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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
Weight of evidence: Two studies are available and can be used in a weight of evidence. Few doses were tested, few examinations were performed and they did not correctly follow a internationally accepted guideline. However, theobromine effects in the testes were identified in both studies.
Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20 April 1982- 27 September 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Test method according reproductive assessment by continuous breeding (NTP). GLP study. General lack of information on method.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
other: National Toxicology Program U.S. Reproductive Assessment by Continuous Breeding
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
other: CD-1 (ICR)BR outbred albino
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
120 days mininum.
Frequency of treatment:
Daily with feed.
Dose / conc.:
126 mg/kg bw/day (nominal)
Dose / conc.:
335 mg/kg bw/day (nominal)
Dose / conc.:
630 mg/kg bw/day (nominal)
Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
POST-MORTEM EXAMINATIONS: Yes. Organs examined: kidney weight, liver weight
OTHER: Estrous cycle length, absolute testis, epididymis weight, sex accessory gland weight (prostate, seminal vesicle), epidid. sperm parameters (motility, morphology), determination of affected sex (crossover), hormonal analyses (luternizing hormones, follicle stimulating hormones, prolactin, progesteron, estrogen and testosterone.
Sperm parameters (parental animals):
Parameters examined in all male parental generations: absolute testis, epididymis weight, sex accessory gland weight (prostate, seminal vesicle), epididymis sperm parameters (motility, morphology)
Litter observations:
PARAMETERS EXAMINED: The following parameters were examined in F1 and F2 offspring: litters/pair, live pups/litter, pup wt./litter, cumulative days to litter
GROSS EXAMINATION OF DEAD PUPS :no
Postmortem examinations (parental animals):
SACRIFICEAfter the delivery and analysis of these litters, the first-generation control and high dose animals were killed and necropsied.
Reproductive indices:
Reproductive indices were adversely affected by theobromine exposure. The mean number of litters per pair was decreased at the high dose level by 19%.
Offspring viability indices:
Live pups per litter dropped by 15% for the low dose, 21% for the medium dose, and 66% for the high dose mice. Additionally, the proportion born alive was reduced by 5 and 35% at the middle and high dose groups, respectively, and pup weight adjusted for litter size was reduced at all dose levels by 4, 13, and 27%, for the low, medium, and high doses.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
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 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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): four females died: one control, two middle dose, and one high dose. The variety of postmortem findings and the lack of relationship to dose led to the conclusion that these deaths were not treatment related.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): no alteration

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): no alteration

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): Absolute testis weight decreased by 12% at the high dose. Although epididymal sperm density and motility did not differ between the control and 0.5% theobromine groups, the treated animals had approximately 4-fold more abnormally shaped sperm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): it clearly reduced female reproductive capability: there was a 74% reduction in the number of live pups delivered of treated females mated to control males, the proportion of those pups born alive was reduced by 50%, and the weight of those pups adjusted for litter size was reduced by 28%.

ORGAN WEIGHTS (PARENTAL ANIMALS): adjusted liver weights increased for both sexes at the high dose by 12% for males and 9% for females.

OTHER FINDINGS (PARENTAL ANIMALS). Hormonal analysis: No differences were seen between the treated and control mice in the levels of luternizing hormones, follicle stimulating hormones, prolactin, progesterone, estrogen, and testosterone.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
<= 126 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
General toxicity
Effect level:
< 630 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Critical effects observed:
yes
Lowest effective dose / conc.:
630 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.:
630 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
one control, two middle dose, and one high dose. The variety of postmortem findings and the lack of relationship to dose led to the conclusion that these deaths were not treatment related.
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Remarks:
general toxicity
Generation:
F1
Effect level:
>= 630 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Dose descriptor:
NOAEL
Remarks:
toxicity to reproduction
Generation:
F1
Effect level:
>= 630 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
126 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Tables of results. Summary: NTP Reproductive Assessment by Continuous Breeding Study (theobromine):

Dose concentration

0.10%

0.25%

0.5%

F0 generation

General toxicity

Male, female

Male, female

Male, female

Body weight

No change, No change

No change, No change

No change, No change

Kidney weight

No observation

No observation

No observation

Liver weight

No observation

No observation

Statistically significant change (p<0.05), Statistically significant change (p<0.05)

Mortality

No change, No change

No change, No change

No change, No change

Feed consumption

No change, No change

No change, No change

No change, No change

Water consumption

No observation

No observation

No observation

Clinical signs

No change, No change

No change, No change

No change, No change

 

