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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) has unequivocal mutagenic activity on Salmonella typhimuriumTA100 and TA1535 strains whose auxotrophy caused by base-pair substitution in the absence and also presence of exogenous metabolic activation under the test conditions used in the study OECD 471 (Ames test positive).

P-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), tested up to the maximum cytotoxic concentrations 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 (OECD 473 negative).

For the test item p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1), no in vitro mammalian gene cell mutation test was performed. However, data is available for the two individual components of the test item (p-Nitrobenzoic acid and 2,2’,2”-nitrilotriethanol). The substance p-Nitrobenzoic acid (CAS 62-23-7) was examined for mutagenic activity in V79 Chinese hamster cells with two independent experiments. p-Nitrobenzoic acid does not induce gene mutations in the HGPRT test with V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described (OECD 476 negative). The substance 2,2’,2”-Nitrilotriethanol (CAS 102-71-6) was non mutagenic for mouse lymphoma L5178Y cells when evaluated in the absence and presence of an externally supplied metabolic activation (S9) system under the experimental conditions described (OECD 476 negative). Both components do not induce gene mutations in mammalian cell (OECD 476 negative for both substances). Therefore, it can be concluded that the mixture 1:1 of both substances should not induce gene mutations in mammalian cells.

In conclusion, the test item p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) is not classified with genetic toxicity according to the CLP regulation 1272/2008/EC.

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
Chromatid and chromosome type aberrations (gaps, deletions and exchanges), in metaphase cells.
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
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.
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.

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


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.


Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 March, 2017 - 05 April, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, 2012
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are
defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot
repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100)
carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA
excision repair.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/¿-naphthoflavone-induced rats
Test concentrations with justification for top dose:
S9 Mix: 3200; 1600; 500; 250; 160; 50 and 16 µg/plate;
+S9 Mix: 5000; 1600; 1000; 500; 160; 50 and 16 µg/plate
Vehicle / solvent:
ultrapure water (ASTM Type 1)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
The tester strains arrived to the test facility in a form of disc cultures. The origin of the following tester strains: Salmonella typhimurium TA98, TA100, TA1535,
TA1537 and Escherichia coli WP2 uvrA:
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures

Storage of Tester Strains

The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO is added as a cryoprotective agent.


The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity
(uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Procedure for Bacterial Cultures

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the
overnight cultures in the assay. The cultures were incubated for approximately 10-14 hours in a 37oC Benchtop Incubator Shaker.

The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Media

The details of the used chemicals (supplier/manufacturer, batch/lot number and expiry/retest date) are summarised in Table 3. This report contains the media composition in the sections 5.4.2 - 5.4.5, 5.5.2 and 5.5.3 referring to 1000 mL.

The Minimal Glucose Agar (MGA) Plates

Ready-to-use minimal glucose agar (MGA) plates were used in the study. The origin of the ready-to use MGA plates:
Supplier: VWR International;
Manufacturer: Merck Life Science GmbH, Germany.
Certificates of Analysis* were obtained from the supplier.
Typical composition (g/1000 mL) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g
* Batch No.: 142584; Expiry date: 22 May 2017; (used in the Informatory Toxicity Test)143118; Expiry date: 09 July 2017; (used in the Initial and the Confirmatory Mutation Tests)

Nutrient Broth No. 2

Nutrient broth No. 2. 25.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.

Nutrient Agar

Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.

Top Agar for Salmonella typhimurium Strains

Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 mL
Sterilization for 20 minutes was performed at 121°C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 mL
Agar solution 900.0 mL

Top Agar for Escherichia coli Strain

Tryptophan solution (2 mg/mL):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 mL
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth by (Section: 5.4.2) 50.0 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution by (Section: 5.4.4) 947.5 mL

Metabolic Activation System

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented
post-mitochondrial fraction (S9).

Rat Liver S9 Fraction

The S9 fraction of Phenobarbital (PB) and ß-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH
(Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier1). The original Quality Control & Production
Certificates of rat liver S9 are stored in the Laboratory of TOXI-COOP ZRT.

