Tris(pentane-2,4-dionato-O,O')vanadium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The parent compound Vanadium-tris-acetylacetonate is rapidly hydrolysed to 2,4 -pentanedione (CAS no. 123 -54- 6) and Vanadyl acetylacetonate (CAS no. 3153 -26 -2) in the presence of water or moisture (> 80% hydrolysis after 1h at pH 1.2, 4, 7 and 9). Hence, the half life is < 1 h under neutral and acidic conditions. Accordingly, reliable data of the hydrolysis products 2,4-Pentadione (Cas no. 123-54-6) and Vanadyl acetylacetonate (3153-26-2) or comparable inorganic Vanadium compounds are used to address the endpoint, which is entirely appropriate to draw conclusions on the genetic toxicity of Vanadium-tris-acetylacetonate.

gene mutation bacterial cells

V₂O₅ S. typhimurium TA97, TA98, TA100, TA102, TA1535, Ames (OECD TG 471): negative

Acetylacetonate, S. typhimurium TA1535, Ames (OECD TG 471): negative

clastogenicity

V₂O₅, human lymphocytes, CA (OECD TG 473): negative

V₂O₅, human lymphocytes, SCE (OECD TG 479): negative

V₂O₅, CHO, MNA (OECD TG 487): positive

V₂O₅, CHO, SCE (OECD TG 479): negative

Acetylacetonate, CHO, SCE (OECD TG 479): positive

gene mutation mammalian cells

NH₄VO₃, CHO, h(g)prt (OECD TG 476): negative

VOSO₄, CHO, h(g)prt (OECD TG 476): negative

V₂O₅, CHO, h(g)prt (OECD TG 476): negative

Acetylacetonate, CHO, hprt (OECD TG 476): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The study was conducted scientifically reasonable equivalent to OECD 476 and is sufficiently documented to conclude that the study was well-performed. Positive and negative controls gave the appropriate response. Hence, the results are considered sufficiently reliable to assess the mutagenic potential of the test item in mammalian cells. In this experimental system ammonium metavanadate has no mutagenic effect. This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Executive summary:

The mutagenic potential of ammonium metavanadate was assessed in a study equivalent to OECD TG 476 (forward gene mutation test, hprt-assay) in V79 cells. The test item was tested in concentrations of 0, 0.5, 1, 2, and 5 mM with and without metabolic activation, EMS and DMN served as positive controls.

The results obtained with V79 mammalian cells showed a toxic effect of vanadium compounds. The increase of survival observed in cells treated with metavanadate in the presence of the S9 fraction as compared to the absence of metabolic activation seems to agree with the hypothesis that the NADPH of the S9 fraction may contribute to the reduction of V5 to vanadyl, which is less toxic. In fact, the survival percent in cells treated with metavanadate in the presence of S9 fraction is similar to that of cells treated with vanadyl without S9 fraction at the maximum concentration used. Vanadyl is probably complexed with proteins of S9 hepatic fraction since the survival is near 100 % in all concentrations used. In this experimental system no mutagenic effect is observed, positive and negative control gave the appropriate response. The study was classified as acceptable.

This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The study was conducted scientifically reasonable equivalent to OECD 476 and is sufficiently documented to conclude that the study was well-performed. Positive and negative controls gave the appropriate response. Hence, the results are considered sufficiently reliable to assess the mutagenic potential of the test item in mammalian cells. In this experimental system Vanadyl sulfate has no mutagenic effect. This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Executive summary:

The mutagenic potential of Vanadyl sulfate was assessed in a study equivalent to OECD TG 476 (forward gene mutation test, hprt-assay) in V79 cells. The test item was tested in concentrations of 0, 1, 2, 5 and 7.5 mM with and without metabolic activation, EMS and DMN served as positive controls.

The results obtained with V79 mammalian cells showed a toxic effect of vanadium compounds. The increase of survival observed in cells treated with metavanadate in the presence of the S9 fraction as compared to the absence of metabolic activation seems to agree with the hypothesis that the NADPH of the S9 fraction may contribute to the reduction of V5 to vanadyl, which is less toxic. In fact, the survival percent in cells treated with metavanadate in the presence of S9 fraction is similar to that of cells treated with vanadyl without S9 fraction at the maximum concentration used (see table above). Vanadyl is probably complexed with proteins of S9 hepatic fraction since the survival is near 100 % in all concentrations used.

In this experimental system no mutagenic effect is observed, positive and negative control gave the appropriate response. The study was classified as acceptable.

This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

no metabolic activation system, controls not specified

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Lymphocyte cultures were prepared by adding heparinized blood from non-smoking healthy male, 28 years old, to McCoy's 5A medium and phyothaemagglutinin M

Additional strain / cell type characteristics:

not specified

Untreated negative controls:

yes

Remarks:

distiiled water

Negative solvent / vehicle controls:

yes

Remarks:

distiiled water

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 72 h
- Exposure duration: 72 h
- Expression time (cells in growth medium): none

SPINDLE INHIBITOR (cytogenetic assays): colchichine 0.4 µg/mL

STAIN (for cytogenetic assays): 5-bromodeoxyuridine (BrdU)

NUMBER OF REPLICATIONS: 2, the same healthy blood donor was used in both the experiment and its replicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were made by dropping single drops of the cell suspension, airdrying, staining with Hoechst-33258, immersed in KCl (0.075 M) and irradiating with UV light. These slides were then incubated in 2 × SSC and stained with Giemsa.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): minimum of 100 well-spread first-division metaphases were analyzed for structural and numerical aberrations, 90 metaphases were analyzed for satellite associations, 1000 cells were analyzed for mitotic index (MI). SCE was analyzed in 60 second-division mitoses.

DETERMINATION OF CYTOTOXICITY
mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

For each concentration and each experiment a minimum of 100 well-spread first-division metaphases were analysed for structural and numerical aberrations, 90 metaphases were analysed for satellite associations, 1000 cells were analysed for mitotic index (MI). SCE was analysed in 60 second-division mitoses.
The types of aberrations scored included chromatid-type aberrations such as chromatid and isochromatid gaps, chromatid breaks, chromatid interchanges, chromosome-type aberrations such as breaks and rings.
Numerical aberrations scored included only polyploidy.
The average generation time (AGT) was calculated by the method proposed by Ivett and Tice (1982).

Key result

Species / strain:

lymphocytes: human primary lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

The treatment of human lymphocytes with V2O5 does not induce sister-chromatid exchanges.

Executive summary:

Cytogenicity of vanadium pentoxide was tested in in vitro sister chromatide exchange assay in human lymphocytes (similar to OECD Guideline 479). The data show that treatment of human lymphocytes with V₂O₅ does not induce sister-chromatid exchanges. However, the treatment did affect cell division and the structure and behavior of the spindle apparatus.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

less than 300 metaphases scored per concentration, no metabolic activation system

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Lymphocyte cultures were prepared by adding heparinized blood from non-smoking healthy male, 28 years old, to McCoy's 5A medium and phyothaemagglutinin M

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

Vanadium pentoxide (dissolved in distilled water) was added at concentrations of 2, 4, and 6 µg/ml (selected on the basis of preliminary experiments).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water (distilled)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 72 h
- Exposure duration: 72 h
- Expression time (cells in growth medium): none

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.4 µg/mL

STAIN (for cytogenetic assays): 5-bromodeoxyuridine (BrdU)

NUMBER OF REPLICATIONS: 2, the same healthy blood donor was used in both the experiment and its replicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were made by dropping single drops of the cell suspension, airdrying, staining with Hoechst-33258, immersed in KCl (0.075 M) and irradiating with UV light. These slides were then incubated in 2 × SSC and stained with Giemsa.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): minimum of 100 well-spread first-division metaphases were analyzed for structural and numerical aberrations, 90 metaphases were analyzed for satellite associations, 1000 cells were analyzed for mitotic index (MI). SCE was analyzed in 60 second-division mitoses.

