2,2,6,6-tetramethylpiperidinooxy

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro genotoxicity tests were conducted with the test substance; all three - an Ames test, a micronucleus test and a mouse lymphoma assay were positive with and without metabolic activation (Gallez et al., 1992; Guo et al., 2013). Guo et al., observed that S9 activation increased the cytotoxicity and genotoxicity of Tempo, indicating that metabolites of Tempo are more cytotoxic and genotoxic than Tempo itself. The mode of action is considered to be related to the nitroxyl radical, the formation of reactive oxyen species and the depletion of gluthathione species.

Gallez B, De Meester C, Debuyst R, Dejehet F, Dumont P, 1992. Mutagenicity of nitroxyl compounds: structure-activity relationships. Toxicology Letters 63:35-45.

Guo et al., 2013. Nitroxide TEMPO: A genotoxic and oxidative stress inducer in cultured cells. Toxicology in Vitro 27, 1496–502.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Strain TA100 used only.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Strain TA100 used only.

Principles of method if other than guideline:

The test was carried out according to Maron and Ames (1983, Mutat. Res. 113, 173-215) using Salmonella typhimurium strain TA 100 with and without S9 mix.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.06, 0.6, 1.5, 3.0, 6.0, 15.0, and 30.0 μmol

Vehicle / solvent:

DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-aminoanthracene when S9 mix was used

Details on test system and experimental conditions:

Aliquots of TEMPO (0.1 ml of a dilution in DMSO corresponding to 0.06, 0.6, 1.5, 3.0, 6.0, 15.0, and 30.0 μmol) were added into the top agar (plate incorporation assay), or into the top agar supplemented with S9 mix (500 μl corresponding to 50 μI of S9). The colonies grown on the minimal glucose-agar plates at 37°C were counted after 48h. Each assay was performed in duplicate with three plates for each concentration.

Statistics:

t-test

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

180 to 210 revertants/plate, statistically significant

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 30 µmol/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Remarks:

140 (mean) revertants/plate

Positive controls validity:

valid

Remarks:

380 (mean) revertants/plate

Additional information on results:

A cytotoxic effect was observed at 30 μmol/plate. A weak but statistically significant (p<0.01) increase of the number of revertants (180 to 210 revertants/plate) was observed between 6 and 15 μmol/plate (assay repeated three times). Thus, a cytotoxic effect could mask partly the mutagenic activity at higher concentrations.

Conclusions:

The test compound is positive for gene mutations in bacterial tester strain Salmonella typhimurium TA100.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Principles of method if other than guideline:

The L5178Y/Tk+/- -3.7.2C mouse lymphoma cell line was used for the assay. Cells were grown and pre-existing Tk-/- mutants were cleansed periodically. The basic medium was Fischer’s medium for leukemic cells of mice with L-glutamine supplemented with pluronic F68 (0.1%), sodium pyruvate (1 mM), penicillin (100 units/ml), and streptomycin (100 µg/ml). The treatment medium (F5p), growth medium (F10p), and cloning medium (F20p) were the basic medium supplemented with 5%, 10%, and 20% heat-inactivated horse serum, respectively. The cultures were gassed with 5% (v/v) CO2 in air and were maintained in a shaker incubator at 37°C.

TEMPO working solutions (100x) were prepared just prior to use by dissolving TEMPO in DMSO. 6 x 10exp6 cells in a total volume of 10 ml F5p were exposed to different concentrations of TEMPO in the absence and in the presence of 1% S9 (1 - 3 mM TEMPO without S9 and 1–2 mM TEMPO with S9) for 4 h at 37°C. Positive controls were 0.3 µg/ml benzo[a]pyrene with S9 and 0.1 µg/ml 4-NQO without S9. After treatment, the cells were centrifuged and washed twice with fresh medium, and then were resuspended in F10p at a density of 3 x 10exp5 cells/ml. The culture tubes were gassed with 5% CO2 in air and placed on a roller drum (15 rpm) in a 37°C incubator to begin the standard 2-day phenotypic expression.

