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Genetic toxicity in vitro

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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:
other: OECD guideline study (OECD 471/472) conducted in compliance with GLP regulations
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
and OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay) (May 1983/Revised Draft, March 1996)
Deviations:
yes
Remarks:
Compared to OECD TG 471 (1997), only one positive control substance (2AA) used as the sole indicator of the efficacy of the S9-mix, a second control substance is missing.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His +/-; Trp +/-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
The S9 fractions were prepared from the liver of Aroclor 1254-induced male Sprague-Dawley rats.
Test concentrations with justification for top dose:
0; 20; 100; 500; 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
water control
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With S-9 mix: all strains 2-aminoanthracene; without S-9 mix: strains TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine; TA98: 4-nitro-o-phenylendiamine; TA1537: 9-aminoacridine; E.coli WP2 uvrA: N-ethyl-N'-nitro-N-nitrosoguanidine.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation test

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3


METHOD OF APPLICATION: standard plate test (plate incorporation assay)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY (in both assays)
- Method: Toxicity was detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
- reduction in the titer.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met: A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions at doses > 2500 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A slight decrease in the number of revertants was occasionally observed at doses of 5000 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found.

Standard plate test (mean ± SD):

 Dose (µg/plate)  TA1535     TA100     TA1537     TA98        E. coli WP2 uvrA                                                                        
   -S9  +S9  -S9  +S9  -S9  +S9 -S9  +S9 -S9  +S9                                                                         
 0

21±2

23±1

117±6

117±3

13±1

16±2

29±2

45±4

32±2

47±3

 

 

 

 

 

 

                                                           

20 

20±1

20±1

113±7

109±7

13±1

12±1

26±2

35±1

32±4

42±4

 

 

 

 

 

 

                                                           

 100

17±2

18±3

107±11

107±3

13±3

11±1

24±3

33±1

32±5

39±2

 

 

 

 

 

 

                                                           

 500

19±3

20±1

109±15

115±19

11±2

9±2

23±2

31±3

34±4

34±1

 

 

 

 

 

 

                                                           

 2500

19±2

19±1

104±10

123±9

11±2

8±1

24±3

28±1

31±3

33±3

 

 

 

 

 

 

                                                           

 5000

6±1

17±3

100±8

115±13

12±1

11±1

22±2

29±2

28±2

36±5

 

 

 

 

 

 

                                                           

 2 -AA

-

149±25

-

1243±212

 -

110±5

1127±86

 -

206±23

 

 

 

 

 

 

                                                           

 MNNG

1048±64

-

1116±75

-

-

 -

-

 -

-

-

 

 

 

 

 

 

                                                           

 AAC

-

-

-

-

732±64

-

-

-

-

-

 

 

 

 

 

 

                                                           

 NOPD

 -

-

-

-

-

-

728±62

-

 

 

 

 

 

 

                                                           

 ENNG

 -

-

-

-

-

-

-

-

749±69

-

 

 

 

 

 

 

                                                           

Preincubation-test (mean ± SD):

 Dose (µg/plate)

 TA1535   

 TA100   

 TA1537   

 TA98   

 E. coli WP2

uvrA   

 

 

 

 

 

 

 

                                                       

 

 -S9

 +S9

 -S9

 +S9  -S9  +S9 -S9  +S9  -S9 +S9                                                                       
 0

21±2

20±2

129±9

150±17

12±1

13±2

31±1

46±6

33±2

49±5

 

 

 

 

 

 

 

                                                       

20

20±3

19±1

121±8

166±12

10±1

10±1

31±1

43±2

32±3

45±5

 

 

 

 

 

 

 

                                                       

 100

18±3

18±2

123±11

162±11

10±1

11±1

31±3

42±4

31±4

40±2

 

 

 

 

 

 

 

                                                       

 500

15±2

18±1

117±17

159±13

9±1

8±1

26±3

41±3

26±4

41±3

 

 

 

 

 

 

 

                                                       

 2500

13±1

16±3

145±16

160±13

9±2

7±2

23±3

31±1

26±7

37±2

 

 

 

 

 

 

 

                                                       

 5000

9±2

15±2

126±7

163±15

6±1

8±1

24±2

31±1

22±2

31±1

 

 

 

 

 

 

 

                                                       

2-AA

-

170±29

-

1593±105

-

135±32

-

1380±205

-

233±

13

 

 

 

 

 

 

 

