Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in-vitro genetic toxicity studies have been conducted all of which determined the registered substance to be non-mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 August 2012 to 24 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully GLP compliant and in accordance with current test guidelines
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Human lymphocyte cultures
Species / strain / cell type:
lymphocytes: Human lymphocyte cultures
Details on mammalian cell type (if applicable):
Blood from three healthy, non-smoking male volunteers from a panel of donors at Covance was used for each experiment. No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication.
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Positive controls
4-Nitroquinoline 1 oxide (NQO): Stock concentration, 0.250 and 0.500 mg/mL and final concentration, 2.50 and 5.00 µg/mL
Cyclophosphamide (CPA): Stock concentration, 0.625 and 1.250 mg/mL and final concentration, 6.25 and 12.50 µg/mL

Vehicle / solvent:
dimethyl formamide (DMF)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl formamide (DMF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Remarks:
For positive control concentrations see test concentrations section
Details on test system and experimental conditions:
No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL pre-warmed (in an incubator set to 37 ± 1°C) HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin/streptomycin, so that the final volume following addition of S-9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1°C for approximately 48 hours and rocked continuously.
Range-finder - S-9 mix or KCl (0.5 mL per culture) was added appropriately. Cultures were treated with the test article, vehicle or untreated control (0.1 mL per culture). as follows: 3 hour treatment and 17 hour recovery test had 4 vehicle control cultures, 2 with S-9 and 2 without, 2 untreated controls, 1 with S-9 and 1 without, 2 with test article, 1 with S-9 and 1 without and no positive controls. The 20 hour treatment test had 2 vehicle controls both without S-9, 1 untreated control without S-9 and 1 with test article without S-9. Positive control treatments were not included. The final culture volume was 10 mL. Cultures were incubated at 37 ° 1°C for the designated exposure time. For removal of the test article, cells were pelleted (approximately 300 g, 10 minutes), washed twice with sterile saline (pre-warmed in an incubator set to 37 ± 1°C), and resuspended in fresh pre-warmed medium containing foetal calf serum and penicillin/streptomycin.
Harvesting - Approximately 2 hours prior to harvest, colchicine was added to give a final concentration of approximately 1 µg/mL to arrest dividing cells in metaphase. At the defined sampling time cultures were centrifuged at approximately 300 g for 10 minutes; the supernatant was carefully removed and cells were resuspended in 4 mL pre-warmed hypotonic (0.075 M) KCl and incubated at 37 ± 1ºC for 15 minutes to allow cell swelling to occur. Cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, two to three minutes) until the cell pellets were clean.
Slide preparation - Lymphocytes were kept in fixative at 2-8ºC prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of 45% (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred on to clean microscope slides labelled with the appropriate study details. Slides were flamed, as necessary, to improve quality. Slides were dried on a hot plate (set to approximately 80 - 100°C) then stained in filtered 4% (v/v) Giemsa in pH 6.8 Gurr’s buffer for 5 minutes. The slides were rinsed, dried and mounted with coverslips using DPX.
Slide analysis - For each treatment regime, two vehicle control cultures were analysed for chromosome aberrations. As the proportions of cells with structural aberrations in the vehicle and untreated cultures fell within current historical vehicle control (normal) ranges in Experiment 1, it was considered not necessary to analyse the untreated control cultures in Experiment 2.
Slides from NQO and CPA positive control treatments were checked to ensure that the system was operating satisfactorily. One concentration from each positive control, which gave satisfactory responses in terms of quality and quantity of mitoses and extent of chromosomal damage, was analysed.
All slides for analysis were coded using randomly generated letters by an individual not connected with the scoring of the slides. Labels with only the study number, assay type, experiment number, the sex of the donor and the code were used to cover treatment details on the slides.
Where appropriate, one hundred metaphases from each code were analysed for chromosome aberrations. Where 10 cells with structural aberrations (excluding gaps) were noted on a slide, analysis may have been terminated. Only cells with 44 to 48 chromosomes were considered acceptable for analysis. Any cell with more than 48 chromosomes (that is, polyploid, hyperdiploid or endoreduplicated cells) observed during this evaluation was noted and recorded separately. Structural aberrations were classified according to the ISCN scheme (ISCN, 1995).
Experiment 1 treatment scheme - Experiment one only had a 3 hour treatment and 17 hour recovery test. 8 cultures for vehicle control were used, 4 with S-9 and 4 without. 4 untreated controls, 2 with S-9 and 2 without. 4 treated cultures, 2 with S-9 and 2 without.4 Positive controls, 2 with S-9 and 2 without.
Experiment 2 treatment scheme - For the 3 hour treatment and 17 hour recovery test there were 4 vehicle controls all with S-9, 2 untreated controls with S-9, 2 treated cultures with S-9 and 2 positive controls with S-9. For the 20 hour treatment and 0 hour recovery test there were 4 vehicle controls without S-9, 2 untreated controls without S-9, 2 treated control without S-9 and 2 positive controls without S-9.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures.
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p < 0.05).
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments, or effects occurring only at high or very toxic concentrations, and the types and distribution of aberrations.
Species / strain:
lymphocytes: Human lymphocyte cultures
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes
- Effects of osmolality: No marked changes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Mitotic Index Determinations - Range-Finder Experiment