For mutant selection the cells were counted and the densities were adjusted daily using fresh F10p following exposure. After 2 days of expression, mutants were enumerated by adding 3 µg/ml of TFT to cells suspended in F20p at a concentration of 1 x 10exp4 cells/ml. The cells then were seeded into four 96-well flat-bottom microtiter plates using 200 µl per well. For the determination of plating efficiency, the cultures were adjusted to 8 cells/ml in F20p without TFT, and aliquoted at 200 µl per well into two 96-well flat-bottom microtiter plates. All plates were incubated at 37°C in a humidified incubator with 5% CO2 in air. After 11 days of incubation, the colonies were counted by visual inspection and the mutant colonies were categorized as small colony (SC) or large colony (LC) mutants. SCs are defined as those occupying <25% the diameter of the well. Mutant frequencies (MFs) were calculated using the Poisson distribution. Cytotoxicity was measured using relative total growth (RTG), which includes measurements of cell growth during the treatment (4 h), expression (2-day), and cloning (11-day) phases of the assay.

GLP compliance:

no

Type of assay:

other: mouse lymphoma test

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

-3.7.2C

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1 - 3 mM TEMPO without S9
1 - 2 mM TEMPO with S9

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Rationale for test conditions:

Based on cytotoxicity in dose range-finding test: RTG about 20% in highest dose.

Evaluation criteria:

After 11 days of incubation, the colonies were counted by visual inspection and the mutant colonies were categorized as small colony (SC) or large colony (LC) mutants. SCs are defined as those occupying <25% the diameter of the well.

Statistics:

Mutant frequencies (MFs) were calculated using the Poisson distribution.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

In the absence of S9 activation, the relative total growth (RTG) produced by 3.0 mM TEMPO was approx. 20%, with the mutant frequencies (MF) (208 x 10exp6) marginally exceeding the global evaluation factor (GEF) of 126 x 10exp6. A final concentration of 1% S9 was used in assays testing the influence of metabolic activation on the cytotoxicity and mutagenicity of TEMPO. In the presence of S9, TEMPO resulted in dose-responsive decreases in RTG and increases in MF, and MFs were positive by GEF for TEMPO doses of 1.25–2 mM. Compared with the treatment conducted without S9, the treatment of 2.0 mM TEMPO with S9 induced significantly higher levels of cytotoxicity and mutagenicity (p < 0.05). MFs with S9 were not determined for doses higher than 2 mM due to the induction of severe cytotoxicity and low plating efficiency.

Conclusions:

As a result, under the conditions of this study TEMPO is mutagenic to mammalian cells with and without metabolic acitivation. However, S9 activation increased the cytotoxicity and genotoxicity of TEMPO, indicating that metabolites of TEMPO are more cytotoxic and genotoxic than TEMPO itself. In a preliminary study with the MLA, mouse lymphoma cells were treated for 24 h in the absence of S9. Both the 4 and 24 h treatments with TEMPO produced similar, low levels of mutagenicty but the 24 h exposure produced a marked increase in cytotoxicity. These results provide further evidence that the mutagenicity of TEMPO mainly is due to metabolic activation by S9 enzymes.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

no

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Remarks:

TK6

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

1.4 - 2.3 mM TEMPO without S9
0.9 - 1.5 mM TEMPO with S9

Dose range-finding studies indicated that 0.9 - 2.3 mM TEMPO produced a useful range of cytotoxicity for conducting the MN assay in TK6 cells.

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

other:

Details on test system and experimental conditions:

The MN analysis was performed using the in vitro MicroFlow™ Kit (Litron Laboratories, Rochester, NY). The samples were analyzed using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA).

Evaluation criteria:

Positive responses were characterized by a concentration-related increase in MN frequency, at least one response in excess of the 95% confidence interval for the historical negative control (0.59 ± 0.40% MN with S9; 0.60 ± 0.36% MN without S9).

Statistics:

Differences between groups were evaluated by the one-way analysis of variance (ANOVA) followed by pairwise comparisons to the vehicle control.

