Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance did not induce genetic toxicity in an in vitro HPRT-test and in two Ames Assays, except a positive result in one Ames assay in Salmonella typhimurium strain TA 1535 without metabolic activation applying the pre-incubation method.

Link to relevant study records

Referenceopen allclose all

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)
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:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT )-gene in mouse lymphoma cells
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
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).
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'.

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

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.

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 14 FEB 2017 to 21 APR 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21. July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 31. May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Purity: >99%
Species / strain / cell type:
other: S. typhimurium LT2 strains TA97a, TA98, TA100, TA102 and TA1535
Additional strain / cell type characteristics:
other: Genetic characteristics of each test bacteria strain such as amino acid requirement, UV sensitivity, membrane mutation and drug resistance were verified in advance and bacteria strains possessing the required characteristics were used.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male rat liver S9 mix
Test concentrations with justification for top dose:
plate incorporation test (experiments 1 a): 0, 0.05, 0.15, 0.5, 1.5, and 5 µL/plate
plate incorporation test (experiments 1 b):0, 0.015, 0.05, 0.15, 0.5, 1.5, and 5 µL/plate
pre-incubation (experiment 2a): TA97a, TA98, TA100 and TA1535: 0, 0.05, 0.09, 0.19, 0.38, 0.75 and 1.5 µL/plate, top dose reduced to 1.5 µL/plate due to toxicity observed in experiment 1
pre-incubation (experiment 2a ): TA102: 0, 0.16, 0.31, 0.63, 1.3, 2,5 and 5 µL/plate
pre-incubation (experiment 2b): TA1535: 0, 0.05, 0.09, 0.19, 0.38, 0.75 and 1.5 µL/plate, top dose reduced to 1.5 µL/plate due to toxicity observed in experiment 1
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: test item was sufficiently soluble in Ethanol, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol, DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4- Nitro-1,2-phenylene diamine (CASRN 99-56-9) without S9 mix for strains TA97a, TA98, and TA102; 2-amino-anthracene (CASRN 613-13-8) with S9 mix for strains TA97a, TA100, TA102, and TA1535
Remarks:
4- Nitro-1,2-phenylene Diamine is not a standard positive control according to OECD guideline 471 but is recommended according to Ames et al. [D.M. Maron, B.N. Ames: “Revised methods...”, Mutation Research 113 (1983) 173- 215, Elsevier Biomedical Press].
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar
experiment 1: plate incorporation
experiment 2: preincubation

DURATION
- Preincubation period: only experimt 2 - 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates per concentration tested

DETERMINATION OF CYTOTOXICITY
- Method: other: Performed in experiment 1 only, analogously to the titre control (see below) with the maximum dose of test item with and without S9 mix on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1°C.
- Titer control: The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.
Rationale for test conditions:
The test material was sufficiently soluble in Ethanol. Testing, applying the plate incorporation method, was performed to the maximal test concentration mentioned in the testing guideline where test material showed signs of toxicity towards strains TA97a, TA98, TA100 and TA1535 in both the absence and presence of metabolic activation at the highest cconcentration of 5 µL/plate. Based on this the experiment was repeated for the bacteria strains TA97a, TA98, TA100 and TA1535 with an additional concentration.
Evaluation criteria:
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Statistics:
The colonies were counted visually and the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
The test item induced an increase in the number of revertants colonies in experiment 2a and 2b without metabolic activation, applying the pre-incubation method
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the experiment 1a and b, the test item caused cytotoxicity in the highest concentration (5 µL/plate), only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the experiment 1a and b, the test item caused cytotoxicity in the highest concentration (5 µL/plate), only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the experiment 1a and b, the test item caused cytotoxicity in the highest concentration (5 µL/plate), only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the experiment 1a and b, the test item caused cytotoxicit in the highest concentration (5 µL/plate), only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the experiment 1a and b, the test item caused cytotoxicity in the highest concentration (5 µL/plate), only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the experiment 1a and b, the test item caused cytotoxicity in the highest concentration (5 µL/plate), only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the experiment 2, the test item caused cytotoxicity in the highest concentration (5 µL/plate), only
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test item induced a dose-related increase in the number of revertant colonies in the bacteria strain TA1535 at 1.5 and 0.75 µL per plate, in the absence of metabolic activation applying the pre incubation method, but was negative in the presence of metabolic activation. None of the tested concentrations of the test item induced an increase of revertant colonies number with and without metabolic activation applying the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. The test item Tri-n-Butylamine showed signs of toxicity towards strains TA97a, TA98, TA100 and TA1535 at the highest concentration, in both the absence and presence of metabolic activation while strain TA102 was not affected.
Remarks on result:
other:
Remarks:
pre incubation method
Conclusions:
Testing the test item tri-n-butylamine for it’s mutagenic potential in the reverse bacterial mutation assay (AMES Test according to OECD TG471 and GLP; plate incorporation and pre-incubation method) showed a positive result in one strain of five tested.

