Coffee, bean, roasted, ext.

Endpoint summary

Description of key information

ORAL

LD50: >2000 mg/kg bw (Coffea Canephora robusta)

LD50: 220 - 412 mg/kg bw (Caffeine)

INHALATION

LC50: 4.94 mg/l (Caffeine)

DERMAL

LD50: >2000 mg/kg bw (Caffeine)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

No further information provided.

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: roasted and ground beans of Coffea canephora robusta
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerated
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The aqueous extract was obtained by infusion of roasted and ground beans of Coffea canephora robusta (Ccr). A filter coffee machine of Philips brand Daily collection insulated stainless timer HD7479/20, was used to prepare coffee. The infusion was made with 30 g of roast and ground beans of Ccr in 175 ml of distilled water. The filtrate obtained was evaporated in an oven at a temperature of 60 °C. The crystals obtained were pulverized. The captured fine powder was kept refrigerated in sterile glass jars sealed. This technique yielded 3g of dry extract.
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid: not applicable

OTHER SPECIFICS:
To extract caffeine, one gram of freeze-dried coffee ground powder was transferred to a volumetric flask of 50 ml. An amount of 35 ml of methanol was added and the resulting suspension was immersed in an ultrasonic bath for 10 minutes. The suspension was subsequently cooled to room temperature and filtered on Whatman paper N°4.
The HPLC system consisting of a pump, a UV detector, and controlled by a computer (software) was used for assaying the proportion of caffeine in the extract. Qualitative analysis of caffeine was obtained by comparison of retentions times of the compounds eluted to the retention times of the reference solutions. The concentrations were determined from the average of the peak areas of the reference solutions. Caffeine content being the average of 3 tests, the concentration of caffeine in the extract is given by the following formula:
SC = S area x CW/ W area, where

SC = sample concentration
CW = Concentration witness
W area = peak area witness
S area = peak area sample

Species:

rat

Strain:

Wistar

Remarks:

albino

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Pasteur Institute Adipodoumé, Abidjan, Ivory Coast
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8-10 weeks
- Weight at study initiation: between 100 and 120 g
- Fasting period before study: not specified
- Housing: wire cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 3ºC
- Humidity (%): 50%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12 hours

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Test solutions and control solution were administered by a stomach tube.

Doses:

2000 and 5000 mg/kg bw

No. of animals per sex per dose:

3 female rats

Control animals:

yes

Remarks:

control animals received distiled water

Details on study design:

- Duration of observation period following administration: 14 days

- Frequency of observations and weighing:
Clinical signs were observed at 30 minutes, 1, 4, 8 , 24 and 48 hours.
The weight of each animal was determined before administration and then every third day.

- Necropsy of survivors performed: yes

- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other:
Organs such as the kidney, liver and heart were removed and weighed.
The biochemical parameters such urea, blood sugar, creatinine, transaminases ASAT and ALAT and hematological parameters such as leukocytes, erythrocytes and platelets counts, hemoglobin, hematocrit, MCV, MCHC were determined.

Statistics:

The statistical analysis was performed using the Graph Pad Prism 5 software. The analysis of variance ANOVA (One-way ANOVA) followed by the Tukey-Kramer test was used for comparison of results. The difference is considered statistically significant when P <0.05.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 - < 5 000 mg/kg bw

Based on:

test mat.

Mortality:

Two deaths occured at the dose of 5000 mg/kg bw after 24 and 48 hours. No deaths related to the extract administration were recorded at the dose of 2000 mg/kg bw.

Clinical signs:

other: During the first hour after administration of the extract, the rats from experimental batches seemed agitated. This excitation was attributed to caffeine, which increases, neuromuscular transmission and increases the neural excitability reducing the disch

Gross pathology:

No effects were observed on liver, heart and kidney in experimental and control animals.

Other findings:

Caffeine content in the aqueous extract of roasted and ground beans of Coffea Canephora robusta was determined at 7.5%.

Effects on the organs weigh in animals receiving 2000 mg/kg of bw of the extract.

Organ (g)	Control rats	Experimental rats
Liver	4.08±0.41	4.17±0.28
Heart	0.43±0.26	0.47±0.14
Kidney	0.64±0.025	0.67±0.02

 

Effects on biochemical parameters in animals receiving 2000 mg/kg of bw of the extract.

