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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Test method according to OECD Guideline 414 with deviations. GLP study.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1986
Report Date:
1985

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
the exposure started later than specified, the number of animals is lower than required, clinical observation schedule not specified
Principles of method if other than guideline:
Preliminary study: the oral embryonic/foetal LDs0 was determined using a minimum of ten bred rabbits,group. On days 6-29 of gestation the rabbits were given by gavage suspensions of theobromine in 0.2% CMC (I ml suspension,,kg body weight) that provided doses of 0, 25, 75, 125 or200 mg TBR/kg body weight. The stability, homogeneity and concentrations of the suspensions were determined on days 5, 6 and 29 of gestation and found to be acceptable. Body weight, food consumption and clinical observations were recorded on days 0 and 6-30 of gestation. Autopsies were performed on all females that aborted or died during the study. All remaining does were killed by CO 2 asphyxiation on day 30 of gestation; the ovaries were examined and the number of corpora lutea recorded. Each litter was evaluated for numbers of live, dead and resorbed foetuses: all foetuses were weighed and examincd for anomalies by external inspection and the viscera were examined using a dissecting microscope. The sex of each foetus was determined and all foetuses were evaluated for skeletal anomalies.The effect of dietary theobromine on teratogenesis was evaluated in study 4. A minimum of 15 pregnant rabbits/group were given 0, 0.0625, 0.125 or 0.1875% theobromine in the diet (approximately 0, 21, 41 or 63 mg theobromine/kg body weight/day) on days 6-29 of gestation.
GLP compliance:
yes
Remarks:
Conducted in accordance with US FDA GLP, current at the time the study was conducted.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Theobromine- Physical state: white crystalline powder- Analytical purity: >99%

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: preliminary study: Hills of Home Ranch, Jamul, CAmain test: Lesser's Rabbitry, Union Grove, WI- Age at study initiation:4-5 months old- Weight at study initiation:preliminary study: 3.2-4.6 kgmain test: 3.4-5.1 kg- Fasting period before study: no data- Housing: stainless-steel cages
- Diet (e.g. ad libitum): Purina Certified Rabbit Chow no. 5322 (Ralston Purina, St Louis, MO)
- Water (e.g. ad libitum): tap waterENVIRONMENTAL CONDITIONS- Temperature (°C): 13.33-21.11
- Humidity (%): 30-70% relative humidity
- Photoperiod (hrs dark / hrs light): 12h light/dark cycle

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:DIET PREPARATION
- Mixing appropriate amounts with (Type of food): the animals were given 0, 0.0625, 0.125 or 0.1875% of the test substance mixed with the basal diet.
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: cohoused
Duration of treatment / exposure:
From days 6 to 29 of gestation.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
21 mg/kg bw/day
Dose / conc.:
41 mg/kg bw/day
Dose / conc.:
63 mg/kg bw/day
No. of animals per sex per dose:
A minimum of 15 pregnant rabbits/group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Results of the preliminary study (embryonic/foetal LDs0 ) indicated that exposure to 200 mg theobromine/kg body weight/day administered by gavage on days 6-29 of gestation resulted in 40% maternal mortality and it was inferred that maternal toxicity indirectly affected foetal development as indicated by foetal weight, retarded skeletal ossification and increased embryolethality. On this basis, the highest theobromine dose used in the major feeding study of theobromine was less than 125 mg theobromine/kg/day.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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: the ovaries were examined.
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: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: weighed, external inspection, sex determination.
- Soft tissue examinations: Yes: the viscera were examined using a dissecting microscope.
- Skeletal examinations: Yes: [all per litter]
Statistics:
Data were analysed on a DECl0 computer using standardized programs from the Statistical Package for the Social Sciences (Nie, Jenkins, Steinbrenner & Bart, 1975), and a Fortran Applications Program for the Gladen jack-knife test (Gladen, 1979) and modified Jonckheere analyses (Lin & Haseman, 1976). All data were independently verified and cross-checked for accuracy and statistical analyses were based on the litter as the experimental unit.Maternal death (preliminary study only) was analysed by the Fisher's exact test (Siegel, 1956). The total weight gain and diet consumed by does during specified intervals were analysed using a two-way analysis of variance (Snedecor & Coehran, 1967). The numbersof implantation sites and corpora lutea and litter size were analysed by analysis of variance. Foetal body weights were compared by analysis of covariance (Snedecor & Cochran, 1967), followed by a Dunnett's apriori test (Steel & Torrie, 1960). Embryolethality was assessed by both a modified Jonckheere test andKruskal-Wallis analysis (Conover, 1971). Foetal anomalies and developmental variants were analysed using the Kruskal-Wallis test to ascertain differences between treatment and control groups and by Gladen's test which compared group proportions and incorporated provisions for litter size. A foetus was statistically classified as a runt when its body weight was reduced by 30% or more of the control group's grand mean (Leuschner & Czok, 1973).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Details on results:
In the dietary theobromine study, anorexia, diarrhoea, soft stools and various discharges were observed in all groups Food consumption and theobromine intake decreased in all groups during gestation. Theobromine consumption was in the range of 18-23, 33-47 and 50-70 mg/kg body weight/day at doses of 0.0625, 0.125 and 0.1875% theobromine, respectively. There were no treatment-related effects on maternal body-weight change or the mean number of corpora lutea. There were no maternal deaths but three does aborted. These abortions were not considered to be treatment related. The predominant compound found in the serum after theobromine ingestion was unchanged theobromine. Two minor metabolites observed at lower concentrations were 3- and 7-methylxanthine.

