4-chlorophenol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-08-14 to 2019-10-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-chlorophenol

EC Number:

203-402-6

EC Name:

4-chlorophenol

Cas Number:

106-48-9

Molecular formula:

C6H5ClO

IUPAC Name:

4-chlorophenol

Test material form:

solid

Details on test material:

Batch (Lot) Number: 0011968273
Expiry date: 23 December 2019
Physical Description: Colourless to beige blocks
Purity/Composition: 99.46%
Storage Conditions: In refrigerator (2-8°C)

Method

Species / strain

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium, supplemented with heat-inactivated fetal calf serum, L-glutamine, penicillin/streptomycin and heparin.
- Cells obtained from healthy non-smoking human volunteers who had not knowingly either been exposed to high levels of radiation or hazardous chemicals, or had a viral infection.
- The cell-cycle time was determined using BrdU (bromodeoxyuridine) and the average generation time (AGT) calculated. The AGT was 12.7-14.0h.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B: prior to cell harvest at final concentration of 5 μg/mL for 24 hours

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

- Preliminary toxicity test: The dose range of test item used was 31 to 1285 μg/mL (0.01M).
- Experiment I: 50, 250, 350, 400, 450, 500, 550 and 600 μg/mL (-S9); 50, 250, 350, 400, 450 and 500 μg/mL (+S9)
- Experiment II: 1, 10, 15, 20, 50, 60, 70 and 80 μg/mL, without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was soluble in DMSO at all tested concentrations, solubility checks were performed in-house

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Solvent for positive control: Hank's balanced salt solution without Ca or Mg.

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Solvent for positive control: Hank's balanced salt solution without Ca or Mg.

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Solvent for positive control: Hank's balanced salt solution without Ca or Mg.

Positive control substance:

colchicine

Remarks:

without metabolic activation

Details on test system and experimental conditions:

ACTIVATION: The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed into culture media, was 1.8% (v/v).

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment I: 3 hours, with and without metabolic activation
Experiment II: 24 hours, without metabolic activation
- Expression time (cells in growth medium):
Experiment I: 24 hours with Cytochalasin B, with and without metabolic activation
Experiment II: 24 hours with Cytochalasin B, without metabolic activation

CYTOKINESIS BLOCK: Cytochalasin B (5 μg/mL)
STAIN (for cytogenetic assays): 6.7% Giemsa for 10-30 minutes, rinsed, dried and a cover slip applied using mounting medium

NUMBER OF REPLICATIONS: duplicate cell cultures (A and B) for each dose level

NUMBER OF CELLS EVALUATED: At least 1000 cells per culture


CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for identifying micronuclei were that they were round or oval in shape, nonrefractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)

Evaluation criteria:

The frequency of binucleate cells with micronuclei in the vehicle control cultures will normally be within the range of the laboratory historical control data. The frequency of spontaneous background micronuclei may be slightly elevated above the normal range and the experiment still considered valid.
All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

Statistics:

A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with micronuclei which was reproducible.

Results and discussion

Test results

Species / strain:

lymphocytes: Human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant change on pH was remarked.
- Effects of osmolality: osmolality did not increase by more than 50 mOsm
- Precipitation: no precipitation of the test material was observed at any of the test concentrations

RANGE-FINDING/SCREENING STUDIES:


CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See Table 1, 2, 3, 4 and 5.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: A minimum of 1000 cells per culture were scored.
- Indication whether binucleate or mononucleate where appropriate: See table 1, 2, 3, 4 and 5. The vehicle control cultures had frequencies of binucleate cells with micronuclei within the expected range. The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See tables 1, 2, 3, 4 and 5. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.
- Negative (solvent/vehicle) historical control data: The frequency of spontaneous binucleate cells with micronuclei in the vehicle control cultures was within the range of the laboratory historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI cytokinesis-block method (see tables 1, 2, 3, 4 and 5).

Any other information on results incl. tables

Table 1

Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with 4-chlorophenol in the First Cytogenetic Assay

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI		% cytostasis
0	1.53	-	1.56	1.55		0
10	1.53	-	1.54	1.54		2
50	1.49	-	1.52	1.50		8
200	1.41	-	1.42	1.42		24
250	1.40	-	1.42	1.41		25
300	1.36	-	1.39	1.37		32
0.25 MMC-C	1.30	-	1.34	1.32		42
0.38 MMC-C	1.26	-	1.30	1.28		48
0.1 Colch	1.14	-	1.16	1.15		73
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI		% cytostasis
0	1.57	-	1.60	1.59		0
50	1.53	-	1.55	1.54		8
250	1.42	-	1.43	1.43		28
350	1.37	-	1.40	1.38		35
400	1.36	-	1.37	1.37		38
450	1.30	-	1.32	1.31		47
500	1.26	-	1.27	1.27		54
15 CP	1.21	-	1.23	1.22		62
17.5 CP	1.19	-	1.19	1.19		67

Note: All calculations were performed without rounding off.

Table 2

Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with 4-chlorophenol in the Cytogenetic Assay 1A

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI		% cytostasis
0	1.39	-	1.43	1.41		0
50	1.42	-	1.45	1.44		-6
250	1.34	-	1.38	1.36		12
350	1.29	-	1.34	1.31		24
400	1.24	-	1.27	1.26		38
450	1.21	-	1.23	1.22		47
500	1.13	-	1.13	1.13		67
550	1.11	-	1.13	1.12		70
600	1.00	-	1.02	1.01		98
0.25 MMC-C	1.24	-	1.28	1.26		36
0.38 MMC-C	1.20	-	1.22	1.21		48
0.1 Colch	1.01	-	1.02	1.01		97

Table 3

Number of Mononucleated or Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with 4-chlorophenol in the First Cytogenetic Assay and Cytogenetic Assay 1A

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	1	0	1	2		4	6
50	-6	2	0	2	0		3	3
350	24	1	0	1	2		5	7
450	47	4	2	6^*	7		4	11
500	67	3	2	5	3		8	11
0.25 MMC-C	36	3	0	3	22		17	39^***
0.1 Colch	97	31	25	56^***	20^2)		2^2)	22^***

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei 1)			Number of binucleated cells with micronuclei 1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	1	0	1	2		3	5
50	8	2	2	4	3		3	6
400	38	1	3	4	5		2	7
500	54	1	1	2	2		3	5
15 CP	62	3	1	4	41		36	77***

*)Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

¹⁾1000 – 1010 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

²⁾931 and 781 binucleated cells were scored for the presence of micronuclei, respectively.

Table4

Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with4-chlorophenol in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/mL	CBPI			Mean CBPI		% cytostasis
0	1.57	-	1.58	1.58		0
1	1.49	-	1.50	1.49		15
10	1.44	-	1.47	1.46		21
15	1.38	-	1.44	1.41		28
20	1.42	-	1.50	1.46		20
50	1.38	-	1.40	1.39		33
60	1.37	-	1.38	1.38		35
70	1.28	-	1.29	1.29		50
80	1.31	-	1.32	1.31		46
0.15 MMC-C	1.25	-	1.30	1.27		53
0.23 MMC-C	1.21	-	1.23	1.22		61
0.05 Colch	1.01	-	1.01	1.01		98

Note: All calculations were performed without rounding off.

Table 5

Number of Mononucleated or Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with 4-chlorophenol in the Second Cytogenetic Assay

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration (µg/mL)	Cytostasis (%)	Number of mononucleated cells with micronuclei^1)			Number of binucleated cells with micronuclei^1)
		1000	1000	2000	1000		1000	2000
		A	B	A+B	A		B	A+B
0	0	1	1	2	3		3	6
1	15	0	1	1	3		4	7
15	28	2	2	4	2		1	3
70	50	0	1	1	1		3	4
0.15 MMC-C	53	5	4	9^*	27		21	48^***
0.05 Colch	98	21^2)	17	38^***	0^3)		2^3)	2^**

*)Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

¹⁾1000 bi- and mononucleated cells were scored for the presence of micronuclei.

Duplicate cultures are indicated by A and B.

²⁾917 mononucleated cells were scored for the presence of micronuclei.

³⁾43 and 70 binucleated cells were scored for the presence of micronuclei, respectively.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation

Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

The study was performed to the requirements of OECD TG 487 : guidelines under GLP conditions to assess within the in vitro cell micronucleus assay the clastogenic and aneugenic potential of the test item to the nuclei of normal human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 3-hour exposure in the presence and absence of a standard metabolizing system (S9, at a 1.8% final concentration). Experiment 2, used a 24 -hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the main experiments were selected using data from the preliminary toxicity test. The dose levels were as follows: 3 -hour without S9 -Mix: 50, 250, 350, 400, 450, 500, 550 and 600 µg/mL culture medium; 3 -hour with S9 -Mix (1.8%): 50, 250, 350, 400, 450 and 500 µg/mL culture medium. In a second experiment the dose levels were 24 -hour without S9: 1, 10, 15, 20, 50, 60, 70 and 80 µg/mL culture medium. All vehicle (dimethyl sulphoxide) controls had frequencies of binucleate cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. 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 micronuclei, in either of the two experiments, using a dose range that included a dose level that induced approximately a 50% reduction in CBPI in all three exposure groups. Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.