2,5-di-tert-butylhydroquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames (OECD471): non-mutagenic

Chromosome Aberration test (OECD473): non-clastogenic

Gene Mutation test (OECD476): non-mutagenic

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The test substance is identified as di-tert-butyl hydroquinone. The purity is 99.6%. The physical state/appearance of material is white solid. The expiry date of material is 1 March February 2019. The substance can be stored at room temperature in the dark conditions.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

the E. coli tester strain contains a uvrA- DNA repair deficiency

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

With and without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.For With Metabolic Activation system the procedure followed was same mentioned above in addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
With and without Metabolic Activation

0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate. For With Metabolic Activation system the procedure followed was same mentioned above in addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Some manual counts were performed due to spreading colonies to ensure an accurate count.

Test for Mutagenicity: Confirmatory Experiment – Pre-Incubation Method
As Experiment 2 was concluded to be positive in TA1535 only, a third, confirmatory experiment was performed using the pre-incubation method in the presence and absence of metabolic activation.


Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Some manual counts were performed due to spreading colonies to ensure an accurate count

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

other: Weakly

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Di-tert-butyl hydroquinone was considered to be weakly mutagenic under the conditions of this test only in S. typhimurium strain TA1535 when utilizing the pre-incubation method. The test item di-tert-butyl hydroquinone was considered not to be mutagenic in other S. typhimurium strains or E. coli under the conditions of this test.

Executive summary:

The OECD GLP study was conducted to evaluate the mutagenicity of di-tert-butyl hydroquinone by a reverse mutation test using Salmonella typhimurium and Escherichia coli strains. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with di-tert-butyl hydroquinone using both the Ames plate incorporation and pre-incubation methods, both with and without the addition of a rat liver homogenate metabolizing system. The dose range for experiment was 15 to 5000 μg/plate.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the mutation tests; plate incorporation method and pre-incubation method. However, the test item induced statistically significant and reproducible increases in the frequency of TA1535 revertant colonies both with and without metabolic activation (S9-mix) in pre-incubation method. In pre-incubation method, statistically significant increases in revertant colony frequency were observed at and above 500 μg/plate in the absence of S9-mix and at and above 150 μg/plate in the presence of S9-mix. In the confirmatory experiment, statistically significant increases were observed at and above 500 μg/plate (1500 to 5000 μg/plate and 500 μg/plate) in the absence of S9-mix and at all dose levels tested in the presence of S9-mix (all dose levels were). di-tert-butyl hydroquinone was weakly mutagenic under the conditions of this test only in S. typhimurium tester strain TA1535 when utilizing the pre-incubation method. The test item di-tert-butyl hydroquinone was considered not to be mutagenic in the remaining S. typhimurium strains or E. coli under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 October 2019 - 08 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Name of Test Item : Yapox 2245
Chemical Name (IUPAC) : 2,5-Di-tertiary butyl hydroquinone
CAS No. : 88-58-4
Physical Appearance (with color) : White to light tan crystalline powder
Batch No. : 20015011219
Purity (Declared by sponsor and/or as per Certificate of Analysis) : 99.37%
Batch Produced by (Name and Address) : YASHO INDUSTRIES LIMITED
Plot No. 2514/2515, Phase IV,
G.I.D.C., Vapi-396195, Gujarat, India
Date of Manufacture : June 2019
Date of Expiry : May 2021
Storage Conditions : Ambient (21 to 29 ºC )
Test Item Code by Test Facility : D819-002

Species / strain / cell type:

primary culture, other: primary cell cultures derived from healthy human donor.

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human peripheral lymphocytes from the blood of healthy, young, non-smoking donors 24 (Female) and 29 (Male)with no known recent exposure to genotoxic chemicals or radiation were used.
- Suitability of cells: The primary cell cultures of human whole blood were selected on the basis of growth ability in culture, stability of the karyotype. This provides the opportunity to test using the same test system which the in vitro test is predictive of in vivo genotoxic events. Further as per the regulatory requirements the human peripheral blood lymphocytes is one of the recommended test system.