1) Lot Number: 3634; Expiry date: May 12, 2018; Protein content: 40.3 mg/mL
(used in the Informatory Toxicity Test);
Lot Number: 3662; Expiry date: July 07, 2018; Protein content: 40.5 mg/mL
(used in the Informatory Toxicity Test);
Lot Number: 3712; Expiry date: November 03, 2018; Protein content: 34.3 mg/mL
(used in the Confirmatory Mutation Test);
Lot Number: 3727; Expiry date: December 01, 2018; Protein content: 33.7 mg/mL
(used in the Initial and Confirmatory Mutation Tests).

The S9 Mix (with Rat Liver S9)

Salt solution for S9 Mix Final concentration in S9 Mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 mL
Sterilized by filtration through a 0.22 µm membrane filter.

The complete S9 Mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 mL
Rat liver homogenate (S9) 100 mL
Salt solution for S9 Mix 400 mL
The S9 Mix was kept in an ice bath before it was added to the culture medium.

Sodium Phosphate Buffer (0.2 M, pH 7.4)

Solution A:
Na2HPO4 x 12H2O 71.63 g
Ultrapure water ad 1000 mL
Solution B:
NaH2PO4 x H2O 27.6 g
Ultrapure water ad 1000 mL

Solution A 880 mL
Solution B 120 mL*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture
of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter.
Rationale for test conditions:
Justification of concentrations:
Choice of the concentrations was done on the basis of a Solubility Test and a concentration Range Finding Test (Informatory Toxicity Test)

Based on the solubility test, a stock solution with a concentration of 50 mg/mL was prepared in ultrapure water and diluted in 6 steps by factor of
approximately v10.
Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial Mutation Test:
±S9 Mix: 5000; 1600; 500; 160; 50; 16 and 5 µg/plate.
Unequivocal, demonstrative positive results were obtained in the Initial Mutation Test (Plate Incorporation Test) in the Salmonella typhimurium TA100 and TA1535 strains in the absence and also in the presence of exogenous metabolic activation (±S9 Mix); therefore modification of the originally proposed study procedure was considered as necessary. To investigate and prove the reproducibility of the obtained positive results as Confirmatory Mutation Test a Repeated Plate Incorporation Test was performed in Salmonella typhimurium TA100 and TA1535 strains, and the investigated refined concentration levels were the
followings:
S9 Mix: 3200; 1600; 500; 250; 160; 50 and 16 µg/plate;
+S9 Mix: 5000; 1600; 1000; 500; 160; 50 and 16 µg/plate.
Evaluation criteria:
Evaluation of Experimental Data

The colony numbers on the untreated, vehicle control, positive control and the test item treated plates were determined visually by manual counting,
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without
metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times
higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not
regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean values and appropriate standard deviations and mutation rates were calculated by EXCEL software.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
E. coli WP2 uvr A
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:
Validity of the Performed Experiments

The tester strains used in this study demonstrated the specific phenotype characteristics, were in line with the corresponding historical control data ranges, and showed the adequate strain culture titer.
Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system .
Each of the investigated reference mutagens showed the expected increase (at least a3-fold increase) in induced revertant colonies over the mean value of
the respective vehicle control in both main experimental phases and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in the main experimental phases, in all tester strains.
The spontaneous revertant colony numbers of the ultrapure water (ASTM Type 1) vehicle control plates showed characteristic mean numbers agreed
with the actual historical control data ranges in all strains in both main experimental phases.
Seven concentration levels were investigated in the Informatory Toxicity Test and in the main mutation experiments (Initial and Confirmatory Mutation Tests) in the absence and presence of exogenous metabolic activation (±S9 Mix) (see in Tables of Appendices I-IV).
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have therefore been met.

Controls

In the performed Initial and Confirmatory Mutation Test multiple test items were tested with reference values from the common parallel controls.
In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the ultrapure water (ASTM Type 1) vehicle control plates with and without S9
Mix were in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.
The revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in different experimental phases were slightly higher or lower
than the ultrapure water control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges
In summary, the actual values of untreated, vehicle and positive controls were in line with the criteria for validity of the assay.