                                                       

 MNNG

2029±73

-

2281±169

-

-

-

-

-

-

-

 

 

 

 

 

 

 

                                                       

 AAC

-

-

-

-

644±58

-

-

-

-

-

 

 

 

 

 

 

 

                                                       

NOPD

-

-

-

-

-

-

871±140

-

-

-

 

 

 

 

 

 

 

                                                       
 ENNG -  -  -

 - -  - - 764±5  -                                                                      

X: reduced background growth

2-AA: 2-aminoanthracene

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

ENNG; N-ethyl-N-nitro-N-nitrosoguanidine

NOPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

Under the conditions tested 2-[2-(dimethylamino)ethoxy]ethanol is non mutagenic in the bacterial AMES-test.

The positive control gave the expected response.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study (OECD 473) and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008 was conducted in compliance with GLP regulations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
In accordance with UK GLP standards with one exception - no analysis was carried out for homogeneity, concentration or stability of the test item formulation as it was formulated within two hours of it being applied to the test system.
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positve controls.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37ºC with 5% CO2 in humidified air.
- Properly maintained: yes
- Sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors was determined to be approximately 16 hours under typical experimental exposure conditions.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 - the final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2% in the Preliminary Toxicity Test and Experiment 1 and 1% in Experiment 2.
Test concentrations with justification for top dose:
Experiment 1:
4-hr exposure with and without S9 (2%) followed by 20-hr culture: 41.6, 83.3, 166.5, 333, 666 and 1332 µg/ml

Experiment 2:
24-hr continuous exposure without S9: 41.6, 83.3, 166.5, 333, 666 and 1332 µg/ml
4-hr exposure with S9 (1%) followed by 20-hr culture: 41.6, 83.3, 166.5, 333, 666 and 1332 µg/ml
Vehicle / solvent:
The test item was dissolved in Eagle'sminimal essential medium with HEPES buffer (MEM). There was no significant change in pH when the test item was dosed into MEM and the osmolality did not increase by more than 50 mOsm.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Eagle'sminimal essential medium with HEPES buffer (MEM)
True negative controls:
no
Positive controls:
yes
Remarks:
Without S9, mitomycin C (MMC) - 0.4 and 0.2 µg/ml in Experiment 1 and 2 respectively. With S9, cyclophosphamide (CP) - 5 µg/ml in both experiments.
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
The test item was formulated within two hours of it being applied to the test system. No analysis was conducted to determine the homogeneity, concentration or stability of the test formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Vehicle and positive controls were used in parallel with the test item.

METHOD OF APPLICATION: In medium - duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components: 8.05 - 9.20 ml MEM, 10% (FBS); 0.1 ml Li-heparin; 0.1 ml phytohaemagglutinin; 0.60 - 0.75 ml heparinised whole blood. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).


DURATION
- Preincubation period: approximately 48-hrs

- Exposure duration: Experiment 1: 4 hours (with and without S9 metabolic activation. Experiment 2: 4 hours (with S9 metabolic activation), 24 hours (without S9 metabolic activation)

- Expression time (cells in growth medium):
-- With S9 Metabolic Activation: 20 hours: the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 ml wash
of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37ºC in 5% CO2 in humidified air.
-- Without S9 Metabolic Activation: 20 hours (Experiment 1 only): the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

- Selection time (if incubation with a selection agent): N/A

- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 ug/ml. Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4ºC to ensure complete fixation.

STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF REPLICATIONS: Duplicate cultures were established for each dose level.

NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index - A preliminary toxicity test was performed on cell cultures using a 4-hour exposure time with and without metabolic activation followed by a 20-hour recovery period, and a continuous exposure of 24 hours without metabolic activation. The dose range of test item used was 0, 5.2, 10.4, 20.8, 41.6, 83.3, 166.5, 333, 666 and 1332 µg/ml. Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.
- Determination of endoreplication: If the chromosomes were arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell was scored as
endoreduplicated (E).
- Other: N/A

Evaluation criteria:
Scoring of Chromosome Damage:
- Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Classification:
- Multiple Aberrations: If many aberrations are present in one metaphase, the exact details may not be scorable. This is particularly the case when chromosome pulverisation occurs. If the number of aberrations was 10 or more then the cell was classified as X.