Treatment
(mg/mL)

Mitotic index (%)

3+17 hours, -S-9

3+17 hours, +S-9

20+0 hours, -S-9

A

B

MIH*

A

B

MIH*

A

B

MIH*

 

 

 

 

 

 

 

 

 

 

Vehicle

6.0

6.0

-

2.8

5.9

-

3.3

3.2

-

UTC

6.9

NT

-

8.3

NT

-

5.3

NT

-

1.451

NS

NT

-

NS

NT

-

NS

NT

-

2.419

NS

NT

-

NS

NT

-

NS

NT

-

4.031

NS

NT

-

7.4

NT

0

5.2

NT

0

6.718

7.9

NT

0

5.7

NT

0

2.7

NT

17

11.20

7.3

NT

0 E

6.5

NT

0

3.7

NT

0 H

18.66

6.8

NT

0 E

3.4

NT

22 E

2.5

NT

23 H

31.10

4.2

NT

30 EH

2.1

NT

52 EH

0.3

NT

91 H

51.84

0.3

NT

95 EH

NE

NT

- EH

NE

NT

- H

86.40

0.4

NT

93 EH

NE

NT

- EH

NE

NT

- H

144.0

1.1

NT

82 PEH

NE

NT

- PEH

NE

NT

- PH

240.0

0.4

NT

93 PEH

NE

NT

- PEH

NE

NT

- PH

400.0

0.9

NT

85 PEH

NE

NT

- PEH

NE

NT

- PH

 

 

 

 

 

 

 

 

 

 

 

UTC = Untreated control

NT = Not tested 

NS = not scored 

NE = Not evaluated – noscoreablecells on slide

P = Precipitation observed at treatment

E = Precipitation observed at the end of treatment incubation

H = Precipitation observed at harvest

 

*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

 

Table 2: Mitotic Index Determinations - Experiment 1


Treatment
(mg/mL)

Mitotic index (%)

3+17 hours, -S-9

3+17 hours, +S-9

A/C

B/D

MIH*

A/C

B/D

MIH*

 

 

 

 

 

 

 

Vehicle

11.1/12.3

9.7/10.3

-

11.7/9.6

11.6/13.5

-

UTC

12.2

13.4

-

11.2

10.9

-

1.000

NS

NS

-

NS

NS

-

2.000

NS

NS

-

NS

NS

-

4.000

NS

NS

-

11.5

11.8

0

6.000

12.3

12.1

0 E

8.3

11.6

14

8.000

10.5

11.4

0 E

10.1

10.4

12

10.00

8.5

8.4

22 E

11.5

10.0

7 #

12.50

11.4

10.8

0 E

9.3

10.0

17 E

15.00

9.7

10.0

9 # E

8.8

10.0

19 E

17.50

12.4

8.3

5 E

10.4

7.4

23 E

20.00

9.1

7.5

24 # E

8.3

9.9

22 # E

30.00

7.4

5.3

41 # EH

7.3

9.4

28 EH

40.00

3.2

3.1

71 EH

6.2

5.6

49 # EH

50.00

0.7

0.7

94 PEH

3.0

2.7

75 PEH

 

