Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance TiBTD (CAS No. 3064-73-1) was not mutagenic in the strains S. typhimurium TA98, TA100, TA1535 and TA1537, and E. coli WP2 uvr A in the presence and absence of phenobarbital/β- naphthoflavone-induced rat liver S9 metabolic activation. (OECD 471/GLP).

Read-across to Vanlube-7723 (CAS No. 10254-57-6) - In vitro cytogenicity (chromosome aberration) study in mammalian cells: the substance Vanlube-7723 was concluded to be negative for the induction of chromosome aberrations in the presence and absence of phenobarbital/β- naphthoflavone-induced rat liver S9 metabolic activation in human peripheral blood lymphocyte cell cultures. (OECD 473/GLP)

Read-across to Vanlube-7723 (CAS No. 10254-57-6) -Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in L5178Y/TK+/- -3.7.2C mouse lymphoma cells  exposed to Vanlube-7723  in the presence or absence of phenobarbital/β- naphthoflavone-induced rat liver S9 mammalian metabolic activation (OECD 476/GLP).

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
Study period:
24 March 2017 - 26 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
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: Willing New Material Technology Co.,Ltd.; 23161211201
- Expiration date of the lot/batch: 10 December 2017
- Purity: 97%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 ºC)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Range Finding Test: 5000; 2500; 1000; 316; 100; 31.6 ,10 μg/plate
Main Test 1: 5000, 1581, 500, 158.1, 50, 15.81 μg/plate
Main Test 2: 5000, 1581, 500, 158.1, 50, 15.81, 5 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: N,N-Dimethylformamide (DMF)
- Justification for choice of solvent/vehicle:
The solubility of the test item was examined using Distilled water, N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). Test item was insoluble at 100 mg/mL concentration using Distilled water. Partial dissolution was observed at the same concentration using DMSO. The test item was soluble at this concentration using DMF. DMF was selected as vehicle (solvent) for the study
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMF
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
Remarks:
TA98: 4-nitro-1,2-phenylenediamine (NPD) without activation; All strains: 2-aminoanthracene (2AA) with activation
Details on test system and experimental conditions:
ETHOD OF APPLICATION: Preliminary Concentration Range Finding Test as well as in the first Main Test, the plate incorporation method was used. In the second main test, the pre-incubation method was used.

DURATION
- Preincubation period: 20min at 37ºC
- Exposure duration: 48±1 hours

NUMBER OF REPLICATIONS: Triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in background lawn
Evaluation criteria:
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positiveresponse for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and
Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and
TA1537 bacterial strains.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a
reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.



Statistics:
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Species / strain:
other: All stains
Remarks:
Main test 1
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:
valid
Positive controls validity:
valid
Species / strain:
other: All strains
Remarks:
Main test 2
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate/slight precipitate was detected on the plates in the main tests in all examined strains with and without metabolic activation at higher concentrations


RANGE-FINDING/SCREENING STUDIES:
In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample (including the controls) was tested in triplicate.

Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). Precipitate/slight precipitate was observed in the Preliminary Concentration Range Finding Test in both bacterial strains with and without metabolic activation at 5000, 2500 and 1000 μg/plate concentrations. Inhibitory or toxic effects of the test item were not detected in the preliminary Experiment. Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate. The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 (Appendix 2) and in Appendix 3.


HISTORICAL CONTROL DATA
- Positive historical control data: Yes with ranges, means and standard deviation (Period of 2011-2015) Appendix 6
- Negative (solvent/vehicle) historical control data: Yes with ranges, means and standard deviation (Period of 2011-2015) Appendix 6
Conclusions:
TiBTD has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.
Executive summary:

In a reverse gene mutation assay in bacteria (17_053-007M), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to TiBTD (97%) in DMF at concentrations of 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.58 μg/plate (direct plate incorporation; experiment 1) and 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Phenobarbital/β- naphthoflavone-induced rat liver S9).

TiBTD was tested up to the limit concentration (5000 µg/plate). 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please see read-across justification.
Reason / purpose:
read-across source
Species / strain:
other: human peripheral blood lymphocyte
Remarks:
Experimant 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
120 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: Human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
120 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
In an in vitro chromosome aberration study, Vanlube 7723 was non-clastogenic to human peripheral blood lymphocytes.
Executive summary:

In a mammalian cell cytogenetics assay (860-109), human peripheral blood lymphocyte cell cultures were exposed to Vanlube-7723 in DMSO at concentrations of up to 0, 7.5, 15, 30, 60, 120 µg/ml with and without Phenobarbital/β-naphthoflavone-induced rat liver S9.

Vanlube-7723 was tested up to the lowest precipitating dose level (120 µg/ml). Positive controls induced the appropriate response. The test material did not induce any statistically significant increases in the frequency of cells with aberrations/number of polyploid cells in the presence or absence of metabolic activation.