No increase of revertants was observed in the plate incorporation assay in any strain, neither in the presence nor in the absence of metabolic activation. Also no increase of revertants was observe in the pre-incubation assay in the strains TA97a, TA98, TA100 and TA 102 in the presence and absence of metabolic activation. An increase in the number of revertant colonies was observed in strain TA1535 in the two highest concentrations in the absence - but not in the presence - of metabolic activation in the preincubation test.
Executive summary:

Reverse mutation testing of the test material, tri-n-butylamine, was conducted according to OECD testing guideline 471 and GLP using 5 strains of bacteria: Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535. The test material (dissolved in Ethanol) was tested up to concentrations of 5μL/plate in the absence and presence of S9-mix in two experiments (experiment 1: plate incorporation method; experiment 2: pre-incubation method).

In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5μL/plate. In experiments applying the plate incorporation method, the test item showed cytotoxicity at the highest concentration towards bacteria strains TA97a, TA98, TA100, and TA1535 (reduction of number of revertants). Based on the observed cytotoxicity the test item was tested up to concentrations of 5  µL/plate in the absence and presence of S9-mix in the bacteria strain TA102 and tested up to concentrations of 1.5 µL/plate in the absence and presence of S9-mix in the bacteria strains TA97a, TA98, TA100 and TA1535, using the pre-incubation method. For strain TA102 the test item showed cytotoxicity at the highest concentration applying pre incubation method (reduction of number of revertants).

The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 10EXP9 bacteria/mL. All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory. Thus indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Since all criteria for acceptability have been met, the study is considered valid.

No increase of revertants was observed in the plate incorporation assay in any strain, neither in the presence nor in the absence of metabolic activation. Also no increase of revertants was observed in the pre-incubation assay in the strains TA97a, TA98, TA100 and TA 102 in the presence and absence of metabolic activation. An increase in the number of revertant colonies was observed in strain TA1535 in the two highest concentrations in the absence - but not in the presence - of metabolic activation in the preincubation test.

Under the conditions of this test the test item tri-n-butylamine was not mutagenic in any of the tested strains with and without metabolic activation in the plate incorporation assay. Also, no mutagenicity was observed in the pre-incubation assay in the strains TA97a, TA98, TA100, TA102 with and without metabolic activation and in strain TA1535 with metabolic activation. Mutagenicity was only observed in the pre-incubation assay in strain TA1535 in the absence of metabolic activation.

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

Genetic toxicity in vivo

Description of key information

The substance did not induce micronuclei in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-04-24 to 1989-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study (OECD 474)
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 28.9 (26-34) g; females: 24.1 (22-28) g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: groups of 5 animals in Macrolon cages
- Diet (ad libitum): Altromin 1324, altromin GmbH, Lage/Lippe, Germany
- Water (ad libitum): tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
sesame oil
Duration of treatment / exposure:
single exposure
Frequency of treatment:
single exposure
Post exposure period:
animals were sacrificed 24, 48 and 72 h post exposure
Remarks:
Doses / Concentrations:
150 mg/kg bw (single dose) in sesame oil (10 mL/kg bw)
Basis:
nominal conc.
No. of animals per sex per dose:
5 per sex
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide, dose 50 mg/kg bw
Tissues and cell types examined:
polychromatic erythrocytes from femoral bone marrow
Details of tissue and slide preparation:
For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.

