Theobromine

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Similar to OECD 212. No data on GLP and analytical monitoring.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 212

GLP compliance:

not specified

Analytical monitoring:

not specified

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: To produce a defined and standardized vehicle for these experiments, 10% Hank’s balanced salt solution (made from cell-culture tested, powdered Hank’s salts, without sodium bicarbonate, Cat. No H6136-10X1L, Sigma-Aldrich, St Louis, MO)was used at a concentration 0.98 g/L in Milli-Q water (resistivity =18.2 MΩpercm), with the addition of sodium bicarbonate at 0.035 g/L (Cell culture tested, Sigma Cat S5761), and adjusted to pH 7.46.- Controls: yes, vehicle (buffer)
- Evidence of undissolved material (e.g. precipitate, surface film, etc): at the highest concentration (1000 mg/L), compounds were precipitated out of solution.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish- Strain: AB wild type
- Source: Selecta Aquarium Speciaalzaak (Leiden, the Netherlands) who obtain stock from Europet Bernina International BV (Gemert-Bakel, the Netherlands).
- Method of breeding: First were kept at a maximum density of 100 individuals in glass recirculation aquaria (L 80 cm; H 50 cm, W 46 cm) on a 14 h light : 10 h dark cycle. Water and air were temperature controlled (25 ± 5ºC and 23ºC, respectively)- Food type: O.S.L. Marine Lab., Inc., Burlingame, USA and Dutch Select Food, Aquadistri BV, the Netherlands
- Frequency: twice daily with ‘Sprirulina’ brand flake (OSL) food and twice a week with frozen food (Dutch)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

25 ± 0.5ºC

pH:

7.46 in buffer (adjusted)

Nominal and measured concentrations:

Nominal: 0, 30, 60, 120, 240 and 480 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass recirculation aquaria- Material, size, headspace, fill volume: length: 80 cm, heigth: 50 cm, width: 46 cm
- No. of organisms per vessel: maximum density of 100 individuals per glass recirculation aquaria.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-Q water (resistivity = 18.2 MΩcm)

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: 14h light: 10 h dark cycle (lights on at 08.00)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality after 96 hours exposure.

TEST CONCENTRATIONS
Range finding study:
- Test concentrations: 0, 1, 10, 100 and 1000 (mg/L)
- Results used to determine the conditions for the definitive study: Not specified.

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

150.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: ±1.83

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.83 mmol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: ± 0.01

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: in Table 1, for the highest logarithmic series concentration percentage mortality was found but, theobromine was precipitated out of solution.

Reported statistics and error estimates:

LC50 (expressed in mg/L of buffer) was determined based on cumulative mortality obtained from three independent experiments at 120 hpf using Regression Probit analysis with SPSS Statistics for windows version 17.0 (SPSS Inc., Chicago, USA).Statistical analyses were performed using GraphPad Prism for Windows (version 5.03) or R (v. 2.12). One way ANOVA and Newman-Keuls Multiple Comparison test was employed for survival rate. Correlation and ANCOVA models were used to investigate the relationship between LC50 in zebrafish embryos and published LD50 values in rodents.

Sublethal observations / clinical signs:

Table 1:

Cumulative % mortality after 96 h exposure with theobromine
Logarithmic series (mg/L)					Geometric series± SEM
0	1	10	100	1000	0	30	60	120	240	480
0	0	0	50	100*	0±0	13±2	15±2	38±0	58±4	100±0

* percentage of mortality was found but at these high concentrations, theobromine was precipitated out of solution.

Table 2: Zebrafish embryo LC50 values for theobromine found in this study, and the corresponding rodent LD50 values based on the literature

Zebrafish Embryo LC50 (mg/L± SEM)	Zebrafish Embryo LC50 (mmol/L± SEM)	Rodent LD50 (mg/kg)	Rodent LD50 (mmol/kg)
150.4±1.83	0.83±0.01	530(*)	2.94

(*)from Chemical Identification/Dictionary database at http://toxnet.nlm.nih.gov/cgibin/sis/search/

Validity criteria fulfilled:

not specified

Conclusions:

The 96h-LC50 for theobromine in zebrafish was 150.4 ± 1.83 (mg/L ± SEM).

Executive summary:

A short-term toxicity study was carried out with a method similar to OECD Guidelines 212. The test was conducted with zebrafish embryos.To determine a suitable range of concentrations for testing, a range-finding test was performed using a logarithmic series (0.0, 1.0, 10.0, 100.0 and 1000 mg/L) as recommended in standard protocols. After the range finding experiments, a series of concentrations lying in the range between 0% and 100% mortality were selected. The concentrations were 0, 30, 60, 120, 240 and 480 mg/L, each concentration was repeated three times (in total 48 embryos per concentration).The embryos were exposed to the test substance for 96 h. 96h-LC50 (expressed in mg/L or mmol/L.) was determined based on cumulative mortality obtained from three independent experiments. The results showed that the LC50 for theobromine was 150.4 ± 1.83 (mg/L ± SEM) or 0.83 ± 0.01 (mmol/L ± SEM).

Description of key information

Weight of evidence: The available 96-LC50 comes from a test method similar to OECD 212. No data on GLP. The 96h-LC50 for theobromine was 150.4 mg/L in zebrafish embryo.
The other study reports a 96h-LC0 of 158 mg/kg-bw in Cyprinus carpio.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

150.4 mg/L

Additional information

Weight of evidence: The available LC50 comes from a test method similar to OECD 212.The test was conducted with zebrafish embryos in three independent experiments. A total of 48 zebrafish embryos were exposed to test concentrations of 0, 30, 60, 120, 240 and 480 mg/L based on range-finding studies. The embryos were exposed to the test substance for 96 h. The 96h-LC50 for theobromine was 150.4 ± 1.83 mg/L. The other study reported a 96h-LC0 of 158 mg/kg-bw in Cyprinus carpio.This data is consistent with the 96 -LC50 in embryo zebrafish.