Registration Dossier

Diss Factsheets

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria (OECD 471): negative

- Gene mutation in mammalian cells (OECD 476): negative

- Cytogenicity / micronucleus study in mammalian cells (OECD 487): negative (3-h treatment; -/+ S9); positive (24-h treatment; -S9)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 - 22 Oct 2009
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:
Adopted in 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S. typhimurium strains)
trp operon (E. coli)
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:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague-Dawley rats treated with Phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
First experiment (dose setting): 2.29, 6.86, 20.6, 61.7, 185, 556, 1667 and 5000 µg/plate with and without metabolic activation in all tested strains.
Second experiment (main): 313, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation in all strains except S. typhimurim TA 1537.

With strain TA 1537, growth inhibition occured in the dose setting study, so doses in the main study were adjusted accordingly, as follows:
TA 1537 (-S9): 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
TA 1537 (+S9): 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle/solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-furyl)3-(5-nitro2-furyl)acrylamide [AF-2] in DMSO; 2-Aminoanthracene [2-AA] in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation was utilized as the application method in both the dose setting and the main studies.
- Cell density at seeding (if applicable): for use in studies viability required to be ≥1 x 10E9 cells/mL.

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 h at 37 °C

NUMBER OF REPLICATIONS: triplicate in each of the dose setting and main studies

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition (antibacterial activity) and examination of the bacterial background lawn.
Evaluation criteria:
Results were considered positive if the relevant colony count increased in tandem with dose concentration, and the increase was at least double that of the negative control, and the result was reproducible.

Results were considered negative if none of the positive results were observed.
Statistics:
No statistics were used for the data analysis.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
precipitation was observed from 556 µg/plate with/without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
main study (experiment 2) without S9: ≥156 µg/plate; main study (experiment 2) with S9: ≥313 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Remarks:
precipitation was observed from 556 µg/plate with/without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Remarks:
precipitation was observed from 556 µg/plate with/without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
no cytotoxicity
Remarks:
precipitation was observed from 556 µg/plate with/without metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was noted at ≥556 µg/plate of test substance with and without metabolic activation in both studies.


Test Period: From Nov 2007 to Jun 2008

Negative Control (Revertants per plate)
Bacterial Strain TA 100 TA 1535 WP2uvrA TA 1537 TA 98
Strain S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 102 107 12 12 69 97 9 12 17 22
SD 8 11 3 3 10 13 3 4 4 2
Min 78 81 4 2 44 61 2 0 5 16
Max 126 132 19 22 93 134 15 24 30 28


Positive Control (Revertants per plate)
Bacterial Strain TA 100 TA 1535 WP2uvrA TA 1537 TA 98
Strain S9-mix -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Mean 640 1523 360 289 1064 901 558 240 479 508
SD 42 115 39 21 167 65 106 31 39 48
Min 552 1224 268 225 646 760 338 163 364 395
Max 728 1823 451 354 1482 1042 778 317 593 620
Conclusions:
Under the conditions of this OECD Guideline 471 study, the test substance AF-317, was found to be negative for mutagenicity with and without metabolic activation.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 May 2019 - 25 June 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 using the Hprt and xprt genes)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hprt gene
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: V79 (Lot No. 15H003), obtained from ECACC (European Collection of Cell Cultures), UK
- Suitability of cells: Cell line is well established; the stability of the karyotype and morphology makes this cell line suitable for gene mutation studies, with a low background spontaneous mutation rate

For cell lines:
- Number of passages if applicable: from 3rd to 4th passages
- Methods for maintenance in cell culture: For each experiment with duplicate cultures in the absence and presence of S9 one vial was thawed rapidly; the cells were diluted in DMEM and incubated at 37°C in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of Petri dishes.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Dulbecco’s Modified Eagle’s medium (DMEM) with 4.5 g/L of glucose supplemented with L-glutamine, 10% FCS (not during treatment), 100 units/mL penicillin and 100 µg/mL streptomycin
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not used
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Sprague Dawley male rats weighing approximately 322 g
- method of preparation of S9 mix: Animals were pretreated with 20-methylcholanthrene to stimulate mixed function oxidases in the liver. 20-methylcholanthrene was administered via intraperitoneal injection at 80 mg/kg bw 5 days prior to sacrifice. Preparation of S9 fraction (protein content 50 mg/L)was performed according to literature and stored in liquid nitrogen.
- concentration or volume of S9 mix and S9 in the final culture medium: The S9-mix was prepared with the following composition: 3 mL of S9 fraction; 1 mL of 40 mM NADP; 1 mL of 50 mM glucose-6-phosphate; 1 mL of 330 mM KCl; 1 mL of 50 mM MgCl2; 2 mL of 20 mM HEPES buffer; 1 mL of deionised H2O (total volume 10 mL). It was added to the culture medium at a final concentration of 10%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Activity of S9 fraction was determined in bacterial reverse gene mutation test on Salmonella typhimurium strains TA 98 with a positive control of 2-aminoanthracene at 0.005 mg/plate.
Test concentrations with justification for top dose:
- Experiment 1 (-S9): 0, 1, 5, 7.5, 10, 15, 20 and 30 µg/mL (30 µg/mL was disregarded due to unacceptable cytotoxicity)
- Experiment 2 (+S9): 0, 5, 10, 15, 25, 30, 35, 40 and 45 µg/mL (40 and 45 µg/mL was disregarded due to unacceptable cytotoxicity)
The highest concentration tested was limited by cytotoxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO in the presence of emulsifier Tween 80
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated solubility in DMSO in the presence of the emulsifier Tween 80 at the final concentration of at least 200 µg/mL.

