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Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro
Bacterial reverse mutation assays (Ames)

A reverse mutation assay was conducted according to OECD guideline 471 with theobromine. Salmonella typhimurium strains TA97a, T100, TA102 and TA104 were exposed to theobromine at concentrations ranging from 1 to 20000 µg/plate for 48 hours with and without metabolic activation, DMSO was used as the solvent. Positive and solvent controls were included however positive control for TA104 was not available. A total of four plates were used for each test concentration and control. Strains TA102 and TA104 showed very weak mutagenic effect in few concentrations with metabolic activation. Some more concentrations (5, 50 and 500 µg/plate) were included for these strains. Theobromine at different concentrations produced weak but significant increases in revertants colonies in TA 102 and TA104 with metabolic activation. Moreover theobromine was toxic to diverse bacteria strains at 10000 and 20000 µg/plate

Another bacterial mutagenicity assay was performed according to the standard NTP protocol which is equivalent to OECD guideline 471. Strains TA100, TA1535, TA197 and TA98 of S. typhimurium were exposed to concentrations ranging from 1 to 10000 µg/plate of the test substance theobromine. The preincubation method was employed. The study included metabolic activation with different concentrations of S9 preparations from hamster and rat liver. The test substance resulted to be non-mutagenic to the tester strains in concentrations up to 10000 µg/plate with and without metabolic activation.

An additional bacterial reverse mutation assay was performed in accordance to the method described by Ames et al.(1975) which is equivalent to OECD 471 Guideline. Overnight cultures of TA1535, TA100, TA1537, TA1538 and TA98 of Salmonella typhimurium were exposed to the test chemical with and without metabolic activation (S9) through the standard plate incorporation method. As a positive control, 2- aminoanthracene was used and distiled water was used as the solvent. Theobromine gave no evidence of mutagenicity up to the highest concentration still soluble of 0 .25% (2 .5 mg/ml) with and without metabolic activation.

Another reverse mutation assay was conducted following the test procedures described by Ames et al.(1975) which are similar to OECD guideline 471 in compliance with U.S. FDA Good Laboratory Practices regulations. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were exposed to theobromine at concentrations ranging from 0.5 to 5000 µg/plate with and without metabolic activation, DMSO was used as the solvent. Positive and solvent controls were included. No evidence of mutagenicity was observed.

Gene mutation assay

A mammalian cell gene mutation assay was performed following the test procedures described by Clive and Spector (1975) and Clive et al. (1979) which are similar to OECD 490 MLA. The study was conducted in compliance with U.S. FDA Good Laboratory Practices regulations. Cultures containing 3x 106L5178Y TK+/- mouse lymphoma cells were exposed to the test chemical for 4 hours at preselected concentrations according to a preliminary cytotoxicity test, then were washed and placed in growth medium for 2 days to allow recovery, growth and expression of the induced BrdU-resistant phenotype (presumptive TK -/- mutants) . Cell counts were determined daily and appropriate dilutions were made to allow optimal growth. At the end of expression period, 3x106cells for each selected dose were seeded in soft agar dishes with selection medium containing BrdU (1x106cells/dish) and BdrU resistant colonies (mutant) were counted after approximately 10 days incubation. The activation portion of the test includes the addition of Aroclor-induced S9 fraction of rat liver homogenate. The results showed small increases in mutant frequencies in the presence and absence of metabolic activation. The active concentration range for activation test conditions was 1250-1500 µg/mL and 2000 -2500 µg/mL for non-activation test conditions. The mutagenic response was observed at the highest dose tested with only a suggestion of a dose-response effect. The mutagenic responses were associated with high levels of toxicity.

Cytogenetic assay

A chromosome aberration study in CHO cells was performed according to OECD guideline 473 in compliance with U.S. FDA Good Laboratory Practices regulations. CHO cells were exposed to concentrations ranging from 62.5 to 1000µg/mL of theobromine in deionized water in presence and absence of metabolic activation. Solvent and positive (triethylene-melamine and cyclophosphamide) controls were included. Colcemid was added the last 2 hours of incubation and metaphase cells were collected, fixed, stained with Giemsa and mounted. 100 cells per concentration were analyzed for aberrations. Chromatid and isochromatid gaps were noted but were not included in the totals for aberrations. Data was analyzed using a Student's t test. No increases were observed for any type of chromosome aberration in CHO cells treated with theobromine up to 1000µg/mL with and without metabolic activation.