For lymphocytes:
- Sex, age and number of blood donors: 24 (Female) and 29 (Male)
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: pooled


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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- method of preparation of S9 mix : Sodium phenobarbitone and β-Naphthoflavone induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar Rats induced with intraperitoneal injection of sodium phenobarbtone 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.
- 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:

Initial cytotoxicity test:
Based on the results of solubility, precipitation and pH tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the chromosomal aberration test. The concentrations selected for initial cytotoxicity test were 0.0156, 0.0312, 0.0625, 0.125 and 0.25 µL/mL.
Set No. Metabolic activation Treatment details Duration of treatment for Initial cytotoxicity test
1 +S9 Vehicle control/Test item 3 Hours & 19 minutes
2 - S9 Vehicle control/Test item 3 Hours & 19 minutes
3 - S9 Vehicle control/Test item 21 Hours and 49 minutes

Cell cultures treated with Yapox 2245 at the concentrations of 0.0156, 0.03125, 0.0625, 0.125 and 0.25 mg/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) showed reduction in mitotic index and the observed values were 48.02%, 66.07%, 71.00%, 80.71% and 97.26% respectively. In the absence of metabolic activation (short term treatment 3 to 6 hours) the obtained reduction in mitotic index was 48.36%, 66.04%, 73.11%, 80.81% and 97.47% respectively. In the absence of metabolic activation (long term treatment 20 to 24 hours) the obtained reduction in mitotic index was 48.45%, 66.62%, 70.49%, 81.83% and 97.81% at 0.0156, 0.03125, 0.0625, 0.125 and 0.25 mg/mL respectively.

Chromosomal aberration test:
Based on the results of cytotoxicity test, the concentrations selected for the chromosomal aberration test were 0.0039, 0.0078 and 0.0156 mg/mL of 2,5-di-tert-butylhydroquinone as low, mid and high concentrations respectively.
Set No. Metabolic Activation Treatment Duration
1. +S9 Vehicle control/test item/positive control (10 µg/mL of Cyclophosphamide Monohydrate) 3 Hours and 24 minutes
2. -S9 Vehicle control/test item/positive control (0.05 µg/mL Mitomycin-C) 3 Hours and 24 minutes
3. -S9 Vehicle control/Test item/Positive control (0.05 µg/mL Mitomycin-C) 21 Hours and 20 minutes

Vehicle / solvent:

- Vehicle:dimethyl sulphoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:

- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:

- Exposure duration/duration of treatment: both with metabolic activation (+S9) (set 1) for 3 to 6 hours, without metabolic activation (-S9) - set 2 for 3 to 6 hours and without metabolic activation (-S9) for 20 to 24 hours set 3 at 37±1ºC and 5±1% CO2

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Before 1 to 3 hours of harvesting, colchicine of concentration 0.3 µg/mL was added to all the tubes of set 1, 2 and 3. Post incubation of 1 to 3 hours with colchicine, cell suspension was collected to pre labeled tubes and centrifuged for 10 minutes at 1500 rpm.
Pellets were mixed with 4 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 4 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 2200 rpm for 10 minutes. The procedure was repeated twice by adding 3 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 was air dried.
Minimum of 3 slides were prepared for each treatment replicate. Slides were stained using 5% Giemsa stain for 15 minutes.


SLIDE EVALUATION
• Coding of slides was not carried out for initial cytotoxicity test. For each replicate minimum of 500 cells were scored.
• Percent mitotic index (MI %) was determined by the following formula
Mitotic Index
MI% = Number of Mitotic cells × 100
Total number of cells scored
Percent reduction in mitotic index was obtained by using the formula:
= [(Percentage MI of VC - Percentage MI of treated)/Percentage MI of VC] ×100
VC: Vehicle Control, MI: Mitotic Index.