Results of Initial and Confirmatory Mutation Tests


Dose-related changed, significant, biologically relevant revertant colony number increases, positive results were obtained in the Initial Mutation Test following
treatment with p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) in Salmonella typhimurium TA100 and TA1535 strains (whose auxotrophy
caused by base-pair substitution) at the concentrations of 1600 and 500 µg/plate, in the absence ( S9 Mix) and at 5000 and 1600 µg/plate in the presence of exogenous metabolic activation (+S9 Mix). Borderline positive results were obtained in S. typhimurium TA100 at 500 µg/plate in the presence of metabolic
activation (+S9 Mix) and the increased revertant colony numbers were above the corresponding historical control data range; however remained below the threshold for being positive in
S. typhimurium TA1535 at 500 µg/plate (+S9 Mix).The obtained revertant colony number increases (in comparison with the revertant colony numbers of the
vehicle control) were also above the corresponding historical control data range in S. typhimurium TA98 at the concentration of 1600 µg/plate ( S9 Mix).
This latter increase was rather unique, did not accompanied with clear dose-related tendency.
In the other examined strains in S. typhimurium TA1537 and in E. coli WP2 uvrA in the absence and presence of metabolic activation (±S9 Mix) and also in
case S. typhimurium TA98 ( S9 Mix) further revertant colony number increases were not observed.
In the Initial Mutation Test, unequivocal inhibitory effect of the test item on bacterial growth was noticed in S. typhimurium TA100, TA1535 and TA1537 strains at the concentration of 5000 µg/plate ( S9 Mix); furthermore in TA1537 at 5000 µg/plate (+S9 Mix), at 1600 µg/plate ( S9 Mix).
The inhibitory effect in the above cases was indicated by absent revertant colony counts and affected background lawn development: reduced or slightly
reduced background lawn.
All of the further observed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system.
Due to the unequivocal, demonstrative positive results obtained in the Initial Mutation Test (Plate Incorporation Test) in the Salmonella typhimurium TA100 and TA1535 strains, in the absence and also in the presence (±S9 Mix) of exogenous metabolic activation; modification of the originally proposed
study procedure was considered as necessary.
To investigate and prove the reproducibility of the obtained positive results, the plate incorporation procedure was repeated as Confirmatory M
utation Test in Salmonella typhimurium TA100 and TA1535 strains in the absence and also in the presence of exogenous metabolic activation (±S9 Mix).
The other strains (Salmonella typhimurium TA98, TA1537 and Escherichia coli WP2 uvrA) were not further examined.

The positive mutagenicity results were unequivocally repeated confirmed in both strains at 1600 and 500 µg/plate, in the absence of metabolic activation ( S9 Mix), and at 1600 µg/plate, in the presence of metabolic activation (+S9 Mix). Further positive results were noticed in both strains at 1000 µg/plate (+S9 Mix) and in S. typhimurium TA1535 at 500 µg/plate (+S9 Mix).
The increased revertant colony numbers were above the corresponding historical control data ranges; however remained below the thresholds for being
positive in both strains at 250 µg/plate ( S9 Mix), and in S. typhimurium TA1535 at 5000 and 160 µg/plate (+S9 Mix).
All of the further observed higher revertant colony numbers (when compared to the revertant colony numbers of the vehicle control) were of minor intensity, far below the corresponding thresholds for being positive and remained within the biological variability range of the applied test system.
In the Confirmatory Mutation Test, unequivocal inhibitory effect of the test item was noticed in both strains at 5000 µg/plate ( S9 Mix).
The inhibitory effect was indicated by decreased revertant colony counts (when compared to the revertant colony counts of the vehicle control, that in
S. typhimurium TA100 were below the corresponding historical control data range) and slightly reduced background lawn.
In the performed experiment no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration
level (±S9 Mix).

Summary Table of the Results of the Concentration Range Finding Test

Range Finding Test (Informatory Toxicity Test)

 

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

TA 98

TA 100

-S9

+S9

-S9

+S9

Mean values of revertants per plate and
Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

 

Untreated Control

21.7

0.82

25.7

0.95

103.7

1.10

121.0

1.24

 

DMSO Control

23.3

1.00

25.3

1.00

93.3

1.00

 

Ultrapure Water Control

26.3

1.00

27.0

1.00

94.0

1.00

97.7

1.00

 

5000

48.3

1.84

36.3

1.35

#

#

614.0

6.29

 

1600

44.7

1.70

26.0

0.96

764.3

8.13

272.7

2.79

 

500

25.3

0.96

25.7

0.95

320.0

3.40

156.3

1.60

 

160

22.0

0.84

25.7

0.95

131.7

1.40

127.0

1.30

 

50

21.3

0.81

25.0

0.93

111.3

1.18

114.7

1.17

 