- Chromosome Number: If the chromosome (centromere) number was 44-48 then it was classified as a diploid cell and scored for aberrations. If less than 44 chromosomes were counted then the cell was ignored under the assumption that some chromosomes may have been lost for technical reasons. If greater than 44 chromosomes were scored and the number is a multiple of the haploid count then the cell was classified as a polyploid cell. If the chromosomes were
arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell was scored as endoreduplicated (E).

Evaluation Criteria:
- A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship was generally required and appropriate statistical tests may have been applied in order to record a positive response.

Historical Control Data:
- The current in-house historical aberration ranges were provided in the study report.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-clastogenic
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-clastogenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
41% mitotic inhibition at 666 ug/ml without S9; 50% mitotic inhibition at 1332 ug/ml with S9
Additional information on results:
Experiment 1: All vehicles (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Experiment 2: Inhibition of mitotic index was observed - 41 % mitotic inhibition was observed at 666 µg/ml in the absence of S9 and 50% mitotic inhibition was achieved at 1332 µg/ml in the presence of S9. The maximum dose level selected for metaphase analysis was therefore, 666 and 1332 µg/ml in the absence and presence of S9, respectively.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups. See results below (Tables 1 - 4).
Remarks on result:
other: Experiment 2

2-[2-(dimethylamino)ethoxy]ethanol (DMEE): CHROMOSOME ABERRATION TEST IN HUMAN LYMPHOCYTES IN VITRO

Table 1. Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9). Treatment Period = 4 hours.

S9 mix Concentration µg/ml   Number and Percentage of Cells Showing Structural Chromosome Aberrations (%) g Cell Growth th Index Number and Percentages of Cells Showing Numerical Abberations (%)
  Observed ctb cte csb cse Others Total Mitotic Index (%) Observed Polyploids Others Total
- Negative Control (MEM)                                   0  A 100 1 0 0 0 0 1 0 4.40 101 1 0 1
B 100 1 0 0 0 0 1 1 3.70 100 0 0 0
Total  200 2 0 0 0 0 2 1   201 1 0 1
% (100) (1.0) 0.0 0.0 0.0 0.0 (1.0) (0.5) (100)       (0.5)
- 333 A 100 0 0 0 0 0 0 0 3.50 100 0 0 0
B 100 0 0 0 0 0 0 0 3.45 100 0 0 0
Total  200 0 0 0 0 0 0 0   200 0 0 0
% (100) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 (86)       0.0
- 666 A 100 1 0 0 0 0 1 1 3.75 100 0 0 0
B 100 1 0 0 0 0 1 1 3.65 100 0 0 0
Total  200 2 0 0 0 0 2 2   200 0 0 0
% (100) (1.0) 0.0 0.0 0.0 0.0 (1.0) (1.0) (91)       0.0
- 1332 A 100 0 1 0 0 0 1 0 3.90 100 0 0 0
B 100 0 0 0 0 0 0 3 4.00 101 1 0 1
Total  200 0 1 0 0 0 1 3   201 1 0 1
% (100) 0.0 (0.5) 0.0 0.0 0.0 (0.5) (1.5) (98)       (0.5)
- Positive Control (MMC)                               0.4 A 50a 11 9 0 0 0 16 1 0.85 50 0 0 0
B 50a 13 2 2 0 0 16 1 2.40 50 0 0 0
Total  100 24 11 2 0 0 32*** 2   100 0 0 0
% (100) (24.0) (11.0) (2.0) 0.0 0.0 (32.0) (2.0) (40)       0.0

MMC = Mitomycin C

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

*** = P < 0.001

MEM = Eagle's minimal essential medium with HEPES buffer (MEM)

Table 2. Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9). Treatment Period = 4 hours.

