1,3-dimethyl-3-phenylbutyl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
4-methyl-4-phenylpentan-2-ol (source chemical) is the hydrolysis product of 1,3-dimethyl-3-phenylbutyl acetate (target chemical). Hydrolysis of 3-dimethyl-3-phenylbutyl acetate is expected to occur in the human body, with enzyme activity playing an important role. As the hydrolysis is expected to occur, read across from the hydrolysis product 4-methyl-4-phenylpentan-2-ol is considered to be appropriate.

2. SOURCE AND TARGET CHEMICAL(S)
4-methyl-4-phenylpentan-2-ol is expected to be one of the hydrolysis products of 1,3-dimethyl-3-phenylbutyl acetate, along with acetic acid (acetate ion). The target substance is the acetate of the test substance 4-methyl-4-phenylpentan-2-ol. In 1,3-dimethyl-3-phenylbutyl acetate, the hydroxyl group in the 2 position of the pentane backbone is replaced by an acetate group (O-C(CH3)=O).
These two substances have similar melting point, boiling point, density, surface tension and vapour pressure properties. Although there is a difference in water solubility, both substances are soluble to some extent. Partition coefficient values are similar, at 2.82 and 3.55 for the source and target substance respectively. Neither of the substances would be considered to be bioaccumulative. As both substances have flash points >60 ºC they are not considered to be flammable.
3. ANALOGUE APPROACH JUSTIFICATION
The read across justification is based on the fact that 4-methyl-4-phenylpentan-2-ol is expected to be one of the hydrolysis products of 1,3-dimethyl-3-phenylbutyl acetate, along with acetic acid (acetate ion). Hydrolysis would only be expected to occur at high and low pH values. Initial studies have shown low hydrolysis at pH 4 (< 3 % after 120 hours) and moderate hydrolysis at pH 9 (ca 27 % after 120 hours). The test item showed a moderate hydrolysis rate (t1/2 ≤ 30 d) at pH 9 and 50 °C. At pH 9 at 20 and 30 °C only a slow hydrolysis (t1/2 > 30 d) was observed and at 20 °C the half live was > 1 year indicating no significant hydrolysis of the test item in a study according to OECD Guideline 111 and EC Method C.7 (Lange 2015).
While no data is available for the hydrolysis of 1,3-dimethyl-3-phenylbutyl acetate at gastric pH values (pH 1.2), hydrolysis data is available for two related esters, namely butyl acetate (CAS No. 123-86-4, EC no. 204-658-1) and phenylethyl acetate (CAS no. 103-45-7, EC 203-113-5) in artificial gastric fluid at 37 ºC (Longland et al, 1977). The acid hydrolysis half-life for these two esters was 318 and 300 minutes for butyl acetate and phenylethyl acetate, respectively. In the same study, hydrolysis in artificial pancreatic juice adjusted to pH 7.5 was measured to be 66 and 29.7 minutes respectively, for butyl acetate and phenylethyl acetate. In rat liver and small intestinal mucosa preparations the hydrolysis half-life for butyl acetate was 8.13 minutes and 1.8 minutes respectively. The study showed that enzyme activity was a major contributing factor in the hydrolysis of the two esters and that studies employing liver and small intestine preparations reflect more accurately the hydrolytic fate of esters in in vitro toxicological evaluations.
1,3-dimethyl-3-phenylbutyl acetate would react similarly to butyl acetate and phenylethyl acetate and therefore would be expected to hydrolyse rapidly to 4-methyl-4-phenylpentan-2-ol and acetate ion. A review of the human health data from the sodium acetate registration dossier indicates that there are no reported hazards associated with exposure to a variety of acetate ions for any of the toxicological endpoints. Consequently, it can be concluded that the acetate ion would not be expected to contribute to any of the potential toxicological endpoints required under REACH.