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

In the main study, TEMPO induced dose-dependent increases in MN frequency, with positive increases measured at doses ≥1.4 mM (without S9) and ≥1.0 mM (with S9). The 1.8 mM treatment without S9, which produced an RPD (relative population doubling, presenting cytotoxicity) of 51%, induced a 4.5-fold increase in MN frequency over the vehicle control and a net increase of 1.95% micronuclei. 1.3 mM TEMPO with S9 resulted in a mean RPD of 45% and a 5.4-fold increase in MN frequency over the control with a net increase of 2.63% micronuclei. Based on evaluation criteria of OECD TG487, TEMPO produced positive responses in the TK6 cell MN assay, both with and without S9.

Furthermore, TEMPO treatment increased the frequency of hypodiploid nuclei, with the induction being greater in the presence than in the absence of S9. Applying similar criteria for positive responses recommended by TG487 for MN induction, TEMPO was positive for the induction of hypodiploid nuclei in TK6 cells.

Conclusions:

As a result, under the conditions of this study TEMPO induced increases in the frequence of micronuclei in TK6 human lymphoblastoids in vitro with and without metabolic acitivation. However, S9 activation increased the cytotoxicity and genotoxicity of TEMPO, indicating that metabolites of TEMPO are more cytotoxic and genotoxic than TEMPO itself.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in vivo genotoxcity tests are available conducted with the read-across source 4-Hydroxy-Tempo. Both the micronucleus test in the mouse and the UDS test in the rat were negative indicating that the in vitro mode of genotoxicity is not relevant in vivo, presumably due to the presence of protection systems against oxidative stress.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
4-Hydroxy-2,2,6,6-tetramethylpiperidinoxyl (4-Hydroxy-Tempo) is used as source substance for the read-across analogue approach to fulfill the information requirement on in vivo genotoxicity for 2,2,6,6-tetramethylpiperidinooxy (Tempo).

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target and the source chemical are structurally similar; both have a tetramethylpiperidinyl structure carrying a nitroxyl radical. The mode of action for genotoxicity of nitroxyl radicals on the example of tetramethylpiperidinyloxyls has been investigated and publicly described (see below). Thus, 4-Hydroxy-TEMPO is considered to be a suitable source substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
No additional information is available for target and source substances.

3. ANALOGUE APPROACH JUSTIFICATION
Gallez et al. (1992) describe three nitroxyl radicals to be mutagenic in the Ames test: Tempo, 4-Hydroxy-Tempo and Amino-Tempo. This finding is verified by an additional experimental study for 4-Hydroxy-TEMPO, which is reported in the Evonik REACh dossier for 4-Hydroxy-TEMPO. Since it is known that nitroxyl radicals are reduced to their corresponding hydroxylamines by ascorbic acid, Gallez et al. (1992), tested the influence of ascorbic acid on the mutagenicity of nitroxyl radicals in the Ames test. As a result, by the preincubation of the nitroxyl compounds with ascorbic acid mutagenic activity was suppressed leading to the conclusion that the mutagenicity was produced by the free radical species.
Sies and Mehlhorn (1986) reported that the mutagenicity of the nitroxyl radicals decreased in cells carrying a mutation that leads to the induction of enzymes protecting against oxidative stress. At the example of 4-Hydroxy-Tempo they propose the superoxide radical (via the formation of oxidized thiols e.g. reactive glutathione species) to be the active species for the mutagenic effect of nitroxyl radicals. This is supported by findings of Guo et al. (2013) observed in mouse lymphoma cells treated with Tempo. Analysis of reactive oxygen species (ROS) levels revealed a significant increase compared to vehicle control with a maximum at 2 to 4 hours after treatment depending on dose. A significant dose-dependent decrease in relative gluthathione levels was measured after 4 hours of treatment. Furthermore, an analysis of the loss of heterozygocity (LOH) at the thymidine kinase (Tk) locus in Tk mutants from the mouse lymphoma cell assays showed an increased percentage of LOH at the Tk locus. In summary, these results suggest that nitroxyl radicals are genotoxic in mammalian cells, at least partially through the generation of oxidative stress, resulting in large genetic alterations (Guo et al., 2013).

The ascorbic acid reduction rates of Tempo and 4-Hydroxy-Tempo were found to be in a similar range (91 or 72 µmol/min/mmol ascorbic acid, respectively). Moreover, the number of revertants by plate induced by both substances at 15 µmol/plate (the highest non-toxic concentration) were comparable (210 or 240 revertants/plate, respectively). Both findings indicate similar reactivity and mutagenicity of Tempo and 4-Hydroxy-Tempo (Gallez et al., 1992).

