(Z)-2-butene-1,4-diol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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: Non-GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Test concentrations with justification for top dose:

20 - 5000 ug/plate (standard plate test) and (preincubation test)

Vehicle / solvent:

Distilled water

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See study details below

Evaluation criteria:

Doubling of the spontaneous mutation rate
Dose-response relationship
Reproducibility of the results

Statistics:

None

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:

other: Slight, <50% at 5000 mcg/plate with S9 in some strains using titer method, but no reduction in background bacterial lawns on test plates.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Toxicity:

Slight, <50% at 5000 mcg/plate with S9 in some strains using titer method, but no reduction in background bacterial lawns on test plates.

Mutagenicity:

An increase in the number of his+ revertants was not observed both in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

According to the result ot the present study, the test substance 2 -Buten-1,4 -diol is not mutagenic in the Ames test under the experimental conditions chosen here.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2018 - January 2019

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:

29 July 2016

Deviations:

no

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 locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Japanese Collection of Research Bioresources (JCRB), maintained in the Mutagenicity Section at Jai Research Foundation
- Number of passages if applicable: CHO-K1 cell line passage number 26 was used in cytotoxicity test and passage number 25 in the main study.

The cells were grown as monolayer in disposable tissue culture flasks. The cell line free from mycoplasma contamination was used in the study. Cultures were free from any contamination during the conduct of the study.

α-MEM (Minimum Essential Medium, Eagle α-Modification with nucleosides) with nucleosides (Gupta R.S., 1984) with 10% heat inactivated, sterile, fetal bovine serum was used as the culture medium to grow the CHO-K1 cell line. Culture medium was supplemented with antibiotic and antimycotic solution (Penicillin: 50 IU/mL; Streptomycin: 50 µg/mL and Amphotericin B: 0.25-0.5 μg/mL). At the time of selection Minimum Essential Medium Eagle alpha-modification without nucleosides (alpha-MEM w/o NS) with 10% dialyzed fetal bovine serum was used.

The medium to eliminate the existing mutants in the culture for treatment was prepared by addition of 2 mL of reconstituted HAT supplement to 98 mL of α-MEM w/o NS with 5% fetal bovine serum [50X vial of HAT media supplement was reconstituted using 10 mL of sterile α-MEM w/o NS. The reconstituted supplement contains 5 x 10-5 Hypoxanthine, 2 x 10-5 M Aminopterine and 8 x 10-4 M Thymidine].
2-amino-6-mercaptopurine (6-thioguanine) was used as selective agent at a concentration of 5 µg/mL alpha-MEM without nucleosides.

Disposable tissue cultures flasks of 25 cm2 (Nunc during cytotoxicity test) and 75 cm2 culture (Nunc during mutagenicity test) area with canted neck were used to culture the cell line and the treatment was given in the same flask. 60 mm disposable culture dishes (Corning) were used to determine the cloning efficiency. Tissue culture dishes (Corning) of 100 mm were used to select mutant colonies.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

55.1, 110.2, 220.3, 440.6 and 881.2 µg/mL in the absence and presence of metabolic activation based on cytotoxicity test and considering substance purity (correction factor 0.9938). The highest concentration (881.2 µg/mL) is equal to 10 mM recommended by the Guideline.

Vehicle / solvent:

distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

HAT (Hypoxanthine Aminopterine Thymidine media supplement) treated (Nestmann, E.R. et al., 1991) cells showing normal growth were used for preparing cultures for treatment. Approximately 24 hours prior to treatment, 28 culture flask (75 cm2) were prepared by seeding 2025000 cells per flask. The culture flasks prepared for treatment were incubated at 37 ± 1 °C and 5% CO2 in humid air using a CO2 incubator (standard conditions). The day of preparation of culture was recorded as ‘day 0’.

The day of treatment was recorded as day 1 in this experiment. Twenty eight flask (fourteen each for treatment in absence and presence of metabolic activation) culture flasks prepared on ‘day 0’ were observed under an inverted microscope to assess their growth and culture conditions. Cultures free from contamination were used during main study. All test cultures were identified using self-adhesive labels containing a test item code, which identifies the test item, study number, treatment phase and replicate.
The media in culture flasks was removed prior to treatment. Serum free medium was used for the treatment. For treatment in presence of metabolic activation, media containing S9 was used at a final concentration 2% v/v S9 mix.