DETERMINATION OF CYTOTOXICITY
mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

For each concentration and each experiment a minimum of 100 well-spread first-division metaphases were analysed for structural and numerical aberrations, 90 metaphases were analysed for satellite associations, 1000 cells were analysed for mitotic index (MI). SCE was analysed in 60 second-division mitoses.
The types of aberrations scored included chromatid-type aberrations such as chromatid and isochromatid gaps, chromatid breaks, chromatid interchanges, chromosome-type aberrations such as breaks and rings. Numerical aberrations scored included only polyploidy. The average generation time (AGT) was calculated.

Key result

Species / strain:

lymphocytes: human primary lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The mitotic index (recommended cytotoxicity parameter for primary cultures of lymphocytes acc. to OECD TG 473) significantly decreased from 2.7% (control) to 2.0 % (2 µg/ml), 1.12 % (4 µg/ml) and 1.14 (6 µg/ml)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

The treatment of human lymphocytes with V2O5 does not induce chromosomal aberrations.

Executive summary:

Cytogenicity of vanadium pentoxide was tested in in vitro chromosomal aberration assay in human lymphocytes (similar to OECD Guideline 473). The data show that treatment of human lymphocytes with V₂O₅ does not induce chromosomal aberrations. However, the treatment did affect cell division and the structure and behavior of the spindle apparatus.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the test substance was cytotoxic at 10 mg/plate to strain TA1538, and cytotoxic to all strains at 30 mg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The acetylacetonate test substance was cytotoxic to all Salmonella strains, both in the absence and in the presence of S9 at 30 mg/plate, and also at 10 mg/plate to strain TA1538 (-S9). No dose-related increases in the number of mutant colonies were observed in Salmonella strains TA98, TA100, TA1535, TA1537, or TA1538, either in the absence or in the presence S9 metabolic activation system. Solvent and positive control cultures demonstrated appropriate activity in the test system.

Conclusions:

The study was conducted scientifically reasonable equivalent to OECD 471. Positive and negative controls gave the appropriate response. Hence, the results are considered sufficiently reliable to assess the mutagenic potential of the test item in bacteria.
The acetylacetonate test material was not mutagenic to Salmonella and E. Coli strains with and without metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535, TA 1537 and TA1538 of S. typhimurium were exposed to acetylacetonate at concentrations of 0.3, 1.0, 3.0, 10.0 and 30.0 mg/plate in the presence and absence of mammalian metabolic activation (Ames Test according to OECD 471). A dose range-finding study was conducted using tester strain TA100, and dose levels of the acetylacetonate test material ranging from 0.3 to 10 mg/plate were used. In the main study there were two treatment sets for each tester strain, with (+S9) and without (-S9) metabolic activation. Each of the tester strains was dosed with five concentrations of test substance, vehicle controls, and a positive control. Three plates/dose group/strain/treatment set were evaluated. Plates were incubated for 48 -72 hours at 37oC.

The acetylacetonate test material was cytotoxic to all tester strains at 30 mg/plate, and at 10 mg/plate to strain TA1538 in the absence of the metabolic activation system (-S9). No dose-related increase in the number of mutant colonies were observed in all tester strains, either in the absence or in the presence of S9 metabolic activation system. The positive control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response.

Therefore, the acetylacetonate test substance was considered to be non-mutagenic without and with S9 mix in the bacterial reverse mutation test.

This result is also valid for the target substance vanadium-tris-acetylacetonate, since the source substance acetylacetonate is a hydrolysis product of vanadium-tris-acetylacetonate.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Acetylacetonate treatment at 1.0 mg/ml reduced relative survival to <10 % in the absence of S9 and to <50 % in the presence of S9.
Corrected mutation frequencies were 2-3.9 x 10^-6 for solvent controls, 6.0 x 10^-6 for medium controls, and 0-17.3 x 10^-6 for acetylacetonate-treated cultures in the absence of S9. In the presence of S9, corrected mutation frequencies were 0-1.4 x 10^-6, 0, and 0-3.0 x 10^-6 for vehicle, medium, and acetylacetonate-treated cultures.
There was no significant increase in gene mutation in CHO cells treated with acetylacetonate, either in the absence or in the presence of S9.
Increased mutation frequencies were obtained in the test system on treatment with positive control substances.

Conclusions:

Interpretation of results: negative
The acetylacetonate test material was not genotoxic under the conditions of the test.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD Guideline 476, cultured CHO cells were exposed to the test substance acetylacetonate at concentrations of  0.01, 0.05, 0.1, 0.5, 1.0 and 1.5 mg/ml in the presence and absence of a mammalian metabolic activation system. The positive controls induced the appropriate response.  There was no evidence of induced mutant colonies over background, therefore the acetylacetonate test material was not genotoxic under the conditions of the test.

This result is also valid for the target substance vanadium-tris-acetylacetonate, since the source substance acetylacetonate is a hydrolysis product of vanadium-tris-acetylacetonate.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

sister chromatid exchange

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Significant increases in the frequencies of SCE were observed both in the absence and in the presence of S9 activation. In the absence of S9, the increases were 1.2-fold at the low dose (0.02 mg/ml), 1.4-fold at the middose (0.03 mg/ml), and 4.2-fold at the high dose (0.10 mg/ml). In the presence of S9 activation, the increases were 1.2-fold at the low dose (0.03 mg/ml), 1.3-fold at the mid-dose (0.10 mg/ml), and 1.5-fold at the high dose (0.3 mg/ml).

Conclusions:

Acetylacetone was positive in vitro in Sister Chromatide Exchange Assay in CHO cells. This indicates that the acetylacetone moiety of vanadium-tris-acetylacetonate has clastogenic properties. This result is also valid for the target substance vanadium-tris-acetylacetonate, since the source substance acetylacetonate is a hydrolysis product of vanadium-tris-acetylacetonate.

Executive summary:

Acetylacetone was tested in Sister Chromatid Exchange Assay similar to OECD 479. Significant increases in the frequencies of SCE were observed both in the absence and in the presence of S9 activation. In the absence of S9, the increases were 1.2-fold at the low dose (0.02 mg/ml), 1.4-fold at the middose (0.03 mg/ml), and 4.2-fold at the high dose (0.10 mg/ml). In the presence of S9 activation, the increases were 1.2-fold at the low dose (0.03 mg/ml), 1.3-fold at the mid-dose (0.10 mg/ml), and 1.5-fold at the high dose (0.3 mg/ml). This indicates that the acetylacetone moiety of vanadium-tris-acetylacetonate has clastogenic properties.

This result is also valid for the target substance vanadium-tris-acetylacetonate, since the source substance acetylacetonate is a hydrolysis product of vanadium-tris-acetylacetonate.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The study was conducted scientifically reasonable equivalent to OECD 476 and is sufficiently documented to conclude that the study was well-performed. Positive and negative controls gave the appropriate response. Hence, the results are considered sufficiently reliable to assess the mutagenic potential of the test item in mammalian cells. In this experimental system V2O5 failed to induce gene mutation in V79 cells. This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Executive summary:

The mutagenic potential of ammonium metavanadate was assessed in a study equivalent to OECD TG 476 in V79 cells. The test item was tested in concentrations of 0, 1, 2, 3 and 4 µg/ml without metabolic activation, MEM and MNNG served as solvent and positive controls, respectively.

