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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

Chromosome aberration test (similar to OECD 473): negative in CHL/IU cells without metabolic activation; inconclusive with metabolic activation

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:
23 Mar - 2 Apr 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no data on historical controls provided
GLP compliance:
yes
Remarks:
according to the Japanese GLP Standard (as described on JECDB)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in closed bottle at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 2.71%, easily soluble in DMSO and acetone


Target gene:
his operon, trp operon
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, prepared from the livers of rats treated with phenobarbital and 5-6-benzoflavone
Test concentrations with justification for top dose:
TA100 and TA1535, with and without metabolic activation: 39.1, 78.1, 156, 313, 652, 1250 and 2500 μg/mL (tested up to cytotoxic concentrations)
WP2uvrA, TA98 and TA1537, with and without metabolic activation: 156, 313, 652, 1250, 2500 and 5000 μg/mL (tested up to the limit dose)
Vehicle:
- 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.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: aminofluorene for TA100 and TA98, N-ethyl-N'-nitrosogunanidine for WP2uvrA, 2-aminoanthracene for strains with S9
Details on test system and conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS:3 replications each in 2 independent experiments


Evaluation criteria:
Evaluation:
A test substance was considered positive in the Ames test if:
a) a more than 2-fold increase in the number of revertants was observed
b) the increase was dose-related
c) the positive result was reproducible.

A test substance was considered negative in the Ames test if none of the above criteria were observed under all experimental conditions.

Statistics:
Mean values and standard deviations were calculated.
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
TA100/ TA1535/ TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
starting at 1250 µg/mL +/-S9 in TA 100 and TA 1535; starting at 2500 µg/mL +/- S9 in WP2 uvrA, TA 98 and TA 1537
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary test was performed with test concentrations ranging from 1.22 - 5000 μg/mL with/without S9. No increase in the number of revertants was observed. Furthermore, cytotoxicity was observed starting at 1250 μg/mL in TA100 and TA1535, and at 5000 μg/mL in WP2urvA, TA98 and TA1537 with and without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: number of revertant colonies


Table 1. Test results for 2 -chlorphenol (Trial 1)

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

± standard deviation

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2urvA

TA98

TA1537

0

112 ± 7

 10 ± 3

24 ± 2

22 ± 5

7 ± 2

39.1

123 ± 5

8 ± 1

-

-

-

78.1

104 ± 18

12 ± 1

-

-

-

156

116 ± 15

11 ± 5

22 ± 3

24 ± 2

6 ± 2

313

103 ± 12

8 ± 2

33 ± 3

21 ± 3

7 ± 2

625

101 ± 12

12 ± 4

22 ± 2

23 ± 4

7 ± 2

1250

84 ± 9*

7 ± 2*

22 ± 2

20 ± 2

5 ± 1

2500

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

5000

-

-

0 ± 0*

0 ± 0*

0 ± 0*

Positive controls

AF-2

NaN3

ENNG

AF-2

9-AA

Mean No. of colonies/plate

± SD

541 ± 38

377 ± 12

821 ± 29

375 ± 29

351 ± 39

+

0

113 ± 4

7 ± 2

27 ± 5

26 ± 4

13 ± 5

+

39.1

111 ± 9

8 ± 1

-

-

-

+

78.1

110 ± 15

11 ± 3

-

-

-

+

156

122 ± 6

9 ± 2

25 ± 4

28 ± 3

10 ± 2

+

313

117 ± 9

7 ± 2

29 ± 9

29 ± 10

12 ± 3

+

625

109 ± 11

9 ± 2

32 ± 7

28 ± 7

14 ± 3

+

1250

85 ± 5*

12 ± 3*

31 ± 4

27 ± 6

12 ± 4

+

2500

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

+

5000

-

-

0 ± 0*

0 ± 0*

0 ± 0*

+

Positive controls

2-AA

2-AA

2-AA

2-AA

2-AA

Mean No. of colonies/plate

± SD

1161 ± 32

168 ± 7

1313 ± 38

443 ± 9

140 ± 6

AF-2= 2 -aminofluorene

NaN3 = sodium azid

ENNG = N-ethyl-N'-nitro-N'-nitrosogunanidine

9-AA = 9-aminoacridine

2AA = 2-aminoanthracene

SD = standard deviation

* = growth inhibition/cytotoxicity

Table 2. Test results for 2 -chlorphenol (Trial 2)