S9 mix Concentration µg/ml   Number and Percentage of Cells Showing Structural Chromosome Aberrations (%) g Cell Growth th Index Number and Percentages of Cells Showing Numerical Abberations (%)
  Observed ctb cte csb cse Others Total Mitotic Index (%) Observed Polyploids Others Total
+ Negative Control (MEM)                                   0  A 100 0 0 0 0 0 0 0 3.65 100 0 0 0
B 100 1 0 0 0 0 1 0 3.45 100 0 0 0
Total  200 1 0 0 0 0 1 0   200 0 0 0
% (100) (0.5) 0.0 0.0 0.0 0.0 (0.5) 0.0 (100)       0.0
+ 333 A 100 0 0 0 0 0 0 0 4.25 100 0 0 0
B 100 0 0 0 0 0 0 1 5.35 100 0 0 0
Total  200 0 0 0 0 0 0 1   200 0 0 0
% (100) 0.0 0.0 0.0 0.0 0.0 0.0 (0.5) (135)       0.0
+ 666 A 100 2 1 0 1 0 2 0 3.75 100 0 0 0
B 100 0 0 0 0 0 0 0 3.65 100 0 0 0
Total  200 2 1 0 1 0 2 0   200 0 0 0
% (100) (1.0) (0.5) 0.0 (0.5) 0.0 (1.0) 0.0 (104)       0.0
+ 1332 A 100 0 0 0 0 0 0 1 3.80 100 0 0 0
B 100 0 0 0 0 0 0 0 3.55 101 0 0 1
Total  200 0 0 0 0 0 0 1   201 0 0 1
% (100) 0.0 0.0 0.0 0.0 0.0 0.0 (0.5) (104)       0.0
+ Positive Control (CP)                               5 A 50a 14 8 0 0 1 16 3 0.45 50 0 0 0
B 50a 16 9 1 0 0 19 1 0.80 50 0 0 0
Total  100 30 17 1 0 1 35*** 4   100 0 0 0
% (100) (30.0) (17.0) (1.0) 0.0 (1.0) (35.0) (4.0) (18)       0.0

CP = Cyclophosphamide

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

*** = P < 0.001

MEM = Eagle's minimal essential medium with HEPES buffer (MEM)

Table 3. Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation (S9). Treatment Periiod = 24 hours.

S9 mix Concentration µg/ml   Number and Percentage of Cells Showing Structural Chromosome Aberrations (%) g Cell Growth th Index Number and Percentages of Cells Showing Numerical Abberations (%)
  Observed ctb cte csb cse Others Total Mitotic Index (%) Observed Polyploids Others Total
- Negative Control (MEM)                                   0  A 100 0 0 0 0 0 0 0 4.80 100 0 0 0
B 100 0 0 0 0 0 0 0 4.08 100 0 0 0
Total  200 0 0 0 0 0 0 0   200 0 0 0
% (100) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 (100)       0.0
- 166.5 A 100 1 0 0 0 0 1 2 5.05 100 0 0 0
B 100 0 0 0 0 0 0 1 4.10 100 0 0 0
Total  200 1 0 0 0 0 1 3   200 0 0 0
% (100) (0.5) 0.0 0.0 0.0 0.0 (0.5) (1.5) (103)       0.0
- 333 A 100 1 0 1 0 0 2 0 4.20 100 0 0 0
B 100 0 0 0 0 0 0 1 3.80 100 0 0 0
Total  200 1 0 1 0 0 2 1   200 0 0 0
% (100) (0.5) 0.0 (0.5) 0.0 0.0 (1.0) (0.5) (90)       0.0
- 666 A 100 0 1 0 0 0 0 0 2.45 100 0 0 0
B 100 0 0 0 0 0 0 1 2.75 102 2 0 2
Total  200 0 1 0 0 0 0 1   202 2 0 2
% (100) 0.0 (0.5) 0.0 0.0 0.0 0.0 (0.5) (59)       (1.0)
- Positive Control (MMC)                               0.2 A 50a 12 11 0 0 1 21 2 0.55 50 0 0 0
B 50a 9 8 2 0 0 17 4 0.70 50 0 0 0
Total  100 21 19 2 0 1 38*** 6   100 0 0 0
% (100) (21.0) (19.0) (2.0) 0.0 (1.0) (38.0) (6.0) (14)       0.0

MMC = Mitomycin C

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

*** = P < 0.001

MEM = Eagle's minimal essential medium with HEPES buffer (MEM)

Table 4. Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation (S9). Treatment Period = 4 hours.

