Theobromine

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

Test method according to OECD 407. No data on GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Several parameters not analysed.

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:

TEST ANIMALS
- Source: A.R.S. Sprague-Dawley, Madison, Wisconsin.
- Age at study initiation: Mature rats: 13 weeks old and females: 15 weeks old.Immature rats: 27 days
- Weight at study initiation: Mature rats: Males (22% casein diet) approximately 350 g; males (10% casein diet) approximately 310 g; females (22% casein diet) approximately 240 gImmature rats: Males (22% casein diet) approximately 75 g; males ( 10% casein diet) approximately 65 g; females ( 22% casein diet) approximately 70 g
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.
- Acclimation period: To the diet: Mature rats: 6 weeks "Purina rat chow" diet, 1 week purified diet.Immature rats: 1 week purified diet

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Remarks:

In diet

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONSDIET PREPARATION
Mature rats were fed Purina rat chow for 6 weeks. Then were fed the purified diet in agar form prepared according to the method of Shank and Newberne for 1 week. The nutritional composition was: A.N.R.C. Reference casein 22%; Vitamin mix A.O.A.C., 1% ; Mineral mix U.S.P. XVIII, 5%; corn oil (Mazola), 10 %; cellulose, 1%; and glucose monohydrate, 61%. One group of male rats was given a modified diet composed of A.N.R.C. Reference casein, 10%; corn oil, 8%, glucose monohydrate, 75%, the other ingredients being of the same concentration as indicated above.Immature rats were fed the purified diet for 1 week.Groups of rats were divided in subgroups, each of which was given diets similar to the purified but containing different concentrations of theobromine.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

0.2 other: %

Remarks:

Basis:nominal in diet

Dose / conc.:

0.4 other: %

Remarks:

Basis: nominal in diet

Dose / conc.:

0.6 other: %

Remarks:

Basis:nominal in diet

Dose / conc.:

0.8 other: %

Remarks:

Basis:nominal in diet

Dose / conc.:

1 other: %

Remarks:

Basis:nominal in diet

No. of animals per sex per dose:

10 rats/sexe/dose

Control animals:

yes, plain diet

Details on study design:

Male and female, mature and immature rats were fed different concentrations of theobromine in diet (22% casein).In order to compare effects of diet, male, mature and immature rats were fed with a low protein diet (10% casein) with the same concentrations of theobormine as for the first experiment.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes. Twice a week
FOOD CONSUMPTION AND COMPOUND INTAKE: Food intake was monitored twice a week
FOOD EFFICIENCY: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: No.
HISTOPATHOLOGY: Yes

Other examinations:

At the conclusion of the 28-day feeding period, each rat was weighed, killed by decapitation and exsanguinated. The following organs were removed and weighed: brain, thymus, liver, kidneys, testes of male rats, and reproductive tract of female rats. Segments of these organs and of lung, heart, adrenal glands, pancreas, stomach, duodenum, jejunum, terminal ileum, and colon were fixed in 10% neutral buffered formaldehyde solution for histological examination. Sections were stained with hematoxylin-eosin.

Statistics:

t Tests were performed on all data according to the method of Snedecor and Cochran (1971).

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

General decreases in body weight.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

As the investigation proceeded it became clear that feeding high levels of theobromine to rats produced marked decreases in food intake.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

General decrease in thymus and, in many groups, testicular weights.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Few deaths occurred during the experiments with male and female immature rats given the theobromine in 22% casein diet but were random and not correlated with a specific dietary level of theobromine.The combination of higher levels of theobromine and 10% casein diet decreased survival. None of the male rats in the group of 1 % dietary theobromine and 10% casein survived the 4 week period.

BODY WEIGHT AND WEIGHT GAIN
The prominent effect of increasing concentration of dietary theobromine were anorexia (except female rats given the 22 % casein diet), decreases in bodyweight in mature rats, growth retardation in immature rats.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
As the investigation proceeded it became clear that feeding high levels of theobromine to rats produced marked decreases in food intake. ORGAN WEIGHTSChanges in organ weights are presented as tables.General decrease in thymus weights. Significant decreases in testicular weights ocurred in the male rats at higher theobromine concentrations (22 % casein diet) with earlier and greater decreases in male rats given the 10% protein diet.

