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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
- genetic toxicity in vitro: - positive, OECD TG 471; Support / Sokolowski / 2011/ bacterial reverse mutation test - positive, OECD TG 473; Support / Buskens / 2010 / mammalian chromosome aberration test (peripheral human lymphocytes) - genetic toxicity in vivo: - negative, OECD TG 474; Key / Buskens / 2010 / chromosome aberration micronucleus assay in mouse bone marrow cells - negative, OECD TG 486; Key / Brown / 2012 / liver unscheduled DNA synthesis (UDS) assay / rat
Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-05-10 to 2012-06-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according guideline (OECD 486) under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes (incl. QA statement)
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
other: Wistar HanTM (RCCHanTM:WIST)
Sex:
male
Details on test animals or test system and environmental conditions:
Sufficient Wistar HanTM (RCCHanTM: WIST) rats were obtained from Harlan Laboratories UK Ltd., Oxon, UK. At the start of the main test the male rats weighed 176 to 218 g and were approximately seven to twelve weeks old. After a minimum acclimatisation period of at least five days the animals were selected at random and given a number unique within the study. The animals were housed in groups of up to four in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Harlan Teklad 2014 Rodent Pelleted Diet from Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study. The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
Route of administration:
other: oral
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not reported, but corn oil is a standard vehicle in toxicologic studies
- Concentration of test material in vehicle: 1000 or 2000 mg/kg (Experiment 1 and 2)
- Amount of vehicle (if gavage or dermal): 10 mL/kg (dose volume)
- Lot/batch no. (if required): Sigma MKBF8603V
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- dimethyl itaconate (DMI) suspended in corn oil
-For the purpose of this study the test item was freshly prepared as required as a solution at the appropriate concentration in corn oil (Sigma MKBF8603V).
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration.

Duration of treatment / exposure:
Experiment 1:
Two groups of four rats were dosed orally with test item at 2000 and 1000 mg/kg. In addition, two further groups of rats were included in the experiment, one group (four rats) was dosed orally with the vehicle alone (Corn Oil) and the second group (four rats) was dosed orally with 2AAF as a positive control. The perfusion of livers from all animals commenced approximately 16 hours after dosing.

Experiment 2:
The dose groups and group sizes were as for Experiment 1 except that the positive control was NDHC and perfusion of the livers commenced approximately 4 hours after dosing.
Frequency of treatment:
single treatment (oral)
Post exposure period:
not applicable, animals sacrificed for derivation of liver cells 4 or 16 h post dosing
Remarks:
Doses / Concentrations:
1000 or 2000 mg/kg in the main UDS test
Basis:
other: oral treatment
Remarks:
Doses / Concentrations:
2000 mg/kg in the confirmatory range-finding toxicity test
Basis:
other: oral treatment
No. of animals per sex per dose:
- 2 males per dose in the range finding toxicity test
- 4 males per dose in the main UDS test
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive Controls:
2-Acetylaminofluorene (2AAF) was used for Experiment 1; freshly prepared as a suspension at the appropriate concentration in arachis oil.
N,N'-Dimethylhydrazine dihydrochloride (NDHC) was used for Experiment 2; freshly prepared as a suspension in sterile distilled water.
- Justification for choice of positive control(s): not given
- Route of administration: oral
- Doses / concentrations:
dose level 50 mg/kg and dose volume 10 mL/kg (2AAF)
dose level 40 mg/kg and dose volume 10 mL/kg (NDHC)
Tissues and cell types examined:
Hepatocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A confirmatory range-finding toxicity test was performed to determine a suitable dose level for the UDS Assay. For ethical reasons the range finding test was carried out using two male animals to confirm that the maximum recommended dose of 2000 mg/kg, determined in the Harlan CCR study 1470900 via the oral route did not cause any significant toxicity or death in the sub-strain of Wistar Rats used in the UK. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

Isolation of hepatocytes:
The isolation of the hepatocytes was carried out using a two stage in situ method, and was performed immediately after termination of the animal. The post-aortic vena cava was ligatured, and then cannulae were introduced into the hepatic portal vein and superior vena cava. This allowed a flow of liquid through the liver and out through the heart. Livers were first perfused with a buffered medium containing chelating agents to remove metal ions; this was followed by a digest medium containing collagenase and calcium causing the liver to disassociate into a single cell population.
The liver was removed from the body and the capsule opened and the liver cells were suspended in attachment medium. The suspended cells were then passed through nylon gauze to remove large particles and debris. The cells were centrifuged and washed three times using a buffered medium and finally suspended in attachment medium at 1 x 10E5 viable cells/ml. Cell viability and cell concentrations were estimated using a haemocytometer slide and Trypan blue exclusion dye. They were then seeded onto cover slips in 35 mm six-well plates at 3 ml/well (six cover slips per animal). The plates were incubated at 37 °C in 5 % CO2:95 % air in a humidified incubator for 1.5 to 2 hours to allow cell attachment.

