Cocoa, ext.

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

multi-generation reproductive toxicity

Remarks:

based on test type (migrated information)

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: FDA guideline study, to GLP. Reasonably well-reported publication.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

other: Three generation study conducted according to FDA guideline [no specific guideline mentioned]

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Purchased from Charles River Breeding Laboratories (Kingston, NY, USA).
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: rats were housed individually in raised wire-stainless steel cages
- Diet (e.g. ad libitum): ad libitum access to certified Purina rodent meal
- Water (e.g. ad libitum): ad libitum access to deionised-distilled water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±1oC
- Humidity (%): 45 ± 10%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): standard diet (Purina rodent meal)
- Storage temperature of food: 4 ± 2 °C (finished diet)

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 7 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing, the first male was replaced by another in the same treatment group
- Further matings after two unsuccessful attempts: no data
- After successful mating, pregnant females were returned to raised wire cages for days 0-14 of gestation and were then transferred to polypropylene bins with bedding

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diets analysed for methylxanthine content to determine homogeneity and stability [no further details given].

Duration of treatment / exposure:

F0 males: 12 weeks prior to mating, then continuously until sacrifice “after the production of the F1b generation”
F0 females: 2 weeks prior to mating, then continuously until sacrifice “after the production of the F1b generation” [presumably at lactation day 21]
F1 and F2 generations: continuous (in utero up until sacrifice at 21 days old (F1a, F2a), or until next generation produced (F1b, F2b))
F3 generation: no clear data, but presumably treated similarly to the F1 and F2 generations

Frequency of treatment:

Daily (in diet)

Details on study schedule:

- Selection of parents from F1 and F2 generation when pups were 0 days of age (F1b and F2b rats were mated, F1a, F2a and F3a rats were sacrificed as 21-day-old pups, F3b rats were not apparently mated)
- Age at mating of the animals in the study: 12 weeks

Remarks:

Doses / Concentrations:
0, 1.5%, 3.5% or 5% [about 0, 1110, 2670 or 3850 mg/kg bw/day for males, and 0, 1330, 3190 or 4670 mg/kg bw/day for females (from reported methylxanthine doses of 30, 72 and 104 mg/kg bw/day for males, and 36, 86 and 126 mg/kg bw/day for females.
Basis:
nominal in diet

No. of animals per sex per dose:

F0: 35
Subsequent litters culled to 10 pups each.

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: no data

Positive control:

None used

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice-daily mortality checks, with at least 6 hours between checks. Clinical observations, including gross signs of toxicity and behavioral effects, were conducted weekly throughout the study

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during growth phase (12 weeks pre-mating), then in pregnant rats on days 0, 7, 14 and 21 of gestation, and on days 0 (parturition), 4, 7, 14 and 21 of lactation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): food consumption was monitored once a week for the 12-week pre-mating period. Food consumption data on pregnant rats was collected on days 0, 7, 14 and 21 of gestation, and on days 0 (parturition), 4, 7, 14 and 21 of lactation
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in F0 and F1 male parental generations:
The testes, epididymis, seminal vesicles and prostate were examined microscopically in the control and top-dose F0 and F1 males. The testes were also examined microscopically in the mid-dose F1 males.

Parameters examined in F0, F1 and F2 male parental generations:
Testis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes (culled to 10 pups)

PARAMETERS EXAMINED
The following parameters were examined in offspring: gross external examination on days 0, 4, 7, 14 and 21 of lactation. Pup body weights measured at 0, 4, 7, 14 and 21 days, then weekly. Number of viable pups measured at parturition and lactation days 4 and 21

GROSS EXAMINATION OF DEAD PUPS: No data

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving F0, F1b and F2b animals after the last litters in each generation were produced.
- Maternal animals: All surviving F0, F1b and F2b animals after the last litters in each generation were produced [presumably after weaning at lactation day 21]

GROSS NECROPSY
- Conducted on the sacrificed F0, F1b and F2b animals.
- The thymus, spleen, kidneys, vagina, ovaries, testes, epididymis, seminal vesicles, prostate and any abnormal tissues were obtained.

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights assessed in the sacrificed F0, F1b and F2b rats.
Organ weights determined for the ovaries, testes, thymus and spleen.

Histopathology conducted on the F0 and F1b animals.
The thymus, spleen, kidneys, vagina, ovaries, testes, epididymis, seminal vesicles, prostate and abnormal tissues were examined microscopically for the control and 5% groups. For the F1b generation, microscopic analysis was additionally performed on the testes of animals given 3.5% dietary cocoa powder. Histopathology was not performed on the tissues of the F2b generation.