16

23.0

0.87

29.3

1.09

96.3

1.02

109.3

1.12

 

5

23.0

0.87

26.7

0.99

95.7

1.02

101.3

1.04

 

NPD (4mg)

218.7

9.37

 

SAZ (2mg)

1944.0

20.68

 

2AA (2mg)

1746.7

68.95

2360.0

25.29

 

MR:Mutation Rate

NPD:4-Nitro-1,2-phenylenediamine

SAZ:Sodium azide

2AA:2-aminoanthracene

Remarks:   Ultrapure water was applied as vehicle of the test item and the positive control substance SAZ; the DMSO was applied as vehicle for positive control substances NPD and 2AA. The mutation rate of the test item, SAZ and untreated control is given referring to the ultrapure water; the mutation rate of NPD and 2AA is given referring to DMSO.

#:   The exact evaluation of the revertant colony numbers in S. typhimurium TA100 at the concentration of 5000 µg/plate (-S9 Mix) was equivocal by unaided eye. In microscope at 40X magnification a not affected background lawn development and high number of colonies were seen.

 

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichiacoli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

17.7

1.08

23.0

0.75

100.0

1.11

109.0

1.04

10.0

0.88

14.3

1.05

7.3

0.85

7.3

0.88

32.0

0.88

39.3

1.16

DMSO Control

17.3

1.00

25.0

1.00

92.3

1.00

14.0

1.00

6.0

1.00

7.7

1.00

33.3

1.00

Ultrapure Water Control

16.3

1.00

30.7

1.00

90.0

1.00

104.3

1.00

11.3

1.00

13.7

1.00

8.7

1.00

8.3

1.00

36.3

1.00

34.0

1.00

5000

30.0

1.84

20.7

0.67

0.0

0.00

562.0

5.39

0.0

0.00

78.3

5.73

0.0

0.00

0.0

0.00

24.0

0.66

31.3

0.92

1600

38.0

2.33

20.3

0.66

456.7

5.07

329.3

3.16

157.7

13.91

106.3

7.78

0.0

0.00

8.3

1.00

27.3

0.75

31.3

0.92

500

26.3

1.61

18.3

0.60

286.7

3.19

212.0

2.03

45.0

3.97

33.7

2.46

10.0

1.15

10.0

1.20

27.3

0.75

31.3

0.92

160

19.7

1.20

22.7

0.74

146.7

1.63

123.3

1.18

22.7

2.00

19.7

1.44

6.7

0.77

7.7

0.92

29.0

0.80

32.3

0.95

50

17.7

1.08

24.3

0.79

104.0

1.16

112.7

1.08

12.3

1.09

12.7

0.93

10.0

1.15

8.3

1.00

27.3

0.75

34.3

1.01

16

18.3

1.12

22.0

0.72

94.0

1.04

119.3

1.14

11.3

1.00

11.7

0.85

9.3

1.08

10.0

1.20

30.3

0.83

32.7

0.96

5

18.0

1.10

26.3

0.86

100.0

1.11

121.3

1.16

10.3

0.91

14.0

1.02

9.0

1.04

6.7

0.80

28.7

0.79

38.3

1.13

NPD (4mg)

202.7

11.69

SAZ (2mg)

1234.7

13.72

765.3

67.53

9AA (50mg)

676.0

112.67

MMS (2mL)

918.7

25.28

2AA (2mg)

2429.3

97.17

1632.0

17.68

228.3

16.31

239.3

31.22

2AA (50mg)

197.3

5.92

MR:Mutation Rate;          NPD:4-Nitro-1,2-phenylenediamine;SAZ: Sodium azide;9AA:9-Aminoacridine;MMS:Methyl methanesulfonate;2AA: 2-aminoanthracene

Remarks:           Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and MMS; the DMSO was applied as vehicle for positive control substances NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.


Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Repeated Plate Incorporation Test)

 

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

TA 100

TA 1535

-S9

+S9

-S9

+S9

Mean values of revertants per plate and
Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

 

Untreated Control

98.3

1.06

97.0

1.04

12.7

0.79

14.7

1.22

 

DMSO Control

92.7

1.00

10.3

1.00

 

Ultrapure Water Control

92.7

1.00

93.7

1.00

16.0

1.00

12.0

1.00

 

5000

134.3

1.43

26.7

2.22

 

3200

48.3

0.52

3.0

0.19

 

1600

491.3

5.30

252.7

2.70

208.7

13.04

131.7

10.97

 

1000

218.7

2.33

95.7

7.97

 

500

365.3

3.94

134.0

1.43

69.0

4.31

44.7

3.72

 

250

161.7

1.74

33.3

2.08

 

160

120.3

1.30

104.3

1.11

21.7

1.35

31.0

2.58

 

50

103.3

1.12

109.0

1.16

13.0

0.81

14.0

1.17

 

16

91.7

0.99

98.7

1.05

14.3

0.90

15.0

1.25

 

SAZ (2mg)

1874.7

20.23

1784.0

111.50

 

2AA (2mg)

2562.7

27.65

246.0

23.81

 

MR:Mutation Rate;        SAZ:Sodium azide;2AA:2-aminoanthracene

Remarks:             Ultrapure water was applied as vehicle of the test item and the positive                control substance SAZ; the DMSO was applied as vehicle for positive                               control substance 2AA. The mutation rate of the test item, SAZ and                               untreated control is given referring to the ultrapure water; the mutation              rate of 2AA is given referring to DMSO.

Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

 

Bacterial strains

Historical control data of untreated control

-S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.0

105.0

10.5

8.1

25.4

SD

3.7

25.7

1.4

2.3

5.2

Minimum

9

66

3

2

11

Maximum

39

155

23

19

45

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.5

117.1

11.8

9.0

33.9

SD

4.3

18.1

1.4

1.9

5.2

Minimum

12

75

4

2

17

Maximum

46

166

23

20

56

 

Bacterial strains

Historical control data of DMSO

control

-S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

20.4

100.1

10.3

7.9

24.7

SD

3.6

24.8

1.3

2.4

4.6

Minimum

10

64

3

2

11

Maximum

38

147

23

20

45

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

26.5

113.8

11.8

8.8

33.7

SD

4.1

18.3

1.5

1.9

5.0

Minimum

15

71

3

3

16

Maximum

47

162

25

20

57

 

Bacterial strains

Historical control data of Water

control

-S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.9

104.7

10.5

7.6

26.1

SD

3.7

25.9

1.5

2.2

5.5

Minimum

12

68

3

2

12

Maximum

35

154

24

16

48

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.4

117.3

11.4

8.7

34.9

SD

4.0

18.5

1.3

2.2

4.9

Minimum

15

83

4

3

18

Maximum

43

167

22

16

57

 

Bacterial strains

Historical control data of positive controls

-S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

260.1

977.2

847.3

478.6

724.5

SD

31.8

150.6

126.3

104.5

65.0

Minimum

123

521

359

110

320

Maximum

664

1970

1855

1601

1313

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

1222.7

1436.4

164.1

147.0

257.7

SD

274.9

318.3

33.1

20.1

72.5

Minimum

386

583

85

69

140

Maximum

2676

2988

498

399

477

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,

                               TA1537;E. coli:Escherichia coliWP2uvrA

                               SD: Standard deviation; DMSO: Dimethyl sulfoxide

Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item caused effects were above the
genotoxicological threshold of biological relevance, above the threshold for being positive in the Salmonella typhimurium TA100 and TA1535 strains, examined.
In conclusion, the test item p-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1) has unequivocal mutagenic activity on Salmonella typhimurium TA100 and TA1535 strains whose auxotrophy caused by base-pair substitution in the absence and also presence of exogenous metabolic activation, under the test
conditions used in this study.
Executive summary:

The test itemp-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains ofSalmonella typhimurium(Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. In the Confirmatory Mutation Test (Repeated Plate Incorporation Test) theSalmonella typhimuriumTA100 and TA1535 strains were examined, only.

The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Repeated Plate Incorporation Test).

Based on the results of the Solubility and the Concentration Range Finding Tests the test item was dissolved in ultrapure water (ASTM Type 1). This vehicle was compatible with the survival of the bacteria and the S9 activity and appropriate historical control database is available in the testing laboratory.

Based on the results of the preliminary Concentration Range Finding Test the following concentrations of the test item were prepared and investigated in the Initial Mutation Test:

±S9 Mix: 5000; 1600; 500; 160; 50; 16 and 5 µg/plate.