 

 

 

 

 

 

 

UTC = Untreated control

NS = Not scored

P = Precipitation observed at treatment

E = Precipitation observed at the end of treatment incubation

H = Precipitation observed at harvest

 

*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

 

# Highlighted concentrations selected for analysis.

 

Table 3: Mitotic Index Determinations - Experiment 2


Treatment
(mg/mL)

Mitotic index (%)

20+0 hours, -S-9

3+17 hours, +S-9

A/C

B/D

MIH*

A/C

B/D

MIH*

 

 

 

 

 

 

 

Vehicle

5.4/4.2

5.0/3.8

-

10.5/10.8

8.8/8.7

 

UTC

8.9

10.9

-

NS

NS

-

0.5000

5.0

4.4

0

NT

NT

-

1.000

6.5

6.8

0

NT

NT

-

2.000

5.2

4.0

0

9.0

10.2

1

3.000

6.3

4.5

0

NT

NT

-

4.000

4.2

6.9

0

8.6

9.9

5 #

5.000

5.9

6.0

0

NT

NT

-

6.000

5.6

6.0

0

8.6

7.2

19 #

7.000

5.3

5.7

0

NT

NT

-

8.000

4.1

5.1

0

7.1

9.0

17

10.00

5.7

4.4

0 #

10.3

6.6

13

12.50

3.4

3.5

25 #

8.7

7.3

18 #

15.00

3.9

3.5

20 H #

8.0

7.7

19 E #

17.50

NT

NT

-

8.2

8.3

15 E

20.00

4.2

4.0

11 H

8.0

9.9

8 E

25.00

NT

NT

-

7.6

7.0

25 E

30.00

NT

NT

-

6.7

6.6

31 EH

40.00

NT

NT

-

5.5

8.7

27 EH

 

 

 

 

 

 

 

 

UTC = Untreated control

NT = Not tested 

NS = Not scored

E = Precipitation observed at the end of treatment incubation

H = Precipitation observed at harvest

 

*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

 

# Highlighted concentrations selected for analysis.

 

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that TOFA_TETA_PAA_BADGE_CGE_Adduct did not induce biologically relevant increases in the frequency of structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of solubility in culture medium in both the absence and presence of S-9.
Executive summary:

TOFA_TETA_PAA_BADGE_BGE_Adduct was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced animals. The test article was formulated in dimethylformamide (DMF). The highest concentrations analysed in the Main Experiments were limited by the observation of post-treatment precipitate and were determined following a preliminary cytotoxicity Range‑Finder Experiment.

Treatments were conducted (as detailed in the following summary tables) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for chromosome analysis were selected by evaluating the effect of TOFA_TETA_PAA_BADGE_BGE_Adduct on mitotic index. In each experiment, chromosome aberrations were analysed at three or four concentrations and a summary of the chromosome aberration data is presented in Table 1 and Table 2.

Table 1: Experiment 1 – Results summary

Treatment

Concentration (mg/mL)

Cytotoxicity(%)$

% Cells with Chromosome Aberrations (Excluding Gaps)

Historical
(%)#

Statistical Significance

 

 

 

 

 

 

3+17 hour -S-9

Vehiclea

-

0.50

0-3

-

 

UTC

-

0.50

 

NR

 

15.00

9

1.00

 

NR

 

20.00

24

1.00

 

NR

 

30.00

41

3.50

 

NR

 

*NQO, 2.50

ND

25.00

 

p<0.001

 

 

 

 

 

 

