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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was negative in Ames tests in Salmonella typhimurium and Echerichia coli strains. In addition in vitro gene mutation and chromosome aberration studies were negative.

Link to relevant study records

Referenceopen allclose all

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:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
No details on the concentrations used
Qualifier:
no guideline followed
Principles of method if other than guideline:
Method described in Trosko et al., 1984 (In Handbook of Carcinogen Testing) is based on metabolic cooperation between 6-thioguanine sensitive (HGPRT+) cells and 6-thioguanine resistant (HGPRT-) cells in vitro.
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):
- Type and identity of media: Modified Eagle's medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
other: with and without HGPRT deficiency
Metabolic activation:
without
Test concentrations with justification for top dose:
not indicated
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 12-tetra-decanoyl-1-phorbol-13-acetate (TPA)
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
only without metabolic activation tested
Principles of method if other than guideline:
Method according to Dean et al.: Mut. Res., 153, 57-77, (1985)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 17589
Species / strain / cell type:
hepatocytes:
Details on mammalian cell type (if applicable):
Rat hepatocytes (RL4)
Metabolic activation:
without
Test concentrations with justification for top dose:
100 - 400 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Key result
Species / strain:
other: rat hepatocytes RL4
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
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
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 1x106 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 µg/mL in the absence and presence of an externally supplied metabolic activation system (S9). 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 S9 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 S9 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 10E6 while this value in the treated cultures ranged from 0.4 - 1.46 x 10E6. 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
Based upon the results of Assays 1 and 2, it was concluded that monoethanolamine (MEA) did not induce a mutagenic response in the mouse lymphoma forward mutation assay in the absence or presence of metabolic activation.
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:
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AQ01
Target gene:
his
Species / strain / cell type:
S. typhimurium, other: TA98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA1538
Species / strain / cell type:
E. coli, other: WP2uvrA and WP2uvrA/pKM101
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:
9-aminoacridine
Remarks:
see "Any other information on materials"
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
see "Any other information on materials"
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
see "Any other information on materials"
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-amino anthracene (2AA)
Remarks:
see "Any other information on materials"
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: pyruvic aldehyde (PA)
Remarks:
see "Any other information on materials"
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: bleomycin (BLM)
Remarks:
see "Any other information on materials"
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
see "Any other information on materials"
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro2-furyl)acrylamide (2AF)
Remarks:
see "Any other information on materials"
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 0.1 M 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
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

-

-

-

-

-

-

-

-

-

-

-

-

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

Genetic toxicity in vivo

Description of key information

Negative results were observed in the in vivo micronucleus test with mice.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Tank B705

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, N2-conditions





Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, Wiga, Sulzfeld, Germany
- Weight at study initiation: mean 27 g
- Assigned to test groups randomly: yes, under following basis: computorized
- Fasting period before study: none
- Housing: Makrolon cages, individually
- Diet: standardized pellet feed, Kliba, Klingentalmuehle, Kaiseraugst, Switzerland ad libitum
- Water: ad libitum
- Acclimation period: about 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was dissolved at dose levels of 375, 750 and 1500 mg/kg immediately before administration
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
single application
Post exposure period:
none
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males, 5 females used for each dose level of the test substance;
5 animals in total were used for each positive control substance
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide or vincristine
- Route of administration: orally or ip
- Doses / concentrations: 20 mg/kg (cyclophosphamide) or 0.15 mg/kg (vincristine)
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Pretests for dose selection
In a pretest for the determination of the acute oral deaths were observed down to a dose of 1,750 mg/kg body weight whereas 1,500 mg/kg body weight were survived by all animals. Therefore, a dose of 1,500 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 750 mg/kg and 375 mg/kg body weight were administered as further doses.

The substance to be administered per kg body weight was dissolved in purified water.

- Test group 3 was given 375 mg test substance/kg body weight or 10 mL/kg body weight of a solution with a concentration of 3.75 g/100 mL.
- Test group 4 was given 750 mg test substance/kg body weight or 10 mL/kg body weight of a solution with a concentration of 7.5 g/100 mL.
- Test groups 5 and 6 were given 1,500 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 15.0 g/100 mL.


Preparation of, the bone marrow
The bone marrow was prepared according to the method described by Schmid, W (1976,1977).
- The two femora were prepared from the animals, and all soft tissues were removed.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 mL/femur )
- The suspension was mixed thoroughly with a pipette, centrifuged at 1,500 rpm for 5 minutes, the supernatant was removed except for a few drops, and the precipitate was resuspended.
- 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

Staining of the slides
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in purified water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were embedded in Corbit-Balsam.
Evaluation criteria:
Microscopic evaluation
In general, 1,000 polychromatic erythrocytes (PCE) from each of the male and female animals of every test group are evaluated and investigated for micronuclei (MN). The normochromatic erythrocytes (NCE), which occur, are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei

The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normochromatic erythrocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes. This ratio indicates an influence of the test substance specifically on the bone marrow.
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus, D = cell diameter)
The size of micronuclei may give an indication on the possible mode of action of the test substance, i.e. a clastogenic or a spindle poison effect. Slides were coded before microscopic analysis.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF AG). The number of micronuclei in polychromatic erythrocytes was analyzed. A comparison of the dose group with the vehicle control was carried out using the Wilcoxon test for the hypothesis of equal medians. Here, the relative frequencies of cells with micronuclei of each animal were used. If the results of this test were significant, labels (+ for p ≤ 0.05, ++ for p ≤ 0.01) were printed with the group means in the tables. This test was performed one-sided. This analysis was done separately for each sex and combined for both sexes.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2 females of the 48h 1500 mg/kg bw dosing group died
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF PRETEST FOR DOSE SELECTION
In a pretest for the determination of the acute oral deaths were observed down to a dose of 1,750 mg/kg body weight whereas 1,500 mg/kg body weight were survived by all animals.

