2-(methylamino)ethanol, compound with sulphur dioxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-methylaminoethanol

EC Number:

203-710-0

EC Name:

2-methylaminoethanol

Cas Number:

109-83-1

IUPAC Name:

2-(methylamino)ethanol

Details on test material:

- Name of test material (as cited in study report): N-Methylaminoethanol (MMEA). It was received on 26 October 2009

- Substance type: organic
- Physical state: a clear liquid with a molecular weight of 75.11.
- Analytical purity: 99.8%
- Impurities (identity and concentrations): no
- Composition of test material, percentage of components: no
- Isomers composition: no
- Purity test date: 20.10.2009
- Lot/batch No.: T2568
- Expiration date of the lot/batch: 24 months from date of production (19 October 2011)

- Stability under test conditions: stable
- Storage condition of test material: under desiccant at 15-25C in the dark.
- Other: N Methylaminoethanol (MMEA) was soluble in water for injection (purified water) at a concentrations of at least 72.16 mg/mL. The solubility limit in culture medium was at least 7216 µg/mL. A maximum concentration of 751.1 µg/mL was selected for the cytotoxicity Range Finder Experiment, in order that treatments were performed up to 10 mM [5]. Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range-Finder Experiment.

-Molecular formula: C5H13NO2
-Purity test date: Not specified
-Stability under test conditions: Not specified
-Storage condition of test material: Not specified
-Other: Soluble in sterile water (purified water) at concentrations up to at least 72.16 mg/mL

Method

Target gene:

- Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Mouse lymphoma L5178Y cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

L5178Y tk +/- (3.7.2C) mouse lymphoma cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of tk- mutants, checked for spontaneous mutant frequency and that it was mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1: 50 to 600 µg/mL in the absence of S-9 and from 100 to 750 µg/mL in the presence. 7 days after: 500 µg/mL was the highest concentration analysed to determine viability and 6TG (6-thioguanine) resistance in the absence and 600 µg/mL in the presence of S9.
Experiment 2: 50 to 600 µg/mL in the absence of S-9 and from 100 to 700 µg/mL in the presence. 7 days after: 450 µg/mL was the highest concentration analysed to determine viability and 6TG (6-thioguanine) resistance in the absence and 500 µg/mL in the presence of S9.

Range-finder: 23.47, 46.94, 93.89, 187.8, 375.6 and 751.1 µg/mL
Experiment 1: 50, 100, 200, 250, 300, 350, 400, 450, 500 and 600 µg/mL (without S-9), and 100, 200, 300, 350, 400, 450, 500, 550, 600 and 750 µg/mL (with S-9)
Experiment 2: 50, 100, 200, 300, 350, 400, 450, 500, 550 and 600 µg/mL (without S-9), and 100, 200, 300, 400, 450, 500, 550, 575, 600 and 700 µg/mL (with S-9)

Vehicle / solvent:

- Justification for choice of vehicle: Preliminary solubility data indicated that N-Methylaminoethanol (MMEA) was soluble in sterile water for injection (purified water) at concentrations up to at least 72.16 mg/mL. The solubility limit in culture medium was in excess of 7216 µg/mL, as indicated by a lack of precipitation at this concentration 3 hours after test article addition. A maximum concentration of 751.1 µg/mL was selected for the cytotoxicity range-finder experiment, in order that treatments were performed up to 10 mM. Concentrations for the Mutation Experiments were selected based on the results of this cytotoxicity range-finder experiment.
Test article solutions were prepared by formulating N-Methylaminoethanol (MMEA) under subdued light conditions in purified water (with the aid of vortex mixing, as required) immediately prior to assay to give the maximum required treatment solution concentration

Controls

Negative solvent / vehicle controls:

yes

Remarks:

purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

without S9 -mix: 4-nitroquinoline-N-oxide (NQO), final concentration: 0.10 and 0.15 µg/mL; with S9 -mix: benzo(a)pyrene (B[a]P), final concentration: 0.2 and 0.3 µg/mL. Historical control data were included in the report.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in suspension: fluctuation protocol
DURATION
- Preincubation period: 3h
- Exposure duration:
- Expression time (cells in growth medium): 7 days
NUMBER OF CELLS EVALUATED: 20,000
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth: In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S 9, ranging from 23.47 to 751.1 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to give >10% relative survival (RS) was 375.6 µg/mL, which gave 30% and 45% RS in the absence and presence of S 9, respectively.

OTHER EXAMINATIONS:
- Other: survival (relative cloning efficiency, viability and 6TG resistance

Metabolic activation system
Rat liver S-9 fraction from male Sprague Dawley rats induced with Aroclor 1254. Batches were stored at –80ºC prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P- 450-catalysed enzyme activities.
Treatment was carried out both in the absence and presence of S-9, prepared in the following way:
Glucose-6-phosphate (G6P: 180 mg/mL), β-Nicotinamide adenine dinucleotide phosphate (NADP: 25 mg/mL), Potassium chloride (KCl: 150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. Cultures treated in the absence of S-9 received 1 mL KCl (150 mM). The final concentration of the liver homogenate in the test system was 2%.

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
- the mutant frequencies in the negative (vehicle) control cultures fell within the normal range (not more than three times the historical mean value)
- at least one concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).

