Phenol, C14-18-alkyl derivs.

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:

21 August 2014 to 25 October 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

C14-16-18 Alkyl phenol

EC Number:

931-468-2

Cas Number:

1190625-94-5

Molecular formula:

For substance information - see Section 1.2

IUPAC Name:

C14-16-18 Alkyl phenol

Test material form:

other: slightly viscous liquid

Details on test material:

- Appearance: Clear amber liquid
- Storage conditions of test material: At room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Blood samples: Blood was collected from healthy adult, non-smoking, male volunteers by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20 % (v/v) heat-inactivated (56 °C; 30 minutes) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) and 30 U/mL heparin.
- Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
- Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were humid atmosphere of 80 to 100 % (actual range 60 to 91 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.0 to 37.4 °C).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

- Range-finding test: 0.54, 1.7, 5.4, 17, 52, 164 and 512 µg/mL (without metabolic activation; 24 hour exposure time, 24 hour fixation time; 48 hour exposure time, 48 hour fixation time)
- First cytogenetic assay: 5.4, 17 and 52 µg/mL (with and without metabolic activation; 3 hour exposure time, 24 hour fixation time)
- Second cytogenetic assay: 10, 30, 40, 50, 60 and 70 µg/mL (without metabolic activation; 24 hour exposure time, 24 hour fixation time; 48 hour exposure time, 48 hour fixation time)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol absolute. The final concentration of the solvent in the exposure medium was 0.5 % (v/v).

Controls

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:

METHOD OF APPLICATION: in medium. The cytogenetic assay was carried out as described by Evans, 1984 with minor modifications.
- Cytogenetic assay 1A: Lymphocytes were cultured for 48 ± 2 hours and thereafter exposed to selected doses for 3 hours in the absence and presence of S9-mix. After 3 hours of exposure, the cells were separated from the exposure medium by centrifugation (5 minutes, 365 x g). The supernatant was removed and the cells were rinsed once with 5 mL Hank’s Balanced Salt Solution (HBSS). After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 to 22 hours (24 hour fixation time).
- Second cytogenetic assay: Lymphocytes were cultured for 48 ± 2 hours and thereafter exposed to selected doses for 24 and 48 hours in the absence of S9-mix. The cells were not rinsed after exposure but were fixed immediately after 24 and 48 hours (24 and 48 hour fixation time).

SPINDLE INHIBITOR: colchicine
STAIN: Giemsa

NUMBER OF REPLICATIONS: Experiments were carried out in duplicate

CHROMOSOME PREPARATION
During the last 2.5 to 3 hours of the culture period, cell division was arrested by the addition of colchicine (0.5 µg/mL medium). Thereafter the cell cultures were centrifuged for 5 minutes at 365 x g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 minute treatment with hypotonic 0.56 % (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).

PREPARATION OF SLIDES
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 to 30 minutes with 5 % (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated coverslipper.

NUMBER OF CELLS EVALUATED: One hundred metaphase chromosome spreads per culture were examined

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5 %). At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50 % or above whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also, cultures treated with an intermediate dose were examined for chromosome aberrations. In case the test material was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analysed at the 3 hour exposure time was determined by the solubility in the culture medium. If dose related cytotoxicity was observed, the highest concentration analysed at the 24 and 48 hour continuous exposure times was based on toxicity irrespective of the solubility of the test material in the culture medium. However, the extent of precipitation should not interfere with the scoring of chromosome aberrations.

ANALYSIS OF SLIDES
One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was = 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

Evaluation criteria:

A test material is considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test material was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:
¿² = [(N – 1)(ad – bc)²] / [(a+b) (c+d) (a+c) (b+d)]
where:
b = the total number of aberrant cells in the control cultures
d = the total number of non-aberrant cells in the control cultures
n0 = the total number of cells scored in the control cultures
a = the total number of aberrant cells in treated cultures to be compared with the control
c = the total number of non-aberrant cells in treated cultures to be compared with the control
n1 = the total number of cells scored in the treated cultures
N = sum of n0 and n1

If P[¿² = [(N – 1)(ad – bc)²] / [(a+b) (c+d) (a+c) (b+d)]] (one-tailed) is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95 % confidence level.
Biological relevance is evaluated against historical control ranges; any increase in aberrant cells remaining within the historical control data ranges is considered to be not biologically relevant.

Results and discussion

Test results

Species / strain:

lymphocytes: Cultured peripheral human 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:

DOSE RANGE FINDING TEST / FIRST CYTOGENETIC ASSAY
At a concentration of 52 µg/mL, the test material precipitated in the culture medium. The pH and osmolarity at a concentration of 17 µg/mL were determined to be 7.7 and 371 mOsm/kg, respectively (compared to 7.8 and 281 mOsm/kg in the solvent control).
Table 1 shows the mitotic index of cultures.
All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix, the test material did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Table 2).
Both in the absence and presence of S9-mix, the test material did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY
Table 1 shows the mitotic index of cultures. Based on these observations, the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 10, 40 and 50 µg/mL culture medium (24 hour exposure time, 24 hour fixation time); 10, 30 and 40 µg/mL culture medium (48 hour exposure time, 48 hour fixation time).
The test material did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Table 3).
The test material did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

