p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August - 03 September

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21st July, 1997, corrected 26th June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

November 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

impurity 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

impurity 2

Reference substance name:

Acetone

EC Number:

200-662-2

EC Name:

Acetone

Cas Number:

67-64-1

Molecular formula:

C3H6O

IUPAC Name:

acetone

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Constituent 1

Reference substance name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

EC Number:

269-198-6

EC Name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Cas Number:

68189-42-4

Molecular formula:

C6H6NO6P.2C4H11NO3

IUPAC Name:

4-nitrophenyl dihydrogen phosphate - 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Test material form:

solid

Details on test material:

Batch / Lot No. 46587100

Specific details on test material used for the study:

Batch-No.: 46587100
Storage: refrigerator (2-8 °C)

Method

Target gene:

histidine locus (Salmonella typhimurium strains) and tryptophan locus (Escherichia coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

bacteria

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

bacteria

Metabolic activation:

with and without

Metabolic activation system:

10 % (v/v) rat liver S9 mix

Test concentrations with justification for top dose:

Initial Mutation Test and Confirmatory Mutation Test: 5000, 2500, 1600, 500, 160 and 50 µg/plate.
For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate.

Concentration Range Finding Test (Informatory Toxicity Tests):
The revertant colony numbers (RCN) and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (S. typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
The RCN of vehicle control plates with and without S9 Mix were in line with the corresponding historical control data ranges; however, in the case of S. typhimurium TA98 the RCN of the ultrapure water vehicle control plates were above the corresponding historical control data range of the ultrapure water (actual value: 41, historical control data range: 10-35), in the absence of exogenous metabolic activation (-S9). Due to the informatory character of this test phase, the higher value was considered as an acceptable.
The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
In the performed informatory toxicity test unequivocal inhibitory effect of the test item was not observed, the colony and background lawn development was not affected in any case. All of the obtained slight RCN decreases or increases (compared to vehicle control) remained within the biological variability range of the applied test system.
In the case of S. typhimurium TA98, RCNs in the concentration range of 1600-5 µg/plate (-S9) were above the corresponding ultrapure water historical control data range.
These data were considered as acceptable and informative at the concentration choice for later follow-up experiments.
No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9).

Vehicle / solvent:

- Vehicle/solvent used: Ultrapure water (ASTM Type I)
- Justification for choice of solvent/vehicle: In the solubility test the test item behaviour was investigated in the applied test system. The test item was adequately soluble in ultrapure water (ASTM Type I); therefore, it was dissolved in ultrapure water, accordingly. The obtained solutions with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine, NPD

Remarks:

Positive Control concentration 4 µg/plate for TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

positive control concentration: 2 µg/plate for TA100 and TA 1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Positive control concentration: 50 µg/plate for TA1537

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control concentration: 2 µL/plate for WP2 uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

Positive Control concentration with metabolic activation: 2 µg/plate for S. typhimurium strains; 50 µg/ plate for E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION
initial mutation test: in agar (plate incorporation)
confirmatory test: in agar (plate incorporation) with preincubation
- Bacterial cultures: The frozen bacterial cultures were thawed at room temperature and 200 µL in oculum 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 10-12 hours in a 37°C Benchtop Incubator Shaker.
- Molten top agar was prepared and kept at 45°C. Two mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45°C).
The content of the tubes:
top agar 2000 µL
vehicle or solution of test item or positive controls 100 µL
overnight culture of test strain* 100 µL
phosphate buffer (pH: 7.4) or S9 mix 500 µL
*: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA.
This solution was mixed and poured on the surface of the properly labeled minimal agar plates. For incubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 Mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each with the addition of negative and positive controls. The plates were incubated at 37°C for about 48 hours.
- Exposure duration: incubated at 37°C for about 48 hours
- Preincubation period (in confirmatory test): Before the overlaying of the test item, the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to allow direct contact between bacteria and the test item (in its vehicle). These tubes were gently mixed and incubated for 20min at 37ºC in a shaking incubator. After the incubation period, two mL of molten top agar was addedto the tubes, and the content was mixed and poured onto minimal glucose agar plates. After preparation the plates were incubated at 37°C for about 48 hours.