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
There were no treatment-related effects on the mean number of corpora lutea but three females aborted. These abortions were not considered to be treatment related. There were no differences between the groups in the mean number of implantations, litter size or embryolethality. The percentage of dead and resorbed foetuses was greater in the control group than in any of the treated groups because five does resorbed their entire litters.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant reduction in foetal body weight was noted in the 0.125% and 0.1875% groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
The incidence of sternebral variations in the 0.125 and 0.1875% TBR dose groups was significantly higher than in the controls.
Details on embryotoxic / teratogenic effects:
Neither the percentage of live foetuses nor the sex ratios was affected at any treatment level but a significant reduction in foetal body weight was noted in the 0.125% and 0.1875% groups. The foetal observations presented a variety of malformations and skeletal variations scattered throughout the groups, irrespective of treatment. Runting was again the most common observation. The most frequent developmental variations noted were incompletely ossified or absent sternebrae, metacarpals or phalanges. The incidence of sternebral variations in the 0.125 and 0.1875% TBR dose groups was significantly higher than in the controls. Many other frequently occurring skeletal variations were observed in all groups and these were judged to be unrelated to treatment.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
41 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes

Any other information on results incl. tables

The foetal observations show a variety of malformations and skeletal variations scattered throughout the groups, irrespective of treatment. (Not all malformations or variations are shown.) Runting was again the most common observation. The most frequent developmental variations noted were incompletely ossified or absent sternebrae, metacarpals or phalanges. The incidence of sternebral variations in the 0.125 and 0.1875% theobromine dose groups was significantly higher than in the controls. When the data from the nonbled and bled groups were combined the incidences of incompletely ossified or absent sternebrae, metacarpals and phalanges were significantly increased in comparison with the controls. There was no correlation between the serum theobromine concentrations of the does on day 28 of gestation and the incidence of affected foetuses, and no bioaccumulation of theobromine during gestation. Many other frequently occurring skeletal variations were observed in all groups and these were judged to be unrelated to treatment.

Table 1: Summary of foetal data for rabbits given theobromine in the diet on days 6 -29 of gestation

 

Values for groups given theobromine at dietary levels (%) of:

Observation

0

0.0625

0.125

0.125B*

0.1875

0.1875B

No. pregnant does

15

13

16

15

15

14

No. of litters examined (no. of foetuses)

10 (75)

12 (87)

15 (111)

14 (94)

14 (92)

14 (95)

No. of implantations

112

97

128

112

116

114

No. of implantations/litter (mean ±SD)

7.5 ± 3.0

8.1 ± 1.4

8.5 ± 2.4

8.0 ± 1.8

7.7 ± 2.0

8.1 ± 2.5

Percentage of live foetuses

67.0

89.7

86.7

83.9

79.3

83.3

No. of live foetuses/litter (mean ± SD)

50 ± 3.8

7.3 ± 1.5

7.4 ± 2.1

6.7 ± 2.3

6.1 ± 3.1

6.8 ± 2.3

Foetal weight (g. mean ±SD):