As a conclusion, the underlying mechanism of the genotoxic effects of the nitroxyl radicals has been identified, and 4-Hydroxy-Tempo shows similar characteristics than Tempo so that it appears well suited as source substance for the read-across approach to fulfill the information requirement on in vivo genotoxicity for Tempo.

4. DATA MATRIX
4-Hydroxy-2,2,6,6-tetramethylpiperidinoxyl (4-Hydroxy-Tempo) is used as source substance. The relevant study records are linked in the target record field ‘Cross-reference’.

REFERENCES
Gallez B, De Meester C, Debuyst R, Dejehet F, Dumont P, 1992. Mutagenicity of nitroxyl compounds: structure-activity relationships. Toxicology Letters 63:35-45.
Guo X, Mittelstaedt RA, Guo L, Shaddock JG, Heflich RH, Bigger AH, Moore MM, Mei N, 2013. Nitroxide TEMPO: A genotoxic and oxidative stress inducer in cultured cells. Toxicology in Vitro 27, 1496–502.
Sies H, Mehlhorn R, 1986. Mutagenicity of Nitroxide-Free Radicals. Archives of Biochemistry and Biophysics 251:393-96.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1993

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: young adults
- Body weight at study initiation: 29.9 ±6.0 g
- Housing: conventional, 5 mice/sex in Macrolon cages type III
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3°C
- Humidity:30 - 70 %
- Air changes: 15 per h
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 1995-05-03 To: 1995-05-12 (toxicity test); 1995-05-15 To: 1995-05-22 (main study)

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: physiol. saline
- Amount of vehicle: 10 mL/kg bw

Frequency of treatment:

single exposure

Post exposure period:

24 or 48 hrs

Dose / conc.:

1 200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 100 mg/kg bw in 10 mL/kg bw

Tissues and cell types examined:

femoral bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
MTD (limit dose: 2000 mg/kg bw): highest dosage causing signs of toxicity but no mortality within 48 hrs of test compound administration), determined in pre-test

DETAILS OF SLIDE PREPARATION:
After sacrifice, bone marrow was suspended in FCS and erythrocytes were purified in a cellulose chromatography column. The eluate was centrifuged and the pellet was re-suspended in FCS/EDTA. Two slides were prepared per animal and stained with May-Grünwald Giemsa.

METHOD OF ANALYSIS:
Fully automated scoring: counting micronuclei in a total of appr. 2000 PCE (i.e. 100000 PCE per treatment group). Based on 2000 PCE scored, the PCE/NCE index was calculated.

Evaluation criteria:

A test compound is considered an inducer of micronuclei in PCE, if at least one group of mice treated with the test compound reveals a statistically and biologically relevant increase in micronucleated PCE (as compared to the negative control group of the same sampling time).

Statistics:

Heterogeneity Chi square calculated first. If homogenous: Pearson's contingency 2x2 chi square test (DF = 1), including a Yates correction factor. If inhomogenous: transformation and t-test (one sided, two sample). The latter also for evaluation of PCE/NCE ratios.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 400, 750, 1000, 1250, 1500, 1750 or 2000 mg/kg bw
- Solubility: soluble in vehicle up to limit concentration (2000 mg/kg bw)
- Clinical signs of toxicity in test animals: clinical symptoms (hunched posture, sedation, piloerrection and closed eyes), death at high dosages
- Evidence of cytotoxicity in tissue analyzed: not reported

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No
- Ratio of PCE/NCE: unaltered compared to controls

Conclusions:

The genetic toxicity in vivo of 4-Hydroxy-TEMPO was tested in a GLP compliant OECD 474 guideline study (mouse micronucleus test). As a result, the test item was not genotoxic under the conditions tested.