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
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Please see read-across justification.
Reason / purpose:
read-across source
Species / strain:
other: L5178Y/TK+/- -3.7.2C mouse lymphoma cells
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: L5178Y/TK+/- -3.7.2C mouse lymphoma cells
Remarks:
Experiment 2 (24 hrs)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 μg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: 100 μg/ml
Remarks:
Experiment 2 (3 hrs)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
VANLUBE 7723 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

In an in vitro gene mutation study in mammalian cells (499691), L5178Y/TK+/- -3.7.2C mouse lymphoma cells were exposed to Vanlube-7723 (99%) in DMSO at concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 μg/ml (experiment 1) for 3 hrs with and without Phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation and 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 μg/ml (experiment 2) for 3 and 24 hours in the presence/absence of metabolic activation respectively.

Vanlube-7723 was tested beyond the limit of the solubility to obtain adequate cytotoxicity data.The positive controls induced the appropriate response. 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 476 for in vitro cytogenetic gene mutation data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 September 2004 to 10 February 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across
Qualifier:
according to
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: R.T. Vanderbilt Company, Inc; 4C018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
Species / strain / cell type:
other: Human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human volunteer
- Cell cycle length, doubling time or proliferation index: Average Generation Time (AGT) for regular donors used in the lab was approx. 17 hours.
- Whether whole blood or separated lymphocytes were used if applicable: The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of PHA at 90 ug/ml final concentration.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagls's MEM with HEPES buffer with L-glutamin, pen/strep, amphotericin B nad 15% FCS.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 16.48 – 4220 µg/ml

The dose selection for the main studies used the lowest precipitating dose level as the maximum dose level and was 120 µg/ml for all exposure groups both with and without metabolic activation.

Experiment 1: 0, 7.5, 15, 30, 60, 120 µg/ml
Experiment 2: 0, 7.5, 15, 30, 60, 120 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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: 4 hrs and 24 hr (-S9); 4 hrs (+S9)
- Expression time (cells in growth medium): 48 hours

STAIN (for cytogenetic assays): 5% Gurrs Giemsa

NUMBER OF REPLICATIONS: Duplicate lymphocyte cultures per dose

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The lymphocytes were re-suspended in several ml of fresh fixative before centrifugation and resuspension in a small amount of fixative. Several drops of this suspension were dropped into clean, wet microscope slides and left to air dry. When the slides were dry, they were stained with 5% Gurrs Giemsa for 5 mins, rinsed, dried and coverslipped using mounting medium.

NUMBER OF CELLS EVALUATED: Up to a total of 2000 lymphocyte cell nuclei were counted

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 100


DETERMINATION OF CYTOTOXICITY: Mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, observed and recorded.
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test.
Species / strain:
other: Human peripheral blood lymphocytes
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
up to 120 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: Human peripheral blood lymphocytes
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
up to 120 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH & osmolality: In the preliminary test, there was no significant change in pH when the test material was dosed into media and the osmolality did not increase by more than 50mOsm.
- Precipitation: The dose selection for the main studies used the lowest precipitating dose level from the preliminary test as the maximum dose level and was 120 µg/ml for all exposure groups both with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: The test material induced no evidence of toxicity in any of the exposure groups up to 4220 µg/ml (Table 1)

HISTORICAL CONTROL DATA
- Positive historical control data: Yes with ranges, means and standard deviation (Appendix 1)
- Negative (solvent/vehicle) historical control data: Yes with ranges, means and standard deviation (Appendix 1)
Conclusions:
In an in vitro cytogenicity (chromosome aberration) study in mammalian cells , Vanlube 7723 was non-clastogenic to human peripheral blood lymphocytes.
Executive summary:

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells (860/109), human peripheral blood lymphocyte cell cultures were exposed to Vanlube-7723 in DMSO at concentrations of up to 0, 7.5, 15, 30, 60, 120 µg/ml with and without Phenobarbital/β-naphthoflavone-induced rat liver S9.

Vanlube-7723 was tested up to the lowest precipitating dose level (120 µg/ml). Positive controls induced the appropriate response. The test material did not induce any statistically significant increases in the frequency of cells with aberrations/number of polyploid cells in the presence or absence of metabolic activation.

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
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: R.T. Vanderbilt Company, Inc; 2011063698
- Expiration date of the lot/batch: 10 February 2016 (Retest date)
- Purity: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark
Target gene:
Thymidine Kinase (TK)
Species / strain / cell type:
other: L5178Y/TK+/- -3.7.2C mouse lymphoma cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Invitrogen), 1 mM sodium pyruvate (Sigma) and 2 mM L-glutamin (Invitrogen Corporation). Horse serum (Invitrogen Corporation) was inactivated by incubation at 56°C for at least 30 minutes. All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 – 100% (actual range 47 – 98%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.9 – 38.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 34.9 - 36.0°C), humidity (with a maximum of 27%) and CO2 percentage (with a maximum of 1%) that occurred were caused by opening and closing of the incubator door, the duration of these deviations did not exceed 4 hours. Based on laboratory historical data these deviations are considered not to affect the study integrity

- Periodically 'cleansed' against high spontaneous background: Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10-4 M hypoxanthine (Sigma), 2 x 10-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10-5 M thymidine (Merck) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment

- Periodically checked for Mycoplasma contamination: Yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and ß-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 3.33, 10, 33.3, 100, 333 μg/ml
Experiment 1: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 μg/ml
Experiment 1: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO

- Justification for choice of solvent/vehicle:The test substance was dissolved in dimethyl sulfoxide (SeccoSolv, Merck Darmstadt, Germany). VANLUBE® 7723 concentrations were used within 1.5 hours after preparation..The final concentration of the solvent in the exposure medium was 0.8% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1 – 3 hrs with and without 8% S9.;
Experiment 2 – 3 hrs (with 12% S9) and 24 hrs (without S9)

- Expression time (cells in growth medium): 48 hrs

SELECTION AGENT (mutation assays): Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).