The results obtained with V79 mammalian cells showed a toxic effect of vanadium compounds as measured by the number of binucleated cells in the presence of Cyt B after 24 h treatment. An increase in the number of mononucleated cells, with a significant decrease in binucleated cells, at the concentrations 1-3 µg/ml, indicate that V₂0₅ inhibits mitosis even at low concentrations. The percent relative survival decreased with increased concentrations (see Table above) are also consistent with the cytotoxicity of V₂O₅ in a concentration-dependent response.

V₂O₅ was tested for the ability to induce gene mutation at the HPRT locus in V79 cells. None of the 3 concentrations tested significantly increased the frequency of TGr mutants; no dose response or minimum 3-fold increase over background was demonstrated.

In this experimental system no mutagenic effect is observed, positive and solvent control gave the appropriate response. The study was classified as acceptable. This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Reference 9

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Cytotoxicity Test

The data indicate that with increases in concentration of the test substance, the number of binucleated cells significantly decreases from 89 % in the control to 23 - 32 % at the range of concentrations tested (3 - 12 µg/ml).

Induction of MN in V79 cells following V₂O₅ treatment

A clear concentration-related increase in MN frequencies was observed (see table below). In the concentration range from 1 to 3 µg/ml, the incidence of MN increases from 4.15 to 7.55 %. The dose-response relationship was statistically significant. At the concentration of 3 µg/ml, a more than 3 -fold increase in the frequency of MN cells over the control value was elicted.

The results of immunofluorescence staining of kinetochores in MN with anticentromer antibody showed that almost one half (49 %) of micronuclei in untreated cells contained kinetochores (KN); however more than 69 % of micronuclei induced by V₂O₅ (1 µg/ml) reacted positively with the antibody. The frequency of KC+ MN also showed a concentration-related increase in the treated cells.

No increease in the number of KC- micronucleated cells and no decrease in NDI was noted in the cultures treated with V₂O₅ .

Conclusions:

The study was conducted scientifically reasonable equivalent to OECD 487 and is sufficiently documented to conclude that the study was well-performed. Positive and negative controls gave the appropriate response. Hence, the results are considered sufficiently reliable to assess the mutagenic potential of the test item in mammalian cells.The test substance is cytotoxic to the V79 cells and inhibits mitosis even at low concentrations. Furthermore the results of the antikinetochore antibody study indicate that V2O5 is an aneuploidogenic agent. This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Executive summary:

The induction of micronucleus formation (MN) in V79 cells following vanadium pentoxide treatment and the immunofluorescent staining of kinetochores was assessed in a study equivalent to OECD TG 487. The test substance was tested in concentrations of 0, 1, 2, and 3 µg/ml without metabolic activation, Mitomycin C and Vincristine sulfate served as positive controls.

In this experimental system a clear concentration-related and statistically significant increase in MN frequencies was observed in the concentration range from 1 to 3 µg/ml, positive and negative control gave the appropriate response. The results of the antikinetochore antibody study indicate that MN formation in V79 cells exposed to vanadium pentoxide is due to spindle dysfunction, leading to chromosome lagging and the improper execution of mitosis. Apparently, vanadium pentoxide is an aneuploidogenic agent. The study was classified as acceptable. This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Reference 10

Endpoint:

genetic toxicity in vitro, other

Remarks:

other: sister-chromatid exchange

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

The study was conducted scientifically reasonable equivalent to OECD 479 and is sufficiently documented to conclude that the study was well-performed. Positive and negative controls gave the appropriate response. Hence, the results are considered sufficiently reliable to assess the mutagenic potential of the test item in mammalian cells. In this experimental system V2O5 failed to induce SCE in V79 cells. This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Executive summary:

The induction of sister-chromatid exchange (SCE) in V79 cells following vanadium pentoxide treatment was assessed in a study equivalent to formerly OECD TG 479. The test substance was tested in concentrations of 0, 1, 2, 3 and 4 µg/ml without metabolic activation, Trinitrofluorenone served as positive control.

In this experimental system no significant increase in SCE frequency was observed with the V₂O₅ concentration tested, positive and negative control gave the appropriate response. A decrease in the replicative index was found in the V₂O₅ -treated cells. The concentration of 4 µg/ml was overly toxic to the cells. The frequencies of endoreduplication of chromosomes were 6 %, 8 % and 19 % at 1 µg/ml, 2 µg/ml and 3 µg/ml of V₂O₅ respectively. The study was classified as acceptable.

This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight toxicity at highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Vanadium pentoxide (0.03 to 333.00 μg/plate) was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100, TA102, or TA1535 with or without induced rat or hamster liver S9 enzymes.

Conclusions:

Vanadium pentoxide (0.03 to 333.00 μg/plate) was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100, TA102, or TA1535 with or without induced rat or hamster liver S9 enzymes. This indicates the vanadium, which is released after hydrolysis of the target substance vanadium-tris-acetylacetonate might be nutagenic.

Executive summary:

Vanadium pentoxide was tested similar to OECG Guideline 471 (preincubation method) in S. typhimurium strain TA97, TA98, TA100, TA102, or TA1535 with or without induced rat or hamster liver S9 enzymes at 8 concentrations ranging between 0.03 and 333 µg/plate. As a result, all tests were negative.

This result is also valid for the target substance vanadium-tris-acetylacetonate, since vanadium is a hydrolysis product of vanadium-tris-acetylacetonate. Therefore, data on several vanadium compounds are taken into account.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

V₂O₅,mouse, ip, single dose, COMET: positive

Na₃VO₄, mouse, oral, 5 weeks, COMET (OECD TG 489): positive

Na₃VO₄, mouse, oral, 5 weeks, MNA (OECD TG 474): positive

Na₃VO_4, mouse, ip, single dose, aneuploidy in sperm cells: positive

Na₃VO_4,mouse, ip, single dose, MNA (OECD TG 474): negative

2,4 -pentanedione, rat and mouse, inhalation, 5 days, MNA (OECD TG 475): negative

2,4 -pentanedione, rat and mouse, inhalation, 5 days, CA (OECD TG 474): negative

2,4 -pentanedione, rat, inhalation, 5 days, dominant lethal assay (OECD TG 478): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Figures 1 and 2 and table 1 represent the effect of vanadium pentoxide or saline on the DNA damage index on testicular germ cells, calculated after the measurements by COMET assay. DNA migration increased significantly depending on the dose of vanadium in both large and small cells. The degree of DNA damage in large and small cells was larger in those animals injected with the higher dose of V2O5. The results presented herein show that vanadium treatment induced SSB in DNA of testis cells.
In combination with the reproductive toxicity study (please refer to section 7.8.1), the results presented herein show that vanadium treatment induced SSB in DNA of testis cells, sperm head abnormalities and a low frequency of DLM (dominant lethal mutations). The DLM in males was measured by determining the frequency of live vs. dead and resorbed fetuses occurring after mating chemical-treated males with untreated females.

