Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro and in vivo genotoxicity studies were conducted with the dissociation products LAS and MEA. Overall, results from these studies indicate no evidence of genotoxic potential of the tested substances.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
Directive 84/449/EEC, B.14 Mutagenicity (Salmonella typhimurium - reverse mutation assay)" 1984; equivalent to OECD 471
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
also TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Arochlor-induced S9 fraction
Test concentrations with justification for top dose:
8, 40, 200, 1000 and 5000 ug/plate
Vehicle / solvent:
Water solution at 50 g/L
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
yes
Positive controls:
yes
Remarks:
aminoanthracene
Positive control substance:
other: nitrofluorene, sodium azide and aminoacridine
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
with and without activation
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TA 1538 also tested negative. During the pre-incubation test, signs of toxicity were noted at concentrations as low as 125 ug/plate. No precipitation of the product was observed at any concentration tested.
Conclusions:
LAS is not mutagenic in the Ames test.
Executive summary:

A bacterial mutagenicity study (Ames test) was conducted on LAS and was found to be negative for mutagenicity.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 16, 1995-June 30, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 from aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
0, 0.6, 1, 1.8, 3, 6 ug/ml without S9
0, 6, 10, 18, 30, 60 ug/ml with S9
Vehicle / solvent:
none
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
yes
Remarks:
(H0 medium)
Positive controls:
yes
Positive control substance:
other: ethyl methane sulfonate; 3-(20-)methylcholanthrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 1 week
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 6 days at 37 degree C for cloning efficiency study, 9 days for mutation assay

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
A test substance was considered mutagenic if a statistically significant dose-related increase in mutant frequency was found in concentrations with greater than 20% survival rate. The mean mutant frequency must also be significantly above the maximum spontaneous mutant frequency
Statistics:
Statistical significance was determined by the t-test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity preliminary test showed cytotoxicity at >= 50 ug/ml without S9, and >= 100 ug/ml with S9.
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: In both the studies with and without S9, the mutant frequencies in the treated groups were statistically significantly higher than in the concurrent negative controls. However, the mutant frequencies in the treated groups were not significantly increased when compared to historical negative controls. There was also no dose-response relationship. The increased mutant frequency in treated groups was therefore not considered to be biologically significant.
Remarks on result:
other: all strains/cell types tested

Results of Test 1 ¿ Without S9 Mix            

Concentration (ug/ml)

Absolute cloning efficiency (%)

Mutant frequency ( x 106)

0

82

3 ± 2

0.6

86

7 ± 1

1

85

3 ± 2

1.8

78

5 ± 2

3

86

1 ± 1

6

83

0 ± 1

EMS

83

277 ± 17

Results of Test 1 ¿ With S9 Mix     

Concentration (ug/ml)

Absolute cloning efficiency (%)

Mutant frequency ( x 106)

0

90

2 ± 1

6

88

1 ± 1

10

84

9 ± 4

18

78

5 ± 3

30

89

3 ± 2

60

89

7 ± 2

MCA

81

91 ± 9

Results of Test 2 ¿ Without S9 Mix

Concentration (ug/ml)

Absolute cloning efficiency (%)

Mutant frequency ( x 106)

0

96

1 ± 1

0.6

92

2 ± 3

1

95

1 ± 1

1.8

93

5 ± 2

3

90

2 ± 1

6

91

6 ± 6

EMS

90

309 ± 20

Results of Test 2 ¿ With S9 Mix     

Concentration (ug/ml)

Absolute cloning efficiency (%)

Mutant frequency ( x 106)

0

90

2 ± 1

6

92

7 ± 3

10

88

9 ± 2

18

94

2 ± 1

30

93

2 ± 2

60

90

5 ± 1

MCA

95

89 ± 6

Conclusions:
The test substance is not mutagenic in either the presence or absence of metabolic activation.
Executive summary:

This study examined the potential of the test substance to cause mutations in mammalian cells. Chinese Hamster Ovary (CHO) cells were exposed to concentrations of 0, 0.6, 1, 1.8, 3, and 6 ug/ml without S9, and 0, 6, 10, 18, 30, and 60 ug/ml with S9. The cells were then examined for cytogenicity and mutation frequency. Ethyl methane sulfonate and 3-(20-)methylcholanthrene were used as positive control substances. Preliminary tests show the test substance was cytogenic at concentrations of 50 ug/ml or greater with metabolic activation, and 100 ug/ml or above without metabolic activation. There was no biologically significant increase in mutation frequency in the treated groups. The test substance is considered not mutagenic to CHO cells both in the presence and absence of S9.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This test has been held according to the Notification stipulating the standard provided by the Minister of Labour based on the Industrial Safety and Health Law Article 57-2 Paragraph 1 (Notification No.77 of the Ministry of Labour, dated September 1, 1988) and related Notice (No.603, dated September 16, 1988) .
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
His
Species / strain / cell type:
S. typhimurium, other: TA98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA1538 bacteria
Species / strain / cell type:
E. coli, other: rat liver S9 (Sodium phenobarbital and 5,6-benzofravone-induced)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 (Sodium phenobarbital and 5,6-benzofravone-induced)
Test concentrations with justification for top dose:
50 - 5000 μg/plate
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-(2-furyl)-3-(5-nitro2-furyl)acrylamide, bleomycin and 2-amino anthracene
Details on test system and experimental conditions:
The preincubation procedure was performed as described previously (Matsushima et al., 1980).The test compound dissolved in 0.05 or 0.1 ml of solvent was supplemented with 0.5 ml of S9 mix (metabolic activation method) or 0.1M phosphate buffer pH 7.4 (direct method) and 0.1 ml of tester strains which had been cultured in nutrient broth. The mixture was incubated for 20 min at 37°C, then rapidly mixed with 2 ml of molten top agar containing 0.05 μmol/ml of L-histidine and biotin for the Salmonella test. In the E. coli test 0.05 μmol/ml of L-tryptophan was used instead of L-histidine and biotin. Then the top agar mixture was rapidly poured onto a 30 ml of Vogel -Bonner minimal agar plate.All plates were incubated for 48 hours at 37°C and the numbers of revertant colonies were scored.
Evaluation criteria:
Two-hold rule criteria was used for data evaluation (Ames et al., 1975). The chemicals are considered to be mutagenic when a dose-related increase in revertant colony count is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when a reproducibility of test result is observed. Mutagenic potency was calculated by following equation and maximum value of mutagenic potency was expressed as a specific acitivity on the data sheet: mutagenic potency (induced revertants / mg test substance) _(number of induced revertants on the dose X - number of revertant on the solvent control) = mg of test chemical on the dose X .
Key result
Species / strain:
S. typhimurium, other: TA98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA1538 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=2000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli, other: WP2uvrA and WP2uvrA/pKM101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=2000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

control values (mean± SD):

