Theobromine

Toxicity to reproduction

Administrative data

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.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: National Toxicology Program U.S. Reproductive Assessment by Continuous Breeding

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Theobromine

EC Number:

201-494-2

EC Name:

Theobromine

Cas Number:

83-67-0

Molecular formula:

C7H8N4O2

IUPAC Name:

theobromine

Details on test material:

- Name of test material (as cited in study report): Theobromine

Test animals

Species:

mouse

Strain:

other: CD-1 (ICR)BR outbred albino

Sex:

male/female

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

126 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

335 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

630 mg/kg bw/day (nominal)

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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

2

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

Target system / organ toxicity (P0)

1

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

2

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

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

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

2

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

Overall reproductive toxicity

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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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).