4,4'-bis(diethylamino)benzophenone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a reliable study the potential of the substance and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichiacoli (E. coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9) was determined. The substance did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  

Read-across studies:

In a guideline (OECD 473) study conducted with GLP certification, the test material (EC 444-860-9) was shown to not be genotoxic, but did exhibit a clastogenic response indicating cytotoxicity at 120 µg/L. The test was carried out over a range of concentrations (20 -200 µg/l) in Chinese hamster lung fibroblasts with and without metabolic activation. The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

An in vitro gentoxicity study was undertaken to evaluate the substance for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity of the substance was tested in two independent experiments.In the first cytogenetic assay, the substance was tested up to 125 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. The substance precipitated in the culture medium at this dose level.  In the second cytogenetic assay, the substance was tested up to 125 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 90 µg/ml for a 48 h continuous exposure time with a 48 h fixation time, both in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed in the first cytogenetic assay both in the absence and presence of S9-mix and in the second cytogenetic assay at the 24 h exposure time. However, it was noted that the substance increased the number of polyploid cells in the absence of S9-mix at the 48 h exposure time at the highest concentration tested. This may indicate that the substance has the potential to inhibit mitotic processes.

In a guideline (OECD 476) study conducted with GLP certification, the test material (EC: 444-860-9) was considered to be non-genotoxic. The HPRT test was conducted in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Based on the solubility properties of the test substance and according to an initial rangefinding cytotoxicity test doses up to 250 µg/ml were tested in presence and absence of a metabolic activation system.

In a guideline (OECD 476) study conducted with GLP certification, the test material (EC 444-860-9) was determined not to be genotoxic. The test was conducted on mice (10 mice per sex per dose), with the substance administered via gavage in a single dose (nominal concentrations: 500, 1000 and 2000 mg/kg bw). The bone marrow of the test subjects was sampled at 24 and 48 hours. The results of the study indicate that the test material does not meet the criteria to be considered mutagenic under the EU Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2018 - 18 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:

Adopted July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Appearance: White to pale yellow green powder
Batch: 171116
Substance storage: At room temperature protected from light
Stable under storage conditions until: 16 November 2019 (expiry date)

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:

Rat liver microsomal enzymes (S9 homogenate) prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
Direct plate assay and Pre-Incubation Assay: 5.4, 17, 52, 164 and 512 μg/plate.
Pre-Incubation Assay 2: 5.4, 17, 52, 164, 512, 1600 μg/plate.
The highest concentration of the test item used in the mutation assays was the level at which the test item exhibited limited solubility in the dose-range finding test.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-Aminoanthracene

Details on test system and experimental conditions:

Direct Plate Assay: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the substance in DMSO and either 0.5 ml S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Pre-Incubation Assay: Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays), 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the substance in DMSO. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the test facility.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The potential of the substance and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichiacoli (E. coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9) was determined.  Following a dose-range finder study employing concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate, the test was then performed in three independent experiments. A direct plate assay and a pre-incubation assay was performed employing substance concentrations of 5.4, 17, 52, 164 and 512 μg/plate. A second pre-incubation assay was performed employing substance concentrations of 5.4, 17, 52, 164, 512, 1600 μg/plate. The substance did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation.  Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)