S9 mix Concentration µg/ml   Number and Percentage of Cells Showing Structural Chromosome Aberrations (%) g Cell Growth th Index Number and Percentages of Cells Showing Numerical Abberations (%)
  Observed ctb cte csb cse Others Total Mitotic Index (%) Observed Polyploids Others Total
+ Negative Control (MEM)                                   0  A 100 0 0 1 0 0 1 2 2.45 100 0 0 0
B 100 0 0 0 0 0 0 0 3.40 100 0 0 0
Total  200 0 0 1 0 0 1 2   200 0 0 0
% (100) (1.0) 0.0 (0.5) 0.0 0.0 (0.5) (1.0) (100)       0.0
+ 333 A 100 3 0 0 0 0 3 0 3.20 100 0 0 0
B 100 0 0 0 0 0 0 0 2.2 100 0 0 0
Total  200 3 0 0 0 0 3 0   200 0 0 0
% (100) (1.5) 0.0 0.0 0.0 0.0 (1.5) 0.0 (92)       0.0
+ 666 A 100 2 1 2 0 0 3 5 2.80 100 0 0 0
B 100 0 0 0 0 0 0 0 3.15 100 0 0 0
Total  200 2 1 2 0 0 3 5   200 0 0 0
% (100) (1.0) (0.5) (1.0) 0.0 0.0 (1.5) (2.5) (102)       0.0
+ 1332 A 100 0 0 0 0 0 0 0 2.40 100 0 0 0
B 100 0 0 0 0 0 0 0 0.55 101 1 0 1
Total  200 0 0 0 0 0 0 0   201 1 0 1
% (100) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 (50)       (0.5)
+ Positive Control (CP)                               5 A 50a 13 0 2 0 0 15 4 0.95 50 0 0 0
B 50a 23 2 0 0 0 24 5 0.60 50 0 0 0
Total  100 36 2 2 0 0 39*** 9   100 0 0 0
% (100) (36.0) (2.0) (2.0) 0.0 0.0 (39.0) (9.0) (26)       0.0

CP = Cyclophosphamide

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

*** = P < 0.001

MEM = Eagle's minimal essential medium with HEPES buffer (MEM)

Conclusions:
The test item, 2-[2-(dimethylamino)ethoxy]ethanol (DMEE), was considered to be non-clastogenic to human lymphocytes in vitro.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-17 to 2012-09-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was well conducted and described in accordance with GLP standards and compatible with an OECD guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
other: Japanese METI/MHLW guidelines for testing new chemical substances
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK +/- locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Metabolic activation:
with and without
Metabolic activation system:
S9 = 20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1. In Experiment 2, 10% S9-mix (i.e. 1% final concentration of S9), was added.
Test concentrations with justification for top dose:
Preliminary toxicity test: 0; 5.20; 10.41; 20.81; 41.63; 83.25; 166.5; 333; 666; 1332 µg/ml with and without metabolic activation (4-hour exposure) and without metabolic activation (24-hour exposure)
Experiment 1: 0; 20.81; 41.63; 83.25; 166.5; 333; 666; 999; 1332 µg/ml with and without metabolic activation (4-hour exposure)
Experiment 2: 0; 25; 50; 100; 200; 300; 400; 500; 600 µg/ml without metabolic activation (24-hour exposure)
Experiment 2: 0; 20.81; 41.63; 83.25; 166.5; 333; 666; 999; 1332 µg/ml with metabolic activation (4-hour exposure)

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: R0 medium (see section "any other information on materials and methods incl. tables" cell cultures)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984)

DURATION
- Exposure duration: Experiment 1: 4 hours with and without metabolic activation; Experiment 2: 4 hours with metabolic activation and 24 hours without metabolic activation
- Incubation period: Experiment 1: The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood; Experiment 2: The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.
- Expression time (cells in growth medium): 48 hours (The cultures were incubated at 37 °C with 5% CO 2 in air and subcultured every 24 hours
for the expression period of two days by counting and diluting to 2 x 105 cells/ml)


SELECTION AGENT (mutation assays): 4 µg/ml 5-trifluorothymidine (TFT)
STAIN (for cytogenetic assays): MTT (to assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.)


NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: On Day 2 of the experiment, the cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.)

OTHER EXAMINATIONS:
- Measurement of survival, viability and mutant frequency
- Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).
- Plate scoring: the number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick.

OTHER:
Evaluation criteria:
For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.
Statistics:
Test for linear trend
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no (performed in a separate test: Harlan Laboratories Ltd. Project No. 41202878;Chromosome Aberration Test)
- Effects of osmolality: no (performed in a separate test: Harlan Laboratories Ltd. Project No. 41202878;Chromosome Aberration Test)
- Evaporation from medium:
- Water solubility: the solubility test data from which was considered acceptable for use in this study were determined in a separate test (Harlan Laboratories Ltd. Project No. 41202878;Chromosome Aberration Test)
- Precipitation: no
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: In the 4-hour exposure groups in both the absence and presence of metabolic activation, there was evidence of moderate dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. However, a much greater dose-related reduction in %RSG was observed in the 24-hour exposure group. Precipitate of the test item was not observed at any of the dose levels. Based on the %RSG values observed, the maximum dose level in the subsequent Mutagenicity Test was the 10 mM limit dose of 1332 µg/ml for the 4-hour exposure groups and limited by test item-induced toxicity for the 24-hour exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Table 1: Summary of Results Experiment 1

