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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

MDEA was evaluated for potential genotoxic activity using the Salmonella/microsome reverse gene mutation test (similar to OECD 471, no GLP), the CHO/HGPRT forward gene mutation test (similar to OECD 476, no GLP), and a sister chromatid exchange test in cultured CHO cells (similar to OECD 479, no GLP). MDEA did not produce any significant or dose-related increases in the frequencies of gene mutations, chromosome aberrations, sister chromatid exchanges or micronuclei. These results indicate that MDEA is not genotoxic in the tests conducted.

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Endpoint:
in vitro gene mutation study in bacteria
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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Method according to Ames et al (1975)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine gene
Species / strain / cell type:
other: Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 liver homogenate, isolated from Aroclor 1254 induced rat


Test concentrations with justification for top dose:
0.1, 0.3, 1, 3, 5, 10 mg/plate
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitrophenylenediamin
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracen
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48-72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: inhibition of growth of the background lawn




Evaluation criteria:
At least twice the solvent control for at least one concentration and there was evidence of a concentration-related increase in the number of revertant colonies.
Statistics:
no data
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9 mix: from 3 mg/plate onwards; +S9-mix: at 10 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9 mix: from 3 mg/plate onwards; +S9-mix: at 10 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9 mix: from 3 mg/plate onwards; +S9-mix: at 10 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9 mix: from 3 mg/plate onwards; +S9-mix: at 10 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-S9 mix: from 3 mg/plate onwards; +S9-mix: at 10 mg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No mutagenic activity was observed in any of the 5 strains mutants over the range of concentrations tested in the absence or the presence of S9 activation, either by evidence of a dose-response relation or a doubling of the mean number of colonies over the vehicle control value.

Table 1: Results:

 Chemical   TA98      TA100     TA1535     TA1537     TA1538   
  Dose (mg/plate)   -S9  +S9  -S9  +S9  -S9 +S9   -S9  +S9  -S9  +S9
 water  100  22±5  14±5  123±24  127±27  10±2  11±2  7±2  6±2  8±2  11±4
 4 -NPD  0.01  683±43  -  -  -  -  -  -  -  794±50  -
 NaN3  0.01  -  -  1087±60  -  917±194  -  -  -  -  -
 9 -AA  0.06  -  -  -  -  -  -  240±33  -  -  -
 2 -AA  0.0025  -  1890±34  -  1757±164  - 123±10  -  299±6  -  1655±109
 MDEA  0.1  26±3 21±6   87±13  124±17 15±12 11±4 11±6 5±2  8±1  13±3
 MDEA  0.3  19±4  18±1  113±20  104±4 13±5 13±3 4±2 5±3  12±3  10±4
 MDEA  1  18±3  19±0  106±13  102±6 10±4 11±2 6±3 5±2  8±1  13±5
 MDEA  3  22 (s)  15±2  80±6  101±16 8 (s) 5±2 4±2 4±2  7 (T)  10±4
 MDEA  5    15±3    100±11   11±1    3±1    16±1
 MDEA  10  toxic    toxic    toxic    toxic    toxic  

4-NPD: 4-nitrophenylenediamine;

NaN3: sodium azide;

9-AA: 9-aminoacridine;

2-AA: 2-aminoanthracene.

 

Toxic: clearing of background lawn or average number of colonies less than half of the solvent control value.

S: sparse growth of background lawn in one or two of the triplicates tested. Counts not included in the calculation of mean and standard deviations not calculated.

T: toxic to one or two of the triplicates tested.

No mutagenic activity was observed in any of the 5 strains mutants over the range of concentrations tested in the absence or the presence of S9 activation, either by evidence of a dose-response relation or a doubling of the mean number of colonies over the vehicle control value.

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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
CHO/HGPRT forward gene mutation test according to O'Neill (1977)
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Type and identity of media: Modified F12 cell-culture medium
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 homogenate from Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
0.1, 0.3, 0.6, 1.0, 1.5, 2.0, 33.0 mg/mL
Vehicle / solvent:
water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 Mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: dimethylnitrosoamine
Remarks:
with S9 mix
Details on test system and experimental conditions:
Approximately 20-24 h prior to mutation test, 5 x10E5 cells were inoculated into two 25-cm2 culture flasks containing F12-D5 medium, and incubated at 37°C in a 5% CO2 atmosphere. On the day of testing, appropriate concentrations of the test agent were added to duplicate cultures of cells, and cultures were treated for 5 h at 37°C. The cells were allowed a period of 18-24 h of recovery from treatment before chemical-induced cytotoxicity was determined. Treatment of cells in the presence of an S9 metabolic activation system was performed identically, with the exception that F12 medium without serum was used. The colony-forming potential of 100-200 treated cells was used as measure of treatment-induced cytotoxicity.