Evaluation criteria
For valid data, the test substance was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
- the mutant frequency at one or more concentrations was significantly greater than that of the negative control (p ≤ 0.05)
- there was a significant concentration-relationship as indicated by the linear trend analysis (p ≤ 0.05)
- the effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared
with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The highest concentration to give >10% relative survival (RS) was 375.6 µg/mL, which gave 30% and 45% RS in the absence and presence of S-9, respectively

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At the highest concentration tested in the cytotoxicity Range-Finder Experiment (751.1 µg/mL), a marked increase in pH (of ≥1 unit, compared to the concurrent vehicle controls) was observed at 751.1 µg/mL in the absence and presence of S-9. Further pH measurements were therefore made in Experiments 1 and 2.
- Effects of osmolality: At the highest concentration tested in the cytotoxicity Range-Finder Experiment (751.1 g/mL), no marked changes in osmolality, compared to the concurrent vehicle controls, were observed.

RANGE-FINDER EXPERIMENT ON TOXICITY
- In the cytotoxicity range-finder experiment, 6 concentrations were tested in the absence and presence of metabolic activation, ranging from 23.47 to 751.1 μg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to give >10 % relative survival (RS) was 375.6 μg/mL, which gave 30 % and 45 % RS in the absence and presence of metabolic activation, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Mutant frequencies in negative control cultures fell within normal ranges.


Accordingly, for Experiment 1 ten concentrations, ranging from 50 to 600 µg/mL in the absence of S 9 and from 100 to 750 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations tested in the absence of S-9 (600 µg/mL) and in the presence of S 9 (750 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected. The highest concentrations analysed were 500 µg/mL in the absence of S 9 and 600 µg/mL in the presence of S 9, which gave 16% and 7% RS, respectively (see Table 8). In the presence of S-9, no concentration gave 10-20% RS (cultures treated at 550 and 600 µg/mL gave 26% and 7% RS, respectively, therefore both concentrations were analysed).
In Experiment 2 ten concentrations, ranging from 50 to 600 µg/mL in the absence of S 9 and from 100 to 700 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest three concentrations tested in the absence of S-9 (500 to 600 µg/mL) and in the presence of S 9 (575 to 700 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, the lowest concentrations tested in the absence and presence of S-9 (50 and 100 µg/mL, respectively) were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected. The highest concentrations analysed were 450 µg/mL in the absence of S 9 and 500 µg/mL in the presence of S 9, which gave 8% and 17% RS, respectively (see Table 8). In the absence of S-9, no concentration gave 10 20% RS (cultures treated at 400 and 450 µg/mL gave 30% and 8% RS, respectively, therefore both concentrations were analysed).



COMPARISON WITH HISTORICAL CONTROL DATA:
The assay was considered valid if the following criteria were met:
1.the mutant frequencies in the negative (vehicle) control cultures fell within the normal range (not more than three times the historical mean value)
2.at least one concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).


ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The acceptance criteria were met and the study was therefore accepted as valid.

Table 2: Range-finder experiment Treatment (µg/mL) -S-9 %RS +S-9 %RS 0 100 100 23.47 82 86 46.94 81 77 93.89 88 67 187.8 66 84 375.6 30 45 751.1 0 0 %RS    Percentage Relative Survival

 

 Table 3: Experiment 1 (3-hour treatment in the absence and presence of S-9)

Treatment (µg/mL)	-S-9 %RS                 MF§		Treatment (µg/mL)	+S-9 %RS                 M F§
0 50 100 200 250 300 350 400 450 500	100 105 100 80 77 72 54 44 24 16	7.38 6.76     NS 7.31     NS 7.20     NS 9.52     NS   7.62    NS    5.93    NS 7.37    NS 8.74    NS 7.15    NS	0 100 200 300 350 400 450 500 550 600	100 99 93 84 81 82 61 53 26 7	6.97 6.22      NS 8.23     NS 5.65     NS 5.94     NS 6.74     NS 5.52     NS 6.92     NS 6.25     NS 6.64     NS
Linear trend                        7			Linear trend                           NS
NQO 0.1 0.15	68 53	60.53 65.51	B[a]P 2 3	79 57	34.06 53.10

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

% RS             Percent relative survival adjusted by post treatment cell counts

NS                 Not significant

Table 4: Experiment 2 (3-hour treatment in the absence and presence of S-9) Treatment (µg/mL) -S-9 %RS                               MF§ Treatment (µg/mL) +S-9 %RS                           MF§ 0 100 200 300 350 400 450 100 112 100 66 57 30 8        6.05 6.06     NS 3.90     NS 5.64     NS 6.91     NS 5.75     NS 6.13     NS   0 200 300 400 450 500 550 100 107 88 72 68 55 17        6.08 4.58      NS 5.27      NS 4.81      NS 4.45      NS 5.19      NS 6.87      NS         Linear trend                                           NS       Linear trend                                            NS NQO 0.1 0.15   84 73   29.20 23.62 B[a]P 2 3   73 19   42.89 65.55 §                     6TG resistant mutants/10^6viable cells 7 days after treatment % RS             Percent relative survival adjusted by post treatment cell counts NS                 Not significant

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that N Methylaminoethanol (MMEA) did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).