Dibutyl itaconate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an AMES test, performed according to OECD/EC guidelines and GLP principles, Dibutyl itaconate was found not to be mutagenic with or without metabolic activation.

Furthermore, an in vitro micronucleus study was performed according to OECD TG 487 and in accordance with GLP principles. The test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions in this study.

Moreover, an in vitro mouse lymphoma assay was performed according to OECD TG 490 and in accordance with GLP principles. Based on the results in this study, the test item is not mutagenic in the TK mutation test system.

Based on the current data-set, there are no indications that the substance has genotoxic properties.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2021 - 10 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed according to ECHA Decision CCH-D-2114538624-46-01/F with a deadline of 24 October 2022

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Physical description: Clear yellow liquid
Storage conditions: At room temperature
Test item handling: No specific handling conditions required
Solubility in vehicle (ethanol): completely
Stability in vehicle (ethanol): Stable

Species / strain / cell type:

lymphocytes: Peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: peripheral human lympocytes from healthy adult, non-smoking volunteers (aged 25 to 29 years)
- Suitability of cells: Peripheral human lymphocytes are recommended in the international OECD guideline.
- Normal cell cycle time (negative control): not reported

For lymphocytes:
- Age and Average Generation Time (AGT) of donors: Dose-range finding study: age 27, AGT = 14.3 h; First cytogenetic assay: age 25, AGT = 13.1 h; Cytogenetic assay 1A: age 29, AGT = 14.6 h; Second cytogenetic assay: age 27, AGT = 14.3 h
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: No
- Mitogen used for lymphocytes: yes, phytohaemagglutinin

MEDIA USED
- Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.

ENVIRONMENTAL CONDITIONS
-Temperature: 37.0 ± 1.0°C (actual range 35.0 - 37.8°C)
-Humidity: 80 - 100% (actual range 48 - 94%)
-CO2 percentage: 5.0 ± 0.5% CO2

Cytokinesis block (if used):

cytochalasin B (5 μg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague Dawley rats
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES.
The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin).
- concentration S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported

Test concentrations with justification for top dose:

In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. The test item was tested in the absence and presence of S9-mix. Lymphocytes were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B was added to the cells simultaneously with the test item at the 24 hours exposure time. At the 3 hours exposure time, Cytochalasine B was added after the removal of the exposure medium containing the test chemical. A vehicle control was included at each exposure time.
The highest tested concentration was determined by the solubility of the test item in the culture medium.
Cytotoxicity of the test item in the lymphocyte cultures was determined using the cytokinesis-block
proliferation index (CBPI index).
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of
55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control (3 hours and 24 hours exposure time in absence of S9-mix) and was determined by the solubility (3 hours exposure time in presence of S9-mix).

Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay:
Without and with S9-mix : 50, 100, 300, 350, 400, 450 and 500 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).

The following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 50, 100, 125, 150, 175, 200, 225 and 250 μg/mL culture medium (24 hours exposure time, 24 hours harvest time).

Vehicle / solvent:

- Vehicle used: ethanol

- Justification for choice of solvent/vehicle: the test item was stable and soluble in the selected vehicle

- Justification for percentage of solvent in the final culture medium: not provided. The vehicle concentration in the final culture medium was 0.5%. and a vehicle control is included in the study design.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Remarks:

Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS)

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not applicable
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: not applicable
- Exposure duration/duration of treatment: 3 or 24 hours
- Harvest time after the end of treatment (sampling/recovery times): 24 or 27 hours


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: not applicable
- If cytokinesis blocked method was used for micronucleus assay: cytochalasin B (5 μg/mL)
- Methods of slide preparation: To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride solution. Immediately after, ethanol: acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue.
At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.
- Number of cells spread and analysed per concentration: at least 1000 cells per culture
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
The following criteria for scoring of binucleated cells were used:
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

The following criteria for scoring micronuclei were adapted from Fenech, 1996:
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): not applicable
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).
Three analyzable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. At the 3 hours and 24 hours exposure time without S9-mix, the highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined. At the 3 hours exposure time in the presence of S9-mix, the test item was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium.

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

based on cytotoxicity of 55 ± 5% (3 hours and 24 hours exposure time in absence of S9-mix); based on solubility (3 hours exposure time in presence of S9-mix).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

FORMULATION ANALYSIS
- Accuracy: In the vehicle, no test item was detected. The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
- Homogeneity: The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%).
- Stability: Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.868 (compared to 7.987 in the solvent control)
- Data on osmolality: 336 mOsm/kg (compared to 368 mOsm/kg in the solvent control)
- Precipitation and time of the determination: not precipitating at the highest tested concentration

DOSE-RANGE-FINDER
In the dose-range finding study, at the 3 hours and 24 hours exposure time, blood cultures were treated with 15.6, 31.3, 62.5, 125, 250 and 500 μg test item/mL culture medium with and without S9-mix. At a concentration of 500 μg/mL the test item precipitated in the culture medium at 3 hours exposure time.