Two culture flasks were maintained for each test concentration, negative and positive controls. The cultures were incubated at 37 ± 1 °C and 5% CO2 in humidified air for 4 hours. At the end of the exposure period, the treatment medium was removed from the flasks and the cell surface washed using Dulbecco’s Phosphate Buffered Saline (DPBS). The cells were trypsinized and suspended in complete medium to obtain single cells. The cell concentration was determined using a hemocytometer and adjusted accordingly with complete medium. The cell concentration in the flasks was adjusted to 1 - 2 x 10^5 cells/mL. A sample taken from the cell suspension was serially diluted with complete medium to approximately 1000 - 2000 cells/mL.

An aliquot of 100 µL was then dispensed onto the center of 60 mm tissue culture dishes and 5 mL of complete medium added. The plates were incubated for 8 days to determine relative survival and also to demonstrate the cytotoxic effect of selected test concentrations.

An appropriate volume was transferred to fresh twenty eight tissue culture flasks to receive approximately 2 x 10^6 cells for each treatment, negative and positive control. These flasks were kept as expression flasks for mutation. All plates and flasks were incubated in a CO2 incubator at 37 ± 1 °C and 5% CO2.

At the end of the 8 day incubation period the cytotoxicity plates were removed and the medium decanted. The colonies were fixed, stained and washed. The colonies formed on each plate were counted and recorded for calculation of relative survival/cloning efficiency following treatment.

The cultures for mutation expression were subcultured on days 3 and 5, following culture processing on day 1. During the expression period (7 - 9 days) the cell concentration in flasks was adjusted by subculturing on day 3 (one flask per replicate) and 5 (one flask per replicate). The cells in each flask were trypsinized and the cell concentration adjusted to approximately 2 x 10^6 cells/culture, provided with fresh complete medium and incubated under standard conditions.

On day 8, the cells in the flasks (expression flasks) were trypsinized for each test concentration and negative and positive controls, they were then counted and plated for determination of survival and mutant frequency.
The cell concentration was adjusted to approximately 1 - 2 x 10^5 cells/mL for survival plating and 2 x 10^5 cells/mL for selection of mutants. A sample taken from the cell suspension was diluted to approximately 1000 - 2000 cells/mL by serial dilution. An aliquot of 100 µL was then dispensed on the center of 60 mm culture dish and 5 mL of complete medium added. Duplicate plates were maintained for each test concentration, negative and positive controls. The plates were then incubated for 8 days for the determination of survival, at this stage, to calculate the mutant frequency.

1 mL of suspension (2 x 105 cells/mL) (Gupta R.S., 1984) was added into 100 mm culture dish with 10 mL of selective medium (α-MEM without nucleoside - complete medium containing 5 µg/mL of 6-thioguanine). For each treatment 12 dishes were maintained per replicate, i.e. 24 dishes/concentration. The plates were incubated at 37 ± 1 °C and 5% CO2 in humidified air using a CO2 incubator for 8 days.

On day 16, the plates were removed from the incubator and the medium decanted, colonies were then fixed using 3.7% formaldehyde for 10 minutes. The fixative was removed and the colonies stained using 0.4% methylene blue for 10 minutes. The number of colonies formed on each replicate plate was counted and recorded. The number of colonies was used to calculate the absolute cloning efficiency at the time of selection, number of clonable cells and the mutant frequency per 1 x 10^6 clonable cells.

Rationale for test conditions:

According to the OECD Guideline 476

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item was considered to be clearly positive if all the following criteria are met, in any of the experimental conditions examined:
a. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
b. The increase is concentration-related when evaluated with an appropriate trend test.
c. Any of the results are outside the distribution of the historical negative control data (e.g. Poisson based 95% control limit).
When all of these criteria are met, the test item was then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test item was considered clearly negative if, in all experimental conditions examined:
d. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
e. There is no concentration-related increase when evaluated with an appropriate trend test.
f. All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limit).
The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

There is no requirement for verification of a clearly positive or negative response.

Statistics:

Weighted regression analysis was performed to evaluate the dose response relationship (Li, A.P. et al., 1987; Hsie, A.W. et al., 1981) on treatment groups against the negative control group (excluding positive controls).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

SOLUBILITY, PRECIPITATION, pH, OSMOMOLALITY
The test item was found to be soluble in distilled water at 88120 µg/mL. Therefore, distilled water was selected as the vehicle for this study.