Rationale for test conditions:

Based on the results of cytotoxicity test, the concentrations selected for the chromosomal aberration test were 0.0039, 0.0078 and 0.0156 mg/mL of Yapox 2245 as low, mid and high concentrations respectively.

Evaluation criteria:

Acceptance of a test is based on the following criteria:
• The concurrent negative/vehicle controls should ideally be within the 95% control limits of the distribution of the laboratory’s historical negative/vehicle control database.
• Concurrent positive controls should produce a statistically significant increase compared with the concurrent negative/vehicle control and it positive controls should induce responses that are compatible with those generated in the historical positive control data base.
• Adequate number of cells (at least 300 well spread metaphases per concentration) and concentrations (at least three analyzable concentrations) was analyzed.
• The criteria for the selection of concentrations for chromosomal aberration test was fulfilled.

Interpretation of Results:
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
• The increase is dose-related when evaluated with an appropriate trend test.

The test item is then considered to be able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all experimental conditions examined:
• None of the test item concentrations exhibits a statistically significant increase compared with the concurrent negative/vehicle control.
• There is no concentration-related increase when evaluated with an appropriate trend test.
• All results are inside the distribution of the historical vehicle control data.
The test item is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

Statistics:

Data (Percentage of cells with aberrations) 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) and the statistical significance was designated by the superscripts in the report as stated below:
* Statistically significant (P<0.05) change than the vehicle control group.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Heavy precipitation was observed at 0.5, 1 and 2 mg/mL in the precipitation test. In the initial cytotoxicity test over 50% cytotoxicity was observed at and above 0.03125 mg/mL.

Vehicle controls validity:

valid

Untreated negative 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 concentration tested.
- Data on osmolality: not available
- Possibility of evaporation from medium: not evaluated
- Water solubility: Test item was soluble in dimethyl sulphoxide at 200 mL/mL.
- Precipitation and time of the determination: Precipitation test was conducted at 0.0156 0.0312, 0.0625, 0.125, 0.25, 0.5, 1 and 2 µL/mL. Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 21 hours 15 minutes of incubation, heavy precipitation was observed at 0.5, 1 and 2 mg/mL. Mild and moderate precipitation was observed at 0.125 and 0.25 mg/mL respectively. No precipitation was observed in 0.03125 mg/mL and 0.0625 mg/mL.

RANGE-FINDING/SCREENING STUDIES (if applicable):
Initial cytotoxicity test
Cell cultures treated with Yapox 2245 at the concentrations of 0.0156, 0.03125, 0.0625, 0.125 and 0.25 mg/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) showed reduction in mitotic index and the observed values were 48.02%, 66.07%, 71.00%, 80.71% and 97.26% respectively. In the absence of metabolic activation (short term treatment 3 to 6 hours) the obtained reduction in mitotic index was 48.36%, 66.04%, 73.11%, 80.81% and 97.47% respectively. In the absence of metabolic activation (long term treatment 20 to 24 hours) the obtained reduction in mitotic index was 48.45%, 66.62%, 70.49%, 81.83% and 97.81% at 0.0156, 0.03125, 0.0625, 0.125 and 0.25 mg/mL respectively.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
Positive control, 10 µg/mL of Cyclophosphamide Monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 10.00% of aberrated cells which was statistically significant compared to the vehicle control (0.67%). The reduction in mitotic index was 7.14% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 10.67% of aberrated cells which was statistically significant to the vehicle control (1.00%). The reduction in mitotic index observed was 8.14% when compared with the vehicle control for short term treatment.
Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (1.00%). The reduction in mitotic index observed was 6.78% when compared with the vehicle control for long term treatment.

Conclusions:

Based on the results obtained, the test item, 2,5-di-ter-butylhydroquinone is considered as non-clastogenic up to the concentration of 0.0156 mg/mL both in the presence and absence of metabolic activation under the presented test conditions.