FIRST CYTOGENETIC ASSAY
Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay:
Without and with S9-mix : 50, 100, 300, 350, 400, 450 and 500 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).

In the absence of S9-mix no appropriate dose levels could be selected for scoring of micronuclei since at the concentration of 100 μg/mL not enough cytotoxicity was observed (22%), whereas the next higher concentration of 300 μg/mL was too toxic for scoring (77%).

The experiment was repeated in cytogenetic assay 1A. The following dose levels were selected for the first cytogenetic assay:
Without S9-mix : 50, 100, 150, 175, 200, 225, 250 and 300 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).

The following dose levels were selected for scoring of micronuclei:
Without S9-mix : 50, 200 and 250 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).
With S9-mix : 50, 350 and 500 μg/mL culture medium (3 hours exposure time, 27 hours harvest time).

Both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei (see Table 1 and 2 under 'Any other information on results incl. tables'). In the first cytogenetic assay (3 hours exposure time with S9-mix), also mononucleated cells were scored. The test item did not induce a statistically significant or biologically relevant increase in the number of mononucleated cells with micronuclei in this part of the study. These results are not reported and do not influence the outcome of the study.


SECOND CYTOGENETIC ASSAY
The following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 50, 100, 125, 150, 175, 200, 225 and 250 μg/mL culture medium (24 hours exposure time, 24 hours harvest time).

The following dose levels were selected for the scoring of micronuclei:
Without S9-mix : 50, 125 and 200 μg/mL culture medium (24 hours exposure time, 24 hours harvest time).

The test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei (See Table 3 under 'Any other information on results incl. tables').

For CBPI of both assays and dose-range finding test see attachment.

ACCEPTABILITY
The number of binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database.
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of binucleated cells with micronuclei in at least one experiment. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Table 1. First cytogenetic assay (without S9-mix, 3 h exposure time, 27 h harvest time)

Concentration (μg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei	Number of binucleated cells with micronuclei	Number of binucleated cells with micronuclei(1)
		1000	1000	2000
		A	B	A+B
0	0	2	4	6
50	20	4	2	6
200	38	2	2	4
250	54	3	2	5
0.25 MMC-C	37	28	27	65****
0.1 Colch	92	7(2)	7(2)	14*

* Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001
or **** P < 0.0001.
(1) 1000 binucleated cells were scored for the presence of micronuclei. Duplicate cultures are indicated by A and B.
(2) 864 and 798 binucleated cells were scored for the presence of micronuclei, respectively.

 

Table 2 - First cytogenetic assay (with S9-mix, 3 h exposure time, 27 h harvest time)

Concentration (μg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei	Number of binucleated cells with micronuclei	Number of binucleated cells with micronuclei (1)
		1000	1000	2000
		A	B	A+B
0	0	1	1	2
50	6	0	3	3
350	27	0	1	1
500 (2)	16	0	2	2
15 CP	63	32	25	57****

* Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.
(1) 1000 binucleated cells were scored for the presence of micronuclei. Duplicate cultures are indicated by A and B.
(2) precipitation of test item

 

Table 3 - Second cytogenetic assay (without S9-mix, 24 h exposure time, 24 h harvest time)

Concentration (μg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei	Number of binucleated cells with micronuclei	Number of binucleated cells with micronuclei (1)
		1000	1000	2000
		A	B	A+B
0	0	2	2	4
50	18	0	0	0
125	36	1	1	2
200	50	0	1	1
0.25 MMC-C	52	8	9	17*
0.1 Colch	99	21 (2)	25 (2)	46****

* Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.
(1) 1000 binucleated cells were scored for the presence of micronuclei. Duplicate cultures are indicated by A and B.
(2) 168 and 221 binucleated cells were scored for the presence of micronuclei, respectively.

Conclusions:

An in vitro micronucleus test with human peripheral lymphocytes was performed in accordance with OECD TG 487 and GLP principles. In conclusion, this test is valid and the test substance is not clastogenic or aneugenic in human lymphocytes under the experimental conditions in this study.

Executive summary:

An in vitro micronucleus test with human peripheral lymphocytes was performed in accordance with OECD TG 487 and GLP principles. The objective of this study was to evaluate the test item for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments.

