1H-1,2,4-triazole, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is one ames OECD 471 study (Haddouk, 2005) and one  OECD 473 mutation gene study (Haddouk, 2006)  which were negative.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Dec. 2, 2005 to Jul. 27, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 473)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human lymphocytes

Details on mammalian cell type (if applicable):

- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver microsomes

Test concentrations with justification for top dose:

1st experiment - S9 (3 hours): 0, 78.13, 156.3, 312.5, 625, 937.5, 1250, 2500, and 5000 µg/mL
2nd experiment -S9 (20 hours): 0, 312.5, 625, 937.5, 1250, 1875, and 2000 µg/mL
2nd experiment - S9 (44 hours): 0, 312.5, 625, 937.5, 1250, 1875, and 2000 µg/mL
1st experiment + S9 (3 hours): 0, 78.13, 156.3, 312.5, 625, 937.5, 1250, 2500, and 5000 µg/mL
2nd experiment + S9 (3 hours): 0, 312.5, 625, 937.5, 1250, 1875, and 2000 µg/mL
2nd experiment + S9 (3 hours): 0, 312.5, 625, 937.5, 1250, 1875, and 2000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water for injections

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water for injections

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: with S9 mix

Details on test system and experimental conditions:

1st experiment: Lymphocyte cultures were exposed to test article or control for 3 hours both in the absence or presence of S9.
Harvest time was 20 hours after the beginning of treatment.

2nd experiement: Without S9, cells were exposed continuously to the test or control item until harvest. With S9, cells were exposed to the test or control item for 3 hours then rinsed. Harvest time were 20 hours and 44 hours after the beginning of testing.

NUMBER OF REPLICATIONS: 2 independent experiments.

NUMBER OF CELLS EVALUATED: 200 metaphases/dose level.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:

The cytotoxicity of the test item was evaluated using the mitotic index. Analysis of 200 metaphases/dose level was made. The following structural aberrations were recorded for each metaphase: gaps, chromatid and chromosome breaks and exchanges, multiple aberrations and pulverizations.

Statistics:

Chi-square test.

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

decrease of mitotic index

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

-Experiments without S9 :
Cytotoxicity : Following the 3-hour treatment, a slight to strong toxicity was induced at dose-levels > or = 2500 µg/ml, as shown by 33-100% decrease in mitotic index. Following the 20-hour treatment, a slight to moderate toxicity was induced at dose-levels > or = 1875 µg/ml, as shown by 23-45% decrease in mitotic index. Following the 44-hour treatment, no decrease in mitotic index was noted.
Metaphase analysis : The dose-levels selected for metaphase analysis were : 312.3 , 625 and 1250 µg/ml for the 3-hour and 20-hour treatments, higher dose-levels inducing a significant increase in pH value; 1250 µg/ml for the 44-hour treatment, higher dose-levels inducing a significant increase in pH value. No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3-, 20- as well as 44-hour treatments.

-Experiment with S9 :
Cytotoxicity : At the 20-hour harvest time, a slight to strong toxicity was induced at dose-levels > 2000 µg/ml, as shown by 32-100% decrease in mitotic index. At the 44-hour harvest time, no decrease in mitotic index.
Metaphase analysis : the dose-levels selected for metaphase analysis were 312.5 , 625 and 1250 µg/ml for the 20-hour harvest time in both experiments, higher dose-levels inducing a significant increase in pH value; 1250 µg/ml for the 44-hour harvest time higher dose-levels inducing a significant increase in pH value.
No significant increase in the frequency of cels with structural chromosomal aberrations was noted in either experiment and at either harvest time.
The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. the study was therefore considered valid.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

The test item was freely soluble in the vehicle (water for injections) at 100 mg/mL.

No precipitate was noted at the end of the treatement period, at any tested dose.

Due to the increase in pH observed in the treated dose-levels, pH measurements were peformed int reated medium at a time equivalent to the end of the treatment period. At 5000 µg/mL (the highest dose), the pH was approximately 9.7 (7.6 for the vehicle control) and the osmolarity equal to 390 mOsm/kg H2O (292 for the vehicle control).

Conclusions:

Interpretation of results (migrated information):
negative

1,2,4-triazole did not induce chromosome aberrations in cultured human lymphocytes.The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. the study was therefore considered valid.

Executive summary:

The study was performed according to the international guidelines (OECD 473) and in compliance with the principles of GLP regulations. 1,2,4 -triazole sodium salt was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254.

