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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
2009-12-14 to 2010-05-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study (OECD 476)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): tributylamine
- Substance type: clear colourless liquid
- Physical state: liquid
- Analytical purity: 99.52%
- Purity test date: 2009-10-30
- Lot/batch No.: 50000014974
- Expiration date of the lot/batch: September 2010
- Stability under test conditions: responsibility of the Sponsor
- Storage condition of test material: at 15-25°C, protected from light and under nitrogen

Method

Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT )-gene in mouse lymphoma cells
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI10
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-extract from male Sprague Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
(P): Precipitate observed at time of treatment
Cytotoxicity range-finder experiment:
0, 57.91, 115.8, 231.6, 463.3 (P), 926.5 (P), and 1853 (P) µg/mL in the absence and presence of S 9
Experiment 1:
0, 50, 100, 150, 200, 250, 300, 350, 400 (P), 450 (P) and 500 (P) µg/mL in the absence of S 9
0, 100, 200, 300, 350, 400 (P), 450 (P), 500 (P), 550 (P) and 750 (P) µg/mL in the presence of S 9
Experiment 2:
0, 100, 200, 300, 400 (P), 450 (P), 550 (P), 600 (P), and 750 (P) µg/mL in the absence of S 9
0, 200, 300, 450 (P), 500 (P), 600 (P), 700 (P), and 800 (P) µg/mL in the presence of S 9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): 6-thioguanine (6TG)

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p 2. There was a significant concentration relationship as indicated by the linear trend analysis (p 3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance (according to UKEMS guidelines, reference stated).

Results and discussion

Test results
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH were observed in the Range-Finder experiment at the highest concentration tested, compared to the concurrent vehicle controls
- Effects of osmolality: No marked changes in osmolality were observed in the Range-Finder experiment at the highest concentration tested, compared to the concurrent vehicle controls
- Precipitation: at >/= 400 µg/mL (details see under "test concentrations", above)
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S 9 ranging from 57.91 to 1853 µg/mL (equivalent to 10 mM at the highest concentration tested). Upon addition of the test article to the cultures, precipitate was observed at 463.3 µg/mL and above but no precipitate was observed following the treatment incubation period. The highest concentrations to provide >10% relative survival (RS) were 231.6 and 463.3 µg/mL in the absence and presence of S 9, which gave 49% and 33% RS, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:Yes,

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

RANGE-FINDING STUDIES ( (P): Precipitate observed at time of treatment; RS: relative survival):

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

57.91

72

84

115.8

63

97

231.6

49

77

463.3 P

4

33

926.5 P

0

0

1853 P

0

0

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

1.65

 

0

 

100

5.04

 

50

 

108

1.38

NS

100

 

110

4.56

NS

100

 

64

1.57

NS

200

 

67

5.80

NS

150

 

105

1.09

NS

300

 

72

4.08

NS

200

 

92

2.45

NS

350

 

75

2.70

NS

250

 

74

1.44

NS

400

P

79

6.31

NS

300

 

75

1.55

NS

450

P

74

3.65

NS

350

 

95

1.52

NS

500

P

64

4.16

NS

400

P

90

2.13

NS

550

P

55

2.49

NS

450

P

63

2.25

NS

750

P

52

4.00

NS

500

P

65

3.24

NS

 

 

 

 

 

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

93

18.78

 

2

 

80

46.36

 

0.15

 

76

13.84

 

3

 

74

41.89

 

 

 

 

 

 

 

 

 

 

 

 

 

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

P   Precipitation observed at time of treatment

%RS  Percent relative survival adjusted by post treatment cell counts

NS    Not significant

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

4.72

 

0

 

100

9.11

 

100

 

84

4.91

NS

200

 

90

4.19

NS

200

 

68

5.76

NS

300

 

71

5.88

NS

300

 

54

6.66

NS

450

P

68

7.56

NS

400

P

52

7.17

NS

500

P

64

3.58

NS

450

P

50

4.26

NS

600

P

61

3.86

NS

550

P

36

6.32

NS

700

P

46

6.30

NS

600

P

33

6.81

NS

800

P

10

5.79

NS

750

P

6

6.66!

NS

 

 

 

 

 

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

84

15.14

 

2

 

80

45.81

 

0.15

 

89

23.96

 

3

 

34

74.85

 

 

 

 

 

 

 

 

 

 

 

 

 

§ 6‑TG resistantmutants/106viable cells 7 days after treatment

P  Precipitation observed at time of treatment

%RS  Percent relative survival adjusted by post treatment cell counts

NS    Not significant

Comparison of controls with historical means

Treatment

Current

Historical

Ratio

C/H

 

MF

MFi-MFc

MF

MFi-MFc

 

Negative control

1.65

 

11.11

 

0.148

0.1   NQO

18.78

17.13

43.78

32.67

0.524

0.15  NQO

13.84

12.20

63.04

51.93

0.235

Treatment

Current

Historical

Ratio

C/H

 

MF

MFi-MFc

MF

MFi-MFc

 

Negative control

5.04

 

11.80

 

0.427

2     B[a]P

46.36

41.31

68.01

56.21

0.735

3     B[a]P

41.89

36.85

111.61

99.81

0.369

Applicant's summary and conclusion

Conclusions:

It is concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study, neither in the presence nor in the absence of metabolic activation.
Executive summary:

Tributylamine 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 (ranging from 57.91 to 1853 µg/mL

) 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 ethanol.

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

Range-Finder Experiment: The highest concentrations to provide >10% relative survival (RS) were 231.6 µg/mL in the absence of S-9 and 463.3 µg/mL in the presence of S-9, which gave 49% and 33% RS, respectively.

Experiment 1: ten concentrations, ranging from 50 to 500 µg/mL in the absence of S-9 and from 100 to 750 µg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations selected to determine viability and 6TG resistance were 500 µg/mL in the absence of S-9 and 750 µg/mL in the presence of S‑9, which gave 65% and 52% RS, respectively. In the absence and presence of S-9, no concentration gave 10‑20% RS.

Experiment 2: ten concentrations, ranging from 100 to 750 µg/mL in the absence of S-9 and from 100 to 1000 µg/mL in the presence of S-9, were tested. Sevendays after treatment the highest concentrations selected to determine viability and 6TG resistance were 750 µg/mL in the absence of S-9 and 800 µg/mL in the presence of S‑9, which gave 6% and 10% RS, respectively.In the absence of S-9, no concentration gave 10‑20 % RS (cultures treated at 600 and 750 µg/mL gave 33% and 6% RS, respectively, therefore both concentrations were analysed).

Negative (vehicle) and positive control treatments were included in each mutation experiment in the absence and presence of S‑9. Mutant frequencies in negative 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 tributylamine at any concentration tested in the absence and presence of S‑9 and there were no significant linear trends.

In Experiment 1, the highest concentrations tested (500 µg/mL in the absence of S-9 and 750 µg/mL in the presence of S-9) gave only 65% and 52% RS, respectively. This was unexpected, based on the Range-Finder toxicity data. However, concentrations giving a suitable level of toxicity (including one concentration giving <10% RS in the absence of S-9) were observed in Experiment 2 and there was no evidence of mutagenic activity in either experiment, therefore this did not affect the interpretation of the data, which were considered valid.

It is concluded that tributylamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study.