The vehicle of the test item was ethanol. In the vehicle, no test item was detected. The concentrations analyzed in the dose formulation samples were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The dose formulation samples were homogeneous (i.e. coefficient of variation ≤ 10%). Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

In the first cytogenetic assay, the test item was tested up to 250 μg/mL and 500 μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-mix, respectively. In the absence of S9-mix, appropriate toxicity was reached at this dose level. In the presence of S9-mix, the test item precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test item was tested up to 200 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. The positive control chemical colchicine produced a statistically significant increase in the number of binucleated cells with micronuclei in at least one experiment. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

The test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, this test is valid and the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 October 2021 - 4 January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study performed according to ECHA Decision CCH-D-2114538624-46-01/F, with a deadline of 24 October 2022.

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Physical description: clear yellow liquid
Storage conditions: at room temperature
Test item handling: no specific handling conditions required
Solubility in vehicle: ethanol: completely
Stability in vehicle: ethanol: stable

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y/TK+/--3.7.2C mouse lymphoma cells; American Type Culture Collection, (ATCC, Manassas, USA)
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD)
- Normal cell cycle time (negative control): not reported

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: not reported
- Methods for maintenance in cell culture: All incubations were carried out in a humid atmosphere (80 - 100%, actual range 26 - 101%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 32.4 - 38.6 °C).
- Cell cycle length, doubling time or proliferation index : not reported
- Modal number of chromosomes: not reported
- Periodically checked for karyotype stability: not reported
- Periodically ‘cleansed’ of spontaneous mutants: yes


MEDIA USED
- Hourse serum: was inactivated by incubation at 56°C for at least 30 minutes.
- Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.
- Exposure medium: Cells will be exposed to the test material in basic medium supplemented with 5% to 10% (v/v) heat-inactivated horse serum.
- Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT)
- Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight)
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration S9 in the final culture medium: 4% (v/v)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported

Test concentrations with justification for top dose:

Dose-range Finding Test:
- Without metabolic activation: 0.01 to 120 µg/mL
- With metabolic activation: 1 to 1000 µg/mL

First mutagenicity test:
- Without metabolic activation: 2.5, 5, 10, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL exposure medium
- With metabolic activation: 10, 20, 40, 80, 90, 100, 110, 120, 130, 140, 150, 160 and 170 μg/mL exposure medium

Second mutagenicity test:
- Without metabolic activation: 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL exposure medium

Vehicle / solvent:

- Vehicle used: ethanol

- Justification for choice of solvent/vehicle: not reported

- Justification for percentage of solvent in the final culture medium: not reported

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single (solvent control in duplicate)
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: Per culture 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: not applicable
- Exposure duration/duration of treatment: 3 or 24 hours
- Harvest time after the end of treatment: not reported

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection time (if incubation with a selective agent): 11 or 12 days in selective medium (TFT-selection)
- Fixation time (start of exposure up to fixation or harvest of cells): 1.5-2 hours by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
- Method used: microwell plates for the mouse lymphoma assay.
- Selective agent: 5 µg/mL trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: For determination of the mutant frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).
- Criteria for small (slow growing) and large (fast growing) colonies: The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: see calculations under 'Any other information on materials and methods incl. tables'

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: mutation factor (MF)
- Any supplementary information relevant for genotoxicity: see calculations under 'Any other information on materials and methods incl. tables'

Evaluation criteria:

A test material is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test material is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test material is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutant frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING TEST

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.13
- Data on osmolality: 0.360 Osm/kg

RANGE-FINDING/SCREENING STUDIES (if applicable):
- 3-hour incubation: In the absence of S9-mix, the relative suspension growth was 33% at the test material concentration of 60 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test material concentration of 120 μg/mL.
In the presence of S9-mix, the relative suspension growth was 59% at the test material concentration of 1000 μg/mL compared to the relative suspension growth of the solvent control.
- 24 hour incubation: The relative suspension growth was 14% at the test material concentration of 60 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test material concentration of 120 μg/mL.

