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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 August - 14 September, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29th July, 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: cytogenetic test, which detects structural chromosome aberrations in somatic and/or germ cells

Test material

Constituent 1
Chemical structure
Reference substance name:
p-nitrobenzoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)
EC Number:
230-989-6
EC Name:
p-nitrobenzoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)
Cas Number:
7394-38-9
Molecular formula:
C7 H5 N O4 . C6 H15 N O3
IUPAC Name:
p-nitrobenzoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)
Test material form:
solid: particulate/powder
Details on test material:
Lot no. 2601LP2609
Storage: At room temperature, protected from light

Method

Target gene:
Chromatid and chromosome type aberrations (gaps, deletions and exchanges), in metaphase cells.
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79: Chinese hamster lung male
Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
This cell line was purchased from ECACC (European Collection of Cells Cultures).
Cytokinesis block (if used):
Colchicine (0.2 µg/mL)
Metabolic activation:
with and without
Metabolic activation system:
liver microsome preparations (S9 mix)
Test concentrations with justification for top dose:
Cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below:

Experiment A with 3/20 h treatment/sampling time
without: 250, 500, 1000 and 2000 *g/mL test item
with S9 mix: 500, 1000 and 2000 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 *g/mL test item
Vehicle / solvent:
DME (Dulbecco’s Modified Eagle’s) medium
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
other: DME (Dulbecco’s Modified Eagle’s) medium
Details on test system and experimental conditions:
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male).
This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in liquid nitrogen. Checking for mycoplasma infections was carried out. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 ¿C in an incubator with a humidified atmosphere, set at 5 % CO2. The V79 cells for this study was grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with
L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 ¿g/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.

Mammalian Microsomal Fraction S9 Mix

An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary. Many substances only develop mutagenic potential when they are metabolised by the mammalian organism. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix). The protein concentrations of the S9 batch used in the experiments were 33.7 and 33.8 mg/mL.

Rat Liver S9 Fraction

The S9 fraction of phenobarbital (PB) and ß-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2, D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA). Certificate of Analysis was obtained from the supplier. The Certificate of Analysis of rat liver S9 mix is stored in the laboratory.

The S9 Mix (with Rat Liver S9)

The complete S9 Mix was freshly prepared containing components with the following ratios:
S9 fraction 3 mL
HEPES* 20 mM 2 mL
KCl 330 mM 1 mL
MgCl2 50 mM 1 mL
NADP** 40 mM 1 mL
Glucose-6-phosphate 50 mM 1 mL
DME medium 1 mL
*= N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid
**= ß-Nicotinamide Adenine Dinucleotide Phosphate
Before adding to the culture medium the S9 mix was kept in an ice bath.

Rationale for test conditions:
Acceptability of the Assay

The Chromosome Aberration Assay is considered acceptable because it meets the following criteria:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data, .
– concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically
significant increase compared with the concurrent negative control,
– cell proliferation in the solvent control is adequate,
– adequate number of cells and concentrations are analyzable,
– all requested experimental conditions were tested unless one resulted in a positive result
– the criteria for the selection of top concentration is adequate.
Evaluation criteria:
Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– There were no equivocal results in this study.
– pH and Osmolality data were summarised in tabular form.

Interpretation of Results

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative because:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data

Statistics:
For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility and Dose Selection

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was dissolved in DME (Dulbecco’s Modified Eagle’s). A clear solution was obtained up to a concentration of 50 mg/mL. There was no precipitation in the medium at any concentration tested. Concentration selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays (Experiment A and B), both in the absence and in the presence of a metabolic activation system (rodent S9 mix). Toxicity was determined by cell counting and results noted. Based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. Detailed results of the cytotoxicity assay with P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) are presented in Table 2A - 2C. These results were used to select concentrations of test item for the Chromosome Aberration Assays.
Based on the results of the cytotoxicity assay the following concentrations were selected for the Chromosome Aberration Assay.
All concentrations were run in duplicates (incl. negative and positive controls) and at least 300 (150-150) well-spread metaphases were assessed:

Experiment A with 3/20 h treatment/sampling time
without: 250, 500, 1000 and 2000 *g/mL test item
with S9 mix: 500, 1000 and 2000 *g/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 *g/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 *g/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 *g/mL test item

Chromosome Aberration Assay

In Experiment A, P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) did not induce an increase in the number of cells with aberrations without gaps at any examined concentration, neither in the absence nor in the presence of metabolic activation.There were no statistically significant differences between test item treatment and control groups and no dose-response relationship was noted.

