N-(5-chloro-2-methoxyphenyl)-2-[(2-methoxy-4-nitrophenyl)azo]-3-oxobutyramide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria. Key study. Method according to OECD guideline 471, GLP study. The test item was found to be not mutagenic with or without metabolic activation in the Salmonella typhimurium strainsTA 1535, TA 1537, TA98, TA100 and E. coli WP2 uvrA pKM101.

In vitro micronucleus study in mammalian cells. Key study. Method according to OECD guideline 487, GLP study. The test item was found as non aneugenic and non clastogenic in human lymphocytes up to the concentration of 0.0625 mg/mL both in the presence and absence of metabolic activation under the test conditions.

In vitro mammalian cell gene mutation test: Key study: Test method according to the OECD Guideline 476 with GLP study. The test item was found as non-mutagenic up to the concentration of 0.0625 mg/mL, both in the presence and absence of metabolic activation under the test conditions.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 AUG 1998 to 13 AUG 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 471) with Prival modifications for azo-dyes, GLP compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC directive 92/69, L 383 A, Annex B14

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9; hamster liver S9

Test concentrations with justification for top dose:

0, 50, 160, 500, 1600, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 100, TA1535), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98), 1-methyl-3-nitro-1-nitrosoguanidine - MNNG (WP2uvrA)

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains)

Remarks:

with metabolic activation (rat liver S9 mix)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (TA 100, TA 1535, TA 1537, WP2uvrA), congo red (TA 98)

Remarks:

with metabolic activation (hamster liver S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- plate incorporation assay without and with induced rat liver S9 mix (10% (v/v); induction with Aroclor 1254)
- preincubation assay without and with uninduced hamster liver S9 mix (30% (v/v))

DURATION
- Preincubation period: ca. 30 minutes at 30 °C
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including controls

Evaluation criteria:

A test compound is classified as mutagenic if it has either of the following effects:
- it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
- it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn

Statistics:

Arithmetic means and standard deviation of the counted colonies were calculated, as well as the respective dose/control ratio.

Key result

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:

in a dose range of 160 and 500 µg/plate and above without metabolic activation and in a dose range of 500 to 1600 µg/plate and above with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in a dose range of 160 and 500 µg/plate and above without metabolic activation and in a dose range of 500 to 1600 µg/plate and above with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: visible precipitation of the test itemat 160 µg/plate and above

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- in a dose range of 160 and 500 µg/plate and above without metabolic activation and in a dose range of 500 to 1600 µg/plate and above with metabolic activation (thinning of bacterial lawns and in some cases also a reduction in the number of colonies)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.

Conclusions:

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and preincubation assay according to Prival) with and without metabolic activation.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and E. coli WP2 uvrA pKM101 with (induced rat liver S9 mix) and without metabolic activation at concentrations of 0, 50, 160, 500, 1600, and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 0, 50, 160, 500, 1600, and 5000 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2022 - 17 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test method according OECD Guideline 487. GLP study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: peripheral blood lymphocytes from human donors.
- Suitability of cells: healthy, young, non-smoking donors with no known recent exposure to genotoxic chemicals or radiation were used. Blood from individual was collected in sodium heparin vacutainer and analyzed using Advia 2120. All the parameters were within the acceptable range.
- Normal cell cycle time (negative control): 12 to 15 hours.

For lymphocytes:
- Sex, age and number of blood donors: One male of 28 and one female of 24 years old were used, one for the initial cytotoxicity and the other for the micronucleus test.
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: No
- Mitogen used for lymphocytes: Phytohemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin-Streptomycin), 5±1% CO2, 37±1ºC.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B (CytoB)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver of male Wistar rat induced with sodium phenobarbtone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively.
- method of preparation of S9 mix: 1 mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM Nicotinamide Adenine Dinucleotide Phosphate (NADP), 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS) at pH 7.3.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL (10% v/v) of S9 mix and 0.05 mL (1% v/v) of S9 homogenate.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 tester strain.

Test concentrations with justification for top dose:

0.015625, 0.03125 and 0.0625 mg/mL.
Justification for top dose: Based on the results of precipitation and pH tests, the initial cytotoxicity test was conducted at concentrations of 0.0078125, 0.015625, 0.03125, 0.0625 and 0.125 mg/mL. At 0.125 mg/mL slides were not able to evaluate as more particles were present (particles were interfering with cells). The % cytotoxicity ranged from 1.61 to 4.92 at 0.0625 to 0.0078125 mg/mL. As the % cytotoxicity was not more than 55±5% at 0.0625 mg/mL, the same was selected as the highest concentration for the micronucleus test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test item formed suspension in DMSO at 50 mg/mL, precipitation and pH tests were conducted at the concentrations of 0.015625, 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL. Post incubation, heavy precipitation was observed at 0.25 and 0.5 mg/mL, mild and moderate precipitation at 0.0625 and 0.125 mg/mL and no precipitation was observed at 0.015625, 0.03125 mg/mL. No pH change was observed at any of the other concentrations tested at 0.015625, 0.03125, 0.0625, 0.125, 0.25, and 0.5 mg/mL. Hence 0.125 mg/mL was selected as highest concentration for the initial cytotoxicity test.