Test item solutions were prepared by formulating AF-317 in vehicle immediately prior to dosing. The stock solution was membrane filter-sterilised (Millipore filter, 0.20 µm pore size) and subsequent dilutions made using DMSO. 0.05 mL of test item solution was added to 5 mL of serum free medium. The test item solutions were used within 60 min of initial formulation of the test item.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments : one with and one without metabolic activation

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): At least 1E+06 cells
- Test substance added in medium: yes

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 24 h
- Exposure duration/duration of treatment: 3 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: 10 µg/mL 6-thioguanine
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2E+05 cells/dish; cells were counted by eye

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS; plating efficiency)
- Any supplementary information relevant to cytotoxicity: The cells were plated into 5 Petri dishes/culture (3E+02 cells/dish) and grown in DMEM with serum in order to determine plating efficiency. The plates were incubated at 37 ± 1ºC in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (7 days). Colonies of viable cells were stained and counted.

Evaluation criteria:
The assay is considered valid if the following criteria were met:
• Number of spontaneous mutants of the solvent control cultures are within the range of historical control data.
• The number of induced mutants of positive controls are within the expected range.
• A minimum of four analysable concentrations with mutant frequency data are required.
• The maximum dose tested was either limited by solubility (assessed in post treatment medium); cytotoxicity (%RPE reduced to 10 to 20%) or 10 mM or 2000 µg/mL (which ever was the lower).

For valid data, the test item is considered to induce forward mutation at the hprt locus in Chinese hamster lung V79 cells if:
• At least one of the test concentrations exhibits a statistically significant increase in mutant frequency compared with concurrent negative control. The induced mutant frequency is at least 3-fold greater than that of the negative control.
• The increase is concentration-related when evaluated with an appropriate trend test.
• Any of the results are outside the distribution of the historical negative control data.
Statistics:
For each test concentration summary statistics (average, standard deviation, coefficient of variation, minimum, maximum) was calculated. The same was done for negative and positive control data. Two sample comparison was carried out to identify the test concentration for which median of the observed frequency data is significantly different from the median in the negative control group. Mann Whitney (Wilcoxon) W test was applied for this purpose. Significance level of 0.05 was adopted among statistical analyses to make appropriate conclusions. Statgraphics TM Centurion software (version XV) was employed for statistical data evaluation.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: at 30 µg/mL; Experiment 2: at 40 and 45 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No marked changes of pH of treatment media were observed in the mutation experiments at concentrations up to 45 µg/mL tested as compared to the concurrent vehicle controls.
- Data on osmolality: The osmolality values were within the physiological range for these cells.
- Precipitation and time of the determination: No precipitation observed.

RANGE-FINDING/SCREENING STUDIES (if applicable): Treatment and post-treatment dilution of cell cultures for the cytotoxicity Range-Finder Experiment were performed. In the cytotoxicity experiment, single cultures were used. Final treatment volume of 5 mL was used for Petri dishes (diameter of 90 mm). Following treatment, cells were washed twice with DPBS, trypsinised, adjusted to 3 x 10^2 cells/plate in DMEM with serum and plated out in Petri dishes (5/culture) for determination of plating efficiency. The plates were incubated at 37 ± 1ºC in a humidified incubator gassed with 5% v/v CO2 in air for 7 days. After 7 days plates were removed from the incubator and immediately stained with 1% methylene blue in purified water (0.2 mL for 20 minutes) and viable colonies were identified by eye and counted.

Test concentrations in the range-finding study:
-S9: 0, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24 µg/mL
+S9: 0, 1, 2, 4, 6, 8, 12, 16, 20, 24, 30 µg/mL
+S9: 0, 12.5, 25, 50, 75, 100, 150, 175 µg/mL

The highest concentration tested which gave acceptable survival (measured by relative plating efficiency [RPE] in the range of 10-20 %) was 24 µg/mL in the absence of S9, which gave ~11%. The survival (measured by relative plating efficiency [RPE]) at 50 µg/mL tested in the presence of S9 was ~4 %. Higher concentrations did no longer result in detectable survival.
Therefore, test concentrations up to 30 µg/mL and 45 µg/mL were chosen for the mutation experiments without and with metabolic activation, respectively.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : valid; please refer to the result tables (Table 1 and 2).

