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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Available literature data, in vitro studies as well as in vitro and in vivo studies on the read-across nucleoside L-Thymidine were considered in the evaluation of the potential genetic toxicity of Thymidine. The literature data was limited in relevance, adequacy as well as reliability and results were inconclusive. Of the three studies carried out with Thymidine a positive result was obtained in the forward gene mutation assay, only. Three in vitro studies and an in vivo micronucleus test carried out using the isomer L-Thymidine were negative. Therefore, based on the information available Thymidine is considered non genotoxic.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: See read-across justification attached.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
Duration of treatment / exposure:
Single treatment
Post exposure period:
Bone marrow preparations obtained 24 and 48 hours postdose were assessed.
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Each treated and control group must include at least 5 analysable animals per sex.
Control animals:
yes
Positive control(s):
Appropriate positive controls were used.
Tissues and cell types examined:
Bone marrow preparations.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.
A test substance for which the results do not meet the above criteria is considered nonmutagenic in this test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
No evidence of chromosome damage occurred in this in vivo micronucleus test.
Executive summary:

A study was carried out according to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test). This in vivo micronucleus test was performed in CD-1 mice given single oral doses of 0, 500, 1000, or 2000 mg/kg bw (limit dose). The numbers of micronuclei in bone marrow preparations obtained at 24 and 48 h postdose were determined. The test item was prepared as a solution in DMSO and appropriate negative and positive controls were used. No evidence of chromosome damage occurred in this mouse micronucleus test following single oral doses up to 2000 mg/kg bw.

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

Additional information

Additional information from genetic toxicity in vivo:

The genetic toxicity potential of the nucleoside Thymidine was evaluated in a step-wise approach. First, existing data published in the literature was assessed. In a second step a series of in vitro GLP-studies was carried out. Remaining data gaps were filled using in vitro and in vivo data available for the L-isomer of Thymidine in a read-across approach. The read-across approach is justified. Not further testing is required.

Literature data

Several authors have published studies on the genotoxic potential of Thymidine. Relevant in vitro data include Ames tests, forward gene mutation tests, sister-chromatid exchange studies, DNA inhibition studies as well as a cell transformation assay. In addition data from an in vivo micronucleus test was considered. Notably, all published data is of limited relevance, adequacy and reliability.

Published in vitro data

Marquadt et al. (1988) investigated the genotoxic activity of various 5-substituted pyrimidine 2'-deoxyribosides in different in vitro test systems. Thymidine was non-mutagenic in Salmonella Typhimurium strains TA 97 and TA 100 treated in a preincubation assay at concentrations of up to 2000 ug/mL without and with metabolic activation. No genotoxic potential was observed in 8-Azaguanine resistant V-79 Chinese hamster cells treated at concentrations of up to 500 ug/mL without metabolic activation. Thymidine did not induce unscheduled DNA synthesis in primary rat hepatocytes, but in contrast protected cells against damage by mutagenic compounds.

The negative results obtained by Marquadt et al. were reproduced by Klungland et al. (2001) in 6-Thioguanine resistant in V-79 Chinese hamster cells following treatment with 0.003 to 0.1 mmol without metabolic activation. In contrast, previously published studies by Anderson et al. (1981) and Bradley et al. (1981) revealed that Thymidine can produce an increase in V79 cells resistant to 8-Azaguanine, Ouabain and 6-Thioguanine at non-cytotoxic and low concentrations without metabolic activation.

Potential induction of sister chromatid exchanges was investigated in different cell types. Perry (1983) showed that addition of Thymidine to the culture medium of Chinese hamster ovary cells causes a dose-dependent increase and at 100 µM concentration, the sister chromatid exchange frequency is approximately doubled. The positive results were confirmed by Suzuki and Yosida (1984), who observed chromatid breaks and sister chromatid exchanges in Chinese hamster D-6 cells at concentrations of 500 umol/L. Further, Rainaldi et al. (1984) revealed sister chromatid exchanges in V-79/AP4 Chinese hamster cells at concentrations of 3 mmol/L. In contrast, studies by Zakharov and Bairamjan (1980) on the potential to induce sister chromatid exchanges were not conclusive.

