Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: yellow-brown liquid
Details on test material:
Name: TOFA_TETA_PAA_BADGE_CGE_Adduct
Synonyms: Euredur 450 BD and Aradur 450 BD
CAS number: 186321-96-0
Batch number: WA520
Molecular weight: 755
Date of receipt: 24 August 2012
Storage details: stored at 15 - 25°C, protected from light
Purity: 100%
Expiry date: February 2014

Method

Target gene:
Human lymphocyte cultures
Species / strain
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)
Controls
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.

Results and discussion

Test results
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'.

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion

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.