Executive summary:

In an in vivo mouse micronucleus assay (95-0250-DGM), male and female NMRI mice were orally (gavage) exposed to a single dose of 4 -Hydroxy-TEMPO at a concentration of 1200 mg/kg bw (vehicle: saline solution). The maximum tolerated dose (MTD) was determined prior in a toxicity test. The post exposure period was 24 or 48 hrs. The following main signs of toxicity were observed: hunched posture, sedation, in males: staggering gait, convulsions. One male of the 48 h sampling group and one male of the satellite group died. No statistically or biologically significant increase in micronucleated polychromatic erythrocytes at the sampling times of 24 and 48 h in male and female animals. No alterations of the ratio between polychromatic and normochromatic erythrocytes (PCE/NCE ratio) were detected. There was no indication of a clastogenic effect. The positive control induced the appropriate responses. The study satisfies the requirements of Test Guideline OECD 474 for the evaluation of in vivo mutagenicity (resp. clastogenicity: induction of micronuclei).

Reference 2

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
4-Hydroxy-2,2,6,6-tetramethylpiperidinoxyl (4-Hydroxy-Tempo) is used as source substance for the read-across analogue approach to fulfill the information requirement on in vivo genotoxicity for 2,2,6,6-tetramethylpiperidinooxy (Tempo).

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target and the source chemical are structurally similar; both have a tetramethylpiperidinyl structure carrying a nitroxyl radical. The mode of action for genotoxicity of nitroxyl radicals on the example of tetramethylpiperidinyloxyls has been investigated and publicly described (see below). Thus, 4-Hydroxy-TEMPO is considered to be a suitable source substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
No additional information is available for target and source substances.

3. ANALOGUE APPROACH JUSTIFICATION
Gallez et al. (1992) describe three nitroxyl radicals to be mutagenic in the Ames test: Tempo, 4-Hydroxy-Tempo and Amino-Tempo. This finding is verified by an additional experimental study for 4-Hydroxy-TEMPO, which is reported in the Evonik REACh dossier for 4-Hydroxy-TEMPO. Since it is known that nitroxyl radicals are reduced to their corresponding hydroxylamines by ascorbic acid, Gallez et al. (1992), tested the influence of ascorbic acid on the mutagenicity of nitroxyl radicals in the Ames test. As a result, by the preincubation of the nitroxyl compounds with ascorbic acid mutagenic activity was suppressed leading to the conclusion that the mutagenicity was produced by the free radical species.
Sies and Mehlhorn (1986) reported that the mutagenicity of the nitroxyl radicals decreased in cells carrying a mutation that leads to the induction of enzymes protecting against oxidative stress. At the example of 4-Hydroxy-Tempo they propose the superoxide radical (via the formation of oxidized thiols e.g. reactive glutathione species) to be the active species for the mutagenic effect of nitroxyl radicals. This is supported by findings of Guo et al. (2013) observed in mouse lymphoma cells treated with Tempo. Analysis of reactive oxygen species (ROS) levels revealed a significant increase compared to vehicle control with a maximum at 2 to 4 hours after treatment depending on dose. A significant dose-dependent decrease in relative gluthathione levels was measured after 4 hours of treatment. Furthermore, an analysis of the loss of heterozygocity (LOH) at the thymidine kinase (Tk) locus in Tk mutants from the mouse lymphoma cell assays showed an increased percentage of LOH at the Tk locus. In summary, these results suggest that nitroxyl radicals are genotoxic in mammalian cells, at least partially through the generation of oxidative stress, resulting in large genetic alterations (Guo et al., 2013).

The ascorbic acid reduction rates of Tempo and 4-Hydroxy-Tempo were found to be in a similar range (91 or 72 µmol/min/mmol ascorbic acid, respectively). Moreover, the number of revertants by plate induced by both substances at 15 µmol/plate (the highest non-toxic concentration) were comparable (210 or 240 revertants/plate, respectively). Both findings indicate similar reactivity and mutagenicity of Tempo and 4-Hydroxy-Tempo (Gallez et al., 1992).

As a conclusion, the underlying mechanism of the genotoxic effects of the nitroxyl radicals has been identified, and 4-Hydroxy-Tempo shows similar characteristics than Tempo so that it appears well suited as source substance for the read-across approach to fulfill the information requirement on in vivo genotoxicity for Tempo.

4. DATA MATRIX
4-Hydroxy-2,2,6,6-tetramethylpiperidinoxyl (4-Hydroxy-Tempo) is used as source substance. The relevant study records are linked in the target record field ‘Cross-reference’.