NUMBER OF REPLICATIONS
Triplicates

METABOLIC ACTIVATION SYSTEM:
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).

VALIDITY CRITERIA
The tests (Initial and Confirmatory Mutation Tests) are considered valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM 101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers are in the 10exp9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three nontoxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.
- For soluble, non-toxic test compounds the maximum test concentration is 5 mg/plate or 5 µL/plate. For test compounds that are not soluble at 5 mg/plate or 5 µL/plate and that are not toxic at levels lower than an insoluble level, the highest doses tested is at least one insoluble concentration in the final treatment mixture under the actual conditions of the test at the start of the experiment. Insolubility is assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye.
- The test has to be included five analyzable concentrations (where the precipitate does not interfere with the scoring) and a minimum of three non-precipitated dose levels.

Evaluation criteria:

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 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.

Statistics:

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

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: slight inhibitory tendency was noticed in Salmonella typhimurium TA98 (+S9), in TA100 (+S9), in TA1535 (-S9), in TA1537 (±S9) strains at the highest concentration level of 5000 µg/plate, in TA98 (+S9) and in TA1537 (±S9) at 2500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Validity Criteria: All criteria for the validity of the performed experiments were met.
- Controls: The positive control reference items (diagnostic mutagens) induced the expected, biological relevant increases (more than 3-fold increase) in revertant colonies and the number of revertant colonies was within the historical control data range per strain, thereby meeting the criteria for a valid positive control in all experimental phases (in the confirmatory mutation test together with the additional confirmatory mutation test) and all tester strains.
The spontaneous revertant colony numbers of the ultrapure water (ASTM Type I) vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in the experiments (in the confirmatory mutation test with the additional
confirmatory mutation test).
In the concentration range finding test (informatory toxicity test) in the case of Salmonella typhimurium TA98 the revertant colony numbers of the ultrapure water vehicle control plates were above the corresponding historical control data range of the ultrapure water (actual value: 41, historical control data range: 10-35), in the absence of exogenous metabolic activation (-S9). Due to the informatory character of this test phase, the higher value was considered as acceptable.
In the confirmatory mutation test in the case of Salmonella typhimurium TA100 the revertant colony numbers of the ultrapure water (ASTM Type I) vehicle control plates (as well as the revertant colony numbers of the untreated control) were far below the corresponding historical control data range in the absence of exogenous metabolic activation (-S9). In this case the reference mutagen treatment (Sodium azide positive control) showed biological relevant increase (more than 3.0-fold increase) in induced revertant colonies and over the mean value of the respective vehicle control, but the number of revertants was far below the the corresponding historical control range of Sodium azide. The obtained lower revertant colony numbers in the respective controls invalidated this experimental part. In this experimental part, at the test item treatments the obtained revertant colony numbers were low, all of them were below the corresponding historical control data range of the ultrapure water vehicle control. The validation criterion regarding the number of analysable concentration
levels were not fulfilled, either.
In summary, the actual values of positive, vehicle and untreated controls were in line with the criteria for validity of the assay.