Executive summary:

The test item,Yapox 2245 obtained fromYasho Industries Limited was evaluated for chromosomal aberrations in human lymphocytes, as per the OECD guideline for the testing of chemicals, No. 473 “In vitro Mammalian Chromosomal AberrationTest” adopted on 29^thJuly 2016.

Test item was miscible in dimethyl sulphoxide at 200 mg/mL.Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 22 hours and 15 minutes of incubation, heavy precipitation was observed at 0.5, 1 and 2 mg/mL, mild and moderate precipitation was observed at 0.125 and 0.25 mg/mL respectively. No precipitation was observed at 0.03125 and 0.0625 mg/mL. No change in pH was observed in any of the concentration tested hence, 0.25 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.0156, 0.3125, 0.0625 and 0.125 mg/mL of test item.

In initial cytotoxicity test,the percentage reduction in Mitotic Index was in the range of 66.04% to 98.71% at 0.03125, 0.0625, 0.0125 and 0.25 mg/mL. The percentage reduction in Mitotic Index was in the range of 48.02%  to 48.45% at 0.0156mg/mL. As the percentage reduction in MI was not more than 45±5% at 0.0156 mg/mL, same has been selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.0039 mg/mL and 0.0078 mg/mL.

In the chromosomal aberration test, the cells were treated with Yapox 2245 at the concentrations of 0.0039, 0.0078 and 0.0156 mg/mL using DMSO as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mitomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, thecells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.

The observed mean percent aberrated cells at 0.0039, 0.0078 and 0.0156 mg/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 0.67, 1.00 and 1.00 respectively. Similarly, the observed mean percent aberrated cells at 0.0039, 0.0078 and 0.0156 mg/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 1.00, 1.34 and 1.00 respectively.

The observed mean percent aberrated cells at 0.0039, 0.0078 and 0.0156 mg/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.00, 1.33 and 1.67 respectively.

Positive control, 10 µg/mL of Cyclophosphamide Monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 10.00% of aberrated cells which was statistically significant compared to the vehicle control (0.67%). The reduction in mitotic index was 7.14% when compared with the vehicle control for short term treatment.

Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 10.67% of aberrated cells which was statistically significant to the vehicle control (1.00%). The reduction in mitotic index observed was 8.14% whencompared with the vehicle control for short term treatment.

Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (1.00%). The reduction in mitotic index observed was 6.78% when compared with the vehicle control for long term treatment.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 October 2019 - 08 December 2019

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)

GLP compliance:

yes

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:

Name of Test Item : YAPOX 2245
Chemical Name (IUPAC) : 2,5-Di-tertiary butyl hydroquinone
CAS No. : 88-58-4
Physical Appearance (with color) : White to light tan Crystalline powder
Batch No. : 20015011219
Purity As per Certificate of Analysis) : 99.37%
Batch Produced by (Name and Address) : YASHO INDUSTRIES LIMITED
Plot No. 2514/2515, Phase IV,
G.I.D.C., Vapi – 396195,
Gujarat, India
Date of Manufacture : June 2019
Date of Expiry : May 2021
Storage Conditions : Ambient (21 to 29°C)
Test Item Code by Test Facility : D819-002

Target gene:

Hprt locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO AA8 , American Type Culture Collection (ATCC)
- Suitability of cells: CHO AA8 cells are one of the recommended test systems by regulatory agencies for conducting In vitro Mammalian gene mutation Test.

For cell lines:
- Absence of Mycoplasma contamination: Cells free of mycoplasma will be used for the experiment.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: 10% FBS with antibiotics (1% streptomycin and penicillin) and incubated at 37±1°C and 5±1% CO2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Based on the results of solubility, pH and precipitation tests, an initial cytotoxicity test was conducted for the selection of test concentrations for the gene mutation test. Five concentrations of 0.0078125, 0.015625, 0.03125, 0.0625 and 0.125 mg/mL of the test item were tested in an initial cytotoxicity test.