OTHER: HAEMATOLOGY AND CLINICAL CHEMISTRY
Apparently performed on F0, F1b and F2b animals.
After an overnight fast, blood was collected from 10/sex/group after the last litters in each generation were produced [presumably at lactation day 21 in females]. See table 2 in “any other information on materials and methods” for details.

Postmortem examinations (offspring):

SACRIFICE
The offspring not selected as parental animals (i.e. F1a, F2a [and presumably F3a]) were sacrificed at lactation day 21
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of a gross visceral examination

HISTOPATHOLOGY / ORGAN WEIGHTS
Abnormal tissues were prepared for microscopic examination

Statistics:

Non-count parametric data were analysed using one-way analysis of variance (ANOVA) followed by the Newman-Keuls test. Nonparametric data were assessed by the Kruskal-Wallis test. Bartlett’s test was used to determine homogeneity. Gladen’s estimated proportion test or the chi-square test was used to analyse count-type raw data. Fisher’s exact test was used when differences were detected by chi-square analyses. Probability values of p ≤ 0.05 were considered statistically significant.

Reproductive indices:

Probably assessed in the F0, F1b and F2b generations [data appears to be presented as effects on pups – i.e. no results are attributed to the F0 generation].
Mating (number of copulations/number of females exposed to fertile males)
Fertility (number of females conceiving/number of females exposed to fertile males)
Conception (number of pregnancies/number of copulations)

Offspring viability indices:

Assessed in the F1b, F2b and F3b pups.
Gestation (number of females with live litters/number of pregnant females)
Viability (viable pups at lactation day 4/viable pups at parturition)
Lactation (viable pups at lactation day 21/pups retained lactation day 4)

Clinical signs:

not specified

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No data [presumably no effect].

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
For all three generations of rats selected as parents, treatment had little or no effect on food consumption. The exception was the F2b males in the 5% cocoa powder group, who consumed about 10% less than corresponding controls.
Final (week 12) mean body weight was statistically significantly lower (up to about 20%) in male rats given 3.5 or 5%, compared to controls, in both the F1b and F2b generations. Female rats in the F1b and F2b generations exhibited similar effects (up to about 10% lower final body weight) in the 5% group. No effect on weight gain was seen in the F0 generation. It was suggested that these effects on growth could be due to differences in protein bioavailability or utilisation, or other nutritional or metabolic perturbations.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Not relevant.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No significant increase in incidence of incomplete spermatogenesis.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
[The results are presented in such a way that parental reproductive effects appear to be given as an effect on the offspring – i.e. no results are given for the F0 generation. The F3 generation don’t appear to have been bred – fertility results for these animals probably relate to an effect on F2b rats; likewise, F1 fertility data probably relates to F0 rats, and F2 data to F1 rats.]

Few statistically significant changes were observed. Treatment was associated with increases in fertility and conception indices in the F1 generation. These unexpected (beneficial) findings probably resulted from unusually low rates of conception (86.2% and 68.6%) among the control groups in the F1a and F1b generations.
A statistically significant decrease in the conception index was noted in the F3b rats in the 1.5% test group (86% compared to 100% in controls), but not in those given higher doses.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No significant changes attributable to treatment were evident in the weights of the thymus, spleen or ovaries/testes in the F1, F1b and F2b rats. A minor, but statistically significant, decrease in spleen weight was noted in F0 generation males in the 3.5% group, but this effect was not observed in subsequent generations or in groups exposed to higher levels of dietary cocoa powder.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No data [presumably any adverse effect would have been reported].

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological examinations of tissues from F0 and F1b generation rats indicated that dietary cocoa powder exposure did not cause any striking lesions. A few sporadic findings, suggested the testes (male) and kidneys (male and female) as possible target organs (see table 3 in “remarks on results including tables and figures” for details). Effects on the testes were not statistically significant at any dose level. Renal pelvic mineralisation was observed in both male and female rats but a statistically significant increase in the incidence of this lesion occurred only in F0 generation males in the 5.0% group.