Parameter	Control rats	Experimental rats
Urea (g/L)	0.19±0.01	0.18±0.02
Blood sugar (g/L)	0.95±0.16	1.05±0.20
Creatinine (mg/L)	8.67±0.95	7.67±1.04
Transaminase ASAT (U/L)	35.33±3.06	32.0±4.57
Transaminase ALAT (U/L)	39.33±1.67	37.67±2.62

 

Effects on leukocytes parameters in animals receiving 2000 mg/kg of bw of the extract.

Parameter	Control rats	Experimental rats
Polymorphonuclear neutrophils (%)	14.0±2.65	12.67±1.15
Polymorphonuclear eosinophils (%)	1.67±0.58	1.33±0.58
Polymorphonuclear basophils (%)	0.0±0.00	0.0±0.00
Lymphocytes (%)	79.33±3.08	81.0±2.00
Monocytes (%)	5.0±1.00	5.0±1.00

 

Effects on hematological parameters in animals receiving 2000 mg/kg of bw of the extract.

Parameter	Control rats	Experimental rats
Leukocytes (10^9/L)	8.43±2.21	10.53±1.77
Erythrocytes (10^12/L)	6.93±0.63	7.17±0.24
Hemoglobin (g/dL)	13.07±0.57	13.73±0.40
Hematocrit (%)	37.1±1.81	39.47±1.40
MCV (fl)	51.60±1.15	54.37±1.30
MCH (pg)	17.87±0.75	18.57±1.07
MCHC (g/dL)	34.40±1.02	34.67±0.85
Platelets (10^9/L)	728±109	753±127

Administration of roasted and ground beans of Coffea canephora robusta extract did not have any effect on biochemical and hematological parameters. Normal values of urea and creatinine suggest that this extract did not alter the structure and renal functions. In addition, the levels of transaminases (ALAT) and (ASAT) were not altered during the experiment indicating that liver and to a lesser degree, muscles were not affected.

Interpretation of results:

GHS criteria not met

Conclusions:

In an acute oral toxicity study in rats, conducted according to OECD test guideline 423, a LD50 value was determined between 2000 and 5000 mg/kg body weight for aqueous extract of roasted and ground beans of Coffea canephora robusta containing 7.5% of caffeine.

Executive summary:

The acute oral toxicity study in rats, conducted according to OECD test guideline 423 was performed to assess the acute toxicity potential of aqueous extract of roasted and ground beans of Coffea canephora robusta.

Two sets of 3 animals received Coffea canephora robusta extract at 2000 mg/kg body weight, another set of 3 animals received a dose of 5000 mg/kg body weight and a control group were administered distilled water, by stomach tube.

Clinical signs including diarrhea, lethargy, excitability and death were observed at 30 minutes, 1, 4, 8, 24 and 48 hours. The relative weights of organs (kidneys, liver and heart) were recorded at the beginning of the study, and then every third day. At the end of the study, animals were sacrificed and gross pathology was performed. Additionally, the biochemical and hematological parameters were determined. 

No deaths were observed at 2000 mg/kg body weight, two deaths occurred at the dose of 5000 mg/kg body weight after 24 and 48 hours. There were no significant effects on the body weight in animals receiving coffee extract in comparison to control animals. Additionally, all animals from control and treatment groups gained weight during the experiment.

The administration of aqueous extract of roasted and ground beans of Coffea Canephora robusta did not have any significant effect on the relative weights of organs and biochemical and hematological parameters

The value of the lethal dose 50 (LD50) in this study has been determined between 2000 and 5000 mg/kg body weight, indicating that the aqueous extract of roasted and ground beans of Coffea Canephora robusta containing 7.5% of caffeine has relatively low acute toxicity and does not meet GHS criteria for acute toxicity classification.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

NCTR Standard Procedure, similar to OECD guideline 401

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine, purity was 99.9% (analyzed)
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: a stock solution of caffeine/sodium benzoate was prepared on April 6 and 7, 1981. Sodium benzoate was added to enhace the solubility of caffeine. Caffeine stock solutionn contained 160 mg/ml caffeine and 160 mg/ml sodium benzoate. Serial dilutions were made with a sodium benzoate water solution so that final concentrations of the sodium benzoate reminded contstant and caffeine concentrations decreased by one-half.
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available

OTHER SPECIFICS: none

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

other: sodium benzoate water solution

Details on oral exposure:

Caffeine was administered one time by oral exposure (gavage) in an aqueous sodium benzoate solution. Sodium benzoate increased the solubility of caffeine.