At 2- to 3-day intervals after treatment, cells were subcultured. After a period of at least 7 days to allow the expression of the mutant phenotype, cells were dissociated with 0.075% trypsin, counted and plated. The colony-forming ability determined by the viable fraction of the plated cells was used to correct mutant frequency for the individual treated cultures and to detect variations in the growth ability of the cells.
Statistics:
Statistical analysis of the mutation data for this test been described by Slesinski et al.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
While the positive control substances, ethylmethane sulfonate and dimethylnitrosamine, both produced increased mutant frequencies, MDEA treatment did not produce a reproducible dose-related increase in the number of mutants over the range of concentrations tested either with or without an S9 metabolic activation system. Some small and sporadic numerical in creases in the mutation frequencies were observed, these increases were within the historical control variability for this test system and they were statistically different from the concurrent control.
 
Therefore, MDEA was not considered to be mutagenic in this in vitro gene mutation test.
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
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 -liver cell line RL4 is a epithelial-type cell line derived by Dean et al. following the procedure descibed by Williams et al. 1971. RL4 was derived from a 10 -day old Wistar rat in 1978 (Dean and Hodson-Walker, 1979).
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
Details on test system and experimental conditions:
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 analysed microscopically.

Cytotoxicity assay
Monolayer cultures of rat-liver cells were prepared in multi-well tissue culture trays. The cultures were incubated at 37°C for 24 h to commence active growth before treatment with the test compounds. After 24 h exposure the cell monolayers were stained and the growth inhibition effects noted. The concentrations selected for the chromosome assay were 0.5, 0.25 and 0.125 of the concentration at which growth was inhibited to 50 % (GI50).
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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

MDEA was evaluated for potential genotoxic activity in an in vivo peripheral blood micronucleus test in Swiss-Webster mice (similar to OECD 474, no GLP). MDEA did not produce any significant or dose-related increases in the frequencies of micronuclei.

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Method according to Schmid (1975) and McGregor (1980)
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-8 weeks


Route of administration:
intraperitoneal
Duration of treatment / exposure:
single application
Frequency of treatment:
once
Post exposure period:
30, 48 and 72 hours
Dose / conc.:
175 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Dose / conc.:
560 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg
- 30 h post dosing
Tissues and cell types examined:
peripheral blood erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
based on pretest: about 80, 50 and 25 % of the LD50

DETAILS OF SLIDE PREPARATION:
Slides of blood smears were stained with Gurr's R-66 Giemsa diluted in phosphate buffer, coded and read without knowledge of treatment group to prevent bias.

METHOD OF ANALYSIS:
The polychromatic/normochromatic erythrocyte ratio for approximately 1000 total cells for each animal was calculated to provide an estimate of cytotoxicity.

Evaluation criteria:
A positive result was concluded if at least one statistical significant increase above vehicle control was an indication of a dose-related effect.
Statistics:
Data were compared for significant differences using the Fisher's Exact Test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There were no significant differences in the polychromatic erythrocyte to normochromatic erythrocyte ratios at any dosage. Furthermore, no significant increases in the incidence of micronucleated polychromatic erythrocyte were observed at any sampling time. Therefore, MDEA is not considered to be inducer of micronuclei under the condition of this in vivo test.