CONTROLS
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Mitotic Index

Test Material Concentration (µg/mL)	± Metabolic Activation	Exposure Time (hours)	Fixation Time (hours)	Number of Metaphases*				Percentage of Control
				Absolute		Number of Cells Scored
Range-finder
Control	-S9	24	24	78	-	1042	-	100
0.54				52	-	1008	-	67
1.7				69	-	999	-	88
5.4				64	-	1005	-	812
17				79	-	1002	-	101
52¹				24	-	1005	-	31
164²				16	-	1005	-	21
512¹				2	-	1009	-	3
Control	-S9	48	48	33	-	1004	-	100
054				25	-	1007	-	76
1.7				34	-	1012	-	103
5.4				54	-	1004	-	164
17				46	-	1020	-	139
52¹				17	-	1002	-	52
164¹				2	-	1007	-	6
512¹				1	-	1003	-	3
Assay 1A
Control	-S9	3	24	93	86	1003	1003	100
5.4				94	85	1011	1016	100
17				88	73	1001	1013	90
52¹				99	77	1003	1011	98
MMC 0.5				62	61	1008	1005	69
MMC0.75				42	59	1006	1002	56
Control	+S9	3	24	74	77	1008	1004	100
5.4				74	50	1002	1015	82
17				73	79	1002	1003	101
52¹				72	87	1004	1011	105
CP 10				43	47	1005	1004	60
Second Cytogenetic assay
Control	-S9	24	24	76	73	1000	1000	100
10				71	72	1002	1001	96
30				65	62	1000	1003	85
40¹				51	58	1000	1010	73
50¹				38	30	1004	1002	46
60¹				19	14	1000	1000	22
70¹				6	7	1000	1000	9
MMC 0.2				36	39	1000	1004	50
MMC 0.3				24	21	1000	1004	30
Control	-S9	48	48	70	69	1005	1041	100
10				60	69	1001	1004	93
30				57	55	1000	1000	81
40¹				31	37	1005	1000	49
50¹				18	21	1002	1006	28
60¹				12	10	998	1002	16
70¹				5	4	1000	1000	6
MMC 0.1				37	31	1000	1004	49
MMC 0.15				31	25	1003	1000	40

*Duplicate cultures with the exception of the range-finding test

¹The test material precipitated in the culture medium

² The test material precipitated in the culture medium. Mean of 2 scorings (28 metaphases in 1004 cells and 4 metaphases in 1006 cells)

MMC = Mitomycin C

CP = Cyclophosphamide

 

Table 2: Chromosome Aberrations in the First Cytogenetic Assay

	Concentration (µg/mL)
	3 Hour Exposure Time, 24 Hour Fixation Time (-S9)					3 Hour Exposure Time, 24 Hour Fixation Time (+S9)
	Control	5.4	17	52	MMC 0.5	Control	5.4	17	52	CP 10
Culture	A + B	A + B	A + B	A + B	A + B	A + B	A + B	A + B	A + B	A + B
MI (%)	100	100	90	98	69	100	82	101	105	60
No. of cells scored	200	200	200	200	200	200	200	200	200	200
No. of cells with aberrations (+ gaps)	0	0	1	0	65***	2	0	0	4	69***
No. of cells with aberrations (- gaps)	0	0	0	0	65***	1	0	0	3	69***
Total aberrations (+ gaps)	0	0	1	0	88	2	0	0	4	105
Total aberrations (- gaps)	0	0	0	0	86	1	0	0	3	105

MMC = Mitomycin C

CP = Cyclophosphamide

MI = Mitotic index

***Significantly different from the control group (Chi-square test), p< 0.001

 

Table 3: Chromosome Aberrations in the Second Cytogenetic Assay

	Concentration (µg/mL)
	24 Hour Exposure Time, 24 Hour Fixation Time (-S9)					48 Hour Exposure Time, 48 Hour Fixation Time (-S9)
	Control	10	40	50	MMC 0.2	Control	10	30	40	MMC 0.1
Culture	A + B	A + B	A + B	A + B	A + B	A + B	A + B	A + B	A + B	A + B
MI (%)	100	96	73	46	50	100	93	81	49	49
No. of cells scored	200	200	200	200	200	200	200	200	200	200
No. of cells with aberrations (+ gaps)	0	0	2	2	61***	1	2	3	1	66***
No. of cells with aberrations (- gaps)	0	0	1	1	60***	1	2	2	1	66***
Total aberrations (+ gaps)	0	0	2	2	73	1	2	3	1	81
Total aberrations (- gaps)	0	0	1	1	71	1	2	2	1	78

MMC = Mitomycin C

CP = Cyclophosphamide

MI = Mitotic index

***Significantly different from the control group (Chi-square test), p< 0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this study the test material is not clastogenic in human lymphocytes with and without metabolic activation.

Executive summary:

The ability of the test material to induce chromosome aberrations in cultured peripheral human lymphocytes was evaluated in a study conducted in accordance with the standardised guidelines OECD 473 and EU Method B.10 under GLP conditions.

The study investigates the effect of the test material on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix).

Experiments were carried out in duplicate and concurrent solvent (ethanol) and positive controls took place.

The dose levels tested were as follows:

- Range-finding test: 0.54, 1.7, 5.4, 17, 52, 164 and 512 µg/mL (without metabolic activation; 24 hour exposure time, 24 hour fixation time; 48 hour exposure time, 48 hour fixation time)

- First cytogenetic assay: 5.4, 17 and 52 µg/mL (with and without metabolic activation; 3 hour exposure time, 24 hour fixation time)

- Second cytogenetic assay: 10, 30, 40, 50, 60 and 70 µg/mL (without metabolic activation; 24 hour exposure time, 24 hour fixation time; 8 hour exposure time, 48 hour fixation time)

In the first cytogenetic assay, the test material precipitated in the culture medium at 52 µg/mL. In the second cytogenetic assay, appropriate toxicity was reached at the dose levels.

The test material did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No effects of the test material on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions used.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The positive controls both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Under the conditions of this study the test material is not clastogenic in human lymphocytes with and without metabolic activation.