Unequivocal, demonstrative positive results were obtained in the Initial Mutation Test (Plate Incorporation Test) in theSalmonella typhimuriumTA100 and TA1535 strains in the absence and also in the presence of exogenous metabolic activation (±S9 Mix); therefore modification of the originally proposed study procedure was considered as necessary. To investigate and prove the reproducibility of the obtained positive results as Confirmatory Mutation Test a Repeated Plate Incorporation Test was performed inSalmonella typhimuriumTA100 and TA1535 strains, and the investigated refined concentration levels were the followings:

-S9 Mix: 3200; 1600; 500; 250; 160; 50 and 16 µg/plate;

+S9 Mix: 5000; 1600; 1000; 500; 160; 50 and 16 µg/plate.

The selection of the concentration range for the Initial Mutation Test was based on the Concentration Range-Finding Test results and on the recommendations in OECD 471 guideline for non-toxic, soluble test compounds; accordingly the test item was investigated up to and including the concentration level of 5000 µg/plate. Based on the Initial Mutation Test results, the examined concentration ranges were adequately modified in the Confirmatory Mutation Test.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.

Unequivocal inhibitory effect of the test item on bacterial growth was observed in the Initial Mutation Test in the examinedSalmonella typhimuriumTA100, TA1535 and TA1537 strains, in the Confirmatory Mutation Test in TA100 and TA1535 strains.


 

The inhibitory effect was indicated by decreasedrevertant colony counts (when compared to the revertant colony counts of the vehicle control) or even absent revertant colony counts and affected background lawn development: reduced or slightly reduced background lawn.

The revertant colony numbers of vehicle control (ultrapure water, ASTM Type I) plates with and without S9 Mix demonstratedthe characteristic mean number of spontaneous revertantsthat was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant coloniesand the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin all experimental phases, in all tester strains.

Biologically relevant revertant colony number increases were observed in revertant colony numbers ofSalmonella typhimuriumTA100 and TA1535 strainsfollowing treatment withp-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)in the absence and in the presence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item caused effects were above the genotoxicological threshold of biological relevance, above the threshold for being positive in theSalmonella typhimuriumTA100 and TA1535 strains.

In conclusion, the test itemp-Nitrobenzoic acid, compound with 2,2’,2”-nitrilotriethanol (1:1)has unequivocal mutagenic activity onSalmonella typhimuriumTA100 and TA1535 strains whose auxotrophy caused by base-pair substitution in the absence and also presence of exogenous metabolic activation,under the test conditions used in this study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Supplier, City, State (Lot, Reference Number): BASF Corporation, Ludwigshafen, Germany (Lot #000STD77L0).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
0, 50, 100, 250, 500, 1000 and 1500 µg/ml
Vehicle / solvent:
distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
10 and 15 µg/ml of culture medium in the nonactivation assay
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other:
Remarks:
20-methylcholanthrene (20-MCA) for metabolic activation assay
Details on test system and experimental conditions:
Two independent mutation assays were conducted to assess the mutagenic potential of the test material.

Treatment Procedure
Cells from logarithmically growing stock cultures (maintained in an orbital shaker at 37 C) were treated in individual 50 ml culture tubes. To these cells, 4 ml of either S9 mix or medium was added along with the test material. Following the addition of the test compounds, the tubes were incubated for approximately 4 hours at 37°C in a roller drum (approximately 25-35 orbits/minute). At the end of the incubation period, the cells were pelleted, rinsed with medium and resuspended. The tubes were returned to the roller drum and maintained at 37°C during a standard expression period of two days.

Toxicity Assay
This assay was conducted for selecting concentrations of the test material to be used in the gene mutation assay. The cells were treated with various concentrations of the test material (one replicate per dose) in the absence and presence of S9. At approximately 24 hours after treatment (day 1), the test cultures were counted. If the treated cells failed to multiply to a density of 4 x 10E05 on the first day following treatment, the culture was returned to the incubator without any dilution. On day 2, cultures were again counted for cell density. From these cell counts, the following indices were calculated:
Day 1 SG = Suspension Growth over first day
Day 2 SG = Suspension Growth during second day
CSG = Cumulative Suspension Growth during first 2 days
Day 2 RSG (%) = Cumulative relative suspension growth over first two days

