reaction products of 1,4-butanediol with 1,3-chloro-2,3-epoxypropane, esters with prop-2-enoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several genotoxicity tests were performed to evaluate the mutagen potential of butane-1,4-diylbis(oxy-2-hydroxypropane-3,1-diyl) bisacrylate. Negative results were observed in the Ames test (OECD 471) and in the HPRT (OECD 476), however the in vitro micronucleus assay (OECD 487) is positive.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted May 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: 8 g Difco nutrient broth + 5 g NaCI/liter
- Properly maintained: yes

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: 8 g Difco nutrient broth + 5 g NaCI/liter
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 rat liver induced S9 mix

Test concentrations with justification for top dose:

20µg, 100µg, 500µg, 2500µg, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: low water solubility of test substance, DMSO had been demonstrated to be suitable in bacterial lreverse mutation tests and historical control data are available

Untreated negative controls:

other: sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

with S9

Untreated negative controls:

other: sterility contol

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for TA 1535, TA 100; 4-nitro-o-phenylendiamine (NOPD) for TA 98; 9-aminoacridine (AAC) for TA 1537; 4-nitroquinoline-N-oxide (4-NQO) for E . coli WP2 uvrA

Remarks:

without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; with and without preincubation

DURATION
- Preincubation period: 20min
- Exposure duration: 48h,72h

SELECTION AGENT (mutation assays): lack of tryptophane in soft agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (=reduced his- or trp- background
growth ); reduction in the titer

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met :
• A dose-related and reproducible increase in the number of revertant colonies, i .e . about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if :
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other .

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight at >= 2500µg/ml (standard) or >= 500 - 2500µg/ml (preincubation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight at >= 2500µg/ml (standard) or >= 500 - 2500µg/ml (preincubation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight at >= 2500µg/ml (standard) or >= 500 - 2500µg/ml (preincubation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight at >= 2500µg/ml (standard) or >= 500 - 2500µg/ml (preincubation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No increase in the number of his + or trp+ revertants in both, the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.
Cytotoxicity was observed in Salmonella strains in the preincubation assay at and above 500 µg/plate - 2500 µg/plate depending on strain.

Conclusions:

Thus, under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic agent in a bacterial reverse mutation test.

Executive summary:

In a gene mutation assay in bacteria (Ames) according to OECD471 and GLP (2002), 1,4 -butanediylbis[oxy(2 -hydroxy-3,1 -propanediyl)] diacrylate did not lead to an increase in the number of his + or trp+ revertants in both, the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Concentrations up to 5000µg/plate were tested in Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli 1412 uvrA. Cytotoxicity was observed in Salmonella strains in the preincubation assay at and above 500µg/plate - 2500µg/plate depending on strain. S9 fraction was prepared from aroclor 1254 induced rat liver.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adoted May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

adopted Aug. 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

4h treatment without S9: 1.3, 2.5, 5.0, 10.0µg/ml
4h treatment with S9: 20, 40, 80, 160, 240µg/ml
24h treatment without S9: 0.5, 1.0, 2.0, 3.0, 4.0µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium):7
- Selection time (if incubation with a selection agent): 7-10 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
- The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

Evaluation of Results
- A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item is classified as mutagenic, if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

Statistics:

linear regression (least squares) to assess dose-dependency

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=10µg/ml (4h exposure), >=4µg/ml (24h exposure)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=240µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Additional data :
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 10.0 µg/mL and above without metabolic activation and at 240 µg/mL and above with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 4.0 µg/mL without metabolic activation and at 240 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with metabolic activation. Without metabolic activation the relative cloning efficiency went down to 24.9% which is sufficiently close to the upper limit of approximately 20%.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. An increase of the induction factor reaching or exceeding the induction factor of three times the mutation frequency of the corresponding solvent control was observed in both cultures of the first experiment with metabolic activation at 240 µg/mL. However, the increased values of the mutation frequency described above were judged as biologically irrelevant as the absolute values of the mutation frequency remained within the historical range of solvent controls (3.4 to 36.6 mutant colonies per 106 cells) at all of the data points mentioned above and the increase was not reproduced in the second experiment with metabolic activation. The level of cytotoxicity was severe at 240.0 µg/mL in the first experiment with metabolic activation with a relative cloning efficiency I right at the lower limit of 10% (9.8% in both cultures). Therefore, the moderate increase of the mutation frequency within the historical range of solvent controls is based on cytotoxicity. The threshold was also exceeded in the second culture of the second experiment at 80.0 and 160.0 µg/mL. This effect however, is based on the low solvent control of just 4.3 mutant colonies per 106 cells and of no biological relevance at all.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was detected in both cultures of the first experiment with metabolic activation. This trend however, was judged as irrelevant as the mutation frequency remained within the historical range of solvent controls at all of the data points.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.3 up to 16.0 mutants per 106 cells; the range of the groups treated with the test item was from 5.9 up to 33.9 mutants per 106 cells.
EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.


TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: only above the concentrations used in the presence of S9 mix (>= 312.5µg/ml)

RANGE-FINDING/SCREENING STUDIES:
Strong cytotoxicity was observed at 20µg/ml and above after 4h exposure and at and above 2.5µg/ml after 24h exposure in the absence of metabolic activiation. In the presence of metabolic activation strong toxic effects occured at and above 312.5µg/ml after 4h exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: valid

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

The test substance was also not mutagenic in an HPRT assay with mammalian cells according to OECD 476 and GLP. Two independent experiments were carried out, both with and without the addition of phenobarbital and ß-naphthoflavone induced rat liver S9 mix. The maximum doses assessed were limited by the cytotoxic properties of the test substance (the highest dose chosen produced excessive cytotoxicty in all experiments). After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of 7 days and a selection period of 7 -10 days followed. The colonies of each test group were stained with methylene blue and counted. All test groups resulted in mutant frequencies within historical control data and in no reproducible increase above concurrent control frequencies. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

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:

other: OECD 487 - In vitro Mammalian Cell Micronucleus Test, adopted July 2010

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM containing an L-glutamine source supplemented with 10% FCS, 1% penicillin/streptomycin, 1% amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

1st experiment
Without S9: 3.13, 6.25, 12.5µg/ml
With S9: 25, 50, 100µg/ml
2nd experiment
Without S9: 1.88, 3.75, 7.5µg/ml
With S9: 15, 30, 60µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: good solubility of the test substance and availability of historical control data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium): 20h
- Fixation time (start of exposure up to fixation or harvest of cells): 24h

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 2000 per dose group

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC), proliferation index

OTHER EXAMINATIONS:
- Other: morphology, attachment to the slides

Evaluation criteria:

The in vitro micronucleus assay 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.
• The number of cells containing micronuclei in the vehicle control was within the range of the historical negative control data.
• The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells.

A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
• The number of micronucleated cells exceeds both the value of the concurrent vehicle control and the range of the historical negative control data.

A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the dose groups is not significant increased above the concurrent vehicle control value and is within the range of the historical negative control data.

Statistics:

The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 15µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