OTHER FINDINGS (PARENTAL ANIMALS)
Treatment had no effect on the haematology of males in the F0, F1b or F2b generations.
Clinical chemistry analyses revealed that mean plasma cholesterol levels in F1b generation male rats were increased over control values by 27, 35 and 49% in the 1.5, 3.5 and 5.0% groups respectively. In female rats, a 31% increase in mean plasma cholesterol concentration was observed, but only in the F1b generation rats in the 5.0% group. These changes are probably an adaptive response of the rats to a high fat diet (cocoa powder contains about 15% fat) compared to the control diet, rather than an adverse effect.

Dose descriptor:

NOAEL

Effect level:

ca. 3 190 mg/kg bw/day

Sex:

female

Basis for effect level:

other: Mean body weight significantly lower than in controls at week 12 in animals given at least 2670 mg/kg bw/day.

Remarks on result:

other: Generation: F1b, F2b (migrated information)

Dose descriptor:

NOAEL

Effect level:

ca. 1 110 mg/kg bw/day

Sex:

male

Basis for effect level:

other: Mean body weight significantly lower than in controls at week 12 in animals given at least 2670 mg/kg bw/day.

Remarks on result:

other: Generation: F1b, F2b (migrated information)

Dose descriptor:

NOAEL

Effect level:

ca. 3 850 mg/kg bw/day

Sex:

male

Basis for effect level:

other: No adverse effects on fertility of parental animals in any tested group.

Remarks on result:

other: Generation: F0, F1b, F2b (migrated information)

Dose descriptor:

NOAEL

Effect level:

ca. 4 670 mg/kg bw/day

Sex:

female

Basis for effect level:

other: No adverse effects on fertility of parental animals in any tested group.

Remarks on result:

other: Generation: F0, F1b, F2b (migrated information)

Dose descriptor:

LOAEL

Effect level:

ca. 1 330 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Significantly lower birth weight in all treatment groups, compared with controls

Remarks on result:

other: Generation: F1b, F2b and F3a pups (migrated information)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

The following refer to results from pups only. Details on pups raised to adulthood for use as parents are available in the “details on results (parental animals)” section of this robust study summary.

VIABILITY (OFFSPRING)
Reductions in gestation index were seen in several 5.0% groups but none of these changes were statistically significant compared with the concurrent controls.
A slight (2.5%) but statistically significant decrease in the lactation index was observed in the F3a pups in the 1.5% group, but not in those given higher doses.
The viability index reflecting survival at postpartum day 4 was modestly reduced (3-6%) in the 5.0% pups in both the F2a and F3a generations and also in the 3.5% group of F3b pups. Effects were not seen in the F2b generation. These marginal effects were considered to “probably represent chance occurrences not related to dietary [cocoa powder] exposure”.

CLINICAL SIGNS (OFFSPRING)
No data [presumably any adverse effect would have been reported].

BODY WEIGHT (OFFSPRING)
In the F1b, F2b and F3a generations, statistically significant decreases in mean pup birth weight were observed in all treated groups [data not given]. These reductions were overcome in the 1.5 and 3.5% groups by lactation day 4. In the groups given 5%, birth weight was significantly reduced in F1, F2 and F3 pups (by up to about 15%), persisting until at least lactation day 21 in the F1a, F2b, F3a and F3b groups. As the lactation indices were similar to those of control, these effects in the 5% groups did not affect pup survival). It was suggested that these effects on growth could be due to different protein bioavailability or utilisation, or other nutritional or metabolic perturbations.

SEXUAL MATURATION (OFFSPRING)
Not examined.

ORGAN WEIGHTS (OFFSPRING)
Not examined.

GROSS PATHOLOGY (OFFSPRING)
No gross malformations were seen in pups from any test group. Common gross visceral lesions were observed in a small proportion of pups examined on day 21 but none were judged to be treatment-related.

HISTOPATHOLOGY (OFFSPRING)
Not examined.

Dose descriptor:

LOEL

Generation:

F1b

Effect level:

ca. 1 110 mg/kg bw/day

Sex:

male

Basis for effect level:

other: In treated F1b males, cholesterol levels were significantly higher than those seen in controls. However, these changes are likely an adaptive response, rather than an adverse effect. No effects reported for F0 generation.

Reproductive effects observed:

not specified

Conclusions:

In a three-generation FDA guideline study, performed to GLP, treatment with cocoa powder at up to 5% in the diet [about 3850 or 4670 mg/kg bw/day in males and females, respectively] had no significant adverse effect on fertility or gross malformations. Slight reductions in body weights of pups in all treated groups and of dams at the top dose were seen, possibly as a result of nutritional deficiencies.