The highest dose-level for treatment in the first experiment was selected in the basis of pH, osmolarity and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index in the first experiment was also taken into account. In the first experiment, lymphocyte cultures were exposed to the test or control item (with or without S9 mix) for 3 hours then rinsed. Cekks were harvested 20 hours after the beginning of treatment. The second experiment was performed as follow : without S9 mix, cells were exposed continuously to the test or control item until harvest ; and with S9 mix, cells were exposed to the test or control item for 3 hours and then rinsed.Cells were harvested 20 hours and 44 hours after treatment.

One and a half hours before harvest, each cuture was treated with a colcemid solution to block cells at the metaphase-stage of mitosis. 1,2,4 -triazole sodium salt was dissolved in water for injections. Positive controls were used.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest time, without and with S9 mix. The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. the study was therefore considered valid.

1,2,4-triazole sodium salt did not induce chromosome aberrations in cultured human lymphocytes.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 6, 2004 to Dec. 19, 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 471) with acceptable restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

- 2-anthramine was the only positive control compound used to test the efficacy of the S9 fraction

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver microsomes

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500, 3750 and 5000 µg/plate

Vehicle / solvent:

The vehicle was water for injections, batch No. FVCOIA (Frésenius-Kabi, Sèvres, France).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See Table 1

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48-72 hours
- Expression time (cells in growth medium): revertants were scored with an automatic counter
- Fixation time (start of exposure up to fixation or harvest of cells): no data

NUMBER OF REPLICATIONS: 2 independent experiments, 3 plates/dose
NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: no data on used method

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Evaluation criteria:

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

no

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate-to-marked toxicity at > 2500 µg/plate (+/- S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate-to-marked toxicity at > 2500 µg/plate (+/- S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A moderate to marked toxicity was generally observed, both with and without S9 mix, mainly at dose-levels = 2500 µg/plate, depending on the tester strains. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.

Conclusions:

Interpretation of results
negative with and without metabolic activation

Under the experimental conditions, 1,2,4 -triazole sodium salt did not show any mutagenic activity in the bacterial reverse mutation test.

Executive summary:

The study was performed according to the international guidelines (OECD 471, Commission Directive No. B 13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels of 1,2,4-triazole sodium salt to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Five strains of bacteria Salmonella Typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. 1,2,4-triazole sodium saltwas dissolved in water for injections.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

The selected treatment-levels were: 312.5, 625, 1250, 2500, 3750 and 5000 gg/plate, for both mutagenicity experiments with and without S9 mix.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.

A moderate to marked toxicity was generally observed, both with and without S9 mix, mainly at dose-levels 2500 gg/plate, depending on the tester strains.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There is one OECD 474 in vivo micronucleus study (Allen , 1985) which was negative.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Study period:

From Jul. 5, 1985 to Nov. 7, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 474). no GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK.
- Age : no data
- Weight at study initiation: 22 to 24 g.
- Housing: Animals kept in a plastic disposable cage.
- Diet (e.g. ad libitum): Scientific Feeds (Labsure) LAD 1 diet ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ºC
- Humidity : no data
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Route of administration:

oral: gavage

Vehicle:

Aqueous 1% methyl cellulose

Details on exposure:

All animals in all groupes were dosed with the standard volume of 20 ml/kg bw.

Duration of treatment / exposure:

Single dose administration

Frequency of treatment:

Single dose administration

Post exposure period:

Animals were sacrificed 24, 48, and 72 hours after administration.

Dose / conc.:

1 200 mg/kg bw/day (nominal)

Remarks:

Doses / Concentrations:
1200 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

15 animals/sexe in control (vehicle) and test article group.
5 animals/sexe in positive control group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Route of administration: intraperitoneal injection
- Doses / concentrations: 4 mg/kg bw (0.2 mg/mL)

Tissues and cell types examined:

Bone marrow cells.

Details of tissue and slide preparation:

The animals were killed and both femurs were dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone. A direct bone marrow smear was made onto a slide (one smear/femur) and the prepared smears were air-dried, fixed in methanol, and stained.

Evaluation criteria:

The stained smears were examined by light microscopy to determine the incidence of micronucleated cells/1000 polychromatic erythrocytes/animal. The ratio of polychromatic to normochromatic erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes.

Statistics:

Wilcoxon test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A preliminary toxicity test had been carried out to determine the toxicity of 1,2,4-triazole. From the results it was estimated that a dosage of 1200 mg/kg would be expected to kill approximately 10% of the animals within 72 hours of dosing.

Conclusions:

Interpretation of results : negative
No evidence of mutagenic potential or bone marrow cell toxicity was noted in this study.