Tester strain

Solvent control

positive control

- S9

+ S9

- S9

+ S9

TA100

138 ± 27

139 ± 29

728 ± 196

1011 ± 210

TA1535

14 ± 5

13 ± 4

300 ± 81

255 ± 70

TA98

20 ± 8

26 ± 7

413 ± 82

404 ± 118

TA1538 (2NF)

16 ± 3

23 ± 5

376 ± 79

556 ± 216

TA1538 (4NQO)

16 ± 3

23 ± 5

343 ± 34

556 ± 216

TA1537

8 ± 2

11 ± 4

497 ± 254

196 ± 70

TA102

260 ± 49

317 ± 52

758 ± 175

1676 ± 562

TA104

269 ± 40

332 ± 48

1973 ± 755

1196 ± 252

WP2uvrA

29 ± 11

34 ± 11

273 ± 126

879 ± 177

WP2uvrA/pKM101

141 ± 43

198 ± 49

2080 ± 884

928 ± 250

 

results with 2 -aminoethanol

conc (µg/plate)

Number of revertants/plete (mean of 2 Experiments)

 

Base-substitution

Frame-shift

Strain

TA100

TA1535

WP2uvrA

TA98

TA1537

TA1539

Metabolic acitvation

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

pos control

942

462

258

172

274

1071

293

301

307

255

296

227

neg control

205

197

17

11

23

22

23

31

11

9

9

16

50

203

215

26

12

29

26

19

38

9

22

11

13

100

198

208

30

10

15

28

21

32

9

13

9

15

200

199

218

17

12

23

23

16

32

10

14

9

20

500

205

220

22

12

19

21

19

30

6

20

7

15

1000

188

191

15

19

26

26

22

28

6

17

8

8

2000

72

148

12

15

15

29

14

21

4

8

6

8

5000

0

0

0

0

0

0

0

0

0

0

0

0

Judgement

-

-

-

-

-

-

-

-

-

-

-

-

Conclusions:
Monoethanolamine was found to be non-mutagenic in all the tested strains (Salmonella typhimurium strains, TA98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA1538 and Escherichia coli strains WP2uvrA and WP2uvrA/pKM101) with and without metabolic activation.
Executive summary:

The bacterial reverse mutation test of Monoethanolamine was conducted following methods similar to the OECD guideline 471 (Bacterial Reverse Mutation Test) and according to Japan Guidelines.

Salmonella typhimurium strains, TA98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA1538 and Escherichia coli strains WP2uvrA and WP2uvrA/pKM101 were employed in this study.

The preincubation procedure was used to evaluate the mutagenicity potential of this test substance in the presence liver S9 (Sodium phenobarbital and 5,6-benzofravone-induced) and absence of metabolic activation system. For preincubation method, test compound dissolved in 0.05 or 0.1 ml of solvent was supplemented with 0.5 ml of S9 mix (metabolic activation method) or 0.1M phosphate buffer pH 7.4 (direct method) and 0.1 ml of tester strains which had been cultured in nutrient broth. The mixture was incubated for 20 min at 37°C, then rapidly mixed with 2 ml of molten top agar containing 0.05 μmol/ml of L-histidine and biotin for the Salmonella test. In the E. coli test 0.05 μmol/ml of L-tryptophan was used instead of L-histidine and biotin. Then the top agar mixture was rapidly poured onto a 30 ml of Vogel -Bonner minimal agar plate. All plates were incubated for 48 hours at 37°C and the numbers of revertant colonies were scored.

In conclusion, the test substance, monoethanolamine was found to be non-mutagenic in all strains both in presence and absence of metabolic activation.

This bacterial reverse mutation test is classified as acceptable and is comparable to guideline requirements of the OECD 471 method and Japan Guidelines.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(test concentrations were tested only up to 2000 µg/plate)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus
Histidine locus of following Salmonella typhimurium tester strains served as a specific target gene:
- TA 1535 and TA 100: Base-pair substitutions
- TA 1537, TA1538 and TA 98: Frame shift mutations

Tryptophan locus
Tryptophan locus of following E.coli strains served as a specific target gene:
-Escherichia coli WP 2 and WP 2 uvrA
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 (Arcoclor 1254 induced)
Test concentrations with justification for top dose:
0.2, 2, 20, 500 and 2000 µg/plate
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
7,12-dimethylbenzanthracene
sodium azide
benzo(a)pyrene
cyclophosphamide
methylmethanesulfonate
other: neutral red
Remarks:
Benzo[a]pyrene, 4-nitroquinoline N-oxide and dimethylbenzanthracene were dissolved in dimethyl sulphoxide (DMSO); the remaining positive control compounds were prepared as aqueous solutions.
Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS: quadruplicate-triplicates/dose level
- Number of independent experiments: 2

METHOD OF APPLICATION:
TEST ORGANISM: All Salmonella typhimurium strains (TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were procured from Professor B.N. Ames, University of California and both Escherichia coli WP 2 and WP 2 uvrA strains were obtained from Dr. M.H.L. Green, University of Sussex.

METHOD OF APPLICATION:
Plate-incorporation assay

The method used was basically that described by Ames et al. (1975), using S9 microsomal fraction obtained from a rat liver homogenate from rats pre-treated with Aroclor 1254. From 1975 to 1980, a range of amounts of each compound was tested (0.2, 2, 20, 500 and 2000 μg/plate) both in the presence and in the absence of S9 mix. Since 1980, a preliminary cytotoxicity assay has been carried out to assess both the cytotoxicity of the test compound and its solubility in the top agar. The amounts to be used in the mutation assays were selected on this basis. 0.1 mL of a dilution (1:20000) of an overnight bacterial culture was added to 2 mL top agar, together with 20 µL test compound to give final amounts of 125, 250, 500, 1000, 2000 and 4000 μg/plate and 0.5 mL S9 mix (± S9) or 0.5 mL pH 7.4 phosphate buffer (- S9). The top agar was poured onto nutrient agar plates and an assessment of cytotoxicity was made after 24 hours incubation at 37°C. In the mutation assays control plates were set up with the solvent alone and with a known positive control compounds. All tests were carried out in quadruplicate (until 1980) or triplicate. Two replicate assays were carried out on different days in order to confirm the reproducibility of the results.