 Treatment [µg/ml]        4 -hours-S-9    Treatment [µg/ml]          4 -hours+S-9
   %RSG  RTG  MF§      %RSG  RTG  MF§
 0  100  1.00 127.63   0 100   1.00  148.06
 20.81 Ø  95      20.81  Ø 91     
 41.63 Ø  94      41.63  Ø  87    
 83.25  96  0.90  133.06  83.25  86  0.91  146.46
 166.5  94 0.89   130.89  166.5  84  0.83  161.56
 333  94  0.97  98.27  333  84  0.89  129.41
 666  82  0.85  90.59  666  70  0.73  114.88
 999  51  0.61  120.71  999  53  0.53  157.57
 1332  33  0.32  137.59  1332  46  0.41  178.80
       Linear trend NS  Linear trend        NS 
EMS        CP       
 400  71  0.52  1181.32  2  50  0.27  1454.01

 

Table 2: Summary of Results Experiment 2

 Treatment [µg/ml]        24 -hours-S-9    Treatment [µg/ml]          4 -hours+S-9
   %RSG  RTG  MF§      %RSG  RTG  MF§
 0  100  1.00 167.68   0 100   1.00  193.81
 25  91  1.00 175.32   20.81  Ø 97     
 50  89  0.96 170.21   41.63  Ø  94    
 100  80  0.81  184.06  83.25  105  1.10  180.21
 200  82 1.08  135.02  166.5  102  1.09  178.20
 300  43  0.54  185.55  333  92  1.03  181.89
 400  11  0.13  222.46  666  76  0.77  199.15
 500 Ø  2      999  50  0.45  211.67
 600 Ø  1      1332  58  0.72  147.16
       Linear trend NS  Linear trend        NS 
EMS        CP       
 150  25  0.22  1356.50  2  46  0.29  1367.85

Ø : no definition found in the study for this symbol

%RSG= Relative Suspension Growth

RTG = Relative Total Growth

MF§ = 5-TFT resistant mutants/106 viable cells 2 days after treatment

NS = Not significant

Conclusions:
Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.
Executive summary:

Introduction:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Methods:

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation. The dose range of test item was selected following the results of a preliminary toxicity test and was 20.81 to 1332 µg/ml in both the absence and presence of metabolic activation for Experiment 1. In Experiment 2 the dose range was 25 to 600 µg/ml in the absence of metabolic activation, and 20.81 to 1332 µg/ml in the presence of metabolic activation.

Results:

The maximum dose level used in the Mutagenicity Test was the 10 mM limit dose of 1332 µg/ml for the 4-hour exposure groups, and limited by test item-induced toxicity in the 24-hour exposure group. Precipitate of the test item was not observed at any of the dose levels in the study. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Negative findings for DMEE were reported in three reliable and well-conducted in vitro studies (i.e., a Salmonella typhimurium/Escherichia coli Reverse Mutation Assay, a Chromosome Aberration Test in human lymphocytes, and an L5178Y Mouse Lymphoma Assay):

 

1. In a Klimisch 1 study conducted under GLP and according to OECD Guideline 471 (Bacterial Reverse Mutation Assay), DMEE did not produce a dose-dependent mutagenic response in any of the S. typhimurium orE. colistrains tested. Under the conditions of this assay, DMEE was not mutagenic at 20, 100, 500, 2500 or 5000 µL/plate dose levels.

 

2. In a Klimisch 1 study conducted under GLP and according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test), doses of 41.6, 83.3, 166.5, 333, 666, or 1332 ug/mL DMEE did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation. DMEE was reported to be non-clastogenic under the conditions of this study.

 

3. In a Klimisch 1 rated Mouse Lymphoma Assay conducted under GLP and according to OECD 476 (In vitro Mammalian Cell Gene Mutation Test), L5178Y mouse lymphoma cells were exposed to doses of DMEE ranging from 20 to 1332 ug/mL. DMEE did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation.

Justification for classification or non-classification

Classification for genetic toxicity is not warranted according to the criteria of EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.