In Experiment B, P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was examined without S9 mix, over a long treatment period and the sampling was made at approximately 1.5 cell cycles (20 hours after treatment start). The cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent solvent and historical controls.

There was no increase in the number of cells with aberrations without S9 mix following exposure over a long treatment period of 20 hours and sampling at approximately 2 cell cycles (28 hours after treatment start).

A 3-hour treatment in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations without gaps compared to the concurrent solvent and historical controls.

In Experiment A and in Experiment B no statistically significant differences between test item treatment and controls (solvent and historical) groups and no dose-response relationships were noted.

No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of P-Nitrobenzoic acid,
compound with 2,2’,2”-nitrilotriethanol (1:1).

pH and osmolality values of control and test item treatment solutions were measured. In Experiments A and B no significant differences between test item
treatment and control groups were observed.

In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was less than 5 %, confirming the suitability of the
cell line used.

The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 *L/mL) and Cyclophosphamide (5 *g/mL) caused the expected biologically relevant increases of cells with structural chromosome
aberrations as compared to solvent controls and were compatible with the historical positive control data (Tables 3, 4, 5, 6 and 7). Thus, the study is considered valid.

Any other information on results incl. tables

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

3-hour treatment without and with S9 mix / 20-hour sampling time

 

Test group

Concentration
(µg/mL)

Parallels

S9-mix

Cell counts

Mean cell counts

Increase in cell counts

RICC (%)

Cytotoxicity
(%)

First count

Second count

Initial cell count

-

A

1950000

2050000

1968750

-

-

-

-

B

1950000

1950000

-

C

2000000

2000000

-

D

1900000

1950000

Solvent control (DME)

-

A

6800000

7000000

7012500

5043750

100,00

0,00

-

B

7100000

7150000

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

250

A

6900000

6900000

6900000

4931250

97,77

2,23

500

A

5900000

6100000

6000000

4031250

79,93

20,07

1000

A

5900000

5600000

5750000

3781250

74,97

25,03

2000

A

4100000

4250000

4175000

2206250

43,74

56,26

EMS 1µL/mL

A

4300000

4600000

4450000

2481250

49,19

50,81

Solvent control (DME) medium

-

A

+

5900000

6250000

6162500

4193750

100,00

0,00

-

B

+

6100000

6400000

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

250

A

+

6150000

6150000

6150000

4181250

99,70

0,30

500

A

+

6000000

5950000

5975000

4006250

95,53

4,47

1000

A

+

5800000

5750000

5775000

3806250

90,76

9,24

2000

A

 

5300000

5600000

5450000

3481250

83,01

16,99

Cycl. 5µg/mL

A

+

4100000

4000000

4050000

2081250

49,63

50,37

RICC=Relative Increase in Cell Counts

Cytotoxicity= 100-RICC

DME: (Dulbecco’s Modified Eagle’s)medium

EMS: Ethyl methanesulfonate (EMS)

Cycl: Cyclophosphamide monohydrate

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

 

20-hour treatment without S9 mix / 20-hour sampling time

 

Test group

Concentration
(µg/mL)

Parallels

S9-mix

Cell counts

Mean cell counts

Increase in cell counts

RICC (%)

Cytotoxicity
(%)

First count

Second count

Initial cell count

-

A

1950000

2050000

1968750

-

-

-

-

B

1950000

1950000

-

C

2000000

2000000

-

D

1900000

1950000

Solvent control (DME)