RESULTS OF DEFINITIVE STUDY
The single oral administration of purified water in a volume of 10 ml/kg body weight led to 1.7 ‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.4 ‰ after the 48-hour sacrifice interval.

After the single administration of the highest dose of 1,500 mg/kg body weight, 1.2 ‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.3 ‰ after 48 hours.

In the two lower dose groups rates of micronuclei of about 2.1 ‰ (750 mg/kg group) and 1.0 ‰ (375 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.

With 11.4 ‰ the positive control substance cyclophosphamide for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing mainly small micronuclei at a dose level of 20 mg/kg body weight.

With 112 ‰ the positive control vincristine for spindle poison effects also led to a clearly enhanced number of micronuclei containing polychromatic erythrocytes with the expected amount of large micronuclei, i.e . 27.2 ‰.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals. Thus, the test substance Monoethanolamine did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the solvent control value at any of the sacrifice intervals.
A slight inhibition of erythropoiesis induced by the treatment of mice with Monoethanolamine was detected at the highest dose of 1,500 mg/kg body weight both after a sacrifice interval of 24 hours and 48 hours.

CLINICAL EXAMINATIONS
The single oral administration of the vehicle in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms. In the treatment groups the following signs of toxicity were observed:
375 mg/kg: Piloerection after about 30 minutes; no symptoms any longer after 3 hours.
750 mg/kg: Piloerection, squatting posture and irregular respiration after about 30 minutes - 2 hours. Piloerection was observed even the day after treatment.
1,500 mg/kg: Piloerection, squatting posture and irregular respiration after about 30 minutes - 5 hours; the general state of the animals was poor. Piloerection was still observed the day after treatment and 4 animals were found dead.

Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

According to the results of the present study, there are thus no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (375 mg/kg, 750 mg/kg and 1,500 mg/kg) or between the two sacrifice intervals (24 and 48 hours). Thus, under the experimental conditions chosen here, the test substance Monoethanolamine has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

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

Additional information

In vitro

Three reliable Ames tests were available for assessment, performed according to protocols similar to OECD guideline 471 and using test substance concentrations up to 5000 μg/plate (JETOC, 1996), up to 2000 μg/plate (Dean BJ, et al., 1985) and up to 10000 µg/plate (NTP, 1982). In the first study, mutagenicity of MEA was tested in Salmonella typhimurium (TA98, TA 100, TA 102, TA 104, TA 1535, TA 1537 and TA1538) strains and Escherichia coli (WP2uvrA and WP2uvrA/pKM101) strains; in the second study, its mutagenicity was tested in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538, and Escherichia coli (WP2 tyr-) strains. In both tests, MEA was found to be not mutagenic, both in the presence and the absence of metabolic activation. Concentrations >= 2000 µg/plate of the test substance were cytotoxic. The substance was also negative when tested up to 10000 µg/plate with and without metabolic activation in Salmonella typhimurium (TA98, TA 100, TA 1535 and TA 1537) (NTP, 1982).

The substance was also negative in a chromosome aberration test in rat hepatocytes, performed according to the protocol similar to OECD guideline 473, at concentrations 100-400 μg/mL in the absence of metabolic activation (Dean et al., 1985).

Also two reliable in vitro gene mutation studies in mammalian cells were available for assessment. In the first one, performed according to OECD guideline 476 and GLP, MEA was tested in the mouse lymphoma (L5178Y TK+/-) forward mutation assay, with and without metabolic activation, using test substance concentrations up to 610 μg/mL (Linscombe et al., 1999). No cytotoxicity was observed, but the highest concentration corresponded to the limit dose of 10 mM. In the second study, MEA was tested in a HGPRT test with Chinese hamster lung fibroblasts (V79) in the absence of metabolic activation (Chen et al., 1984). In both cases, negative results were observed.

In vivo

In an in vivo micronucleus test, performed according to OECD guideline 474 and GLP, 375, 750 and 1500 mg/kg bw of the test substance was administered orally by gavage to groups of 5 male and female NMRI mice (BASF AG, 1995). Signs of toxicity were observed in the mid and high dose level groups. After single application of the test substance, the animals were sacrificed 24 or 48 h post-dosing and bone marrow slides were prepared. There were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups or between the two sacrifice intervals. Based on the results of the study, it was concluded that MEA has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available information on the test item regarding genetic toxicity are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information,the test substance is not classified for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.