2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropanoyl)phenoxy]phenyl]-2-methylpropan-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

2019-01-08 - 2019-02-01 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997
“Bacterial Reverse Mutation Test“

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage: Fridge (2 – 8 °C); keep away from light; keep away from humidity.
- Stability in solvents: H2O: unknown; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

Target gene:

his

Species / strain / cell type:

S. typhimurium, other: TA 97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9 produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.

Test concentrations with justification for top dose:

- 5000 / 1500 / 500 / 150 / 50 µg/plate (plate incorporation method; experiment 1a)
- 5000 / 1500 / 500 / 150 / 50 / 15 µg/plate (plate incorporation method; experiment 1b)
- 5000 / 2500 / 1250 / 625 / 313/ 156 / 78 µg/plate (pre-incubation method; experiment 2)
- top dose was selected according to the guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone. The solid test item was sufficiently soluble in DMSO, only. Based on this non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Untreated negative controls:

yes

Remarks:

H2O

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 min (Pre-incubation method)
- Exposure duration: 48h

SELECTION AGENT (mutation assays):
Minimal histidine agar

NUMBER OF REPLICATIONS: Per strain and dose, three plates with and three plates without S9 mix were used. Two experiments were performed

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

Two independent experiments with variations in methodology should be performed.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Effects of osmolality: none stated
- Evaporation from medium: based on the substance' vapour pressure, evaporation is highly unlikely
- Water solubility: substance is sufficiently soluble
- Precipitation: none stated

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
COMPARISON WITH HISTORICAL DATA
In the following table, the history of the spontaneous revertants and positive controls of the performed experiments with these strains up to 04. Dec. 2018 is stated in comparison with the experiments performed within this study. Only experiments which were performed before the performance of the study were considered.
For the historical data, the plate incorporation method and the pre- incubation method were used.

Table Historical Data of Spontaneous Revertants

Strain TA97a TA98 TA100 TA102 TA1535
Induction - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
Demin.water Mean 88 95 26 27 94 98 286 305 17 17
Min 60 63 6 8 51 64 85 67 6 7
Max 144 138 52 53 147 141 445 587 36 40
SD 16 16 13 12 15 15 61 73 6 6
Exp 1a 79 107 49 42 100 91 421 324 12 13
Exp 1b 97 91 41 42 90 103 293 293 9 11
Exp. 2 77 86 40 47 88 102 297 304 11 9

DMSO Mean 88 98 25 26 90 94 287 298 17 17
Min 58 67 7 8 44 62 79 80 8 6
Max 139 144 50 51 138 199 465 499 35 37
SD 17 16 13 13 15 16 63 65 6 6
Exp 1a 97 94 50 43 107 97 339 324 12 11
Exp 1b 95 97 43 42 81 88 229 293 10 10
Exp. 2 76 80 47 43 102 117 289 291 12 12

Pos.Control* Mean 539 536 388 149 523 809 1119 1214 269 149
Min 264 228 77 39 220 273 491 408 55 45
Max 1165 1181 s.g. 488 1256 1912 2331 6083 515 s.g.
SD 163 173 172 114 205 287 387 517 85 109
Exp 1a 564 456 300 287 1021 s.g. 1208 1184 252 188
Exp 1b 480 539 90 86 716 1003 1267 1320 377 176
Exp. 2 264 431 309 312 1192 1261 1344 1365 176 96

* Different positive controls were used
s.g.= strong growth, too strong for counting of revertants

The incubation of the bacteria strains TA98, TA100 and TA1535 with the respective positive control induced such a high increase in revertant colonies, which overlapped and made counting impossible. So, it could not be determined whether colony count was in the range of historical control, but an >2-fold increase over control was obvious.

Conclusions:

not mutagenic in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535, in the absence and presence of metabolic activation

Executive summary:

A study was conducted to investigate the potential of the test substance to induce reverse mutations in bacteria according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Salmonella typhimurium strains TA 97a, TA 1535, TA 98, TA 102 and TA 100 were treated with the test substance. The study was performed as three experiments in the presence and absence of rat liver S9-mix induced by Aroclor 1254. All negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In experiments 1a and 1b, precipitation of the test substance was observed at the two highest concentrations (5000 and 1500 μg/plate). In experiment 2, precipitation was observed at 5000 and 2500 μg/plate. In all three experiments, the test substance caused cytotoxicity towards the bacterial strains. The confirmation tests of the genotype did not show irregularities. The control of the titre was above the required value of 109 bacteria/mL. The means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Positive control revertant colony numbers were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability were met, the study was considered valid. Under the conditions of the reverse mutation assay, the test substance was considered to be non-mutagenic with and without metabolic activation (Andres, 2019). 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QSAR prediction from a well-known and acknowledged tool. See below under "attached justification section" for QMRF and QPRF

