1,4-bis(isopropylamino)anthraquinone

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:

3 SEPTEMBER 2018 to 26 OCTOBER 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD 471 Guideline for Testing of Chemicals. ‘Bacterial Reverse Mutation Tests’. 21st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis(isopropylamino)anthraquinone

EC Number:

238-101-9

EC Name:

1,4-bis(isopropylamino)anthraquinone

Cas Number:

14233-37-5

Molecular formula:

C20H22N2O2

IUPAC Name:

1,4-bis[(propan-2-yl)amino]-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Method

Target gene:

The Ames test evaluates the potential of the test item to revert mutations present in amino acid-requiring bacterial strains. The reversion restores the functional capability of the bacteria to synthesize the essential amino acid thus enabling the bacterial culture to grow in the absence of the amino acid required by the parent bacterial strain.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: All the bacterial strains used in the Ames test carry a mutant gene that prevents them from synthesizing an essential amino acid. These strains may carry additional mutations which increase their sensitivity to different types of mutagens.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: All S. typhimurium strains used in the test carry the rfa mutation. This mutation causes an alteration in the lipopolysaccharide (LPS) layer making the bacteria more permeable to larger molecules.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9

Test concentrations with justification for top dose:

The test item was soluble in DMSO at a concentration of 50 mg/mL (5 mg/plate).
Nevertheless, precipitation signs were observed in the assay final mixture with PBS at concentrations of 50, 16.7 and 1.85 mg/mL.
Therefore, the C5 selected for the cytotoxicity assay was 1.85 mg/mL (0.185 mg/plate), as recommended by the OECD guideline 471.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle:
The test item is soluble in this vehicle.

- Justification for percentage of solvent in the final culture medium:
The test item was soluble in DMSO at a concentration of 50 mg/mL (5 mg/plate).
Nevertheless, precipitation signs were observed in the assay final mixture with PBS at concentrations of 50, 16.7 and 1.85 mg/mL.
Therefore, the C5 selected for the cytotoxicity assay was 1.85 mg/mL (0.185 mg/plate), as recommended by the OECD guideline 471.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 5

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk:
C5- 0.185 mg/plate
C4- 0.62 mg/plate
C3- 0.021 mg/plate
C2- 0.007 mg/plate
C1- 0.002 mg/plate


TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 48h
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

Rationale for test conditions:

The Ames test evaluates the potential of the test item to revert mutations present in amino acid-requiring bacterial strains. The reversion restores the functional capability of the bacteria to synthesize the essential amino acid thus enabling the bacterial culture to grow in the absence of the amino acid required by the parent bacterial strain.
Many chemicals are not mutagenic in their native forms, but are converted into mutagenic substances by metabolism in the liver. Selected bacterial strains do not produce the enzymes required to transform these chemicals. To identify the pro-mutagenic potential of a test item, the metabolic activation system (commercially available post-mitochondrial fraction (S9) from livers of rodents treated with the enzyme-inducing agent Aroclor) is also used in the test.
The mutagenic or pro-mutagenic potential of the test item is assessed by the increase in the number of revertant colonies upon exposure to the test item relative to the number of spontaneously occurring revertant colonies in the controls.

Evaluation criteria:

The criteria used for determining a positive result take into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
A result is considered positive whenever the number of revertants of the test item-treated plates is increased
when compared to the solvent-treated plates according to the following criteria:
Species Strain Mutagenic Ratio (R) cut-off point for
considering a positive result
S. typhimurium TA98 2 fold
S. typhimurium TA100 2 fold
E. coli WP2(pKM101) 2 fold
S. typhimurium TA1535 3 fold
S. typhimurium TA1537 3 fold
Biological relevance of the results was also considered

Ames test acceptance criteria
The bacterial reverse mutation test for the test item 1,4-bis(isopropylamino)anthraquinone was considered
valid as the following criteria were met:
− The mean solvent control counts complied with Vivotecnia historical data for each strain.
− The mean reference item control counts complied with Vivotecnia historical data for each strain.

Statistics:

The number of revertant colonies per plate was counted and recorded by an automatic colony counter.
Average plate counts were presented with the mean and the standard deviation for each set of triplicates per test item concentration and was used to calculate the ratio of colonies per exposed plate compared to the corresponding negative control.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

According to the results disclosed in this report, under these experimental conditions it can be concluded that the test item 1,4-bis(isopropylamino)anthraquinone induces frameshift mutations in the genome of S. typhimurium strain TA98 in the pre-incubation procedure with metabolic activation.
Therefore, the test item 1,4-bis(isopropylamino)anthraquinone at an exposure dose range of 0.021 – 0.185 mg/plate is considered to be NON MUTAGENIC / PRO-MUTAGENIC under the experimental conditions assayed.

Executive summary:

The bacterial reverse mutation test (Ames test) assesses the mutagenic and/or pro-mutagenic potential of the test item 1,4-bis(isopropylamino)anthraquinone in several bacterial strains.
The test was performed in accordance with OECD Guideline 471 for the Testing of Chemicals (Bacterial Reverse Mutation Test. Adopted 21st July 1997).
Cytotoxicity evaluation of the test item was performed in the S. typhimurium TA100 strain by the direct incorporation procedure and without metabolic activation with 5 concentrations of the test item based on its solubility profile (0.02, 0.0067, 0.0022, 0.0007 and 0.0002 mg/plate).
No test item related cytotoxic activity was observed at any of the concentrations tested.
On the basis of these results, 5 test item doses ranging between 0.0002 and 0.02 mg/plate were assayed in the main test. None of the concentrations assayed for the test item showed an increase in the R value either with or without S9 metabolic activation regardless of the procedure.
No dose response for the test item 1,4-bis(isopropylamino)anthraquinone was observed in any of the tested bacterial strains.
Overall interpretation of the study results suggests that the test item does not induce point mutations or frameshifts in the genome of the bacterial strains with or without metabolic activation regardless of the procedure.
Therefore, the test item 1,4-bis(isopropylamino)anthraquinone at an exposure dose range of 0.0002 – 0.02 mg/plate is considered to be NON MUTAGENIC / NON PRO-MUTAGENIC under the experimental conditions assayed.