Executive summary:

In this assessment of the effect of 1,2,4 -triazole on the incidence of micronucleated polychromatic erythrocytes in mice, a dosage of 1200 mg/kg bw was administered by oral gavage. A negative and a positive controls were used. Bone marrow smears were obtained from the negative control and test compound groups at 3 sampling times; these being 24, 48 or 72 hours after dosing.

At all sampling times, mice treated with 1,2,4 -triazole showed no significant increase in the frequency of micronucleated polychromatic erythrocytes. There was no significant decrease in the ratio of polychromatic to normochromatic erythrocytes after treatment of the animals with 1,2,4 -triazole. The positive control produced large highly significant increases in the frequency of micronucleated polychromatic erythrocytes together with decreases in the ratio of polychromatic to normochromatic erythrocytes.

It is concluded from the results obtained that 1,2,4 -triazole shows no evidence of mutagenic potential or bone marrow cell toxicity when administered orally in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Test (in vitro) : Ames test (Haddouk 2005)

The study was performed according to the international guidelines (OECD 471, Commission Directive No. B 13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels of 1,2,4-triazole sodium salt to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Five strains of bacteria Salmonella Typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to six dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. 1,2,4-triazole sodium salt was dissolved in water for injections.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

The selected treatment-levels were: 312.5, 625, 1250, 2500, 3750 and 5000 gg/plate, for both mutagenicity experiments with and without S9 mix.

No precipitate was observed in the Petri plates when scoring the revertants at any dose-level.

A moderate to marked toxicity was generally observed, both with and without S9 mix, mainly at dose-levels 2500 gg/plate, depending on the tester strains.

1,2,4-triazole sodium salt did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Test (in vitro) : Chromosomic aberrations on human lymphocytes (Haddouk 2006)

The study was performed according to the international guidelines (OECD 473) and in compliance with the principles of GLP regulations. 1,2,4 -triazole sodium salt was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254.

The highest dose-level for treatment in the first experiment was selected in the basis of pH, osmolarity and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of mitotic index in the first experiment was also taken into account. In the first experiment, lymphocyte cultures were exposed to the test or control item (with or without S9 mix) for 3 hours then rinsed. Cekks were harvested 20 hours after the beginning of treatment. The second experiment was performed as follow : without S9 mix, cells were exposed continuously to the test or control item until harvest ; and with S9 mix, cells were exposed to the test or control item for 3 hours and then rinsed.Cells were harvested 20 hours and 44 hours after treatment.

One and a half hours before harvest, each cuture was treated with a colcemid solution to block cells at the metaphase-stage of mitosis. 1,2,4 -triazole sodium salt was dissolved in water for injections. Positive controls were used.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in either experiment and at either harvest time, without and with S9 mix. The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. the study was therefore considered valid.

1,2,4-triazole sodium salt did not induce chromosome aberrations in cultured human lymphocytes.

Test (in vivo) : Micronucleus test (Allen 1985) with 1,2,4 -triazole (CAS no.288 -88 -0)

In this assessment of the effect of 1,2,4 -triazole on the incidence of micronucleated polychromatic erythrocytes in mice, a dosage of 1200 mg/kg bw was administered by oral gavage. A negative control group was dosed in an identical manner with the vehicle, aqueous 1% methylcellulose. A positive control group was dosed with mitomycin C by intraperitoneal injection.

Bone marrow smears were obtained from the negative control and test compound groups at 3 sampling times; these being 24, 48 or 72 hours after dosing. Bone marrow smears were obtained from the positive control group 24 hours after dosing. One smear from each animal was examined for the rpesence of micronuclei in 1000 polychromatic erythrocytes.

At all sampling times, mice treated with 1,2,4 -triazole showed no significant increase in the frequency of micronucleated polychromatic erythrocytes. There was no significant decrease in the ratio of polychromatic to normochromatic erythrocytes after treatment of the animals with 1,2,4 -triazole. The positive control produced large highly significant increases in the frequency of micronucleated polychromatic erythrocytes together with decreases in the ratio of polychromatic to normochromatic erythrocytes.

It is concluded from the results obtained that 1,2,4 -triazole shows no evidence of mutagenic potential or bone marrow cell toxicity when administered orally in this in vivo test procedure.

Short description of key information:
Three reliable studies was available for this endpoints : a Ames test, a in vitro chromosomic aberration test, and a in vivo micronucleus test.
All tests show negative results.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to EU Directive 67/584/EEC and EU regulation (EC) No 1272/2008 (CLP) :

1,2,4-triazole sodium salt was not mutagen (negative results in in vitro and in vivo studies).