Table 1: Effects of Vanadium pentoxide on Mouse Testis Cells in the COMET Assay

	Grade of damage in large cells (%)					Grade of damage in small cells (%)
Treatment	None (<5 %)	Low (5-20 %)	Medium (21-40 %)	High (41-95 %)	Total (>95 %)	None (<5 %)	Low (5-20 %)	Medium (21-40 %)	High (41-95 %)	Total (>95 %)
Control	58	22	20	0	0	68	14	14	4	0
5.75***	16	22	16	40	6	4	4	30	56	6
11.50*	14	0	20	56	10	52	30	14	4	0
23.00***	1	0	22	76	0	0	8	26	60	6

*P<0.05 for large cells vs. control

**P<0.05 for small cells vs. control

Conclusions:

The results presented herein show that vanadium treatment induced SSB in DNA of testis cells. It should be noted that gonads contain a mixture of somatic and germ cells. For this reason, positive results in whole gonad (testis) are not necessarily reflective of germ cell damage; nevertheless, they indicate that tested chemical(s) and/or its metabolites have reached the gonad (OECD TG 489). In combination with the reproductive toxicity study (please refer to section 7.8.1), the results presented herein show that vanadium treatment induced SSB in DNA of testis cells, sperm head abnormalities and a low frequency of DLM (dominant lethal mutations). The DLM in males was measured by determining the frequency of live vs. dead and resorbed fetuses occurring after mating chemical-treated males with untreated females. This indicates the vanadium, which is released after hydrolysis of the target substance vanadium-tris-acetylacetonate might be mutagenic.

Executive summary:

Effects of vanadium pentoxide (V2O5) treatment on reproductive function and testicular DNA in male mice were investigated. These functions were evaluated with fertility rate, implants, resorptions, sperm counts, motility, and morphology. The DNA damage in individual testis cells was analyzed by single-cell gel electrophoresis technique (COMET assay). V2O5 treatment resulted in a decrease in fertility rate, implantations, live fetuses, and fetal weight, and an increase in the number of resorptions/dam. Sperm count, motility, and morphology were impaired with the advancement of treatment. Vanadium treatment induced DNA damage depending on the dose in the testis cells that was expressed and detected as DNA migration in the COMET assay. The distribution of DNA migration among cells, a function of dose, revealed that the majority of cells of treated animals expressed more DNA damage than cells from control animals. It is concluded that vanadium pentoxide was a reprotoxic and genotoxic agent in mice. This indicates the vanadium, which is released after hydrolysis of the target substance vanadium-tris-acetylacetonate might be mutagenic.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

COMET assay

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Clinical signs of toxicity in test animals
No lethality or severe toxicity were observed in mice receiving ortho-vanadate in drinking water for 5 weeks, except for one animal administered with the highest concentration of Na3VO4 (1500 mg/L), which died during the 3rd week of treatment. Mild signs of toxicity, viz. lethargy, were observed during the last 2 weeks of treatment in mice treated with 750 and 1500 mg/L Na3VO4

A statistically significant (p < 0.05) increase of tail moment values was observed in splenocytes of mice treated with the highest dose (1500 mg/l); no positive effect was observed at lower doses, nor in bone marrow and testis cells at any tested concentration. Data on vanadium distribution do not explain the greater sensitivity of splenocytes compared to bone marrow observed in this study.

In addition to whole testis cells, also epididymal spermatozoa were isolated from treated animals and tested both in the comet assay and with the sperm chromatin structure assay methodology. While comet assay detects structural DNA damage, the sperm chromatin structure assay measures the susceptibility of DNA to in situ denaturation, which indicates derangements of normal chromatin remodelling and packaging, a hallmark of germ cell damage (Spano and Evenson, 1993). The combination of these two complementary techniques allows a thorough assessment of genotoxicity in sperm cells. The results obtained do not show significant, treatment-related alterations of the parameters investigated, indicating that oral exposure to vanadium pentavalcnt does not result in genotoxic damage in mature spermatozoa.

Flow cytometry

The relative frequencies of the testicular cell types were obtained by DNA flow cytometric analysis. Data show that exposure to Na3VO4 by the oral route did not induce significant changes in the testis cell DNA content distributions, at any tested concentration. These data, together with testis weight values, suggest that oral administration of vanadate at the doses tested does not induce cytotoxic effects on testicular cells.

Conclusions:

The results of this study indicate that penlavalent vanadium is able to exert a significant genotoxic effect in somatic cells in vivo, as shown by the DNA damage detectable by comet assay in splenocytes. However, such effects were unambiguously detectable only in mice receiving high concentrations of pentavalent vanadium in drinking water, corresponding to exposure values in the order of tens of mg/kg body weight. This indicates the vanadium, which is released after hydrolysis of the target substance vanadium-tris-acetylacetonate might be nutagenic.

Executive summary:

In this study, the genotoxic effects produced by the oral intake of sodium ortho-vanadate (Na3VO4) were investigated. Male CD-1 mice were treated for 5 weeks with a range of concentrations of Na3VO4 in drinking water (7 .5-1500 mg/L). Primary DNA lesions as detected by comet assay were assessed in several tissues. A significant increase of comet tail length was observed in splenocytes of mice receiving Na3 VO4 at 1500 mg/L, whereas no effect was observed in bone marrow and testis cells. No treatment-related effect on sperm chromatin structure or on testis cell population was observed. Overall, the results obtained indicate that the genotoxic activity of pentavalent vanadium is expressed in vivo only following high dose exposure, possibly as a consequence of the poor bioavailability of the element. This indicates the vanadium, which is released after hydrolysis of the target substance vanadium-tris-acetylacetonate might be nutagenic.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Clinical signs of toxicity in test animals
No lethality or severe toxicity were observed in mice receiving ortho-vanadate in drinking water for 5 weeks, except for one animal administered with the highest concentration of Na3VO4 (1500 mg/L), which died during the 3rd week of treatment. Mild signs of toxicity, viz. lethargy, were observed during the last 2 weeks of treatment in mice treated with 750 and 1500 mg/L Na3VO4

bone marrow cells
The results of the micronucleus test in mouse bone marrow are summarized in Table 1. In the first experiment, mice received Na3VO4 in drinking water at 7.5, 75, 750 and 1500 mg/L. At the end of the 5-week treatment period, no deviation in the PCE percentual, indicative of bone marrow toxicity, was observed. The incidence of micronucleated PCEs was significantly (p < 0.01) increased in mice treated with the two highest doses. An increase in micronucleated PCEs with lower statistical significance (p < 0.05) was also observed in mice treated with the lowest dose (7.5 mg/L), but not so in mice receiving a 10-fold higher Na3VO4 concentration in drinking water (75 mg/L). In order to clarify the consistency of the experimental findings, a second experiment was carried out with a lower dose range (0.75, 7.5 and 75 mg/L). In the repeated experiment, no statistically significant deviation in the average incidence of micronucleated PCEs was observed, comparing treated and control animals. A clearcut positive response was produced by the positive control MMS, with no evidence of toxicity to bone marrow cells.

blood reticulocytes
The incidence of micronuclei in blood RETs was evaluated in all mice at the beginning of treatment (t0), and 1, 3 and 5 weeks thereafter. The time trend in each experimental group is shown in Fig. 1. An increased frequency of micronucleated reticulocytes was observed in some treated groups at weeks 3 and 5 compared to t0. The difference with to attained statistical significance (p < 0.05, Student's t-test) at the 3rd week for the 75 and 750 mg/L groups, and at the 5th week for 0.75, 7.5 and 750 mg/L groups. However, the interpretation of the biological significance of these findings is uncertain, in view of the lack of relationship with dose, and the uniformly negative results given by the analysis of RETs of animals treated with the highest dose of Na3VO4 (1500 mg/L). On the other hand, a highly significant (p < 0.001) increase in micronucleated RETs was induced by the positive control MMS given by i.p. 24 h before sacrifice (3.5 versus 21.7%c in control and treated mice, respectively). In all cases, no evidence of toxicity to blood cells was provided by the discrimination of younger and older reticulocytes on the basis of their RNA content.