NUMBER OF REPLICATIONS: 1; the solvent control was tested in duplicate

NUMBER OF CELLS EVALUATED:
For determination of the MF a total number of 9.6 x 105 cells/concentration were plated in five
96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10x6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10x6 survivors and ≤ 170 per 10x6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 1x06 survivors, and for CP not below 700 per 10x6 survivors
Statistics:
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 (ref. 12).
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test
Species / strain:
other: L5178Y/TK+/- -3.7.2C mouse lymphoma cells
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: L5178Y/TK+/- -3.7.2C mouse lymphoma cells
Remarks:
Experiment 2 (24 hrs)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
100 μg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: L5178Y/TK+/- -3.7.2C mouse lymphoma cells
Remarks:
Experiment 2 (3 hrs)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: VANLUBE 7723 precipitated in the exposure medium at concentrations of 33 μg/ml and above. VANLUBE 7723 was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range finding test was 333 μg/ml.

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3.33 to 333 μg/ml in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.

Table 1 shows the cell counts of the cultures after 3 hours of treatment with various concentrations of VANLUBE 7723 and after 24 and 48 hours of subculture and the calculated suspension growth and the relative suspension growth.

In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentrations of 333 μg/ml compared to the solvent control.

In the presence of S9-mix, the relative suspension growth was 65% at the test substance concentration of 333 μg/ml compared to the relative suspension growth of the solvent control.
Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of VANLUBE 7723 and after 24 hours of subculture and the calculated suspension growth and the relative suspension growth.

In the absence of S9-mix, the relative suspension growth was 48% at the test substance concentration of 333 μg/ml compared to the relative suspension growth of the solvent control

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay from January 2009 to January 2012 (See APPENDIX 3, Table 12).

- Negative (solvent/vehicle) historical control data: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range from January 2009 to January 2012. (See APPENDIX 3, Table 11).


Conclusions:
VANLUBE 7723 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

In an in vitro gene mutation study in mammalian cells (499691), L5178Y/TK+/- -3.7.2C mouse lymphoma cells were exposed to Vanlube-7723 (99%) in DMSO at concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 μg/ml (experiment 1) for 3 hrs with and without Phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation and 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 μg/ml (experiment 2) for 3 and 24 hours in the presence/absence of metabolic activation respectively.

Vanlube-7723 was tested beyond the limit of the solubility to obtain adequate cytotoxicity data.The positive controls induced the appropriate response. 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 476 for in vitro cytogenetic gene mutation data.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test)

There is one gene mutation study (Bacterial Reverse Mutation Assay/Ames test) with TiBTD available.

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to TiBTD (97%) in DMF at concentrations of 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.58 μg/plate (direct plate incorporation; experiment 1) and 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Phenobarbital/β- naphthoflavone-induced rat liver S9). TiBTD was tested up to the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In vitro cytogenicity (chromosome aberration) study in mammalian cells

There is no in vitro cytogenicity (chromosome aberration) study in mammalian cells with TiBTD available. One read-across study for in vitro cytogenicity (chromosome aberration) study in mammalian cells with Vanlube-7723 (CAS No. 10254-57-6) is available.

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells (OECD 473/GLP), human peripheral blood lymphocyte cell cultures were exposed to Vanlube-7723 in DMSO at concentrations of up to 0, 7.5, 15, 30, 60, 120 µg/ml with and without Phenobarbital/β-naphthoflavone-induced rat liver S9. Vanlube-7723 was tested up to the lowest precipitating dose level (120 µg/ml). Positive controls induced the appropriate response. The test material did not induce any statistically significant increases in the frequency of cells with aberrations/number of polyploid cells in the presence or absence of metabolic activation.

Gene mutation (mammalian cell gene mutation assay)

There is no gene mutation (mammalian cell gene mutation assay) study with TiBTD available. One read-across study for gene mutation (mammalian cell gene mutation assay) with Vanlube-7723 (CAS No. 10254-57-6) is available.

In an in vitro gene mutation study in mammalian cells (OECD 476/GLP), L5178Y/TK+/- -3.7.2C mouse lymphoma cells were exposed to Vanlube-7723 (99%) in DMSO at concentrations of 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 μg/ml (experiment 1) for 3 hrs with and without Phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation and 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 μg/ml (experiment 2) for 3 and 24 hours in the presence/absence of metabolic activation respectively. Vanlube-7723 was tested beyond the limit of the solubility to obtain adequate cytotoxicity data.The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

All three studies are negative and the results are suitable for use in the human health hazard assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance TiBTD (CAS No. 3064-73-1) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.