Radiolabeling of cells:
After cell attachment, the medium was aspirated using aseptic techniques. The hepatocytes were washed with serum-free medium which was replaced with 2 ml of serum-free medium containing 10 μCi/ml (370 kBq/ml) of [methyl-3H] thymidine. The cultures were then incubated for a further 3 hours at 37°C in 5 % CO2:95 % air. Cultures were washed three times with serum-free medium containing 0.25 mM unlabelled thymidine solution. This was a ‘cold chase’ procedure to remove excess radiolabel from the cultures. The cells were incubated overnight with 2 ml of ‘cold chase’ medium.

Autoradiography:
The medium was aspirated and the cells washed with phosphate buffer solution. The cells were fixed in freshly prepared 1:3 acetic acid:methanol, three changes of fixative were used. Finally cells were washed with distilled water and the cover slips allowed to air dry and then mounted onto the ends of glass slides, cell side up, and left to dry overnight in a dust free environment. The cover slips were coated with autoradiographic emulsion and incubated at approximately 4 °C for 7 to 14 days in a sealed light proof container. The emulsion is sensitive to the emission of radioactive particles, causing a deposit of silver, visualised as black grains when development is complete. Following the exposure period the slides were processed using photographic developer and fixative, and then the cells were stained using haematoxylin/eosin. When the cells were dry a cover slip was applied using a mounting medium. The slides were then assessed for obvious signs of toxicity, reduced numbers of cells and poor labelling. Two good quality slides from each animal were selected and coded using a code supplied by the ‘Grain’ computer programme.
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION:

METHOD OF ANALYSIS:

OTHER:
Evaluation criteria:
The coded slides were scored using an automated image analysis system linked to a computer programme (Grain) which followed the UKEMS guidelines for statistical analysis. The method used to score the slides was an area counting method which complies with the UKEMS guidelines. Two slides for each animal were scored with a maximum of 50 cells per slide giving an accumulative total of 100 cells per animal, to meet the OECD Guidelines. The number of silver grains within the nucleus were first observed and recorded as the Nuclear Grain Count (N). Three cytoplasmic areas (each equal to the nuclear area) were also scored to give the Mean Cytoplasmic Grain Count (C). A Net Nuclear Grain Count (N-C) was then calculated. Cells in ‘S’-phase were not scored for grain counts, these were easily recognisable due to the dense ‘graining’ appearance of the nucleus. Mean values (N), (C), (N-C) and percentage cells in repair (% R) were calculated. Values of (N-C) are typically in the range of -6 to -2 for vehicle controls, although variations in experimental conditions can give values outside of this range. The UKEMS guidelines suggest that in positive responses at least 20% of all cells assessed should be in repair, ie undergoing unscheduled DNA synthesis, having a (N-C) value of +5 or greater.
Statistics:
no statistics reported
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2000 mg/kg one animal died in Experiment 1
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
1. Mortality data

Confirmatory Range Finding Toxicity Test:
The clinical sign hunched posture was observed in both animals dosed with the test item at 2000 mg/kg.
With no excessive toxicity or severe clinical signs, the maximum recommended dose (MRD) of the test item, 2000 mg/kg, was selected for use in the main test, with 1000 mg/kg as the lower dose level.

Main USD Test:
There were no premature deaths in any of the dose groups. With the exception of one animal in Experiment 1 (animal 7), the clinical sign hunched posture was observed in the 2000 mg/kg dose groups in Experiments 1 and 2 after approximately 16 and 4 hours exposure, respectively. A summary of results for both experiments is presented under other information on results, where group mean net nuclear grain count values and percentage cells in repair data are presented.