For all test methods and criteria for data analysis and interpretation:
- Statistical analysis; p-value if any : 0.05

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
Experiment 1: Seven days after treatment, six concentrations in the absence of S9 were selected to determine viability (at the end of treatment and at the point of mutant frequency determination) and 6TG resistance. The highest concentration plated for viability assessment (% RPE) was 20 µg/mL in the absence of S9, which gave ~18 % and ~13 % RPE, respectively.
Experiment 2: Seven days after treatment, seven concentrations in the presence of S9 were selected to determine viability (at the end of treatment and at the point of mutant frequency determination) and 6TG resistance. The highest concentration plated for viability assessment (% RPE) was 40 µg/mL in the presence of S9. At this level almost complete cytotoxicity (RPE values ~8 % and ~6 %, respectively) was observed. The next highest concentration plated for viability assessment which gave acceptable RPE was 35 µg/mL (~14% and ~11% RPE).

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures : at least 1E+06 cells / petri dish; expression period: 3E+02 cells/dish (5 Petri dishes/culture); sub-culturing was performed as required with the aim, where possible, of retaining at least 2 to 3E+05 cells/dish.
o Number of cells plated in selective and non-selective medium : 2E+05 cells/dish

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data (mutant frequencies (1E-05 ))
EMS: average: 23.49, range: 30.84, SD: 6.36, 95% confidence interval for average: 23.4882 ± 0.126239 [22.2258; 24.7506]; 95% confidence interval for SD: [5.58601; 24.7506]; 10 experiments with a total of 100 counts.
DMBA: average: 34.25, range: 45.60, SD: 11.07, 95% confidence interval for average: 34.2522 ± 2.1972 [32.055; 36.4494]; 95% confidence interval for SD: [9.72247; 12.8636]; 10 experiments with a total of 100 counts.
- Negative (solvent/vehicle) historical control data (mutant frequencies (1E-05)): average: 1.48, range: 4.43, SD: 0.91, 95% confidence interval for average: 1.48356 ± 0.126836 [1.35672; 1.6104]; 95% confidence interval for SD: [0.828354; 1.00871]; 20 experiments with a total of 200 counts.

In Experiment 1 with culture A in the absence of S9, a statistically significant increase (p < 0.05) in mutant frequency (MF) was observed following treatment with AF-317 at concentration of 7.5 µg/mL only. At this level mutant frequency of 19.71 resistant mutants/1E+06 viable cells was obtained, compared to a value of 11.72 resistant mutants /1E+06 viable cells for the vehicle control. The increase in MF was not greater than 3-fold above that of the concurrent vehicle control, with no evidence of an increase in MF at any of the other concentrations tested. At concentration of 7.5 µg/mL cytotoxicity (expressed in terms of RPE at the end of treatment) was reduced to ~59 %. Based on this, the statistical significance at this concentration was considered biologically irrelevant.

In Experiment 1 with culture B in the absence of S9, no statistically significant increases in mutant frequency were observed following treatment with AF-317 at any concentration tested.

In Experiment 2 with culture A in the presence of S9, no statistically significant increases in mutant frequency were observed following treatment with AF-317 at any concentration tested.

In Experiment 2 with culture B in the presence of S9, statistically significant increase (p<0.05) in mutant frequency was observed at concentration of 15 µg/mL only. At this level mutant frequency of 28.43 resistant mutants/1E+06 viable cells was obtained, compared to a value of 18.17 resistant mutants/1E+06 viable cells for the vehicle control. The increase in mutant frequency was not greater than 3-fold above that of the concurrent vehicle control, with no evidence of an increase in MF at any of the other concentrations tested. At concentration of 15 µg/mL level RPE was reduced to ~94%. Mutant frequencies in vehicle controls were within the acceptable range and clear increases in mutant frequency were observed by the positive controls, ethyl methanesulphonate (without S9) and 7,12-dimethyl-benz(a)anthracene (with S9). The assay system was therefore considered to be both sensitive and valid.

Table 1: Experiment I (A, B)a – 3-h exposure without metabolic activation

Concentration

[µg/mL]

% RPE

Mutants per 1E+06 surviving cells

0b

A

100.0

11.72

1

A

103.4

12.94

5

A

80.9

6.50

7.5

A

58.5

19.71*

10

A

54.6

9.27

15

A

19.4

8.06

20

A

17.8

7.50

EMS (600)

A

92.5

191.84*

0b

B

100.0

19.77

1

B

99.6

14.95

5

B

79.7

12.14

7.5

B

53.0

17.08

10

B

41.6

21.94

15

B

17.1

6.98

20

B

12.8

9.53

EMS (600)

B

94.9

201.04*

aA or B - codes for two replicate cultures

bDMSO with Tween 80

*Statistically significant (p<0.05) (Mann-Whitney W test)

Table 2: Experiment II (A, B)a – 3-h exposure with metabolic activation

Concentration

[µg/mL]

% RPE

Mutants per 1E+06 surviving cells

0b

A

100.0

18.64

5

A

96.8

22.32

10

A

94.6

23.55

15

A

91.1

21.62

25

A

46.9

15.28

30

A

20.8

20.89

35

A

13.7

16.07

40

A

8.3c

28.87*

DMBA (5)

A

92.5

173.01*

0b

B

100.0

18.17

5.

