and high dose rats

Histopathological findings: neoplastic:

not specified

Details on results:

ANALYTICAL ANALYSES:
- Actual dose level: 33, 167 and 662 mg/m3 (100-101% of target)
- Homogeneity (uniformity): relative standard deviation <1% of mean value

- Hematology: No differences between the exposed rats in any group of either sex when compared to their respective control group.
- Clinical chemistry: triglycerides decreased in males at 33 (34%) and 167 mg/m3 (22%) only.
- Gross lesions: few gross lessions observed with no evidence of changes attributed to exposure to glycerol.
- Histopathology: minimal squamous metaplasia of the epiglottis in 2/25, 1/19, 4/20 and 10/21 rats at 0, 33, 167 and 662 mg/m3; 1 male at 662 mg/m3 showed mild squamous metaplasia.
No differences in morphology of the Clara cells in control and high dose rats

STATISTICAL RESULTS: all effects mentioned showed statistical significance (squamous metaplasia only significant at high concentration)

Dose descriptor:

NOAEC

Effect level:

662 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEC

Effect level:

167 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on local irritant effects on the upper respiratory tract.

Critical effects observed:

no

No additional information available.

Conclusions:

Because the metaplasia of the squamous epithelium of the larynx seen in rats at 662 mg/m3 with glycerol aerosol was only minimal to slight, is not interpreted as adverse.

The NOEC of 165 is mg/m3 for local toxicity was based on local irritant effects on the upper respiratory tract.
The NOAEC for local and systemic toxicity is 662 mg/m3.

Executive summary:

The subchronic toxicity of glycerol was examined following aerosol exposure.

Because the metaplasia of the squamous epithelium of the larynx seen in rats at 662 mg/m3 with glycerol aerosol was only minimal to slight, is not interpreted as adverse.

The NOEC of 165 for local toxicity is mg/m3 was based on local irritant effects on the upper respiratory tract.
The NOAEC for local and systemic toxicity is 662 mg/m3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

662 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information meets the information requirements under REACH

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5 040 mg/kg bw/day

Study duration:

subchronic

Species:

rabbit

Additional information

Study via the dietary, dermal and respiratory route demonstrate the low level of concern about glycerol.

In the available evaluations performed by official bodies (MAK 2007, OECD 2002, EFSA 2017) a number of additional repeated dose toxicity studies were evaluated and summarised.

 

Repeated dose toxicity oral
In the OECD SIDS document the study reported by Hine et al. was identified as the most relevant key study. This study is included in the REACH dossier in detail as RSS.
A number of other studies have been incorporated in a table in the OECD SIDS document. These studies are considered less reliable indicators of the systemic effects of glycerol following repeated administration, mainly because of limited toxicity assessments and/or deficient experimental design. The effects they do report are consistent with those observed in the key studies and as such they may contribute to the overall assessment of toxicity of glycerol.

EFSA conclusion: Short-term or subchronic studies were not performed according to current test guidelines. In a subchronic toxicity study (in drinking water) in rats, the effects reported were observed with doses in the range of the LD50 for glycerol. The Panel considered that the local irritating effects of glycerol in the gastrointestinal tract reported in some gavage studies in rat (100% glycerol at 2,800 mg/kg bw per day, the lowest dose tested (Staples et al., 1967)), and dogs (100% glycerol at 5,600 mg/kg bw per day (Staples et al., 1967)) was likely due to the hygroscopic and osmotic effects of glycerol.

 

From the available chronic toxicity and carcinogenicity studies, glycerol was not carcinogenic in mice and rats and did not show evidence of adverse effects in a 2-year chronic toxicity study. The Panel noted that no adverse effects were reported in rats receiving doses up to 10,000 mg/kg bw per day for 1 year, the highest dose tested. The Panel also noted that there was no increase in the tumours incidences in rats receiving doses up to 5,000 mg/kg bw per day for 2 years, the highest dose tested.The Panel considered that none of the animal studies available identified an adverse effect for glycerol.

 

Repeated dose toxicity inhalation
The key studies identified and evaluated by the OECD are compiled as RSS in the dossier (Renne 1992). Based on an increased incidence of “minimal” to “mild” squamous metaplasia of the epiglottis, the NOAEC for local irritant effects to the upper respiratory tract was derived at 165 mg/m3 and 662 mg/m3 for systemic effects.

The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has re-evaluated glycerol [56-81-5]in 2016, considering the endpoints irritation of the respiratory tract.
Based on the results of an expert workshop, the NOAEC value for local toxicity was re-evaluated. Because the metaplasia of the squamous epithelium of the larynx seen in rats at 662 mg/m3 with glycerol aerosol was only minimal to slight, is not interpreted as adverse (Kaufmann et al. 2009). Furthermore it has been taken into account, that the response does not increase with the exposure duration.

The NOAEC in rats for local and systemic effects after 13-week exposure is 662 mg/m3.

 

Glycerin [MAK Value Documentation in German language, 2007]

The MAK Collection for Occupational Health and Safety 2017, Vol 2, No 2

Re-evaluation of glycerol (E 422) as a food additive, EFSA Journal 2017;15(3):4720

Glycerol, CAS Number 56-81-5, OECD SIDS Initial Assessment Report For SIAM 14 Paris, France, 26-28 March 2002

Kaufmann W, Bader R, Ernst H, Harada T, Hardisty J, Kittel B, Kolling A, Pino M, Renne R, Rittinghausen S, Schulte A, Wöhrmann T, Rosenbruch M (2009) 1st International ESTP Expert Workshop: “Larynx squamous metaplasia”. A re-consideration of morphology and diagnostic approaches in rodent studies and its relevance for human risk assessment. Exp Toxicol Pathol 61: 591–603

 

 

Justification for classification or non-classification

There is no justification for classification based on data from available studies.