Two additional in vitro studies are available. Cohen and Studzinski (1967) revealed DNA synthesis inhibition at high concentrations (250 mg/L) in human HeLa cells. Heidelberger et al. (1983) revealed no effects of Thymidine in a mouse cell transformation assay.

Published in vivo data

Sato et al. (1993) assessed the potential of Thymidine to induce micronucleated polychromatic erythrocytes (MNPCEs) in a micronucleus test. The test item was administered in single i.p. injections at concentrations of 370 and 740 mg/kg bw to groups of six mice each of the BALB/c, C57BL/6 and DBA/2 strains. After 30 hours the number MNPCEs in 1000 PCEs and also the number of PCEs in 1000 erythrocytes were counted for each mouse. Thymidine increased the frequency of MNPCEs significantly in the high dose group of BALB/c mice only. No increase in frequency was noted for the low BALB/c dose group and the two other mouse strains. Further, no marked influence on bone marrow cell proliferation was observed. Whereas the effects on the BALB/c strain may be attributed to a lower rate of DNA repair and cell kinetics may contribute to strain differences the overall result on the genotoxic potential of Thymidine is considered inconclusive.

In summary, there is a wide range of in vitro data on the genotoxic potential of Thymidine available. However, all data is of limited relevance, adequacy and reliability. Therefore, the data published in the literature is considered inconclusive.

Thymidine studies (GLP)

A standard battery of in vitro GLP-studies was initiated to further assess the genetic toxicity potential of Thymidine. The test program included a bacterial reverse mutation assay (Ames test) according to OECD Guideline 471, a chromosome aberration assay according to OECD Guideline 473 and a forward gene mutation (HPRT) test according to OECD Guideline 476. All studies were carried out according to good laboratory practice (GLP). Tests were run up to limit or cytotoxic concentrations, without and with metabolic activation and appropriate negative and positive controls were included.

Thymidine revealed no mutagenic activity in the microbial mutagenicity assay. In the chromosome aberration test no evidence of clastogenicity was noted. In the in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells no genotoxic effects were observed following exposure over five hours without and with metabolic activation, however, when treatment without metabolic activation was prolonged, significant increases in mutant frequency were observed.

L-Thymidine studies (GLP; read-across)

Following evaluation of the available data published in the literature and based on the results obtained in a standard in vitro test program, further data on genetic toxicity potential is required. However, instead of initiating additional animal studies, studies using L-Thymidine were considered in a read-across approach. The read-across between the D-isomer and the synthetic nucleoside L-isomer is justified (see Thymidine read-across justification for details). Results of three in vitro assays and one in vivo test are available (EMEA, 2007). As for the Thymidine studies, all studies using L-Thymidine were highly relevant, adequate and reliable (all studies according to Good Laboratory Practice).

L-Thymidine in vitro studies

In vitro studies on L-Thymidine include an Ames test and two chromosome aberration tests, one using Chinese hamster ovary cells and one human peripheral blood lymphocytes. Reverse mutation potential was determined in Salmonella Typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 and Escherichia coli strain WP2uvrA. The assay was negative in all five bacterial strains tested up to limit concentrations (5000 ug/plate), without and with metabolic activation. The in vitro chromosomal aberration assays were performed at up to limit concentrations (Chinese hamster ovary cells) and up to cytotoxic concentrations (human peripheral blood lymphocytes), without or with metabolic activation. Both chromosome aberration tests showed no evidence of clastogenicity.

L-Thymidine in vivo study

An in vivo micronucleus test was performed in CD-1 mice given single oral doses of 0, 500, 1000, or 2000 mg/kg bw. The numbers of micronuclei in bone marrow preparations obtained at 24 and 48 h post dose were determined. There was no evidence of chromosome damage this mouse micronucleus test up to the limit dose tested.