Induction of micronucleus in peripheral erythrocytes:

 Sex  Mean PCE/1000 NCE (±SD)            
   Water  TEM  MDEA (mg/kg)      
       175  350  560
30 h postdosing          
  Male  52±24  36±8  55±17  64±11  70±12
  Female  35±10  36±9  41±8  48±9  41±14
48 h postdosing          
  Male  43±13    45±17  43±12  36±4
  Female  31±6    34±18  32±12  38±4
72 h postdosing          
  Male  39±7    38±12  36±14  32±14
  Female  32±9    31±11  39±9  24±7

 Sex  Mean MN-PCE/1000 NCE (±SD)            
   Water  TEM  MDEA (mg/kg)      
       175  350  560
30 h postdosing          
  Male  5.4±3.2  42.0±8 b  4.8±2.4  6.6±2.9 4.8±3.5 
  Female  2.6±1.7  43±15 b  4.2±2.8  3.0±0.7  1.6±1.5
48 h postdosing          
  Male  3.8±3.1   4.2±2.8   5.0±4.4  3.0±2.0
  Female  2.4±1.5    3.8±2.4  2.2±1.3  2.6±2.7
72 h postdosing          
  Male  5.0±2.0    2.6±2.1  7.0±5.6  2.7±1.2
  Female  3.2±1.1   2.2±1.3   3.0±2.8  2.8±1.9

There were no significant differences in the polychromatic erythrocyte to normochromatic erythrocyte ratios at any dosage. Furthermore, no significant increases in the incidence of micronucleated polychromatic erythrocyte were observed at any sampling time. Therefore, MDEA is not considered to be inducer of micronuclei under the condition of this in vivo test.

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

Additional information

In vitro

MDEA was evaluated for mutagenicity at concentrations of 0.1, 0.3, 1, 3, 5, and 10 mg/plate in the Salmonella/microsome reverse gene mutation test (similar to OECD 471, no GLP). The strains tested were S. typhimurium TA98, TA100, TA1535, TA1537 and TA1538. Cytotoxicity was observed from 3 mg/plate onwards in absence of S9 mix, and at 10 mg/plate in presence of S9 mix. No mutagenic activity was observed in any of the 5 strains tested in the absence or the presence of S9 activation, either by evidence of a dose-response relationship or a doubling of the mean number of colonies over the vehicle control value. MDEA was therefore considered not mutagenic under the conditions of this in vitro mutagenicity test (Leung & Ballantyne, 1997).

In another Ames test (also similar the OECD 471), MDEA was tested in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 (with and without metabolic activation) using concentrations of 33, 100, 333, 1000, 2000, 3333 and 10000 µg/plate (Zeiger et al, 1987). No mutagenic effects were observed. Cytotoxicity was observed from 3333 µg/plate.

In a CHO/HGPRT forward gene mutation test (similar to OECD 476, no GLP), MDEA treatment (0.1, 0.3, 0.6, 1.0, 1.5, 2.0, 33.0 mg/mL) did not produce a reproducible dose-related increase in the number of mutants over the range of concentrations tested either with or without an S9 metabolic activation system. Cytotoxicity was not observed. MDEA was not considered to be mutagenic in this in vitro gene mutation test (Leung & Ballantyne, 1997).

In a SCE test (similar to OECD 479, no GLP), statistically significant increases in the mean number of SCEs were observed in one culture at 0.3 mg/mL MDEA, and in duplicate cultures at 0.6 and 1.0 mg/mL MDEA in the absence of S9 metabolic activation. These increases were small, being less than 1.5-fold those of untreated controls, and they were not dose-related. In the presence of S9 metabolic activation, MDEA failed to produce any statistically significant increase in SCEs in comparison to concurrent controls. There were no increases in the numbers of first division cells which suggests that the dose ranges used were appropriate. Therefore, MDEA was not considered to induce reciprocal chromatid exchanges under the condition of this in vitro test (Leung & Ballantyne, 1997).

No chromosome aberration assay is available for MDEA. Therefore, read-across to the structural analogous substance MEA was performed. MEA is 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).

In vivo

In the mouse micronucleus test (similar to OECD 474, no GLP), mice were dosed by a single intraperitoneal injection and observed for mortality for 72 h. Three dose levels of 175, 350, and 560 mg/kg bw (about 25, 50 and 80% of the LD50) were selected for the micronucleus test. There were no major gender differences in mortality responses. The LD50 (combined sexes) for MDEA was about 696 mg/kg bw. Selection of the top test dose was based on the lethality response rather than on bone marrow suppression. There were no significant differences in the polychromatic erythrocyte to normochromatic erythrocyte ratios at any dosages. Furthermore, no significant increases in the incidence of micronucleated polychromatic erythrocyte were observed at any sampling time. Therefore, MDEA was not considered to be an inducer of micronuclei under the conditions of this in vivo test (Leung & Ballantyne, 1997).

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.