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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-across to TBBC - Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance TBBC was not mutagenic in the strains Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA in the presence and absence of phenobarbital and 5,6-benzoflavone-induced rat liver S9 metabolic activation. (Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD (471 and 472))/GLP).

Read-across to TBBC - in vitro cytogenicity (chromosome aberration) study in mammalian cells: the substance TBBC was concluded to be negative for the induction of chromosome aberrations in the presence and absence of phenobarbital and 5,6-benzoflavone-induced rat liver S9 metabolic activation in Chinese hamster lung fibroblasts (CHL/IU) cells. (Similar or equivalent to OECD 473/GLP).

In vitro gene mutation study in mammalian cells: Negative read-across studies from TBBC are available (reverse gene mutation assay in bacteria and in vitro mammalian chromosome aberration test). UV1084 is predicted to be negative based on the results of these studies also. In a 2-year dietary NTP bioassay of TBBC (NTP TR 435, 1994) there was no evidence of carcinogenic activity in male or female F344/N rats or in male or female B6C3F1 mice under the conditions of the study. Overall, based on this data from TBBC, UV1084 is not predicted to be genotoxic or carcinogenic and therefore the in vitro gene mutation study in mammalian cells is waived.

Link to relevant study records

Referenceopen allclose all

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:
comparable to guideline study
Remarks:
Read-across, Original study in Japanese
Qualifier:
according to guideline
Guideline:
other: Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD (471 and 472)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sumitomo Chemical Co., Ltd; 40701
- Purity: >98%

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:
Phenobarbital and 5,6-benzoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Dose-range finding: 0, 50, 150, 500, 1500, 5000 µg/plate.

Main tests:
-S9 mix, 0, 0.781, 1.56, 3.13, 6.25, 12.5, 25 and 50 μg/plate (TA100, TA1535 and TA1537), 0, 3.13 - 200 μg/plate (TA98) and 0, 313 - 5000 μg/plate (WP2)

+S9 mix, 0, 12.5, 25, 50, 100, 200, 400 and 800 μg/plate (TA100, TA1535, TA98 and TA1537) and 0, 313 - 5000 μg/plate (WP2)
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
-S9 mix: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, WP2, TA98), Sodium azide (TA1535)and 9-Aminoacridine (TA1537); +S9 mix, 2-Aminoanthracene (five strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
A concentration-related increase over 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.
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>12.5 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>400 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In main test 1, precipitate was observed at 50μg/plate -S9 and at 400μg/plate +S9 in all S. typhimurium strains. Precipitate was observed at all concentrations in the E. coli strain.

In main test 2, precipitate was observed at 50μg/plate -S9 and at 200μg/plate +S9 in all S. typhimurium strains. Precipitate was observed at all concentrations in the E. coli strain.



Conclusions:
Under the conditions described for this study, it is concluded that TBBC is non-mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA.
Executive summary:

In a reverse gene mutation assay in bacteria, strains of Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA were exposed to TBBC (>98%) in DMSO at concentrations of 0, 0.781, 1.56, 3.13, 6.25, 12.5, 25 and 50 μg/plate (TA100, TA1535 and TA1537), 0, 3.13 - 200 μg/plate (TA98) and 0, 313 - 5000 μg/plate (WP2) and 0, 12.5, 25, 50, 100, 200, 400 and 800 μg/plate (TA100, TA1535, TA98 and TA1537) and 0, 313 - 5000 μg/plate (WP2) in the absence and presence of mammalian metabolic activation respectively (Phenobarbital and 5,6-benzoflavone-induced rat liver S9).

Toxicity was observed at 12.5 μg/plate (TA100 and TA1537), 25 μg/plate (TA1535) and 100 μg/plate (TA98) without S9 mix, and 400 μg/plate (TA100, TA1535 and TA1537) and 500 μg/plate (TA98) with S9 mix. No toxicity was observed in WP2 either without S9 mix or with S9 mix. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Read-across, Original study in Japanese
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sumitomo Chemical Co., Ltd; 40701
- Purity: >98%
Species / strain / cell type:
other: Chinese hamster lung fibroblasts (CHL/IU)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Research Resource Bank (JCRB)
- Number of passages if applicable: February 1988, at passage: 4th passage, now 12th

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle’s MEM (Nissui Pharmaceutical Co., Ltd) with 10% FCS (Biocell); 5% CO2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and 5,6-benzoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Dose range finding: -S9: 0-0.01 mg/ml; +S9: 0-0.2mg/ml

Main test:
S9 mix (24, 48 hrs): 0, 0.00050, 0.0010, 0.0020 mg/ml
-S9 mix (6 hrs): 0, 0.00023, 0.00045, 0.00090 mg/ml
+S9 mix (6 hrs): 0, 0.0050, 0.010, 0.020 mg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 hrs, 24 hrs, 48 hrs
- Expression time (cells in growth medium): 6 hr treatment – 18 hours

STAIN (for cytogenetic assays):
Two hours before the end of the culture, colcemid was added to the culture solution to a final concentration of about 0.1 μg / ml. Chromosome specimens were prepared according to a conventional method. Six slide specimens were prepared for each dish. The prepared specimens were stained with 3% Giemsa solution.

NUMBER OF REPLICATIONS:1

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Analysis of chromosomes was performed based on the classification method by the Japan Society of Environmental Mutagenesis, Mammalian Examination (MMS) Subcommittee 1), and the presence or absence of structural abnormality such as chromosome type or chromosome type gap, The presence or absence of cells (polyploid) was observed.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 metaphase cells per group and 800 division metastatic cells for ploidy cells.