4. DATA MATRIX
See 'Attached justification'.

Reason / purpose for cross-reference:

read-across source

Type of assay:

mammalian cell gene mutation assay

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at high doses

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity observed (phase separation) at 900 ug/mL without metabolic activation and at 450 ug/mL with activation

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity observed in main experiment I at 337.5 and 450 ug/mL

The test medium was checked for precipitation visible to the naked eye at the end of each treatment period just prior to removal of the test item. No precipitation was noted in the main experiments at the evaluated concentrations. In the first experiment relevant toxic effects indicated by a relative cloning efficiency of 1 or a relative total growth of less than 50 % of survival were observed at 112.5 μg/mL and above with and without metabolic activation. In the second experiment relevant toxic effects as described above were noted at 56.0 μg/mL and above without metabolic activation (24 h treatment), and 160 μg/mL and above with metabolic activation. The recommended toxic range of approximately 10 – 20% of survival or RTG was covered in all experimental parts.

The data generated in experiment I at 225.0 μg/mL without metabolic activation (culture I) and at 225.0 μg/mL with metabolic activation (culture II) are not considered valid since both parameters of toxicity (survival and RTG) remained below the threshold of 10 %. In experiment II, culture I with metabolic activation, the data at the maximum concentration of 240 μg/mL were also not considered valid due to exceedingly strong toxic effects.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments up to the maximum concentration with and without metabolic activation at both treatment intervals. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software.

A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the first experiment without metabolic activation. Since the mutation frequency neither exceeded the historical range of solvent controls and the threshold as indicated above, nor showed a dose-response relationship, the statistical result is considered as a biologically irrelevant fluctuation. In this study the range of the solvent controls was from 57 up to 139 mutant colonies per 10e6 cells; the range of the groups treated with the test item was from 50 up to 204 mutant colonies per 10e6 cells.

Methane methyl sulphonate (MMS) at 19.5 μg/mL in experiment I and 13.0 μg/mL in experiment II and cyclophosphamide (CPA) at 3.0 μg/mL and 4.5 μg/mL in both main experiments were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity at at least one of the concentrations of the controls.

Table 1: Summary of results

			relative	relative	mutant		relative	relative	mutant
	Conc. µg/mL	S9 mix	Cloning efficiency 1	Total growth	Colonies/10^6cells	threshold	Cloning efficiency 1	Total growth	Colonies/10^6cells	threshold
column	1	2	3	4	5	6	7	8	9	10
Experiment I/ 4 h treatment			Culture I				Culture II
Solvent control with DMSO		-	100.0	100.0	91	217	100.0	100.0	103	229
Positive control with MMS	19.5	-	86.8	69.4	270	217	91.8	50.4	334	229
Test item	14.1	-	100.0	116.8	72	217	93.1	73.6	121	229
Test item	28.1	-	125.8	120.5	121	217	112.3	66.0	130	229
Test item	56.3	-	100.0	96.5	79	217	91.8	56.8	106	229
Test item	112.5	-	67.7	73.8	81	217	72.1	43.0	119	229
Test item	225.0	-	2.6	2.5	122	217	16.0	10.9	204	229
Test item	337.5	-	0.0	Culture not continued #			2.7	Culture not continued #
Test item	450.0	-	0.0	Culture not continued #			1.3	Culture not continued #
Solvent control with DMSO		+	100.0	100.0	96	222	100.0	100.0	104	230
Positive control with CPA	3.0	+	55.1	48.7	227	222	57.9	56.3	269	230
Positive control with CPA	4.5	+	37.9	36.3	258	222	40.9	35.0	362	230
Test item	14.1	+	71.4	80.3	135	222	91.5	93.7	110	230
Test item	28.1	+	83.2	84.6	166	222	78.1	98.1	100	230
Test item	56.3	+	64.5	62.8	179	222	64.9	106.4	128	230
Test item	112.5	+	60	39.9	159	222	64.9	95.4	92	230
Test item	225.0	+	14	11.3	170	222	5.0	5.0	108	230
Test item	337.5	+	2.9	Culture not continued #			0.4	Culture not continued #
Test item	450.0	+	1.1	Culture not continued #			0.0	Culture not continued #

 