HISTOPATHOLOGY: NON-NEOPLASTIC
The progressive decreases in thymus and testicular weights with increasing doses of dietary theobromine concentrations in male rats given the 22% casein diet were accompanied by striking changes in tissue morphology as viewed by light microscopy. There was a decreased lymphocyte density and blurring of the demarcation between cortex and medulla in the thymus glands from rats given diets containing 0.6% theobromine. At the 0.8 % dietary theobromine concentration, the cortex of the thymus was almost devoid of lymphocytes and zonal demarcations were absent. Thymic remnants from rats given diets containing 1.0% theobromine were composed only of stromal elements and scattered medullary lymphocytes. Testicular cytology was normal in rats fed diets containing up to and including 0.4% theobromine. An abrupt change was obvious in testes taken from rats given 0.6% dietary theobromine and was characterized by seminiferous tubular cell degeneration, by distortion of the normal cellular relationships and the presence of multinucleate cells. Testicular tissue from rats given the 0.8% dietary theobromine showed extensive tubular cell degeneration and necrosis, oligospermia, and may multinucleated cells, some of which resembled giant cells. In the testes of rats given 1% theobromine-containing diets, there was obvious atrophy, aspermia, widespread tubular cell degeneration and necrosis, and the presence of many multinucleated cells, some of which were in various stages of degeneration and some obviously necrotic.Although the weights of the thymus glands were significantly reduced in female rats given dietary theobromine at the 0.2 and 0.4% concentrations, significant histopathologic changes were only observed in the thymus glands from female rats fed 0.6% and higher levels of theobromine. At 0.6 and 0.8 %, there was a marked decrease in lymphocyte density and in the depth of the thymic cortex but zonal demarcations were distinct.The thymus in adult female rats given the 1.0% theobromine-containing diets was severely atrophied, almost depleted of lymphocytes and zonal demarcations were either faint or more often completely absent. Morphologic lesions in mature male rats given the 10% casein diet occurred in the same glands: thymus and testes with a comparable but not identical sequence as in the mature male rats given the 22% casein diet. The testes of adult male rats given the 10% casein diet with 0.6 % theobromine contained many multinucleate cells, some of which resembled giant cells, against a background of seminiferous tubular cell degeneration and necrosis. At the higher levels of dietary theobromine, both tubular cell destruction and multinucleate cell formation and degeneration were exaggerated and were accompanied by oligospermia or aspermia.

Key result

Dose descriptor:

LOAEL

Remarks:

Mature rats

Effect level:

<= 349 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

no NOAEL identified

Dose descriptor:

LOAEL

Remarks:

Mature rats ( 10% casein diet)

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Remarks:

Mature rats ( 22 % casein diet)

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Remarks:

Immature rats (22 % casein diet)

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

349 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Critical effects observed:

yes

Lowest effective dose / conc.:

349 mg/kg bw/day (nominal)

System:

immune system

Organ:

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1. Average theobromine intake by adult rats during last 3 weeks (linearity) of a 4-week treatment

Sex	Dietary protein	Theobromine intake from dietary theobromine levels (%) (mg/kg/day)
		0.2	0.4	0.6	0.8	1.0
Male	22	94	215	310	422	492
Female	22	110	236	355	489	596
Male	10	144	291	382	425	562

Each group consisted of 10 animals

 

Table 2. Thymus, kidney and testicular weights in adult male rats given varying levels of theobromine in a 22% casein diet

Organ	Organ weight at dietary theobromine levels (%)(% body weight)
	0.0	0.2	0.4	0.6	0.8	1.0
Thymus	0.177 ± 0.006	0.106 ± 0.011	0.088 ± 0.006^a	0.081 ± 0.009^a	0.053 ± 0.004^a	0.039 ± 0.003^a
Kidneys	0.344 ± 0.004	0.336 ± 0.003	0.343 ± 0.005	0.368 ± 0.007	0.378 ± 0.005	0.447 ± 0.022^a
Testes	0.456 ± 0.007	0.458±0.012	0.433 ± 0.006	0.451 ± 0.008	0.393 ± 0.008^a	0.279 ± 0.011^a

 ^ap < 0.01 when compared with controls,ttest.

 

 Table 3    Thymus, kidney and testicular weights in adult female rats given varying levels of theobromine in a 22% casein diet

Organ	Organ weight at dietary theobromine levels (%)(% body weight)
	0.0	0.2	0.4	0.6	0.8	1.0
Thymus	0.120 ± 0.010	0.063 ± 0.003^a	0.057 ± 0.006^a	0.057 ± 0.006^a	0.043 ± 0.003^a	0.033 ± 0.003^a
Kidneys	0.364 ± 0.008	0.334 ± 0.005	0.362 ± 0.005	0.386 ± 0.006	0.407 ± 0.009^a	0.422 ± 0.013^a
Reproductive system	0.473 ± 0.042	0.709±0.138	0.686 ± 0.081	0.938 ± 0.105	0.803 ± 0.096	0.619 ± 0.105

 ^ap < 0.01 when compared with controls,ttest.