Executive summary:

A three-generation study, performed to GLP and apparently conducted in compliance with FDA guidelines, was conducted to investigate the reproductive toxicity of cocoa powder in Sprague-Dawley rats.

Animals were given cocoa powder in the diet at 0, 1.5, 3.5 or 5% [corresponding to about 0, 1110, 2670 or 3850 mg/kg bw/day in males, and 0, 1330, 3190 or 4670 mg/kg bw/day in females]. Each generation was mated twice, with one set of offspring killed at day 21 of lactation, and the other chosen as parents of the next generation. For the F0 generation, consisting of 35/sex/dose, males were fed for 12 weeks prior to mating. Females were concurrently kept on a control diet for 10 weeks, then treated for the last 2 weeks prior to mating. Subsequent females were not subject to the 10-week control diet period.

Statistically significant reductions were seen in the mean final (week 12) body weight of male and female F1b and F2b rats, in males given at least 3.5% and in high-dose females. No significant body weight effects were seen in the F0 generation. Food consumption was reduced by about 10% in F2b high-dose males.Mean plasma cholesterol levels were seen in F1b male rats in all groups, and in high-dose F1b females, possibly an adaptive response to a cocoa diet higher in fat than the controls[i1] .

No dose-related adverse effects were seen on the fertility of parental animals, and no significant adverse effects on the weight or histopathology of the reproductive organs were observed. A statistically significant increased incidence of kidney lesions was                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           seen in the high-dose F0 males only.

No increases in gross malformations were seen in the offspring of treated animals, but viability was modestly reduced in the high-dose F2a and F3a pups, and also in the mid-dose F3b pups. As effects were not seen in the F2b generation, or in the F3b high-dose pups,these marginal effects were considered to “probably represent chance occurrences not related to dietary [cocoa powder] exposure”. Statistically significant decreases in birth weight were seen in all treated F1b, F2b and F3a groups. In the low and mid-dose groups, effects resolved within four days. The high dose was associated with reduced birth weight for all F1, F2 and F3 groups, with effects, in the majority of cases, persisting until at least day 21.

The study authors state that “exposure of rats to dietary [cocoa powder] at concentrations of up to 5.0% of the diet for three generations caused no consistent effect on any of the reproductive indices monitored”. The study found no treatment-related adverse effect on fertility (NOAELs of 3850 and 4670 mg/kg bw/day for males and females, respectively), or on the incidence of gross malformations in offspring. Reduced birth weight was seen in the offspring of treated animals (LOAEL: 1330 mg/kg bw/day). Maternal growth was also reduced at higher concentrations (NOAEL: 3190 mg/kg bw/day). It was suggested that these effects on growth could be due to different protein bioavailability or utilisation, or other nutritional or metabolic perturbations.

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 850 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Dietary studies available to the Consortium included a reliable 3-generation reproductive toxicity study, a poorly-described one-generation reproduction study, and a reliable chronic study with microscopic analysis of the reproductive organs.
The NOAEL was 4670 mg/kg bw/day for females . [Please see discussion below].

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Effects on Fertility:

In a GLP three-generation study male and female Sprague-Dawley rats were given orally cocoa powder at concentrations of 0, 1.5, 3.5 and 5% in the animal feed. No consistent dose-related effects on any of the reproductive indices (mating, fertility, conception, gestation, viability and lactation) were observed over the three generations. Non-reproductive toxicity included decreased body weight gain at 3.5 and 5% probably due to components of cocoa powder altering protein bioavailability/utilization (i.e., oxalic acid); and renal mineralization in F₀generation males only at 5% cocoa powder but had no effect on pup survival. The NOAEL was stated to be 5% cocoa powder in feed, equivalent to 3850mg/kg bw/day cocoa powder for males and 4670mg/kg bw/day cocoa powder for females (Hostetler et al., 1990).

NOAEL for fertility effects:3850mg/kg bw/day (males) and 4670mg/kg bw/day (females) cocoa powder, oral route.

Short description of key information:
Available studies on the reproductive toxicity of dietary cocoa powder include a reliable 3-generation study, in which no adverse effects on fertility or reproductive organs of male and female rats given 5% dietary cocoa powder [about 3850 or 4670 mg/kg bw/day, respectively] was observed. When rats from the third generation of this study were treated chronically, a statistically significant increase in the incidence of histopathological lesions in the testes was observed in males given 5% in the diet [about 2000 mg/kg bw/day].
In a one-generation reproductive toxicity, presumably similar to that described by OECD Guideline 415, rats were given diets containing an unspecified amount of irradiated, non-irradiated or fumigated cocoa bean powder, or a control diet, for an unspecified period of time. Offspring were assessed until weaning.