STUDY RESULTS
First mutagenicity assay:
- The dose levels selected to measure mutant frequencies at the TK-locus were:
Without S9-mix: 5, 10, 30, 40, 50, 60, 80 and 90 μg/mL exposure medium.
With S9-mix: 10, 20, 40, 90, 100, 110, 120 and 130 μg/mL exposure medium.
In the absence of S9-mix, relative total growth was 13% and 47% at the dose levels of 90 and 80 µg/mL, respectively.
In the presence of S9-mix the RTG was 7% and 50% at the dose levels of 130 and 120 µg/mL, respectively.
- No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test material either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity assay:
- The dose levels selected to measure mutant frequencies at the TK-locus were:
5, 10, 20, 30, 40, 60, 80 and 90 µg/mL exposure medium.
The relative total growth was 9% and 18% at the dose levels of 90 and 80 µg/mL, respectively.
- No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test material. The numbers of small and large colonies in the test material treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

CONTROLS
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database (see Table 6). It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
The suspension growth over the two-day expression period for cultures treated with ethanol was between 16 and 21 (3 hour treatment) and 96 and 108 (24 hour treatment)

DOSE FORMULATION ANALYSIS
In the vehicle, no test item was detected. The concentrations analysed in the dose formulation samples were in agreement with target concentrations (i.e., mean accuracies between 90% and 110%). The dose formulation samples were homogeneous (i.e., coefficient of variation ≤ 10%). Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

Conclusions:

A mouse lymphoma assay has been performed according to OECD TG 490 and in accordance with GLP principles. Based on the results, Dibutyl itaconate is not mutagenic in the TK mutation test system under the experimental conditions in this study.

Executive summary:

A mouse lymphoma assay has been performed according to OECD TG 490 and in accordance with GLP principles.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

In the first experiment, the test material was tested up to concentrations of 90 and 130 µg/mL in the absence and presence S9-mix, respectively. In the absence of S9-mix, relative total growth (RTG) was reduced to 13% and 47% at concentrations of 90 and 80 µg/mL, respectively. In the presence of S9-mix the RTG was reduced to 7% and 50% at concentrations of 130 and 120 µg/mL, respectively.

In the vehicle, no test item was detected. The concentrations analysed in the dose formulation samples were in agreement with target concentrations (i.e., mean accuracies between 90% and 110%). The dose formulation samples were homogeneous (i.e., coefficient of variation ≤ 10%). Analysis of the dose formulation samples after storage yielded a relative difference of ≤ 10%. The dose formulation samples were found to be stable during storage at room temperature under normal laboratory light conditions for at least 4 hours.

In the second experiment, the test material was tested up to concentrations of 90 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 9% and 18% at concentrations of 90 and 80 µg/mL, respectively.

The mutant frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency.

In conclusion, Dibutyl itaconate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

The study deviates from OECD 471 (2020) in that it uses 2-Aminoanthracene (2AA) as the sole indicator of the efficacy of the S9-mix. Each batch of S9 should also have been characterised with a mutagen that requires metabolic activation by microsomal enzymes, e.g., benzo(a)pyrene, dimethylbenzanthracene. However, since a significant increase in revertant was seen in all strains when exposed to 2AA, and the S9 mixture was produced and used as described in OECD 471, the efficacy of the S9 mixture is probably adequate and thus this deviation is unlikely to affect the study integrity. Moreover, the test item is not structurally similar to benzo(a)pyrene, 9,10-Dimethylanthracene, Cyclophosphamide or 7,12-Dimethylbenzanthracene, as such, it is unlikely to require metabolic activation by microsomal enzymes to exert a potential genotoxic effect like BAP and DMBT. In other words, even if the microsomal enzymes in the s9 were not having proper activity, which (as discussed above) is already unlikely, it would not result in a false negative.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1:
Preliminary test (without and with 5% (v/v) S9-mix) (TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study (TA1535, TA1537 and TA98):
Without S9-mix: 5.4, 17, 52, 164, 512 and 1600 µg/plate
With 5% (v/v) S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Experiment 2:
TA1535, TA1537 and TA100:
Without S9-mix: 87, 154, 275, 492, 878 and 1568 μg/plate
With 10% (v/v) S9-mix: 492, 878, 1568, 2800 and 5000 μg/plate
TA98 and WP2uvrA:
Without S9-mix: 275, 492, 878, 1568, 2800 and 5000 μg/plate
With 10% (v/v) S9-mix: 492, 878, 1568, 2800 and 5000 μg/plate

Doses were selected based on the dose range finding test (observed precipitation and toxicity).