Vehicle / solvent:

- Vehicle: DMSO
- Justification for choice of solvent/vehicle: The test substance was dissolved in DMSO. Test substance concentrations were used within 2 hours after preparation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

3 µg/ml

Positive control substance:

benzo(a)pyrene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/ml

Positive control substance:

4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Each treatment group was maintained with tetra plate cultures. Cells were exposed to the test item for 3 hours for both with exogenous metabolic activation and without exogenous metabolic activation respectively in the gene mutation test at 37±1oC with 5±1% CO2.
The treatment schedule for the experiment was maintained as indicated in the following table:
Set No. MetabolicActivation Treatment Duration
1 +S9 Vehicle Control/Test item/Positive Control (3 µg/mL of Benzo(a)pyrene) 3 hours
2 - S9 Vehicle Control/Test item/Positive Control (1 µg/mL of4 Nitroquinoline N-oxide) 3 hours
Tetra plate treatments were pooled into a pre labeled tube and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded and cell pellet was retained.
Each treatment replicate was plated in triplicate with cell concentration of 200 cells/5 mL media in 25 cm2 flasks and incubated at 37±1oC with 5±1% CO2 for 8 days.
The replicate cultures were subcultured in duplicates at a density of 1×106 cells/culture flask. Cells were incubated at 37±1oC with 5±1% CO2, followed by sub culturing with an interval of 2 to 3 days for the remaining 8 days of expression period.
Post expression period of 8 days of mutant phenotype, each replicate treatment cultures were pooled and sub cultured in quintuplicates at a density of 4×105 cells per 25 cm2 flask with culture media containing 10 µM of 6-Thioguanine and 200 cells /25 cm2 flask in triplicates without 6-Thioguanine for determination of cloning efficiency. Flasks were incubated at 37±1°C with 5±1% CO2 for 10 days. Post incubation period, medium from each dish was aspirated and stained with 5% Giemsa stain, number of colonies formed were counted manually.


DETERMINATION OF CYTOTOXICITY:
For tests with exogenous metabolic activation, 1 mL of S9 mix was added to all the flasks. A volume of 100 µL of vehicle/different concentrations of test item was added to tetra plate cultures to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours and 5 minutes at 37±1oC with 5±1% CO2.
For tests without exogenous metabolic activation, a volume of 100 µL of vehicle/different concentrations of test item was added to tetra plate culture to get the required test concentration per mL of the test medium and volume of medium was made up to 10 mL. Cells were exposed to the test item for 3 hours and 5 minutes at 37±1oC with 5±1% CO2.
Post incubation period (Set 1 and 2), medium from each flask was aspirated and monolayer was washed with DPBS. Cells were trypsinized by adding trypsin-EDTA. Trypsinization was stopped by adding culture media followed by collecting the media with cells.
Tetra plate treatments were pooled and collected in prelabelled tubes and centrifuged at 800 rpm for 10 minutes. Supernatant was discarded and cell pellet was retained and resuspended in culture media.
Each treatment replicate was plated in triplicate with cell concentration of 200 cells / 5 mL media in 25 cm2 flasks and incubated at 37±1oC with 5±1% CO2 for 9 days.
Post incubation period, medium from each culture flask was aspirated and stained with 5% Giemsa stain. Number of colonies formed was counted manually

Evaluation criteria:

Acceptance of a test is based on the following criteria:
• The concurrent vehicle control is considered acceptable for addition to the laboratory historical vehicle control database as described in OECD guidelines for testing of chemicals, No. 476.
• Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative/vehicle control.
• Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
• Adequate number of cells and concentrations are analysable (according to OECD guidelines for testing of chemicals, No. 476).
• The criteria for the selection of top concentration are consistent with those described in OECD guidelines for testing of chemicals, No. 476.

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups using SPSS Software version 22 at a 95% level (p<0.05) of significance. The statistical significance was designated by the superscripts in the report as stated below:
* Statistically significant (p<0.05) change than the vehicle control group.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

there was no evidence of excessive cytotoxicity (˂10% RS) at and up to 0.125 mg/mL in both presence of metabolic activation and absence of metabolic activation when compared to vehicle control.