Experiment II2/ 4 h treatment			Culture I				Culture II
Solvent control with DMSO		-	100.0	100.0	139	265	100.0	100.0	97	223
Positive control with MMS	13.0	-	12.8	13.3	502	265	21.1	11.3	451	223
Test item	3.5	-	141.1	Culture not continued ##			100.0	Culture not continued ##
Test item	7.0	-	87.1	Culture not continued ##			96.4	Culture not continued ##
Test item	14.0	-	118.3	125.7	126	265	101.9	67.1	116	223
Test item	28.0	-	84.5	79.8	95	265	94.6	49.9	120	223
Test item	56.0	-	88.4	45.8	122	265	78.7	38.7	196	223
Test item	112.0	-	70.0	42.9	146	265	54.4	18.5	119	223
Test item	168.0	-	55.8	22.5	130	265	47.2	15.8	122	223
Solvent control with DMSO		+	100.0	100.0	57	183	100.0	100.0	124	250
Positive control with CPA	3.0	+	50.7	45.8	162	183	50.3	51.1	283	250
Positive control with CPA	4.5	+	42.2	23.0	276	183	40.5	22.7	434	250
Test item	40.0	+	71.3	Culture not continued ##			60.4	Culture not continued ##
Test item	80.0	+	73.6	80.6	58	183	84.8	103.2	113	250
Test item	160.0	+	59.4	40.7	78	183	64	66	117	250
Test item	180.0	+	35	36.7	64	183	62.2	53.6	105	250
Test item	200.0	+	27.4	30.2	50	183	39.3	51.1	92	250
Test item	220.0	+	21.7	20.3	60	183	27.2	27.9	137	250
Test item	240.0	+	8.4	5.0	130	183	9.5	10.7	99	250

threshold = number of mutant colonies per 106cells of each solvent control plus 126

# not determined, culture not continued due to exceedingly strong toxic effects

## culture was not continued since a minimum of four concentrations is required by the guidelines

The values printed in bold are judged as invalid, since the acceptance criteria are not met.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the read-across substance, 4-methyl-4-phenylpentan-2-ol, did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of the read-across substance, 4-methyl-4-phenylpentan-2-ol, to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

 

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

 

The highest concentration (1800 μg/mL) applied in the pre-experiment was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. The concentration range of both main experiments was limited by cytotoxic effects of the test item.

 

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

 

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix: 14.1; 28.1; 56.3; 112.5; and 225.0 μg/mL

with S9 mix: 14.1; 28.1; 56.3; 112.5; and 225.0 μg/mL

 

Experiment II:

without S9 mix: 14.0; 28.0; 56.0; 112.0; and 168.0 μg/mL

with S9 mix: 80.0; 160.0; 180.0; 200.0; 220.0; and 240.0 μg/mL

 

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the read-across substance, 4 -methyl-4 -phenylpentan-2 -ol, did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, 1,3-dimethyl-3-phenylbutyl acetate is also considered to be non-mutagenic in mammalian cells.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methyl-4-phenylpentan-2-ol

EC Number:

218-002-7

EC Name:

4-methyl-4-phenylpentan-2-ol

Cas Number:

2035-93-0

Molecular formula:

C12H18O

IUPAC Name:

4-methyl-4-phenylpentan-2-ol

Method

Target gene:

thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Experiment I:
without S9 mix: 14.1, 28.1, 56.3, 112.5, and 225.0 μg/mL
with S9 mix: 14.1, 28.1, 56.3, 112.5, and 225.0 μg/mL

Experiment II:
without S9 mix: 14.0, 28.0, 56.0, 112.0 and 168.0 μg/mL
with S9 mix: 80.0, 160.0, 180.0, 200.0, 220.0 and 240.0 μg/mL

Vehicle / solvent:

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

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMOS

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In medium; in agar (plate incorporation); pre-incubation; in suspension; as impregnation on paper disk.

In the mutation experiment 1 10e7 cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3 % horse serum (15 % during 24 h exposure) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. Positive and solvent controls were performed in parallel.After 4 h (24 h in the second experiment without metabolic activation) the test item was removed by centrifugation (425 g, 10 min) and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium and incubated for an expression and growth period of totally 48 h.
The cell density was determined each day and adjusted to 3x10e5 cells/mL , if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector (1). One sample of the cells was taken at the end of the expression time (4 and 24 h, respectively), diluted and seeded into microtiter plates, to determine the viability of the cells after treatment (cloning efficiency 1).
After the expression period the cultures were seeded into microtiter plates. Cells from each experimental group were seeded into 2 microtiter plates so that each well contains approximately 4x10e3 cells in selective medium (see below) with TFT (Serva, 69042 Heidelberg, Germany). The viability (cloning efficiency 2) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 37° 1.5 °C in 4.5 % CO2/95.5 % humidified air for 10 - 15 days.
Then the plates were evaluated.

