Dibutyl itaconate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl itaconate

EC Number:

218-451-9

EC Name:

Dibutyl itaconate

Cas Number:

2155-60-4

Molecular formula:

C13H22O4

IUPAC Name:

1,4-dibutyl 2-methylidenebutanedioate

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

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.