- Justification for percentage of solvent in the final culture medium:

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

Remarks:

3 h treatment, without metabolic activation (0.03 µg/mL)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

24 h treatment, without metabolic activation (0.075 µg/mL)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

3 h treatment, with metabolic activation (10 µg/mL)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 44 to 48 hours, with phytohaemagglutinin (PHA) to induce cell division prior to exposure.
- Exposure duration/duration of treatment: 3 h (short term treatment, +S9 and -S9); 24 h (long term treatment, -S9).
- Harvest time after the end of treatment (sampling/recovery times): 20 to 24 hours (1.5- 2 cell cycle length).

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Cytokinesis was blocked using Cytochalasin B at 6 µg/mL. For the short treatment, cytoB was added at the end of treatment and incubated for 20-24 h. For the long treatment, cytoB was added at the beggining of the treatment.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After treatment, cell suspension was mixed with 3 mL of freshly prepared warm 0.56% Potassium chloride. Cell suspension was incubated for 10 minutes at room temperature and later it was centrifuged at 1800 rpm for 10 minutes. Supernatant was discarded and cell pellet was mixed with 3 mL of freshly prepared cold acetic acid:methanol fixative (1:3). Cell suspension was incubated for 10 minutes at room temperature and later suspension was centrifuged at 2000 rpm for 10 minutes. The procedure was repeated twice by adding 4 mL of cold acetic acid: methanol fixative (1:3). Clean slides were stored in a beaker with distilled water and kept in the refrigerator for at least 1 hour before use. The cell suspension was mixed using a pipette and few drops of the suspension were aspirated and dropped onto the chilled slide pre labeled with study number, with (+S9) or without metabolic activation (-S9), treatment/group and slide number. The slides were air dried. Minimum of 3 slides were prepared for each treatment replicate. Smear was stained using Acridine Orange stain by allowing the stain to retain for 5 minutes.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Measurement of the relative frequencies of mononucleate, binucleate and multi-nucleate cells in the culture was made to determine cell proliferation and the cytostatic activity of the treatment to ensure that only cells that divided during or after treatment were scored. Slides were evaluated for the presence of micronuclei in at least 2000 binucleates per concentration (at least 1000 per culture).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: CBPI was determined from 500 cells per culture.

Evaluation criteria:

Positive result if, in any of the experimental conditions examined:
a. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b. The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
c. Any of the results are outside the distribution of the historical negative control data.

Negative result if, in all experimental conditions examined:
a. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b. There is no concentration-related increase when evaluated with an appropriate trend test.
c. All results are inside the distribution of the historical negative control data.

Statistics:

Data was analyzed using SPSS Software version 22 for differences among solvent/vehicle control, positive control and test item groups using ANOVA following Dunnett’s test at a 95% level of confidence (p<0.05).

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Based on precipitation and pH tests, 0.125 mg/mL was selected as the highest concentration for the initial cytotoxicity test and other concentrations tested in the initial cytotoxicity test were 0.0078125, 0.015625, 0.03125 and 0.0625 mg/mL.
In the initial cytotoxicity test, at 0.125 mg/mL slides were not able to evaluate as more particles were present (particles were interfering with cells). The % cytotoxicity was in the range of 1.61 to 4.92 at 0.0625 to 0.0078125 mg/mL. As the % cytotoxicity was not more than 55±5% at 0.0625 mg/mL, the same was selected as the highest concentration for the micronucleus test. Other concentrations tested were 0.015625 mg/mL and 0.03125 mg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The positive controls resulted increase of the micronuclei frequencies with statistical significance at 95% level of confidence (p<0.05) under identical conditions, when compared with the vehicle control. This demonstrated the sensitivity of test system towards positive control and confirmed that the test conditions were adequate and within the range of historical control.

Micronucleus test in mammalian cells:
The test item induced cytotoxicity at 0.0625 mg/mL (3.28 to 6.56%) when compared to vehicle control. There was no statistically significant increase in the percentage of micronuclei in binucleated cells were observed in any of the tested concentrations when compared to the vehicle control.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
The micronucleus frequencies observed for the test item treatments fell within acceptable ranges with regard to in-house historical control data.