B

102.0

11.26

10

B

92.8

24.67

15

B

94.3

28.43*

25

B

56.3

14.06

30

B

22.0

11.92

35

B

10.5

17.45

40

B

6.4c

29.44*

DMBA (5)

B

80.7

203.30*

aA or B - codes for two replicate cultures

bDMSO with Tween 80

cData disregarded due to unacceptable cytotoxicity

*Statistically significant (p<0.05) (Mann-Whitney W test)

Conclusions:
Under the conditions of this OECD Guideline 476 study, the test substance AF-317, was found to be negative for mutagenicity with and without metabolic activation.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 June - 8 Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted in 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Slovak National Accreditaiton Service, Bratislava, Slovak Rebublic
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Lot No. 15H003; obtained from ECACC (European Collection of
Authenticated Cell Cultures), UK
- Suitability of cells: well established, stability of the karyotype and morphology makes this cell line suitable for micronucleus scoring, with high sensitivity for clastogenic and aneugenic chemicals
- Normal cell cycle time (negative control): 12 - 16 h

For cell lines:
- Absence of Mycoplasma contamination: Yes.
- Number of passages if applicable: Cells were subcultured for a maximum of 8 passages and then used to initiate cultures for the assay.
- Methods for maintenance in cell culture: When the cells were growing well, subcultures were established in an appropriate number of Petri dishes. Subculturing was performed 2-3 times a week with the aid of trypsin (0.25%)/EDTA solution and cells were counted in suspension using Bürker’s chamber.
- Cell cycle length, doubling time or proliferation index : 10 - 14 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: Yes
- Specification by DNA bar-coding sequencing of the mitochondrial cytochrome c oxidase (COX) subunit 1 gene. The availability of published PCR primer sequences for this region has made this the gene of choice for speciation using this method. The pass criteria are that the top 5 matches must be the same as the expected species and the percentage matches must be above 95%. Result: Pass
- Sterility testing of Cell Banks: No growth

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Dulbecco’s Modified Eagle’s medium (DMEM) with 4.5 g/L of glucose supplemented with L-glutamine, penicillin (100 units / mL) / streptomycin (100 µg/mL). Cells were incubated at 37°C in a humidified atmosphere of 5% v/v CO2 in air.
- 3 h-treatments with and without S9 were carried out using serum free culture medium.
- 24 h-treatments without S9 were carried out using complete medium containing 2.5% fetal bovine serum.
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Sprague Dawley male rats weighing approximately 322 g
- method of preparation of S9 mix : Animals were pre-treated with 20-methylcholanthrene to stimulate mixed function oxidases in the liver. The 20-methylcholanthrene was administered via intraperitoneal injection at 80 mg/kg bw 5 days prior to sacrifice. The S9 fraction (batch number MCH250418, protein content 44 mg/mL) was performed according to literature and stored in liquid nitrogen. The S9-mix was prepared with the following composition: 3 mL of S9 fraction; 1 mL of 40 mM NADP; 1 mL of 50 mM glucose-6-phosphate; 1 mL of 330 mM KCl; 1 mL of 50 mM MgCl2; 2 mL of 20 mM HEPES buffer; 1 mL of deionised H2O (total volume 10 mL).
- concentration or volume of S9 mix and S9 in the final culture medium: The S9-mix was added to the culture medium at a final concentration of 10%.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Activity of S9 fraction was determined in bacterial reverse gene mutation test on Salmonella typhimurium strains TA 98 with a positive control of 2-aminoanthracene at 0.005 mg/plate.
Test concentrations with justification for top dose:
- 3 h, -S9: 0.05, 0.1, 0.5, 1, 2 and 2.5 µg/mL (evaluated: 0.05, 0.5 and 2 µg/mL)
- 3 h, +S9: 1, 2.5, 5, 10, 15, 20, 25, 27, 30, 50, 80, 100 and 200 µg/mL (evaluated: 5, 10 and 20 µg/mL)
- 24 h, -S9: 0.1, 0.5, 1, 1.25, 1.5 and 1.75 µg/mL (evaluated: 1, 1.5, and 1.75 µg/mL)

Selection of concentrations for micronucleus analysis:
If the maximum concentration is based on cytotoxicity, the highest concentration should aim to achieve 55 ± 5% cytotoxicity using the recommended cytotoxicity parameters (reduction in Cytokinesis-Block Proliferation Index (CBPI) to 45 ± 5% of the concurrent negative control). Care should be taken in interpreting positive results only found in the higher end of this 55 ± 5% cytotoxicity range. Slides from the highest selected concentration and two lower concentrations test item were taken for micronuclei analysis.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO in the presence of emulsifier Tween 80

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that AF-317 is soluble in DMSO in the presence of emulsifier Tween 80 at the final concentration of at least 200 µg/mL. A highest test concentration in Range Finding Test (3h-treatment with metabolic activation) of 200 µg/mL did not result in visible precipitation.