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals

Principles of method if other than guideline:

The mutagenicity potential of the test substance at the hypoxanthine‐guanine phosphoribosol transferase (HGPRT) locus in the CHO cells was predicted using the 'Danish_QSAR_DB_Mutations_in_HGPRT_locus_in_CHO_cells_QSARmodels.food.dtu.dk v1' QSAR model. Since the test substance is a mono-constituent (concentration range: ≥99-<100%) with ≤1% of unidentified impurities, the prediction was performed only for the test substance using the SMILES code as input parameter.

GLP compliance:

no

Remarks:

Not relevant for QSAR modelling

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Test substance: 2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropionyl) phenoxy]phenyl]-2-methylpropan-1-one
CAS No: 71868-15-0
EC No: 472-110-0
Composition: 2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropionyl) phenoxy]phenyl]-2-methylpropan-1-one (>=99 - <100 % (w/w)); unknown impurities (>0 - <=1 % (w/w)
SMILES code used for the QSAR modelling: C(C)(C(=O)C1=CC=C(C=C1)OC2=CC=C(C=C2)C(=O)C(C)(C)O)O

Target gene:

hypoxanthine‐guanine phosphoribosol transferase (HGPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

not specified

Genotoxicity:

negative

Remarks:

in domain

Additional information on results:

Using the model ‘Danish_QSAR_DB_Mutations_in_HGPRT_locus_in_CHO_cells_QSARmodels.food.dtu.dk v1’ 2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropionyl) phenoxy]phenyl]-2-methylpropan-1-one is predicted to be non-mutagenic in the HGPRT assay using CHO cells. The prediction is within the AD (see Section 3.3 of the QPRF for further details). This result is in agreement with:
-the results of the Ames test on the test substance;
-the prediction performed by the model ‘Danish_QSAR_DB_Mutations_in_thymidine_kinase_locus_in_Mouse_lymphoma_cells_in_vitro_QSARmodels.food.dtu.dk v1’;
-the profiling of the test substance with respect to DNA binding structural alerts obtained via the OECD QSAR Toolbox v.4.4.1.(refer to the attached OECD toolbox in the 'attached justification' section).
Based on the available information, the prediction of the test substance in the HGPRT assay is considered to be reliable with low to moderate degree of uncertainty.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Overall, the prediction for the test substance using the Danish EPA QSAR model for HGPRT assay can be considered to be reliable with moderate to high confidence.

Executive summary:

The gene mutation potential of the test substance at the HGPRT locus in Chinese hamster ovary (CHO) was predicted using the ‘Danish_QSAR_DB_Mutations_in_HGPRT_locus_in_CHO_cells_QSAR models.food.dtu.dk v1’ QSAR model. Since the test substance is a mono-constituent (concentration range: ≥99-<100%) with ≤1% of unidentified impurities, the prediction was performed only for the test substance using the SMILES code as input parameter. Using the Danish EPA QSAR model, the test substance was predicted to be non-mutagenic and within the applicability domain of the model (Danish EPA, 2021). However, considering that only one nearest neighbour was identified from the training set, the reliability of the prediction can be considered to be moderate. Nevertheless, the HGPRT assay prediction was in agreement with (a) the results of the Ames test on the test substance; (b) the predictions from the statistical QSAR model ‘Danish_QSAR_DB_Mutations_in_thymidine_kinase_locus_in_Mouse_lymphoma_cells_in_vitro_QSAR models.food.dtu.dk v1’ QSAR model for the same endpoint; and (c) the profiling of the test substance with respect to DNA binding structural alerts obtained via the OECD   QSAR Toolbox v.4.4.1. Based on all the available information, the overall uncertainty in the predictions can be considered to be low to moderate. Therefore, the prediction for the test substance using the Danish EPA QSAR model for HGPRT assay can be considered to be reliable with moderate to high confidence.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

QSAR prediction from a well-known and acknowledged tool. See below under "attached justification section" for QMRF and QPRF

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs: Chapter R.6. QSARs and grouping of chemicals

Principles of method if other than guideline:

The mutagenicity potential of the test substance at the thymidine‐kinase (TK) locus in the in Mouse lymphoma cells was predicted using the 'Danish_QSAR_DB_Mutations_in_thymidine_kinase_locus_in_Mouse_lymphoma_cells_in_vitro_QSARmodels.food.dtu.dk v1’ QSAR model. Since the test substance is a mono-constituent (concentration range: ≥99-<100%) with ≤1% of unidentified impurities, the prediction was performed only for the test substance using the SMILES code as input parameter.