2. Main USD Test (Experiment 1):

A summary of the group mean net nuclear grain counts and percentage cells in repair data as well as the results obtained from the slides for the 16-hour harvest time point are presented under other information on results. As can be seen from the data, all four animals from the vehicle control group were successfully processed to give slides suitable for scoring; four out of four animals from the 1000 mg/kg test item group and four out of four animals from the 2000 mg/kg test item dose group were processed to provide slides suitable for scoring. All four positive control group animals were processed to provide slides suitable for scoring. It should be noted that whilst the cell viability values were low (see under other information on results) the group mean was above 50 % for the vehicle control group. It is possible that the digest media was very potent causing marked damage to the cell membranes during the perfusion stage. However, it was considered not to affect the purpose or integrity of the study. It should be noted that for the animals in the vehicle and test item dose groups the mean net nuclear grain count values are all less than zero, whilst for the animals in the positive control group the values are markedly greater than zero, indicating increased repair was occurring. The test item did not induce any marked increases in the incidence of cells in repair at either dose level. The net nuclear grain counts (N-C) were slightly outside the typical range of -2 to -6 in the vehicle and test item dose groups, however these values were considered acceptable and due to experimental variation. It should be noted that in the test item dose groups, the mean net grain count of cells in repair increases with dose level when compared to the vehicle control group. With no marked increase in the number of cells in repair it was considered this effect was due to toxicity rather than a true genotoxic response. This effect coupled with slight reductions in cell viability indicates a toxic response to the test item. This response observed is considered to be a cytotoxic mechanism rather than a true genotoxic mechanism. The positive control 2-Acetylaminofluorene (2AAF) induced a marked increase in the percentage of cells in repair as expected with the elevated N-C values demonstrating that the test method was operating satisfactorily.

2. Main USD Test (Experiment 2):

A summary of the group mean net nuclear grain counts and percentage cells in repair data as well as the results obtained from the slides for the 4-hour harvest time point are presented under other information on results. As can be seen from the data, all four animals from the vehicle control group were successfully processed to give slides suitable for scoring; four out of four animals from the 1000 mg/kg test item dose group and three out of four animals from the 2000 mg/kg test item dose group were processed to provide slides suitable for scoring. All four positive control group animals were processed to provide suitable slides for scoring. It was considered that the loss of one animal (perfusion failure) from the 2000 mg/kg test item group did not affect the integrity of the study because the minimum number of animals specified in the test guideline was achieved in the vehicle control and the test item dose groups. As was seen in Experiment 1 it should be noted that whilst the cell viability values were low (see under other information on results) the group mean was above 50 % for the vehicle control group. It is possible that the digest media was very potent causing marked damage to the cell membranes during the perfusion stage. However, it was considered not to affect the purpose or integrity of the study. The net nuclear grain count data from this experiment complemented the results from Experiment 1 and indicated marked repair was only occurring in the positive control animals. The test item did not induce any marked increases in the incidence of cells in repair at either dose level. The net nuclear grain counts (N-C) were marginally outside the typical range of -2 to -6 in the vehicle and test item dose groups, however these values were considered acceptable and due to experimental variation. As in Experiment 1, it should be noted that in the test item dose groups, the net grain count of cells in repair increases when compared to the vehicle control group, indicating a toxic response/repair mechanism. This effect coupled with very slight reductions in cell viability indicates a toxic response to the test item. This response observed is considered to be a cytotoxic mechanism rather than a true genotoxic mechanism. It should also be noted that the response in Experiment 2 was less pronounced than in Experiment 1, due to the reduced exposure time of 4 hours. The positive control 2 N,N’-dimethylhydrazine dihydrochloride (NDHC) induced a marked increase in the percentage of cells in repair and elevated N-C values, demonstrating that the test method was operating satisfactorily.




Group Mean Net Nuclear Grain Count Values and % Cells in Repair:

Experiment 1:

Dose Level

(mg/kg)

Net Nuclear Grain Count

(N-C)

Net Nuclear Grain Count of Cells in Repair

Percentage of Cellsin Repair (N-C ≥ 5)

 

 

Mean

SD

Mean

SD

Mean

SD

0

Vehicle

-1.3

1.1

6.1

1.0

2.5

1.7

1000

DMI

-1.4

0.4

8.8

4.0

2.5

1.3

2000

DMI

-1.6

0.7

11.3

3.8

1.3

1.9

50

2AAF

12.5

4.9

26.1

4.8

48.8

14.4

Experiment 2:

Dose Level

(mg/kg)

Net Nuclear Grain Count

(N-C)

Net Nuclear Grain Count of Cells in Repair

Percentage of Cellsin Repair (N-C ≥ 5)

 

 