REFERENCES
Gallez B, De Meester C, Debuyst R, Dejehet F, Dumont P, 1992. Mutagenicity of nitroxyl compounds: structure-activity relationships. Toxicology Letters 63:35-45.
Guo X, Mittelstaedt RA, Guo L, Shaddock JG, Heflich RH, Bigger AH, Moore MM, Mei N, 2013. Nitroxide TEMPO: A genotoxic and oxidative stress inducer in cultured cells. Toxicology in Vitro 27, 1496–502.
Sies H, Mehlhorn R, 1986. Mutagenicity of Nitroxide-Free Radicals. Archives of Biochemistry and Biophysics 251:393-96.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, UK
- Age at study initiation: 7 - 10 wks
- Body weight at study initiation: 166 - 254 g (males); 150 - 191 g (females); only males used in main study
- Housing: conventional, 4 rats/sex in solid floored cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8 - 9 days (main study); 2 - 13 days (pre-test)

ENVIRONMENTAL CONDITIONS
- Temperature: 19.6- 23.1°C
- Humidity: 49 - 78 %
- Air changes: 15 per h
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: not specified

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: water
- Concentration of test material in vehicle: 70 -200 mg/mL
- Amount of vehicle: 10 mL/ kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in purified water, vortex mixed or ultrasonicated where required. Preparations were protected from light and used within 2:45 hrs.

Frequency of treatment:

single dose

Post exposure period:

experiment 1: 12 - 14 hours;
experiment 2: 2 - 4 hours

Dose / conc.:

800 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 males (except for one of positive controls: DMN, 3 males)

Control animals:

yes, concurrent vehicle

Positive control(s):

- 2-acetylaminofluorene (2-AAF, experiment 1); dimethylnitrosamine (DMN, experiment 2)
- Route of administration: oral, gavage
- Doses / concentrations: 7.5 mg/mL 2-acetylaminofluorene, 1.0 mg/mL dimethylnitrosamine

Tissues and cell types examined:

rat hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Clinical signs of toxicity and body weights from pre-experiment were base for MTD of main study.

DETAILS OF SLIDE PREPARATION: Hepatocytes were prepared from at least three of the four animals in each dose group. The liver was perfused with suitable buffers, digested with collagenase and hepatocytes then taken into culture. Hepatocyte suspension (approximately 1.5 x 105 viable cells/mL) was radiolabelled with 10 mCi/mL [3H] thymidine and fixed on slides with standard methods.

METHOD OF ANALYSIS: Autoradiography/Grain counting

1. only cells with normal morphology were scored
2. isolated nuclei with no surrounding cytoplasm were not scored
3. cells without nuclear and/or cytoplasmic graining were not scored
4. cells with unusual staining artefacts were not scored
5. heavily labelled cells in S-phase were not scored
6. all other normal cells, 100 per animal were scored
7. all slides were analysed blind (coded).

The following were calculated for each slide, animal and dose point:
1. the population average NNG and standard deviation (SD)
2. the percent of cells responding or in repair (ie ≥ 5 NNG)
3. the population average cytoplasmic and nuclear grain count.

Evaluation criteria:

The test article would be considered as positive if, at any dose and at either time point:
1. the test article yielded group mean NNG values greater than 0 NNG and 20% or more of cells responding (mean NNG values ≥ 5)
2. an increase above solvent control levels was seen in both NNG and the percentage of cells in repair.

Cytoplasmic and nuclear grain count values as well as the concurrent negative control data would be considered in relation to the overall NNG values of cultures from treated animals. If the test article failed to induce UDS at any dose tested after both 2-4 and 12-14 hours exposure, it would be considered clearly negative in this system.

Statistics:

The following were calculated for each slide, animal and dose point:
1. the population average NNG and standard deviation (SD)
2. the percent of cells responding or in repair
3. the population average cytoplasmic and nuclear grain count.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 700- 2000 mg/kg bw
- gender: males and females
- Clinical signs of toxicity in test animals: lack of faecal pellets noted (3F), weight loss (3M3F)

For the main study, a lower dose of 800 mg/kg (40% of the maximum dose) was also selected. As no substantial gender differences in toxicity were observed, male animals only were treated in the main study.