There were no effects on mating performance and fertility.
Due to limited reporting (without dietary doses), it was not possible to define the NOAEL for fertility, or the LOAELs for maternal and developmental toxicity. [Please refer to Hostetler et al. (1990) and Tesh et al. (1982)].

Effects on developmental toxicity

Description of key information

Reliable developmental toxicity studies involving dietary exposure of pregnant rabbits and rats (2 studies) to cocoa powder at up to about 2765 or 6095 mg/kg bw/day, respectively, are available. Skeletal variations (indicative of delayed osteogenesis) were seen when the offspring were examined in utero. No gross malformations were seen in three studies in which the offspring were raised to weaning, but adverse effects on pup growth and survival were noted, and the birth weight of treated pups was statistically significantly lower than that of controls in a three-generation reproduction study.
No standard developmental toxicity studies are available on laboratory animals treated via inhalation or by the dermal route.

Link to relevant study records

Reference

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).

Reason / purpose for cross-reference:

reference to other study

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

Species:

rabbit

Strain:

New Zealand White

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

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

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.

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].

Dose descriptor:

NOAEL

Effect level:

ca. 2 765 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

ca. 1 865 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

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.]

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 720 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Dietary studies available to the Consortium included reliable developmental toxicity studies in rabbits and rats, and a 3-generation study in rats.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Developmental Toxicity:

Tarka et al., (1986a) evaluated the teratogenic potential of cocoa powder in a GLP study conducted in New Zealand white rabbits. Cocoa powder was administered at 2.5, 5 and 7.5% of the diet, equivalent to about 925, 1865 or 2680mg cocoa powder/kg bw/day during days 6-29 of gestation. Neither foetotoxicity nor teratogenicity was associated with cocoa powder ingestion at any dose level. However, due to incomplete ossification observed at 7.5% cocoa powder, the NOAEL was stated as 5.0% or ca 1865mg/kg bw/day cocoa powder.

NOAEL for developmental toxicity (rabbit): 1865mg/kg bw/day (cocoa powder rabbit, oral route).

Tarka et al., (1986b) also evaluated the perinatal, postnatal and teratogenic potential of cocoa powder in Sprague-Dawley rats. In the peri/postnatal study, rats were fed diets containing 0, 2.5, 5 and 7.5% cocoa powder daily throughout gestation and lactation. In the teratology study rats were given diets containing 0, 2.5 and 5% cocoa powder on days 6-19 of gestation. No malformations occurred, however, there was a slight increase in a delay of osteogenesis that could be explained by significant reductions in food intake on gestation days 13-19 in the 2.5 and 5% cocoa powder groups throughout gestation (days 6-19). In the light of this it was concluded that cocoa powder was not embryotoxic or teratogenic.

NOAEL for developmental toxicity (rat): 3720mg/kg bw/day cocoa powder (rat, oral route).

Justification for selection of Effect on developmental toxicity: via oral route:
Good quality developmental toxicity study on rabbits given cocoa powder in the diet from GD6 to GD29

Justification for classification or non-classification

According to DSD (67/548/EEC) and CLP (1272/2008) regulations, cocoa extract would not be classified as a reproductive or developmental toxicant. Notably no adverse effects on fertility were observed in good-quality studies (including a 3-generation study in rats) and no teratogenicity was seen in reliable developmental studies in rats and rabbits.

Skeletal variations (delays in ossification) were seen in the developmental toxicity studies in rabbits and rats. However, these are not considered to constitute an adverse effect that requires classification and labelling. Delayed (or incomplete) ossification is one of the most common skeletal variations encountered in regulatory guideline developmental toxicity studies. Delays in ossification are not thought to persist – the postnatal skeleton having considerable capacity to remodel – and are thought to be more indicative of a generalised foetal growth delay that can be influenced by e.g. maternal malnutrition or reduced feed intake. A delay in ossification is not thought to have general predictive value for teratogenesis. Indeed, no increases in malformations were seen in these developmental toxicity studies or the 3-generation reproductive study.

On this basis, the reported increased incidence of skeletal variations is indicative of a minor delay in skeletal ossification, and is thought to be a transient effect that is completely reversible postnatally, not justifying classification of cocoa extract for toxicity to reproduction.