Conclusions:

In an in vitro micronucleus test, the test item was found as non aneugenic and non clastogenic in human lymphocytes up to the concentration of 0.0625 mg/mL both in the presence and absence of metabolic activation under the test conditions.

Executive summary:

The test item was evaluated for formation of micronuclei in human lymphocytes according to OECD Guideline 487, following the Principles of GLP. Based on the results of the precipitation and pH tests, the test item was formulated in DMSO as vehicle and 0.125 mg/mL was selected as the highest concentration in the Initial Cytotoxicity Test. This test was conducted at concentrations of 0.0078125, 0.015625, 0.03125, 0.0625 and 0.125 mg/mL. In the initial cytotoxicity test,  at 0.125 mg/mL slides were not able to evaluate as more particles were present (particles were interfering with cells). The % cytotoxicity was in the range of 1.61 to 4.92 at 0.0625 to 0.0078125 mg/mL. As the % cytotoxicity was not more than 55±5% at 0.0625 mg/mL, the same has been selected as the highest concentration for the main test. Cytochalasin B (cytoB) was used as cytokinesis blocker. Cytotoxicity was assessed by Cytokinesis-Block Proliferation Index (CBPI). Cultured human peripheral blood lymphocytes previously incubated for 44 -48 h with PHA in RPMI medium were treated with test item in DMSO at the concentrations of 0.015625, 0.03125 and 0.0625 mg/mL. The treatment was carried out in duplicates for the short term period (3 h) both in the presence and absence of metabolic activation (S9 mix) and for the long term period (24 h) in the absence of metabolic activation. The presence of micronuclei was evaluated in at least 2000 binucleate cells per concentration (at least 1000 per culture). Cyclophosphamide Monohydrate (+S9 for short term) at 10 µg/mL, Colchicine (-S9 for short term) at 0.03 µg/mL and Mytomycin C at 0.075 µg/mL (-S9 long term) were used as positive controls. In the micronucleus test, the test item induced cytotoxicity at 0.0625 mg/mL (3.28 to 6.56%) when compared to vehicle control. There was no statistically significant increase in the percentage of micronuclei in binucleated cells observed in any of the tested concentrations when compared to the vehicle control. Values obtained with concurrent vehicle and positive controls were within the 95% control limits of the distribution of their respective laboratory’s historical control databases. All acceptability criteria were met. Based on these results, it is concluded that the test item is non clastogenic and non aneugenic in cultured human lymphocytes at and up to 0.0625 mg/mL both in short term and long term treatments (presence and absence of metabolic activation).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 May 2022 to 16 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO AA8 cells procured from American Type Culture Collection (ATCC)
- Suitability of cells: The CHO AA8 cells are one of the recommended test systems by regulatory agencies for conducting in vitro mammalian gene mutation test.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Alpha Minimal Essential Medium (MEM) without ribonucleosides containing 10% Fetal Bovine Serum (FBS) and antibiotics (1% Penicillin and streptomycin) were used. The pH of the culture medium was 7.2 to 7.4. The media was stored at 2 to 8ºC till use thawed to room temperature before use.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: liver of male Wistar rats.
- method of preparation of S9 mix: The S9 homogenate was prepared from male wistar rats induced with intraperitoneal injection of sodium phenobarbitone and β-naphthoflavone at 16 mg/mL and 20 mg/mL respectively for 3 days prior to sacrifice. The S9 homogenate was prepared and stored in the test facility at -80±10ºC until use. One mL of S9 homogenate was thawed immediately before use and mixed with 9 mL of co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in Phosphate Buffer Saline (PBS).
- concentration or volume of S9 mix and S9 in the final culture medium:
1 mL, 10% (v/v) S9 mix and 0.1 mL, 1% (v/v) S9.
- quality controls of S9: Batch of S9 homogenate was assessed for sterility, protein content and for its ability to metabolize the promutagens 2-Aminoanthracene and Benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain.

Test concentrations with justification for top dose:

0.0078125, 0.015625, 0.03125 and 0.0625 mg/mL.
The top dose was selected based on preliminary solubility and precipitation tests as well as an initial cytotoxicity test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on the solubility results.
- Percentage of solvent in the final culture medium: 1% v/v.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 (cytotoxicity and cloning efficiency in non-selective medium) and 5 (cloning efficiency of mutant colonies in selective medium)
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approx. 2 x 10^6 cells/culture flask (Initial cytotoxicity test and Gene mutation test).
- Test substance added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours at 37±1ºC with 5±1% CO2.
- Harvest time after the end of treatment (sampling/recovery times): 7 days

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 8 days
- Selection time (if incubation with a selective agent): 8 days.
- Method used: monolayer cultures.
- Selective agent: 10 μM of 6-Thioguanine. Flasks were incubated at 37±1°C with 5±1% CO2 for 8 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 200 x 3 per group (cytotoxicity and cloning efficiency in non-selective medium) and 4x10^5 x 5 per group (cloning efficiency of mutant colonies in selective medium). Flasks were incubated at 37±1°C with 5±1% CO2 for 8 days. Post incubation period, medium from each dish was aspirated and stained with 5% Giemsa stain, number of colonies formed were counted manually.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS)
- Any supplementary information relevant to cytotoxicity: An initial cytotoxicity test was carried out at five different concentrations (0.00390625, 0.0078125, 0.015625, 0.03125 and 0.0625 mg/mL of the test item). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival according to formulas included in annex 2 of OECD TG 476.

