Extract obtained from defatted powder of Theobroma cacao (Malvaceae) by extraction with ethanol

Endpoint summary

Description of key information

Acute toxicity, oral in mice: LD50 > 3000 mg/kg bw (read-across from cocoa powder, non-GLP, K, Rel. 2)

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The behavioural effects of cocoa powder were assessed in male mice. Ambulation was measured for 3 hours after treatment with 100, 300, 1000 or 3000 mg/kg bw (and was also measured for 30 minutes before treatment). Avoidance was measured in lever-press and shuttle tests carried out immediately after treatment with 100, 300 or 1000 mg/kg bw. In all cases, the extent of the examination was not given – only behavioural results were reported.

GLP compliance:

no

Test type:

standard acute method

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): cocoa powder
- Physical state: presumably solid

Species:

mouse

Strain:

other: dd strain for ambulation tests and ddY for avoidance tests

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Gunma University School of Medicine (dd strain) and Japan Laboratory Animals (ddY strain)
- Age at study initiation: 7 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: standard breeding cages, with 10 animals per cage
- Diet (e.g. ad libitum): ad libitum access to solid diet
- Water (e.g. ad libitum): ad libitum access to tap water
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1 °C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Route of administration:

oral: gavage

Vehicle:

physiological saline

Details on oral exposure:

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

Doses:

Ambulation test: 0 (saline), 100, 300, 1000 or 3000 mg/kg bw
Avoidance tests: 0 (saline), 100, 300 or 1000 mg/kg bw

No. of animals per sex per dose:

Ambulation test: 10-20 [possibly per dose, but may be the total number tested. Mice were ‘drug-naïve’, so different mice were probably tested at each dose]
Avoidance tests: 9 (lever-press), 10 (shuttle) [possibly per dose, but appears to be the total number tested, with the same group of mice given each dose]

Control animals:

yes

Details on study design:

- Duration of observation period following administration: at least 3 hours (ambulation).

For the avoidance tests, “drug testing sessions were repeated at intervals of 3-4 days”. It is not clear if each drug testing session constitutes a single treatment with a single dose, or whether each concentration was tested simultaneously on different groups of mice. Cocoa testing was followed by theobromine (at 0, 3, 10, 30, 100, 300 mg/kg bw) and caffeine (at 0, 1, 3, 10, 30, 100 mg/kg bw) testing, apparently on the same animals. Assuming these cocoa contituents would not improve health, the observation period for mortality due to cocoa is equivalent to at least 6 days.
- Frequency of observations and weighing: no data
- Necropsy of survivors performed: no
- Other examinations performed: behaviour (ambulation and avoidance)

Statistics:

Mean overall ambulatory activity count, and mean response and avoidance rates calculated. Data were analysed by analysis of variance (ANOVA). If significant overall effects were seen, individual data were compared using a two-tailed t-test and / or a Cochran-Cox test.

Sex:

male

Dose descriptor:

LD50

Effect level:

> 1 000 mg/kg bw

Remarks on result:

other: With an observation period of at least 6 days (during which theobromine and caffeine were also apparently administered to the same animals), no mortalities were reported in mice given cocoa at up to 1000 mg/kg bw.

Sex:

male

Effect level:

> 3 000 mg/kg bw

Remarks on result:

other: With an observation period of 3-4 hours, no mortalities were reported in mice given cocoa at up to 3000 mg/kg bw

Mortality:

Mortality was not specifically assessed, but would presumably have been reported. No deaths were noted within 3-4 hours of treatment with up to 3000 mg/kg bw (in the ambulation experiment) or within at least 6 days of treatment with up to 1000 mg/kg bw (in the avoidance tests).

Clinical signs:

Clinical signs were not specifically assessed. Presumably overt clinical signs would have been reported. No such effects are noted.

Body weight:

No data

Gross pathology:

Not examined

Other findings:

- Behaviour: cocoa at 1000 mg/kg bw significantly increased ambulatory activity, but no significant effects were seen at the top dose of 3000 mg/kg bw.
No significant effect was seen in the lever-press avoidance or in the shuttle avoidance test. However, the response rate was significantly increased in mice given the top dose of 1000 mg/kg bw in the shuttle avoidance test (about 6 responses per minute, compared with 5 per minute in control animals given saline).

