Cocoa, ext.

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study comparable to guideline, with acceptable restrictions (only 12 and 11 litters examined in control and low-dose groups, respectively).

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Evaluation of the teratogenic potential of cocoa powder and theobromine in New Zealand White rabbits

Author:

Tarka S.M., Applebaum R.S. and Borzelleca J.F.

Year:

1986

Bibliographic source:

Food and Chemical Toxicology 24, 363-374

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

[groups of at least 14 pregnant rabbits used, but groups of about 20 recommended; as a result, only 11 and 12 litters examined in the low-dose and control groups, respectively]

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cocoa powder

IUPAC Name:

cocoa powder

Constituent 2

Reference substance name:

Cocoa, ext.

EC Number:

283-480-6

EC Name:

Cocoa, ext.

Cas Number:

84649-99-0

Molecular formula:

Not applicable (i.e., UVCB substance)

IUPAC Name:

Cocoa, ext.

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): cocoa powder
- Substance type: UVCB
- Physical state: solid/powder
- Analytical purity: 100%
- Composition of test material, percentage of components: contained 2.46% theobromine and 0.19% caffeine
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data
- Other: cocoa powder was obtained from a specific cocoa-bean bland prepared according to defined roasting parameters

Test animals

Species:

rabbit

Strain:

New Zealand White

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): Purina Certified Rabbit Chow no. 5322
- Storage temperature of food: no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from each dose level taken from top, middle and bottom of the lot and analysed by high performance liquid chromatography (HPLC) to assess stability, homogeneity and concentration.

Details on mating procedure:

Impregnation procedure: no data

Duration of treatment / exposure:

Gestational days (GDs) 6 to 29

Frequency of treatment:

Daily

Duration of test:

Until autopsy at GD30

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 2.5, 5.0 or 7.5% in diet [925, 1865 or 2680 mg/kg bw/day, replicate groups:5.0% or 7.5% consuming about 1655 or 2765 mg/kg bw/day, Doses were said to supply about 0, 25, 50 and 75 mg methylxanthines/kg bw/day
Basis:
nominal in diet

No. of animals per sex per dose:

“At least” 14 pregnant females, with additional groups of pregnant females treated with 5.0% and 7.5% (included for serum metabolite analysis)
The following numbers of litters were examined:
0 (control): 12
2.5%: 11
5.0%: 13 ‘non-bled’, 12 ‘bled’ (total 25)
7.5%: 15 ‘non-bled’, 14 ‘bled’ (total 29)

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: no data

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily [no details on effects checked]

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: GDs 0, 6, 9, 12, 15, 18, 25 and 30

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, daily
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 30
- Organs examined: “autopsied using standard teratological procedures”

OTHER: Blood was collected about 3 hours after dosing from the replicated 5 and 7.5% groups on days 7, 14, 21 and 28 of gestation for serum metabolite analysis

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data [“number of resorbed foetuses” assessed]
- Number of late resorptions: No data [“number of resorbed foetuses” assessed]

Fetal examinations:

- External examinations: Yes [all per litter]
- Soft tissue examinations: No data
- Skeletal examinations: Yes [all per litter]
- Head examinations: No data

Statistics:

Data were analysed for differences between non-bled and control groups, and between bled and non-bled animals given the same dose. Data were also analysed with non-bled and bled groups combined.
Maternal weight gain and food consumption was assessed using two-way analysis of variance (ANOVA). ANOVA was also used to analyse number of implantation sites and corpora lutea, and litter size. Foetal body weights were compared using analysis of covariance, then Dunnett’s apriori test. A modified Jonckheere test and Kruskal-Wallis analysis assessed embryolethality. Foetal anomalies and developmental variants were analysed using the Kruskal-Wallis test to compare treated and control animals, and Gladen’s test to compare group proportions. Probabilities of p ≤ 0.05 were considered statistically significant; this was reduced to p ≤ 0.017 for Gladen’s test to correct for multiple comparison procedures.

Indices:

Not calculated.

Historical control data:

Not presented.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No significant treatment-related adverse effects on maternal growth or mortality were observed in treated groups [clinical toxicity was not discusses, presumably any adverse effect would have been reported].