3+17 hour +S-9

Vehiclea

-

1.00

0-3

-

 

UTC

-

0.50

 

NR

 

10.00

7

0.00

 

NR

 

20.00

22

1.00

 

NR

 

40.00

49

1.50

 

NR

 

*CPA, 12.50

ND

52.63

 

p<0.001

 

 

 

 

 

 

 

UTC                       Untreated control

a              Vehicle control was DMF

*             Positive control

#             95thpercentile of the observed range

$              Based on mitotic index

NR         Not required as there were no concentrations analysed where both cultures demonstrated aberrant cell       frequencies (excluding gaps) that exceeded historical vehicle control ranges

ND         Not determined

 

Table 2: Experiment 2 – Results summary

Treatment

Concentration (mg/mL)

Cytotoxicity(%)$

% Cells with Chromosome Aberrations (Excluding Gaps)

Historical
(%)#

Statistical Significance

 

 

 

 

 

 

20+0 hour -S-9

Vehiclea

-

2.50

0-3

-

 

10.00

0

4.00

 

NS

 

12.50

25

0.50

 

NS

 

15.00

20

1.00

 

NS

 

*NQO, 2.50

ND

42.55

 

p<0.001

 

 

 

 

 

 

3+17 hour +S-9

Vehiclea

-

1.00

0-3

-

 

4.000

5

0.00

 

NR

 

6.000

19

0.50

 

NR

 

12.50

18

0.00

 

NR

 

15.00

19

1.00

 

NR

 

*CPA, 12.50

ND

44.94

 

p<0.001

 

 

 

 

 

 

 

a              Vehicle control was DMF

*             Positive control

#             95thpercentile of the observed range

$              Based on mitotic index

NS          Not significant

NR         Not required as there were no concentrations analysed where both cultures demonstrated aberrant cell       frequencies (excluding gaps) that exceeded historical vehicle control ranges

ND         Not determined


Appropriate negative (vehicle and untreated) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in the vehicle and untreated cultures fell within current historical vehicle control (normal) ranges in Experiment 1 and it was considered not necessary to analyse the untreated control cultures in Experiment 2. 4‑Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

All acceptance criteria were considered met and the study was accepted as valid.

Treatment of cultures with TOFA_TETA_PAA_BADGE_BGE_Adduct for 3+17 hours in the absence of S-9 in Experiment 1 resulted in frequencies of cells with structural aberrations that were generally similar to those observed in concurrent vehicle controls. Numbers of aberrant cells (excluding gaps) in treated cultures fell within the 95thpercentile of the observed range (0-3%) with the exception of one culture at the highest concentration analysed (30.00 µg/mL) in which 5% aberrant cells (excluding gaps) were observed, which fell within the observed range of 0-5%. Furthermore, no such increase was observed in the replicate culture at 30.00 µg/mLor at any other concentration analysed in this experiment, therefore the observation was considered not biologically relevant.

Treatment of cultures for 20+0 hours in the absence of S-9 in Experiment 2 resulted in frequencies of cells with structural aberrations that were generally similar to those observed in concurrent vehicle controls. Numbers of aberrant cells (excluding gaps) in treated cultures marginally exceeded the 95thpercentile of the observed range (0-3%) in both cultures at the lowest concentration analysed (10.00 µg/mL) in which 4% aberrant cells (excluding gaps) were observed, which fell within the observed range of 0-5%. However, this was not statistically higher than the concurrent vehicle controls and there was clearly no evidence of a concentration-related response, therefore the observation was considered of little or no biological relevance.

Treatment of cultures in the presence of S-9 in Experiments 1 and 2 resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent vehicle controls. Numbers of aberrant cells (excluding gaps) in all treated cultures in both experiments fell within the 95th percentile of the observed range.