Conclusions:

The genetic toxicity in vivo of 4-Hydroxy-TEMPO was tested in a GLP compliant OECD 486 guideline study (unscheduled DNA synthesis). As a result, the test item was not genotoxic under the conditions tested.

Executive summary:

In an in vivo/in vitro unscheduled DNA synthesis (UDS) assay (2003-6674-DGM), male rats were orally (gavage) exposed to a single dose of 4 -Hydroxy-TEMPO at a concentration of 800 or 2000 mg/kg bw (vehicle: water). The maximum tolerated dose was determined prior in a toxicity test. The post exposure period was 24 or 48 hrs (experiment 1) or 2 to 4 hrs (experiment 2). No clinical signs of toxicity or loss of body weight were observed in the main study. The study was valid according to the criteria defined. The data indicate that treatment of male rats dosed once via oral gavage with 800 or 2000 mg/kg test substance did not result in increased UDS in hepatocytes isolated approximately 12-14 or 2-4 hours after dosing. The study satisfies the requirements of Test Guideline OECD 486 for the evaluation of in vivo mutagenicity (unscheduled DNA synthesis).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Gallez et al. (1992) describe three nitroxyl radicals to be mutagenic in the Ames test: Tempo, 4-Hydroxy-Tempo and Amino-Tempo. This finding is verified by an additional experimental study for 4-Hydroxy-TEMPO, which is reported in the Evonik REACh dossier for 4-Hydroxy-TEMPO. Since it is known that nitroxyl radicals are reduced to their corresponding hydroxylamines by ascorbic acid, Gallez et al. (1992), tested the influence of ascorbic acid on the mutagenicity of nitroxyl radicals in the Ames test. As a result, by the preincubation of the nitroxyl compounds with ascorbic acid mutagenic activity was suppressed leading to the conclusion that the mutagenicity was produced by the free radical species. Sies and Mehlhorn (1986) reported that the mutagenicity of the nitroxyl radicals decreased in cells carrying a mutation that leads to the induction of enzymes protecting against oxidative stress. At the example of 4-Hydroxy-Tempo they propose the superoxide radical (via the formation of oxidized thiols e.g. reactive glutathione species) to be the active species for the mutagenic effect of nitroxyl radicals. This is supported by findings of Guo et al. (2013) observed in mouse lymphoma cells treated with Tempo. Analysis of reactive oxygen species (ROS) levels revealed a significant increase compared to vehicle control with a maximum at 2 to 4 hours after treatment depending on dose. A significant dose-dependent decrease in relative gluthathione levels was measured after 4 hours of treatment. Furthermore, an analysis of the loss of heterozygocity (LOH) at the thymidine kinase (Tk) locus in Tk mutants from the mouse lymphoma cell assays showed an increased percentage of LOH at the Tk locus. In summary, these results suggest that nitroxyl radicals are genotoxic in mammalian cells, at least partially through the generation of oxidative stress, resulting in large genetic alterations (Guo et al., 2013).

Two in vivo genotoxcity tests are available conducted with the read-across source 4-Hydroxy-Tempo. Both the micronucleus test in the mouse and the UDS test in the rat were negative indicating that the in vitro mode of genotoxicity is not relevant in vivo, presumably due to the presence of protection systems against oxidative stress.

References

Gallez B, De Meester C, Debuyst R, Dejehet F, Dumont P, 1992. Mutagenicity of nitroxyl compounds: structure-activity relationships. Toxicology Letters 63:35-45.

Guo X, Mittelstaedt RA, Guo L, Shaddock JG, Heflich RH, Bigger AH, Moore MM, Mei N, 2013. Nitroxide TEMPO: A genotoxic and oxidative stress inducer in cultured cells. Toxicology in Vitro 27, 1496–502.

Sies H, Mehlhorn R, 1986. Mutagenicity of Nitroxide-Free Radicals. Archives of Biochemistry and Biophysics 251:393-96.

Justification for classification or non-classification

Since in vivo genotoxcity tests are available showing that the in vitro mode of genotoxicity is not relevant in vivo, according to Regulation (EC) 1272/2008 the data are conclusive but not sufficient for classification.