Pre-incubation assay
The method used was that described by Brooks and Dean (1981). Bacteria (0.5 mL) and S9 mix or pH 7.4 phoshate buffer (2.5 mL) were incubated at 37°C with the test solution (0.1 ml) or solvent for 30 minutes before incorporation of 0.5 mL of this pre-incubation mixture into 2 mL of top agar. All assays were carried out at least in triplicate (i.e. 3 plates per data data point).
Evaluation criteria:
- A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value.
- If the test substance does not induce a statistically significant, dose-dependent increase in revertant frequency, but does induce a revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal.
- A negative result is defined as the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants.
Statistics:
A variety of statistical techniques were applied to bacterial data to detect weak positive responses. These includes standard t-test, the Control Chart method and the Quadrant Sum method.
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
other: all strains/cell types tested Migrated from field 'Test system'.
Conclusions:
Monoethanolamine was found to be non-mutagenic in all the tested strains (Salmonella typhimurium strains TA 1535, TA 1537, TA1538, TA 98 and TA 100 and Escherichia coli strains WP 2 and WP 2 uvrA) with and without metabolic activation.
Executive summary:

The bacterial reverse mutation test of Monoethanolamine was conducted following methods similar to the OECD guideline 471 (Bacterial Reverse Mutation Test).

Salmonella typhimurium strains (TA 1535, TA 1537, TA1538, TA 98 and TA 100) and Escherichia coli strains WP 2 and WP 2 uvrA were employed in this study. Pre incubation and plate incorporation methods were used to evaluate the mutagenicity potential of this test substance in the presence (S-9 mix from the livers of rats induced with Aroclor-1254) and absence of metabolic activation. Each test concentration, including the positive and negative controls, was tested in quadruplicate or triplicates.

For pre incubation method, 0.1 mL of test solution was preincubated with the 0.5 mL of test strain and 2.5 mL of sterile phosphate buffer (pH 7.4) (for -S9) or the metabolic activation mixture (+S9) for 30 minutes at 37°C prior to mixing with the overlay agar and poured onto the surface of a 2.0 mL agar plate. All tests were carried out at least in triplicate. For plate incorporation method, 0.1 mL of an overnight bacterial culture was added to 2 mL top agar, together with 20 µL of the test solutions and 0.5 mL of sterile phosphate buffer (pH 7.4) (for –S9) or the metabolic activation mixture (for +S9). The top agar was poured onto nutrient agar plates and an assessment of cytotoxicity was made after 24 hours incubation at 37°C. All tests were carried out in quadruplicate or triplicate. Two replicate assays were carried out on different days in order to confirm the reproducibility of the results.

Various statistical techniques like standard t-test, the Control Chart method and the Quadrant Sum method were applied to evaluate mutagenicity. No positive response was observed in all tester strains with and without S9 mix.

In conclusion, the test substance, monoethanolamine was found to be non-mutagenic in all strains both in presence and absence of metabolic activation.

This bacterial reverse mutation test is classified as acceptable and satisfies the guideline requirements of the OECD 471 method.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test), 1997
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
38.1, 76.3, 152.5, 305, 610 μg/mL (represents the limit dose of 10mM)
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: MMS (-S9) and 20-MCA (+S9)
Details on test system and experimental conditions:
Treatment procedure
Cells from logarithmically growing stock cultures (maintained in an orbital shaker at 37°C) were treated in individual 50 mL culture tubes. Each tube consisted of 6 mL of a cell suspension containing lx106 cells/mL in a 1:1 mix of FoP and conditioned medium. To these cells, 4 mL of either S-9 mix or FoP was added along with the test material. The treatment consisted of duplicate cultures per concentration with the exception of positive controls where only one replicate was used. Following the addition of the test compounds, the tubes were incubated for approximately 4 hours at 37°C in a roller drum (approximately 25-35 orbits/minute). At the end of the incubation period, the cells were pelleted, rinsed with FoP and resuspended in 20 mL F10P. The tubes were returned to the roller drum and maintained at 37°C during a standard expression period of 2 days.
Evaluation criteria:
Mutant frequencies were evaluated based upon statistical analysis as well as a number of other criteria (Clive et al., 1995). The test chemical is considered positive when the conditions listed below are met:
a) there is a statistically significant, dose related, reproducible increase in mutant frequency with a range of dose levels yielding >20% relative total growth.
b) the mutant frequency in at least one dose level of the treated cultures (resulting in >20% relative total growth) is 100 x 10-6 above concurrent solvent controls (assuming these to be in the range of 20-80 x 10-6).
The biological and scientific plausibility of the observed response is also taken into consideration in the final interpretation of the data.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Assay

In a preliminary toxicity assay, the test material was evaluated at targeted concentrations of 2.38, 4.77, 9.53, 19.1, 38.1, 76.3, 153, 305, and 610 pg/mL in the absence and presence of an externally supplied metabolic activation system (S-9). The test material did not exhibit toxicity at any concentrations as determined by Day 2 relative suspension growth (RSG). Based upon the results of this assay, concentrations of 38.1, 76.3, 152.5, 305, and 610 µg/mL were selected for the gene mutation assay with and without S9. The highest concentration selected (610 µg/mL) represents the generally accepted limit dose (10 mM) for these assays.

Mutation Assays
A total of 2 independent assays were conducted for the non-activated and S-9 activated portions of the study. In Assay 1 none of the concentration levels evaluated induced undue toxicity as measured by the relative, total growth (RTG). There were no significant increases in the mutant frequency at any of the dose levels evaluated in this assay. Cultures treated with the positive control chemical had significantly higher mutant frequencies compared to the solvent control. Negative control mutant frequencies were within the range of laboratory historical data. All other criteria for a valid assay were satisfied in this assay.

In the confirmatory assay (Assay 2), the test material was again evaluated at concentrations ranging from 38.1 to 610 µg/mL. Without activation, the relative total growth in the test material treated cultures varied from 38 to 132%. Although statistical analyses indicated a significant trend, pairwise comparisons of the treated VS negative control did not identify significantly different mutant frequencies at any of the test material concentrations. Based upon the weight of evidence (absence of a significant effect in pair-wise comparisons along with the observation of negative results in Assay 1) it was interpreted that MEA did not elicit a mutagenic response in the absence of S-9 in assay 2.

The portion of Assay 2 with S-9 activation showed no evidence of toxicity. The relative total growth in the test material treated cultures varied from 125 to 393%. The larger calculated relative total growth values (RTG >100%) in the treated cultures is attributable to lower suspension growth of the negative cultures on the day following treatment. For example, the cell number in the negative control on Day 1 was approximately 0.3 x 106 while this value in the treated cultures ranged from 0.4 - 1.46 x 106. However, by Day 2, all the cultures, including the negative controls, had similar suspension growth. Furthermore, there were no remarkable differences in the relative plating efficiencies of the test material treated Cultures (75-115% relative to the negative control value). Based upon these data, it was concluded that the test material did not exhibit any toxicity in this assay and the apparently higher calculated RTG values did not have any consequence on the interpretability of the data on mutant frequencies. There was no evidence of an increase in mutant frequency in the treated cultures as compared to the negative control values.

