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Genetic toxicity in vitro

Description of key information

Ames assay:

The chemical was tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 at multiple doses ranging from 1 to 1000 µg/plate, with and without Aroclor-induced rat liver microsomal S9 cell fraction. Dose levels were selected based on preliminary cytotoxicity studies. The results in the main study were evaluated against solvent control data. The test chemical was conclusively non-mutagenic in all strains with and without metabolic activation up to 1000 µg/plate. It was not specified whether positive controls were included or not, however, a number of other chemicals that were investigated in the study tested positive for mutagenicity in the presence, but not in the absence, of the metabolic activation system. This indicates that the metabolic activation system was functioning.

In vitro mammalian chromosome aberration study:

The chemical was tested for clastogenic effects in Chinese hamster ovary cells at 0 (solvent control), 1511, 2159 and 3084 µg/ml in the presence and absence of UV light (at 1500 mJ/cm2). UV light was used as a metabolic inducer because sulisobenzone is used as a sunscreen. The top dose of 3084 µg/ml corresponded to 10 mM in culture medium. 4-Nitroquinoline-1-oxide and 8-methoxypsoralen served as positive controls in the absence and presence of UV light respectively. One hundred metaphases from each test were scored and categorised as follows: cells with structural aberration including gaps (category 1), cells with structural aberrations excluding gaps (category 2), and polyploid, endoreduplicated or hyperdiploid cells (category 3).The test chemical failed to produce any significant increase in the frequency of cells with structural aberrations compared to the solvent control data. The positive controls produced expected increases in structural aberrations, thus confirming the validity of the assay.

In vitro mammalian cell gene mutation assay:

The chemical was tested for mutagenic effects in Chinese hamster ovary cells at 0 (solvent control), 1, 2.5, 5 and 10 mM with and without metabolic activation. 7,12-dimethylbenz(a) anthracene and N-ethyl-N-nitrosourea were employed as positive controls with and without metabolic activation respectively. Treatment with N-ethyl-N-nitrosourea produced a significant increase in the number of revertant colonies whereas 7,12-dimethylbenz(a) anthracene did not. As such, the performance of the metabolic activation system could not be ascertained in the experiment. Without metabolic activation, the test chemical failed to produce a significant increase in the number of revertant colonies when compared to control data.No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from publication.
Qualifier:
according to guideline
Guideline:
other: As mentioned below
Principles of method if other than guideline:
To evaluate mutagenic effects of the given test chemical by the Ames Salmonella /Mammalian-Microsomal Assay.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver microsomal S-9 cell fraction
Test concentrations with justification for top dose:
1.0 - 1000 µg/plate
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Justification for choice of solvent/vehicle: The chemical is soluble in DMSO.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
No data
Rationale for test conditions:
No data
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
S. typhimurium, other: TA98, TAl00, TA1535, TA1537, and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Preliminary cytotoxicity studies determined the dose range of the compound to be used.
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical tested negative for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 in the presence and absence of S9 activation system.
Executive summary:

The chemical was tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 at multiple doses ranging from 1 to 1000 µg/plate, with and without Aroclor-induced rat liver microsomal S9 cell fraction. Dose levels were selected based on preliminary cytotoxicity studies. The results in the main study were evaluated against solvent control data. The test chemical was conclusively non-mutagenic in all strains with and without metabolic activation up to 1000 µg/plate. It was not specified whether positive controls were included or not, however, a number of other chemicals that were investigated in the study tested positive for mutagenicity in the presence, but not in the absence, of the metabolic activation system. This indicates that the metabolic activation system was functioning.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Data is from study report.
Qualifier:
according to guideline
Guideline:
other: EC Scientific Committee for Cosmetology Guideline CSC/803-5/90 (1990)
Principles of method if other than guideline:
To evaluate clastogenic effects of the given test chemical by in vitro mammalian chromosome aberration test.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Details on mammalian cell line Type and identity of media: McCoy's 5A medium including 10% (v/v) foetal calf serum (FCS), and 100 μg/mL gentamycin
- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
other: CHO cells have low number of chromosomes making scoring relatively easy
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
UV light was used as metabolic inducer.
Test concentrations with justification for top dose:
1511, 2159, 3084 µg/mL. The top dose corresponded to 10 mM in culture medium.
Vehicle / solvent:
Vehicle- Vehicle(s)/solvent(s) used: Sterile ROHP water for test material and negative control; DMSO for positive control
- Justification for choice of solvent/vehicle: No data available
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-l-oxide (in absence of UV light) 8-methoxypsoralen (8-MOP) (in presence of UV light)
Details on test system and experimental conditions:
Details on test system and conditions
METHOD OF APPLICATION: in medium
DURATION- Preincubation period: 15 mins
- Exposure duration: 3 hrs
- Expression time (cells in growth medium): 17 hrs
- Selection time (if incubation with a selection agent): 17 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hrs
SELECTION AGENT (mutation assays): gentamycin
SPINDLE INHIBITOR (cytogenetic assays): colchicines 1µg/mL
STAIN (for cytogenetic assays): 4%(v/v) filteredGiemsa stain in pH 6 .8 buffer
NUMBER OF REPLICATIONS: Duplicate (with and without irradiation)
NUMBER OF CELLS EVALUATED: 100D
ETERMINATION OF CYTOTOXICITY- Method: mitotic index
OTHER EXAMINATIONS:- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Other:
Evaluation criteria:
The test article was to be considered as positive in this assay if-1)A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurred at one or more concentrations, and 2) The proportion of cells with structural aberrations at such doses exceeded the normal range.3) Statistically significant increases in cells with chromosome aberrations were induced in the presence of UV but not in itsabsence, and4) Cells with chromosomal aberrations occurred at lower doses in the presence of UV, or with significantly higher frequencies than the total of aberration frequencies observed in the irradiated solvent control plus the aberration frequency in the non-irradiated sample at that concentration.
Statistics:
The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.The proportion of cells for each test treatment condition is compared with the proportion in concurrent negative controls using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: Yes
- Precipitation: No precipitation observed in culture medium
- Other confounding effects: No data available
RANGE-FINDING/SCREENING STUDIES: A preliminary range-finding experiment, covering a broad range of doses, was performed in the presence of two doses of UV light to investigate the phototoxicity of the chemical and to determine the dose range to be used in the main study. The highest dose level used in the range-finder, 3083 μg/mL, was a concentration of 10 mM in culture medium. The doses of UV used were 1500 and 750 mJ/cm2.In the phototoxicity range-finder, there was no marked mitotic inhibition shown following irradiation at either of the two levels (1500 and 750 mJ/cm2), and only the higher UV dose was used in the main study. The concentration of 3084 μg/mL (approximately 10 mM) was chosen as the top dose for the main study and a range of doses from this concentration were used in the absence and presence of UV light. The test article dose levels for chromosome analysis from the irradiated cultures were selected by evaluating the effect of 2-hydroxy-4 methoxybenzophenone-5-sulfonic acid (sulisobenzone) on mitotic index. Chromosome aberrations were analysed at 3 consecutive dose levels. The equivalent doses were taken for analysis from the cultures treated in the absence of UV light.The highest concentration chosen for analysis, 3084 μg/mL, did not induce mitotic inhibition in either the absence or presence of UV light.
COMPARISON WITH HISTORICAL CONTROL DATA:Historical solvent controls were incorporated in the study and a comparison was made to check whether the proportion of cells with structural aberrations fell within the solvent control ranges.
ADDITIONAL INFORMATION ON CYTOTOXICITY:No data available
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical tested negative for clastogenic effects in cultured CHO cells at doses up to 10 mM in the absence and presence of UV light.
Executive summary:

The given test chemical was used to detect whether it is capable of causing chromosomal aberrations. In vitro mammalian chromosome aberration test was carried out on cells obtained from Chinese hamster Ovary. As some test chemicals may become potent clastogens when photosensitised, the clastogenic potential of the test article in this study was assessed by its effects on the chromosomes of CHO cells treated in the absence and presence of UV light. A preliminary range-finding experiment, covering a broad range of doses, was performed in the presence of two doses of UV light to investigate the phototoxicity of the chemical and to determine the dose range to be used in the main study. The doses of UV used were 1500 and 750 mJ/cm2. There was no marked mitotic inhibition shown following irradiation at either of the two levels (1500 and 750 mJ/cm2), and only the higher UV dose was used in the main study. The highest concentration of the test material chosen for analysis was 3084 ug/ml (10mM) and a range of doses from this concentration were used in the absence and presence of UV light. Appropriate negative (solvent) and positive controls were included in the test system in the presence and absence of UV light. Treatment of cultures with chemical in the absence of UV light resulted in frequencies of cells with structural aberrations that were similar to and not significantly different from the frequency seen in concurrent, non irradiated, solvent control cultures. Aberrant cell frequency was within the historical negative control range. Treatment of cultures with test chemical in the presence of UV light resulted in frequencies of cells with structural aberrations that were not increased compared to the concurrent, irradiated, solvent control cultures. The test chemical tested negative for clastogenic effects in cultured CHO cells at doses up to 10 mM in the absence and presence of UV light.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The purpose of this study was to assess toxic and genotoxic effects of the given test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot. This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
0, 1.0, 2.5, 5.0 or 10.0 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Test chemical was easily dissolved in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: N-ethyl-N-nitrosourea (ENU) (2.5 mM) without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium with pre-incubation
DURATION- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)
SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet
NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated
DETERMINATION OF CYTOTOXICITY- Cytotoxicity test: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
Rationale for test conditions:
No data
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
not determined
Remarks:
The metabolic activation system could not be ascertained as the positive control failed to produce a significant increase in the number of revertant colonies.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
not valid
Additional information on results:
No data

Table 1A.Effect of 2-Hydroxy-4-methoxybenzophenone-5-sulfonic acid exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO2. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO2. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

 

With S9

Without S9

 

with TG

without TG

with TG

without TG

Neg. control

0/4

835/4

0/4

683/4

Pos. control

1/4

814/4

31/4

831/4

1.0 mM

0/4

777/4

0/4

820/4

2.5 mM

0/4

927/4

0/4

881/4

5.0mM

0/4

922/4

0/4

889/4

10.0 mM

0/4

843/4

0/4

839/4

 

 

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to 2-Hydroxy-4-methoxybenzophenone-5-sulfonic acid in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

 

With S9

Without S9

Neg. control

N/A

N/A

Pos. control

-2.61x10-4

3.84x10-4

1.0 mM

N/A

N/A

2.5 mM

N/A

N/A

5.0mM

N/A

N/A

10.0 mM

N/A

N/A

 

Conclusions:
Without metabolic activation, the test chemical tested negative for mutagenicity in Chinese hamster ovary (CHO) cells at doses up to 10 mM. No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data.
Executive summary:

The chemical was tested for mutagenic effects in Chinese hamster ovary cells at 0 (solvent control), 1, 2.5, 5 and 10 mM with and without metabolic activation. 7,12-dimethylbenz(a) anthracene and N-ethyl-N-nitrosourea were employed as positive controls with and without metabolic activation respectively. Treatment with N-ethyl-N-nitrosourea produced a significant increase in the number of revertant colonies whereas 7,12-dimethylbenz(a) anthracene did not. As such, the performance of the metabolic activation system could not be ascertained in the experiment. Without metabolic activation, the test chemical failed to produce a significant increase in the number of revertant colonies when compared to control data. No conclusion could be drawn regarding the mutagenicity of the test chemical in CHO cells in the presence of metabolic activation because of invalid positive control data.

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

At present, there is not a sufficient amount of data to make a classification or non-classification for Germ Cell mutagenicity. The test chemical needs to be evaluated for chromosomal aberration effects in the presence of metabolic activation (e.g. OECD 473). Also, because the metabolic activation system could not be validated in the above-mentioned OECD 476 study, the chemical needs to be re-evaluated for mutagenic effects in a mammalian cell line in the presence of metabolic activation (e.g. OECD 476).