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Toxicological information

Toxicity to reproduction

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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 guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 / concentrationsopen allclose all
Dose / conc.:
126 mg/kg bw/day (nominal)
Dose / conc.:
335 mg/kg bw/day (nominal)
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)

open allclose all
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

Target system / organ toxicity (P0)

open allclose all
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

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)

open allclose all
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

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).