GLP compliance:

no

Remarks:

Not relevant for QSAR modelling

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Test substance: 2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropionyl) phenoxy]phenyl]-2-methylpropan-1-one
CAS No: 71868-15-0
EC No: 472-110-0
Composition: 2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropionyl) phenoxy]phenyl]-2-methylpropan-1-one (>=99 - <100 % (w/w)); unknown impurities (>0 - <=1 % (w/w)
SMILES code used for the QSAR modelling: C(C)(C(=O)C1=CC=C(C=C1)OC2=CC=C(C=C2)C(=O)C(C)(C)O)O

Target gene:

thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

not specified

Genotoxicity:

negative

Remarks:

in domain

Additional information on results:

Using the model Danish_QSAR_DB_Mutations_in_thymidine_kinase_locus_in_Mouse_lymphoma_cells_in_vitro_QSARmodels.food.dtu.dk v1’, 2-hydroxy-1-[4-[4-(2-hydroxy-2-methylpropionyl) phenoxy]phenyl]-2-methylpropan-1-one is predicted to be non-mutagenic in the mouse lymphoma assay. The prediction is within the AD (see Section 3.3 of the QPRF for further details). This result is in agreement with:
-the results of the Ames test on the test substance;
-the prediction performed by the model ‘Danish_QSAR_DB_Mutations_in_thymidine_kinase_locus_in_Mouse_lymphoma_cells_in_vitro_QSARmodels.food.dtu.dk v1’;
-the profiling of the test substance with respect to DNA binding structural alerts obtained via the OECD QSAR Toolbox v.4.4.1.(refer to the attached OECD toolbox in the 'attached justification' section).
Based on the available information, the prediction of the test substance in the HGPRT assay is considered to be reliable with low to moderate degree of uncertainty.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

Overall, the prediction for the test substance using the Danish EPA QSAR model for HGPRT assay can be considered to be reliable with moderate to high confidence.

Executive summary:

The gene mutation potential of the test substance at the TK locus in mouse lymphoma cells was predicted using the ‘Danish_QSAR_DB_Mutations_in_thymidine_kinase_locus_in_Mouse_lymphoma_cells_in_vitro_QSARmodels.food.dtu.dk v1’ QSAR model. Since the test substance is a mono-constituent (concentration range: ≥99-<100%) with ≤1% of unidentified impurities, the prediction was performed only for the test substance using the SMILES code as input parameter. Using the Danish EPA QSAR model, the test substance was predicted to be non-mutagenic and within the applicability domain of the model (Danish EPA, 2021). However, considering that only one nearest neighbour was identified from the training set, the reliability of the prediction can be considered to be moderate. Nevertheless, the HGPRT assay prediction was in agreement with (a) the results of the Ames test on the test substance; (b) the predictions from the statistical QSAR model ‘Danish_QSAR_DB_Mutations_in_HGPRT_locus_in_CHO_cells_QSARmodels.food.dtu.dk v1’ QSAR model for the same endpoint; and (c) the profiling of the test substance with respect to DNA binding structural alerts obtained via the OECD   QSAR Toolbox v.4.4.1. Based on all the available information, the overall uncertainty in the predictions can be considered to be low to moderate. Therefore, the prediction for the test substance using the Danish EPA QSAR model for mouse lymphoma assay can be considered to be reliable with moderate to high confidence.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cytogenicity (B10)

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster V79 Ovary Cells

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 130 ... 150 μg/ml
Concentration range in the main test (with metabolic activation): 140 μg/ml
Concentration range in the main test (with metabolic activation): 150 μg/ml
Concentration range in the main test (without metabolic activation): 80 ... 100 μg/ml
Concentration range in the main test (without metabolic activation): 90 μg/ml
Concentration range in the main test (without metabolic activation): 95 μg/ml
Concentration range in the main test (without metabolic activation): 100 μg/ml

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 4 hours

Expression time:
4 h
Fixation time:
20 h

Species / strain:

other: Chinese Hamster V79 Ovary Cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(250 μg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Chinese Hamster V79 Ovary Cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(125 μg/ml)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Chinese Hamster V79 Ovary Cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(140 μg/ml)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

other: Chinese Hamster V79 Ovary

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(90 μg/ml)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Observations:
CLASTOGENICITY:
in the main experiment without metabolic activation the aberration rates of highest dose groups (95 ug/mL and 100 ug/mL) were clearly increased as compared to the negative and solvent control. A dose-response relationship was indicated. In the main experiment with metabolic activation the highest groups evaluated (130, 140 and 150 ug/mL) shown an increase of aberration rates again with a dose-response relationship.