- Percentage of test item in solvent in the final culture medium: 1%
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 (single, duplicate, triplicate) : duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5E5 (3 - 5th passage)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 24 h
- Exposure duration/duration of treatment: 3 h (-S9 / +S9); 24 h (-S9)
- Harvest time after the end of treatment (sampling/recovery times): 3-h treatment: After the treatment, the medium was removed and cells were washed with Dulbecco´s phosphate buffered saline (DPBS). The treatment medium was replaced with fresh medium containing cytochalasin B to block the cytokinesis and then cultures were incubated for another 21 h (period corresponding to 1.5 - 2 normal cell cycle lengths after the beginning of treatment). 24-h treatment: Cells were harvested 24 h after exposure with the test substance or controls and cytochalasin B started.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.: Cytochalasin B; 3 µg/mL; incubation period 3-h treatment: 21 h (after test substance treatment); incubation period 24-h treatment: 24 h (simultaneously with test substance treatment).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): After the cytochalasin B incubation, the cells were trypsinized, collected by centrifugation (800 rpm for 8 min), swelled in cold hypotonic solution (5 mL, KCl 0.075M), and fixed in methanol : glacial acetic acid (3:1, 5 mL) fixative. The cells were centrifuged and the fixation process was repeated. Fixed cells were added to pre-cleaned cold microscope slides, dried, and after 24 h stained with 5% Giemsa-Romanowski solution for 8 min.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2000 binucleated cells per concentration (1000 binucleated cells per duplicate culture) were analysed.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction CBPI of the treated cells in the non-activated and activated systems

OTHER:
Acceptance criteria:
The assay was to be considered valid if the following criteria were met:
1. At least 3 dose levels for each test item had a level of cytotoxicity below 60%.
2. The frequency of mononuclei of binucleated cells (MNBN) in negative controls fell within the normal ranges.
3. The positive control test item induced statistically significant increases in the proportion of MNBN cells compared to the negative control cultures.
4. At least of 50% of cells have gone through at least one cell division (a measured by mononucleate + binucleate + multinucleate cell counts) in negative control cultures.
Evaluation criteria:
Criterion for positive response:
The test item was considered to have caused a positive response in this assay if the test item showed a statistically significant increase (p < 0.05) over that of the solvent control group in the frequency of micronucleated cells (MNBN) at one or more concentrations.
A concentration-related increase in the proportion of MNBN cells was observed in at least one experimental condition when evaluated with an appropriate trend test.
The test item is considered as positive in this assay if all the above criteria were met.

Criterion for negative response:
A negative response was claimed if no statistically significant increases in the number of micronucleated cells above concurrent solvent control frequencies were observed at any dose level, and there was no evidence of a dose-response relationship.
Statistics:
The micronucleus frequencies observed in test item at defined concentrations were compared to those of the concurrent negative control applying Chi-squared statistical procedure. Prospective dose-response relationship was evaluated by the means of Logistic Regression and Poisson Regression. Following statistical software was applied to process the experimental data: STATGRAPHICS Centurion XV, (version 15.2.14) and Study Size (version 2.0).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
3-h treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity at the evaluated concentrations < 56%; at higher concentrations cytotoxicity was observed and no slides were prepared for this concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
3-h treatment
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity at the evaluated concentrations < 54%; at higher concentrations cytotoxicity was observed and no slides were prepared for this concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity at the evaluated concentrations < 56%; at higher concentrations cytotoxicity was observed and no slides were prepared for this concentrations.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No marked changes of pH of treatment media were observed in the experiments at any concentration tested as compared to the concurrent solvent controls.
- Data on osmolality: The osmolality values were within the physiological ranges for these cells.
- Precipitation and time of the determination: No precipitation observed.

RANGE-FINDING/SCREENING STUDIES (if applicable):
In order to determine the toxicity of the test item, a preliminary Range Finding Test was performed. The cytotoxicity of the test item was assessed by determining the Cytokinesis-Block Proliferation Index (CBPI) of the treated cells. The CBPI indicates the average number of nuclei per cell, and was calculated according to the following formula:
CBPI = [(No. mononucleated cells) + ( 2 x No. binucleate cells) + (3 x multinucleate cells)] / Total number of cells
Cytotoxicity = 100 - 100x(CBPT (Test Item) - 1) / (CBPI (Control) - 1)

Concentrations tested in Range Finding Experiments:
- 3 h; -S9: 0.05, 0.1, 0.5, 1, 2.5, 5, 7.5, 10, 15 and 20 µg/mL
- 3 h; +S9: 1, 5, 10, 15, 20, 25, 30, 50, 80, 100 and 200 µg/mL
- 24h; -S9: 0.001, 0.005, 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.5, 2, 10 and 50 µg/mL

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The percentage of cells in the negative control that undergo cell division were in the range of ~52% and ~60%.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : 24-h treatment, -S9: There was a concentration-related increase (Logistic Regression and Poisson Regression) and the data fulfilled the criteria for a positive response.
- Statistical analysis; p-value if any : 24-h treatment, -S9: Statistically significant increases in frequencies of MNBN cells compared with the negative control (p < 0.001).