Small, sporadic increases in the frequency of cells with numerical aberrations, which marginally exceeded the concurrent vehicle controls and the normal ranges, were observed in cultures treated with TOFA_TETA_PAA_BADGE_BGE_Adduct in the absence and presence of S-9 in Experiments 1 and 2. However, numerical aberrations were not assessed quantitatively and the assay is not specifically designed to evaluate the potential to induce polyploidy, therefore these increases can be considered of highly questionable biological relevance.

It is concluded that TOFA_TETA_PAA_BADGE_BGE_Adduct did not induce biologically relevant increases in the frequency of structural chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of solubility in culture medium in both the absence and presence of S-9.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 February 2013 to 14 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully GLP compliant and in accordance with current test guidelines
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
mouse lymphoma L5178Y cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 +/- 1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) from male Sprague Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Positive controls
4-nitroquinoline 1-oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no metabolic activation
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL with metabolic activation

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 9.375 to 300 µg/mL (limited by solubility in culture medium).

In Experiment 1, eleven concentrations, ranging from 0.2083 to 12.5 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 2.083 to 50 μg/mL, were tested in the presence of S-9.
In Experiment 2, eleven concentrations, ranging from 0.5 to 10 µg/mL in the absence of S-9 and from 5 to 80 µg/mL in the presence of S-9, were tested.
Vehicle / solvent:
vehicle DMF diluted 100 fold in the treatment medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMF diluted 100 fold in the treatment medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Remarks:
For positive control concentrations please see test concentrations section.
Details on test system and experimental conditions:
Range-finder
Treatment of cell cultures for the cytotoxocity range-rinder experiment were as described below in the mutation experiment. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g), washed with tissue culture medium and resuspended in 20 mL RPMI 10.
Cell concentrations were adjusted to 8 cells/mL abd, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate fore determination of relative survival. The plates were incubated at 37±1°Cin a humidified incubator gassed with 5±15 v/v carbon dioxide in air for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.
Mutation assays - At least 10EO7 cells in a volume of 18.8 mL of RPMI 5 were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, culture medium (for UTC), test article or positive control solution was added. S-9 mix or 150 mM KCl was added. Each treatment, in the absence or presence of S-9 was in duplicate (single cultures for positive controls) and the final volume was 20 mL.
After 3 hours' incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed and resuspended in 20 mL RPMI 10. Cell densities were determined using a Coulter Counter and , where sufficient cells survived, the concentrations adjusted to 2 x 10EO5 cells/mL. Cells were transferred to flasks for growht throughout the expression period or were diluted to be plated for survival.
Plating for survival - Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v carbon dioxide in air until scoreable (7 days). Wells containing viable clones were identified by eye using background illumunation and counted.
Expression period - Cultures were maintaied in flasks for a period of 7 days during which the hprf mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. Form observations on recovery and growth of the cultures during the expression period, cultures were selected to be plated for viability and 6TG resistance.
Plating for viability - Using a multichanneled pipette, 0.2 mL of the final concentration of each culture was placed into each well. The plates were incubated at 37±1°C in 5±1 v/v carbon dioxide in air until scorable (8 to 11 days).
Plating for 6TG resistance - Using a multichanneled pipette, 0.2 mL of each suspension was placed into 4 x 96-well microtitre plates (384 wells at 2 x 10EO4 cells/well). Plates were incubated at 37±1°C in a humidified incubator gassed with 5±1% v/v carbon dioxide in air until scorable (12 days).
Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p < 0.05).
3. The effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Not required
Species / strain:
mouse lymphoma L5178Y cells
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
Additional information on results:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 9.375 to 300 µg/mL (limited by solubility in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations in the absence and presence of S-9 (150 and 300 µg/mL). Following the 3-hour treatment incubation period, precipitate was observed at the highest four concentrations in the absence and presence of S-9 (37.5 to 300 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded.
In Experiment 1, eleven concentrations, ranging from 0.2083 to 12.5 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 2.083 to 50 μg/mL, were tested in the presence of S-9. Upon addition of the test article to the cultures and following the 3-hour treatment incubation period, precipitate was observed at the highest concentration in the presence of S-9 only (50 µg/mL). All cultures were retained post-treatment in the absence and presence of S-9. Seven days after treatment, the highest four concentrations in the absence of S-9 (7.083 to 12.5 μg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. However, an intermediate concentration in the presence of S-9 (12.5 µg/mL) was subsequently excluded from data analysis as there were sufficient concentrations to define an appropriate toxicity profile. The highest concentrations selected for analysis were 5.833 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 12% and 40% RS, respectively.
In Experiment 2, eleven concentrations, ranging from 0.5 to 10 µg/mL in the absence of S-9 and from 5 to 80 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations in the presence of S-9 (60 and 80 µg/mL). Following the 3-hour treatment incubation period, precipitate was observed at the highest six concentrations (40 to 80 µg/mL) in the presence of S-9. The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the presence of S-9 was retained and higher concentrations were discarded. All cultures were retained post-treatment in the absence of S-9. Seven days after treatment, the highest five concentrations in the absence of S-9 (6.5 to 10 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition the lowest concentration tested in the absence of S-9 (0.5 µg/mL) was not selected as there were sufficient concentrations to define an appropriate toxicity profile. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 6 µg/mL in the absence of S-9 and 40 µg/mL in the presence of S-9, which gave 15% and 72% RS, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: RS Values - Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