The positive controls had significantly higher number of mutants than the negative controls. The negative control values were within the range of laboratory historical data. All other criteria for a valid assay were satisfied in this trial.

Analytical values of MEA concentration in the stock dosing solutions ranged from 86 to 100% of the targeted values in Assay 1 and from 85 to 102% of the targeted doses in Assay 2.
Conclusions:
2-Aminoethanol, when tested upto 610 μg/mL (with and without metabolic activation) was considered non-mutagenic in mouse lymphoma thymidine kinase locus assay using L5178Y cell lines.
Executive summary:

The in vitro mammalian gene mutation test of 2 -Aminoethanol was performed according to the OECD guideline 476. The study was performed to evaluate the mutagenic potential of test susbtance to induce mutations at the mouse lymphoma thymidine kinase (TK+/-) locus using the cell line L5178Y.

A preliminary toxicity assay using test substance was performed at 2.38, 4.77, 9.53, 19.1, 38.1, 76.3, 153, 305 and 610 pg/mL in absence as well as presnce of external metabolic activation system (S-9). As test material did not exhibited any toxicity at tested concentrations (based on day 2 relative suspension growth), these concentrations were selected for main experiment.

Water was used as vehicle and served as negative control. Methylmethanesulphonate (MMS; without metabolic activation) and 20-methylcholanthrene (20 -MCA; with metabolic activation) served as positive controls. Duplicate cultures were preapared at each test point, with the exception of positve controls which were prepared in the single culture.

No significant increase in mutant frequency were observed following treatment with the test susbtance at any dose-level, in the absence/presence of S9 metabolism, at any treatment time.

The mutant frequencies in negative control fell within the range of laboratory historical data.

Significantly high number of mutant frequencies were observed in the positive control as compared to the negative control indicating that the tests were sensitive and valid.

2-Aminoethanol, when tested upto 610 μg/mL (with and without metabolic activation) was considered non-mutagenic in mouse lymphoma thymidine kinase locus assay using L5178Y cell lines.

This mammalian gene mutation test is classified as acceptable, and satisfies the guideline requirements for the OECD guideline 476 method.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Principles of method if other than guideline:
The aim of this study was to assess the effect of 2-aminoethanol on inhibition of colony forming and metabolic cooperation ability. Metabolic cooperation assay is an in vitro screening assay for potential teratogens, tumor promoters and reproductive toxicants. The principle of the assay is based on the transfer, via gap junctions, of a phosphorylated metabolite of 6TG added to a co-culture of 6TG sensitive [hypoxanthine guanine phosphoribosyltranferase (HGPRT+) positive] and 6TG resistant (HGPRT-) cells. Colony forming assay (in vitro) was used to determine cytotoxicity. Inhibition of metabolic cooperation of linked to inhibition of gap junctional intercellular communication.
GLP compliance:
no
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Wild type V79 (6TGs, HGPRT+) and mutant (6TGr, HGPRT-) Chinese hamster lung fibroblast cells
- Suitability of cells: no data

For cell lines:
- Absence of Mycoplasma contamination: yes, checked using the Hoechst 33258 staining
- Methods for maintenance in cell culture: The cell cultures were grown at 37°C in a humidified incubator with a 5% CO2 atmosphere. Under the study conditions, the cells grew with a generation period of approximately 12 hours. Approximately 24 hours prior to each test, both cell lines were subculture to ensure a log-phase condition in the cells at the time of the assay.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Cells were grown in modified Eagle's medium, Eagle's balanced salt solutions with a 50% increase of vitamins and essential amino acids, except glutamine, supplemented with non-essential amino acids (100% increase), sodium pyruvate and 3% fetal calf serum. The medium was further supplemented with penicillin G (100 units/mL) and streptomycin (100 /µg/mL) during the experiments.

Metabolic activation:
without
Test concentrations with justification for top dose:
Test substance was tested over a graded concentration range. No further details were available regarding test concentrations.
Vehicle / solvent:
No
Untreated negative controls:
yes
Remarks:
(alcohol)
Positive controls:
yes
Positive control substance:
other: 12-Tetradecanoylphorbol- 13-acetate (TPA) [1ng TPA / mL alcohol]
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 5 parallel plates for negative and positive controls as well as test concentrations
- Number of independent experiments: one for each colony-forming and metabolic cooperation assay

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Colony-forming ability assay
- Procedure: The preliminary test involves a rough determination of the test substance’s effect on cell division or morphological changes in 60×15 mm plates containing 1E+05 cells. Repeated tests with narrower ranges of concentrations were performed to establish dose-dependent cytotoxicity curve. One hundred 6TG resistant cells were inoculated into 60×15 mm disposable plastic tissue culture dishes, having a final volume of 5 mL of medium. After 4-6 hours the test chemical was carefully delivered, followed shortly by the addition of 10 µg/mL of 6TG. After three days the medium was decanted and replaced with fresh medium minus the test chemical. At the end of another 3-4 days of incubation the medium was decanted, plates were washed with physiological saline, fixed and stained with solution of 10% ethanol and 1.0% crystal violet. Colonies were scored visually.
- Any supplementary information relevant to cytotoxicity: Cytotoxicity of a chemical, as determined by the colony-forming assay, using relatively few target cells, will not be identical to the cytotoxicity of that chemical at the same concentration in the metabolic cooperation assay which utilizes 4E+05 cells.

METHODS FOR MEASUREMENTS OF GENOTOXICIY: The ability to inhibit metabolic cooperation was indicated by the number of colonies recovered over the negative control. A colony was scored if it contained at least 50 cells. The test substance was determined to be positive if a dose response effect result in at least a doubling of the recovery over the negative control was observed.


Details on test substance sampling

Test samples were directly added into dishes containing medium and cells. After addition of test samples, mild mix of the medium was taken in order to ensure the uniform distribution and to prevent localized killing of cells and dissolution of the plastic plate.

METABOLIC COOPERATION ASSAY
The assay is based on the transfer, via gap junctions, of a phosphorylated metabolite of 6TG added to a coculture of 6TG sensitive (HGPRT +) and 6TG resistant (HGPRT-) cells. Under these conditions, many 6TG r cells will not form colonies because of "metabolic cooperation." The assay procedures were identical to the cytotoxicity assay, with the exception that 4E+05 6TG sensitive cells were co-cultivated with 100 6TG resistant cells instead of 1E+05 cells.
Evaluation criteria:
Plating efficiency of the 6TG resistant cells was determined by a series of plates containing 100 6TG resistant cells. The ability of any compound to inhibit metabolic cooperation was indicated by the number of colonies recovered over the negative control. A minimum of 50 cells colony needs to be scored. Test substance was determined positive if a dose response effect result in at least a doubling of the recovery over the negative control was observed.
Statistics:
No information
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
2-aminoethanol was not found to inhibit the metabolic cooperation assay in Chinese hamster lung fibroblasts (V79) in absence of metabolic activation. Therefore, 2-aminoethanol has no impact on gap-junctional intercellular communication which play critical role in the regulation of cell proliferation and differentiation.
Executive summary:

The aim of this study was to assess the effect of 2-aminoethanol on inhibition of colony forming and metabolic cooperation ability.