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
Cytotoxicity (> 55 ± 5%) was evaluated (and thus no slides prepared) at the following concentrations:
- 3-h treatment, -S9: 3, 4 and 5 µg/mL
- 3-h treatment, +S9: 30, 35, 40 and 45 µg/mL
- 24-h treatment, -S9: 2, 2.25 and 2.5 µg/mL

HISTORICAL CONTROL DATA: Not given in the study report.

Table 1: Frequency of MNBN Cells obtained for V79 cells treated with AF-317 (treatment: 3 h, -S9)

Test Item Concentration (µg/mL) Replicate No. of BN Cells Scored No. of MNBN Cells Scored Frequency of MNBN Cells / Cells Scored (%) Significance
NC A 1000 8 0.80 /
B 1000 8 0.80
Total 2000 16 0.80
0.05 A 1000 9 0.90 NS
B 1000 7 0.70
Total 2000 16 0.80
0.5 A 1000 9 0.90 NS
B 1000 8 0.80
Total 2000 17 0.85
2.0 A 1000 9 0.90 NS
B 1000 6 0.60
Total 2000 15 0.75
MMC A 1000 27 2.70 p < 0.001
B 1000 27 2.70
Total 2000 54 2.70

NC, negative control, DMEM with 1% DMSO and Tween 80%

MMC, Mitomycin C (0.1 µg/mL)

BN Cells, binucleated cells

MNBN Cells, binucleate cells with micronuclei

NS, not significant

Table 2: Frequency of MNBN Cells obtained for V79 cells treated with AF-317 (treatment: 3 h, +S9)

Test Item Concentration (µg/mL) Replicate No. of BN Cells Scored No. of MNBN Cells Scored Frequency of MNBN Cells / Cells Scored (%) Significance
NC A 1000 7 0.70 /
B 1000 10 1.00
Total 2000 16 0.85
5 A 1000 9 0.90 NS
B 1000 9 0.90
Total 2000 18 0.90
10 A 1000 9 0.90 NS
B 1000 7 0.70
Total 2000 16 0.80
20 A 1000 14 1.40 NS
B 1000 10 1.00
Total 2000 24 1.20
CP A 1000 25 2.50 p < 0.001
B 1000 24 2.40
Total 2000 49 2.45

NC, negative control, DMEM with 1% DMSO and Tween 80%

CP, Cyclophosphamide (40 µg/mL)

BN Cells, binucleated cells

MNBN Cells, binucleate cells with micronuclei

NS, not significant

Table 3: Frequency of MNBN Cells obtained for V79 cells treated with AF-317 (treatment: 24 h, -S9)

Test Item Concentration (µg/mL) Replicate No. of BN Cells Scored No. of MNBN Cells Scored Frequency of MNBN Cells / Cells Scored (%) Significance
NC A 1000 8 0.80 /
B 1000 7 0.70
Total 2000 15 0.75
1.0 A 1000 37 3.70 p < 0.001
B 1000 28 2.80
Total 2000 65 3.25
1.5 A 1000 47 4.70 p < 0.001
B 1000 40 4.00
Total 2000 87 4.35
1.75 A 1000 72 7.20 p < 0.001
B 1000 66 6.60
Total 2000 138 6.90
MMC A 1000 24 2.40 p < 0.001
B 1000 25 2.50
Total 2000 49 2.45

NC, negative control, DMEM with 1% DMSO and Tween 80%

MMC, Mitomycin C (0.1 µg/mL)

BN Cells, binucleated cells

MNBN Cells, binucleate cells with micronuclei

NS, not significant

Conclusions:
Under the conditions of this OECD Guideline 487 study, the test substance AF-317, was found to be positive in the extended (24-h) treatment of V79 cells.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

No study available. Testing proposal for an in vivo micronucleus test (OECD 474) is included due to the positive result in the in vitro micronucleus test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study planned
Study period:
2020
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Tetra methyl butyl hydroquinone

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies : There are no available in vivo GLP studies that address the potential of the substance to cause cytogenetic damage.
- Available non-GLP studies : No in vivo non-GLP studies are available that address the potential of the substance to cause cytogenetic damage.
- Historical human data : There is no historical human data that is relevant to the endpoint “Genetic toxicity in vivo”.
- (Q)SAR : It is not possible to reliably estimate the in vivo potential for the test substance to cause cytogenetic damage using (Q)SAR calculation(s) or profilers.
- In vitro methods : Column 2 of the REACH Regulation (EC) No. 1907/2006, Annex VIII, 8.4, states that “Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII”. The test substance was found to be positive in an in vitro micronucleus test according to OECD guideline 487 in the extended (24-h) treatment of V79 cells. Therefore, using in vitro methods is not adequate to generate the necessary information.
- Weight of evidence : No data suitable for a Weight of Evidence approach to meeting the data requirements for the endpoint “Genetic toxicity in vivo” is available.
- Grouping and read-across : Grouping and read-across may not be used to meet the data requirements as there are no adequate analogue substances with suitable data.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- The “Specific rules for adaptation from Column 1" that are given in Column 2, 8.4 of the REACH Regulation (EC) No. 1907/2006, Annex VIII, 8.4, states that “Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII”. In an in vitro micronucleus test according to OECD guideline 487 performed with the test substance, the result was positive in the extended (24-h) treatment of V79 cells without metabolic activation and negative in the short-term (3-h) treatment of V79 cells with and without metabolic activation. There are no other specific adaptation possibilities of Annex VI to X (and Column 2 thereof) of the REACH Regulation that allow the waiving of an in vivo genotoxicity study under the above-mentioned circumstances.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
AF-317 was found to be positive in an in vitro micronucleus test according to OECD Guideline 487 (hameln rds, 2019) in the extended (24-h) treatment of V79 cells. The in vivo Mammalian Erythrocyte Micronucleus Test (according to OECD Guideline 474) is an adequate test to further investigate the potential of AF-317 to induce structural or numerical chromosome aberrations in vivo.
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
other: mouse or rats
Route of administration:
oral: gavage
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation study in bacteria