Reproductive toxicity

Litters/pair

No change

No change

Statistically significant change (p<0.05)

# Live pups/litter; pup wt./litter

Statistically significant change (p<0.05), Statistically significant change (p<0.05)

Statistically significant change (p<0.05), Statistically significant change (p<0.05)

Statistically significant change (p<0.05), Statistically significant change (p<0.05)

Cumulative days to litter

Statistically significant change (p<0.05)

Statistically significant change (p<0.05)

Statistically significant change (p<0.05)

Absolute testis, epididymis weight

No observation

No observation

Statistically significant change (p<0.05), No change

Sex accessory gland weight (prostate, seminal vesicle)

No observation

No observation

No change, No change

Epidid. sperm parameters (#, motility, morphology)

No observation

No observation

No change, No change, Statistically significant change (p<0.05)

Estrous cycle length

No observation

No observation

No observation

 

 

Determination of affected sex (crossover)

Male

Female

Both

Dose level

No observation

0.5%

No observation

 

Dose concentration

No observation

No observation

No observation

F0 generation

General toxicity

Male, female

Male, female

Male, female

Body weight

No observation

No observation

No observation

Kidney weight

No observation

No observation

No observation

Liver weight

No observation

No observation

No observation

Mortality

No observation

No observation

No observation

Feed consumption

No observation

No observation

No observation

Water consumption

No observation

No observation

No observation

Clinical signs

No observation

No observation

No observation

 

Reproductive toxicity

Litters/pair

No observation

No observation

No observation

# Live pups/litter; pup wt./litter

No observation

No observation

No observation

Cumulative days to litter

No observation

No observation

No observation

Absolute testis, epididymis weight

No observation

No observation

No observation

Sex accessory gland weight (prostate, seminal vesicle)

No observation

No observation

No observation

Epidid. sperm parameters (#, motility, morphology)

No observation

No observation

No observation

Estrous cycle length

No observation

No observation

No observation

 

Summary information

Affected sex?

Female

Study confounders:

None

NOAEL reproductive toxicity:

≤ 0.10%

NOAEL general toxicity:

< 0.5%

F1 more sensitive than F0?

Unclear

Postnatal toxicity:

Unknown

 

Conclusions:
The NOAEL for reproductive toxicity was ≤ 0.10% (126 mg/kg bw) and the NOAEL general toxicity was <0.5% (630 mg/kg bw).
Executive summary:

A Toxicity to Reproduction study was performed with theobromine according to NTP reproductive assessment by continuous breeding method, which is similar to OECD Guideline 416. Data on food and water consumptions, body weights, and clinical signs from the dose-range-finding study were used to set exposure concentrations for the continuous cohabitation phase (126, 335 and 630 mg/kg/day). In the main test, four females died, these deaths were not treatment related. Reproductive indices were adversely affected by theobromine exposure. The mean number of litters per pair was decreased at the high dose level by 19%. Live pups per litter dropped by 15% for the low dose, 21% for the medium dose, and 66% for the high dose mice. Additionally, the proportion born alive was reduced by 5 and 35% at the middle and high dose groups, respectively, and pup weight adjusted for litter size was reduced at all dose levels by 4, 13, and 27%, for the low, medium, and high doses. The mean study day on which each group delivered each litter was increased in all dosed groups. This delay ranged up to 9 days. In the high dose females, significant lengthening was seen even at the first litter. While theobromine consumption did not affect the percent of mice mating or delivering a litter, it clearly reduced female reproductive capability: there was a 74% reduction in the number of live pups delivered of treated females mated to control males, the proportion of those pups born alive was reduced by 50%, and the weight of those pups adjusted for litter size was reduced by 28%. After the delivery and analysis of these litters, the first-generation control and high dose animals were killed and necropsied. While terminal body weights were unchanged, adjusted liver weights increased for both sexes at the high dose by 12% for males and 9% for females. Absolute testis weight decreased by 12% at the high dose. Although epididymal sperm density and motility did not differ between the control and 0.5% theobromine groups, the treated animals had approximately 4-fold more abnormally shaped sperm. At the time of necropsy, blood was taken for hormonal analysis. No differences were seen between the treated and control mice in the parameters checked. In summary, there was evidence that theobromine is a possible a reproductive toxicant (producing fewer live pups per litter and reducing pup weight) at exposure levels that did not affect body weight or mortality, with the female being more sensitive than the male. The NOAEL reproductive toxicity was ≤ 0.10% (126 mg/kg bw) and the NOAEL general toxicity was 0.5 %. (630 mg/kg bw).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
126 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
The selected study was conducted with a standardized protocol and the test subtance theobromine. The other available study was conducted with coca powder with analytical determination of theobromine.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

