3-methyl-1-benzofuran-5-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 15 January 2014 and 15 April 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 473 using a in vitro mammalian chromosome aberration method and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1-benzofuran-5-ol

EC Number:

807-272-7

Cas Number:

7182-21-0

Molecular formula:

C9H8O2

IUPAC Name:

3-methyl-1-benzofuran-5-ol

Test material form:

other: solid

Details on test material:

Identification: IFF TM 11-0230
Storage Conditions: Approximately 4 °C, in the dark
Physical state/Appearance: Off white solid

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The molecular weight of the test item was given as 148; therefore, the maximum dose level was 1480 μg/mL, which was calculated to be equivalent to 10mM. The purity of the test item was 100% and was not accounted for in the test item formulations.

The test item was insoluble in culture media at 14.8 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 148 mg/mL in solubility checks performed in-house. The test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

In the absence of S9, mitomycin C was used at 0.4 and 0.2 μg/mL for cultures in Experiment 1 and 2 respectively dissolved in Minimal Essential Medium. In the presence of S9, cyclophosphamide was used at 5 μg/mL in both experiments dissolved in DMSO.

Details on test system and experimental conditions:

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The mean value of the AGT for the pool of regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.

Culture Conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:

9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

With Metabolic Activation (S9) Treatment
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1.

In Experiment 2, 1 mL of 10% S9-mix (i.e. 1% final concentration of S9 in standard co-factors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

Without Metabolic Activation (S9) Treatment
In Experiment 1, after approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.

After 4 hours at approximately 37 ºC, 5% CO2 in humidified air the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.

The preliminary toxicity test was performed using both of the exposure conditions as described for Experiment 1 and for Experiment 2 in the absence of metabolic activation only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4 hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4 hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

The dose range of test item used was 5.78 to 1480 μg/mL.

Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1
Two exposure groups were used for Experiment 1:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 23.13 to 555 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 11.56 to 3702 μg/mL.

Experiment 2:
Two exposure groups were used for Experiment 2:
i) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 5.78 to 370 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (1%) followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 11.56 to 370 μg/mL.

Cell Harvest:
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator. Supplementary slides were coded manually

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosomal Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. In addition, the incidence of cells with endoreduplicated chromosomes was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Evaluation criteria:

The following criteria were used to determine a valid assay:

Negative Control
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normally be within the laboratory historical control data range.

Positive Control
All the positive control chemicals must induce a clear positive response (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 5.78 to 1480 μg/mL. The maximum dose was the 10 mM concentration. The test item was observed to darken the media in the parallel blood-free cultures at the end of the exposure, at and above 185 μg/mL, in the 4(20)-hour exposure groups and at and above 46.25 μg/mL in the continuous exposure group.

Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase,cells were present up to 370 μg/mL in the 4(20)-hour exposures in both the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour
continuous exposure was also 185 μg/mL.

The mitotic index data indicate the test item induced evidence of dose-related toxicity in all three exposure groups. Increases in mitotic index values over the vehicle control seen in the 4(20)-hour group in the absence of S9 may indicate cell cycle delay induced by test item toxicity.
The selection of the maximum dose level was by toxicity in all exposure groups in both experiments.

Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test item are given below:

20- hour without S9 at final concentration of 0*, 23.13, 46.25, 92.5*, 185*, 370*, 555, MMC 0.4*
20-hour with S9 (2%) at final concentration 0*, 11.56, 23.13*, 46.25, 92.5*, 185*, 370, CP 5*

where: * indicates dose levels selected for metaphase analysis
MMC Mitomycin C
CP Cyclophosphamide

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 370 μg/mL in both exposure groups.

The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 41% mitotic inhibition was achieved at 370 μg/mL in the absence of S9. In the presence of S9 a dose-related inhibition of mitotic index was observed and that 79%, 59% and 32% mitotic inhibition was achieved at 370, 185 and 92.5 μg/mL respectively.

The selection of maximum dose level for metaphase analysis was based on toxicity and was 370 μg/mL in the absence of S9 and 185 μg/mL in the presence of S9.

From the chromosome aberration data are all of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations in the absence of metabolic activation. In the presence of metabolic activation a very modest statistically significant increase in the frequency of cells with aberrations was observed at the maximum dose assessed of 185 μg/mL. This dose level with 59% mitotic inhibition exceeded the optimum level of toxicity of 50%. The response was predominantly due to the ‘B’ culture which was more toxic, consisted of only break-type aberrations with no exchange-type aberrations, was compared to low vehicle control value and only marginally exceeded the historical control range for this exposure group. It was therefore considered that the response was most likely due to a cytotoxic mechanism and of no toxicological significance.

From the polyploid cell frequency data the test item did not induce statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test item are given below:

24- hour without S9 at final concentration of 0*, 5.78, 11.56*, 23.13*, 46.25*, 92.5*, 185, 370, MMC 0.2*
20-hour with S9 at final concentration 0*, 11.56*, 23.13, 46.25*, 92.5, 185*, 370, CP 5*

where: * indicates dose levels selected for metaphase analysis
MMC Mitomycin C
CP Cyclophosphamide

The qualitative assessment of the slides determined that there were metaphases suitable for scoring present 92.5 μg/mL in the absence S9, and up to 370 μg/mL in the presence of S9.

The mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 68% and 31% mitotic inhibition was achieved at 92.5 and 46.25 μg/mL respectively in the absence of S9. In the presence of S9 a plateau of mitotic index was observed with 54% to 37% mitotic inhibition being observed between 23.13 and 185 μg/mL.

The maximum dose level selected for metaphase analysis was 185 μg/mL in the presence of S9, the same as Experiment 1. However, in the 24-hour continuous exposure group it was based on toxicity and limited to 92.5 μg/mL with no metaphases being observed at dose levels above this concentration.

From the chromosome aberration data all of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item induced modest but statistically significant increases in the frequency of cells with chromosome aberrations in both the absence and presence of metabolic activation. However, as was seen in Experiment 1 the responses were only seen in the maximum dose level assessed giving a ‘hockey stick’ dose response curve. In the absence of S9 the response was only marginally above the historical vehicle control range, compared to a low vehicle control value and at a dose level that markedly exceeded the limit of acceptable toxicity. In the presence of metabolic activation the response was predominantly in the ‘A’ culture where greater toxicity was observed, only marginally exceeded the historical vehicle control range for this exposure group, was compared to a very low vehicle control value and as was seen in Experiment 2 only involved break-type aberrations. It was therefore considered that the response was driven by a cytotoxic mechanism and, therefore, of no biological or toxicological significance.

From the polyploid cell frequency data the test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mitotic Index - Preliminary Toxicity Test

4-Hours Treatment, 20-Hour Recovery -S9

4-Hours Treatment, 20-Hour Recovery +S9

24-Hour Treatment -S9

Dose Level (µg/mL)	4(20)-Hour Without S9		4(20)-Hour With S9		24-Hour Without S9
	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	5.15	100	7.35	100	6.10	100
5.78	-	-	-	-	-	-
11.56	-	-	-	-	-	-
23.13	-	-	-	-	3.30	54
46.25	7.55	147	5.80	79	3.75	61
92.5	6.45	125	3.70	50	2.20	36
185	6.15	119	3.75	51	0.50	8
370	4.10	80	1.35	18	NM	NM
740	NM	NM	NM	NM	NM	NM
1480	NM	NM	NM	NM	NM	NM

-      = Not assessed for mitotic index

NM   = No metaphases

Mitotic Index - Experiment 1

Dose Level (mg/mL)	4(20)-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	4.55	3.45	4.00	100	4.35	5.15	4.75	100
11.56	NA	NA	NA	NA	-	-	-	-
23.13	-	-	-	-	3.05	3.10	3.08	65
46.25	-	-	-	-	2.60	2.60	2.60	55
92.5	3.20	4.25	3.73	93	3.80	2.70	3.25	68
185	2.45	3.90	3.18	79	2.20	1.65	1.93	41
370	2.95	1.80	2.38	59	1.30	0.70	1.00	21
555	NM	NM	NM	NM	NA	NA	NA	NA
MMC 0.4	0.70	1.20	0.95	24	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	0.45	0.60	0.53	11

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

Mitotic Index - Experiment 2

Dose Level (µg/mL)	24-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	2.70	2.10	2.40	100	5.65	4.70	5.18	100
5.78	-	-	-	-	NA	NA	NA	NA
11.56	1.90	3.05	2.48	103	4.10	4.35	4.23	82
23.13	1.50	2.25	1.88	78	2.65	2.15	2.40	46
46.25	2.10	1.20	1.65	69	2.50	2.75	2.63	51
92.5	0.85	0.70	0.78	32	3.15	2.35	2.75	53
185	NM	NM	NM	NM	2.45	4.05	3.25	63
370	NM	NM	NM	NM	0.25	0.20	0.23	4
MMC 0.2	0.55	1.25	0.90	38	NA	NA	NA	NA
CP 5	NA	NA	NA	NA	0.40	0.40	0.40	8

MMC = Mitomycin C

CP = Cyclophosphamide

NA = Not applicable

- = Not assessed for mitotic index

NM = No metaphases

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce any toxicologically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was therefore considered to be nonclastogenic to human lymphocytes in vitro, according to OECD Test Guideline 473 using an in vitro mammalian chromosome aberration test method.

Executive summary:

The chromosome aberration potential of the test item, TM 11-0230 was tested in cultured peripheral human lymphocytes, in the presence and absence of S9-mix, according to OECD 473 guideline and GLP principles.

 

Four treatment conditions were used: In experiment 1, 4 hr in the presence or absence of S9 mix (2%) with cell harvest after a 20 -hr expression period. In experiment 2, the 4 hr exposure with addition of S9 (1%) was repeated, whilst in the absence of S9 the exposure time was increased to 24 hr. The dose levels of test substance in the main experiments were selected using data from the preliminary toxicity test results.

Reliable positive and negative controls were included. All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The test item did not induce any toxicologically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition.

Finally, it is concluded that the substance is not clastogenic in human lymphocytes.