OTHER EXAMINATIONS:
- Determination of polyploidy:Yes
Evaluation criteria:
Cases in which significant differences were observed in both of the above tests were defined as positive. Cases in which no significant differences were observed in trend tests were defined as false positive. Cases with less than 100 structural aberrations and cases with fewer than 400 polyploid cells were defined as not assessable due to cytotoxicity.
Statistics:
The frequency of the appearance of cells with chromosome aberrations was tested with Fischer's exact probability test between the vehicle background data and between the test substance treatment groups with reference to the method of Hayashi using a familywise significance level of 5% while taking into consideration multiplicity. If a significant difference was observed with Fischer's exact probability test, the Cochram-Armitage trend test was performed with regard to dose dependence (p<0.05).
Species / strain:
other: Chinese hamster lung fibroblasts (CHL/IU)
Remarks:
6 hrs
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: Chinese hamster lung fibroblasts (CHL/IU)
Remarks:
24 and 48 hrs
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
TBBC did not induce chromosomal abnormalities in Chinese hamster lung fibroblasts (CHL/IU) cells in vitro under the test conditions described above.
Executive summary:

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells, Chinese hamster lung fibroblasts (CHL/IU) cell cultures were exposed to TBBC (>98%) in DMSO at concentrations of up to 0, 0.00050, 0.0010, 0.0020 mg/ml (with Phenobarbital and 5,6-benzoflavone-induced rat liver S9 metabolic activation for 24 and 48 hrs), 0, 0.00023, 0.00045, 0.00090 mg/ml (without metabolic activation for 6 hrs) and 0, 0.00050, 0.0010, 0.0020 mg/ml (without metabolic activation for 24 and 48 hrs).

Positive controls induced the appropriate response. There was no evidence of chromosome aberrations induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline [In vitro mammalian cytogenetics [Chromosome aberration]] OECD 473 for in vitro cytogenetic mutagenicity data.

Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
[Specific explanation in addition to field 'Justification for data waiving']
Negative read-across studies from TBBC are available (reverse gene mutation assay in bacteria and in vitro mammalian chromosome aberration test). UV1084 is predicted to be negative based on the results of these studies also.
In a 2-year dietary NTP bioassay of TBBC (NTP TR 435, 1994) there was no evidence of carcinogenic activity in male or female F344/N rats or in male or female B6C3F1 mice under the conditions of the study. Overall, based on this data from TBBC, UV1084 is not predicted to be genotoxic or carcinogenic and therefore the in vitro gene mutation study in mammalian cells is waived.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

There is no in vitro or in vivo genotoxicity data available for UV1084. There is a read-across reverse gene mutation assay in bacteria from TBBC and a read-across in vitro cytogenicity (chromosome aberration) study in mammalian cells from TBBC available.

In a reverse gene mutation assay in bacteria (Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD (471 and 472)/GLP), strains of Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA were exposed to TBBC (>98%) in DMSO at concentrations of 0, 0.781, 1.56, 3.13, 6.25, 12.5, 25 and 50 μg/plate (TA100, TA1535 and TA1537), 0, 3.13 - 200 μg/plate (TA98) and 0, 313 - 5000 μg/plate (WP2) and 0, 12.5, 25, 50, 100, 200, 400 and 800 μg/plate (TA100, TA1535, TA98 and TA1537) and 0, 313 - 5000 μg/plate (WP2) in the absence and presence of mammalian metabolic activation respectively (Phenobarbital and 5,6-benzoflavone-induced rat liver S9). Toxicity was observed at 12.5 μg/plate (TA100 and TA1537), 25 μg/plate (TA1535) and 100 μg/plate (TA98) without S9 mix, and 400 μg/plate (TA100, TA1535 and TA1537) and 500 μg/plate (TA98) with S9 mix. No toxicity was observed in WP2 either without S9 mix or with S9 mix. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells (Similar to equivalent to OECD 473/GLP), Chinese hamster lung fibroblasts (CHL/IU) cell cultures were exposed to TBBC (>98%) in DMSO at concentrations of up to 0, 0.00050, 0.0010, 0.0020 mg/ml (with Phenobarbital and 5,6-benzoflavone-induced rat liver S9 metabolic activation for 24 and 48 hrs), 0, 0.00023, 0.00045, 0.00090 mg/ml (without metabolic activation for 6 hrs) and 0, 0.00050, 0.0010, 0.0020 mg/ml (without metabolic activation for 24 and 48 hrs). Positive controls induced the appropriate response. There was no evidence of chromosome aberrations induced over background.

In vitro gene mutation study in mammalian cells: Negative read-across studies from TBBC are available (reverse gene mutation assay in bacteria and in vitro mammalian chromosome aberration test). UV1084 is predicted to be negative based on the results of these studies also. In a 2-year dietary NTP bioassay of TBBC (NTP TR 435, 1994) there was no evidence of carcinogenic activity in male or female F344/N rats or in male or female B6C3F1 mice under the conditions of the study. Overall, based on this data from TBBC, UV1084 is not predicted to be genotoxic or carcinogenic and therefore the in vitro gene mutation study in mammalian cells is waived.

Based on this data, UV-1084 is not genotoxic. These results are suitable to use in the human health risk assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance UV1084 (CAS No. 14516-71-3) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied, based on the available read-across studies from TBBC (CAS No. 96-69-5).