WoE

Study 1: A Toxicity to Reproduction study was performed with theobromine according to NTP reproductive assessment by continuous breeding method, which is similar to OECD Guideline 416. Data on food and water consumptions, body weights, and clinical signs from the dose-range-finding study were used to set exposure concentrations for the continuous cohabitation phase (126, 335 and 630 mg/kg/day). Reproductive indices were adversely affected by theobromine exposure. The mean number of litters per pair was decreased at the high dose level by 19%. Live pups per litter dropped by 15% for the low dose, 21% for the medium dose, and 66% for the high dose mice. Additionally, the proportion born alive was reduced by 5 and 35% at the middle and high dose groups, respectively, and pup weight adjusted for litter size was reduced at all dose levels by 4, 13, and 27%, for the low, medium, and high doses. The mean study day on which each group delivered each litter was increased in all dosed groups. This delay ranged up to 9 days. In the high dose females, significant lengthening was seen even at the first litter. While theobromine consumption did not affect the percent of mice mating or delivering a litter, it clearly reduced female reproductive capability: there was a 74% reduction in the number of live pups delivered of treated females mated to control males, the proportion of those pups born alive was reduced by 50%, and the weight of those pups adjusted for litter size was reduced by 28%. After the delivery and analysis of these litters, the first-generation control and high dose animals were killed and necropsied. While terminal body weights were unchanged, adjusted liver weights increased for both sexes at the high dose by 12% for males and 9% for females. Absolute testis weight decreased by 12% at the high dose. Although epididymal sperm density and motility did not differ between the control and 0.5% theobromine groups, the treated animals had approximately 4-fold more abnormally shaped sperm. At the time of necropsy, blood was taken for hormonal analysis. No differences were seen between the treated and control mice in the parameters checked.

In summary, theobromine was found to be a reproductive toxicant (producing fewer live pups per litter and reducing pup weight) at exposure levels that did not affect body weight or mortality, with the female being more sensitive than the male. The NOAEL reproductive toxicity was ≤ 0.10% (126 mg/kg bw) and the NOAEL general toxicity was 0.5 %. (630 mg/kg bw)

Study 2:

A Three-Generation Reproduction Toxicity study was carried with a method similar to OECD Guidelines 416 (two generation).

Male and female Sprague-Dawley rats were continuously exposed to dietary cocoa powder at levels of 0.0, 1.5, 3.5 or 5.0% for three generations. During the initial 12-week growth periods for the F0, F1b and F2b generation rats, mean methylxanthine exposures (mg/kg/day) for males/females were 30/36, 72/86 and 104/126 for the 1.5, 3.5 and 5.0% cocoa powder groups, respectively, No consistent dose-related effects on any of the monitored reproductive indices (mating, fertility, conception, gestation, viability or lactation) were noted over three generations. Minor reductions in mean body weight relative to controls at week 12 were observed in male rats exposed to 3.5 or 5.0% cocoa powder and female rats exposed to 5.0% cocoa powder in the F1b and F2b generations. Renal tubular mineralization in the F0-generation male rats in the 5.0% cocoa powder group was the only statistically elevated histomorphological lesion observed. Plasma cholesterol concentrations in F1b-generation rats were elevated, but cocoa powder did not affect this parameter consistently across multiple generations. Thus, continuous cocoa powder consumption by rats at levels as high as 5.0% (approximately 115 mg /kg-bw/day) of the diet was without effect on reproductive capacity under the conditions of a standard three-generation evaluation.


Short description of key information:
Endpoint selection: Test method according to reproductive assessment by continuous breeding (RACB) protocol (NTP) which is similar to OECD 416. Diverse effects regarding the reproductive performance were found.

The other available study evaluated the reproductive toxicity of cocoa powder, the level of theobromine was determined.

Justification for selection of Effect on fertility via oral route:
The study has a acceptable quality (Klimish score=3) and was conducted with the test subtance theobromine.