Doses:

50, 100, 200, 400, and 800 mg/kg bw

No. of animals per sex per dose:

6

Control animals:

no

Details on study design:

Caffeine, dissolved in an aqueous solution containing 160 mg/ml sodium benzoate, was administered by oral intubation in an approximate volume of 0.5 ml per 100 g body weight. The five dosage groups ( each with six male and six female rats) were administered caffeine at a dose of either 800, 400, 200, 100 or 50 mg/kg body weight. A single oral dose was given in the morning after a period of 18 hours of fasting. Animals were observed, clinical signs recorded and dead animals removed in the morning and afternoon of each day following administration of the compound for a period of 14 days. Rats were removed from cages and individually observed during the morning and afternoon observation periods (approximately 9-10 a.m. and 2-3 p.pm.). All animals were subjected to a routine necropsy. Gross observations were recorded and tissues with apparent lesions were preserved and subjected to routine histological evaluation.

Statistics:

The SAS probit procedure was used to estimate LD50 and LD10 values, as well as the upper and lower 95% confidence limits on these two values. Male and female rats were combined in making these estimates. Estimates of LD10 values were determined from the plots of the results.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

ca. 220 mg/kg bw

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

ca. 240 mg/kg bw

Mortality:

All deaths occured within the first three days of the experiment and in the two highest dosage groups.

Clinical signs:

other: Clinical symptoms occured on the days 1-3 after gavaging and frequently disappeared by the end of the 14 day observation period. They included: discharge from the mouth, letargy, labored or slow respiration, discolored skin, walk-toe, sunken eyes.

Gross pathology:

Hemorrage and autolysis of the thymus in one of the female rat. It was concidered coincidental and not dose-related.

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

In an acute oral toxicity study in Fischer 344 male and female rats, conducted according to a conventional method, a LD50 value for caffeine was determined at approximately 220 and 240 mg/kg body weight for male and female rats respectively.

Executive summary:

The acute oral toxicity study in Fisher 344 male and female rats conducted according to conventional method was performed to assess the acute toxicity potential of caffeine.

Groups of 6 rats per sex were administered the test substance by gavage at dose levels of 50, 100, 200, 400, and 800 mg/kg body weight. The LD50 was determined to be between 200 and 400 mg/kg body weight for male and female rats. However, the slope of the the LD50 curve was rather steep, especially for males, therefore, the value of LD50 determined from the plotted data were approximetely 220 and 240 mg/kg body weight for male and female rats respectively, allocating caffeine in Category 3 based on GHS criteria for acute toxicity classification.

Reference 3

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine, purity of 98.5 - 100 % w/w
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not available
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available

OTHER SPECIFICS: none

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5%

Details on oral exposure:

Rats were given a single dose of the test substance

Doses:

178, 261, and 383 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

Animals were observed for 14 days

Key result

Dose descriptor:

LD50

Effect level:

>= 261 - <= 383 mg/kg bw

Mortality:

Three males and all females of the high dose group died within the first day after dosing.

Gross pathology:

Pathology revealed general congestive hyperemia in the animals that died; and no abnormalities were found in the survivors.

Interpretation of results:

Category 3 based on GHS criteria

Conclusions:

In an acute oral toxicity study in Wistar male and female rats, conducted according to a conventional method, a LD50 value was between 261 and 383 mg/kg body weight for caffeine.

Executive summary:

The acute oral toxicity study in male and female rats conducted according to conventional method was performed to assess the acute toxicity potential of caffeine.

Groups of 5 male and 5 female Wistar rats were given a single dose of the test substance at dose levels of 178, 261, and 383 (by gavage; dissolved in 0.5% aqueous carboxymethyl cellulose) and were observed for 14 days.

Three males and all females of the high dose group died within the first day after dosing. Pathology revealed general congestive hyperemia in the animals that died; and no abnormalities were found in the survivers.

The value of the lethal dose 50 (LD50) in this study has been determined between 261 and 383 mg/kg body weight, allocating caffeine in Category 3/4 based on GHS criteria for acute toxicity classification.

Reference 4

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

GLP compliance:

no

Test type:

standard acute method

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not available
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not available

OTHER SPECIFICS: none

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Route of administration:

oral: gavage

Details on oral exposure:

Animals were deprived of food for 18-20 hours prior to dosing by oral gavage. After dosing, the animals were permitted access to food ad libitum.