Rationale for test conditions:

A precipitation test was conducted at 0.015625, 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg /mL. Post 3 hours of incubation, no precipitation was observed at the tested concentrations of 0.015625 and 0.03125, moderate precipitation was observed at 0.0625 and 0.125 mg/mL and heavy precipitation was observed at 0.25 and 0.5 mg/mL. No major change in pH was observed in any of the test concentrations.

On the basis of these results, 0.0625 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Thus, the initial cytotoxicity test was conducted at the concentrations of 0.00390625, 0.0078125, 0.015625, 0.03125 and 0.0625 mg/mL.

The results of the initial cytotoxicity test showed that the Relative survival is more than 10% at the concentrations of 00390625, 0.0078125, 0.015625, 0.03125 and 0.0625 mg/mL both with metabolic and without metabolic activation, when compared with the vehicle control. Hence 0.0625 mg/mL was considered as the highest concentration for the gene mutation test.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
1) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) The increase is concentration-related when evaluated with an appropriate trend test.
3) Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.

A test chemical is considered clearly negative if, in all experimental conditions examined:
1) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2) There is no concentration-related increase when evaluated with an appropriate trend test.
3) All results are inside the distribution of the historical negative/vehicle control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula: Y=(X+A)eB, where Y = transformed mutant frequency, X = observed mutant frequency and A, B = constants (viz. A = 1 and B = 0.15).
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0. If the analysis of variance was significant at p < 0.05, Dunnett’s test would be conducted, comparing each treatment group and the positive control to the vehicle control.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO AA8 Cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No change in pH was observed in any of the test concentrations.
- Possibility of evaporation from medium: no
- Precipitation and time of the determination: moderate precipitation was observed at 0.0625.

RANGE-FINDING/SCREENING STUDIES (if applicable):

The results of the initial cytotoxicity test showed that the Relative survival is more than 10% at the test item treated concentrations of 00390625, 0.0078125, 0.015625, 0.03125 and 0.0625 mg/mL in both with metabolic and without metabolic activation, when compared with the vehicle control. Hence 0.0625 mg/mL was considered as highest concentration for the gene mutation test.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: There was statistically significant increase in mutant frequencies for positive controls when compared with the vehicle control in both metabolic activation and absence of metabolic activation.

Gene mutation tests in mammalian cells:

- Genotoxicity results:
There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
Treatment with test item resulted in mutant frequencies which fell within acceptable ranges with regard to historical controls.

Conclusions:

In an in vitro HPRT gene mutation test using CHO AA8 cells, the test item is considered as non-mutagenic up to the concentration of 0.0625 mg/mL, both in presence and absence of metabolic activation.

Executive summary:

A gene mutation study at HPRT gene using CHO AA8 cells was performed for the test item, with and without metabolic activation (±S9), according to OECD 476 Guideline (GLP study). Based on the results of the solubility, pH and precipitation tests, the test item was formulated in DMSO as vehicle and 0.0625 mg/mL was selected as the highest concentration in an initial cytotoxicity test. This test was conducted at concentrations of 0.00390625, 0.0078125, 0.015625, 0.03125 and 0.0625 mg/mL. There was no evidence of cytotoxicity (>10% RS) at and up to 0.0625 mg/mL in both presence and absence of metabolic activation when compared to vehicle control. Therefore, 0.0625 mg/mL was selected as the highest concentration in the gene mutation test. The main test was conducted at the concentrations of 0.0078125, 0.015625, 0.03125 and 0.0625 mg/mL with an exposure time of 3 h and 2 min both in the presence and absence of metabolic activation and 6-Thioguanine as the selective agent. DMSO alone was tested as solvent control and Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as positive controls. Parallel cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival. There was no statistically significant increase in number of mutant colonies at any of the concentrations tested when compared with the vehicle control. All results were inside the distribution of the historical vehicle control data. Positive controls resulted in mutant frequencies, which were statistically significant when compared with the vehicle control and within the historical positive control data. Based on these results, the test item is considered as non-mutagenic up to the concentration of 0.0625 mg/mL, both in the presence and absence of metabolic activation under the tested conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.