Gene Mutation Assay
Each dose level was set up in duplicate from the time of treatment until the completion of the assay with the exception of positive controls where only one replicate was used. At the end of treatment, cells were returned to the incubator for phenotypic expression. At 24 hours following
treatment (day 1), the test cultures were counted and diluted to a concentration of approximately 3 x 10E05 cells/ml. If the treated cells failed to multiply to a density of 4 x 10E05 cells/ml on the first day following treatment, the culture was returned to the incubator without any dilution. On day 2 (48 hours following treatment) cultures were again counted and treatment levels with desired levels of toxicity were selected for cloning. Cultures with < 10% day 2 RSG were not
cloned. A total sample size of 3 x 10E06 cells from each culture was suspended in cloning medium with trifluorothymidine (TFT) and plated into three petri dishes (100 mm), allowed to gel for approximately 15 minutes at 0-6°C, and returned to the incubator for approximately 13 days to allow for mutant colony formation. The cloning efficiency was determined by serially diluting the sample in cloning medium without 1 µg/ml TFT and then plating the cells into three petri dishes
(100 mm) at a concentration of approximately 200 cells per dish. The dishes were returned to the incubator for approximately 13 days before counting the number of colonies per dish.
An image analyzer (LAI High-Resolution Colony Counting System, Loats Associates, Inc., Westminster, Maryland) was used to count and size colonies. The separation of small and large colonies was determined by inspection of colony sizing histograms of each culture. Mutant colonies form a bimodal distribution and the cutoff between the two distributions was set manually.
The parameter relative total growth (RTG) was used to determine the cytotoxicity of various treatments.
Initially, plates for cloning efficiency were counted and the RTG values calculated. Only those cultures with RTG values = 10% were counted for mutants. However, one exception occurred in the positive control cultures in the presence of S9 in the initial gene mutation assay where the RTG values were less than 10% and the mutant plates were evaluated. These plates were deemed suitable for counting in the initial assay and the data was confirmed in the repeat assay using a lower concentration of the positive control.
The activation and non-activation assays were considered independent assays with their own solvent and positive controls. For an assay to be considered acceptable,
(1) the mutation frequency of positive controls should have been significantly higher than the solvent controls and
(2) the mutant frequency of the solvent controls should have been within 35-140 x 10-6.
The solvent controls must have had an average absolute cloning efficiency between 65-120% and a cumulative suspension growth greater than eight.
Evaluation criteria:
Mutant frequencies were evaluated based upon biological significance criteria. The test chemical was considered positive when the conditions listed below were met:
a) the average mutant frequency in at least one dose level of the treated cultures (resulting in = 10% relative total growth) was 90 x 10-6 above the average of the concurrent solvent controls (assuming these to be in the range of 35-140 x 10-6).
b) there was a positive dose related linear trend. This was tested using a linear trend test at alpha = 0.05, provided the above criteria was met.

The test material was considered negative in this assay if the following condition was met:
a) there was no evidence of increase in mutant frequency at RTG values = 10%.

The test material was considered equivocal in this assay if the following conditions were met:
a) there was a significant increase in mutant frequency only at RTG values > 10% and < 20%.
b) there was no evidence of increase in mutant frequency at RTG values = 20%.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In a preliminary toxicity assay, the test material was evaluated at concentrations ranging from 5.9 to 1500 µg/ml. In the absence of S9, little to no toxicity was observed with day 2 relative suspension growth (RSG) values ranging from 81.2 to 124.2%. In the presence of S9, the corresponding day 2 values ranged from 80.7 to 101.5%.
There were no biologically significant increases in the mutant frequencies in the test materialtreated cultures compared to the solvent control cultures in either absence or presence of S9. The results of this in vitro mouse lymphoma (L5178Y TK+/-) forward gene mutation assay with triethanolamine indicate that under the conditions of this study, the test article was nonmutagenic when evaluated in the absence and presence of an externally supplied metabolic activation (S9) system.
Conclusions:
Under the conditions of this study, the test article was nonmutagenic when evaluated in the absence and presence of an externally supplied metabolic activation (S9) system.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May 18th, 1988 - August 10th, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
April, 4th 1984
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Storage conditions: dark at 4 °C
Target gene:
HGPR gene
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of the "Genetic Toxicology" allowing the repeated use of the same cell culture batch for many experiments. Consequently, the parameters of the experiments remain similiar because of the identical characteristics of the cells.
Thawed stock cultures are kept at 37 °C and 5 % C02 in 75 cm2 plastic-flasks.
Seeding was done with about 1 x 106 cells per flask in 15 ml of MEM-Medium supplement with 10 % FCS (fetal calf serum) contained 2 mM L-glutamine and 0.01 % neomycinsulfate. The cells were subcultured twice a week.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- method of preparation and storages of S9 mix:
Male Sprague Dawley rats (200-300 g) receive a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at 0 to 4 °C using cold sterile solution and glassware. The livers from at least 5-6 animals are removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers}. The washed livers are cut into small pieces and homogenized in three volumes of KCl. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S9 fraction. It is divided into small portions,
rapidly frozen and storage at -80 °C for not longer than three months.