Metabolic cooperation assay is an in vitro screening assay for screening of potential teratogens, tumor promoters and reproductive toxicants. The principle of the assay is based on the transfer, via gap junctions, of a phosphorylated metabolite of 6TG added to a co-culture of 6TG sensitive [hypoxanthine guanine phosphoribosyltranferase (HGPRT +) positive] and 6TG resistant (HGPRT-) cells. Colony forming assay (in vitro) was usedtodeterminecytotoxicity. The inhibition in this assay is linked to inhibition of gap junctional intercellular communication.

Cell cultures were grown at 37°C in a humidified incubator with a 5% CO2 atmosphere. Positive (TPA) and negative control (alcohol) were used in the study. One hundred 6TG resistant cells were inoculated into 60×15 mm disposable plastic tissue culture dishes consisting of 1E+05 cells (for colony forming assay) and 4E+05 cells (for metabolic cooperation assay), having a final volume of 5 mL of modified Eagle's medium. Test substance was added followed by addition of 10/µg/ml of 6TG. The medium was decanted and was replaced with fresh medium without containing test substance and was kept for 3- 4 days of incubation. Cells were fixed and stained with solution of 10% ethanol and 1.0% crystal violet and colonies were scored visually.

There was no increase in recovery in test substance treated plates as compare to the negative control. Test system was valid as TPA (positive control) resulted in a high recovery of 6-TG resistant colonies and negative control resulted in very low recovery.

Based on above results,2-aminoethanol was not found to inhibit the metabolic cooperation assay in Chinese hamsterlung fibroblasts(V79) in absence of metabolic activation. Therefore, 2-aminoethanol has no impact on gap-junctional intercellular communication which play critical role in the regulation of cell proliferation and differentiation.

 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Principles of method if other than guideline:
The aim of study was to evaluate the genotoxicity potential of monoethanolamine in an in vitro chromosome assay using cultured rat-liver cells (RL 1 and RL4). Epithelial-type cell line were incubated at 37°C for 24 hours and then test substance was exposed to the cell lines over a period of 22 hours. After exposure, colcemid was added to each culture. After 2 hours, the cultures were harvested and a hypotonic solution was added to the cell suspension. After the hypotonic treatment the suspension was centrifuged, the solution was decanted and the cells fixed in 3 changes of fixative solution methanol: acetic acid 3:1). Chromosome preparations were made on microscope slides and stained with Giemsa stain. The preparations were randomly coded and 100 cells from each cultured were analyzed microscopically. The detailed method is provided in Dean et al.: Mut. Res., 153, 57-77, (1985).
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Rat hepatocytes (RL4)
Details on mammalian cell type (if applicable):
CELL LINES: RL1 was initiated in 1973 from a 10-day old Carworth Farm E rat and RL 4 was derived
from a 10-day old Wistar rat in 1978.
PREPARATION OF CELL LINES: The liver from a 10-day old rat was collected into Hanks' basic salt solution, the tissue was then minced using scissors, and the fragments were dissociated by trypsinisation for two periods of 40 minutes using 0.25% trypsin solution (Wellcome Reagents Ltd., Beckenham, Kent). The resulting cells were suspended in Minimal Essential Medium (Wellcome Reagents Ltd.) supplemented with 10% foetal calf serum (Flow Laboratories Ltd., Irvine, Scotland) and 1% non-essential amino acids (Flow Laboratories) in an 8-oz. (200-mL) glass prescription bottle. This culture (designated No. 1) was incubated at 37°C for 20 minutes and then the cell suspension was transferred to asecond bottle (Culture No. 2) and also incubated for 20 minutes. The cell suspension was transferred to a third bottle (No. 3), in which it was incubated for 2 hand the cells remaining in suspension at the end of this time were transferred to the fourth and final bottle. 30 mL of culture medium were added to each bottle after removal of the cell suspension and the cultures were incubated until approx. 50% confluent. In general, the fibroblasts become attached to the growing surface in the first two bottles and culture number 3 or 4 produces an almost pure culture of epithelial-type cells. Any remaining fibroblasts in these latter cultures are spontaneously eliminated during subsequent subculture. The RL1 cell line was derived from the third bottle.

KARYOTYPE OF THE RL1 CELL LINE: Near-diploid, having a chromosome number of 44--45 (compared to the normal rat karyotype of 42) and the extra chromosomes are small metacentrics belonging to groups.
Metabolic activation:
without
Metabolic activation system:
not applicable
Test concentrations with justification for top dose:
Not provided
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
7,12-dimethylbenzanthracene
sodium azide
benzo(a)pyrene
cyclophosphamide
ethylmethanesulphonate
methylmethanesulfonate
other: neutral red
Remarks:
Benzo[a]pyrene, 4-nitroquinoline N-oxide and dimethylbenzanthracene were dissolved in dimethyl sulphoxide (DMSO); the remaining positive control compounds were prepared as aqueous solutions.
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS: No data

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 24 hours
- Exposure duration/duration of treatment: 22-24 hours
- Harvest time after the end of treatment (sampling/recovery times): 2 hours after adding colcemid

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Colcemid was added at a final concentration of 0.3 µg/ml just 2 hours before harvesting.
- Number of cells spread and analyzed per concentration: 100
- Criteria for scoring chromosome aberrations: Not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: An estimate of the cytotoxicity was done based on cell density. The cells were exposed to a range of concentrations of the chemical to determine the appropriate concentrations of the test chemical. Firstly, the monolayer cultures of rat liver cells were incubated at 37°C for 24 hours to commence active growth before a range of freshly prepared solutions of the compound were added. After a further 24-hours incubation the cell monolayers were stained, and growth inhibition effects were noted. The concentration selected for the chromosome assay were 0.5, 0.25 and 0.125 of the GI50 (50% growth inhibition)

METHODS FOR MEASUREMENTS OF GENOTOXICIY: Chromatid gaps, chromatid deletions, chromatid exchanges and chromosome aberrations

CHROMOSOME ABERRATION ASSAY.