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

- Data on pH: pH tested at 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 and 2 mg/mL concentrations. No change in pH was observed in any of the test concentrations.
- Precipitation and time of the determination: The precipitation was tested at 0.03125, 0.0625, 0.125, 0.25, 0.5 and 1 and 2 mg/mL concentrations. Post 3 hours and 4 minutes of incubation, no change in precipitation was observed at the concentrations tested at 0.03125, 0.0625 mg/mL, mild precipitation was observed at 0.125 mg/mL, moderate precipitation was observed at 0.25 mg/mL and heavy precipitation was observed at 0.5, 1 and 2 mg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the initial cytotoxicity test, there was no evidence of excessive cytotoxicity (˂10% RS) at and up to 0.125 mg/mL in both presence of metabolic activation and absence of metabolic activation when compared to vehicle control. In the presence of metabolic activation, the RS values ranged from 65.55% to 91.60% and in the absence of metabolic activation, the RS values ranged from 58.41% to 93.81% in the concentrations of 0.0078125, 0.015625, 0.03125, 0.0625, and 0.125 mg/mL.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)

Vehicle - DMSO
With Metabolic Activation Without Metabolic Activation
(3 to 6 hours) (3 to 6 hours)

Mean Data of Mutant Frequency/2x106 Cells 24.51 25.43
Standard
Deviation 2.81 1.89
Margin of Error 1.95 1.31
Upper bound 26.46 26.74
Lower bound 22.56 24.12

Positive Control - Benzo(a)pyrene and 4- Nitroquinoline N-oxide
With Metabolic Activation [Benzo(a)pyrene] Without Metabolic Activation
(3 to 6 hours) (3 to 6 hours)

Mean Data of Mutant Frequency/2x106 Cells 261.94 264.60
Standard
Deviation 27.28 18.52
Margin of Error 17.82 12.10
Upper bound 279.76 276.70
Lower bound 244.12 252.50

Conclusions:

Based on the results obtained, the test item, YAPOX 2245 is considered as non-mutagenic at and up to the concentration of 0.125 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test item YAPOX 2245 was evaluated for gene mutation test in CHO AA8 cells, as per the OECD guideline for the testing of chemicals,No. 476 “In vitro Mammalian Cell Gene Mutation Tests using the hprt and xprt genes” adopted on 29^thJuly 2016. The test item was found soluble in dimethyl sulphoxide (DMSO) at 200 mg/mL. Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL. Post 3 hours and 4 minutes of incubation, no change in precipitation was observed at the tested concentrations at 0.03125, 0.0625 mg/mL, mild precipitation was observed at 0.125 mg/mL, moderate precipitation was observed at 0.25 mg/mL and heavy precipitation was observed at 0.5, 1 and 2 mg/mL. No change in pH was observed in any of the test concentrations. On the basis of the results 0.125 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.0078125, 0.015625, 0.03125, 0.0625 and 0.125 mg/mL using DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 to 6 hours).

The results of the initial cytotoxicity test indicated that the Relative Survival is greater  than 10% at 0.125 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.125 mg/mL was selected as highest concentration for gene mutation test.

In the gene mutation test, the cells were treated with test item at the concentrations of 0.015625, 0.03125, 0.0625 and 0.125mg/mL using DMSO as the vehicle in tetra plate cultures both in the presence of metabolic activation and absence of metabolic activation. The test item, resulted in mutant frequencies of 23.08 to 25.84 per 2×10⁶ cells in the presence of metabolic activation with 25.81 per 2×10⁶cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 24.72 to 25.84 per 2×10⁶cells were observed with 23.33 per 2×10⁶cells in the vehicle control. There was no statistically significant increase in the mutant frequencies observed when compared with vehicle control at any of the tested concentrations. There was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 72.97 to 93.69% and in the absence of metabolic activation the RS values ranged from 76.32% to 91.23% respectively. Application of the positive control, 3 µg/mL of Benzo (a) pyrene, resulted in a RS value of 71.17% in the presence of metabolic activation, and a mutant frequencyof 273.08 per 2×10⁶ cells which wasstatistically significant when compared with the vehicle control.

