Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation studies performed according to OECD 471 in both 1983 and 1994 indicated that the test item was not mutagenic in the presence or absence of S9 activation.

A study performed in 1994, according to OECD 473, on Chinese hamster lung fibroblasts, suggested possible clastogenic properties of the test item; however this study has been discounted due to being a potential false-positive arising from an unsuitable test system. To confirm this, an OECD487 study was performed in 2019, concluding that the substance is non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study carried out using scientifically valid protocols equivalent to relevant guidelines published in the peer reviewed literature, non-GLP; adapted for this endpoint according to REACH Regulation (EC) 1907/2006: Annex XI - section 1.1.2
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Tested strains: Salmonella: TA98, TA100, TA1535 and TA1537 only with and without metabolic activation
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Tested strains: Salmonella: TA98, TA100, TA1535 and TA1537 only with and without metabolic activation
GLP compliance:
no
Remarks:
Well documented study carried out using scientifically valid protocols equivalent to relevant guidelines published in the peer reviewed literature, non-GLP; adapted for this endpoint according to REACH Regulation (EC) 1907/2006: Annex XI - section 1.1.2
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not applicable.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 (the precise activation system varied by laboratory, however trends in activation to different chemicals was one focus of the study)
Test concentrations with justification for top dose:
Range finding test (pre-incubation method): TA100 strain: 10000 µg/plate or the limit of solubility (i.e. up to 10 mg/plate or solubility limit, whichever lower).
- If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate

Main test (pre-incubation method): All strains: series of 5 doses up to 10000 µg/plate or the solubility limit (i.e. up to 10 mg/plate or solubility limit, whichever lower)
- At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each strain in the presence of S-9 mix or buffer.
Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.

Confirmatory test: All strains: series of 5 doses up to 10000 µg/plate or the solubility limit (i.e. up to 10 mg/plate or solubility limit, whichever lower)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:choice was distilled water; dimethyl sulfoxide (DMSO) was used if the chemical was insoluble or not sufficiently soluble in water. Ethanol (95%) or acetone was used if the chemical was not soluble or stable in DMSO.
- Justification for choice of solvent/vehicle: Depending on solubility (determined prior to testing).
Untreated negative controls:
yes
Remarks:
choline chloride, glycerol, glycine, mannitol, and sodium phosphate, tested at random concurrently
Negative solvent / vehicle controls:
yes
Remarks:
concurrent Solvent Controls
True negative controls:
not specified
Positive controls:
yes
Remarks:
concurrent Positive Controls
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: with metabolic activation: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation in agar (pre-incubation method)
0.5 ml of S-9 mix or 0.1M PO4 buffer was dispensed into an appropriate number of 13 x 100 mm culture tubes maintained at 37°C in a dry-bath. Then, 0.05 ml of cells and 0.05 ml of solvent or chemical dilution were added to each tube. The mixture was vortexed and allowed to incubate with shaking in the early tests or standing (dependent on laboratory) in line with earlier tests, for 20 min at 37°C.
The protocol was later changed to eliminate the shaking procedure, because the commercial shakers available would not fit in the Class 11 Type B hoods and, for the purposes of laboratory safety, it was inadvisable to incubate the chemicals at 37°C in the open laboratory.
Following the preincubation period, 2.5 ml (EGG) or 2.0 ml (CWR, SRI) of molten top agar (45°C) supplemented with 0.5 mM L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 ml of minimal glucose bottom agar [Vogel and Bonner, 1951] in a 15 x 100 mm plastic petri dish. After the overlay solidified, the plates were inverted and incubated at 37°C for 48 h

DURATION
- Expression time (cells in growth medium): 48h at 37 degrees Celsius

NUMBER OF REPLICATIONS: 3

Range finding test (pre-incubation method): TA100 strain: 10000 µg/plate or the limit of solubility (i.e. up to 10 mg/plate or solubility limit, whichever lower).
- If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate

Main test (pre-incubation method): All strains: series of 5 doses up to 10000 µg/plate or the solubility limit (i.e. up to 10 mg/plate or solubility limit, whichever lower)
- At least five doses of test chemical, in addition to the concurrent solvent and positive controls, were tested on each strain in the presence of S-9 mix or buffer.
Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.

Confirmatory test: All strains: series of 5 doses up to 10000 µg/plate or the solubility limit (i.e. up to 10 mg/plate or solubility limit, whichever lower)
Evaluation criteria:
A positive response was indicated by a reproducible, dose-related increase, whether it be two-fold over background.
Statistics:
A statistical analysis was performed. As a result of statistical analyses, a number of calls were changed from the original “negative” to “equivocal.” The statistical analysis did not result in any “positive” or “equivocal” calls being called “negative, under the conditions of the study (dated, 1983).
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Remarks:
No negative control substances yielded a positive
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test item was considered not to be mutagenic in the presence and absence of S9 activation. The test item induced no observed gene mutations in strains of S. typhimurium used.
Executive summary:

The study was performed using methods equivalent or similar to the requirements of OECD Guideline 471, to evaluate the potential mutagenicity of two hundred and fifty substances in an inter laboratory study which included the test item. The method used was the pre-incubation method bacterial reverse mutation assay using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, in both the presence and absence of S-9 mix. The test item was a "practical" purity grade sample obtained from a commercial source. The test item was tested up to 10000 µg/plate or the limit of solubility, whichever lower using distilled water or dimethyl sulfoxide (DMSO). If the chemical was insoluble or not sufficiently soluble in water. Alternatively, ethanol (95%) or acetone was typically used if the chemical was not soluble or stable in DMSO. Appropriate numbers of positive control substances with and without metabolic activation (rat liver S9) were included and randomly allocated concurrent true negative controls and concurrent vehicle controls. Under the conditions of the study, the test item gave a negative, i.e. non-mutagenic response in Salmonella typhimurium strains TA98, TA100, TA1535 and/or TA1537 in both the presence and absence of S-9 mix.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed to OECD and national guidelines under GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not applicable.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Main test (pre-incubation method): All strains: series of half log doses up to 10000 µg/plate or the limit of solubility (i.e. up to 10 mg/plate or solubility limit, whichever lower).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: Not clearly reported; however applicant assessment indicates DMSO is a standard accepted choice for AMES tests, and the authors remarked on the solubility of the test item in DMSO, implying it being the optimal/most-suitable choice.
Untreated negative controls:
yes
Remarks:
Not specified. Concurrent Negative Controls were run.
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48h at 37 degrees Celsius

NUMBER OF REPLICATIONS: 2
Statistics:
A statistical analysis was not performed or reported.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed at more than 1000 µg/plate (TA100, TA1535, TA98, TA1537 without S9; TA100, TA1535 with S9). Observed at more than 2000 µg/plate (TA98, TA1537 with S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Observed at more than 2000 µg/plate (with and without S9).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test item was considered not to be mutagenic in the presence or absence of S9 activation. The test item induced no observed gene mutations in the strains of S. typhimurium and E. coli utilzed.
Toxicity was observed at more than 1000 µg/plate (TA100, TA1535, TA98, TA1537) and at 2000 µg/plate (WP2 uvrA) without S9 mix, and at more than 1000 µg/plate (TA100, TA1535) and at 2000 µg/plate (WP2 uvrA, TA98, TA1537) with S9 mix.
Executive summary:

The study was performed according to the requirements of OECD Guideline 471 and the Japanese Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan), equivalent or similar to EU Method B.13/14, to evaluate the potential mutagenicity of the test item. The method used was the plate-incubation method bacterial reverse mutation assay using Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains in both the presence and absence of S-9 mix. The test item was tested up to 10000 µg/plate or the limit of solubility, whichever lower. The vehicle used for the test item was DMSO. Appropriate numbers of positive control substances with and without metabolic activation (rat liver S9) were included and randomly allocated concurrent true negative controls and concurrent vehicle controls. Under the conditions of the study, the test item gave a negative, i.e. non-mutagenic response in Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains in either the presence or absence of S-9 mix.

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.
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
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
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
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
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.
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).

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 micronuclei1)

Number of binucleated cells with micronuclei1)

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***

202)

22)

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 micronuclei1)

Number of binucleated cells with micronuclei1)

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

212)

17

38***

03)

23)

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.

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
1994
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Remarks:
Unsuitable test system, providing erroneous false-positive result.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
: Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable (chromosome aberration test)
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Remarks:
"Chinese Hamster lung (CHL/IU) -derived fibroblast cell line"
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, derived from rat liver, induced with phenobarbital and 5,6-benzonflavone
Test concentrations with justification for top dose:
The maximum dose level was 400 µg/mL, chosen as the dose at which cell proliferation was inhibited by >50%. pH and osmality are not recorded in the study report.

I. Preliminary toxicity test: 0 (control), 25, 50, 100, 200, 300, 400, and 500 μg/mL
Within two exposure groups:
i) 6-hours exposure to the test item without S9-mix.
ii) 6-hours exposure to the test item with S9-mix.

II. Main Test:
Experiment 1
6(18)-hour without S9: 0, 50, 100, 200, 300, 400 μg/ml and MNNG 2.5
6(18)-hour with S9: 0, 50, 100, 200, 300, 400 μg/ml and BP 10
where:
MNNG = 1-methyl-3-nitro-1-nitrosoguanidine
BP = 3,4-benzo[a]pyrene
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Physiological saline solution, possibly also including DMSO (translation from Japanese is ambiguous).
- Justification for choice of solvent/vehicle: Test item was soluble in the solution. The test item was formulated as required, immediately prior to application to the test system.
Untreated negative controls:
other:
Remarks:
Vehicle control served as the negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation
Untreated negative controls:
other:
Remarks:
Vehicle control served as the negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: Other:
Duplicate (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: Eagle-MEM, 10% (FBS).