Other in vitro genotoxicity assays

A sister chromatid exchange study in CHO cells was performed according to EU method B.19 in compliance with U.S. FDA Good Laboratory Practices regulations. CHO cells were exposed to concentrations ranging from 62.5 to 1000 µg/mL of theobromine in presence and absence of metabolic activation. 5 -Bromo-2'-deoxyuridine (10 µM final concentrations) was added and cells were incubated for 27 hours. Colcemid was added for last 2 hours of incubation and metaphase cells were collected, fixed and stained with a modified fluorescent plus Giemsa (FPG) technique which includes Hoechst 33258 and Giemsa stains. The data was evaluated using a Student’s t test. Theobromine induced significant increases in sister-chromatid exchanges without metabolic activation over a range of 100 - 1000 µg/mL in a dose-related manner. With metabolic activation the results were equivocal and not dose-related.


Other 2 independent trials of sister chromatid exchange analysis in cultured human lymphocytes were performed according to EU method B.19 in compliance with U.S. FDA Good Laboratory Practices regulations. Lymphocytes were exposed to concentrations ranging from 0 to 1000 µg/mL of theobromine in deionized water. 5 -Bromo-2'-deoxyuridine (25 µM final concentrations) was added and cells were incubated for 46 hours. Colcemid was added for last 2.5 hours of incubation, then cells were collected, fixed and stained with a modified fluorescent plus Giemsa (FPG) technique which includes Hoechst 33258 and Giemsa stains. The data was evaluated using a Student’s t test. The results were positive in a dose-related manner. The dose range showing Sister Chromatid Exchange activity was 100-500 µg/mL. A dose response was obtained in both trials.

An in vitro cell transformation assay was performed with procedures based on that reported by Kakunaga (1973) which are according to EU method B.21 in compliance with U.S. FDA Good Laboratory Practices regulations included. The assay included 4 trials. Mouse cells Balb/ c-3T3 were exposed to concentrations ranging from 62 to 5000 µg/mL of theobromine for 24 hours (trials b and d) and from 0.5 to 750 µg/mL for 72 hours (trials a and c), cells were washed and incubation continued for 4 weeks, then cell monolayers were fixed and stained with Giemsa. The stained dishes were examined by eye and microscope to determine the number of foci of transformed cells. The dose levels used were based upon preliminary toxicity tests. The 24-h LC 50 was estimated to be 1000 µg/mL and the 72-h LC50 near 500 µg/mL. Theobromine did not produce convincing evidence of an ability to transform mouse Balb/c-3T3 cells in vitro.

There are several in vitro and in vivo genotoxicity studies with theobromine, there are positive and negative findings both with and without metabolic activation, in a weight of evidence approach, the positive findings are not relevant to germ cell mutagenicity since a rodent dominant lethal test with acceptable quality (K2) gave no indications that theobromine produces heritable gross chromosomal aberrations or lethal gene mutations in germ cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo
An in vivo sister chromatid exchanges assay in bone marrow cells of mice was conducted with theobromine. 5 Swiss albino male mice were used for the test per dose group. Paraffin-coated BdrU tablets (approximately 50mg) were implanted subcutaneously in the flank of mice under anaesthesia. Theobromine was administered by intraperitoneal injection at three doses (12.5, 25, 50 mg/kg b.w.) with DMSO as vehicle to different groups of mice 1 hour after the tablet implantation. Negative control mice were injected with 75 µL of DMSO while mitomycin- C was used as a positive control at a dose of 1.5 mg/kg of body weight. Colchicine (4 mg/kg.) was injected i.p. 22 h after BrdU tablet implantation. Two hours later, slides of bone marrow cells were prepared for examination and chromosomes were differentially stained with fluorescence-plus-Giemsa technique. All the slides were coded and 30 division metaphase cells (40±2 chromosomes) per animal were scored for sister chromatid exchange frequencies, i.e., a total of 150 cells were scored from five animals per dose tested. Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first, second and third division metaphases. Results indicated that theobromine can induce significant increases in sister chromatid exchanges in bone marrow cells of mice after an in vivo exposure from concentrations of 12.5 mg/kg b.w to 50 mg/kg b.w. No significant changes in replicative index were observed for any of the three tested doses.

An in vivo micronucleus test was conducted in Chinese hamsters according to the standard procedure of Schmid, 1973 which is equivalent to OECD 474. 2 equal doses of 20, 30 and 40 mg corresponding to 666, 1000 and 1333 mg/kg b.w. were administered suspended in corn oil by stomach tube to groups of 6 animals per dose with equal number of males and females, 30 and 6 hours before the animals were killed . Theobromine caused highly positive results compared to the control but only at a dose of 2 x 1333 mg/kg b.w.

An in vivo chromosomal aberration test was conducted according to the technique of Swarzacher and Wolf, 1974 which is equivalent to OECD 475 in Chinese hamsters, bone marrow cells were evaluated. 6 animals with equal number of males and females were exposed to theobromine suspended in corn oil at doses of 666, 1000 and 1333 mg/kg b.w. 24 hours before the bone-marrow cells were collected. Colchicine was injected subcutaneously at a dose of 1mg/kg 2 hours before the animals were killed. 300 metaphases per animal were analysed .Theobromine did not increase the number of gaps or chromatid breaks when compared to the control up to 1333 mg/kg b.w. Higher doses were toxic.

An in vivo SCE (sister chromatid exchange) study was performed with theobromine in Chinese hamsters. The test was performed with 5-bromodeoxyuridine tablets (Boehringer, Mannheim) which were implanted subcutaneously into the experimental animals. In the test, 50 mg tablets/animal were used, the BrdU-treatment time was of 26 h, 4 animals per dose were used and a total of 50 metaphases/animals were analysed. The concentrations used in the study were: 0 (control), 83, 167, 333, 500 and 667 mg/kg bw. Colchicine was injected s.c. at a dose of 1 mg/kg bw 2 hours before the animals were killed. In the tested system, theobromine produced dose-related increases in SCE activity. Results were considered positive at the tested doses of 333, 500 and 667 mg/kg bw.

A genotoxicity/teratogenicity study was conducted according to a method similar to a Rodent Dominant Lethal Assay (OECD 478) with theobromine. Sub-lethal dosages of the compound were intraperitoneally administered to JCL: ICR female mice on day 9 or 12 of gestation (nominal concentrations of: 450 and 500 mg/kg). Fetuses were grossly and skeletally examined on day 18 of gestation. In this examination, the number of litter resorption, implants and fetuses (size and body weight) were recorded. Malformations were also observed in fetuses. The results showed that theobromine induced significantly high frequencies of malformed fetuses mainly with axial skeletal defects when they were administered on day 9 of gestation.

A dominant lethal testing of theobromine was conducted in rats with a method which is in accordance with the OECD guideline 478.

Subacute doses of theobromine (50, 150 and 450 mg/kg bw) in a 0.2 % aqueous solution of carboxymethyl cellulose were administered once a day for 5 consecutive days to groups of 20 sexually mature male rats, a control group of similar number of male rats was included in the study design. On the third day after completion of dosing, one untreated female was cohoused with one male for 5 consecutive nights and days. At the end of this period each female was removed and individually housed. On day 13 after the midweek of presumptive mating, all 80 females were killed by CO2 inhalation and dissected. Total implantations (living and dead) and corpora lutea were counted in each female. The mating procedure was repeated with virgin female rats every week for a total of 10 weeks to sample all the stages of spermatogenesis. Weekly tabulations of dead implants per total implants and preimplantation loss per total corpora lutea were analyzed statistically by the estimated proportion of Gladen. Theobromine did not induce dominant lethal mutations or adverse effects on pregnancy rate when given at subacute doses up to 450 mg/kg bw to male rats by the oral route.


Discussion

The only positive response in the bacterial reverse mutation assay was a very weak one in strains TA102 and TA104 although these responses were not dose related and a positive control for the strain 104 was not included, moreover these strains are not recommended by the OECD guideline 471 (1997). In contrast this guideline suggest the strains of S. typhimurium TA1535, TA1537, TA97, TA98 and TA100 due to reliability and reproducibility, all of these recommended strains plus TA1538 and TA97a gave negative results with and without metabolic activation.

The results from the mouse lymphoma assay showed small but reproducible increases in mutant frequencies in the presence and absence of metabolic activation. The active concentration range for nonactivation test conditions was 2000-2500 µg/ml and for activation test conditions was 1250-1500 µg/ml. The mutagenic response was observed at the highest dose tested with only a suggestion of a dose-response effect. The mutagenic responses were associated with high levels of toxicity (percent relative growth values of 3.3, 4.9, 4.9 and 67.5). The 67.5 value was the result of an almost normal relative suspension growth (81% of control vs. 10-15% for the other theobromine treatments) and an unexpectedly high cloning efficiency (83% vs. 25-35% for the other theobromine treatments). Absolute increases in TK mutant counts were not generated in the non-activation studies but were observed in the activation trials. An absolute increase in mutant counts is a good indication that the increase in mutant frequency is real.

 

Positive results were obtained from diverse in vivo and in vitro sister chromatid exchange assays. The in vitro SCE were assessed using CHO cells and cultured human lymphocytes. The results in CHO cells were positive, in a dose-related fashion from 100 to 1000 µg/mL without S-9 metabolic activation. With the S-9 enzyme system, the results were equivocal and not dose-related. The SCE assay in cultured human lymphocytes did not include metabolic activation and the range of dose-related increases in SCEs was 100-500µg/mL.
The in-vivo SCE assays used Chinese hamsters and Swiss albino mice. With hamsters as model the results were considered positive from 200 mg/kg bw forward and with mice the results were positive from 12.5 mg/kg bw forward. Dose related responses were found in both assays.
The in-vivo results should be considered before the in vitro results; moreover the result of the in vitro SCE assay using CHO cells was different with and without metabolic activation.
The lowest tested dose at the in vivo SCE assay with Chinese hamsters was 83 mg/kg b.w. while with Swiss albino mice was 12.5 mg/kg bw.
These responses can be related to the observation of Levi Viktorya et al., 1978 that methylxanthines were able to inhibit poly(ADP-ribose) polymerase (ADPRT), a chromatin-bound enzyme involved in the DNA repair process, moreover it has been shown that inhibition of ADPRT is associated with induction of SCE.

 

The positive result of the rodent dominant lethal by intraperitoneal injection shows that the test substance has an intrinsic mutagenic property but it cannot be ruled out that the positive test is relevant to humans since the factors such as distribution/metabolism should be taken into account, moreover both intraperitoneal dominant lethal assays have low quality in contraposition to the higherquality dominant lethal testing of theobromine by the oral route, which showed that theobromine did not induce dominant lethal mutations or adverse effects on pregnancy rate, remarked that the testing doses were comparable (the i.p tests employed concentrations up to 600 mg/kg bw and the oral test up to 450 mg/kg bw).

In a weight of evidence approach theobromine gave positive results in diverse in vitro and in vivo assays when somatic cells were examined, the positive findings are mainly related to the induction of SCE activity which molecular mecanism has already been clarified. The relevance of such positive findings on the germ cell mutagenicity is doubtful, given that the oral dominant lethal assay by the oral route did not give indications that genetic effects are transmitted through the germline, the overall conclusions is that theobromine does not cause concerns for heritable mutations in the germ cells.


The dominant lethal assay by the oral route is considered relevant to the assessment of genotoxicity because has a good quality (K2) and its results are contributed by in vivo factors such as metabolism, pharmacokinetics and active DNA-repair processes, moreover the study directly assess the genotoxicity in germ cells in contraposition to the genotoxicity in somatic cells which were evaluated in other in vitro and other in vivo tests.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on available data, Theobromine is not classified for germ cel mutagenicity according to CLP Regulation no. 1272/2008. A weight of evidence in somatic cells with the supporting information of a negative outcome in germ cells indicate that theobromine is not expected to produce heritable mutations.