The treatment with the positive control, 1 µg/mL of 4-Nitroquinoline N-oxide, resulted in a RS value 69.30% in the absence of metabolic activation and a mutant frequency of 255.26 per 2×10⁶ cells and was statistically significant when compared with that of vehicle control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

AMES TEST

Bacterial mutagenicity test was conducted as per the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia colistrain WP2uvrA were treated with di-tert-butyl hydroquinone using both the Ames plate incorporation and pre-incubation methods at up to dose range of 5000 μg/plate. The test item induced statistically significant and reproducible increases in the frequency of TA1535 revertant colonies both with and without metabolic activation in preincubation method. No significant increases in the frequency of revertant colonies were recorded in any of the remaining bacterial strains.

Di-tert-butyl hydroquinone was considered to be weakly mutagenic under the conditions of this test only in S. typhimurium strain TA1535 when utilizing the pre-incubation method. The test item di-tert-butyl hydroquinone was considered not to be mutagenic in other S. typhimurium strains or E. coli under the conditions of this test.

CYTOGENICITY TEST

The test item,Yapox 2245 obtained fromYasho Industries Limitedwas evaluated for chromosomal aberrations in human lymphocytes, as per the OECD guideline for the testing of chemicals, No. 473 “In vitroMammalian Chromosomal AberrationTest” adopted on 29^thJuly 2016.

Test item was miscible in dimethyl sulphoxide at 200 mg/mL.Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/mL. Post 22 hours and 15 minutes of incubation, heavy precipitation was observed at 0.5, 1 and 2 mg/mL, mild and moderate precipitation was observed at 0.125 and 0.25 mg/mL respectively. No precipitation was observed at 0.03125 and 0.0625 mg/mL. No change in pH was observed in any of the concentration tested hence, 0.25 mg/mL was selected as highest concentration for testing in the initial cytotoxicity test. The other concentrations selected were 0.0156, 0.3125, 0.0625 and 0.125 mg/mL of test item.

In initial cytotoxicity test,the percentage reduction in Mitotic Index was in the range of 66.04% to 98.71% at 0.03125, 0.0625, 0.0125 and 0.25 mg/mL. The percentage reduction in Mitotic Index was in the range of 48.02%  to 48.45% at 0.0156mg/mL. As the percentage reduction in MI was not more than 45±5% at 0.0156 mg/mL, same has been selected as the highest concentration for the chromosomal aberration test. Other concentrations tested were 0.0039 mg/mL and 0.0078 mg/mL.

In the chromosomal aberration test, the cells were treated withYapox 2245 at the concentrations of 0.0039, 0.0078 and 0.0156 mg/mL using DMSO as the vehicle. The treatment was carried out in duplicates for the short term period (3 to 6 hours) both in the presence and absence of metabolic activation and for the long term period (20 to 24 hours) in the absence of metabolic activation. Cyclophosphamide Monohydrate (+S9 for short term) at the concentration of 10 µg/mL and Mitomycin-C at the concentration of 0.05 µg/mL (-S9 both for short term and long term) were used as positive controls.

The treated cells were harvested at about 1.5 normal cell cycle length after treatment. During harvesting of cultures, thecells were treated with a metaphase-arresting substance (colchicine), harvested, stained and metaphase cells were analysed microscopically for the structural chromosomal aberrations.

The observed mean percent aberrated cells at 0.0039, 0.0078 and 0.0156 mg/mL in the presence of metabolic activation (short term treatment 3 to 6 hours) were 0.67, 1.00 and 1.00 respectively. Similarly, the observed mean percent aberrated cells at 0.0039, 0.0078 and 0.0156 mg/mL in the absence of metabolic activation (short term treatment 3 to 6 hours) were 1.00, 1.34 and 1.00 respectively.