Staininq procedure
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan
Evaluation criteria:
1000 polychromatic erythrocytes were examined for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator. The numbers of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically.

The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically. Actual data were also compared with historical controls.
Statistics:
Statistical analysis was performed using the paired, one-sided or two-sided Wilcoxon test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
increased as well as reduced spontaneous activity, reversible within 5 h post exposure
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The incidence of micronucleated polychromatic erythrocytes of the animals treated with tributylamine was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Tributylamin and was statistically not different from the control values.
Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei in both males and females, indicating.the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extend.
Conclusions:

Under the conditions of this study, the test substance was not mutagenic in the micronucleus test in mice.
Executive summary:

The test compound (purity 99.3%) was administered orally by gavage to male and female NMRI mice (5 per sex). Single doses of 150 mg were tested and the animals were sacrificed 24, 48 and 72 h post exposure. Cyclophosphamide was used as positiv control substance (50 mg/kg bw).

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test substance was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment and was statistically not different from the control values. The positive control substance induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating.the sensitivity of the system. The test substance did not induce micronuclei in vivo. (Hoechst, 1989).

This study, performed according to OECD guideline 474, was judged ro be reliable (RL1) and selected as key study.

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

Mode of Action Analysis / Human Relevance Framework

As there is no evidence for species differences in MoA or species specific activities the available data are regarded as relevant for humans.

Additional information

The mutagenic activity of the test substance has been investigated in several in vitro and in vivo assays, which mainly revealed negative results:

A reliable bacterial reverse mutation assay according to OECD TG 471 and GLP principles has been performed with the Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 (Andres, 2017). The test material (dissolved in Ethanol) was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in two independent experiments (experiment 1: plate incorporation method; experiment 2: pre-incubation method). Under the conditions of this test tri-n-butylamine was not mutagenic in any of the tested strains with and without metabolic activation in the plate incorporation assay. Also, no mutagenicity was observed in the pre-incubation assay in the strains TA97a, TA98, TA100, TA102 with and without metabolic activation and in strain TA1535 with metabolic activation. Mutagenicity was only observed in the pre-incubation assay in strain TA1535 in the absence of metabolic activation.

No mutagenic activity was observed in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 in concentrations up to 10000 µg/plate, without or with metabolic activation in another pre-incubation assay. The test item did not produce a cytotoxic or mutagenic response in bacteria (Zeiger, 1987).

The test substance was assayed for the ability to induce mutation at the hypoxanthineguanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in L5178Y mouse lymphoma cells in concentrations up to 750µg/mL without metabolic activation and 1000µg/mL with metabolic activation. No statistically significant increases in mutant frequency were observed following treatment with tributylamine at any concentration tested and there were no significant linear trends. The highest test concentrations produced cytotoxic effects. Clear increases in mutation were induced by the positive control chemicals 4nitroquinoline 1-oxide (without S9) and benzo(a)pyrene (with S9) (Lloyd, 2010).

In an in vivo mouse micronucleus assay, the test compound was administered orally by gavage to male and female NMRI mice (5 per sex) in single doses of 150 mg and the animals were sacrificed 24, 48 and 72 h post exposure. The incidence of micronucleated poly- or normochromatic erythrocytes of the animals treated with the test substance was within the normal range of the negative control. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment. The positive control substance induced a marked statistically significant increase in the number of polychromatic cells. The test substance did not induce micronuclei in vivo (Müller and Mayer, 1989).

Additionally, an in vivo assay investigating inhibition of DNA synthesis is available. After a single oral intraperitoneal dose (15 -30 % of the LD50) no inhibition of DNA synthesis (examined by the measurement of the nuclear incorporation of 3H-thymidine) in liver and kidney cells of suckling mice was observed (Amlacher, 1981). However, the study was judged not to be reliable (RL3) due to deficiencies in data reporting so that no final conclusions can be drawn.