-Initial and Confirmatory Mutation Tests (Plate incorportation and pre-incubation Tests): No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with 4-NPP, di -TRIS at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.
In the performed experiments, occasional increases in the revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest mean revertant colony number increase was observed in the initial mutation test (plate incorporation test) in the case of Salmonella typhimurium TA1535, at 500 µg/plate, in the absence of exogenous metabolic activation (-S9). The revertant colony numbers at this concentration level were above the corresponding vehicle control data range; however, remained within the corresponding historical control data range and the value (increase) was unique, was not accompanied with dose-relationship; therefore, this higher value was considered rather as being within the biological variability range of the applied system. The mutation rate was 1.50, far below the genotoxicological threshold for being positive.
In the initial mutation test slight, rather equivocal inhibitory effect of the test item on bacterial growth was observed in Salmonella typhimurium TA98 (+S9), in TA100 (±S9), in TA1535 (±S9) and in TA1537 (±S9) strains at the highest concentration level of 5000 µg/plate, in TA1537 (+S9) also at 2500 µg/plate.
In the confirmatory mutation test (in the valid experimental parts) nearly the same, slight inhibitory tendency was noticed in Salmonella typhimurium TA98 (+S9), in TA100 (+S9), in TA1535 (-S9), in TA1537 (±S9) strains at the highest concentration level of 5000 µg/plate, in TA98 (+S9) and in TA1537 (±S9) at 2500 µg/plate.
In the additional confirmatory mutation test in Salmonella typhimurium TA100 slight inhibition was noticed at the highest concentration of 5000 µg/plate (-S9).
The slight cytotoxicity was indicated by affected background lawn development (slightly reduced background lawn) and/or decreased revertant colony counts (revertants below the
corresponding vehicle control data ranges and/or historical control data ranges).
No precipitation of the test item was observed on the plates after about 48 hours incubation in the examined bacterial strains at any examined concentration level (±S9) in the performed
experiments.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item 4-NPP, di -TRIS (CAS No. 68189-42-4) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

Purpose of the study:

The test item 4-NPP, di -TRIS (CAS No. 68189-42-4) was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

Bacterial strains, exogenous metabolic activation:

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/ß-naphthoflavone-induced rats.

Experimental phases:

The study included preliminary solubility investigations, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (plate incorporation test), a confirmatory mutation test (pre-incubation test) and an additional confirmatory mutation test (additional pre-incubation test).

The additional confirmatory mutation test was performed with Salmonella typhimurium TA100, in the absence of exogenous metabolic activation (-S9).

Vehicle, test item concentrations, rationale for dose selection:

Based on the results of the solubility test and the concentration range finding test, the test item was dissolved in ultrapure water (ASTM Type I) and the following concentrations were prepared and investigated in the initial and confirmatory mutation tests in the absence and presence of exogenous metabolic activation: ±S9: 5000; 2500; 1600; 500; 160 and 50 µg/plate.

The selection of the concentration range was in accordance with the OECD 471 guideline. The maximum test concentration was in all strains 5000 µg/plate, as recommended in the guideline for soluble non-toxic compounds.

At the preparation of the test item stock solutions any correction (multiplier) factor was not taken into consideration, the doses were based on the final formulation as is.

Solubility, precipitation:

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

Cytotoxicity results:

In the initial and confirmatory mutation tests slight, but unequivocal inhibitory effect of the test item on bacterial growth was observed in Salmonella typhimurium strains at 5000 µg/plate, in Salmonella typhimurium TA1537 also at 2500 µg/plate. The inhibitory effect appeared in the absence and/or presence of exogenous metabolic activation.

The slight cytotoxicity was indicated by affected background lawn development (slightly reduced background lawn) and/or decreased revertant colony counts (revertants below the corresponding vehicle control data ranges and/or historical control data ranges).

Mutagenicity results:

The revertant colony numbers of vehicle control (ultrapure water, ASTM Type I) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges in all experiments (in the confirmatory mutation test together with the additional confirmatory mutation test). (In the confirmatory mutation test, in the case of Salmonella typhimurium TA100 in the absence of S9 invalid results were obtained (regarding positive, negative controls test item treatments); therefore, this experimental part was repeated as an additional confirmatory

mutation test).

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases (in the confirmatory mutation test together with the additional confirmatory mutation test, see above), in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with 4-NPP, di -TRIS at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

Conclusion:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations in the genome of the strains of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and of Escherichia coli WP2 uvrA.

In conclusion, the test item 4-NPP, di -TRIS (CAS No. 68189-42-4) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.