The observed mean percent aberrated cells at 0.0039, 0.0078 and 0.0156 mg/mL in the absence of metabolic activation, long term (20 to 24 hours) were 1.00, 1.33 and 1.67 respectively.

Positive control, 10 µg/mL of Cyclophosphamide Monohydrate, in the presence of metabolic activation (3 to 6 hours), induced 10.00% of aberrated cells which was statistically significant compared to the vehicle control (0.67%). The reduction in mitotic index was 7.14% when compared with the vehicle control for short term treatment.

Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (3 to 6 hours), induced 10.67% of aberrated cells which was statistically significant to the vehicle control (1.00%). The reduction in mitotic index observed was 8.14% whencompared with the vehicle control for short term treatment.

Positive control, 0.05 µg/mL of Mitomycin-C, in the absence of metabolic activation (20 to 24 hours), induced 10.34% of aberrated cells which was statistically significant to the vehicle control (1.00%). The reduction in mitotic index observed was 6.78% when compared with the vehicle control for long term treatment.

GENE MUTATION TEST

The test itemYAPOX 2245was evaluated for gene mutation test in CHO AA8 cells, as per the OECD guideline for the testing of chemicals,No. 476 “In vitroMammalian Cell Gene Mutation Tests using thehprtandxprtgenes” adopted on 29^thJuly 2016.The test item was found soluble in dimethyl sulphoxide (DMSO) at 200 mg/mL. Precipitation test was conducted at 0.03125, 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL. Post 3 hours and 4 minutes of incubation, no change in precipitation was observed at the tested concentrations at 0.03125, 0.0625 mg/mL, mild precipitation was observed at 0.125 mg/mL, moderate precipitation was observed at 0.25 mg/mL and heavy precipitation was observed at 0.5, 1 and 2 mg/mL. No change in pH was observed in any of the test concentrations. On the basis of the results 0.125 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.0078125, 0.015625, 0.03125, 0.0625 and 0.125 mg/mL using DMSO as a vehicle in tetra plates/group in the presence and absence of metabolic activation (3 to 6 hours).

The results of the initial cytotoxicity test indicated that the Relative Survival is greater  than 10% at 0.125 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.125 mg/mL was selected as highest concentration for gene mutation test.

In the gene mutation test, the cells were treated with test item at the concentrations of 0.015625, 0.03125, 0.0625 and 0.125mg/mL usingDMSO as the vehicle in tetra plate cultures both in the presence of metabolic activation and absence of metabolic activation.The test item, resulted in mutant frequencies of 23.08 to 25.84 per 2×10⁶cells in the presence of metabolic activation with 25.81 per 2×10⁶cells in the vehicle control. In the absence of metabolic activation, mutant frequenciesof 24.72to 25.84per 2×10⁶cells were observed with 23.33 per 2×10⁶cells in the vehicle control. There was no statistically significant increase in the mutant frequencies observed when compared with vehicle control at any of the tested concentrations.There was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 72.97 to 93.69% and in the absence of metabolic activation the RS values ranged from 76.32% to 91.23% respectively.Application of the positive control, 3 µg/mL of Benzo (a) pyrene, resulted in a RS value of 71.17% in the presence of metabolic activation, and a mutant frequencyof 273.08 per 2×10⁶cellswhich wasstatistically significant when compared with the vehicle control.

The treatment with the positive control, 1 µg/mL of 4-Nitroquinoline N-oxide, resulted in a RS value 69.30% in the absence of metabolic activation and a mutant frequencyof255.26per 2×10⁶cellsand wasstatistically significant when compared with that of vehicle control.

Justification for classification or non-classification

Based on the WoE approach (in vitro studies) the substance is not classified for mutagenicity according to the CLP Regulation No. 1272/2008.