Conclusion

In conclusion, available literature data, in vitro studies as well as in vitro and in vivo studies on the read-across nucleoside L-Thymidine were considered in the evaluation of the potential genetic toxicity of Thymidine. The literature data was limited in relevance, adequacy as well as reliability and results were inconclusive. Of the three studies carried out with Thymidine a positive result was obtained in the forward gene mutation assay, only. All three in vitro studies and the in vivo micronucleus test with L-Thymidine were negative.

Therefore, based on the information available Thymidine is considered non genotoxic. No further testing is required.

References

- Anderson D, Richardson CR and Davies PJ (1981) The Genotoxic Potential of Bases and Nucleosides. Mutation Research, 91, 265-272.

- Bradley MO, Bhuyan B, Francis MC, Langenbach R, Peterson A and Huberman E (1981) Mutagenesis by chemical agents in V79 Chinese hamster cells: A review and analysis of the literature: A report of the gene-tox program. Mutation Research/Reviews in Genetic Toxicology, 87(2): 81–142.

- European Medicines Agency (2007) European public assessment report - Scientific discussions. EMEA/H/C/000713.

- Cohen LS and Studzinski GP (1967) Correlation between cell enlargement and nucleic acid and protein content of hela cells in unbalanced growth produced by inhibitors of DNA synthesis. Journal of Cellular Physiology, 69(3): 331–339.

- Heidelberger C, Freeman AE, Pienta RJ, Sivak A, Bertram JS, Casto BC, Dunkel VC, Francis MW, Kakunaga T, Little JB and Schechtman LM (1983) Cell transformation by chemical agents — A review and analysis of the literature ☆: A report of the U.S. environmental protection agency Gene-Tox Program. Mutation Research/Reviews in Genetic Toxicology, 114(3): 283–385.

- Klungland A, Paulsen R, Rolseth V, Yamada Y, Ueno Y, Wiik P, Matsuda A, Seeberg E and Bjelland S (2001) 5-Formyluracil and its nucleoside derivatives confer toxicity and mutagenicity to mammalian cells by interfering with normal RNA and DNA metabolism. Toxicology Letters, 119(1): 71–78.

- Marquadt H, Westendorf J, Schaefer A, Boldt,J, De Clercq E and Marquardt H (1988) Mutagenic and Antimutagenic Effects of 5-Substituted 2'-Deoxyuridines Depending on the Nature of the 5-Substituent; Arzneimittelforschung, 38(12):1820-1824.

- Perry PE (1983) Induction of sister-chromatid exchanges (SCEs) by thymidine and the potentiation of mutagen-induced SCEs in Chinese hamster ovary cells. Mutation Research / Fundamental and Molecular Mechanisms of Mutagenesis, 109(2): 219–229.

- Rainaldi G, Sessa MR and Mariani T (1984) Inhibitors of DNA synthesis induce sister chromatid exchanges at the early S phase of the cell cycle. Chromosoma, 90(1): 46-49.

- Sato S, Takizawa R and Inui N (1993) Mouse strain differences in induction of micronuclei by base analogues and nudeosides. Mutation Research, 301(45): 45-49.

- Suzuki H and Yosida TH (1984) Association of sister chromatid exchanges and chromatid breaks in Chinese hamster D-6 and lung cultured cells treated with hydroxyurea and some other chemicals. Cytologia, 49(3): 667-672.

- Zakharov AF and Bairamjan TL (1980) 5-Bromodeoxycytidine in the study of sister chromatid exchanges in human lymphocytes. Human Genetics, 55(2): 259-263.


Justification for selection of genetic toxicity endpoint
The genetic toxicity potential of the nucleoside Thymidine was evaluated in a step-wise approach. First, existing data published in the literature was assessed. In a second step a series of in vitro GLP-studies was carried out. Remaining data gaps were filled using in vitro and in vivo data available for the L-isomer of Thymidine in a read-across approach. The read-across approach is justified.

Justification for classification or non-classification

Based on the data available the substance is not classified and labeled according to Regulation 1272/2008/EEC (CLP) and Directive 67/548/EEC (DSD).