-

A

6500000

6200000

6362500

4393750

100,00

0,00

-

B

6300000

6450000

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

62.5

A

6400000

6200000

6300000

4331250

98,58

1,42

125

A

6100000

6000000

6050000

4081250

92,89

7,11

250

A

5100000

4950000

5025000

3056250

69,56

30,44

500

A

4500000

4850000

4675000

2706250

61,59

38,41

1000

A

4100000

4100000

4100000

2131250

48,51

51,49

2000

A

3200000

3500000

3350000

1381250

31,44

68,56

EMS 1µL/mL

A

4200000

4000000

4100000

2131250

48,51

51,49

RICC=Relative Increase in Cell Counts

Cytotoxicity= 100-RICC

DME: (Dulbecco’s Modified Eagle’s)medium

EMS: Ethyl methanesulfonate (EMS)

 

Summarized Results of the concentration SELECTION CYTOTOXICITY ASSAY

 

20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time

 

Test group

Concentration
(µg/mL)

Parallels

S9-mix

Cell counts

Mean cell counts

Increase in cell counts

RICC (%)

Cytotoxicity
(%)

First count

Second count

Initial cell count

-

A

1950000

2050000

1968750

-

-

-

-

B

1950000

1950000

-

C

2000000

2000000

-

D

1900000

1950000

Solvent control (DME)

-

A

7850000

7700000

7862500

5893750

100,00

0,00

-

B

8000000

7900000

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

62.5

A

7850000

7700000

7775000

5806250

98,52

1,48

125

A

7500000

7450000

7475000

5506250

93,43

6,57

250

A

6100000

6000000

6050000

4081250

69,25

30,75

500

A

5400000

5750000

5575000

3606250

61,19

38,81

1000

A

4850000

4900000

4875000

2906250

49,31

50,69

2000

A

3850000

3500000

3675000

1706250

28,95

71,05

EMS 1µL/mL

A

4800000

4700000

4750000

2781250

47,19

52,81

Solvent control (DME)

-

A

+

7850000

7650000

7787500

5806250

100,00

0,00

-

B

+

7950000

7700000

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

250

A

+

7750000

7750000

7750000

5768750

99,35

0,65

500

A

+

7550000

7700000

7625000

5643750

97,20

2,80

1000

A

+

7150000

7100000

7125000

5143750

88,59

11,41

2000

A

+

6900000

6750000

6825000

4843750

83,42

16,58

Cycl. 5µg/mL

A

+

5050000

4850000

4950000

2968750

51,13

48,87

RICC=Relative Increase in Cell Counts

Cytotoxicity= 100-RICC

DME: (Dulbecco’s Modified Eagle’s)medium

EMS: Ethyl methanesulfonate (EMS)

Cycl: Cyclophosphamide monohydrate

MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT A

 

 

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/150cells

incl. gaps

excl. gaps

Negative (Solvent) control

-

3 h

20 h

6

3

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

250 µg/mL

-

3 h

20 h

6

4

500 µg/mL

-

3 h

20 h

7

4

1000 µg/mL

-

3 h

20 h

8

3

2000 µg/mL

-

3 h

20 h

8

3

Pos. Control
(
Ethyl methanesulphonate)

-

3 h

20 h

37**

29**

Negative (Solvent) control

+

3 h

20 h

6

3

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

500 µg/mL

+

3 h

20 h

9

4

1000 µg/mL

+

3 h

20 h

7

4

2000 µg/mL

+

3 h

20 h

8

4

Pos. Control (Cyclophosphamide)

+

3 h

20 h

44**

38**

Positive control (-S9): Ethyl methanesulphonate (1.0L/mL)

Positive control (+S9): Cyclophosphamide (5.0g/mL)

** = p < 0.01 to the concurrent negative control and to the historical control


MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B

 

 

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/150cells

incl. gaps

excl. gaps

 

Negative (Solvent) control

-

20 h

20 h

6

3

 

 

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

 

 

125 µg/mL

-

20 h

20 h

6

3

 

 

250 µg/mL

-

20 h

20 h

7

3

 

 

500 µg/mL

-

20 h

20 h

7

4

 

 

1000 µg/mL

-

20 h

20 h

6

3

 

 

Pos. Control
(
Ethyl methanesulphonate)

-

20 h

20 h

45**

35**

 

 

Negative (Solvent) control

-

20 h

28 h

6

3

 

 

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

 

 

125 µg/mL

-

20 h

28 h

6

3

 

 

250 µg/mL

-

20 h

28 h

7

3

 

 

500 µg/mL

-

20 h

20 h

8

4

 