Table 1: Results of micronucleus test in mouse bone marrow after 5 weeks oral exposure to pentavalent vanadium

 

Treatment Na3VO4(mg/L)	Micronucleated PCEs (MnPCEs)		% PCE
	‰Frequency	Total MnPCEs/total PCEs analyzed (MnPCEs/4000 PCEs. indmdual data)
0	3.5 (1.5)^b	139/40000) (11, 14, 12, 13, 27, 13, 16, 12,14, 7)	54.0 (5.9)^b
7.5	5.1 (1.7)^b^,*	102/20000(13, 24, 28, 21, 16)	50.0 (3.9)^b
75	3.1 (1.3)	61/20000(11, 9, 8, 17, 16)	56.2 (3.3)^b
750	6.6 (0.9)^b,^**	133/20000(19, 39, 22, 25, 28)	55.0 (4.2)^b
1500	6.6 (1.3)^b^,**	105/16000(27, 29, 25, 24)	56.1 (3.7)^b
Second experiment
0	4.3 (0.9)^b	87/20000(19, 13, 20, 21, 14)	53.5 (5.3)^b
0.75	4.7 (2.4)^b	94/20000(19, 20, 33, 6, 16)	52.5 (5.8)^b
7.5	5.3 (2.9)^b	106/20000(13, 13, 36, 13, 31)	48.5 (4.2)^b
75	4.6 (0.9)^b	91/20000(19, 26, 11, 18, 17)	52.4 (2.9)^b
Positive control^a	69.8 (35.5)^b^,***	698/10000(122, 72, 91, 162, 251)	52.6(12.8)^b

^aMMS 80 mg/kg b.w.

^bData are mean (S.D.).

^*p < 0.05 (one-tailed Student's /-test).

^**p < 0.01 (one-tailed Student's /-test).

^***p < 0.001 (one-tailed Student's/-test)

Conclusions:

The results of this study indicate that pentavalent vanadium is able to exert a significant genotoxic effect in somatic cells in vivo, as shown by the induction of micronuclei in bone marrow. However, such effects were unambiguously detectable only in mice receiving high concentrations of pentavalent vanadium in drinking water, corresponding to exposure values in the order of tens of mg/kg body weight. This indicates the vanadium, which is released after hydrolysis of the target substance vanadium-tris-acetylacetonate might be mutagenic.

Executive summary:

Male CD-I mice were treated for 5 weeks with a range of concentrations of Na3VO4 in drinking water (0.75 - 1500mg/L). Both micronuclei were assessed in several tissues. Statistically significant increases of micronuclei in bone marrow were observed in mice receiving the two highest concentrations of Na3VO4 (750 and 1500 mg/L). However, such effects were unambiguously detectable only in mice receiving high concentrations of pentavalent vanadium in drinking water, corresponding to exposure values in the order of tens of mg/kg body weight. Overall, the results obtained indicate that the genotoxic activity of pentavalent vanadium is expressed in vivo only following high dose exposure, possibly as a consequence of the poor bioavailability of the element. This indicates the vanadium, which is released after hydrolysis of the target substance vanadium-tris-acetylacetonate might be mutagenic.

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

rats: Significant body weight losses were observed among the males exposed at 800 ppm and both males and females exposed at 600 ppm. Both males and females in the 400 ppm group had depressed body weight gains over the 5-day exposure period

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: rats

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: mice

Additional information on results:

rats
2,4-PD did not produce significant exposure-related increases in the incidence of chromosomal aberrations in the bone marrow of Sprague Dawley® rats exposed, whole body, to 2,4-PD vapor 6 hr/day for 5 consecutive days at target concentrations of 0, 100, 400, and 800 ppm.
The mean percentages of aberrant cells were 1.6 ± 2.6, 5.2 ± 4.6, 1.2 ± 1.8, 2.4 ± 3.3, and 4.4 ± 2.6 for the air-exposed, 100, 400, 600, and 800 ppm males; and 2.4 ± 1.7, 1.6 ± 1.7, 2.0 ± 1.4, and 3.0 ± 3.3 for the air-exposed, 100, 400, and 600 ppm females sampled 6 hr after the 5th exposure. At the 24-hr sampling time, the mean percentages of aberrant cells were 7.6 ± 12.8, 4.0 ± 3.7, 2.0 ± 2.0, 0.4 ± 0.9, 1.2 ± 1.8, and 19.2 ± 3.0 for the air-exposed, 100, 400, 600, 800, and positive control males; and 2.4 ± 2.6, 2.4 ± 0.9, 1.1 ± 1.6, 1.6 ± 0.9, and 28.0 ± 7.9 for the air-exposed, 100, 400, 600, and positive control females.

mice
2,4-PD did not produce significant exposure-related increases in the incidence of chromosomal aberrations in the bone marrow of Swiss Webster mice exposed, whole body, to 2,4-PD vapor 6 hr/day for 5 consecutive days at target concentrations of 0, 100, 400, and 600 ppm. The mean percentages of aberrant cells were 3.6 ± 2.6, 1.6 ± 0.9, 2.0 ± 2.8, and 1.4 ± 2.2 for the air-exposed, 100, 400, 600, and 800 ppm males; and 0.8 ± 1.1, 0, 0.8 ± 1.1, and 0 for the air-exposed, 100, 400, and 600 ppm females sampled 6 hr after the 5th exposure. At the 24-hr sampling time, the mean percentages of aberrant cells were 1.2 ± 1.1, 1.6 ± 1.7, 0.4 ± 0.9, 1.1 ± 1.6, and 28.4 ± 13.0 for the air-exposed, 100, 400, 600, 800, and positive control males; and 2.0 ± 1.4, 0.4 ± 0.9, 1.2 ± 1.8, 1.0 ± 1.4, and 22.8 ± 10.4 for the air-exposed, 100, 400, 600, and positive control females.

Table 12 Effects of 2,4-PD on chromosomal aberration frequencies in the bone marrow of Sprague Dawley® rats (whole body vapour exposure)

2,4-PD (ppm)	Sex	# Cells evaluated	Total* aberrantcells	Mean % aberrant cells ±S.D.)
6-Hr sample
0	M	250	4	1.6±2.6
	F	250	6	2.4±1.7
100	M	250	13	5.2±4.6^c
	F	250	4	1.6±1.7
400	M	250	3	1.2±1.8
	F	250	5	2.0±1.4
600	M	250	6	2.4±3.3
	F	232	7	3.0±3.3
800^a	M	250	11	4.4±2.6
24-Hr sample
0	M	250	19	7.6±12.8
	F	250	6	2.4±2.6
100	M	250	10	4.0±3.7
	F	250	6	2.4±0.9
400	M	250	5	2.0±2.0
	F	228	2	1.1±1.6
600	M	250	1	0.4±0.9
	F	250	4	1.6±0.9
800^a	M	250	3	1.2±1.8
CP^b(30mg/kg)	M	250	48	19.2±3.0^d
	F	250	70	28.0± 7.9^d

^aTarget concentration was lowered to 650 ppm after 2nd exposure day due to unexpected mortalities.