Cultured rat-liver cells were grown on microscope slides contained in petri dishes. Treatment was again for a 24-h period and positive control slides were included. Colcemid was added 2 h before exposure was complete. At exposure completion the cultures were harvested and a hypotonic solution was added to the cell suspension. After the hypotonic treatment the suspension was centrifuged, the solution was decanted and the cells fixed in 3 changes of fixative solution (methanol: acetic acid 3:1). Chromosome preparations were made on microscope slides and stained with Giemsa stain. The preparations were randomly coded and 100 cells from each cultured were analyzed microscopically
Statistics:
No suitable statistical treatment for these types of data has been identified and results were judged on the reproducibility and dose-responsiveness of the aberration frequencies.
Key result
Species / strain:
mammalian cell line, other: No suitable statistical treatment for these types of data has been identified and results were judged on the reproducibility and dose-responsiveness of the aberration frequencies.
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Monoethanolamine was found to be non clastogenic in absence of metabolic activation in both RL1 and RL4 cultured cells.
Executive summary:

This study was conducted to evaluate the potential of monoethanolamine to induce chromosomal aberration using rat liver cell lines.

Dose selection for the chromosomal assay were based on pre-test called as cytotoxicity assay. The monolayer cultures of rat liver cells were incubated at 37°C for 24 hours to commence active growth before a range of freshly prepared solutions of the compound were added. After a further 24-hours incubation the cell monolayers were stained, and growth inhibition effects were noted. The concentration selected for the chromosome assay were 0.5, 0.25 and 0.125 of the GI50 (50% growth inhibition).

Monolayer cultures of rat-liver cells were prepared and treated as in the cytotoxicity assay in 200-mL glass prescription bottles. Positive control cultures (4-nitroquinoline N-oxide, 7,12-dimethylbenzanthracene, sodium azide, benzo[a]pyrene, cyclophosphamide, ethyl methanesulphonate, methyl methanesulphonate and neutal red) were run in parallel. Colcemid® was added to the cultures 22 hours after the exposure. 2 hours later, the cells were treated on the slides in the chambers with hypotonic solution for 20 minutes at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3:1 methanol: glacial acetic acid (v/v) solution. After fixation step the slides were air dried and stained with Giemsa. The preparations were randomly coded and 100 cells from each cultured were analysed microscopically.

Test substance was not found to be clastrogenic. No detailed information regarding the evaluation of clastrogenicity of compound were provided in the study.

In conclusion, the test substance, monoethanolamine was found to be non-clastogenic in absence of metabolic activationin both RL1 and RL4 cultured cells.

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

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented journal article.
Qualifier:
no guideline followed
Principles of method if other than guideline:
A group of 7 male mice was fed a diet containing 0.6% test substance for 9 months. At the end of this period, the animals were each mated with two untreated females. On day 13 of pregnancy, the females were sacrificed, and the ovaries and uteri were examined.
GLP compliance:
no
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
other: JCL-ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CLEA Japan, Inc.
- Age at study initiation: 4 weeks
- Housing: individually, except during breeding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 1
- Humidity (%): 55 +/- 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): feed powder CE-2
Duration of treatment / exposure:
9 months
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0.6%, 300 mg/kg bw d
Basis: nominal in diet
No. of animals per sex per dose:
7
Control animals:
yes
Statistics:
Rohrborn's method.
Key result
Sex:
male
Genotoxicity:
negative
Negative controls validity:
valid
Additional information on results:
There were no significant differences in fertility, the mortality of ova and embryos, the number of surviving fetuses, or the index of dominant lethal induction between the experimental groups and the control group.

Dominant Lethal Assay Results

 

0.6% in Diet

Control

Number of mating females

14

18

Number pregnant

11

12

No. with dead embryos

6

10

Dead embryos per pregnant female

54.6%

83.3%

No. of corpora lutea

156

161

Corpora lutea per pregnant female

14.2

13.4

No. of implants

148

156

Implants per pregnant female

13.5

13.0

Implants per corpora lutea

94.9

96.9

No. of live fetuses

142

143

Live fetuses per pregnant female

12.9

11.9

Live fetuses per corpora lutea

91.0

88.8

Live fetuses per total implants

96.0

91.7

No. of early dead fetuses

4

12

No. of late dead fetuses

2

1

% of dominant lethals

-4.67

-

% of dominant lethals

-8.33

-

Conclusions:
Interpretation of results: negative
The test substance did not cause genetic disorders in mice.
Executive summary:

A group of 7 male mice was fed a diet containing 0.6% test substance for 9 months. At the end of this period, the animals were each mated with two untreated females. On day 13 of pregnancy, the females were sacrificed, and the ovaries and uteri were examined. No increase in dominant lethal induction was seen as compared to controls. The test substance does not cause genetic disorders.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented journal article.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 5 male rats were fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations.
GLP compliance:
no
Type of assay:
mammalian germ cell cytogenetic assay
Species:
rat
Strain:
other: Wistar and SD
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CLEA Japan, Inc.
- Age at study initiation: 4 weeks
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 1
- Humidity (%): 55 +/- 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): feed powder CE-2
Duration of treatment / exposure:
9 months
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0.9%, 450 mg/kg bw d
Basis:nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes
Tissues and cell types examined:
femur bone marrow cells
Details of tissue and slide preparation:
Animals were sacrificed by administration of 1 ml/kg of 1% colchine solution. Femurs were then removed, and bone marrow cells washed into centrifuge tubes. The cells were then treated with 0.075 M KCl solution at 37 degree C for 15 min, and then fixed with an acetic acid 1: ethanol 3 solution. Samples were then flame dried and treated with Giemsa.
Evaluation criteria:
Presence and absence of chromosomal aberrations. 50 metaphases per individual.
Statistics:
Rohrborn's method.
Key result
Sex:
male
Genotoxicity:
negative
Negative controls validity:
valid
Additional information on results:
No increase in chromosome aberrations was noted.

Chromosome Aberrations

 

0.9% in Diet – Wister Rats

0.9% in Diet –

SD Rats

Control

Control

No. of cells with chromatid breaks

0

0

1

0

No. of cells with isochromatid breaks

0

0

0

0

No. of cells with chromatid gaps

3

4

3

4

No. of cells with isochromatid gaps

0

0

0

0

No. of cells with other aberrations

0

0

0

0

Conclusions:
Interpretation of results: negative
The test substance is not clastogenic.
Executive summary:

Groups of 5 male rats were fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations. No increase in chromosome aberrations was seen as compared to controls. The test substance is not clastogenic.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented journal article.
Qualifier:
no guideline followed
Principles of method if other than guideline:
A group of 5 male mice was fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations.
GLP compliance:
no
Type of assay:
mammalian germ cell cytogenetic assay
Species:
mouse
Strain:
other: JCL-ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CLEA Japan, Inc.
- Age at study initiation: 4 weeks
- Housing: individually, except during breeding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 1
- Humidity (%): 55 +/- 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): feed powder CE-2
Duration of treatment / exposure:
9 months
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0.9%, 1170 mg/kg bw d
Basis:nominal in diet
No. of animals per sex per dose:
5
Control animals:
yes
Tissues and cell types examined:
femur bone marrow cells
Details of tissue and slide preparation:
Animals were sacrificed by administration of 1 ml/kg of 1% colchine solution. Femurs were then removed, and bone marrow cells washed into centrifuge tubes. The cells were then treated with 0.075 M KCl solution at 37 degree C for 15 min, and then fixed with an acetic acid 1: ethanol 3 solution. Samples were then flame dried and treated with Giemsa.
Evaluation criteria:
Presence and absence of chromosomal aberrations. 50 metaphases per individual.
Statistics:
Rohrborn's method.
Key result
Sex:
male
Genotoxicity:
negative
Negative controls validity:
valid
Additional information on results:
No increase in chromosome aberrations was noted.