As outlined above, negative results were obtained in all genotoxicity assays except one positive result in one Ames assay in Salmonella typhimurium strain TA 1535 without metabolic activation applying the pre-incubation method. This result is surprising,

a)     Since a negative result was obtained under comparable conditions (Salmonella typh. strain TA1535, pre-incubation method with and without metabolic activation) in the Ames assay reported by Zeiger).

b)     Since a negative result was also obtained in the Salmonella typh.strain TA100 in both assays. Both strains (TA 1535 and TA 100) contain the same base pair substitution mutation hisG46, but differ in the fact that for strain TA100 the plasmid pKM101 is introduced. Due to the enhancement of SOS mutagenesis facilitated by the mucAB genes on the plasmid pKM101, TA100 is normally the most sensitive of all Salmonella typh. tester strains (Prival and Zeiger, 1998).

c)      Since the positive result in TA1535 was only observed in the absence of metabolic activation. Applying the OECD Toolbox V4.0 an alert for DNA binding by OECD arised, based on the assumption that the tertiary amine is metabolized to an iminium ion which can interact with DNA in a so called SN1-reaction (nucleophilic substitution). However, formation of an iminium ion would require metabolic activation and no mutagenic activity was observed in the presence of metabolic activation. No further alerts arised applying the OECD Toolbox (no alert for: carcinogenicity alert by ISS; in vitro mutagenicity (Ames test) alert by ISS; in vivo mutagenicity (Micronucleus) alert by ISS; DNA binding by OASIS V 1.4; DNA alerts for Ames by OASIS V1.4; DNA alerts for CA and MNT by OASIS V1.1; protein binding alerts for Chromosomal aberration by OASIS V1.2).

d)     Since other tertiary amines also obtained negative results in Ames assays. For trimethylamine (CAS: 75-50-3), triethylamine (CAS: 121-448-8), and also for N,N-dimethylethylamine (CAS: 598-56-1) negative results in AMES tests are reported in a SIDS assessment profile (2012). Tripropylamine (CAS: 102-69-2), which has already been registered (registration number: 01-2119987316-26-0003), did not show an increase of revertants per plate in any of the tester strains, neither in the presence nor in the absence of metabolic activation.

As there is no plausible mechanistic explanation for the observed positive result in TA1535 and because this single finding stands in contrast to the negative findings in several other in vitro and in vivo assays it is not regarded as relevant for the overall genotoxic properties of tributylamine in vivo. This interpretation is in good agreement with the findings from Kirkland et al. (2014 a, b) who examined information from several databases to determine whether negative results in mammalian cell tests are associated with the absence of carcinogenic or in vivo genotoxic activity despite a positive AMES test. Based on their observations Kirkland and colleagues concluded ‘Thus, in the case of an AMES-positive chemical, negative results in 2 in vitro mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered as indicative of absence of in vivo genotoxic or carcinogenic potential.’ Therefore, it is concluded that tributylamine has not to be classified as mutagenic according to Regulation (EC) No 1272/2008.

Kirkland et al. (2014a). Can in vitro mammalian cell genotoxicity test results be used to complement positive results in the Ames test and help predict carcinogenic or in vivo genotoxic activity? I. Reports of individual databases presented at an EURL ECVAM Workshop. Mutation Research, 775-776, 55-68. https://doi.org/10.1016/j.mrgentox.2014.10.005

Kirkland et al. (2014b). Can in vitro mammalian cell genotoxicity test results be used to complement positive results in the Ames test and help predict carcinogenic or in vivo genotoxic activity? II. Construction and analysis of a consolidated database. Mutation Research, 775-776, 69-80. https://doi.org/10.1016/j.mrgentox.2014.10.006

Prival, M. J., & Zeiger, E. (1998). Chemicals mutagenic in Salmonella typhimurium strain TA1535 but not in TA100. Mutation Research - Genetic Toxicology and Environmental Mutagenesis, 412(3), 251–260.https://doi.org/10.1016/S1383-5718(97)00196-4

SIDS Initial Assessment Profile: Tertiary Amines, 2012, avaible under http://webnet.oecd.org/hpv/ui/SIDS_Details.aspx?id=5f2ecd05-e202-491a-bcfc-dd01494e6b93

Justification for classification or non-classification

Based on the available information in vitro and in vivo, the test substance has not to be classified according to Regulation (EC) 1272/2008