4-phenylbenzophenone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is mutagenic in the tester strains TA1537, TA1535 and TA98 of the Salmonella typhimurium reverse mutation assay. The substance is not mutagenic in Salmonella typhimurium tester strain TA100 or in the Escherichia coli reverse mutation assay using strain WP2uvrA. The substance is not clastogenic in an in vitro genotoxicity test employing cultured human lymphocyte cells, however the substance may have the potential to inhibit mitotic processes at the prolonged exposure time of 48 h at a concentration of 125 µg/mL. The mammalian cell gene mutation assay was waived in view of the positive finding in the bacterial mutation assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2017 - 07 September 2017

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:

July 21, 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Appearance: White to off-white crystalline powder
Batch: 20161118
Purity/Composition: 99.74%
Test item storage: At room temperature protected from light
Stable under storage conditions until: 17 November 2017 (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.

Test concentrations with justification for top dose:

Dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate.
Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 μg/plate.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

Remarks:

DMSO

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: a: ICR-191 b: 2-aminoanthracene

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 Charles River Den Bosch.
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.

Statistics:

Not applicable.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

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 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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:

cytotoxicity

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:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

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

Additional information on results:

In the absence of S9-mix, the substanced induced dose related increases in the number of revertant colonies in the tester strains TA1537 and TA98. The increases observed in tester strain TA1537 were above the laboratory historical control data range, and were up to 24-fold the concurrent vehicle control. The increases observed in tester strain TA98 were above the laboratory historical control data range and were up to 6.1-fold the concurrent vehicle control.
 
In the presence of S9-mix, the substance induced dose related increases in the number of revertant colonies in the tester strains TA1537 and TA98. The increases observed in tester strain TA1537 were above the laboratory historical control data range, and were up to 12-fold the concurrent vehicle control. The increases observed in tester strain TA98 were above the laboratory historical control data range and were up to 4.6-fold the concurrent vehicle control.

Conclusions:

The substance is mutagenic in the tester strains TA1537 and TA98 of the Salmonella typhimurium reverse mutation assay. The substance is not mutagenic in the other Salmonella typhimurium tester strains (TA1535 and TA100) or in the Escherichia coli reverse mutation assay using strain WP2uvrA.

Executive summary:

The potential of the substance and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonellatyphimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E.coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9) was investigated.  The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. In the first mutation experiment, the substance was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98.  In the second mutation experiment, the substance was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA in the pre-incubation assay.  The substance was mutagenic in the tester strains TA1537 and TA98 of the Salmonella typhimurium reverse mutation assay.  The substance was not mutagenic in the other Salmonella typhimurium tester strains (TA1535 and TA100) or in the Escherichia coli reverse mutation assay using strain WP2uvrA.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2018 - 31 January 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:

July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 20161118
- Expiration date of the lot/batch: 22 November 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in DMSO. The stock solution was treated with ultrasonic waves until the substance had completely dissolved. To protect the substance from light, amber-colored glassware or tubes wrapped in tin-foil were used for substance preparations.

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).

Test concentrations with justification for top dose:

17, 52, 164, 512, 1600 and 5000 µg/plate in tester strains TA1535, TA100 and WP2uvrA.
17, 52, 164, 512, 1000, 1600 and 5000 µg/plate in tester strain TA1537 and TA98.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

yes

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: 2-aminoanthracene

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 Charles River Den Bosch.
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 1535

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

4.1-fold increase

Cytotoxicity / choice of top concentrations:

cytotoxicity

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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:

positive

Remarks:

+S9: 3.6-fold increase in revertants -S9: 13-fold increase in revertants

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

+S9: 4.9-fold increase in revertants. -S9: 10-fold increase in revertants.

Cytotoxicity / choice of top concentrations:

cytotoxicity

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:

cytotoxicity

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The substance is mutagenic in tester strains TA1535, TA1537 and TA98 in the Salmonella typhimurium reverse mutation assay. The test item is not mutagenic in the other Salmonella typhimurium tester strain (TA100) or in the Escherichia coli reverse mutation assay using Escherichia coli strain WP2 uvrA (pKM101) .

Executive summary:

In the in vitro genotoxicity study (Ames test) the substance was tested for mutagenicity in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and in Escherichia coli strain WP2 uvrA (pKM101) in a pre-incubation assay in the presence and absence of a metabolic activation system (rat liver S9 homogenate). Concentrations of up to 5000 μg/plate were tested. No evidence of mutagenic activity was seen at any concentration of the substance in Salmonella typhimurium strain TA100 and Escherichia coli strain WP2 uvrA. It was concluded that the substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed. In the absence of S9-mix, the substance induced increases in the number of revertant colonies in the Salmonella typhimurium tester strains TA1535, TA1537 and TA98. In the presence of S9-mix, the substance induced dose related increases in the number of revertant colonies in the Salmonella typhimurium tester strains TA1537 and TA98.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2017 - 03 August 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:

adopted 29 July 2016

Deviations:

yes

Remarks:

In the first cytogenetic assay (+ S9-mix), 110 metaphases were examined for chromosome aberrations in one of the duplicate cultures of the positive control and a clear positive response was observed.

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

- Batch No.of test material: 20161118
- Expiration date of the lot/batch: 17 November 2017
- Storage condition of test material: At room temperature protected from light

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) are presented below:
Dose-range finding study/ First cytogenetic assay: age 31, AGT = 13.8 h
Second cytogenetic assay: age 35, AGT = 13.9 h

Cytokinesis block (if used):

Yes at 0.5 µg/ml medium.

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose-range finding test. The highest tested concentration was determined by the solubility of Omnirad 4-PBZ in the culture medium. The test item precipitated at concentrations of 125 µg/mL and upwards. Concentrations employed were 7.8, 16, 31, 63, 125 and 250 µg/mL.

Vehicle / solvent:

Dimethyl sulfoxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Evaluation criteria:

One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.
Acceptability Criteria:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations.

Statistics:

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 125 and 250 µg/mL concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

24 and 48 hours exposure - no chromosome aberrations.

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

48 hours exposure - polyploid cells.

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Both in the absence and presence of S9-mix the substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent 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.

Conclusions:

The substance is not clastogenic in human lymphocytes, however the substance may have the potential to inhibit mitotic processes at the prolonged exposure time of 48 h.

Executive summary:

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.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro gene mutation study in bacteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Justification for classification or non-classification

Based on the available in vitro findings, classification of the substance is not justified.