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

± standard deviation

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2urvA

TA98

TA1537

0

105 ± 4

8 ± 2

20 ± 5

23 ± 3

13 ± 1

39.1

106 ± 3

8 ± 2

-

-

-

78.1

103 ± 9

7 ± 1

-

-

-

156

103 ± 5

8 ± 2

29 ± 2

23 ± 2

15 ± 4

313

108 ± 11

11 ± 1

26 ± 4

18 ± 2

12 ± 2

625

94 ± 2

8 ± 1

23 ± 4

18 ± 2

15 ± 1

1250

87 ± 15*

8 ± 2*

19 ± 3

15 ± 2

14 ± 4

2500

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

5000

-

-

0 ± 0*

0 ± 0*

0 ± 0*

Positive controls

AF-2

NaN3

ENNG

AF-2

9-AA

Mean No. of colonies/plate

± SD

524 ± 14

514 ± 46

843 ± 33

505 ± 21

430 ± 34

+

0

105 ± 3

10 ± 2

25 ± 5

25 ± 5

19 ± 2

+

39.1

101 ± 6

9 ± 1

-

-

-

+

78.1

108 ± 10

8 ± 2

-

-

-

+

156

95 ± 8

11 ± 3

28 ± 5

25 ± 4

18 ± 5

+

313

96 ± 6

10 ± 1

27 ± 4

25 ± 6

16 ± 3

+

625

108 ± 10

9 ± 2

28 ± 6

26 ± 2

21 ± 3

+

1250

96 ± 5*

8 ± 3*

29 ± 3

23 ± 0

19 ± 2

+

2500

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

0 ± 0*

+

5000

-

-

0 ± 0*

0 ± 0*

0 ± 0*

+

Positive controls

2-AA

2-AA

2-AA

2-AA

2-AA

Mean No. of colonies/plate

± SD

1010 ± 58

210 ± 9

1477 ± 47

424 ± 12

192 ± 7

AF-2= 2 -aminofluorene

NaN3 = sodium azido

ENNG = N-ethyl-N'-nitro-N'-nitrosogunanidine

9-AA = 9-aminoacridine

2AA = 2-aminoanthracene

SD = standard deviation

* = growth inhibition/cytotoxicity

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
200 metaphases scored, evaluation criteria differ from OECD TG 473 (no statistical analysis performed); limited data on historical data provided, cell proliferation used as parameter for cytotoxicity, no C-charts or X-bar charts provided
Reference:
Composition 1
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
200 metaphases scored, evaluation criteria differ from OECD TG 473 (no statistical analysis performed), limited data on historical data provided (no values/range given), no RPD or RICC values given, no C-charts or X-bar charts provided
GLP compliance:
yes
Remarks:
according to the Japanese GLP Standard (as described on JECDB)
Type of assay:
in vitro mammalian cell micronucleus test
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a closed bottle at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 2.71%, easily soluble in DMSO and acetone
Target gene:
Not applicable
Species / strain:
other: CHL/IU
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: Dainippon Pharmaceutical Co., Ltd.
- Suitability of cells: yes
- Cell cycle length, doubling time or proliferation index: no data given


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle MEM supplemented with 10% fetale calf serum, 0.02% L-glutamine, 0.12% NaHCO3
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Experiment I: with and without metabolic activitaion: 62.5, 125, 250 and 500 μg/mL (tested up to cytotoxic concentrations)
Experiment II: without metabolic activation: 200, 300, 400 and 500 μg/mL (tested up to cytotoxic concentrations)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solibility of the test substance in water, DMSO was selected as the vehicle.
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 4 * 10000 cells/mL. After 3 days of culture, the medium was changed and the cells were exposed to the test substance.

DURATION
- Exposure duration: 6 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (harvest time: 18 h)
h

SPINDLE INHIBITOR (cytogenetic assays): 0.1 µg/mL Colcemid in medium

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates each; +S9 one experiment (Experiment I), -S9: 2 independent experiments (Experiment I and II)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200

Rationale for test conditions:
The test concentrations were selected based on the results of the preliminary tests.
Evaluation criteria:
A test substance is considered positive in the chromosome aberration test if it induced an increase in the aberration frequency of at least 10% . A test substance was considered false positive if it induced an increase in the frequency of aberrant cells in the range of 5 - 10%. A test substance was considered negative if it induced an increase in the aberration frequency of < 5%.
Key result
Species / strain:
other: CHL/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no increase in the aberration frequency in non-cytotoxic concentrations (< 55% cytotoxicity) - S9; inconclusive + S9 (missing statistics and historical data)
Cytotoxicity:
yes
Remarks:
all experimental parts: at the highest tested concentration (MT1, +/- S9: at 500 µg/mL; MT2, -S9: 500 µg/mL) (> 55% cytotoxicity)
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Three range-finding tests were performed to evaluate cytotoxicity of the test material. In the first pre-test, concentrations of 50, 500 and 5000 μg/mL were applied with and without S9 mix. Based on the results in the first test, concentrations of 500, 1000, 2000, 3000, 4000 and 5000 μg/mL were tested in the second pre-test with and without S9 mix. In the second pre-test, reduced cell proliferation > 50% was observed starting at 500 µg/mL with and without S9 mix supplementation. To verify the observed results, a third pre-test was conducted with test concentrations of 12.6, 31.1, 62.5, 125, 250 and 500 µg/mL (no information on results provided).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: cell profilferation (mitotic index)