Doses:

Animals were dosed in groups of 10, usually with a geometric progression of 1.4 between successive dose groups.

Details on study design:

Aminals were observed for a total of 2 weeks after dosing.

Statistics:

Data were analyzed by the PROBIT procedure of the SAS system.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

344 mg/kg bw

Based on:

test mat.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In an acute oral toxicity study in Sprague-Dawley male rats, conducted according to a conventional method, a LD50 value was at 344 mg/kg body weight for caffeine.

Executive summary:

The acute oral toxicity study in Sprague-Dawley male rats conducted according to conventional method was performed to assess the acute toxicity potential of caffeine.

Animals were dosed in groups of 10, with a geometric progression of 1.4 between successive dose groups, and were observed for a total of 2 weeks after dosing.

The value of the lethal dose 50 (LD50) in this study has been determined at 344 mg/kg body weight, allocating caffeine in Category 4 based on GHS criteria for acute toxicity classification.

Reference 5

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

GLP compliance:

no

Test type:

up-and-down procedure

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not available
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not available

OTHER SPECIFICS: none

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Route of administration:

oral: gavage

Details on oral exposure:

Animals were deprived of food for 18-20 hours prior to dosing by oral gavage. After dosing, the animals were permitted access to food ad libitum.

Doses:

The dose for each animal was based on fasted body weight. Animals were dosed singly, at least I day apart. If an animal died or appeared moribund the day after dosing, the dose for the next animal was decreased by a factor of 1/1.3; otherwise the dose for the next animal was increased by a factor of 1.3

Details on study design:

Animals were observed 1 week after dosing

Statistics:

Studies were analyzed by the method of maximum likelihood using the SAS procedure NLIN.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

421 mg/kg bw

Based on:

test mat.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In an acute oral toxicity study in Sprague-Dawley male rats, conducted according to an up-and down method, a LD50 value was at 412 mg/kg body weight for caffeine.

Executive summary:

The acute oral toxicity study in Sprague-Dawley male rats conducted according to an up-and down method was performed to assess the acute toxicity potential of caffeine.

Animals were dosed singly, at least 1 day apart. If an animal died or appeared moribund the day after dosing, the dose for the next animal was decreased by a factor of 1/1.3; otherwise, the dose for the next animal was increased by a factor of 1.3. Animals were observed for 1 week after dosing.

The value of the lethal dose 50 (LD50) in this study has been determined at 412 mg/kg body weight, allocating caffeine in Category 4 based on GHS criteria for acute toxicity classification.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

2 concentrations only

Principles of method if other than guideline:

A third test group (as required by the OECD guideline no. 403) was not included since a third dose level (higher than 4.94 mg/l) was expected to be lethal to all exposed animals.

GLP compliance:

no

Test type:

traditional method

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine, purity of 99.5 - 100% (dried substance)
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not available
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available

OTHER SPECIFICS: none

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

other: Aerosil R 972 at 2%

Details on inhalation exposure:

The rats were exposed for 4 hours and observed for 14 days (day of exposure = day 0).

Concentrations:

Nominal concentrations of 2.48 and 4.94 mg/l

No. of animals per sex per dose:

5

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

ca. 4.94 mg/L air (nominal)

Mortality:

No deaths were observed at the low dose level.
In the high dose group, 6/10 animals died. Late deaths were observed; one male each died at day 0 and 7, respectively; 2, 1, and 1 female died at day 0, 1, and 2, respectively.

Clinical signs:

other: Clinical signs of toxicity included changes in respiration (irregular, accelerated, intermittent, gasping), eyelid closure, salivation, restlessness, attempts to escape, reddish nasal discharge, apathy, and (in the high dose group only) decreased pain ref

Gross pathology:

Pathology revealed general congestion, bloody ulcers in the glandular stomachs, and/or intensified hyperemia in the animals that died. No pathological changes were observed in the survivors.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

In an acute inhalation toxicity study in Wistar male and female rats conducted according to OECD test guideline 403, the LC50 value was determined at 4.94 mg/l for caffeine aerosol.

Executive summary:

The acute inhalation toxicity study in Wistar male and female rats conducted according OECD test guideline 403 was performed to assess the acute toxicity potential of caffeine aerosol.

