Hexamethylene diacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09/2021 - 03/2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

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:

Hexamethylene diacrylate

EC Number:

235-921-9

EC Name:

Hexamethylene diacrylate

Cas Number:

13048-33-4

Molecular formula:

C12H18O4

IUPAC Name:

hexane-1,6-diyl bisacrylate

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

other: Human blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
Blood samples were drawn from healthy non-smoking donors. Blood was collected from a female donor (21 years old) for Experiment 1, from a male donor (20 years old) for Experiment 2 and from a female donor (33 years old) for Experiment 3. Lymphocytes of the respective donors were shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.

MEDIA USED
An 11 % mixture of whole blood in medium within 30 h after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (1:1) supplemented with 200 mM GlutaMAX™. The medium was also supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL), mitogen PHA (1.5% (v/v) as extract (Exp. 1) or 3 µg/mL as solvent lyophilizate (Exp. 2 & 3), 10% FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B (4 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/ beta-naphthoflavone induced rat liver. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test

Test concentrations with justification for top dose:

18.9 - 5000 µg/mL (Experiment 1/ Preliminary Cytotoxicity Test)
0.5 - 70 µg/mL (Experiment 2)
18.9 - 420 µg/mL (Experiment 3)

Vehicle / solvent:

DMSO (1.0 %)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other:

Details on test system and experimental conditions:

TEST MATERIAL PREPARATION
- Stock formulations of the test material and serial dilutions were made in DMSO. The final concentration of DMSO in the culture medium was 1.0 %.
- All formulations were prepared freshly before treatment and used within 2 h of preparation.

PRELIMINARY CYTOTOXICITY TEST
- Performed to determine the concentrations to be used in the main experiment.
- % cytostasis: percentages of reduction in the CBPI in comparison to the controls (by counting 500 cells per culture).
- 10 concentrations of the test material, a solvent and positive control were used.
- Replicates: duplicate
- Exposure time: 4 h with and without S9-mix

MAIN TEST
- Replicates: duplicate
- Test substance incorporation: Added in medium
- Preincubation period, if applicable: 48 h
- Exposure duration/duration of treatment: 4 or 20 h without S9-mix (Experiment 2), 4 h with S9-mix (Experiment 3).
- Removal/ washing: After 20 h cells were spun down by gentle centrifugation for 5 min and supernatant was discarded and the cells were re-suspended in saline G. The washing procedure was repeated once. Cells were re-suspended in complete culture medium containing 10 % FBS (v/v). Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 h until preparation (cytokinesis block).
- Harvest time after the end of treatment: 40 h

DATA EVALUATION
- Methods of slide evaluation: Performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area.
- Number of cells spread and analysed per concentration: At least 1000 binucleate cells per culture.
- Criteria for scoring micronucleated cells: Criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).

ACCEPTABILITY CRITERIA
The micronucleus assay will be considered acceptable if it meets the following criteria:
1) The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% control limit realised as 95% confidence interval)
2) The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase compared with the concurrent solvent control
3) Cell proliferation criteria in the solvent control are considered to be acceptable
4) All experimental conditions described in section ‘Experimental Design and Study Conduct’ were tested unless one exposure condition resulted in a clearly positive result
5) The quality of the slides must allow the evaluation of an adequate number of cells and concentrations
6) The criteria for the selection of top concentration are consistent with those described in section ‘Dose Selection’.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test material is considered to be clearly negative if, in all of the experimental conditions examined:
1) None of the test material concentrations exhibits a statistically significant increase compared with the concurrent solvent control
2) There is no concentration-related increase
3) The results in all evaluated test material concentrations should be within the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval).
Providing that all of the acceptability criteria are fulfilled, a test material is considered to be clearly positive if, in any of the experimental conditions examined:
1) At least 1 of the test material concentrations exhibits a statistically significant increase compared with the concurrent solvent control
2) The increase is concentration-related in at least one experimental condition
3) The results are outside the range of the laboratory historical solvent control data (95% control limit realised as 95% confidence interval)

Statistics:

Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control. A linear regression was performed to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test material were compared to the solvent control groups. A trend is judged as significant whenever the p-value < 0.05. Both, biological and statistical significance were considered together.

Results and discussion

Test results

Key result

Species / strain:

other: Human blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY
In Experiment 1 in the presence of S9 mix and in Experiment 2 in the absence of S9 mix after continuous treatment, clear cytotoxicity was observed at the highest evaluated concentration.
In Experiment 2 in the absence of S9 mix following 4 h treatment, no cytotoxicity was observed up to the highest evaluated concentration. The next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline showed strong cytotoxic effects and was not evaluable for the genotoxic parameter.
In Experiment 3 in the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. The next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline (factor 1.15) showed strong cytotoxic effects and was not evaluable for the genotoxic parameter.

GENOTOXICITY
In Experiment 1 in the presence of S9 mix, all values (1.15, 1.43 and 1.28 % micronucleated cells) after treatment with 102, 178 and 311 µg/mL of the test material were statistically significantly increased and exceeded the 95% control limit of the historical control data (0.00-1.04 % micronucleated cells). The values 1.28% and 1.43% exceeded the min-max range of historical control data, too (0.10-1.18 % micronucleated cells). A concentration related increase in micronucleus formation, as judged by an appropriate trend test was not observed.
In Experiment 2 or 3 none of these findings were confirmed. Notably, in Experiment 3, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test material. The value of 1.05 % micronucleated cells slightly exceeded the 95 % control limit of the historical control data (0.00-1.04 % micronucleated cells) but was clearly within the min-max range of historical control data (0.10-1.18 % micronucleated cells). Since the result in Experiment 1 is not reproducible, the experimental part with S9 mix can be considered as biologically irrelevant. Taken together the outcome of this study is negative.
Positive controls showed distinct increases in cells with micronuclei. The positive control demecolcine used in the continuous treatment experiment led to a clearly statistically significant increase of micronucleated cells. The value was more than three times higher than the respective solvent control value. Although, the rate of micronucleated cells after exposure to the positive control (2.50 %) was slightly below the recent historical control data ranges (2.85-8.30 % micronucleated cells), but the value exceeded clearly the 95 % control limit of the corresponding solvent control (0.06-0.88 % micronucleated cells). Therefore these finding has no detrimental impact on the validity of the study.

Any other information on results incl. tables

Table 2. Summary of results.

Exp.	Preparation interval	Test material concentration in µg/mL	Proliferation index CBPI	Cytostasis in %*	Micronucleated cells in %**	95% Ctrl limit in %
Exposure period 4 h with S9 mix
1	40 h	Solvent control^1/#	2.06		0.65	0.02 – 1.04
		Positive control^4	1.89	16.4	9.80^S
		102^#	2.04	1.8	1.15^S
		178^#	2.00	5.6	1.43^S
		311^#	1.47	55.5	1.28^S
Trend test: p-value 0.221
Exposure period 4 h without S9 mix
2	40 h	Solvent control^1	1.55		0.30	0.00 – 0.99
		Positive control^2	1.46	16.7	13.40^S
		6.8	1.53	3.5	0.45
		10.2	1.59	n.c.	0.15
		15.2	1.59	n.c.	0.40
Trend test: p-value 0.919
Exposure period 20 h without S9 mix
2	40 h	Solvent control^1	1.67		0.80	0.06 – 0.88
		Positive control^3	1.33	50.4	2.50^S
		2.6	1.47	29.5	0.15
		4.5	1.45	32.2	0.30
		10.2	1.30	54.5	0.20
Trend test: p-value 0.357
Exposure period 4 h with S9 mix
3	40 h	Solvent control^1	1.33		0.90	0.02 – 1.04
		Positive control^5	1.39	n.c.	6.55^S
		104	1.39	n.c.	0.65
		209	1.31	5.4	1.00
		276	1.28	16.3	1.05
Trend test: p-value 0.438

* For the positive control groups and the test material treatment groups the values are related to the solvent controls; ** The number of micronucleated cells was determined in a sample of 2000 binucleated cells;

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells;

S The number of micronucleated cells is statistically significantly higher than corresponding control values;

n.c. Not calculated as the CBPI is equal or higher than the solvent control value;

1 DMSO 1.0 % (v/v);

2 MMC 0.8 µg/mL;

3 Demecolcine 100 ng/mL;

4 CPA 17.5 µg/mL;

5 CPA 20.0 µg/mL

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions, the test material did not induce micronuclei as determined by the in vitro micronucleus test in human blood lymphocytes. The test substance is considered to be non-mutagenic in this in vitro micronucleus test when tested up to cytotoxic concentrations or to the highest evaluable concentrations.

Executive summary:

The test material, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in 3 independent experiments (OECD TG 487, GLP). The highest applied concentration in this study was 5000 µg/mL.
In Experiment 2 in the absence of S9 mix following 4 h treatment, no cytotoxicity was observed up to the highest evaluated concentration. The next highest tested concentration, showed strong cytotoxic effects and was not evaluable for the genotoxic parameter. In Experiment 1 in the presence of S9 mix and in Experiment 2 in the absence of S9 mix after continuous treatment, clear cytotoxicity was observed at the highest evaluated concentration. In Experiment 3 in the presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. The next highest tested concentration, showed strong cytotoxic effects and was not evaluable for the genotoxic parameter.
In the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test material. In the absence of S9 mix, the mean percentage of micronuclei in all treated conditions was within the 95 % historical control limits and none of the values were statistically significantly increased, when compared to the solvent control. None of the treatment conditions showed a concentration related trend. In Experiment 1 in the presence of S9 mix, all values (1.15, 1.43 and 1.28 % micronucleated cells) after treatment with 102, 178 and 311 µg/mL of the test material were statistically significantly increased and exceeded the 95 % control limit of the historical control data (0.00-1.04 % micronucleated cells). The values 1.28 and 1.43% exceeded the min-max range of historical control data, too (0.10-1.18 % micronucleated cells). A concentration related increase in micronucleus formation, as judged by an appropriate trend test was not observed. In the confirmatory Experiment 3 none of these findings were confirmed. No relevant increases in the numbers of micronucleated cells were observed after treatment with the test material. Since the result in Experiment 1 is not reproducible, the experimental part with S9 mix can be considered as biologically irrelevant. Taken together the outcome of this study is negative. The positive control substances induced statistically significant increases in cells with micronuclei.
Under the experimental conditions, the test material did not induce micronuclei as determined by the in vitro micronucleus test in human blood lymphocytes. The test substance is considered to be non-mutagenic in this in vitro micronucleus test when tested up to cytotoxic concentrations or to the highest evaluable concentrations.