DURATION
- Exposure duration: 4 hours (24 hours in the case of experiment 2 - Expression time (cells in growth medium): 10-15days
- Selection time (if incubation with a selection agent)
- Fixation time (start of exposure up to fixation or harvest of cells)

POSITVE CONTROLS (mutation assays):
Methyl methane sulphonate (for experiments without metabolic activation) ; cyclophosphamide (for experiments with metabolic activation)

REPLICATES : 2 ; experiments 1 and 2

NUMBER OF CELLS EVALUATED: approx 2 cells seeded per well (96 wells per plate)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative cloning efficiency

(1) D. Clive, J.F.S. Spector
Laboratory procedure for assessing specific locus mutation at the TK locus in cultured
L5178Y mouse lymphoma cells
Mutation Research 31, 17-29, 1975

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at high doses

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity observed (phase separation) at 900 ug/mL without metabolic activation and at 450 ug/mL with activation

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity observed in main experiment I at 337.5 and 450 ug/mL

Any other information on results incl. tables

The test medium was checked for precipitation visible to the naked eye at the end of each treatment period just prior to removal of the test item. No precipitation was noted in the main experiments at the evaluated concentrations. In the first experiment relevant toxic effects indicated by a relative cloning efficiency of 1 or a relative total growth of less than 50 % of survival were observed at 112.5 μg/mL and above with and without metabolic activation. In the second experiment relevant toxic effects as described above were noted at 56.0 μg/mL and above without metabolic activation (24 h treatment), and 160 μg/mL and above with metabolic activation. The recommended toxic range of approximately 10 – 20% of survival or RTG was covered in all experimental parts.

The data generated in experiment I at 225.0 μg/mL without metabolic activation (culture I) and at 225.0 μg/mL with metabolic activation (culture II) are not considered valid since both parameters of toxicity (survival and RTG) remained below the threshold of 10 %. In experiment II, culture I with metabolic activation, the data at the maximum concentration of 240 μg/mL were also not considered valid due to exceedingly strong toxic effects.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments up to the maximum concentration with and without metabolic activation at both treatment intervals. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software.

A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the first experiment without metabolic activation. Since the mutation frequency neither exceeded the historical range of solvent controls and the threshold as indicated above, nor showed a dose-response relationship, the statistical result is considered as a biologically irrelevant fluctuation. In this study the range of the solvent controls was from 57 up to 139 mutant colonies per 10e6 cells; the range of the groups treated with the test item was from 50 up to 204 mutant colonies per 10e6 cells.

Methane methyl sulphonate (MMS) at 19.5 μg/mL in experiment I and 13.0 μg/mL in experiment II and cyclophosphamide (CPA) at 3.0 μg/mL and 4.5 μg/mL in both main experiments were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity at at least one of the concentrations of the controls.