 

Table 4    Thymus, kidney and testicular weights in adult male rats given varying levels of theobromine in a 10% casein diet

Organ	Organ weight at dietary theobromine levels (%)(% body weight)
	0.0	0.2	0.4	0.6	0.8	1.0
Thymus	0.144 ± 0.01	0.086 ± 0.008^a	0.066 ± 0.005^a	0.038 ± 0.002	0.028 ± 0.004	^b
Kidneys	0.304 ± 0.005	0.315 ± 0.003	0.323 ± 0.004	0.402 ± 0.022	0.475 ± 0.02	0.530 ± 0.038^a
Testes	0.481 ± 0.006	0.486±0.063	0.477 ± 0.014	0.340 ± 0.023	0.228 ± 0.03	0.186 ± 0.004

^ap < 0.01 when compared with controls,ttest.

^bThymic remnants too small to weight accurately

Conclusions:

Feeding high levels of theobromine to rats produced not only marked changes in the morphology of certain organs (thymus and testes) but also marked decreases in food intake and bodyweight. The LOEL was 110 mg/kg b.w however the histopathological examination revealed signs of dysfunction at 0.6 % (approximately 349 mg/kg-bw/day).

Executive summary:

A short-term toxicity study was conducted according to OECD guideline 407. Theobromine was given to mature and immature, male and female rats in concentrations of 0, 0.2, 0.4, 0.6, 0.8 and 1.0 % in diet. The diets contained 10 or 22% casein and were fed for 28 days. After this exposure-period animals were killed and different organs were weighed and prepared to histological evaluation. The prominent effects of increasing concentration of dietary theobromine were anorexia (except female rats given 22% casein diet) and atrophy of the thymus gland and testes which became prominent at the 0.6% dietary theobromine level. Many histopathological changes at the thymus gland and testes were detected, including necrosis at high theobromine levels. The low protein diet enhanced the severity of theobromine effects. The daily dose of theobromine which produced retrogressive changes in weight pattern and in the morphology of the thymus in both sexes and of the testes in males was approximately 250 – 300 mg/kg b.w./day (0.4 %) in mature rats and approximately 500 mg/kg b.w./day (0.4%) in inmature rats, although decreases in body weights, food consumption and thymus weights were seen in some mature and immature rats groups given 22 and 10% casein diets with theobromine at 0.2 %, which corresponds to 110 -144 mg/kg b.w./day (mature rats) and 200 mg/kg/day (immature rats), thus the LOEL can be considered 110 mg/kg b.w. However, the histopatological evaluation of the thymus showed adverse findings at 0.6 % (approximately 349 mg/kg-bw/day in mature rats). Given that theobromine produced decreases in food intake, the changes in organ weights and structure could well have been a reflection of decreased food intake rather than any specific effects of theobromine. That is why a series of complementary experiments (pair fed) were conducted in order to obtain some distinction between the effects of decreased food intake and direct effects of theobromine. These experiments are figured as supporting study and its results support the conclusion that it was theobromine which produced the mentioned adverse effects, and not the decrease of food intake.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LOAEL

349 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study has a Klimish 2 and its results are supported by a pair-fed study. There are other studies performed in other animal species which differ from the OECD guidelines in number of animals per dose group or time exposure but they are scientifically consistent with the key study, and can be taken into account in a weight of evidence.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The key study was performed in rats according to OECD guideline 407; a posterior supporting pair-fed study validated the conclusion that histological changes in thymus and testes were a direct effect of dietary theobromine and not a reflection of the reduced food intake induced by theobromine. As a weight of evidence, diverse studies are presented and were performed with other rodent and non-rodent species: Hamsters, mice and rabbits, all of them showing the same target organs (testes and thymus) and reduction in food intake and weight gain. Nevertheless, it was concluded that hamsters and mice were more resistant to toxic effects induced by theobromine than were rats. A posterior supporting study was conducted to determine wheter the observed changes in rat testes were reversible. It was concluded that the theobromine-induced testicular injury appears to be irreversible over time in the affected seminiferous tubules. The relevance of the observed effects in the testes should be evaluated at the reproductive toxicity point. Although decreases in thymus weight were seen, histopatological examination only revealed organ dysfunction at 0.6 % (equivalent to approximately 300 -350 mg/kg/day) in mature rats.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study: Test method accoding to OECD guideline 407. A 28-day study (Klimish 2) was chosen instead of the 90-day (Klimish 4) study for quality facts and because the range of concentrations used in the 28-day study is wider.

Justification for classification or non-classification

The key study is a 28-day repeated-dose (subacute) toxicity study which was performed in mature and immature rats, decreases in thymus weight was seen, however histopatological examination only revealed organ dysfunction at 0.6 % (equivalent to approximately 300 -350 mg/kg/day). Therefore, if the guidance values on classification are corrected by a factor of 3 as recommended by Haber's rule, theobromine would not classify for specific target organ toxicity-repeated exposure with thymus target organs according to CLP Regulation (EC) no. 1272/2008.