 

1000 µg/mL

-

20 h

28 h

7

3

 

 

Pos. Control
(Ethyl methanesulphonate)

-

20 h

28 h

43**

35**

 

Positive control (-S9): Ethyl methanesulphonate (0.4L/mL)

** = p < 0.01 to the concurrent negative control and to the historical control

 


MEAN NUMBER OF CELLS WITH STRUCTURAL
CHROMOSOME ABERRATION(s) EXPERIMENT B

 

 

Concentration
(µg/mL)

S9 mix

Treatment
time

Harvesting time

Mean aberrant cells/150cells

 

incl. gaps

excl. gaps

 

Negative (Solvent) control

+

3 h

28 h

7

4

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

500 µg/mL

+

3 h

28 h

8

2

1000 µg/mL

+

3 h

28 h

7

4

2000 µg/mL

+

3 h

28 h

9

5

Pos. Control (Cyclophosphamide)

+

3 h

28 h

48**

38**

Cyclophosphamide: 5.0g/mL

** = p < 0.01 to the concurrent negative control and to the historical control

 

 


NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS

 

EXPERIMENT A

 

Concentration
(µg/mL)

S9 mix

Treatment/Harvesting
time

Polyploid Cells (mean)

Endoredup-lication (mean)

Negative (Solvent) control

-

3/20 h

0.0

0.0

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

250 µg/mL

-

3/20 h

0.0

0.0

500 µg/mL

-

3/20 h

0.0

0.0

1000 µg/mL

-

3/20 h

0.0

0.0

2000 µg/mL

-

3/20 h

0.0

0.0

Pos. Control
(
Ethyl methanesulphonate)

-

3/20 h

0.0

0.0

Negative (Solvent) control

+

3/20 h

0.0

0.0

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

500 µg/mL

+

3/20 h

0.0

0.0

1000 µg/mL

+

3/20 h

0.0

0.0

2000 µg/mL

+

3/20 h

0.0

0.0

Pos. Control (Cyclophosphamide)

+

3/20 h

0.0

0.0

Ethyl methanesulphonate: 1.0mL/mL

Cyclophosphamide: 5.0g/mL

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.


NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS

 

EXPERIMENT B

 

Concentration
(µg/mL)

S9 mix

Treatment/Harvesting
time

Polyploid Cells (mean)

Endoredup-lication (mean)

Negative (Solvent) control

-

20/20 h

0.0

0.0

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

125 µg/mL

-

20/20 h

0.0

0.0

250 µg/mL

-

20/20 h

0.0

0.0

500 µg/mL

-

20/20 h

0.0

0.0

1000 µg/mL

-

20/20 h

0.0

0.0

Pos. Control

-

20/20 h

0.0

0.0

Negative (Solvent) control

-

20/28 h

0.0

0.0

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

125 µg/mL

-

20/28 h

0.0

0.0

250 µg/mL

-

20/28 h

0.0

0.0

500 µg/mL

-

20/28 h

0.0

0.0

1000 µg/mL

-

20/28 h

0.0

0.0

Pos. Control

-

20/28 h

0.0

0.0

Positive control (-S9):Ethyl methanesulphonate(0.4L/mL)

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

 

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS

 

EXPERIMENT B

 

Concentration
(µg/mL)

S9 mix

Treatment/Harvesting
time

Polyploid Cells (mean)

Endoredup-lication (mean)

Negative (Solvent) control

+

3/28 h

0.0

0.0

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)

500 µg/mL

+

3/28 h

0.0

0.0

1000 µg/mL

+

3/28 h

0.0

0.0

2000 µg/mL

+

3/28 h

0.0

0.0

Pos. Control

+

3/28 h

0.0

0.0

Cyclophosphamide: 5.0g/mL

 

The number of polyploid and endoreduplicated cells was determined in
300 cells of each test group.