UTC

135

88

9.375

0

102

18.75

0

61

37.50

0 (PP)

74 (PP)

75.00

NP (PP)

NP (PP)

150.0

NP (P, PP)

NP (P, PP)

300.0

NP (P, PP)

NP (P, PP)

UTC                       Untreated control

% RS                      Percent relative survival adjusted by post treatment cell counts

P                             Precipitation observed at time of treatment

PP                          Precipitation observed following treatment incubation period

NP                          Not plated for viability due to precipitation

 

Table 2: Summary of mutation data

Experiment 1 (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

3.61

 

0

 

100

2.84

 

UTC

 

93

3.22

NS

UTC

 

91

3.60

NS

0.2083

 

87

1.64

NS

2.083

 

113

2.11

NS

0.4167

 

80

1.49

NS

4.166

 

97

2.60

NS

0.8333

 

104

1.97

NS

8.333

 

97

3.05

NS

1.666

 

88

3.19

NS

16.67

 

105

2.66

NS

3.333

 

70

4.69

NS

20.83

 

83

2.23

NS

4.583

 

53

4.17

NS

25

 

90

1.58

NS

5.833

 

12

2.20

NS

29.17

 

98

2.13

NS

 

 

 

 

 

33.33

 

78

2.50

NS

 

 

 

 

 

50

P, PP

40

2.88

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.15

 

59

12.40

 

2

 

59

12.77

 

0.2

 

54

19.38

 

3

 

68

38.06

 

 

 

 

 

 

 

 

 

 

 

Experiment 2 (3 hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

2.94

 

0

 

100

3.87

 

UTC

 

138

4.26

NS

UTC

 

95

2.03

NS

1

 

108

1.59

NS

5

 

85

1.58

NS

2

 

111

3.17

NS

10

 

83

1.40

NS

4

 

88

1.40

NS

20

 

87

1.02

NS

5

 

51

2.51

NS

30

 

78

1.44

NS

6

 

15

1.97

NS

35

 

75

1.67

NS

 

 

 

 

 

40

PP

72

1.73

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.15

 

96

8.20

 

2

 

70

10.00

 

0.2

 

75

17.86

 

3

 

53

28.32

 

 

 

 

 

 

 

 

 

 

 

UTC                       Untreated control

P                             Precipitation observed at time of treatment

PP                          Precipitation observed at end of treatment incubation period

§                             6TG resistant mutants/106 viable cells 7 days after treatment

% RS                      Percent relative survival adjusted by post treatment cell counts

NS                          Not significant

 

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that TOFA_TETA_PAA_BADGE_CGE_Adduct did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions of this study. These conditions included treatments up to toxic concentrations in the absence of a rat liver metabolic activation system (S-9) and up to precipitating concentrations in the presence of S-9.
Executive summary:

TOFA_TETA_PAA_BADGE_CGE_Adduct was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post mitochondrial fraction (S-9). The test article was formulated in Dimethylformamide (DMF).