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: Toxicity
In all experimental parts without S9 mix supplementation, an increase in the aberration frequency was observed at the highest concentration tested. Due to the observed excessive cytotoxicity at these concentrations, chromosomal damage is considered as artefactual positive result.

Table 1 Test results of Experiment I.

Test item Concentration Mitotic Index Aberrant cells
  [µg/mL] [%] with gaps (cells) without gaps (%)
structural aberrant cells numerical aberrant cells
Exposure period 6 h, fixation time 18h, without S9 mix
DMSO 0.5% (v/v) 100 1 3.5 0.5
MMC 0.1 53 15 95.5 0
Test substance 62.5 97 2 3 0.5
125 75 0 4.5 0
250 67 0 2 2
500 30 T 3 25.5 0
Exposure period 6h, fixation time 18h, with S9 mix
DMSO 0.5% (v/v) 100 2 1.5 0
BP 20 75 7 89.5 0
Test substance 62.5 105 0 1.5 1
125 104 1 6 2
250 90 3 12 0
500 30 T 1 38.6 0

MMC: mitomycin C, BP: benzo(a)pyrene, T: toxicity

Table 2. Test result of Experiment II.

Test item Concentration

Mitotic

Index

Aberrant cells
  [µg/mL] [%] with gaps (cells) without gaps (%)
structural aberrant cells numerical aberrant cells
Exposure period 6 h, fixation time 18h, without S9 mix
DMSO 0.5% (v/v) 100 1 0 0
MMC 0.1 72 9 61.5 0.5
Test substance 200 69 0 2.5 1.5
300 59 1 1 2.5
400 52 1 2.5 0
500 25 T 5 34 0

MMC: mitomycin C, T: toxicity

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

SCE (in vivo, integrated in a 14 day RDT study): negative in testes and bone marrow cells (limited data, SS)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Remarks:
(limited details on study design and results provided)
Reference:
Composition 1
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: A 14-day repeat dose toxicity study with 2-chlorophenol was performed including evaluation of sister chromatid exchanges. Groups of 12 male and female CD-1 mice were exposed to 2-chlorophenol at dose levels of 35, 69 and 175 mg/kg bw/day by oral gavage once daily for 14 consecutive days. Vehicle control animals received the vehicle, corn oil, only. In addition a treatment naive and a positive control group (cyclosphosphamide, 25 mg/kg bw) were included.

- Parameters analysed / observed: SCE (testes and bone marrow).
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay
Test material information:
Composition 1
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Assigned to test groups randomly: yes
- Housing: individually in plastic shoebox cage with hardwood sawdust bedding

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22° +/- 2°C
- Humidity (%): relative humidity 40-60%
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared on day of administration

Duration of treatment / exposure:
14 days
Frequency of treatment:
Once daily
Post exposure period:
none
Dose / conc.:
35 mg/kg bw/day
Remarks:
Doses tested were 35, 69 and 175 mg/kg bw/day. All animals at 175 mg/kg bw/day died and could not be evaluated. Highest evaluated dose was 69 mg/kg bw/day.
Dose / conc.:
69 mg/kg bw/day
Remarks:
Doses tested were 35, 69 and 175 mg/kg bw/day. All animals at 175 mg/kg bw/day died and could not be evaluated. Highest evaluated dose was 69 mg/kg bw/day.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
other: yes, positive control
Positive control(s):
cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 25 mg/kg bw
Tissues and cell types examined:
testes and bone marrow
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
not specified
Additional information on results:
Mortality was observed in the highest dose group. In the mid-dose group, a decrease in body weight was observed. Administration of the low and mid-dose caused hyperactivity in the test animals. No alterations on clinical or haematological parameters were observed. Females revealed reduced liver, brain and spleen weights. No gross anomalies were observed. No biologically or statistically significant compound-related effects on DNA repair were observed in the examined tissues.
Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

2-Chlorophenol was tested for its mutagenic properties in bacterial cells. Furthermore, data on the clastogenic properties were obtained based on in vitro and in in vivo studies.