POLYPLOID CELLS:
No biologically relevant increase in the frequecies of polypoid cells was found after treatment with ESACURE 2006

Conclusions:

Under the experimental conditions, the test substance induced cytotoxic effects and structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in Chinese Hamster V79 Ovary Cells according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. Cells were exposed for 4 h at 130 to 150 μg/mL and 80 to 100 μg/m test substance with and without metabolic activation, respectively. Toxicity was noted at 90 and 100 μg/mL without metabolic activation and 140 and 150 μg/mL with metabolic activation. Structural chromosomal aberrations were observed at 95 and 100 µg/mL without S9 mix and at 130, 140 and 150 µg/mL with S9 mix. Under the experimental conditions, the test substance induced cytotoxic effects and structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Bioservice, 2006).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Micronucleous test in rat bone marrow Annex V B.12 - OECD n.474

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Micronucleous test in rat bone marrow Annex V B.12 - OECD n.474

GLP compliance:

yes

Type of assay:

other: micronucleus assay and chromosome aberration

Species:

mouse

Strain:

NMRI

Route of administration:

intraperitoneal

Vehicle:

cotton seed oil

No. of animals per sex per dose:

Male: 100 mg/kg; No. of animals: 5; Sacrifice time: hours
Male: 250 mg/kg; No. of animals: 5; Sacrifice time: hours
Male: 500 mg/kg; No. of animals: 5; Sacrifice time: hours
Female: 100 mg/kg; No. of animals: 5; Sacrifice times: hours
Female: 250 mg/kg; No. of animals: 5; Sacrifice times: hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice times: hours

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Observations:
The positive control was conducted using Cyclophosphamide (40 mg/kg bw).

Conclusions:

Under the conditions of this study, the test substance at doses up to 500 mg/kg was concluded to be negative in both male and female mice in the Micronucleus assay.

Executive summary:

A study was conducted to investigate the test substance clastogenic activity and/or potential to induce disruption of mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in NMRI mouse, according to OECD Guideline 474 and EU Method B.12, in compliance with GLP. Five animals per sex per dose were treated via the intraperitoneal route at doses of 100, 250 and 500 mg/kg bw. The vehicle was cottonseed oil and the positive control was cyclophosphamide (40 mg/kg bw). Polychromatice erythrocytes (PCE; proportion among total) and micronucleated polychromatic erythrocyte values were in the range of negative control data at all doses, except for the highest doses at which toxicity was observed (known to induce micronuclei independently to any treatment and considered not relevant for the scope of the study). Under the conditions of the study, the test substance at doses up to 500 mg/kg bw was concluded to be negative in both male and female mice in the micronucleus assay (Bioservice, 2006).  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro 

Gene mutation in bacterial cells

A study was conducted to investigate the potential of the test substance to induce reverse mutations in bacteria according to OECD Guideline 471 and EU Method B.13/14, in compliance with GLP. Salmonella typhimurium strains TA 97a, TA 1535, TA 98, TA 102 and TA 100 were treated with the test substance. The study was performed as three experiments in the presence and absence of rat liver S9-mix induced by Aroclor 1254. All negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In experiments 1a and 1b, precipitation of the test substance was observed at the two highest concentrations (5000 and 1500 μg/plate). In experiment 2, precipitation was observed at 5000 and 2500 μg/plate. In all three experiments, the test substance caused cytotoxicity towards the bacterial strains. The confirmation tests of the genotype did not show irregularities. The control of the titre was above the required value of 109 bacteria/mL. The means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Positive control revertant colony numbers of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability were met, the study was considered valid. Under the conditions of the reverse mutation assay, the test substance was considered to be non-mutagenic with and without metabolic activation (Andres, 2019). 