Effects on developmental toxicity

Description of key information
Supporting information: Test method according to OECD 414. No data on GLP. A delay in osteogenesis was the only statistically significant finding, nevertheless the authors suggested that these developmental variations should be considered to be biologically insignificant since other conventional signs of embryotoxicity, such as dose-related reductions in foetal weights and frank teratogenicity were absent in this study.
Disregarded developmental toxicity study: A study which examined developmental effects of theobromine was considered disregarded since only two high concentrations of theobromine were used by the intraperitoneal route.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Supporting study:Only one study is available for the oral route. However the observed effects were present with maternal toxicity. The authors concluded that they should not be regarded as significative.Test method according to OECD 414. No data on GLP. A delay in osteogenesis was observed, however the relevance of such findings is doubtful since other signs of embryotoxicity or frank teratogenicity were absent and the effects were seen with maternal toxicity.Disregarded study: High concentrations, intraperitoneal route.
Additional information

Supporging study: A Prenatal Developmental Toxicity study was carried out according to OECD Guidelines 414. The study was conducted to determine the teratogenic potential of theobromine in rabbits. Theobromine was given either by gavage at dose levels of 0, 25, 75, 125 or 200 mg/kg body weight/day or administered in the diet at 0, 0.0625, 0.125 or 0.1875% (approximately 0, 21, 41 or 63 mg/kg/day, respectively). The duration of exposure was from days 6 to 29 of gestation. Significant maternal mortality (40%) and reduced food consumption were observed at 200mg theobromine/kg/day. Mean foetal weights were similar to those of the control group at 25 or 75 mg theobromine/kg/day, but decreases in foetal body weight and increases in various malformations and developmental variations were observed in groups given 125 or 200 mg/kg/day. Insufficient litters were available for examination in the 200-mg/kg/day dose group because of maternal toxicity/lethality (repetitive exposure by gavage to 200 mg theobromine/kg approached the maternal LD50). No maternal deaths occurred during dietary theobromine exposure. Maternal weight gain and food consumption, and the mean number of corpora lutea were unaffected by the dietary theobromine. Neither foetotoxicity nor teratogenicity was associated with dietary ingestion of theobromine. The foetuses exposed to 0.125% or 0.1875% theobromine had a significantly higher incidence of incompletely ossified or absent sternebrae, indicating a delay in osteogenesis. The predominant compound found in serum after theobromine ingestion was unchanged theobromine, and there was no evidence of bioaccumulation of theobromine in serum during gestation. Based on the delay in osteogenesis of the foetuses exposed to 0.125 and 0.1875 %, the NOAEL for developmental effects would be 0.0625 % in diet which corresponds to 21.24 mg/kg-bw/day, however, the authors suggested that the developmental variations should be considered to be biologically insignificant since other conventional signs of embryotoxicity, such as dose-related reductions in foetal weights and frank teratogenicity were absent in this study.


Justification for selection of Effect on developmental toxicity: via oral route:
No adverse effect observed.

Toxicity to reproduction: other studies

Additional information

Weight of evidence

Study 1: Theobromine was administered by gavage to 4 rats in a concentration of 250 mg/kg for 31 days.The animals were sacrificed on day 32. One testis and epididymis were removed and weighed. The epididymis was saved for the determination of epididymal sperm reserves. The remaining testis was fixed by whole body glutaraldehyde perfusion and processed for morphologic examination. Theobromine caused numerous alterations in the reproductive male tract of the treated rats at a dose level of 250 mg/kg/day.

Study 2: A repeated toxicity study was conducted according to OECD 407, but only the effects on the reproductive organ of the male rats were analyzed. The effects of theobromine on the testis were compared between rats dosed for 2 and 4 weeks to determine whether a 2 -week dosing period is long enough to detect toxicity. Theobromine was administered orally to male Sprague-Dawley rats at dose levels of 250 and 500 mg/kg for 2 weeks starting at the age of 6 or 8 weeks, and for 4 weeks from the age of 6 weeks. Histopathological examination of reproductive organs revealed toxic findings in the testis at 500 mg/kg after 2 weeks of dosing at both ages, and at 250 and 500 mg/kg after 4 weeks of dosing. The primary findings were degeneration/necrosis and desquamation of spermatids and spermatocytes, vacuolization of seminiferous tubules, and multinucleated giant cell formation. It was concluded that the toxic effects of theobromine on the testis can be detected by repeated dosing for 2 weeks as well as for 4 weeks.

Justification for classification or non-classification

In a weight of evidence approach the substance theobromine is not classified for reproductive or developmental toxicity in accordance with CLP Regulation (EC) no. 1272/2008.