A 3-hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 9.375 to 300 µg/mL (limited by solubility in culture medium). In the absence of S-9, complete toxicity (0% relative survival, RS) was observed at all concentrations analysed. In the presence of S-9, the highest concentration analysed was 37.5 µg/mL (limited by post-treatment precipitate), which gave 74% RS.

In Experiment 1, eleven concentrations, ranging from 0.2083 to 12.5 µg/mL, were tested in the absence of S-9 and ten concentrations, ranging from 2.083 to 50 μg/mL, were tested in the presence of S-9. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 5.833 µg/mL in the absence of S-9 and 50 µg/mL in the presence of S-9, which gave 12% and 40% RS, respectively (limited by post-treatment precipitate in the presence of S-9).

In Experiment 2, eleven concentrations, ranging from 0.5 to 10 µg/mL in the absence of S-9 and from 5 to 80 µg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 6 µg/mL in the absence of S-9 and 40 µg/mL in the presence of S-9, which gave 15% and 72% RS, respectively (limited by post-treatment precipitate in the presence of S-9).

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4 nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with TOFA_TETA_PAA_BADGE_CGE_Adduct at any concentration tested in the absence and presence of S-9 and there were no statistically significant linear trends.

It is concluded that TOFA_TETA_PAA_BADGE_CGE_Adduct did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions of this study. These conditions included treatments up to toxic concentrations in the absence of a rat liver metabolic activation system (S-9) and up to precipitating concentrations in the presence of S-9.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 August 2012 to 4 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully GLP compliant and in accordance with current test guidelines
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 10EO8 to 10EO9 cells/mL, based on cell count data from testing of each strain batch
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Positive controls
2-nitrofluorene: Stock concentration; 50 µg/mL, final concentration; 5 µg/mL, Strain; TA 98, without metabolic activation
Sodium azide: Stock concentration; 20 µg/mL, final concentration; 2 µg/mL, Strain; TA100 and TA1535 without metabolic activation
9-aminoacridine: Stock concentration; 500 µg/mL, final concentration; 50 µg/mL, Strain; TA1537 without metabolic activation
Mitomycin C: Stock concentration; 2 µg/mL, final concentration; 0.2 µg/mL, Strain; TA102 without metabolic activation
Benzo[a]pyrene: Stock concentration; 100 µg/mL, final concentration; 10 µg/mL, Strain; TA98 with metabolic activation
2-aminoanthracene: Stock concentration; 50 µg/mL. final concentration; 5 µg/mL, Strain; TA100, TA1535 and TA1537 with metabolic activation
2-aminoanthracene: Stock concentration; 200 µg/mL, final concentration; 20 µg/mL, Strain; TA102 with metabolic activation

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of TOFA_TETA_PAA_BADGE_CGE_Adduct at 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls.
Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. For strain TA98 in the absence of S-9 and strains TA100, TA1535, TA1537 and TA102 in the absence and presence of S-9, the maximum test concentration was reduced to 200 µg/plate and for strain TA98 in the presence of S-9, the maximum test concentration was reduced to 500 µg/plate based on toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 0.8192 – 200 µg/plate or 2.048 – 500 µg/plate
Vehicle / solvent:
Dimethylformamide (DMF)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylformamide (DMF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Remarks:
For positive control concentrations please see test concentrations section
Details on test system and experimental conditions:
Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 108 to 109 cells/mL, based on cell count data from testing of each strain batch. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.
When the plates were set they were inverted and incubated at 37±1°C in the dark for 3 days.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p < 0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.
Statistics:
Dunnett's test used accordingly
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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
Additional information on results:
Following TOFA_TETA_PAA_BADGE_CGE_Adduct treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any TOFA_TETA_PAA_BADGE_CGE_Adduct mutagenic activity in this assay system.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It was concluded that TOFA_TETA_PAA_BADGE_CGE_Adduct did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to toxic concentrations, in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

TOFA_TETA_PAA_BADGE_CGE_Adduct was assayed for mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254‑induced rat liver post-mitochondrial fraction (S‑9), in two separate experiments.