Gene mutation in mammalian cells

Study 1: The gene mutation potential of the test substance at the TK locus in mouse lymphoma cells was predicted using the ‘Danish_QSAR_DB_Mutations_in_thymidine_kinase_locus_in_Mouse_lymphoma_cells_in_vitro_QSARmodels.food.dtu.dk v1’ QSAR model. Since the test substance is a mono-constituent (concentration range: ≥99-<100%) with ≤1% of unidentified impurities, the prediction was performed only for the test substance using the SMILES code as input parameter. Using the Danish EPA QSAR model, the test substance was predicted to be non-mutagenic and within the applicability domain of the model (Danish EPA, 2021). Considering that only one nearest neighbour was identified from the training set, the reliability of the prediction can be considered to be moderate. Nevertheless, the HGPRT assay prediction was in agreement with:

(a) the results of the Ames test on the test substance

(b) the predictions from the statistical QSAR model ‘Danish_QSAR_DB_Mutations_in_HGPRT_locus_in_CHO_cells_QSARmodels.food.dtu.dk v1’ QSAR model for the same endpoint and

(c) the profiling of the test substance with respect to DNA binding structural alerts obtained via the OECD   QSAR Toolbox v.4.4.1.

Based on all the available information, the overall uncertainty in the predictions is low to moderate. Therefore, the prediction for the test substance using the Danish EPA QSAR model for mouse lymphoma assay can be considered to be reliable with moderate to high confidence.

Study 2: The gene mutation potential of the test substance at the HGPRT locus in Chinese hamster ovary (CHO) was predicted using the ‘Danish_QSAR_DB_Mutations_in_HGPRT_locus_in_CHO_cells_QSAR models.food.dtu.dk v1’ QSAR model. Since the test substance is a mono-constituent (concentration range: ≥99-<100%) with ≤1% of unidentified impurities, the prediction was performed only for the test substance using the SMILES code as input parameter. Using the Danish EPA QSAR model, the test substance was predicted to be non-mutagenic and within the applicability domain of the model (Danish EPA, 2021). Considering that only one nearest neighbour was identified from the training set, the reliability of the prediction can be considered to be moderate. Nevertheless, the HGPRT assay prediction was in agreement with:

(a) the results of the Ames test on the test substance

(b) the predictions from the statistical QSAR model ‘Danish_QSAR_DB_Mutations_in_thymidine_kinase_locus_in_Mouse_lymphoma_cells_in_vitro_QSAR models.food.dtu.dk v1’ QSAR model for the same endpoint and

(c) the profiling of the test substance with respect to DNA binding structural alerts obtained via the OECD   QSAR Toolbox v.4.4.1.

Based on all the available information, the overall uncertainty in the predictions is low to moderate. Therefore, the prediction for the test substance using the Danish EPA QSAR model for HGPRT assay can be considered to be reliable with moderate to high confidence.

Chromosome aberration in mammalian cells

A study was conducted to investigate the potential of the test substance to induce chromosome aberrations in Chinese Hamster V79 Ovary Cells according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. Cells were exposed for 4 h at 130 to 150 μg/mL and 80 to 100 μg/m test substance with and without metabolic activation, respectively. Toxicity was noted at 90 and 100 μg/mL without metabolic activation and 140 and 150 μg/mL with metabolic activation. Structural chromosomal aberrations were observed at 95 and 100 µg/mL without S9 mix and at 130, 140 and 150 µg/mL with S9 mix. Under the experimental conditions, the test substance induced cytotoxic effects and structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Bioservice, 2006).

In vivo 

A study was conducted to investigate the test substance clastogenic activity and/or potential to induce disruption of mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in NMRI mouse, according to OECD Guideline 474 and EU Method B.12, in compliance with GLP. Five animals per sex per dose were treated via the intraperitoneal route at doses of 100, 250 and 500 mg/kg bw. The vehicle was cottonseed oil and the positive control was cyclophosphamide (40 mg/kg bw). Polychromatice erythrocytes (PCE; proportion among total) and micronucleated polychromatic erythrocyte values were in the range of negative control data at all doses, except for the highest doses at which toxicity was observed (known to induce micronuclei independently to any treatment and considered not relevant for the scope of the study). Under the conditions of the study, the test substance, at doses up to 500 mg/kg bw, was concluded to be negative in both male and female mice in the micronucleus assay (Bioservice, 2006).  

Overall conclusion

The test substance was negative in an in vitro bacterial mutation assay and was also evaluated to be negative for gene mutation in mammalian cells based on the results of two separate QSAR models. A positive result was obtained for chromosome aberration in mammalian cells in vitro, but a follow-up in vivo micronucleus study showed negative results at doses up to 500 mg/kg bw in both male and female mice.

Overall, the substance is therefore not considered to be genotoxic. 

Justification for classification or non-classification

Based on the available in vitro and in vivo data, the substance is not considered to be mutagenic and does not require classification for this endpoint according to Regulation (EC) No. 1272/2008.