Groups of 5 Wistar rats per sex were exposed to caffeine aerosol at nominal concentrations of 2.48 and 4.94 mg/l using a head-nose inhalation system. The rats were exposed for 4 hours and observed for 14 days. No deaths were observed at the low dose level. In the high dose group, 6/10 animals died. Late deaths were observed; one male each died at day 0 and 7, respectively; 2, 1, and 1 female died at day 0, 1, and 2, respectively.

Clinical signs of toxicity included changes in respiration (irregular, accelerated, intermittent, gasping), eyelid closure, salivation, restlessness, attempts to escape, reddish nasal discharge, apathy, and (in the high dose group only) decreased pain reflex and death. Pathology revealed general congestion, bloody ulcers in the glandular stomachs, and/or intensified hyperemia in the animals that died. No pathological changes were observed in the survivors.

The value of the lethal concentration 50 (LC50) in this study has been determined at 4.94 mg/l, allocaing caffeine in Category 4 based on GHS criteria for acute toxicity classification.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline available

GLP compliance:

no

Test type:

other: behavioral and neurochemical pharmacology study

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine, purity of 99.5 - 100% (dried substance)
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: caffeine was volatilized in the volatilization device.
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not available

OTHER SPECIFICS: The volatilization device consisted of a stainlesssteel tube (length 10 cm, i.d. 0.6 mm) filled with steel wool used in order to keep the caffeine at the top of the tube. The tube was closely connected to a plastic syringe (60 ml) through which a vacuum system was set up (representing the human’s respiratory system). At the end of the tube, a lighter was turn on and caffeine was heated until the vapor generation started.

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: IIBCE animal facilities, Montevideo
- Females (if applicable) nulliparous and non-pregnant: only male used
- Age at study initiation: not available
- Weight at study initiation: 250–310 g
- Fasting period before study: not specified
- Housing: five animals in plastic cages (50 x 37.5 x 21 cm3)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC,
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 h day–night cycle, lights on at 7h00 am
IN-LIFE DATES: From: To: not available

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Details on inhalation exposure:

The caffeine vapor collected in the plastic syringe was injected through a valve placed in one of the walls of a whole-body chamber (inhalation chamber; 23 x 15 x 10 cm3) where animals were separately placed and exposed acutely to different doses of volatilized caffeine for 10 min.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

GC–MS

Duration of exposure:

10 min

Concentrations:

10, 25, and 50 mg

Control animals:

yes

Details on study design:

After the exposure to the volatilized drug—or control—animals were removed from the inhalation chamber and placed in an Open Field paradigm (OF, a square box of 60 9 60 cm2 with red 40-cm-high acrylic sides) associated to the EthoVision XT 7 software (Noldus, The Netherlands). Using this video tracking software, the horizontal locomotor activity defined as the total distance moved in meters (m) and velocity of movement were measured.

Locomotor and exploratory (number of rearings) activities were registered during 30 and 15 min, respectively. During all the experiments the inhalation chamber and the OF were cleaned with alcohol 30 % before placing the following rat. All experiments were done between 9 a.m. and 3 p.m. In order to measure plasmatic caffeine levels, plasma samples of each animal (control and treated) were collected immediately after the end of behavioral recordings.

Animals were killed by decapitation and trunk blood was collected in plastic Falcon tubes containing a solution of citric acid (0.05 mM), sodium citrate (0.09 mM), and sodium fluoride (0.2 mM). Blood was then centrifuged (Centrifuge, Thermo Scientific) for 15 min at 3000 g and 4ºC. The recovered plasma was stored at -80ºC until assay.

Statistics:

Data are given as mean ± standard error of the mean (SEM) and were analyzed by two-way (time and pretreatment) analysis of variance (ANOVA) for repeated measures followed by post hoc Newman- Keuls multiple comparison test and by one-way ANOVA for independent measures (treatment) followed by Newman–Keuls test. Linear regression was applied. Statistical significance was set at P<0.05.