DURATION
- Preincubation period: Not reported.
- Exposure duration:
The preliminary toxicity test was performed using both of the exposure conditions as described for both experiments (below) in the presence and absence of metabolic activation.
I. With Metabolic Activation (S9) Treatment:
- After 3 days incubation, the cultures were transferred to tubes and centrifuged. An aliquot of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and a quantity of the appropriate solution of vehicle control or test item was added to each culture. S9-mix was added to the cultures. After 6 hours incubation in the presence of S9 mix, the treatment medium was replaced with fresh culture medium. The cells were then re-incubated for a further 18 hours.

II. Without Metabolic Activation (S9) Treatment:
- After 3 days incubation, the cultures were transferred to tubes and centrifuged. An aliquot of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and a quantity of the appropriate solution of vehicle control or test item was added to each culture. After 6 hours incubation in the absence of S9 mix, the treatment medium was replaced with fresh culture medium. The cells were then re-incubated for a further 18 hours.

SPINDLE INHIBITOR (cytogenetic assays): Not specified.

NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.

NUMBER OF CELLS EVALUATED: A total of 200 nuclei were counted per dosing-group, and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
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.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. Cells with unexpected numerical discrepancies were scored as polyploid cells and the incidence of polyploid cells (%) including endoreduplicated cells, reported.
- Other: Scoring: Where possible,structural chromosome aberrations such as any breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations in a similar manner to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines.
Evaluation criteria:
Positive response criteria
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration-related or a statistically significant increase is exhibited in the number of cells with structural chromosome aberrations compared to the concurrent negative control.

Negative response criteria
A test item can be classified as non-genotoxic if:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.

In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment.

Statistical analysis is also performed (see: ‘Statistics’). Biological relevance of the results are to be considered first. Statistical methods are used to analyze the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response.
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. Analysis of data from in vitro cytogenetic assays. In Statistical Evaluation of mutagenicity test data: UKEMS sub-committee on guidelines for mutagenicity testing. Report Part III (Ed: Kirkland, D.J.), Cambridge University Press (1989)
Species / strain:
Chinese hamster lung (CHL/IU)
Remarks:
Chinese hamster lung (CHL/IU) -derived fibroblast line
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
aberrations induced at 300 mg/mL (-S9, 6hr exposure); 50 mg/mL (+S9, 6hr exposure), however effects not dose-dependant.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
400 mg/ml (- S9, 6 hr exposure); 300 mg/ml (+S9, 6 hr exposure).
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No mention of pH in the study report.
- Effects of osmolality: No mention of osmolality in the study report.
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation: Not reported.
- Other confounding effects: None reported.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 500 mg/mL. The selection of the maximum dose level was based on toxicity for the main test.

COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle controls had frequencies of cells with aberrations, which produced no special remark from the study authors.
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY: See Table 1 and 2.
Conclusions:
Interpretation of results:
Positive

Under the conditions of this study, the test substance was considered to be clastogenic to CHL/IU-derived fibroblasts in vitro.

Applicant assessment indicates that the choice of cell-line is unsuitable under the test conditions, and is prone to false-positive results. Further studies confirm this (in vitro micronucleus test).

The study is therefore discounted due to methodological issues.
Executive summary:

The study was performed to the requirements of OECD TG 473, equivalent to EU Method B.10 under GLP conditions to assess the potential chromosomal mutagenicity of the test substance, on the metaphase chromosomes of Chinese Hamster Lung-derived fibroblasts cultured mammalian cells. Duplicate cultures of CHL/IU cells, treated with the test item, were evaluated for chromosome aberrations at multiple dose levels, together with vehicle and positive controls. In this study, two conditions were considered; a 6 -hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), with cell harvest after an 18 -hour expression period, and a 6 -hour exposure in the absence of metabolic activation (S9) with an 18 -hour expression period. The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test (Cell Growth Inhibition Test) where the results indicated that the maximum concentration should be limited to 400 mg/mL. The dose levels selected for the Main Test were as follows, experiment: 6(18)-hour with and without S9 -Mix: 0, 50, 100, 200, 300, and 400 mg/mL. All vehicle controls had frequencies of cells with aberrations within the range expected for normal CHL/IU cells. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. Statistically significant increases in the frequency of cells with aberrations were observed, predominately in the presence of metabolic activation. Structural chromosomal aberrations were induced at 300 mg/mL without S9 mix, and at 50, 100 and 200 mg/mL with S9 mix, respectively. Polyploidy was induced at 100 and 200 mg/mL without S9 mix, and at 100 and 200 mg/mL with S9 mix, respectively. Cytotoxicity was observed at 400 mg/mL without S9 mix, and at 300 and 400 mg/mL with S9 mix. The lowest concentration producing cytogenetic effects in vitro was 50 mg/mL (with metabolic activation). Under the conditions of this study, the test item was considered to be clastogenic to CHL/IU cells in vitro. Applicant assessment indicates that the choice of cell-line is unsuitable under the test conditions, and is prone to false-positive results. Further studies confirm this (in vitro micronucleus test). The study is therefore discounted due to methodological issues.

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

Additional information

Justification for classification or non-classification