^bCP = cyclophosphamide monohydrate.

^cSignificant at p < 0.05.

^dSignificant at p < 0.01.

Table 13 Effects of 2,4-PD on chromosomal aberration frequencies in the bone marrow of SwissWebster mice (whole body vapour exposure)

2,4-PD (ppm)	Sex	# Cells evaluated	Total # aberrant cells	Mean % aberrant cells (±S.D.)
6-Hr sample
0	M	250	9	3.6±2.6
	F	250	2	0.8±1.1
100	M	250	4	1.6±0.9
	F	250	0	0
400	M	250	5	2.0 ±2.8
	F	250	2	0.8±1.1
600	M	350	5	1.4±2.2
	F	100	0	0
24-Hr sample
0	M	250	3	1.2±1.1
	F	250	5	2.0±1.4
100	M	250	4	1.6±1.7
	F	250	1	0.4±0.9
400	M	250	1	0.4±0.9
	F	250	3	1.2±1.8
600	M	350	4	1.1 ±1.6
	F	100	1	1.0±1.4
CP^a(40mg/kg)	M	214	53	28.4± 13.0^b
	F	250	57	22.8± 10.4^b

^aCP=cyclophosphamidemonohydrate.

^bSignificant at p < 0.01.

Conclusions:

2,4-pentanedione was not clastogenic in rat and mouse bone marrow cells in chromosome aberration assay after inhalation exposure for 5 days at 6h/day

Executive summary:

The study was similar to OECD Guideline 475. When rats and mice were exposed to 2,4-PD vapour for 6 h/day for 5 consecutive days at target concentrations up to 800 ppm, there were no significant exposure-related increases in the incidences of chromosomal aberrations in bone marrow samples taken 6 and 24 h after the 5th day of exposure in either species.

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

please refer to 'Any other information on results'

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No significant differences i n the proportion of PCE were observed in any exposure group. The mean PCE/1000 erythrocytes ranged from a low of 418.4 for the 400 ppm male mice to a high of 612.4 for the 400 ppm female mice. The mean PCE/1000 erythrocytes ranged from a low of 258.6 for the air-exposed male rats to a high of 420.4 for the 100 ppm female rats. The mean percentages of MNPCE in mice were 0.34 ± 0.20, 0.28 ± 0.18, 0.20 ± 0.10, 0.20 ± 0.16, and 2.66 ± 0.54 for males and 0.14 ± 0.11, 0.10 ± 0, 0.22 ± 0.08, 0.20 ± 0.14, and 3.18 ± 1.50 for females for the air-exposed, low, mid, high, and positive control groups. The mean percentages of MNPCE in rats were 0.30 ± 0.16, 0.38 ± 0.13, 0.24 ± 0.25, 0.46 ± 0.15, and 3.92 ± 1.50 for males and 0.34 ± 0.29, 0.24 ± 0.23,0.11 ± 0.07,0.20 ± 0.07, and 2.86 ± 1.20 for females for the air-exposed, low, mid, high, and positive control groups. 2,4-PD did not produce significant exposurerelated increases in the incidence of MNPCE in bone marrow samples taken 24 hr after the 5th day of exposure in either species.

Toxicity

mice

There were no noteworthy signs of toxicity at 100 or 400 ppm in either male or female mice. Of 5 female mice in the 600 ppm exposure group, 3 died on exposure days 3 or 4. Prior to death, hypoactivity, prostration gasping, slow respiration, and blepharospasm were observed in one or more of the mice. There were no significant body weight changes in mice during the study.

rats

There were no noteworthy clinical signs of toxicity in male or female rats exposed to 100 or 400 ppm 2,4-PD. Of 5 female rats in the 600 ppm group, 3 died on exposure days 2—4. Significant body weight losses were observed in male and female rats at 600 ppm. Females also had significant body weight losses at 400 ppm, whereas males had significantly lowered body weight gains.

Table 11 Effects of 2,4-PD on micronucleus frequencies in the bone marrow of Swiss Webster mice and Sprague Dawley® rats (whole body vapour exposure)

2,4-PD (ppm)	Sex	Mean PCE/1000 erythrocytes	#PCE evaluated	#MNPCE	Mean %MNPCE(±S.D.)
24-Hr sample—Mice
0	M	486.0±150.08	5,000	17	0.34±0.20
	F	559.8±54.22	5,000	7	0.14±0.11
100	M	518.6±110.42	5,000	14	0.28±0.18
	F	624.0 ±66.19	5,000	5	0.10±0
400	M	418.4±91.25	5,000	10	0.20±0.10
	F	612.4±63.28	5,000	11	0.22±0.08
600	M	433.0±106.56	5,000	10	0.20±0.16
	F	543.0±111.72	2,000	4	0.20±0.14
TEM^a(0.3mg/kg)	M	283.4±73.94	5,000	133	2.66± 0.54^b
	F	456.0±69.59	5,000	159	3.18±1.50^b
24-Hr sample—Rats
0	M	258.6±2.17	5,000	15	0.30±0.16
	F	319.4±69.31	5,000	17	0.34±0.29
100	M	379.4±99.60	5,000	19	0.38±0.13
	F	420.4±104.06	5,000	12	0.24±0.23
400	M	381.2±52.69	5,000	12	0.24±0.25
	F	248.8±118.07	4,536	5	0.11 ±0.07
600	M	419.6±95.66	5,000	23	0.46±0.15
	F	308.0±74.95	2,000	4	0.20±0
TEM^a(0.3mg/kg)	M	307.0 ±86.33	5,000	196	3.92± 1.50^b
	F	335.0±65.80	5,000	143	2.86± 1.20^b

^b significant at p < 0.01

^aTEM-triethylenemelamine

Conclusions:

2,4-pentanedione was not clastogenic in rat and mouse bone marrow cells in micronucleus assay after inhalation exposure for 5 days at 6h/day.

Executive summary:

The study was similar to OECD Guideline 474. When rats and mice were exposed to 2,4-PD vapour for 6 h/day for 5 consecutive days at target concentrations up to 600 ppm, there were no significant exposure-related increases in the incidences of micronucleated PCE in bone marrow samples taken 24 h after the 5th day of exposure in either species.

Reference 6

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

aneuloidy in germ cells

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

micronucleated polychromatic erythrocytes

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

The control mice and all animals treated with orthovanadate showed gradual increases in body weights during the experiment. The average body weights at the start of the experiment were 26.12, 27.28. 29.62 and 28.24 g. respectively, for the control group and the groups treated with 5, 15 or 25 mg/kg bw orthovanadate. After 22 days, the average weight of the control animals was 27.24 g as compared to the treated groups with 30.36, 30.64 and 29.18g, respectively.

sperm count and tested weights

Orthovanadate at doses of 5 to 25 mg/kg bw did not significantly affect sperm counts or testes weights.