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

ca. 2 765 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

ca. 1 865 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects
No treatment-related adverse effects were observed on mean implantation or copora lutea number, litter size, embryolethality, percentage of live foetuses or sex ratios or incidence of malformations. Foetal body weight was statistically significantly higher in the 5% non-bled group only compared to controls.
See table 1 in “remarks on results including tables and figures” for details on the developmental variations seen in treated and control foetuses. The only change reported as being statistically significant was an increased incidence of skeletal variations (incompletely ossified or absent metacarpal no. 1 and middle phalange no. 5) in groups given 5 or 7.5% (at least 1655 mg/kg bw/day). Notably, these effects were only reported as statistically significant when the data from both the main and ‘bled’ groups were considered together. In the absence of ‘bled’ data, only the effects on metacarpal no. 1 were statistically significant, and only at the high dose of 2680 mg/kg bw/day.
The incidence of certain other skeletal variations (incomplete ossification of proximal phalange 1, sternebra 2 and sternebra 6, and absence of sternebra 5) increased with dose, but apparently without statistical significance. These effects do not appear to occur at a markedly higher incidence in groups given 2.5% and 5% dietary cocoa powder (about 925 and 1865mg/kg bw/d, respectively).
No correlation was seen between skeletal variations and serum levels of theobromine.
[Although the method states that probabilities of P ≤ 0.05 were considered statistically significant, reduced to P ≤ 0.017 for data analysed by Gladen’s test (used to compare group proportions of foetal anomalies and developmental variants), the table listing malformation and variation incidences implies that statistically significant effects were only seen with P ≤ 0.001. It is not clear whether effects were seen with P ≤ 0.05.]

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a GLP developmental toxicity study on cocoa powder (similar to that described by OECD Guideline 414), no significant maternal toxicity was seen in rabbits given up to about 2765 mg/kg bw/day [the highest tested dose]. Skeletal variations (of questionable biological relevance) were seen in the offspring of rabbits treated with at least 1655 mg/kg bw/day on gestational days (GDs) 6 to 29, resulting in a developmental toxicity NOAEL of 1865 mg/kg bw/day.

Executive summary:

In a developmental toxicity study (similar to that described by OECD Guideline 414 and performed to GLP), groups of “at least” 14 female New Zealand White rabbits were given cocoa powder in the diet at 0, 2.5, 5 or 7.5% [0 or about 925, 1865 or 2680 mg/kg bw/day] on gestational days (GDs) 6 to 29. Replicate ‘bled’ groups (for serum analysis of metabolites) were treated with 5 or 7.5% [consuming about 1655 or 2765 mg/kg bw/day, respectively].

 

Over the course of the study, the food consumption and body weights of dams were monitored, as well as mortality and overt clinical signs of toxicity. Rabbits were killed on GD30, and foetuses were observed for abnormalities, body weight and mortality. The mean number of implantation sites and corpora lutea were also recorded.

 

Treatment had no significant adverse effect on maternal body weight or mortality. No increases in the incidence of malformations were seen. No differences between treated and control animals were detected for pregnancy rate, mean number of implantations, litter size, corpora lutea, live foetuses, embryolethality or sex ratio. The only statistically significant effect on foetuses was an increased incidence of skeletal variations (incompletely ossified or absent metacarpal no. 1 and middle phalange no. 5) in groups given 5 or 7.5% (at least 1655 mg/kg bw/day). Notably, these effects were only statistically significant when the data from both the main and ‘bled’ groups were considered together. In the absence of ‘bled’ data, only the effects on metacarpal no. 1 were significant, and only at the high dose of 2680 mg/kg bw/day. No correlation was seen between skeletal variations and serum levels of theobromine.

 

In this study, developmental effects (skeletal variations) indicative of delayed osteogenesis were seen in the foetal offspring of dams given at least 1655 mg/kg bw/day (NOAEL 1865 mg/kg bw/day), in the absence of apparent maternal toxicity (NOAEL 2765 mg/kg bw/day). Incomplete ossification of sternebrae is thought to be a transient phase of development, rather than a malformation. According to the study authors, these effects should be considered biologically insignificant in the absence of other conventional signs of embryotoxicity (e.g. reduced foetal weight). No malformations were observed.