Gene mutation in bacteria

Mutagenicity of 2-chlorophenol was tested in a GLP-conform bacterial reverse mutation assay equivalent to OECD TG 471 performed with a standard battery of Salmonella typhimurium tester strains including TA 1535, TA 1537, TA 98 and TA 100 and WP2uvrA bacterial cells (reference 7.6.1-1). Based on the results of a preliminary cytotoxicity test, concentrations of up to 2500 or 5000 µg/plate were tested in TA 100 and TA 1535 or WP2uvrA and, TA 98 and TA 1537, respectively. 2-chlorophenol did not exhibit mutagenic properties in the absence or presence of metabolic activation. Toxicity indicated by reduced number of revertants was observed in all strains at least at the highest applied dose. Positive control substances induced a distinct increase in the number of revertants in all strains with and without metabolic activation thereby proving validity of the assay. Based on the results of the conducted study, 2-chlorophenol is not considered to exhibit mutagenic properties in bacterial cells. 

Cytogenicity in vitro

An in vitro mammalian chromosome aberration test was performed with the test substance in Chinese hamster lung cells (CHL/IU) similar to the current OECD TG 473 and GLP (reference 7.6.1-2). Duplicate cultures were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). Based on the results of two range-finding studies, concentrations of 62.5, 125, 250 and 500 μg/mL were applied for 6 h in the first experiment with and without metabolic activation. In the second experiment cells were exposed to 200, 300, 400 and 500 μg/mL without S9 mix for 6 h. Mitomycin C and Benzo(a)pyrene were used as positive control substances, which validated the test conditions and/or the activity of the metabolizing system. Evaluation of 200 well-spread metaphases per concentration revealed no biologically relevant increase in the frequency of chromosome aberrations at non-cytotoxic concentrations without metabolic activation in comparison to the negative controls. The observed increase in the aberration frequency at clearly cytotoxic concentrations determined in every experimental part without metabolic activation is considered as artefactual positive result due to excessive cytotoxicity > 55% as defined in OECD TG 473. In the presence of S9 mix, a dose-dependent increase in the frequency of aberrant cells was observed exceeding the defined threshold level (10%) at a non-cytotoxic concentration. However, due to missing historical data and statistical analysis, this result has to be considered as inconclusive. Thus, based on the results of this study taking into consideration the observed excessive cytotoxicity, 2-chlorophenol is considered to be non-clastogenic to Chinese hamster lung cells in vitro without metabolic activation, whereas no conclusive decision on the clastogenic properties in the presence of metabolic activation can be drawn.

Cytogenicity in vivo

In addition to the in vitro data on cytogenicity, supporting data from a 14-day repeat dose toxicity study including evaluation of sister chromatid exchanges in testes and bone marrow is available for 2-chlorophenol (reference 7.6.2-1). Groups of 12 male and 12 female CD-1 mice were exposed to the test item at dose levels of 35, 69, and 175 mg/kg bw/day by oral gavage once daily for 14 consecutive days. Vehicle control animals received the vehicle, corn oil, only. In addition a treatment naive and a positive control group (cyclosphosphamide, 25 mg/kg bw) were included. Observations and examinations of the animals included clinical signs, body weight, food consumption, haematology, clinical chemistry, liver microsomal activities, immune response, genetic toxicology, certain reproductive toxicology endpoints, activity and behavioral measurements, gross necropsy and organ weights. Mortality was observed in the highest dose group. In the mid-dose group, a decrease in body weight was observed. Administration of the low and mid-dose caused hyperactivity in the test animals. No alterations on clinical or haematological parameters were observed. Females revealed reduced liver, brain and spleen weights. No gross anomalies were observed. No biologically or statistically significant compound-related effects on DNA repair were observed in the examined tissues. Thus, the available data indicate that 2-chlorophenol does not exhibit DNA damaging properties and hence, support the in vitro data, that 2-chlorophenol does not exhibit clastogenic properties.

In summary, based on the available data on genetic toxicity, there is no clear evidence that 2-chlorophenol induces genetic toxicity in bacteria or clastogenic properties in vitro and in vivo. The in vivo data are considered as sufficiently reliable to enable a conclusive decision on the clastogenic properties as basic data are provided which support the inclonclusive finding in the available cytogenicity study.

Justification for classification or non-classification

Based on the available data on genetic toxicity, there is no indication that 2-chlorophenol induces genetic toxicity in bacteria or clastogenic properties in vitro and in vivo. Taking into consideration the harmonized classification, 2-chlorophenol is not expected to exhibit hazardous properties in regard to genetic toxicity. Thus, 2 -chlorophenol does not meet the classification criteria according to Regulation (EC) 1272/2008.