Chromosome Aberrations

 

0.9% in Diet

Control

No. of cells with chromatid breaks

1

2

No. of cells with isochromatid breaks

1

0

No. of cells with chromatid gaps

4

5

No. of cells with isochromatid gaps

0

0

No. of cells with other aberrations

0

0

Conclusions:
Interpretation of results: negative
The test substance is not clastogenic.
Executive summary:

A group of 5 male mice was fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations. No increase in chromosome aberrations was seen as compared to controls. The test substance is not clastogenic.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented publication.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of male mice were given doses of 200, 400, or 800 mg/kg of Benzenesulfonic acid, C10-14-alkyl derivs., sodium salts. At 6, 24, and 48 hrs, 3 of the mice from each dosage group were sacrificed. The bone marrow cells from the femurs were collected and examined for chromosome aberrations. In addition, one group of mice was exposed daily for 5 consecutive days. Additional groups of mice were exposed to commerical detergents containing 19% or 17.1% of the test substance. Mitomycin C was used as a positive control.
GLP compliance:
no
Type of assay:
chromosome aberration assay
Species:
mouse
Strain:
other: ICR/JCL
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CLEA Japan Inc.
- Age at study initiation: 9-11 weeks
- Housing: individually in plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12 hrs

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Samples were diluted to make a 5 ml/kg dose volume

Duration of treatment / exposure:
Single treatment, except for one group which was given 5 consecutive daily exposures to the test substance
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
200, 400, 800 mg/kg
Basis:
nominal conc.
test substance
Remarks:
Doses / Concentrations:
800, 1600, 3200 mg/kg
Basis:
nominal conc.
commercial detergent containing 19% test substance
Remarks:
Doses / Concentrations:
1000, 2000, 4000 mg/kg
Basis:
nominal conc.
commercial detergent containing 17.1% test substance
No. of animals per sex per dose:
9
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C1
- Route of administration: intraperitoneally
- Doses / concentrations: 5 mg/kg
Tissues and cell types examined:
bone marrow cells from femurs
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 3 animals were sacrificed at 6, 24, and 48 hrs after treatment. One group was exposed daily for 5 days prior to sacrifice.

DETAILS OF SLIDE PREPARATION: Cells were placed in Hank's solution, then centrifuged at 1000 rpm for 5 min. Supernatant was discarded, and 5 ml of 0.075 M KCl was added. The mixture then stood for 5 min., then was stirred and centrifuged again. This was repeated several times, before finally placing one or two drops on the slides, and staining with 2% Giemsa solution.

Evaluation criteria:
number of cells with chromatid and chromosome gaps, number of cells with aberrations
50 metaphases per animal (150 total) were imaged at each time point.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No significant differences in the incidence of chromosomal aberrations were observed in any test substance treatment group relative to the controls.

Chromosome Aberrations

 

Total number of cells having aberrations and occurrence (%)

 

6 hrs

24 hrs

48 hrs

5 days

200 mg/kg

0 (0)

0 (0)

0 (0)

1 (0.7)

400 mg/kg

1 (0.7)

0 (0)

0 (0)

0 (0)

800 mg/kg

0 (0)

0 (0)

0 (0)

0 (0)

800 mg/kg of 17.1% detergent

0 (0)

0 (0)

0 (0)

-

1600 mg/kg of 17.1% detergent

0 (0)

1 (0.7)

0 (0)

-

3200 mg/kg of 17.1% detergent

2 (1.3)

2 (1.3)

0 (0)

-

1000 mg/kg of 19% detergent

-

0 (0)

-

-

2000 mg/kg of 19% detergent

-

0 (0)

-

-

4000 mg/kg of 19% detergent

-

0 (0)

-

-

Mitomycin C

16 (10.7)

53 (353)

13 (8.7)

112 (74.7)

Distilled water

0 (0)

0 (0)

0 (0)

0 (0)

untreated

0 (0)

0 (0)

1 (0.7)

0 (0)

Conclusions:
Interpretation of results: negative
The test substance is not clastogenic.
Executive summary:

Groups of male mice were given doses of 200, 400, or 800 mg/kg of Benzenesulfonic acid, C10-14-alkyl derivs., sodium salts. At 6, 24, and 48 hrs, 3 of the mice from each dosage group were sacrificed. The bone marrow cells from the femurs were collected and examined for chromosome aberrations. In addition, one group of mice was exposed daily for 5 consecutive days. Additional groups of mice were exposed to commerical detergents containing 19% or 17.1% of the test substance. Mitomycin C was used as a positive control. None of the treatment groups showed any significant increase in chromosome aberrations as compared to negative controls. The test substance in not clastogenic.

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

Additional information

Genetic toxicity, in vitro 

     

Study 1 (C10-13 LAS): 

     

A bacterial reverse mutation assay was conducted with C10-13 LAS, sodium salt to assess the potential mutagenicity of LAS. The experiment was run using S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, as well as TA1538 at test concentrations of 8, 40, 200, 1000 and 5000 µg/plate. All strains tested negative with and without S9 activation (Schoeberl, 1993).

 

Study 2 (C10-13 LAS): 

     

An in vitro genetic toxicity study was conducted to evaluate the potential of C10 -13 LAS to cause mutations in mammalian cells. Chinese Hamster Ovary (CHO) cells were exposed to concentrations of 0, 0.6, 1, 1.8, 3, and 6 µg/ml without metabolic activation (S9), and 0, 6, 10, 18, 30, and 60 µg/ml with S9. The cells were then examined for cytogenicity and mutation frequency. Ethyl methane sulfonate and 3-(20-)methylcholanthrene were used as positive control substances. Preliminary tests show the test substance was cytogenic at concentrations of 50 ug/ml or greater with metabolic activation, and 100 ug/ml or above without metabolic activation. There was no biologically significant increase in mutation frequency in the treated groups. The test substance is considered not mutagenic to CHO cells both in the presence and absence of S9 (Anon., 1995). 

