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Toxicity to reproduction

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Administrative data

Endpoint:
multi-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
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.
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
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

Test material

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
IUPAC Name:
283-480-6
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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)

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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.

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     

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