Mean

SD

Mean

SD

Mean

SD

0

Vehicle

-1.1

0.6

7.1

0.8

2.5

2.4

1000

DMI

-1.8

1.9

9.0

1.7

1.8

1.3

2000

DMI

-1.4

0.6

9.0

1.7

4.7

2.9

40

NDHC

31.1

21.4

40.0

16.2

70.0

25.4

Experiment 1: Individual Animal Mean Net Nuclear Grain Count Values and Percentage Cells in Repair at 16 Hours

Dose

(mg/kg)

Animal

number

Net Nuclear Grain

Count

(N-C)

% cells

in

repair

Net Grain

Count of cells

in repair

No. of

cells

scored

 

 

mean

SD

( N-C³5 )

mean

SD

 

0

VEHICLE

2

4

11

14

-1.1

-0.5

-2.9

-0.8

0.0

0.1

1.7

0.2

2.0

1.0

5.0

2.0

5.8

5.0

6.0

7.3

-

-

1.5

0.9

100

100

100

100

1000

TEST ITEM

8

9

10

15

-1.0

-1.5

-1.3

-1.9

0.4

1.7

0.4

1.7

4.0

2.0

3.0

1.0

7.8

6.8

14.7

6.0

2.5

0.7

-

-

100

100

100

100

2000

TEST ITEM

5

7

12

16

-1.6

-2.4

-0.7

-1.5

1.3

1.1

0.5

0.6

1.0

0.0

4.0

0.0

14.0

-

8.6

-

-

-

3.6

-

100

100

100

100

50

2AAF

1

3

6

13

8.1

15.3

8.6

17.9

1.2

1.5

3.7

9.3

40.0

49.0

37.0

69.0

20.7

32.4

26.5

24.7

3.8

9.8

14.3

5.7

100

100

100

100

Experiment 2: Individual Animal Mean Net Nuclear Grain Count Values and Percentage Cells in Repair at 4 Hours

Dose

(mg/kg)

Animal

number

Net Nuclear

Grain

count

(N-C)

% of cells

in

repair

Net Grain

Count of cells

in repair

No. of

cells

scored

 

 

mean

SD

( N-C³5 )

mean

SD

 

0

Vehicle

17

20

26

31

-1.1

-0.4

-1.1

-1.9

0.2

1.2

0.6

1.4

2.0

1.0

6.0

1.0

7.7

6.0

7.1

7.7

-

-

1.7

-

100

100

100

100

1000

TEST ITEM

22

24

27

32

-0.7

-4.5

-0.6

-1.4

1.0

0.2

0.7

0.6

3.0

2.0

2.0

0.0

7.4

8.8

10.8

-

1.3

-

-

-

100

100

100

100

2000

TEST ITEM

19

23

29

30

0.0

-0.2

-1.1

ND

0.3

1.1

0.6

ND

3.0

8.0

3.0

ND

9.4

10.5

7.1

ND

6.0

2.9

2.7

ND

100

100

100

ND

40

NDHC

18

21

25

28

12.1

13.4

52.8

46.2

1.9

7.0

14.9

25.1

50.0

47.0

98.0

85.0

24.5

27.5

54.0

53.8

6.5

3.6

16.8

26.9

100

100

100

100

Experiment 1: Individual Animal Bodyweight and Group Mean Cell Suspension Percentage Viability Values – 16-Hour Treatment

Dose mg/kg

Animal number

Body Weight (g)

% Viable Cells

Vehicle

0

2

186

50

4

178

51

11

203

59

14

176

60

Mean

186

55

SD

12.3

5.2

TEST ITEM 1000

8

218

51

9

188

48

10

198

50

15

195

58

Mean

200

52

SD

12.9

4.3

TEST ITEM 2000

5

199

44

7

192

53

12

192

34

16

191

50

Mean

194

45

SD

3.7

8.4

2AAF

50

1

209

21

3

212

61

6

191

42

13

197

48

Mean

202

43

SD

9.9

16.7

Experiment 2: Individual Animal Bodyweight and Group Mean Cell Suspension Percentage Viability Values – 4-Hour Treatment

Dose mg/kg

Animal number

Body

Weight (g)

% Viable Cells

Vehicle        0

17

201

56

20

202

56

26

194

63

31

197

55

Mean

199

58

SD

3.7

3.7

TEST ITEM 1000

22

190

37

24

194

61

27

208

48

32

196

47

Mean

197

48

SD

7.7

9.8

TEST ITEM

2000

19

201

33

23

205

53

29

199

40

Mean

202

42

SD

3.1

10.1

NDHC

40

18

182

35

21

213

59

25

190

63

28

200

55

Mean

196

53

SD

13.4

12.4

Conclusions:
Interpretation of results (migrated information): negative
The test item did not induce any marked or toxicologically significant increases in the incidence of cells undergoing unscheduled DNA synthesis in isolated rat hepatocytes following in vivo exposure for 4 or 16 hours. Therefore the test item was considered to be non-genotoxic under the conditions of this study.
Executive summary:

The measurement of chemically induced DNA repair as unscheduled DNA synthesis (UDS) is currently accepted as an indicator of genotoxicity, and hence potential carcinogenicity. The measurement of UDS is carried out following the in vivo treatment of rats. Measurement of the excision-repair process is made by assessing the incorporation of tritiated thymidine. This occurs after DNA damage which may happen when there is, for example, chemical damage to DNA at the site of covalent binding. The damaged DNA is enzymically excised and replaced with complementary undamaged nucleotides. If the thymidine supplied is tritiated and available to the cells in the medium, the repaired DNA will become radioactive and this can be measured by either radiography or by scintillation spectrometry. The autoradiographic method allows for the measurement of UDS with the simultaneous measurement of cells undergoing DNA replication and may be considered more suitable for the measurement of UDS than using scintillation spectrometry. The study reported here assesses the genotoxic potential of the test item in the in vivo rat hepatocyte UDS assay using autoradiographic assessment. A study was performed to assess the potential of the test item to induce DNA repair in isolated rat hepatocytes following in vivo administration. The method used has been designed to be compatible with the OECD TG 486 and follows the recommendations of the UKEMS Sub-Committee on Guidelines for Mutagenicity Testing: Report, Part II revised (Supplementary Mutagenicity Tests: UKEMS recommended procedures, 1993). A range-finding test was performed to confirm suitable dose levels of the test item. The main test was performed using only male rats, as a previous recent study indicated that there was no difference in toxicity between sexes. The UDS assay was conducted using the oral route of administration using only male animals and with the test item at the maximum dose level of 2000 mg/kg, with 1000 mg/kg as the lower dose level. The study was performed in two parts, in Experiment 1 perfusion of the livers commenced approximately 16 hours after dosing and in Experiment 2 perfusion was performed approximately 4 hours after dosing. Following perfusion the liver hepatocytes were processed to give stained slides which were then scored using a microscope and an automated image analysis system. The method used for scoring the hepatocytes was an area counting method which is compatible with the UKEMS guidelines and OECD test method. Further groups of rats were given a single oral dose of corn oil, or dosed with 2-acetylaminofluorene (2AAF) at 16 hours or N,N’-dimethylhydrazine dihydrochloride (NDHC) at 4 hours to serve as vehicle and positive controls respectively. There were no marked increases in the incidence of unscheduled DNA synthesis in animals dosed with the test item at either time point. The positive controls produced marked increases in net nuclear grain counts and in the incidence of cells in repair, and the vehicle control groups gave acceptable values for net nuclear grain counts. The test item was considered to be non-genotoxic under the conditions of this study.

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

Additional information

Additional information from genetic toxicity in vivo:

In vitro

Two in vitro tests on genetic toxicity are available:

- bacterial reverse mutation test

In bacterial reverse mutation test (Sokolowski, 2011) performed according to OECD TG 471, five amino-acid requiring strains (TA1537, TA98, TA1535, TA100 of Salmonella typhimurium and E. coli WP2 urvA) were treated with the test item at concentrations up to 5000 µg/plate, both in the absence and in the presence of metabolic activation S9 mix. Substantial and dose dependent increases in revertant colony numbers were observed following treatment with test item in strain WP2 uvrA in the presence and absence of S9 mix. The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at 2500 and 5000 µg/plate. This study was therefore considered to have provided evidence of mutagenic activity in this assay system.