2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone (CAS 903-19-5)was investigated for mutagenicity in bacteria (Ames test) in a study performed according to OECD guideline 471 under GLP conditions (Key, 2009). The Salmonella typhimurium strains TA 1535, TA 1537, TA 100 and TA 98 and Escherichia coli WP2 uvr A/pKM101 were exposed to concentrations up to 5000 µg/plate in DMSO with and without the addition of a metabolic activation system (S9 mix), respectively, in two independent experiments (preincubation method). A dose-setting study (experiment 1) was performed using eight doses up to the maximum dose of 5000 μg/plate, following which no two-fold or greater increase in revertant colony count for S. typhimurium TA100, TA1535, TA98 and TA1537, and E. coli WP2uvrA/pKM101 either with (+S9 mix) or without metabolic activation (-S9 mix). Growth inhibition of S. typhimurium TA1537 was observed with and without metabolic activation at 185 µg/plate and higher. Precipitation was noted in all tester strains at≥556 µg/plate and higher. The second experiment was performed with a maximum dose of 5000 μg/plate (tester strains S. typhimurium TA 1535, TA 100 and TA 98 and E. coli WP2 uvr A/pKM101) or with the dose that showed growth inhibition set as the maximum dose (313 µg/plate; tester strain S. typhimurium TA 1537). No two-fold or greater increase in revertant colony count was observed for any of the bacterial strains with or without metabolic activation. In the second experiment growth inhibition at 185 µg/plate and higher was seen for S. typhimurium TA1537 with and without metabolic activation. Precipitation occurred in S. typhimurium TA 1535, TA 100 and TA 98 and E. coli WP2 uvr A/pKM101 at ≥ 625 µg/plate and higher, but did not influence the results. The positive control substances induced a revertant colony count at least double that for the negative control (solvent control) for each bacterial strain, demonstrating that the study had been performed properly. In conclusion, 2,5-bis(2,4,4-trimethylpentan-2-yl)benzene-1,4-diol was found to show no bacterial mutagenicity.

In vitro gene mutation study in mammalian cells

The potential for in vitro mammalian cell gene mutations at the hprt locus was measured using Chinese hamster lung fibroblasts (V79) after treatment with various concentrations of2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone (CAS 903-19-5). The test was performed according to OECD guideline 476and under GLP conditions (Key, 2019, HPRT).The study consisted of a cytotoxicity range-finder experiment followed by main mutation experiments conducted with duplicate cultures (A & B) in the absence and presence of metabolic activation by 20-methylcholanthrene induced rat liver post-mitochondrial fraction (S9). The test item was formulated in DMSO in the presence of emulsifier Tween 80. A 3-h treatment incubation period was used for all experiments. Based on the results of the range-finding study, in Experiment 1, 8 concentrations of test item ranging from 1 to 30 µg/mL were tested in the absence of S9. In Experiment 2, 8 concentrations of test item, ranging from 5 to 45 µg/mL, were tested in the presence of S9. The highest concentration tested in the respective experiment was limited by toxicity. In Experiment 1 with culture A, a statistically significant increase in mutant frequency (MF) was observed at a concentration of 7.5 µg/mL in the absence of S9 only; however the increase in MF was not greater than 3-fold above that of the concurrent vehicle control, with no evidence of an increase in MF at any of the other concentrations tested. At this concentration the relative plating efficiency (RPE, relative survival) was reduced to ~59%. The highest concentration was limited by cytotoxicity, as a survival rate of 10 - 20% (measured by RPE) was achieved at the highest concentration of 20 µg/mL tested. In Experiment 1 with culture B in the absence of S9, no statistically significant increases in mutant frequency were observed following treatment with the test substance at any concentration within the concentration range of 1 - 20 µg/mL tested. Cytotoxicity, shown as survival rate of 10 - 20% (RPE) was also achieved at the highest concentration of 20 µg/mL tested. In Experiment 2 with culture A in the presence of S9, no statistically significant increases in mutant frequency were observed following treatment at any concentration within the concentration range of 1 - 35 µg/mL tested. Cytotoxicity, shown as survival rate of 10 - 20% (RPE) was achieved at the highest concentration of 35 µg/mL tested. In Experiment 2 with culture B in the presence of S9, statistically significant increase in mutant frequency was observed following treatment at concentration of 15 µg/mL only. There was cytotoxicity reduced to ~ 94% RPE. At this concentration the increase in MF was not greater than 3-fold above that of the concurrent vehicle control, with no evidence of an increase in MF at any of the other concentrations tested. Cytotoxicity, shown as survival rate of 10 - 20% (RPE) was achieved at the highest concentration of 35 µg/mL tested. Mutant frequencies in vehicle control were within the acceptable range and clear increases in mutant frequency were observed by the positive controls, ethyl methanesulphonate (without S9) and 7,12-dimethyl-benz(a) anthracene (with S9). The assay system was therefore considered to be both sensitive and valid. It is concluded that the test substance did not induce mutations at the hprt locus of V79 Chinese hamster lung cells when tested under the conditions employed in this study. These conditions included treatments with doses up to 20 and 35 µg/mL in the absence and the presence of a rat liver metabolic activation system (S9), respectively.