Interpretation of results:

other: Not classified

Remarks:

CLP

Conclusions:

Mice were administered cocoa powder orally [by gavage] in tests to assess effects on behaviour. Although not specifically assessed, presumably no mortality of overt effects were seen at 1000 mg/kg bw (in avoidance tests; 6 day observation period), or up to 3000 mg/kg bw (in an ambulation test; 3-hr observation period).

Executive summary:

A non-guideline study has been carried out to assess the behavioural effects of acute oral treatment with cocoa powder on male mice.

 

In an ambulation study, drug-naïve dd mice were given saline, or cocoa powder by stomach tube at 100, 300, 1000 or 3000 mg/kg bw [it is not clear whether 10-20 mice were tested in total, or whether there were 10-20 per concentration]. Saline was administered to 10-20 control animals. Ambulation was assessed for 3 hours after treatment using a tilting-type ambulometer.

 

Lever-press and shuttle avoidance experiments were conducted on 9 or 10 trained ddY mice, respectively. Mice were treated with 0 (saline), 100, 300 or 1000 mg/kg bw by stomach tube. [It appears that the same mice were treated with each concentration.] Apparently the same mice were later tested with the cocoa components theobromine (at 3-300 mg/kg bw) and caffeine (at 1-100 mg/kg bw). Each “drug testing session” was separated by an interval of 3-4 days. [It is not clear whether these “drug testing sessions” comprised all testing with one substance, or each test with an individual concentration.] The minimum observation period after testing with cocoa was therefore 6 days.

 

In each experiment, mortality and clinical observations are not described. Presumably, however, mortality and other overt effects would have been reported, if seen. No significant, dose-related behavioural effects were reported following treatment with cocoa powder.

 

As no deaths were reported, presumably the acute oral LD50 exceeds 3000 mg/kg bw in male mice (3 h observation period). In the experiment using a more reliable observation time of 6 days, the LD50 presumably exceeds 1000 mg/kg bw. While these values are not sufficient to formally conclude on classification for acute oral toxicity, the LD50 for Cocoa powder probably exceeds 2000 mg/kg bw.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Sex:

male

Dose descriptor:

LD50

Effect level:

> 1 000 mg/kg bw

Remarks on result:

other: With an observation period of at least 6 days (during which theobromine and caffeine were also apparently administered to the same animals), no mortalities were reported in mice given cocoa at up to 1000 mg/kg bw.

Sex:

male

Effect level:

> 3 000 mg/kg bw

Remarks on result:

other: With an observation period of 3-4 hours, no mortalities were reported in mice given cocoa at up to 3000 mg/kg bw

Mortality:

Mortality was not specifically assessed, but would presumably have been reported. No deaths were noted within 3-4 hours of treatment with up to 3000 mg/kg bw (in the ambulation experiment) or within at least 6 days of treatment with up to 1000 mg/kg bw (in the avoidance tests).

Clinical signs:

Clinical signs were not specifically assessed. Presumably overt clinical signs would have been reported. No such effects are noted.

Body weight:

No data

Gross pathology:

Not examined

Other findings:

- Behaviour: cocoa at 1000 mg/kg bw significantly increased ambulatory activity, but no significant effects were seen at the top dose of 3000 mg/kg bw.
No significant effect was seen in the lever-press avoidance or in the shuttle avoidance test. However, the response rate was significantly increased in mice given the top dose of 1000 mg/kg bw in the shuttle avoidance test (about 6 responses per minute, compared with 5 per minute in control animals given saline).

Interpretation of results:

other: Not classified

Remarks:

CLP

Conclusions:

Mice were administered cocoa powder orally [by gavage] in tests to assess effects on behaviour. Although not specifically assessed, presumably no mortality of overt effects were seen at 1000 mg/kg bw (in avoidance tests; 6-day observation period), or up to 3000 mg/kg bw (in an ambulation test; 3-h observation period).

Executive summary:

A non-guideline study has been carried out to assess the behavioural effects of acute oral treatment with cocoa powder on male mice.