All

52.9 ± 5.0

52.6 ± 5.6

52.5 ± 5.1

48.1 ± 5.6**

47.5 ± 7.0**

50.0 ± 4.4

M

52.1 ± 5.3

52.2 ± 7.8

53.7 ± 7.4

48.9 ± 6.1

48.7 ± 7.7

48.9 ± 5.5

F

53.9 ± 5.6

51.8 ± 5.4

49.9 ± 7.2

46.9 ± 6.3

46.0 ± 9.6

49.8 ± 4.7

No. of resorbed foetuses

33

7

10

13

18

12

No. of dead foetuses

4

3

7

5

6

7

Percentage of dead and resorbed foetuses/litter

33.1

10.3

13.3

16.1

20.7

16.6

Sex ratio (M:F)

1.3:1.0

0.9:1.0

1.1:1.0

0.9:1.0

1.1:1.0

0.6:1.0

*Replicate group used for serum analyses

**Significantly different from the control value (P<0.01)

Table 2: Incidence of malformations and variations among litters of rabbits given theobromine in the diet on days 6 29 of gestation

 

Incidence (% fetuses affected/litter) in groups given theobromine at dietary levels (%) of:

Observation

0

0.0625

0.125

0.125B*

0.1875

0.1875B*

No. of litters examined …

10

12

15

14

14

14

Malformations

External observations

Runt

2.8

5.2

4.4

8.9

11.8

5.9

Skeletal variations

Both forelimbs

Metacarpa 1

-         Absent

0.0

1.0

1.5

4.1

13.3

13.1

-         Incomplete ossification

0.0

4.7

10.3

14.8

20.8

22.0

Proximal phalange 1

-         Absent

1.7

0.0

1.0

4.5

0.0

5.7

-         Incomplete ossification

0.0

0.0

4.2

3.8

5.7

3.6

Middle phalange 5

-         Absent

1.7

0.0

0.7

0.9

3.6

6.8

-         Incomplete ossification

0.0

10.1

4.6

5.6

13.7

10.2

Thorax**

Sternebra 2

-         Incomplete ossification

4.2

5.3

10.1

6.5

13.3

12.5

Sternebra 5

-         Absent

0.0

5.8

1.4

4.4

6.8

19.7

-         Incomplete ossification

9.8

34.6

42.7

37.1

43.1

43.8

Sternebra 6

-         Absent

0.0

0.0

3.6

6.9

2.2

8.1

-         Incomplete ossification

5.8

12.2

18.2

13.0

26.9

29.3

*Replicate group used for serum analyses

** The incidence of all sternebral variations combined was significantly higher in the 0.125 and 0.1875% dose groups than in the controls (P ≤ 0.001).

Applicant's summary and conclusion

Conclusions:
The developmental variations observed in theobromine studies should be considered to be biologically insignificant since other conventional signs of embryotoxicity, such as dose-related reductions in foetal weights and frank teratogenicity were absent. By assuming the delay in osteogenesis as an adverse effect a NOAEL of 0.0625 % can be established (21 mg/kg-bw/day).

Executive summary:

A Prenatal Developmental Toxicity study was carried out according to OECD Guidelines 414. The study was conducted to determine the teratogenic potential of theobromine in rabbits. Theobromine was given in the diet at 0, 0.0625, 0.125 or 0.1875% (approximately 0, 21, 41 or 63 mg/kg/day, respectively). The duration of exposure was from days 6 to 29 of gestation. No general toxicity to maternal animals was observed. There were no treatment-related effects on the mean number of corpora lutea, abortions, implantations, litter size, embryolethality, dead and resorbed foetuses or sex ratios. However, a significant reduction in foetal body weight was noted in the 0.125% and 0.1875% groups. A variety of malformations and skeletal variations scattered throughout the groups, irrespective of treatment. The incidence of sternebral variations in the 0.125 and 0.1875% TBR dose groups was significantly higher. Based on the delay in osteogenesis of the foetuses exposed to 0.125 and 0.1875 %, the NOAEL for developmental effects would be 0.0625 which corresponds to 21 mg/kg-bw/day. Nevertheless, the authors suggested that the developmental variations should be considered to be biologically insignificant since other conventional signs of embryotoxicity, such as dose-related reductions in foetal weights and frank teratogenicity were absent.