- Preparation of S9-mix:
Sufficient S9 fraction is thawed immediately before each test at room temperature. One volume of S9 fraction is mixed with 9 volumes of the S9 cofactor solution and kept on ice until used. This preparation is termed S9-mix. The concentrations of the different compounds in the S9-mix are:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP+
100 mM phosphate buffer pH 7.4
Test concentrations with justification for top dose:
Without S9-mix: 500, 1000, 1500, 1750 and 2000 ug/ml
With S9-mix: 500, 1000, 1500, and 2000 ug/ml

For toxic subtances a percentage survival rate relative to the solvent control is calculated for each treatment. The dose level which results in a predicted survival of about 30 % is estimated from the results obtained. This dose is chosen as the highest dose level. The lowest doses are chosen of the level from the negativ controls. If the test substance is not sufficiently toxic to reduce survival up to the 30 % level (the maximum of 5 mg/ml or the lowest concentration at which visible precipitation is observed) will be selected as maximal dose.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
the final concentration of organic solvents will not exceed 1 %
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
1.0 mg/ml
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
0.5 %
Positive control substance:
9,10-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Two days old exponentially growing cultures more than 50% confluent are trypsinated and a single cell suspension is prepared. The trypsin concentration is 0.2 % in Ca-Mg-free salt solution. The Ca-Mg-free salt solution is prepared as follows (per litre): NaCl 8000 mg; KCl 400 mg; glucose 1000 mg; NaHC03 350 mg; Trypsin 2000 mg.
Subsequently the cells are replated for mutagenicity testing and for determination of plating efficiency.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: relative survival (RS)
Evaluation criteria:
- the test substance is classified as mutagenic if it induces with one of the test substance concentrations reproducibly a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
-the test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such evaluation may be considered independently from the enhancement factor for induced mutants.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.
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:
The test substance p-Nitrobenzoic acid was assessed for its mutagenic potential in vitro in the HGPRT-test in two independent experiments without metabolic
activation and two independent experiments with metabolic activation. The results of these experiments are presented in tables 3, 4, 5 and 7. No relevant
reproducible enhancement of the mutant colonies or mutant frequency over the range of the negative control was found with any of the concentrations used,
either with or without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell
cultures treated with the positive control substances. The mutation frequency of the negative controls and the solvent control with metabolic
activation in the repeat was higher than the historical controls. This effect as of no toxicological relevance. No relevant reproducible enhancement of
the mutant colonies or mutant frequency over the range of the negative control was found with any of the concentrations used, either with or without metabolic
activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
Conclusions:
The test compound is not mutagenic in the HGPRT-test with cells of the V79 Chinese hamster cell line.
Executive summary:

The test substance p-Nitrobenzoic acid was examined for mutagenic activity in V79 Chinese hamster cells with two independent experiments. The induction of 6-thioguanine resistant mutants after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation

(S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produce a cytotoxic effect at the highest dose group (2000 ug/ml) without metabolic activation.

No indication of cytotoxicity was observed with metabolic activation up to the limit of'solubility (2000 ug/ml).

For mutagenicity testing two independent experiments with and without metabolic activation ( S9-mi x) up to the limit of

solubility were performed.

In the absence and in the presence of S9 metabolic activation dose levels of 500, 1000, 1500 and 2000 ug/ml and in the absence of S9-mix an additional dose level of 1750 ug/ml were used for mutant selection in the main experiments.

The test compound did not induce a significant increase in the number of mutant colonies or in the mutation frequency at any dose level of the test substance. No relevant cytotoxic effect of the compound was observed in the main experiments.

Marked increases in mutation frequency were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion p-Nitrobenzoic acid does not induce gene mutations in the HGPRTtest with V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification