Hexamethylene diacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro:
Gene mutation in bacteria
- comp. OECD 471; S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, with and without metabolic activation (Ames test): negative (BASF, 1989)
- comp. OECD 471, S typhimurium TA1535, TA 1537, TA 98, TA 100 and TA 1538, with and without metabolic activation (Ames test): negative (TNO,1979)

Read-across to CAS No. 42978-66 -5

- OECD 471; S. typhimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA; with and without S9; negative (BASF, 2003).

- OECD 471; S. typhimurium TA 1535, TA 1537, TA 98, TA 100; with and without S9; 20-5000 µg/plate; not mutagenic (BASF, 1989)

- equi. to OECD 471; S. typhimurium. TA 1535, TA 1537, TA 98, TA 100; with and without S9; 0.005-50.0 µl per plate; not mutagenic (LittonBio., 1980)

Gene mutation in mammalian cells

- OECD 478, Human lymphoma cells with and without metabolic activation (Micronucleus test): negative (ICCR Roßdorf, 2022)
-comp. OECD 476, Mouse L5178Y cells with and without metabolic activation (Mouse Lymphoma Assay): negative (Litton Bionetics, 1976)

Read-across to CAS No. 42978-66 -5

- OECD 476; GLP; CHO cells; with and without S9; 0.23-140 µg/ml; not mutagenic (BASF, 2015)

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:

09/2021 - 03/2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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 %)

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.

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted in accordance with the OECD guideline 471. The rate of induced back mutations was tested in the S. typhimurium indicator organisms TA1535, TA1537, TA98 and TA100. However, the mutagenic potential of the test substance was not tested in an additional strain, as recommended in the latest version of OECD guideline 471 (July 1997).

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The mutagenic potential of the test substance was not tested in an additional E. coli strain or in S. typhimurium TA102.

Principles of method if other than guideline:

The rate of induced back mutations was tested in the S. typhimurium indicator organisms TA1535, TA1537, TA98 and TA100 according to OECD guideline 471. However, the mutagenic potential of the test substance was not tested in an additional E. coli strain or in S. typhimurium TA102, as recommended in the latest version of OECD guideline 471 (July 1997).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of male Sprague-Dawley rats (treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice) and mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH 7.4).

Test concentrations with justification for top dose:

Standard plate test:
1st experiment:
0, 20, 100, 500, 2500 and 5000 µg/plate (tested in the strains S. typhimurium T98 and TA100)
2nd experiment:
0, 4, 20, 100, 500 and 1500 µg/plate without S9-mix; 0, 4, 20, 100, 500 and 2500 µg/plate with S9-mix (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537).

Preincubation test:
0, 4, 20, 100, 500 and 1000 µg/plate (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537).

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details on test system

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A standard plate test and a preincubation test were conducted, both with and without metabolic activation (S9 mix). Each test was conducted in triplicates.

STANDARD PLATE TEST:
The experimental procedure is based on the method of Ames et al. (Mut. Res. 31: 347-364, 1975):
Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

PREINCUBATION TEST:
The experimental procedure is based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (Factors modulating mutagenicity in microbial tests. In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980):
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

CONTROLS:
- Negative control:
Parallel with each experiment with and without S-9 mix, a negative control (with vehicle DMSO) is carried out for each tester strain in order to determine the spontaneous mutation rate.
- Positive controls:
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix: 10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535.
without S-9 mix: 5 µg N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535; 10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98; 100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537.

TITER DETERMINATION:
In general, the titer is determined only in the experiments with S-9 mix both without test substance (solvent only) and after adding the two highest amounts of substance. For this purpose, 0.1 ml of the overnight cultures is diluted to 1 x 10exp-6 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial suspension (dilution: 1 x 10exp-6)
0.5 ml S-9 mix
After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37°C for 48 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>500 µg/plate and higher, depending on test strain.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY:
No increase in the number of his+ revertants was observed, neither in the standard plate test nor in the preincubation test, with or without the addition of S9 mix.
The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

SOLUBILITY:
The test substance was completely soluble in DMSO.

TOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed depending on the strain and test conditions from about 500-2500 µg/plate onward in the standard plate test and at doses >=500 µg/plate in the preincubation test.

Table 1: Maximum revertants/plate and corresponding test concentrations in the standard plate test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	18 ± 2	21 ± 2
	Test substance	19 ± 2 [100]	34 ± 1 [500]
	Positive Control	1817 ± 236 [5; MNNG]	171 ± 17 [10; 2 -AA]
S. typhimurium TA100	DMSO	96 ± 7	116 ± 15
	Test substance	102 ± 5 [4]	129 ± 7 [500]
	Positive Control	1550 ± 132 [5; MNNG]	1493 ± 129 [10; 2 -AA]
S. typhimurium TA98	DMSO	22 ± 1	35 ± 5
	Test substance	24 ± 3 [4]	37 ± 7 [4]
	Positive Control	855 ± 35 [10; NOPD]	1110 ± 26 [10; 2 -AA]
S. typhimurium TA1537	DMSO	6 ± 1	13 ± 2
	Test substance	7 ± 1 [4]	12 ± 4 [4]
	Positive Control	418 ± 50 [100; AAC]	148 ± 17 [10; 2 -AA]
2-AA = 2-aminoanthracene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
NOPD = 4-nitro-o-phenylenediamine
AAC = 9-aminoacridine

Table 2: Maximum revertants/plate and corresponding test concentrations in the preincubation test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	17 ± 2	21 ± 2
	Test substance	15 ± 3 [4]	19 ± 3 [500]
	Positive Control	1343 ± 21 [5; MNNG]	149 ± 20 [10; 2 -AA]
S. typhimurium TA100	DMSO	101 ± 3	110 ± 13
	Test substance	97 ± 5 [4]	92 ± 6 [20]
	Positive Control	1091 ± 113 [5; MNNG]	1353 ± 220 [10; 2 -AA]
S. typhimurium TA98	DMSO	19 ± 2	31 ± 3
	Test substance	22 ± 1 [100]	31 ± 3 [100]
	Positive Control	754 ± 113 [10; NOPD]	1283 ± 257 [10; 2 -AA]
S. typhimurium TA1537	DMSO	7 ± 1	8 ± 1
	Test substance	8 ± 3 [4]	7 ± 2 [100]
	Positive Control	268 ± 91 [100; AAC]	189 ± 33 [10; 2 -AA]
2-AA = 2-aminoanthracene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
NOPD = 4-nitro-o-phenylenediamine
AAC = 9-aminoacridine

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The mouse lymphoma assay was conducted as described by Clive and Spector 1975 (Mutation Res 31: 17-29).

GLP compliance:

no

Remarks:

; GLP was not compulsory at the time the study was conducted.

Type of assay:

mammalian cell gene mutation assay

Target gene:

The mouse lymphoma-TK assay detects the mutations at the thymidine kinase locus caused by base pair changes, frameshift and small deletions. Mouse lymphoma L5178Y cells are heterozygous for a specific autosomal mutation at the TK locus (TK+/-). Mutant cells, deficient in TK due to the forward mutation in the TK locus (from TK+/- to TK-/-), are resistant to the cytotoxic effect of pyrimidine analogues such as trifluorothymidine (TFT). The mutagenicity of the test agents is indicated by the increase in the number of TK-/- mutants after treatment with TFT.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
Cells were maintained in Fisher´s medium for leukemic cells of mice, supplemented with 10 % horse serum and sodium pyruvate; Cloning medium consisted of Fisher´s medium supplemented with 10% horse serum, sodium pyruvate and 0.35 % Noble agar; selection medium was made from cloning medium by the addition of 5 ml BrdU to 100 ml Cloning medium
- Periodically "cleansed" against high spontaneous background

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of male Fischer 344 rats (treated with Aroclor 1254) and mixed with cofactors (NADP sodium salt, isocitric acid).

Test concentrations with justification for top dose:

without activation: 0.0016, 0.0031, 0.0062, 0.0094, 0.0125 µl/ml;
with activation: 0.0062, 0.0094, 0.0125, 0.0188, 0.0250 µl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details on test system

Details on test system and experimental conditions:

ASSAYS:
Each test was conducted in triplicates.

- Nonactivation assay:
The procedure used is a modification of that reported by Clive and Spector (Mutation Res 31: 17-29, 1975). Prior to each treatment, cells were cleansed of spontaneous TK-/- by growing them in a medium containing thymidine, hypoxanthine, methotrexate, and glycine (THMG). This medium permits the survival of only those cells that produce TK, and can therefore, utilize the exogenous thymidine from the medium. The test compound was added to the cleansed cells in growth medium at the predetermined doses for 4 hours at 37 °C on a rocker. The treated cells were washed, fed, and allowed to express in growth medium for 2 days. At the end of this expression perlod, TK-/- mutants were detected by cloning the cells in the selection medium for 10 days. Surviving cell populations were determined by plating diluted aliquots in nonselective growth medium.

- Activation assay:
The activation assay differed from the nonactivation assay in the following manner only: The liver was pretested for toxicity to the cells and the appropriate volume (generally ranging from 1.0 ml to 0.5 ml) was added to 1 ml of the cofactors. This activated mixtue was added to the required volume (generally, 8.0 to 8.5 ml) of medium containing the desired number of cleansed cells.

CONTROLS:
Negative control:
The solvent in which the test compound was prepared was used as a solvent (or vehicle) control. A negative control consisting of media only was used to assess the base-line viability of the cells.
Positive Control:
EthylmethaInsulfonate (EMS), which induces mutation by basepair substitution, was dissolved in culture medium and was used as a positive control for the nonactivation studies at a final concentration of 0.5 µl/ml.
Dimethylnitrosamin (DMN), which induces mutation by base-pair substitution and requires metabolic biotransformation by microsomal enzymes, was used as a positive control substance for the activation studies at a final concentration of 0.3 µl/ml.

TOXICITY:
The solubility, toxicity, and doses for all chemicals were determined prior to screening. The effect of each chemical on the survival of the indicator cells was determined by exposing the cells to a wide range of chemical concentrations in complete growth medium. Toxicity was measured as loss in growth potential of cells induced by a 4 hour exposure to the chemical followed by a 24 hour expression period in growth medium. A minimum of four concentrations was selected from the concentration range by using as the highest dose, a level that showed a reduction in growth potential. At least three lower doses, including levels which were below the toxic range were added. Those compounds that were relatively nontoxic to cells growing in suspension were tested at concentrations of up to 10 mg/ml when solubility permitted. Toxicity produced by chemical treatment was monitored during the experiment.

Evaluation criteria:

A compound is considered mutagenic in this assay if:
- A dose-response relationship is observed over 3 of the 5 dose levels employed.
- The minimum increase at the low level of the dose-response curve is at least 2.5 times greater than the solvent and/or negative control values.
- The solvent and negative control data are within the normal range of the spontaneous background for the TK locus.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without activation: cytotoxicity at doses > 0.0094 µl/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity:
- Doses higher than 0.0094 µl/ml without metabolic activation were already toxic to the cultures within 48 hours of treatment. At the highest tested concentration of 0.0125 µl/ml, the relative growth of the cells was 6.2 % compared to solvent control. As this was below the acceptable 10 % limit, this dose level was not used for the assessment of the mutagenic potential of the test substance. The relative growth of the cells at 0.0094 µl/ml was 14.2 % compared to 100 % for the solvent control. The relative growth of the cells at the three lowest concentrations of the test substance was decreased (53.7, 36.6 and 32.9 %), compared to 100 % of the solvent control.

- With metabolic activation, the percent relative growth in the cultures treated with the test compound ranged from 86 % to 49 %.

Mutagenicity:
- Without activation, the mutant frequencies in the cultures treated with the three lowest concentrations of the test compound (0.0016, 0.0031 and 0.0062 µl/ml) were all comparable to the solvent control values. At the concentration of 0.0094 µl/ml, which already reduced the relative growth to 14.2%, the mutant frequency of the cells was increased to 46.8 cells per 1x10exp+6 cells, but did not exceed 2.5 times the average value for the solvent control (21.1 cells per1x10exp+6 cells). One out of three plates at 0.0094 µl/ml was contaminated and the value for mutant frequency was therefore based on two plates only at this concentration. At the concentration of 0.0125 µl/ml the mutant frequency of 118.1 cells per1x10exp+6 cells exceeded 2.5 times the average value for the solvent control (21.1 cells per1x10exp+6 cells), but due to the occuring severe cytotoxicity of greater 90% at this concentration, as explained above, the values obtained for mutant frequency were not considered relevant for assessment.

- With activation, all the mutant frequencies except one at the concentration of 0.0125 µl/ml were less than 2.5 times the average solvent control (background) frequency. Since this activity occurred at only one concentration in the middle of the range of tested concentrations and was associated with the least toxic activity, the authors regarded this result as spurious. The reduced toxicity of the test compound as a function of concentration indicated a reaction with the metabolic activation mix. The authors concluded that this reaction may reduce the mutagenic as well as toxic activity of a given concentration.

The positive control compounds yielded frequencies in the normal range that were in excess of the negative control values.

 Table 1: Summary of the mouse lymphoma assay results

Nonactivation test: without S9-mix
test substance (µl/ml)	relative growth (%)	mutant frequency per 1x10exp+6 cells
0.0016	53.7	14.6
0.0031	35.6	22.3
0.0062	32.9	34.0
0.0094	14.2	46.8
0.0125	6.2	118.1
solvent control	100.0	21.1
negative control	103.5	11.0
positive control (EMS; 0.5)	25.0	236.5
Activation test: with S9-mix
test substance (µl/ml)	relative growth (%)	mutant frequency per 1x10exp+6 cells
0.0062	68.1	44.5
0.0094	49.4	31.9
0.0125	85.7	73.5
0.0185	68.3	53.8
0.0250	68.3	56.5
solvent control	100.0	24.7
negative control	96.8	23.3
positive control (DMH; 0.3)	3.8	610.5

Genetic toxicity in vivo

Description of key information

in vivo

Read-across to CAS No. 42978-66-5
- acc. OECD 474; Mouse, MNT in-vivo: negative (BASF, 2004)
- acc. OECD 474; Mouse, MNT in-vivo: negative (Covance, 2007)
- Mouse, Single cell gel assay and micronucleus assay: negative (Tice, 1997)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Physical state: liquid
- Analytical purity: >95%
- Lot/batch No.: 030061P040
- Stability under test conditions: the stability of the test substance throughout the study period and in the vehicle was verified analytically.
- Storage condition of test material: room temperature, protected from light

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 31 g (mean)
- Assigned to test groups randomly: yes, under following basis: randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages, type MI, housed individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, olive oil was selected as the vehicle, which had
been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 0.875 g/100 ml; 1.75 g/100 ml and 3.5 g/100 ml

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved in olive oil:
- The low dose group was given 87.5 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of
0.875 g/100 ml.
- The intermediate dose group was given 175 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of
1.75 g/100 ml.
- The top dose groups were given 350 mg test substance/kg body weight or 10 ml/kg body weight of a solution with a concentration of 3.5 g/100 ml.

Duration of treatment / exposure:

one single administration

Frequency of treatment:

one single administration

Post exposure period:

24-48 hours

Dose / conc.:

87.5 mg/kg bw/day (nominal)

Dose / conc.:

175 mg/kg bw/day (nominal)

Dose / conc.:

350 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP) and vincristine (VCR) both dissolved in purified water were administered to male animals once intraperitoneally each in a
volume of 10 ml/kg body weight.
- Justification for choice of positive control(s): The stability of CPP and VCR is well-defined under the selected conditions, since both positive control
articles are well-established reference clastogens and aneugens respectively.
- Route of administration: intraperitoneal
- Doses / concentrations: CPP: 20 mg/kg bw for clastogenic effects; VCR: 0.15 mg/kg bw for aneugenic effects

Tissues and cell types examined:

In general, 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored .

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of
350 mg/kg body weight and in the vehicle controls. In the test groups of 175 mg/kg and 87.5 mg/kg body weight and in the positive control groups,
the 24-hour sacrifice interval was investigated only.

DETAILS OF SLIDE PREPARATION:
The two femora were prepared by dissection and removing all soft tissues. After cutting off the epiphyses, the bone marrow was flushed out of the
diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37°C (about 2 ml/femur). The suspension was mixed
thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µl fresh FCS. One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.
The slides were stained in eosin and methylene blue solution for 5 minutes (May Grünwald solution modified = Wrights solution), rinsed in purified
water and then placed in fresh purified water for 2 or 3 minutes. They were finally stained in 7.5% Giemsa solution for 15 minutes.
After being rinsed twice in purified water and clarified in xylene, the preparations were mounted using Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2,000 polychromatic erythrocytes (PCEs) from each of the animals of every test group are evaluated and investigated for micronuclei (MN).
The normochromatic erythrocytes (NCEs) which occur are also scored. The following parameters are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an
index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals shows the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the target. Individual animals with pathological bone marrow depression
may be identified and excluded from the evaluation.
- Number of small micronuclei (d=D/4) (d = diameter of micronucleus, D= cell diameter)
The size of micronuclei may indicate the possible mode of action of the test substance, i .e . a clastogenic or a spindle poison effect.
Slides were coded before microscopic analysis.
Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative
values can occur.

Evaluation criteria:

The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells, i .e. >=2000 polychromatic erythrocytes and a clear differentiation between polychromatic erythrocytes (PECs) and normochromatic erythrocytes (NECs).
-The ratio of PECs/NECs in the untreated animals (negative control) has to be within the normal range of the animal strain.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for
PECs and NECs.
- The two positive control substances have to induce a significant increase in the number of PECs containing small and large micronuclei within the
range of the historical control data or above.

A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.

A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN.
The asymptotic U test according to Mann-Whitney (modified rank test according to Wilcocon) was carried out to clarify the question whether there were significant differerences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes.
The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determinatian for the U test .

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

the test substance led to clinical signs

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

As a negative control, male mice were administered merely the vehicle, olive oil,by the same route, which

gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.
Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.

The administration of the test substance led to clinical signs, namely piloerection and squatting posture.

According to the results of the present study, the single intraperitoneal administration of Tripropylenglykoldiacrylat did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data.

A dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected from about of 87.5 mg/kg body weight onward.

Conclusions:

Thus, under the experimental conditions chosen in the study, the test substance does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Reference 2

Endpoint:

genetic toxicity in vivo, other

Remarks:

chromosome aberration and DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

The test substance was applied dermally to Tg.AC mice (3 times a week for 20 weeks). Peripheral blood leukocytes were evaluated for DNA damage (single-strand breaks, alkali labile sites, DNA crosslinking) at weeks 4, 8, 12, 16, and 20 by using the alkaline (pH > 13) single cell gel (SCG) assay.
Peripheral blood polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were evaluated for the presence of micronuclei at week 20.

GLP compliance:

not specified

Type of assay:

other: Single cell gel assay and micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Tripropylene glycol diacrylate (TPGDA)
- Physical state: liquid
- Analytical purity: 80% pure monomer
- Impurities (identity and concentrations): hydroquinone (<200 ppm)
- Storage condition of test material: in the dark at 4-6 °C

Species:

mouse

Strain:

other: Tg.AC (v-Ha-ras)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic farms
- Age at study initiation: 12 weeks of age
- Assigned to test groups randomly: yes, under following basis: randomly assigned by body weight to a treatment or control group
- Housing: 3 to 5 per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 4 °C (71 +-7 °F)
- Humidity (%): 50 +- 20 %
- Air changes (per hr): 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

dermal

Vehicle:

- Vehicle(s)/solvent(s) used: [acetone]
- Justification for choice of solvent/vehicle: the test substance is immiscible with water
- Concentration of test material in vehicle: 1, 5 and 10 μM in 200 μl vehicle (acetone)
- Amount of vehicle (if gavage or dermal): 200 μl vehicle (acetone)

Details on exposure:

TEST SITE
- Area of exposure: midscapular region to the base of the tail
- % coverage: 8 cm2
- Time intervals for shavings or clipplings: before the first treatment, no further data


REMOVAL OF TEST SUBSTANCE
no data


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1, 5 and 10 μM in 200 μl vehicle (acetone)
- Concentration (if solution): 1, 5 and 10 μM in 200 μl vehicle (acetone)
- Constant volume or concentration used: yes


USE OF RESTRAINERS FOR PREVENTING INGESTION: no data

Duration of treatment / exposure:

20 weeks

Frequency of treatment:

three (Monday, Wednesday, Friday) topical applications/week

Post exposure period:

no data

Dose / conc.:

1 other: µM

Remarks:

Nominal concentration

Dose / conc.:

5 other: µM

Remarks:

Nominal concentration

Dose / conc.:

10 other: µM

Remarks:

Nominal concentration

No. of animals per sex per dose:

3-8

Control animals:

yes, concurrent vehicle

Positive control(s):

12-O-tetra-decanoylphorbol-13-acetate
- Justification for choice of positive control(s): positive control for tumor induction in this transgenic mouse model
- Route of administration: dermal
- Doses / concentrations: 0.002 μM
This positive control substance is not a genotoxicant, but a tumor promotor.

Tissues and cell types examined:

At 4, 8, 12, 16, and 20 weeks of treatment (~ 3-4 hours after the treatment on Wednesday ; 27-28 hours after the treatment on Monday), 1-2 mm of
the terminal portion of the tail was snipped and blood collected for an evaluation of DNA damage in leukocytes or micronuclei in erythrocytes.

Details of tissue and slide preparation:

see any other information on materials and methods

Evaluation criteria:

Criteria for a positive response included a statistically significant trend test or Kruskall-Wallis test with at least one dose significantiy different from the concurcent control, or at least two doses significantly different from the concurcent control.

Statistics:

Significance was based on obtaining p< 0.05. The statistical analysis of micronuclei data was conducted by using a micronucleus assay data
management, and statistical analysis software system.
For single cell gel data, the statistical analysis was based on tail moment, a metric that takes into account both the amount of migrated DNA and
the length of DNA migration.

Sex:

female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

other: valid, but not useful as positive control for DNA damage

Additional information on results:

The positive control substance induced the expected increase in skin papilloma in the transgenic mouse model. Since it is known, that this substance does not cause DNA damage, but has only tumor promoting properties, it gave negative results in the micronucleus and single cell gel assay. Its usefulness as a positive control in this study is questionable.

The extent of DNA migration in leukocytes of mice treated by dermal application with TPGDA at 1, 5, or 10 μmol per mouse was not significantly different, either by trend test analysis or by a pairwise comparison of each treatment dose against the concurrent vehicle control, at any sample time.

TPA (0.002 μmol per mouse), the positive control for the tumorigenicity studies, also failed to significantly alter the extent of DNA migration or its intercellular dispersion in leukocytes of mice treated by dermal application.

After 20 weeks of treatment, the frequency of micronucleated PCE and NCE in blood were not increased in the mice treated with TPGDA or TPA. The percentage of PCE was increased in mice treated with TPGDA. This increase was highly significant. By a pairwise comparison, the lowest effective dose of TPGDA inducing a significant increase in percentage of PCE was 10 μmol per mouse. TPA, at 0.002 μmol per mouse also induced a marginally nonsignificant increase in the percentage of PCE. This observed increase in the rate of erythropoiesis may reflect bone marrow/blood toxicity, a homeostatic mechanism in response to the treatment-induced tumor burden, and/or a hematopoietic response to epidermal keratinocyte cytokines induced by tissue injury.

Conclusions:

In this study, the dermal application of the test substance, a multifunctional acrylate to female Tg.AC mice over a 20-week period, failed to induce a significant increase in DNA damage in circulating leukocytes at multiple sample times or chromosomal damage in proliferating
bone marrow cells. The absence of genotoxic damage in these two cell populations suggests that this acrylate is not genotoxic or that it is genotoxic but not readily absorbed across the skin and systemically distributed throughout the body.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): TPGDA
- Analytical purity: approx. 87 %
- Lot/batch No.: P8960464SAP
- Expiration date of the lot/batch: 19 March 2008

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, USA
- Age at study initiation: Young adults, approx. 8 weeks old
- Weight at study initiation: males: 33.7 to 38.2 g; females: 23.1 to 28.1 g
- Assigned to test groups randomly: yes by using a computer program
- Housing: separated by gender, up to 5 animals per cage
- Diet: PMI Certified Rodent Diet (R) #5002, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17.7 - 26.1 °C
- Humidity: 30-70 %
- Air changes per hr: >/= 10
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil (CAS No. 8001-30-7); Supplier: Welch, Holme & Clarke; Lot No. 12-455; Storage at > 0 °C to 10 °C

Details on exposure:

Dose Preparation
Prior to dosing, the top stock of the test article, TPGDA, was prepared by adding the appropriate volume of the vehicle, corn oil, to a pre-weighed quantity of the test article and mixed, forming a solution. Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature prior to dosing.

Dose Analyses
The Sponsor was responsible for the determination and documentation of the identity, strength, purity, stability and uniformity of the test article and the determination of stability, homogeneity and concentration of the dosing preparations.

Duration of treatment / exposure:

one single administration

Frequency of treatment:

one single administration

Post exposure period:

24 h for all dose groups; additionally 48 h for an additional vehicle control and an additional 2000 mg/kg bw group

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Range-finder and main study

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Range-finder and main study

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Range-finder and main study

No. of animals per sex per dose:

Range-finding study: 3 males and 3 females per dose
Main study: 5 males per dose and harvest time point

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CAS No. 6055-19-2); Supplier: Sigma Aldrich; Lot No. 076K1050; Storage in a refrigerator set to maintain >0 to 10 °C;
the solvent was water

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Extraction of Bone Marrow
The hind limb bones (tibias) were removed for marrow extraction from five surviving animals in each treatment and control group. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).

Preparation of Slides
Following centrifugation to pellet the marrow, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air-dried. The slides were fixed in methanol, stained in May-Grünwald solution and Giemsa, and protected by mounting with coverslips. For control of bias, all slides were coded prior to analysis.

Slide Analysis
Slides prepared from the bone marrow collected from five animals per group at the designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least 500 erythrocytes per animal.

Evaluation criteria:

Acceptable Controls
The vehicle control group mean must lie within the historical control range and will usually be less than 0.4% micronucleated PCEs. There must be a statistically significant elevation of the mean of the positive control group relative to the vehicle control group, and the positive control response must be consistent with historical positive control data.
Acceptable High Dose
Generally the high dose should reach the limit dose or produce some indication of toxicity, e.g., toxic signs and/or mortality in the test article dosed animals and/or a reduction in the PCE:NCE ratio. If there are solubility constraints, the highest dose tested will be the solubility limit or higher doses if a well-dispersed suspension is obtained that does not settle out rapidly.
Assay Evaluation Criteria
The criteria for a positive response is the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose related response. A test article that does not induce both of these responses is considered negative. Statistical significance is not the only determinant of a positive response; the Study Director also considers the biological relevance of the results in the final evaluation.

Statistics:

The following statistical methods were used to analyze the micronucleus data.
• Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogeneous. Ranked proportions were used for heterogeneous variances.
• If the analysis of variance was statistically significant (p ≤ 0.05), Dunnett's t-test (Dunnett, 1955; 1964) was used to determine which dose groups, if any, were statistically significantly different from the vehicle control. Analyses were performed separately for each sampling time.
The 500, 1000, and 2000 mg/kg dose groups, as well as the positive control group, were compared with the vehicle control group at the 5% probability level.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose Range-finding Study:
Survival and Clinical Observations:
All animals appeared normal immediately after dosing and remained healthy until the end of the observation period.
Conclusion:
The high dose of the commercial product reached the maximum allowable dose of 2000 mg/kg bw, based on regulatory guidelines.


Micronucleus Assay:
- Survival and Clinical Observations:
All animals in all the dose groups appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest timepoint.
- Results and Interpretation:
A commercial product of TPGDA did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (500, 1000, or 2000 mg/kg). TPGDA was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose of the test article analyzed.
The vehicle control group had approximately ≤0.09% micronucleated PCEs and the group mean was within the historical control range. The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard error of 1.93 ± 0.21%.

Table 1: Individual animal data after 24 and 48 h treatment

Treatment	Dose	Animal No.	# MN PCE/2000 PCE	Ratio PCE:NCE
vehicle control 24 h	corn oil 10 ml/kg bw	1	1	0.29
	corn oil 10 ml/kg bw	2	2	0.26
	corn oil 10 ml/kg bw	3	2	0.66
	corn oil 10 ml/kg bw	4	1	0.25
	corn oil 10 ml/kg bw	5	3	0.46
vehicle control 48 h	corn oil 10 ml/kg bw	1	3	0.56
	corn oil 10 ml/kg bw	2	1	0.57
	corn oil 10 ml/kg bw	3	3	0.31
	corn oil 10 ml/kg bw	4	1	0.26
	corn oil 10 ml/kg bw	5	1	0.50
positive control	cyclophosphamide 80 mg/kg bw	1	50	0.10
	cyclophosphamide 80 mg/kg bw	2	26	0.36
	cyclophosphamide 80 mg/kg bw	3	45	0.52
	cyclophosphamide 80 mg/kg bw	4	38	0.74
	cyclophosphamide 80 mg/kg bw	5	34	0.57
Test material 24 h	500 mg/kg bw	1	2	0.81
	500 mg/kg bw	2	0	0.44
	500 mg/kg bw	3	0	0.56
	500 mg/kg bw	4	0	0.38
	500 mg/kg bw	5	3	0.20
Test material 24 h	1000 mg/kg bw	1	1	0.30
	1000 mg/kg bw	2	0	0.49
	1000 mg/kg bw	3	0	0.76
	1000 mg/kg bw	4	1	0.42
	1000 mg/kg bw	5	1	0.64
Test material 24 h	2000 mg/kg bw	1	0	0.32
	2000 mg/kg bw	2	1	0.48
	2000 mg/kg bw	3	4	0.29
	2000 mg/kg bw	4	6	0.45
	2000 mg/kg bw	5	1	0.53
Test material 48 h	2000 mg/kg bw	1	0	0.53
	2000 mg/kg bw	2	1	0.36
	2000 mg/kg bw	3	1	0.45
	2000 mg/kg bw	4	2	0.31
	2000 mg/kg bw	5	1	0.26

PCE = Polychromatic erythrocyte

MN PCE = Micronucleated PCE

NCE = Normochromatic erythrocyte

Conclusions:

The test material was evaluated as negative in the mouse bone marrow micronucleus assay under the experimental conditions of this assay chosen.

Additional information

There are valid data available for the assessment of genetic toxicity with 1,6-hexanedioldiacrylate an the read-across substance tripropylene glycol diacrylate (Cas No. 42978-66-5).

In vitro:

Gene mutation in bacteria

1,6-hexanedioldiacrylate was not mutagenic in a standard plate test and in a pre-incubation Ames test with and without metabolic activation, tested up to 5000 μg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98 and TA 100; S9 fraction was from the liver of male Sprague-Dawley rats, treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice and mixed with a series of cofactors (comp. OECD 471). Cytotoxicity was observed >500 µg/plate and higher, depending on test strain (BASF, 1989).

 

In another standard plate Ames test with and without metabolic activation (tested up to 5000 μg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98, TA 100 and TA 1538 metabolic activation, 1,6-hexanedioldiacrylate was not mutagenic. Metabolic activation was from S9 fraction from the liver of male rats, treated with a single dose of Aroclor 1254 seven days before sacrifice and mixed with a series of cofactors (comp. OECD 471). Cytotoxicity was observed at concentrations of > 500 µg/plate and higher, depending on test strain, without S9-mix in Salmonella typhimurium TA1535, TA 1537, TA 98 and TA 100 and in Salmonella typhimurium TA1538 at concentrations >=1000 µg/plate and higher, with or without S9-mix (TNO, 1979).

 

Available data for tripropylene glycol diacrylate (TPGDA, Cas No. 42978-66-5):

The test substance was tested for mutagenicity in an Ames test with and without metabolic activation, at 0, 20, 100, 500, 2500 and 5000 µg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98, TA 100, and in E. coli WP2 uvrA and in a second experiment at 0, 1000, 2000, 3000, 4000 and 5000 µg/plate in TA 1535 only; (acc. OECD 471). An increase in the number of mutant colonies that lacked dose dependency was observed only in the strain S. typhimurium TA1535 after addition of S9 mix from about 500 µg - 1000 µg/plate (factor 1.7 - 1.8) onward with a maximum response at 2500 µg - 3000 µg/plate (factor 2.5 - 2.6) and a reduced mutation rate (factor 2 - 2.1) at higher concentrations. Cytotoxicity was observed at 500 µg/plate and higher, depending on test strain (BASF, 2003).

 

In another standard plate Ames test with and without metabolic activation (tested at 0, 20, 100, 500, 2500 and 5000 µg/plate in Salmonella typhimurium TA1535, TA 1537, TA 98 and TA 100, the test substance was not mutagenic. Metabolic activation was from S9 fraction from the liver of rats, treated with Aroclor 1254 (acc. OECD 471). A slight decrease in the number of his+ revertants was observed in the standard plate test only with TA 98 at doses > 100 µg/plate. In the preincubation assay a weakly bacteriotoxic effect was found depending on the strain and test conditions at doses > 2500 µg/plate (TA 100, TA 1535) or at doses > 500 µg/plate (TA 98) (BASF, 1989).

 

A third Ames test also provided negative results for the test substance with or without metabolic activation, tested in S. typhimurium strains TA 1535, TA 1537, 1538, TA 98 and TA 100, tested at concentrations of 0.005, 0.01, 0.1, 1.0, 5.0, 10.0, 25.0, 50.0 µl per plate. The test material was toxic to the strains TA-1537 and TA-1538 at 10 µl per plate and to TA-100 at 25 µl and 50 µl per plate. The test material was also slightly toxic at 10 µl per plate for TA-1535 and at 25 µl and 50 µl per plate for the strain TA-98 (Litton Bionetics Inc., 1980).

 

Gene mutation in mammalian cells

The test substance was also negative for genotoxicity in a mouse lymphoma assay using L5178Y cells at levels up to 0.0125 µl/ml without metabolic activation and up to 0.0250 µl/ml with metabolic activation (comp. OECD 476). Cytotoxicity was observed at doses > 0.0094 µl/ml without metabolic activation (Litton Bionetics Inc., 1976). In addition, 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 (ICCR Roßdorf, 2022).

 

Available data for tripropylene glycol diacrylate (TPGDA, Cas No. 42978-66-5):

With the the test substance an HPRT assay according to OECD 476 and GLP guidelines was performed. Chinese hamster ovary cells (CHO) were treated with up to 30 µg/mL without S9 mix and up to 140 µg/mL after the addition of a metabolizing system. Due to cytotoxicity, only concentrations up to 8.74µg/mL without S9 and 60µg/mL with S9 could be evaluated. No biologically relevant increase in the mutant frequency was observed in two repeated experiments. The test substance is thus considered non-mutagenic in mammalian cells (BASF, 2015).

 

in vivo:

Genetic toxicity

There are no valid data available to assess the genetic toxicity of 1,6-hexanedioldiacrylate in-vivo. However, there are valid data available which assessed the genetic toxicity toxicity of the structurally related tripropylene glycol diacrylate (Cas No. 42978-66-5) in-vivo.

 

Tripropylene glycol diacrylate showed no mutagenic activity in in-vivo assays with rodents. In a mouse micronucleus assay on polychromatic erythrocytes, tripropylene glycol diacrylate led to a negative result after single oral administration of 87.5, 175, 350 mg/kg bw. Sampling times were 24 and 48 h. As a negative control, male mice were administered merely the vehicle, olive oil,by the same route, which gave frequencies of micronucleated polychromatic erythrocytes within the historical control range. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity. The administration of the test substance led to clinical signs. According to the results of the present study, the single intraperitoneal administration of tripropylene glycol diacrylate did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals and within the range of the historical control data. A dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected from about of 87.5 mg/kg body weight onward (BASF, 2004).

 

Another in vivo micronucleus test was also negative after oral administration of tripropylene glycol diacrylate. In this test doses of 2000, 1000 and 500 mg/kg bw were administered. Sampling time was 24 and 48 h after administration. 5 males per dose and harvest time point were used. All animals in all the dose groups and controls appeared normal immediately after dosing and remained healthy until the appropriate harvest timepoint. TPGDA did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (500, 1000, or 2000 mg/kg). TPGDA was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose of the test article analyzed. The vehicle control group had approximately≤0.09% micronucleated PCEs and the group mean was within the historical control range. The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control (Covance, 2007).

 

In another study the test substance was applied dermally to Tg.AC mice (3 times a week for 20 weeks). Peripheral blood leukocytes were evaluated for DNA damage (single-strand breaks, alkali labile sites, DNA crosslinking) at weeks 4, 8, 12, 16, and 20 by using the alkaline (pH > 13) single cell gel (SCG) assay. Peripheral blood polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were evaluated for the presence of micronuclei at week 20. The extent of DNA migration in leukocytes of mice treated by dermal application with TPGDA at 1, 5, or 10 μmol per mouse was not significantly different, either by trend test analysis or by a pairwise comparison of each treatment dose against the concurrent vehicle control, at any sample time. TPA (0.002 μmol per mouse), the positive control for the tumorigenicity studies, also failed to significantly alter the extent of DNA migration or its intercellular dispersion in leukocytes of mice treated by dermal application.After 20 weeks of treatment, the frequency of micronucleated PCE and NCE in blood were not increased in the mice treated with TPGDA or TPA. The percentage of PCE was increased in mice treated with TPGDA. This increase was highly significant. By a pairwise comparison, the lowest effective dose of TPGDA inducing a significant increase in percentage of PCE was 10 μmol per mouse. TPA, at 0.002 μmol per mouse also induced a marginally nonsignificant increase in the percentage of PCE. This observed increase in the rate of erythropoiesis may reflect bone marrow/blood toxicity, a homeostatic mechanism in response to the treatment-induced tumor burden, and/or a hematopoietic response to epidermal keratinocyte cytokines induced by tissue injury (Tice, 1997).

Justification for classification or non-classification

The test substance was not genotoxic in in vitro experiments using mammalian and bacterial cells.

There are currently no data available to classify 1,6-hexanedioldiacrylate for causing genotoxicity in-vivo; however, results obtained for the structurally similar tripropylene glycol diacrylate (Cas No. 42978-66-5), which were adopted to 1,6 -hexanedioldiacrylate by read-across, were negative for genotoxic effects in vivo.

Based on the available data for genetic toxicity, the test item is no subject to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).