     

 Study 3 (MEA):  

 

A bacterial reverse mutation assay was conducted with monoethanolamine (MEA) according to a method similar to OECD Guideline 471 and according to Japanese test guidelines. Salmonella typhimurium strains, TA98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA1538 and Escherichia coli strains WP2uvrA and WP2uvrA/pKM101 were exposed to the test substance concentration range of exposed to concentrations of 50-5000 µg/plate with and without metabolic activation. MEA was found to be non-mutagenic in all strains both in presence and absence of metabolic activation (JETOC, 1996).

     

 Study 4 (MEA): 

     

A bacterial reverse mutation assay was conducted with monoethanolamine (MEA) according to a method similar to OECD Guideline 471. Salmonella typhimurium strains, TA98, TA 100, TA 1535, TA 1537 and TA1538 and Escherichia coli strains WP2uvrA were exposed to the test substance at concentrations of 0.2, 2, 20, 500 and 2000 µg/plate with and without metabolic activation under pre-incubation and plate incorporation methods. MEA was found to be non-mutagenic in all strains both in presence and absence of metabolic activation (Dean, 1985).  

  

Study 5 (MEA): 

     

An in vitro mammalian gene mutation assay was conducted with monoethanolamine (MEA) according to OECD Guideline 476. The study was performed to evaluate the mutagenic potential of test susbtance to induce mutations at the mouse lymphoma thymidine kinase (TK+/-) locus using the cell line L5178Y. The cells were exposed to MEA at concentrations of 0, 2.38, 4.77, 9.53, 19.1, 38.1, 76.3, 153, 305 and 610 pg/mL in absence as well as presence of external metabolic activation system (S-9). Water was used as vehicle and served as negative control. Methylmethanesulphonate (without metabolic activation) and 20-methylcholanthrene (with metabolic activation) served as positive controls. Duplicate cultures were prepared for each test concentration. No significant increase in mutant frequency were observed following treatment with the test substance at any dose-level, in the absence/presence of S9 metabolism, at any treatment time. The mutant frequencies in negative control fell within the range of laboratory historical data. MEA was considered non-mutagenic in mouse lymphoma thymidine kinase locus assay using L5178Y cell lines (Linscombe, 1999). 

     

Study 6 (MEA): 

     

An in vitro mammalian gene mutation assay was conducted with monoethanolamine (MEA) to assess the effect of the test substance on colony forming and metabolic cooperation ability of the cells. The principle of the assay is based on the transfer, via gap junctions, of a phosphorylated metabolite of 6TG added to a co-culture of 6TG sensitive [hypoxanthine guanine phosphoribosyltranferase (HGPRT +) positive] and 6TG resistant (HGPRT-) cells. Colony forming assay (in vitro) was used to determine cytotoxicity. Appropriate positive and negative controls were used in the study. Under the study conditions, MEA was not found to inhibit the metabolic co-operation assay in Chinese hamster lung fibroblasts(V79) in absence of metabolic activation (Chen, 1984).  

     

Study 7 (MEA): 

     

An in vitro mammalian cell chromosomal aberration assay was conducted with monoethanolamine (MEA) in rat liver cell lines. The monolayer cultures of rat liver cells were incubated at 37°C for 24 h to commence active growth before a range of freshly prepared solutions of the MEA were added. After a further 24 h incubation the cell monolayers were stained, and growth inhibition effects were observed. The concentrations selected for the chromosome assay were 0.5, 0.25 and 0.125 of the GI50 (50% growth inhibition). Positive control cultures (4-nitroquinoline N-oxide, 7,12-dimethylbenzanthracene, sodium azide, benzo[a]pyrene, cyclophosphamide, ethyl methanesulphonate, methyl methanesulphonate and neutal red) were run in parallel. Colcemid was added to the cultures 22 h after the exposure. 2 h later, the cells were treated on the slides in the chambers with hypotonic solution for 20 min at 37°C. After incubation in the hypotonic solution the cells were fixed with 3:1 methanol: glacial acetic acid (v/v) solution. After fixation step the slides were air dried and stained with Giemsa. The preparations were randomly coded and 100 cells from each cultured were analysed microscopically. Under the study conditions, MEA was not found to be clastogenic in rat liver cells in absence of metabolic activation (Dean 1985b).  

 

Genetic toxicity, in vivo 

     

Study 1 (C10-13 LAS):  

 

An in vivo dominant lethal mutation assay was conducted with C10-13 LAS, sodium salt in male mice. A group of 7 male mice was fed a diet containing 0.6% test substance for 9 months. At the end of this period, the animals were each mated with two untreated females. On day 13 of pregnancy, the females were sacrificed, and the ovaries and uteri were examined. No increase in dominant lethal induction was seen as compared to controls. Under the study conditions, LAS was not mutagenic in mice (Masabuchi, 1976a).     

     

Study 2 (C10-13 LAS): 

     

An in vivo chromosomal aberration assay was conducted with C10-13 LAS, sodium salt in rats. In this study, groups of five male rats were fed diets containing 0.9% LAS for 9 months (equivalent to 405 mg/kg bw/day). At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations. No increase in chromosome aberrations was seen as compared to negative controls in either species but no positive controls have been included in this assay. Under the study conditions, LAS was not clastogenic in this assay (Masabuchi, 1976b).

     

Study 3 (C10-13 LAS): 

     

An in vivo chromosomal aberration assay was conducted with C10-13 LAS, sodium salt in male mice. In this study, groups of five male mice were fed diets containing 0.9% LAS for 9 months (equivalent to 1,125 mg/kg bw/day). At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations. No increase in chromosome aberrations was seen as compared to negative controls in either species but no positive controls have been included in this assay. Under the study conditions, LAS was not clastogenic in this assay (Masabuchi, 1976c).

     

Study 4 (C10-14 LAS): 

     

An in vivo chromosomal aberration assay was conducted with C10-14 LAS, sodium salt in male mice. Groups of male mice were given doses of 200, 400, or 800 mg/kg bw of the test substance. At 6, 24, and 48 h, 3 of the mice from each dosage group were sacrificed. The bone marrow cells from the femurs were collected and examined for chromosome aberrations. In addition, one group of mice was exposed daily for 5 consecutive days. Mitomycin C was used as a positive control. None of the treatment groups showed any significant increase in chromosome aberrations as compared to negative controls. Under the study conditions, LAS was not clastogenic (Inoue, 1979).

Justification for classification or non-classification

Based on in vitro and in vivo genotoxicity studies conducted with the dissociation products of MEA-LAS (MEA and LAS), no classification is warranted for this endpoint according to EU CLP (1272/2008/EC) criteria.