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines as solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; 5 µg/plate in saline for TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

without S9; 2.5 µg/plate in DMSO for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene

Remarks:

without S9; 10 µg/plate in DMSO for TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; 650 µg/plate in DMSO for TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9; 10 µg/plate in DMSO for WP2uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

in DMSO; with 5% S9-mix, 2.5 µg/plate for TA1535, TA1537, 1 µg/plate for TA98, TA100, 15 µg/plate for WP2 uvrA; with 10% S9-mix, 2.5 µg/plate for TA1535, 5 µg/plate for TA1537, 1 µg/plate for TA98, 2 µg/plate for TA100, 15 µg/plate for WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies was determined.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Rationale for test conditions:

Recommended test conditions in international guidelines (e.g. OECD, EC).
The first mutation experiment was performed with 5% (v/v) S9-mix. To obtain more information about the possible mutagenicity of Dibutyl itaconate, the second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation observed at highest concentration tested (5000 μg/plate) in experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest tested concentration (5000 μg/plate) in experiment 1; precipitation was observed at 2800 μg/plate and above in experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest tested concentration tested (1600 μg/plate) in experiment 1; precipitation was observed at highest concentration tested (1568 μg/plate) in experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation observed at highest concentration tested (1568 μg/plate) in experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest concentration tested (1568 μg/plate); preciptation was observed at highest tested concentration (1568 μg/plate) in experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation observed at highest tested concentration (5000 μg/plate) in experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation observed at 2800 μg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 878 μg/plate and above; precipitation was observed at highest concentration tested (1568 μg/plate) in experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitation observed at highest concentration tested (5000 μg/plate) in experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation was observed at the highest concentration tested except in tester strain TA98 in the presence of S9-mix, where no precipitation was observed. In addition, in tester strains TA98 and WP2uvrA (absence of S9-mix), precipitation was also observed at 2800 μg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 512 μg/plate and above in the absence of 5% (v/v) S9-mix and at 1600 and 5000 μg/plate in the presence of 5% (v/v) S9-mix, respectively. In tester strain WP2uvrA, toxicity was only observed at the highest concentration (5000 μg/plate) in the absence of S9-mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 1600 µg/plate, with S9: no toxicity
TA1537: without S9: 1568 µg/plate and above, with S9: no toxicity
TA98: without S9: no toxicity , with S9: no toxicity
TA100: without S9: 878 µg/plate and above, with S9: no toxicity
WP2uvrA: without S9: no toxicity , with S9: no toxicity

ACCEPTANCE OF RESULTS:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes

In strains TA1537 (absence and presence of S9-mix, experiment 1+2) and TA98 (absence of S9-mix, experiment 1), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reduction are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.

Conclusions:

In an AMES test, performed according to OECD/EC guidelines and GLP principles, Dibutyl itaconate was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed with Dibutyl itaconate according to OECD/EC guidelines and GLP principles. All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation in two experiments was seen. In the second experiment the test item precipitated on the plates in the absence and presence of S9-mix, except in tester strain TA98 in the presence of S9-mix, where no precipitation was observed. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or bacterial background lawn, was observed in tester strains TA1535, TA1537 and TA100 in the absence of S9-mix. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Dibutyl itaconate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An AMES test was performed with Dibutyl itaconate according to OECD TG 471 and in accordance with GLP principles. All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation in two experiments was seen. In the second experiment the test item precipitated on the plates in the absence and presence of S9-mix, except in tester strain TA98 in the presence of S9-mix, where no precipitation was observed. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or bacterial background lawn, was observed in tester strains TA1535, TA1537 and TA100 in the absence of S9-mix. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Dibutyl itaconate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

 

A valid in vitro micronucleus test with human peripheral lymphoctes was performed according to OECD TG 487 and in accordance with GLP principles. In the first cytogenetic assay, the test item was tested up to 250 μg/mL and 500 μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-mix, respectively. In the absence of S9-mix, appropriate toxicity was reached at this dose level. In the presence of S9-mix, the test item precipitated in the culture medium at this dose level. In the second cytogenetic assay, the test item was tested up to 200 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. The test item did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. In conclusion, this test is valid and the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions in this study.

 

A valid in vitro mouse lymphoma assay has been performed according to OECD TG 490 and in accordance with GLP principles. In the first experiment, the test material was tested up to concentrations of 90 and 130 µg/mL in the absence and presence S9-mix, respectively. In the absence of S9-mix, relative total growth (RTG) was reduced to 13% and 47% at concentrations of 90 and 80 µg/mL, respectively. In the presence of S9-mix the RTG was reduced to 7% and 50% at concentrations of 130 and 120 µg/mL, respectively. In the second experiment, the test material was tested up to concentrations of 90 µg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 9% and 18% at concentrations of 90 and 80 µg/mL, respectively. In the absence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test material did not induce a biologically relevant increase in the mutant frequency. In conclusion, Dibutyl itaconate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the available data, DBI is not classified for genotoxicity according to Regulation EC (No.) 1272/2008 (CLP).