Aneuploidy

A total of 200,416 epididymal sperm were evaluated from 20 animals. Sex ratios were found to be in the same range as the theoretically expected ratio of 1:1 for X-bearing versus Y-bearing sperm. Treatment of male mice with 15 or 20 mg/kg bw of orthovanadate induced hyperhaploid sperm. A significant increase in the frequency of hyperhaploid sperm by a factor of 1.6 was caused by treatment with the two higher doses of orthovanadate, i.e. 0.092 and 0.096 %, respectively, compared with the corresponding control value of 0.058 % (p < 0.01, x2 test). The frequencies of hyperhaploid sperm showed a linear dose-response between 0 and 25 mg/kg bw of orthovanadate, which can be described by the equation y = 0.06 + 0.002 D (r = 0.96). Of the individual classes of aneuploid sperm, only the frequency of sperm with two X-chromosomes was significantly higher in the highest dose group than in the concurrent control (p < 0.01, Mann-Whitney U-test). It is noteworthy that among the sex-chromosome aneuploidies, XX8- and YY8-bearing sperm were significantly more frequent than XY8-bearing sperm (p < 0.05) in the controls as well as in all treatment groups. This observation indicated that mal-segregation occurred preferentially during the second meiotic division by non-disjunction of sister chromatids. Orthovanadate did not significantly increase the frequencies of diploid sperm at any of the doses tested.

Micronucleus assay in somatic cells

Orthovanadate treatment at the doses tested caused no significant increases in the frequencies of micronucleated polychromatic erythrocytes (MNPCE) compared to the solvent control. Thus, the data did not indicate an induction of MN in bone marrow erythrocytes of the male hybrid mice used at the given treatment regimen. The ratio of PCE to total erythrocytes did not indicate cytotoxic effects in any of the treated groups. Since no induction of MNPCE in bone marrow erythrocytes was found with orthovanadate, the distinction between the clastogenic and aneugenic effects by identifying the origin of MN with centromeric DNA probes was omitted.

BrdU-incorporation assay to assess meiotic delay

An average of 10.000 BrdU-positive and BrdU-negative sperm per animal and a total of 374.724 sperm were scored by LSC from 40 animals. Treatment of male mice with 25 mg/kg bw orthovanadate did not cause a meiotic delay.

Conclusions:

Single i.p. administration of 5, 15 or 25 mg/kg bw of orthovanadate did not significantly alter the body weight gains, and testes weights. Moreover, no significant reduction in the sperm counts was observed. In the present experiments, significant increases above the concurrent controls in the frequencies of hyperhaploid sperm were found after treatment with 15 and 25 mg/kg bw of orthovanadate. The induction of hyperploidies followed a linear dose-response between 5 and 25 mg/kg bw of orthovanadate. On the other hand, no significant increases in the frequencies of micronucleated polychromatic erythrocytes (MNPCE) compared to the solvent control were observed. Since the results show that orthovanadate induced non-disjunction during mate meiosis without an accompanying induction of MN in bone marrow erythrocytes under the present experimental conditions and doses, it is concluded that male germ cells (meiosis) are more sensitive to the aneugenic effects of orthovanadate than somatic cells (mitosis).

Executive summary:

The objective of the current study was to investigate the ability of orthovanadate to induce aneuploidy in mouse sperm and micronuclei in mouse bone marrow cells at the same dose levels. The BrdU-incorporation assay was performed to test if the chemical treatment altered the duration of the meiotic divisions. It was found that orthovanadate (25 mg/kg bw) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated by intraperitoneal (i.p.) injection with 5, 15 or 25 mg/kg bw orthovanadate and sperm were sampled from the Caudae epididymes 22 days later. Fluorescence in situ hybridization (FISH) was performed with DNA-probes for chromosomes 8. X or Y. Significant increases in the frequencies of total hyperhaploid sperm (p < 0.01) were found with 15 and 25 mg/kg bw orthovanadate, indicating induced nondisjunction during male meiosis. The dose-response was described best by a linear equation. Orthovanadate did not significantly increase the frequencies of diploid sperm at any of the three doses tested, indicating that no complete meiotic anest occurred. Orthovanadate was investigated also by the micronucleus test at i.p. doses of 1, 5, 15 or 25 mg/kg bw, followed by bone marrow sampling 24 h after treatment. None of the orthovanadate doses caused a significant increase in the rates of micronuclei (MN). Since the results show that orthovanadate induced non-disjunction during male meiosis without an accompanying induction of MN in bone marrow erythrocytes under the present experimental conditions and doses, it is concluded that male germ cells (meiosis) are more sensitive to the aneugenic effects of orthovanadate than somatic cells (mitosis).

Reference 7

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

please refer to Read-across statement attached in section 13

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

ambiguous

Toxicity:

no effects

Remarks:

please refer to remarks

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other:

Remarks:

412 and 694 ppm groups showed a transient reduction in body weight and body weight gain. This may have represented a toxic effect, but may also have been a result of the stress from exposure to high concentrations of a pungent odiferous material. The only other clinical finding was corneal opacification and ulceration. However, there was no clear relationship with vapor concentration.

Conclusions:

Although not statistically significant, the effects at weeks 2,3, and 4 are clearly dose-related, and are considered to be biologically significant. The time period for these reproductive and gestational effects corresponds to utilization of sperm which were at the spermatid stage of spermatogenesis over the 2,4-PD vapor exposure period. Thus, the results are compatible with a transient weak dominant lethal effect at the spermatid stage of spermatogenesis, and accord with the in vitro increases in sister chromatid exchanges and clastogenic effects in CHO cells. The "no-observable effect level" for all effects was 99 ppm. The results, although not statistically significant, are dose-related and compatible with a transient slight dominant lethal effect at the spermatid stage of spermatogenesis.

Executive summary:

Male Fischer 344 rats, 20 per group, were exposed to 2,4-PD vapour concentrations (mean ± SD) of 0, 99.1 ± 2.2, 412 ± 12.6 and 694 ± 9.1 ppm, for 6 hr/day for 5 consecutive days, smiliar to OECD Guideline 478. The day following the final exposure they were bred to unexposed female Fischer 344 rats, 2 per week for 8 consecutive weeks. Weight loss occurred with males during 2,4-PD exposure for the 412 and 694 ppm groups, with compensatory increased weight at 694 ppm, for the first two weeks postexposure. No histopathological change was seen in brain, testes or thymus from high concentration males sacrificed after eight weeks of mating. Minor transient reproductive and gestational effects were present at 412 and 694 ppm. At week 2 there was a reduction, not statistically significant, in the number of corpora lutea and total and viable implants per dam at 694 ppm, and a slight increase in preimplantation loss. At week 3 the number of pregnant females was slightly reduced at 412 and 694 ppm, causing a lowered female fertility index. At week 4 there was a slight reduction in the number of total and viable implants per litter and a significant preimplantation loss at 694 ppm. The dominant lethal factor (FL%) was increased slightly at 694 ppm for weeks 2 and 4. Thus, the "no observable effect" level for dominant lethal effects was 99 ppm. The results, although not statistically significant, are dose-related and compatible with a transient slight dominant lethal effect at the spermatid stage of spermatogenesis.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

2,4 -pentanedione

2,4 -pentanedione was negative in gene mutation assay in bacteria and mammalian cells. However, it has clastogenic potential in vitro, as was shown in a Sister Chromatid Exchange assay (OECD 479) in CHO cells. Significant increases in the frequencies of SCE were observed both in the absence and in the presence of S9 activation (Vergnes 2002).

In an in vivo micronucleus assay 2,4 -pentanedione was also shown to be clastogenic to erythropoetic cells in Swiss Webster mice, when dosed by the IP route. However, when dosed by inhalation, there were no significant exposure-related increases in the incidences of chromosomal aberrations or micronucleated PCE in bone marrow samples taken 24 hr after the 5th day of exposure in rats and mice (Vergnes 2002).