 

In an ambulation study, drug-naïve dd mice were given saline, or cocoa powder by stomach tube at 100, 300, 1000 or 3000 mg/kg bw [it is not clear whether 10-20 mice were tested in total, or whether there were 10-20 per concentration]. Saline was administered to 10-20 control animals. Ambulation was assessed for 3 hours after treatment using a tilting-type ambulometer.

 

Lever-press and shuttle avoidance experiments were conducted on 9 or 10 trained ddY mice, respectively. Mice were treated with 0 (saline), 100, 300 or 1000 mg/kg bw by stomach tube. [It appears that the same mice were treated with each concentration.] Apparently the same mice were later tested with the cocoa components theobromine (at 3-300 mg/kg bw) and caffeine (at 1-100 mg/kg bw). Each “drug testing session” was separated by an interval of 3-4 days. The minimum observation period after testing with cocoa was therefore 6 days.

 

In each experiment, mortality and clinical observations are not described. Presumably, however, mortality and other overt effects would have been reported, if seen. No significant, dose-related behavioural effects were reported following treatment with cocoa powder.

 

As no deaths were reported, presumably the acute oral LD50 exceeds 3000 mg/kg bw in male mice (3 h observation period). In the experiment using a more reliable observation time of 6 days, the LD50 presumably exceeds 1000 mg/kg bw. While these values are not sufficient to formally conclude on classification for acute oral toxicity, the LD50 for Cocoa powder probably exceeds 2000 mg/kg bw. On this basis cocoa extract would not require classification for acute toxicity in accordance with Regulation EC No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

3 000 mg/kg bw

Quality of whole database:

Appropriate for hazard assessment

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute toxicity: via oral route

A non-guideline study has been carried out to assess the behavioural effects of acute oral treatment with cocoa powder on male mice.

 In an ambulation study, drug-naïve dd mice were given saline, or cocoa powder by stomach tube at 100, 300, 1000 or 3000 mg/kg bw [it is not clear whether 10-20 mice were tested in total, or whether there were 10-20 per concentration]. Saline was administered to 10-20 control animals. Ambulation was assessed for 3 hours after treatment using a tilting-type ambulometer.

 Lever-press and shuttle avoidance experiments were conducted on 9 or 10 trained ddY mice, respectively. Mice were treated with 0 (saline), 100, 300 or 1000 mg/kg bw by stomach tube. [It appears that the same mice were treated with each concentration.] Apparently the same mice were later tested with the cocoa components theobromine (at 3-300 mg/kg bw) and caffeine (at 1-100 mg/kg bw). Each “drug testing session” was separated by an interval of 3-4 days. The minimum observation period after testing with cocoa was therefore 6 days.

 In each experiment, mortality and clinical observations are not described. Presumably, however, mortality and other overt effects would have been reported, if seen. No significant, dose-related behavioural effects were reported following treatment with cocoa powder.

 As no deaths were reported, presumably the acute oral LD50 exceeds 3000 mg/kg bw in male mice (3 h observation period). In the experiment using a more reliable observation time of 6 days, the LD50 presumably exceeds 1000 mg/kg bw. While these values are not sufficient to formally conclude on classification for acute oral toxicity, the LD50 for Cocoa powder probably exceeds 2000 mg/kg bw. On this basis cocoa extract would not require classification for acute toxicity in accordance with Regulation EC No. 1272/2008.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Acute toxicity via Oral route:

Based on the available information, the registered substance is:

- not classified according to the Regulation (EC) No. 1272/2008 and GHS.

Acute toxicity via Dermal route:This information is not available

Acute toxicity via Inhalation:This information is not available.

Specific target organ toxicity: single exposure (Oral):

The classification criteria according to the Annex VI of the Regulation (EC) No. 1272/2008 as specific target organ toxicant (STOT) – single exposure, oral are not met since no reversible or irreversible adverse health effects were observed immediately or delayed after exposure and no effects were observed at the guidance value (oral) for a Category 1 classification (C ≤ 300 mg/kg bw) and at the guidance value (oral) for a Category 2 classification (2000 mg/kg bw ≥ C > 300 mg/kg bw). No classification is required.

Specific target organ toxicity: single exposure (Dermal): This information is not available

Specific target organ toxicity: single exposure (Inhalation): This information is not available.

Based on its physical state, the registered substance is not classified for aspiration hazard.