HISTORICAL CONTROL DATA

3h/20h treatment/sampling time without S9-mix

 

 

number of aberrant cells/ 150 cells

negative control

positive control
(Ethyl methanesulfonate)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.65

2.65

40.69

31.42

SD

0.71

0.65

3.44

3.67

Lower confidence interval

4.11

1.24

33.19

23.42

Upper confidence interval

7.19

4.07

48.19

39.43

n

13

13

13

13

n           = number of experiments

SD        = standard deviation

3h/20h treatment/sampling time with S9-mix

 

 

number of aberrant cells/150cells

negative control

positive control
(
Cyclophosphamide)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.85

2.77

46.19

39.54

SD

0.88

0.55

2.23

2.45

Lower confidence interval

3.94

1.56

41.34

34.20

Upper confidence interval

7.76

3.98

51.05

44.88

n

13

13

13

13

n           = number of experiments

SD        = standard deviation


20h/20h treatment/sampling time without S9-mix

 

 

number of aberrant cells/150cells

negative control

positive control
(Ethyl methanesulfonate)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.62

2.77

45.77

38.19

SD

0.96

0.62

2.39

2.03

Lower confidence interval

3.52

1.42

40.57

33.77

Upper confidence interval

7.71

4.12

50.97

42.61

n

13

13

13

13

n           = number of experiments

SD        = standard deviation

 

20h/28h treatment/sampling time without S9-mix

 

 

number of aberrant cells/ 150cells

negative control

positive control
(Ethyl methanesulfonate)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.54

2.73

45.31

36.85

SD

0.62

0.59

2.27

2.84

Lower confidence interval

4.19

1.45

40.36

30.65

Upper confidence interval

6.89

4.01

50.25

43.04

n

13

13

13

13

n           = number of experiments

SD        = standard deviation

 


 

3h/28h treatment/sampling time with S9-mix

 

 

number of aberrant cells/ 150 cells

negative control

positive control
(Cyclophosphamide)

incl. Gaps

excl. Gaps

incl. Gaps

excl. Gaps

Mean

5.65

2.73

45.81

38.58

SD

0.52

0.52

1.99

2.97

Lower confidence interval

4.52

1.60

41.47

32.11

Upper confidence interval

6.78

3.86

50.14

45.04

n

13

13

13

13

n           = number of experiments

SD        = standard deviation


Applicant's summary and conclusion

Conclusions:
P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), tested up to the maximum cytotoxic cconcentrations without mammalian metabolic activation system and up to the maximum recommended concentration with mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.

Thus, the test item is considered as not clastogenic in this system.
Executive summary:

The test item, P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) was tested in a Chromosome Aberration Assay in V79 cells.The test item was dissolved inDME (Dulbecco’s Modified Eagle’s) mediumand the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using rodent S9 mix).In two independent experiments (both run in duplicate with concurrent negative and positive controls) at least 300 (150-150) well-spread metaphase cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below:

Experiment Awith 3/20 h treatment/sampling time

without:                           250, 500, 1000and 2000g/mLtest item

with S9 mix:                   500, 1000 and 2000g/mLtest item

Experiment Bwith 20/20 h treatment/sampling time

without S9 mix:              125, 250, 500 and 1000g/mLtest item

Experiment Bwith 20/28 h treatment/sampling time

without S9 mix:              125, 250, 500 and 1000g/mLtest item

Experiment Bwith 3/28 h treatment/sampling time

with S9 mix:                   500, 1000 and 2000g/mLtest item

 

In the performed Chromosome Aberration Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for lower -cytotoxic substances is 2000 µg/mL (based on the updated OECD Guideline 473 (2016)).

 

Following treatment (exposure) and sampling (expression) time cells were exposed to selection agent Colchicine

(0.2 µg/mL) 2.5 hours prior to harvesting. Following harvesting cells were treated with fixative for ca. 10 min. before being placed on slides and stained. Chromosome aberration frequencies were then scored for at least 300 well-spread metaphase cells.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum cytotoxic concentrations and recomemmended concentrations.There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

 

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent controls, up to the maximum cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.


In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted.The observed chromosome aberration rates were within the ranges of historical control data.

 

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

 

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

 

The number of aberrations found in the solvent controls was in the range of historical laboratory control data.The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 L/mL) and Cyclophosphamide (5 g/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

 

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), tested up to the maximum cytotoxic cconcentrations without mammalian metabolic activation system and up to the maximum recommended concentration with mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells.

 

Thus, the test item is considered as not clastogenic in this system.