- mammalian chromosome aberration test (peripheral human lymphocytes)

Buskens (2010) performed fully reliable in vitro mammalian chromosome aberration test (peripheral human lymphocytes) according to OECD TG 473 and GLP. This report describes the effect of the test item on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and 11-naphthoflavone induced rat liver S9 mix). The test substance was dissolved in dimethyl sulfoxide. In the cytogenetic assay, Dimethyl itaconate (DMI) was tested up to 350 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence of S9-fraction. In the presence of 1.8% (v/v) S9-fraction Dimethyl itaconate (DMI) was tested up to 1100 µg/ml for a 3 h exposure time with a 24 h fixation time. Appropriate toxicity was reached at these dose levels. In the absence and presence of S9-mix, Dimethyl itaconate (DMI) induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations, both when gaps were included and excluded. No effects of Dimethyl itaconate (DMI) on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. It was noted that the test item increased the number of polyploid cells in the presence of S9 mix at the highest tested concentration of 1100 µg/ml. This may indicate that the test item has the potential to disturb mitotic processes and cell cycle progression. Based on these results it is concluded that the test item is clastogenic in human lymphocytes under the experimental conditions. Dimethyl itaconate (DMI) may have the potential to disturb mitotic processes and cell cycle progression in the presence of S9 mix.

In vivo

Two in vivo tests on genetic toxicity are available

- mammalian chromosome aberration test

Buskens (2010) performed fully reliable in vivo chromosome aberration - micronucleus assay in mouse bone marrow cells study conducted according to OECD TG 474 and GLP. The test item was suspended in corn oil. Males and females were shown to be equally susceptible in a pre-study used to derive the dose levels for the main test. Male NMRI mice were dosed by intraperitoneal injection with vehicle or with 1500, 750 and 375 mg of the test item per kg body weight. Two animals in the high dose group and 1 animal in the low dose groups showed clinical signs like: lethargy, closed eyes, hunched posture, rough coat and ataxia. Bone marrow of the groups treated with the test item was sampled 24 or 48 (highest dose only) hours after dosing. A statistically significant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed at the intermediate dose level of 750 mg/kg bw. The number of micronuclei in the vehicle control animals was low (0 - 2) compared to the historical control data range (0 - 5). Moreover, the mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes (3.0 ± 1.0) and the individual number of micronucleated polychromatic erythrocytes per animal was well within the laboratory historical control data range in the group dosed with the 750 mg/kg bw. Therefore the statistically significant increase in the mean frequency of micronucleated polychromatic erythrocytes was considered not biologically relevant. No increase in the mean frequency of micronucleated polychromatic erythrocytes, compared to the vehicle treated animals, was observed in the polychromatic erythrocytes of the bone marrow of all other dose groups treated with the test item. The mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes of each group was within the laboratory historical control data range.

The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this test substance on erythropoiesis. It is concluded that Dimethyl itaconate (DMI) is not clastogenic or aneugenic in the micronucleus test under the experimental conditions described in this report.

The negative result in the in vivo cytogenetic assay overrules the positive result from the respective in vitro test. Therefore, it can be concluded that the test item is not clastogenic or aneugenic.

- liver unscheduled DNA synthesis (UDS) assay

The UDS assay was conducted using the oral route of administration using only male animals and with the test item at the maximum dose level of 2000 mg/kg, with 1000 mg/kg as the lower dose level. The study was performed in two parts, in Experiment 1 perfusion of the livers commenced approximately 16 hours after dosing and in Experiment 2 perfusion was performed approximately 4 hours after dosing. Following perfusion the liver hepatocytes were processed to give stained slides which were then scored using a microscope and an automated image analysis system. The method used for scoring the hepatocytes was an area counting method which is compatible with the UKEMS guidelines and OECD test method. Further groups of rats were given a single oral dose of corn oil, or dosed with 2-acetylaminofluorene (2AAF) at 16 hours or N,N’-dimethylhydrazine dihydrochloride (NDHC) at 4 hours to serve as vehicle and positive controls respectively. There were no marked increases in the incidence of unscheduled DNA synthesis in animals dosed with the test item at either time point. The positive controls produced marked increases in net nuclear grain counts and in the incidence of cells in repair, and the vehicle control groups gave acceptable values for net nuclear grain counts. Therefore, the test item was considered to be non-genotoxic under the conditions of this study.

The negative result in the in vivo UDS assay overrules the positive result from the respective in vitro test. Therefore, it can be concluded that the test item is not genotoxic.


Justification for selection of genetic toxicity endpoint
The both in vivo genetic toxicity studies of Buskens (2010) and Brown (2012) are reliable and show negative result for this endpoint. The two in vitro studies show positive results of genetic toxicity, however they are considered as supporting studies.

Justification for classification or non-classification

Genetic toxicity:

Based on the above stated assessment dimethyl itaconate is not mutagen according to CLP (5th ATP of Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.