In a dominant lethal assay male Fischer 344 rats, 20 per group, were exposed to 2,4-PD vapour concentrations (mean ± SD) of 0, 99.1 ± 2.2, 412 ± 12.6 and 694 ± 9.1 ppm, for 6 hr/day for 5 consecutive days. The day following the final exposure they were bred to unexposed female Fischer 344 rats, 2 per week for 8 consecutive weeks. Minor transient reproductive and gestational effects were present at 412 and 694 ppm. At week 2 there was a reduction, not statistically significant, in the number of corpora lutea and total and viable implants per dam at 694 ppm, and a slight increase in preimplantation loss. At week 3 the number of pregnant females was slightly reduced at 412 and 694 ppm, causing a lowered female fertility index. At week 4 there was a slight reduction in the number of total and viable implants per litter and a significant preimplantation loss at 694 ppm. The dominant lethal factor (FL%) was increased slightly at 694 ppm for weeks 2 and 4. The results, although not statistically significant, are dose-related and compatible with a transient slight dominant lethal effect at the spermatid stage of spermatogenesis (Tyl, 1989). In MAK (2007), on the other hand, it is stated that the statistical significant occurence of preimplanatation, but not postimplanatation losses indicates that unfertilized ova are the reason for these effects. This suggests toxic effects on sperm cells (e.g. reduction of number, inability to penetrate the ovum). Effective germ cell mutagenes, on the other hand, lead to the increase of postimplantation losses. Therefore, this study shows toxic effects, but not mutagenic effects on germ cells (MAK 2007).

Conclusion

All in all, 2,4 -pentanedione shows genotoxic potential in vitro. This is also indicated by a positive outcome of an in vivo micronucleus assay in mice after i.p administration. However, this potential was not observed after inhalative exposure. The dominant lethal assay shows toxic effects in germ cells, but no mutagenic effects. Based on the described study results, no classification of 2,4 -pentanedione as germ cell mutagen or cancerogen is deviated in the MAK report (2007).

Vanadium compounds

in vitro

Vanadium pentoxide was negative in gene mutation assay in bacteria and mammalian cells. Also vanadyl sulfate and ammonium orthovanadate were negative in gene mutation assays in mammalian cells. However, vanadium pentoxide has clastogenic potential in vitro, as was shown in a micronucleus assay (OECD 487) in CHO cells. A clear concentration-related and statistically significant increase in MN frequencies was observed in the concentration range from 1 to 3 µg/ml in the absence of S9 activation (Zhong 1994)

in vivo - effects on somatic and germ cells

Male CD-1 mice were treated for 5 weeks with a range of concentrations of Na₃VO₄ in drinking water (0.75 - 1500 mg/L). Both micronuclei were assessed in several tissues. Statistically significant increases of micronuclei in bone marrow were observed in mice receiving the two highest concentrations of Na₃VO₄ (750 and 1500 mg/L). However, such effects were unambiguously detectable only in mice receiving high concentrations of pentavalent vanadium in drinking water, corresponding to exposure values in the order of tens of mg/kg body weight. In the same study primary DNA lesions as detected by comet assay were assessed in several tissues. A significant increase of comet tail length was observed in splenocytes of mice receiving Na₃VO₄ at 1500 mg/L, whereas no effect was observed in bone marrow and testis cells. No treatment-related effect on sperm chromatin structure or on testis cell population was observed. Overall, the results obtained indicate that the genotoxic activity of pentavalent vanadium is expressed in vivo only following high dose exposure, possibly as a consequence of the poor bioavailability of the element (Leopardi, 2005).

Effects of vanadium pentoxide (V₂O₅) treatment on reproductive function and testicular DNA in male mice were investigated. These functions were evaluated with fertility rate, implants, resorptions, sperm counts, motility, and morphology. The DNA damage in individual testis cells was analyzed by single-cell gel electrophoresis technique (COMET assay). V₂O₅ treatment resulted in a decrease in fertility rate, implantations, live fetuses, and fetal weight, and an increase in the number of resorptions/dam. Sperm count, motility, and morphology were impaired with the advancement of treatment. Vanadium treatment induced DNA damage depending on the dose in the testis cells that was expressed and detected as DNA migration in the COMET assay. The distribution of DNA migration among cells, a function of dose, revealed that the majority of cells of treated animals expressed more DNA damage than cells from control animals. It is concluded that vanadium pentoxide was a reprotoxic and genotoxic agent in mice (Altamirano-Lozano, 1996).

Although this comet assay was carried out with connective tissue, Sertoli cells, all germ cell stages, blood vessels, etc. due to mincing of the entire testes, the study is significant, as obviously enough vanadium entered the testes to produce positive findings in this assay (MAK 2009).

The ability of Na₃VO₄ to induce aneuploidy in mouse sperm and micronuclei in mouse bone marrow cells at the same dose levels was studied. The BrdU-incorporation assay was performed to test if the chemical treatment altered the duration of the meiotic divisions. It was found that orthovanadate (25 mg/kg bw) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated by intraperitoneal (i.p.) injection with 5, 15 or 25 mg/kg bw orthovanadate and sperm were sampled from the Caudae epididymes 22 days later. Fluorescence in situ hybridization (FISH) was performed with DNA-probes for chromosomes 8. X or Y. Significant increases in the frequencies of total hyperhaploid sperm (p < 0.01) were found with 15 and 25 mg/kg bw orthovanadate, indicating induced nondisjunction during male meiosis. The dose-response was described best by a linear equation. Orthovanadate did not significantly increase the frequencies of diploid sperm at any of the three doses tested, indicating that no complete meiotic arrest occurred. Orthovanadate was investigated also by the micronucleus test at i.p. doses of 1, 5, 15 or 25 mg/kg bw, followed by bone marrow sampling 24 h after treatment. None of the orthovanadate doses caused a significant increase in the rates of micronuclei (MN). Since the results show that orthovanadate induced non-disjunction during male meiosis without an accompanying induction of MN in bone marrow erythrocytes under the present experimental conditions and doses, it is concluded that male germ cells (meiosis) are more sensitive to the aneugenic effects of orthovanadate than somatic cells (mitosis) (Attia, 2005)

Conclusion

While the results for vanadium compounds in in vitro studies were ambiguous, vanadium shows genotoxic potential in somatic and germ cell in in vivo studies. It was positive in COMET assay in splenocytes (Leopardi et al. 2005) and in testis cells (Altamirano-Lozano et al. 1996). In addition, sodium orthovanadate had aneugenic effects in male germ cells without an accompanying induction of MN in bone marrow erythrocytes. This leads to the conclusion that male germ cells (meiosis) are more sensitive to the aneugenic effects of orthovanadate than somatic cells (mitosis) (Attia et al. 2005). The MAK Commission concluded that vanadium compounds are to be considered genotoxic in vitro and in vivo (clastogenic and aneugenic). The underlying mechanisms of action show that enzyme inhibition is responsible for the aneugenic effect and the formation of radicals for the clastogenic effect (MAK 2009).

Justification for classification or non-classification

As a result of its genotoxicity in vitro and in vivo, the accumulation of vanadium in the testes, the DNA damage caused in the testes and two positive dominant lethal tests in mice and rats, vanadium and its inorganic compounds are classified in germ cell mutagen Category 2 according to MAK (2009). Considering this and based on the available studies, vanadium-tris-acetylacetonate is classified as germ cell mutagen Category 2 ( H341: Suspected of causing genetic defects) in accordance with Regulation (EC) No 1272/2008.