Table 1: Summary of results

			relative	relative	mutant		relative	relative	mutant
	Conc. µg/mL	S9 mix	Cloning efficiency 1	Total growth	Colonies/10^6cells	threshold	Cloning efficiency 1	Total growth	Colonies/10^6cells	threshold
column	1	2	3	4	5	6	7	8	9	10
Experiment I/ 4 h treatment			Culture I				Culture II
Solvent control with DMSO		-	100.0	100.0	91	217	100.0	100.0	103	229
Positive control with MMS	19.5	-	86.8	69.4	270	217	91.8	50.4	334	229
Test item	14.1	-	100.0	116.8	72	217	93.1	73.6	121	229
Test item	28.1	-	125.8	120.5	121	217	112.3	66.0	130	229
Test item	56.3	-	100.0	96.5	79	217	91.8	56.8	106	229
Test item	112.5	-	67.7	73.8	81	217	72.1	43.0	119	229
Test item	225.0	-	2.6	2.5	122	217	16.0	10.9	204	229
Test item	337.5	-	0.0	Culture not continued #			2.7	Culture not continued #
Test item	450.0	-	0.0	Culture not continued #			1.3	Culture not continued #
Solvent control with DMSO		+	100.0	100.0	96	222	100.0	100.0	104	230
Positive control with CPA	3.0	+	55.1	48.7	227	222	57.9	56.3	269	230
Positive control with CPA	4.5	+	37.9	36.3	258	222	40.9	35.0	362	230
Test item	14.1	+	71.4	80.3	135	222	91.5	93.7	110	230
Test item	28.1	+	83.2	84.6	166	222	78.1	98.1	100	230
Test item	56.3	+	64.5	62.8	179	222	64.9	106.4	128	230
Test item	112.5	+	60	39.9	159	222	64.9	95.4	92	230
Test item	225.0	+	14	11.3	170	222	5.0	5.0	108	230
Test item	337.5	+	2.9	Culture not continued #			0.4	Culture not continued #
Test item	450.0	+	1.1	Culture not continued #			0.0	Culture not continued #

 

Experiment II2/ 4 h treatment			Culture I				Culture II
Solvent control with DMSO		-	100.0	100.0	139	265	100.0	100.0	97	223
Positive control with MMS	13.0	-	12.8	13.3	502	265	21.1	11.3	451	223
Test item	3.5	-	141.1	Culture not continued ##			100.0	Culture not continued ##
Test item	7.0	-	87.1	Culture not continued ##			96.4	Culture not continued ##
Test item	14.0	-	118.3	125.7	126	265	101.9	67.1	116	223
Test item	28.0	-	84.5	79.8	95	265	94.6	49.9	120	223
Test item	56.0	-	88.4	45.8	122	265	78.7	38.7	196	223
Test item	112.0	-	70.0	42.9	146	265	54.4	18.5	119	223
Test item	168.0	-	55.8	22.5	130	265	47.2	15.8	122	223
Solvent control with DMSO		+	100.0	100.0	57	183	100.0	100.0	124	250
Positive control with CPA	3.0	+	50.7	45.8	162	183	50.3	51.1	283	250
Positive control with CPA	4.5	+	42.2	23.0	276	183	40.5	22.7	434	250
Test item	40.0	+	71.3	Culture not continued ##			60.4	Culture not continued ##
Test item	80.0	+	73.6	80.6	58	183	84.8	103.2	113	250
Test item	160.0	+	59.4	40.7	78	183	64	66	117	250
Test item	180.0	+	35	36.7	64	183	62.2	53.6	105	250
Test item	200.0	+	27.4	30.2	50	183	39.3	51.1	92	250
Test item	220.0	+	21.7	20.3	60	183	27.2	27.9	137	250
Test item	240.0	+	8.4	5.0	130	183	9.5	10.7	99	250

threshold = number of mutant colonies per 106cells of each solvent control plus 126

# not determined, culture not continued due to exceedingly strong toxic effects

## culture was not continued since a minimum of four concentrations is required by the guidelines

The values printed in bold are judged as invalid, since the acceptance criteria are not met.

Applicant's summary and conclusion

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the read-across substance, 4-methyl-4-phenylpentan-2-ol, did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of the read-across substance, 4-methyl-4-phenylpentan-2-ol, to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

 

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

 

The highest concentration (1800 μg/mL) applied in the pre-experiment was chosen with regard to the molecular weight of the test item corresponding to a molar concentration of about 10 mM. The concentration range of both main experiments was limited by cytotoxic effects of the test item.

 

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

 

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix: 14.1; 28.1; 56.3; 112.5; and 225.0 μg/mL

with S9 mix: 14.1; 28.1; 56.3; 112.5; and 225.0 μg/mL

 

Experiment II:

without S9 mix: 14.0; 28.0; 56.0; 112.0; and 168.0 μg/mL

with S9 mix: 80.0; 160.0; 180.0; 200.0; 220.0; and 240.0 μg/mL

 

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the read-across substance, 4 -methyl-4 -phenylpentan-2 -ol, did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, 4-methyl-4 phenylpentan-2-ol is considered to be non-mutagenic in this mouse lymphoma assay.