Sex:

male

Dose descriptor:

other: LC50

Effect level:

> 50 other: mg

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

No mortality was observed

Clinical signs:

other: Not observed

Body weight:

Not determined

Gross pathology:

Not determined

Other findings:

Stimulant effect of volatilized caffeine was observed. Distance moved of rats treated with 50 mg of caffeine was significantly higher than control group (P<0.001), caffeine 25 mg (P<0.01), and 10 mg (P<0.01). The effect of lower doses of caffeine (10 and 25 mg) was not significantly different compared to the control group. Distance moved profile was similarly accompanied by the changes in the velocity of movement and rearing behavior. Animals stimulated with caffeine 50 mg were faster than control group (P>0.001), caffeine 25 mg (P<0.01), and caffeine 10 mg (P<0.01). Again, the effects of lower doses of caffeine (10 and 25 mg) were not significantly different from control animals. Caffeine at 50 mg (P<0.05) but not at 10 or 25 mg doses significantly increased the number of rearings.
Additionally, blood analysis showed a dose-dependent stimulant effect and a significant increment in extracellular dopamine levels after the acute treatment of caffeine.

Interpretation of results:

study cannot be used for classification

Conclusions:

No deaths or undesirable effects of caffeine were noted in Wistar male rats exposed to maximum of 50 mg volatilized caffeine during one inhalation session.

Executive summary:

To study the behavioral and neurochemical pharmacology of caffeine, Wistar male rats were exposed to 10, 20 and 50 mg of volatilized caffeine during one inhalation session of 10 min in the whole-body chambers.

The stimulant effect of caffeine was automatically recorded for several hours and plasmatic levels of caffeine were measured. No deaths or undesirable effects of caffeine were noted. A dose-dependent stimulant effect such as distance moved, velocity of movement and rearing behavior, induced by volatilized caffeine was observed and this effect was directly related with caffeine plasmatic levels. A significant increase in the extracellular dopamine was achieved after 50 mg of volatilized caffeine exposure.

The results of this study support the conclusion from the acute inhalation study that pure caffeine is considered of low toxicity to rats by inhalation route.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

not specified

Test type:

fixed dose procedure

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: caffeine (anhydrous powder), purity of 98.5 - 100 % w/w
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not available
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not available

OTHER SPECIFICS: none

Species:

rat

Strain:

Wistar

Sex:

male/female

Type of coverage:

semiocclusive

Vehicle:

water

Details on dermal exposure:

A 24-hour semiocclusive application of a 50% (w/v) aqueous suspension of the test substance. After 24 hours, the patches were removed, and the application sites were washed with water.

Duration of exposure:

24 hours

Doses:

2000 mg/kg

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

Observation period of 14 days post exposure.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Mortality:

No deaths were recorded.

Clinical signs:

other: No signs of toxicity were observed within the 14-day observation period.

Gross pathology:

No local/pathological findings were observed within the 14-day observation period.

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 value in this study has been determined at >2000 mg/kg body weight, indicating that the aqueous caffeine solution has low acute toxicity and it does not meet the classification criteria for skin irritation under the CLP Regulation (EC) No 1272/2008

Executive summary:

The acute dermal toxicity study in male and female Wistar rats was performed to assess the acute toxicity potential of caffeine solution.

A 24-hour semiocclusive application of a 50% (w/v) aqueous suspension of caffeine at 2000 mg/kg body weight was applied on five male and five female Wistar rats. After 24 hours, the patches were removed, and the application sites were washed with water. No deaths, signs of toxicity, or local/pathological findings were observed within the 14-day observation period.

The LD50 value in this study has been determined at >2000 mg/kg body weight, indicating that the aqueous caffeine solution has relatively low acute toxicity and does not meet GHS criteria for acute toxicity classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

ORAL

The acute oral toxicity study in rats, conducted according to OECD test guideline 423 was performed to assess the acute toxicity potential of aqueous extract of roasted and ground beans of Coffea canephora robusta.

Two sets of 3 animals received Coffea canephora robusta extract at 2000 mg/kg body weight, another set of 3 animals received a dose of 5000 mg/kg body weight and a control group were administered distilled water, by stomach tube.

Clinical signs including diarrhea, lethargy, excitability and death were observed at 30 minutes, 1, 4, 8, 24 and 48 hours. The relative weights of organs (kidneys, liver and heart) were recorded at the beginning of the study, and then every third day. At the end of the study, animals were sacrificed and gross pathology was performed. Additionally, the biochemical and hematological parameters were determined. 

No deaths were observed at 2000 mg/kg body weight, two deaths occurred at the dose of 5000 mg/kg body weight after 24 and 48 hours. There were no significant effects on the body weight in animals receiving coffee extract in comparison to control animals. Additionally, all animals from control and treatment groups gained weight during the experiment.