All TOFA_TETA_PAA_BADGE_CGE_Adduct treatments in this study were performed using formulations prepared in Dimethylformamide (DMF).

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S‑9, using final concentrations of TOFA_TETA_PAA_BADGE_CGE_Adduct at 5, 15.81, 50, 158.1, 500, 1581 and 5000 µg/plate, plus negative (vehicle) and positive controls. Following these treatments, evidence of toxicity was observed at 158.1 and/or 500 µg/plate and above in all strains in the absence and presence of S-9. In addition, evidence of toxicity was also observed at 50 mg/plate in strains TA1537 and TA102 in the absence of S-9 only.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. For strain TA98 in the absence of S-9 and strains TA100, TA1535, TA1537 and TA102 in the absence and presence of S-9, the maximum test concentration was reduced to 200 µg/plate and for strain TA98 in the presence of S‑9, the maximum test concentration was reduced to 500 µg/plate based on toxicity observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 0.8192 – 200 µg/plate or 2.048 – 500 µg/plate, in order to examine more closely those concentrations of TOFA_TETA_PAA_BADGE_CGE_Adduct approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S‑9 were further modified by the inclusion of a pre-incubation step. This modification to the standard plate incorporation methodology has been known to identify mutagenic chemicals that could not be detected using the standard plate incorporation assay system. Following these treatments, evidence of toxicity was observed at 80 and/or 200 µg/plate in strain TA98 in the absence of S-9 and strains TA100, TA1535, TA1537 and TA102 in the absence and presence of S-9, and at 500 µg/plate in the presence of S-9 only.

The test article was completely soluble in the aqueous assay system at all concentrations tested, in each of the experiments performed.

Negative (vehicle) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies all fell within acceptable ranges for negative control treatments, and were significantly elevated by positive control treatments.

Following TOFA_TETA_PAA_BADGE_CGE_Adduct treatments of all the test strains in the absence and presence of S‑9, no increases in revertant numbers were observed that were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any TOFA_TETA_PAA_BADGE_CGE_Adduct mutagenic activity in this assay system.

It was concluded that TOFA_TETA_PAA_BADGE_CGE_Adduct did not induce mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to toxic concentrations, in the absence and in the presence of a rat liver metabolic activation system (S‑9).

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

Additional information

The substance TOFA_TETA_PAA_BADGE_CGE_Adduct has been tested in three GLP in-vitro mutagenicity test systems

- Ames test according to OECD 471 (s. typhimurium strains TA98, TA100, TA102, TA1535 and TA1537),

- Chromosome aberration test according to OECD 473 (cultured human peripheral blood lymphocytes) and

- Mouse lymphoma test according to OECD 476 (mouse lymphoma L5178Y cells),

and was found negative in all three in-vitro test systems, with and without metabolic activation.

Thus, the substance is not expected causing gene mutations an no further (in-vivo) tests are required.


Short description of key information:
The substance TOFA_TETA_PAA_BADGE_CGE_Adduct has been tested in three in-vitro mutagenicity test systems (Ames test according to OECD 471, Chromosome aberration test according to OECD 473 and Mouse lymphoma test according to OECD 476, all under GLP) and was found negative in all three in-vitro test systems, with and without metabolic activation. Thus, the substance is not expected causing gene mutations an no further tests are required.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

TOFA_TETA_PAA_BADGE_CGE_Adduct was found negative in three in-vitro mutagenicity test systems (Ames test according to OECD 471, Chromosome aberration test according to OECD 473 and Mouse lymphoma test according to OECD 476, all under GLP) for inducing gene mutations an thus classification according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC) is not required..