In vitro mammalian cell micronucleus test

2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone (CAS 903-19-5) was tested in an in vitro mammalian cell micronucleus test according to OECD guideline 487 and in compliance with GLP, using duplicate cultures of Chinese hamster lung (V79) cells in 3 independent experiments (Key, 2019, MNT). Treatments, covering a broad range of concentrations, were performed both in the absence and presence of metabolic activation (S9) from 20-methylcholanthrene induced male Sprague-Dawley rats. The test item was formulated in dimethyl sulfoxide (DMSO) in the presence of emulsifier Tween 80. A range-finding test in the absence and presence of S9 was conducted before the main test to determine the appropriate test substance concentrations. The potential of the test substance to cause clastogenic effects was assessed in two independent experiments with and without metabolic activation, during an exposure period of 3 h. One experiment without metabolic activation with extended treatment duration (24 h) was carried out in addition. In short-term experiments (3-h treatment) with duplicate cultures (A, B) both in the absence and presence of S9, 6 (0.05 - 2.5 µg/mL) and 7 concentrations (2.5 - 27 µg/mL) were evaluated for cytotoxicity, respectively. In the long-term experiment (24-h treatment) with duplicate cultures (A, B) 6 concentrations (0.1 - 1.75 µg/mL) were evaluated for cytotoxicity in the absence of S9. The test item concentrations for micronucleus analysis were selected by evaluating the effect of the test item on the Cytokinesis-Block Proliferation Index (CBPI). In each experiment, micronuclei were analysed at three concentrations. The highest concentrations chosen for analysis, 2.0 µg/mL (3-h treatment) and 1.75 µg/mL (24-h treatment) in the absence of S9 and 20 µg/mL (3-h treatment) in the presence of S9, induced ~59%, ~56% and ~54% cytotoxicity, respectively. Appropriate negative (solvent) control cultures were included in the test system in each experiment under each treatment condition. Mitomycin C (MMC) was employed as the clastogenic positive control chemical in the absence of rat liver S9, and cyclophosphamide (CP) was used as the clastogenic positive control chemical in the presence of rat liver S9. 1000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei under each experimental condition. In the experiments with short-term treatment (3-h treatment) in the presence and absence of rat liver S9 the test item did not exhibit statistically significant increases in frequencies of micronuclei in binucleated cells (MNBN cells) compared with the negative control. In the experiment with extended treatment (24-h treatment) the test item exhibited statistically significant increases in frequencies of MNBN cells compared with the negative control. The test item induced concentration-response increase (Logistic Regression and Poisson Regression) in the proportion of binucleate cells with micronuclei. The positive controls showed statistically significant increase in MNBN cells. Therefore, under the conditions of this test and according to the criteria set for evaluating the test results, the test substance was positive in the experiment with extended (24 h) treatment without metabolic activation.

Conclusion

2,5-bis(2,4,4-trimethylpentan-2-yl)benzene-1,4-diol was considered to be not mutagenic in the bacterial reverse mutation test (Ames test) with and without metabolic activation, and in the in vitro mammalian cell gene mutation test did not induce mutations at the hprt locus of V79 Chinese hamster lung cells in the absence and presence of a metabolic activation system. In the in vitro mammalian cell micronucleus test, the substance did not exhibit statistically significant increases in frequencies of micronuclei in V79 Chinese hamster lung cells in the experiments with short-term treatment (3-h treatment) in the presence and absence of rat liver S9. However,the test substance was positive in the experiment with extended (24 h) treatment without metabolic activation. This triggers the requirement to perform additional and appropriate in vivo mutagenicity studies as stipulated in REACH Regulation (EC) No. 1907/2006, item 8.4, Column 2. Therefore, an in vivo mammalian cell micronucleus test according to OECD guideline 474 is proposed.

Justification for classification or non-classification

The available data on genetic toxicity are not sufficient for classification according to Regulation (EC) 1272/2008. An in vivo mammalian cell micronucleus test is proposed to verify the positive results in the in vitro micronucleus test. A final decision on the classification will be made based on the result of the in vivo micronucleus test.