The administration of aqueous extract of roasted and ground beans of Coffea Canephora robusta did not have any significant effect on the relative weights of organs and biochemical and hematological parameters

The value of the lethal dose 50 (LD50) in this study has been determined between 2000 and 5000 mg/kg body weight, indicating that the aqueous extract of roasted and ground beans of Coffea Canephora robusta containing 7.5% of caffeine has relatively low acute toxicity and does not meet the criteria set in the CLP Regulation (EC) 1272/2008 for acute toxicity classification.

The physiological effects of coffee are generally attributed to caffeine. Caffeine is a stimulant of the central nervous system, respiration, and skeletal muscles; other activities include cardiac stimulation, coronary dilation, smooth muscle relaxation, and diuresis. Generally, Arabica coffee extracts contain less caffeine.

Several acute oral rat studies for caffeine were published, in which caffeine showed moderate toxicity after oral uptake showing clinical symptoms of toxicity such as dyspnoea and staggering. LD50 values were ranging from 220 to 412 mg/kg body weight.

 

INHALATION

The acute inhalation toxicity study in Wistar male and female rats conducted according OECD test guideline 403 was performed to assess the acute toxicity potential of caffeine aerosol.

Groups of 5 Wistar rats per sex were exposed to caffeine aerosol at nominal concentrations of 2.48 and 4.94 mg/l using a head-nose inhalation system. The rats were exposed for 4 hours and observed for 14 days. No deaths were observed at the low dose level. In the high dose group, 6/10 animals died. Late deaths were observed; one male each died at day 0 and 7, respectively; 2, 1, and 1 female died at day 0, 1, and 2, respectively.

Clinical signs of toxicity included changes in respiration (irregular, accelerated, intermittent, gasping), eyelid closure, salivation, restlessness, attempts to escape, reddish nasal discharge, apathy, and (in the high dose group only) decreased pain reflex and death. Pathology revealed general congestion, bloody ulcers in the glandular stomachs, and/or intensified hyperemia in the animals that died. No pathological changes were observed in the survivors.

The value of the lethal concentration 50 (LC50) in this study has been determined at 4.94 mg/l, allocating caffeine in Category 4 based on criteria set in the CLP Regulation (EC) 1272/2008 for acute toxicity classification.

To study the behavioral and neurochemical pharmacology of caffeine, Wistar male rats were exposed to 10, 20 and 50 mg of volatilized caffeine during one inhalation session of 10 min in the whole-body chambers.

The stimulant effect of caffeine was automatically recorded for several hours and plasmatic levels of caffeine were measured. No deaths or undesirable effects of caffeine were noted. A dose-dependent stimulant effect such as distance moved, velocity of movement and rearing behavior, induced by volatilized caffeine was observed and this effect was directly related with caffeine plasmatic levels. A significant increase in the extracellular dopamine was achieved after 50 mg of volatilized caffeine exposure.

DERMAL

The acute dermal toxicity study in Wistar male and female rats was performed to assess the acute toxicity potential of caffeine solution. A 24-hour semiocclusive application of a 50% (w/v) aqueous suspension of caffeine at 2000 mg/kg body weight was applied on five male and five female Wistar rats. After 24 hours, the patches were removed, and the application sites were washed with water. No deaths, signs of toxicity, or local/pathological findings were observed within the 14-day observation period.

The value of the lethal dose 50 (LD50) in this study has been determined at >2000 mg/kg body weight, indicating that the aqueous caffeine solution has relatively low acute toxicity and does not meet

criteria set in the CLP Regulation (EC) 1272/2008 for acute toxicity classification.

Justification for classification or non-classification

Coffee and its natural preparations are generally recognized as safe for human consumption. Coffee extracts have a wide spectrum of actions and therefore, they are also used in many types of cosmetics. The physiological effects of coffee are generally attributed to caffeine. Due to caffeine’s direct absorption to the blood stream, bypassing the hepatic first-pass effect, its alveolar absorption is not considered to differ from the oral and/or intravenous route, indicating that the physiological effects of caffeine will not vary depending on different routes of exposure.

Based on the low acute oral toxicity of Coffea Canephora robusta aqueous extract (LD50 >2000 mg/kg body weight) and low to moderate acute oral, inhalation and dermal toxicity of caffeine, a minor constituent of Coffee, bean, roasted, ext. no acute oral, inhalation and dermal toxicity is expected for Coffee, bean, roasted, ext. and therefore, no classification for acute toxicity is warranted according to the criteria set in the CLP Regulation (EC) 1272/2008.