close to cytotoxic concentration

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=120µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Addtional data :
In this study, statistically significant increased numbers of micronucleated cells were observed after 4 hours exposure in all experimental parts either without S9 mix or after the addition of a metabolizing system. In the 1st Experiment after 4 hours treatment in the absence of metabolic activation the rate of cells containing micronuclei was dose related, statistically significant increased. The values at 6.25 and 12.5 µg/mL (2.5 and 3.5% micronucleated cells, respectively) exceeded either the concurrent vehicle control value (0.8% micronucleated cells) or our laboratory’s historical negative control data range (0.1 - 1.8% micronucleated cells). In the independent repeat experiment to corroborate this finding, designated 2nd Experiment in the absence of S9 mix, a single statistically significant value at an intermediate concentration of 3.75 µg/mL was found (1.7% micronucleated cells). Although there was a statistical finding compared to the respective vehicle control group (0.6% micronucleated cells) the micronucleus frequencies of all test groups evaluated (0.8, 1.7 and 0.7% micronucleated cells) were within the range of our laboratory’s historical negative control data (0.1 - 1.8% micronucleated cells).
In the presence of S9 mix, in the 1st Experiment after test substance treatment a single statistically significant value was observed at the highest applied concentration of 100.0 µg/mL. This value (2.5% micronucleated cells) exceeded both the concurrent vehicle control value (1.1% micronucleated cells) and our historical negative control data range (0.1 - 1.8% micronucleated cells). In the 2nd Experiment in the presence of metabolic activation when tested up to clearly cytotoxic concentrations a dose-dependent increase in the number of cells containing micronuclei was obtained. The values at 30.0 and 60.0 µg/mL (1.8 and 3.4% micronucleated cells, respectively) were statistically significant increased compared to the respective vehicle control value (1.0% micronucleated cells). Micronucleus rate at the highest concentration evaluated clearly exceeded our laboratory’s historical negative control data range (0.1 - 1.8% micronucleated cells).
The positive control substances EMS (without S9 mix; 500 µg/mL) and CPP (with S9 mix; 2.5 µg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (2.8 – 5.0% micronucleated cells) was clearly above the range of our historical negative control data range (0.1 - 1.8% micronucleated cells) and within our historical positive control data range (2.3 – 23.5% micronucleated cells).

DIscussion of the results :
According to the results of the present in vitro micronucleus assay, the test substance led to a biologically relevant increase in the number of micronucleated cells after the addition of a metabolizing system in two experiments performed independently of each other. The observation of dose-related increased micronucleus frequencies in the 1st Experiment in the absence of metabolic activation was not confirmed in the repeat experiment. However, in the 2nd Experiment a statistically significant value at the border of our laboratory’s historical negative control data range was obtained. In this study, the concentrations showing relevant increased micronucleus rates were all close to the border of severe cytotoxicity. In all cases higher concentrations were not scorable for cytogenetic damage due to strong toxic effects. Thus, it has to be concluded that the test substance is a high toxicity genotoxin.
The number of micronucleated cells in the vehicle control groups were within our historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of micronuclei induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

PROLIFERATION INDEX
The proliferation index (PI) is based on the scoring of 1 000 cells per culture (2 000 cells per test group) for the different test groups without and with metabolic activation and includes the measurement of colony size. In this study, in the absence and the presence of S9 mix no cytotoxicity indicated by reduced PI values was observed at the test groups scored for cytogenetic damage. However, in several test groups scoring was impossible due to low slide quality and/or reduced cell numbers induced by cytotoxicity.

RELATIVE INCREASE IN CELL COUNT
In addition, in both main experiments in the absence of S9 mix growth inhibition indicated by reduced cell counts was observed from about 15.0 µg/mL onward. In the 2nd Experiment in the presence of S9 mix clearly reduced cell numbers were measured about 120.0 µg/mL onward.

CELL MORPHOLOGY
In this study, cell morphology and/or attachment to slides was adversely influenced (grade =3) from 7.50 µg/mL in the experimental parts in the absence of S9 mix and from 120 µg/mL
in the presence of S9 mix in the 2nd Experiment.


TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced
- Effects of osmolality: not influenced
- Precipitation: none observed

RANGE-FINDING/SCREENING STUDIES:
The maximum concentration was limited by cytotoxicity. In the pretest, exceeding cytotoxicity was observed at 54.7µg/mL with S9 and at 27.3µg/mL without S9, the lowest concentration tested.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without S9 mix, cytotoxocity, i.e. RICC values below 45%, were oberserved at and above 15µg/mL. Additional morphological observation showed a first effect on cell attachment (few rounded cells) already at 1.8µg/mL. At and above 7.5µg/mL most cells were rounded and the number of analyzable cells was reduced.
With S9 mix, concentrations of and above 120µg/mL were cytotoxic as determined via RICC. Slightly reduced attachment of the cells to the slides and a few rounded cells were observed at and above 60µg/mL.

Table 1: Summary table - experimental parts without S9 mix

Experiment	Test groups	S9 mix	Prec.*	Genotoxicity Micronucleated cells** [%]	Proliferation index (PI)	RICC*** [%]
1 2	Vehicle control 0.78 µg/mL 1.56 µg/mL 3.13 µg/mL 6.25 µg/mL 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL Positive control Vehicle control 1.88 µg/mL 3.75 µg/mL 7.50 µg/mL 15.00 µg/mL 30.00 µg/mL Positive control	- - - - - - - - - - - - - - - -	n.d. - - - - - - - n.d. n.d. - - - - -  n.d.	0.8 n.d. n.d. 1.5s 2.5s 3.5s n.s. n.s. 2.8s 0.6 0.8 1.7s 0.7 n.s. n.s. 2.8s	2.13 n.d. n.d. 2.14 2.30 2.22 n.s. n.s. 2.27 2.21 2.12 2.20 1.98 n.s. n.s. 2.13	100.0 104.5 115.8 92.5 89.5 62.4 24.1 -7.5 n.t. 100.0 110.3 114.3 101.7 36.0 22.3 n.t.

Table 2: Summary table - experimental parts with S9 mix

Experiment	Test groups	S9 mix	Prec.*	Genotoxicity Micronucleated cells** [%]	Proliferation index (PI)	RICC*** [%]
1 2	Vehicle control 0.78 µg/mL 1.56 µg/mL 3.13 µg/mL 6.25 µg/mL 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL Positive control Vehicle control 1.88 µg/mL 3.75 µg/mL 7.50 µg/mL 15.00 µg/mL 30.00 µg/mL Positive control	+ + + + + + + + + + + + + + + +	n.d. - - - - - - - n.d. n.d. - - - - - n.d.	1.1 n.d. n.d. n.d. n.d. 1.4 1.3 2.5s 3.9s 1.0 1.0 1.8s 3.4s n.s. n.s. 5.0s	2.17 n.d. n.d. n.d. n.d. 2.20 2.20 2.03 1.84 2.24 2.17 2.19 2.27 n.s. n.s. 1.91	100.0 97.7 108.1 93.1 96.0 115.6 109.6 90.8 n.t. 100.0 112.8 95.3 83.5 43.9 8.4 n.t.

* Precipitation in culture medium at the end of exposure period

** Relative number of micronucleated cells per 2000 cells scored per test group

*** Relative increase in cell count (RICC)

S Frequency statistically significant higher than corresponding control values

n.s. Not scorable due to strong cytotoxicity

n.t. Not tested

n.d. Not determined

Table 3: Cell morphology

Without S9 mix
1st experiment				2nd experiment
Test groups	Attachment to slides	Quality of cells		Test groups	Attachment to slides	Quality of cells
Vehicle control* 0.78 µg/mL 1.56 µg/mL 3.13 µg/mL 6.25 µg/mL 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL 500.00 µg/mL EMS 600.00 µg/mL EMS	1 1 1 2 2 3 3 4 1 1	E1 E1 E1 E1 E1 E2 N2 N1 E1 E1		Vehicle control* 1.88 µg/mL 3.75 µg/mL 7.50 µg/mL 15.00 µg/mL 30.00 µg/mL 500.00 µg/mL EMS 600.00 µg/mL EMS	1 2 2 3 3 3 1 1	E1 E1 E1 E2 N2 N1 E1 E1
With S9 mix
1st experiment				2nd experiment
Test groups	Attachment to slides	Quality of cells		Test groups	Attachment to slides	Quality of cells
Vehicle control* 1.56 µg/mL 3.13 µg/mL 6.25 µg/mL 12.50 µg/mL 25.00 µg/mL 50.00 µg/mL 100.00 µg/mL 2.50 µg/mL CPP	1 1 1 1 1 1 1 2 1	E1 E1 E1 E1 E1 E1 E1 E1 E1		Vehicle control* 15.00 µg/mL 30.00 µg/mL 60.00 µg/mL 120.00 µg/mL 240.00 µg/mL 2.50 µg/mL CPP	1 1 1 2 3 3 1	E1 E1 E1 E1 N2 N1 E1

1 = complete attachment, i.e., fibroblast-like cells

2 = slightly reduced attachment, i.e., few rounded cells

3 = reduced attachment, i.e., most cells rounded

4 = complete detachment, i.e., all cells rounded

E1 = sufficient cells of good quality

E2 = reduced number of analysable cells

N1 = evaluation not possible - no or only a few cells for evaluation

N2 = evaluation not possible - no or only a few cells for evaluation and with fragmentations

Conclusions:

Thus, under the experimental conditions chosen here, the conclusion is drawn that the test substance has the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Executive summary:

The substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following doses were tested. Test groups printed in bold type were evaluated.

A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. 2 000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide,

led to the expected increase in the number of cells containing micronuclei.

Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) was observed at least in the highest applied test substance concentration in all experimental parts of this study, except in the 1st Experiment with metabolic activation. Statistically significant increased numbers of micronucleated cells were observed after 4 hours exposure in all experimental parts in the absence and presence of metabolic activation. Biologically relevant increased values exceeding the range of our historical negative control data were observed in the 1st Experiment in the absence of metabolic activation and in both experiments in the presence of metabolic activation. Besides in some cases a dose-related increase in genotoxicity was observed.

Thus, under the experimental conditions described, the test substancehas the potential to induce structural chromosome damage (clastogenic) and/or numerical chromosome aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In order to clarify if the positive results obtained in the in vitro micronucleus test is only due to cytotoxicity and to evaluate the biological relevance, a micronucleus test in vivo was performed. At the end, the test substance does not induce cytogenetic damage in bone marrow cells of mice (OECD 474).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2012 - April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

adopted 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5-8 weekds
- Weight at study initiation: on average 27.9g
- Assigned to test groups randomly: yes
- Housing: single in Makrolon type M II cages
- Diet (e.g. ad libitum): standardized pelleted feed ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: at least 5 days

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

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO (4mL/kg b.w.) + corn oil
- Justification for choice of solvent/vehicle: solubility of the test substance, historic control data is available
- Concentration of test material in vehicle: 25 - 100mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was dissolved by thourough shaking in DMSO (4 mL/kg) and than emulsified in corn oil (up to 10 mL/kg). All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

one admistration

Frequency of treatment:

one admistration

Post exposure period:

24h , 48h (only high dose)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP), Vincristine sulfate (VCR)
- Justification for choice of positive control(s): both substances are well-established reference clastogens and aneugens, respectively
- Route of administration: via gavage in deionized water
- Doses / concentrations: 20mg/kg (CPP), 0.15mg/kg (VCR)

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a pretest to determine the acute oral toxicity in males and females, lethality was observed at the recommended highest dose of 2 000 mg/kg body weight. In addition, clear signs of toxicity were observed: piloerection, hunched posture and reduced general condition. However, there were no distinct differences in clinical observations between male and female animals. Thus, only male animals were used in the main experiment. Based on the data of the pretest a dose of 1 000 mg/kg body weight was defined as MTD (maximum tolerated dose) and was selected as the highest dose in the present cytogenetic study. 500 mg/kg and 250 mg/kg body weight were administered as further doses.

DETAILS OF SLIDE PREPARATION:
One drop of isolated bone marrow cells in FCS was dropped onto a clean microscopic slide, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained with eosin and methylene blue (modified May-Grünwald solution) for about 5 minutes. After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2 - 3 minutes. Subsequently, the slides were stained with Giemsa solution for about 15 minutes. After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 2 000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 10 000 PCEs were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Fraction of polychromatic erythrocytes containing micronuclei (index of clastogenic or aneugenic activity)
• Fraction of normochromatic erythrocytes containing micronuclei (24h value: control value for situation before test substance administration)
• Ratio of polychromatic to normochromatic erythrocytes (indicator that the test substance reached the bone marrow)
• Number of small micronuclei (d < D/4) and of large micronuclei (d = D/4) [d = diameter of micronucleus, D = cell diameter] (differentiation between a clastogenic and spindle poison effect, respectively)

Evaluation criteria:

Acceptance criteria
• The quality of the slides must allow the evaluation of a sufficient number of analyzable cells; i. e. = 2 000 PCEs per animal and a clear differentiation between PCEs and NCEs.
• The ratio of PCEs/NCEs in the concurrent vehicle control animals has to be within the normal range for the animal strain selected.
• The number of cells containing micronuclei in vehicle control animals has to be within the range of the historical vehicle control data both for PCEs and for NCEs.
• The two positive control substances have to induce a distinct increase in the number of PCEs containing small and/or large micronuclei within the range of the historical positive control data or above.

A finding is considered positive, if the number of PCEs containing micronuclei is statistically significant and dose-related increased, and the number of PCEs containing micronuclei exceeds both the concurrent vehicle control value and the range of the historical vehicle control data.

A test substance is considered negative, if the number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

PCE to NCE ratio suggests a slight inhibition of erythropoiesis at 1000mg/kg after 48h

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Negative and positive controls :
Vehicle control male mice showed frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. The single oral administration of the vehicle DMSO/corn oil in a volume of 10 mL/kg body weight led to 0.9‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 0.8‰ after the 48-hour sacrifice interval, respectively.

Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.
The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (16.4‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected.
Vincristine sulfate, a spindle poison, produced a statistically significant increase (24.8‰) in the number of polychromatic erythrocytes containing micronuclei. A significant portion increase, 8.5‰ was attributable to large micronuclei.


Results with the test susbtance:
After the single administration of the highest dose of 1 000 mg/kg body weight, 0.9‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 0.8‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of 1.1‰ (500 mg/kg group) and 0.7‰ (250 mg/kg group) were detected at a sacrifice interval of 24 hours in each case.

The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.
A slight inhibition of erythropoiesis induced by the treatment of mice with test substance was detected at 1 000 mg/kg body weight at 48 hours sacrifice interval. The ratio of polychromatic to normochromatic erythrocytes was influenced compared to the respective vehicle control group which is an indication of target organ toxicity.

Clinical signs :
The administration of the test substance did not lead to clinical signs of toxicity in the main experiment. However, lethality and clear clinical observations were found in the pretest at a two-fold higher dose of 2 000 mg/kg body weight.

Conclusions:

Under the condition of this study, the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Executive summary:

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to 5 male animals per group at dose levels of 250 mg/kg, 500 mg/kg and 1 000 mg/kg body weight. The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours (all dosages and control), and 48 hours (high dose and vehicle control) after administration. The preparations were stained, and 2 000 polychromatic erythrocytes were evaluated per animal. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

Vehicle control male mice showed frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.

According to the results of the present study, there are thus no statistical significances or biologically relevant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control groups and the three dose groups (250 mg/kg, 500 mg/kg and 1 000 mg/kg) or between the two sacrifice intervals (24 and 48 hours). The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the vehicle control values at any of the sacrifice intervals and was within the historical vehicle control data range. Nor were large micronuclei (d = D/4) observed either in the vehicle control group or in the three dose groups treated with test substance.

Based on the pretest, 1 000 mg/kg body weight was defined as maximum tolerated dose (MTD) due to lethality observed at 2 000 mg/kg body weight. Although no signs of toxicity were found after treatment with 1 000 mg/kg body weight in the main experiment, bioavailability of the test substance in the target organ was shown by reduction of polychromatic erythrocytes at 48-hour sacrifice interval.

In this study, after single oral administration of the vehicle DMSO/corn oil the ratio of PCEs/NCEs in the vehicle control animals at both sacrifice intervals was within the normal range for the animal strain selected.

Thus, under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Gene mutation in bacteria (2002)

In a gene mutation assay in bacteria (Ames) according to OECD 471 and GLP. Butane-1,4-diylbis(oxy-2-hydroxypropane-3,1-diyl) bisacrylate did not lead to an increase in the number of his + or trp+ revertants in both, the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Concentrations up to 5000µg/plate were tested in Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli 1412 uvrA. Cytotoxicity was observed in Salmonella strains in the preincubation assay at and above 500µg/plate - 2500µg/plate depending on strain. S9 fraction was prepared from aroclor 1254 induced rat liver.

 

Gene mutation in mammalian cells (2012)

The test substance was also not mutagenic in an HPRT assay with mammalian cells according to OECD 476 and GLP. Two independent experiments were carried out, both with and without the addition of phenobarbital and ß-naphthoflavone induced rat liver S9 mix. The maximum doses assessed were limited by the cytotoxic properties of the test substance (the highest dose chosen produced excessive cytotoxicty in all experiments). After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of 7 days and a selection period of 7 -10 days followed. The colonies of each test group were stained with methylene blue and counted. All test groups resulted in mutant frequencies within historical control data and in no reproducible increase above concurrent control frequencies. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

 

Micronucleus test in vitro (2012)

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) in two independent experiments with and without the addition of phenobarbital and ß-naphthoflavone induced rat liver S9. After an exposure period of 4h, cells were left to recover for 20h and stained. A sample of at least 1 000 cells for each culture were analysed for micronuclei, i.e., 2 000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei.

Without metabolic activation, a dose dependent increase was observed in the first experiment, which could not be reproduced in the second experiment under identical conditions. In the first experiment, mutant frequencies of 1.5, 2.5, and 3.5% were observed at 3.13, 6.25, and 12.5µg/mL, respectively, which exceeded the concurrent vehicle control (0.8%) as well as historical control values (0.1 - 1.3%). Slightly reduced attachment of the cells to the slides was already observed at 3.13µg/mL; at 12.5µg/mL most cells were rounded. In the second experiment, no dose dependency was observed, and the only significantly increased value was detected at the medium concentration of 3.75µg/mL (1.7%).

 With metabolic activation, the highest dose that could still be evaluated showed a significant increase in mutant frequencies in both experiments (2.5% and 3.4%), which also exceeded historical control data (0.1 - 1.8%). Both concentrations did not yet lead to a reduced RICC, though some rounded cells were already detected. A minimal increase to 120 µg/mL led to death of the majority of cells. This fits the cytotoxicity data obtained with the same cells in the HPRT assay.

Thus, the test substance has the potential to induce structural chromosome damage (clastogenic) and/or numerical chromosome aberrations (aneugenic activity) under in vitro conditions in V79 cells at or close to cytotoxic concentrations. In order to clarify, if this effect is only due to cytotoxicity and to evaluate biological relevance, a micronucleus test in vivo was performed.

 

Genetic toxicity in vivo (2013)

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in DMSO and emulsified in corn oil, was administered once orally to 5 male animals per group at dose levels of 250 mg/kg, 500 mg/kg and 1 000 mg/kg body weight. The animals were sacrificed and the bone marrow of the two femora was prepared 24 hours (all dosages and control), and 48 hours (high dose and vehicle control) after administration. The preparations were stained, and 2 000 polychromatic erythrocytes were evaluated per animal. The normocytes with and without micronuclei occurring per 2 000 polychromatic erythrocytes were also recorded.

Vehicle control male mice showed frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range. Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei. A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes as indication of test substance toxicity in the target organ was detected at 48-hour sacrifice interval.

According to the results of the present study, the single oral administration of test substance did not lead to any biologically relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was close to the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.

Thus, the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

 

 

 

Justification for classification or non-classification

Butane-1,4-diylbis(oxy-2-hydroxypropane-3,1-diyl) bisacrylate did not induce gene mutations in bacteria or mammalian cells. In vitro, chromosome aberrations were observed at or close to cytotoxic concentrations. But since this result could not be confirmed in an in vivo experiment (OECD 474) the test substance does not need to be classified as genotoxic according to the Regulation EC n°1272/2008.