A significant change in pH (± 1 unit) or osmolality (≥ 50 mOsm/kg H2O) was not observed at 0 and at 4 h in any of the tested concentrations of 27.5, 55.1, 110.2, 220.3, 440.6 and 881.2 µg/mL of culture medium. No precipitation was observed up to tested concentrations of 881.2 µg/mL.

Considering these results, 881.2 µg/mL was selected as the highest concentration for the cytotoxicity test.

CYTOTOXICITY
Cytotoxicity due to the test item was assessed by calculating the percent relative cloning efficiency following treatment.
The pH and osmolality at the beginning of the treatment at 881.2 µg/mL was 7.36 and 307 mOsm/kg H2O, respectively (compared to 7.31 and 310 mOsm/kg H2O in the negative control) in the absence of metabolic activation, while pH and osmolality at 881.2 µg/mL was 7.37 and 325 mOsm/kg H2O, respectively (compared to 7.31 and 315 mOsm/kg H2O in the negative control), in the presence of metabolic activation. Hence, significant change in the pH or osmolality was not observed up to the dose of 881.2 µg/mL both in the absence and presence of the metabolic activation.

The percent relative cloning efficiency observed was 97.90, 89.67, 92.26, 87.53, 83.58, and 86.11 in the absence of metabolic activation at tested concentration of 27.5, 55.1, 110.2, 220.3, 440.6 and 881.2 µg/mL, respectively. The percent relative cloning efficiency observed was 96.55, 89.75, 94.91, 92.19, 85.41 and 87.30 in the presence of metabolic activation (2% v/v S9), at tested concentrations of 27.5, 55.1, 110.2, 220.3, 440.6 and 881.2 µg/mL in the presence of metabolic activation, respectively.

Based on the observed results, 881.2 µg/mL was selected as the highest concentration in the absence and presence of metabolic activation for the main study experiment.

No relevant influence of the test item on osmolality and pH value was observed in the absence and presence of metabolic activation during main study.

Absolute and Relative Cloning Efficiency/Survival Following Treatment

The mean adjusted absolute cloning efficiency (ACE) and percent relative cloning efficiency/survival following the treatment both in the absence and presence of metabolic activation in main study are summarized as below:

Group (Concentration µg/mL)	Absence		Group ( Concentration µg/mL)	Presence (2% v/v S9 mix)
	Mean Adjusted ACE	RCE(%)		Mean Adjusted ACE	RCE(%)
NC (DW)	4.8839	100	NC (DW)	5.2925	100
T1 (55.1)	4.5644	93.46	T1 (55.1)	4.8684	91.99
T2 (110.2)	4.4491	91.10	T2 (110.2)	4.8935	92.46
T3 (220.3)	4.4523	91.16	T3 (220.3)	4.6112	87.13
T4 (440.6)	4.2180	86.37	T4 (440.6)	4.7483	89.72
T5 (881.2)	4.1895	85.78	T5 (881.2)	4.4916	84.87
PC	3.9267	80.40	PC	4.1977	79.31

Absolute Cloning Efficiency at Selection and Mutant Frequency

The mean absolute cloning efficiency (ACE) at selection and mean mutation frequency per 1 x10⁶cells (MF) in the absence and presence of metabolic activation for main study are provided below:

Group (Concentration in µg/mL)	Absence		Group (Concentration in µg/mL)	Presence (2% v/v S9 mix)
	Mean ACE	Mean MF		Mean ACE	Mean MF
NC (DW)	0.7018	18.14	NC (DW)	0.7065	16.55
T1 (55.1)	0.6602	17.38	T1 (55.1)	0.6744	17.02
T2 (110.2)	0.6512	17.35	T2 (110.2)	0.6584	17.75
T3 (220.3)	0.6192	19.20	T3 (220.3)	0.6423	17.27
T4 (440.6)	0.5996	16.37	T4 (440.6)	0.6212	15.95
T5 (881.2)	0.5820	17.25	T5 (881.2)	0.5659	18.84
PC	0.5890	360.01	PC	0.5840	350.06

Conclusions:

It is concluded that 1,4-Butenediol does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation (2% v/v S9 mix) under the given experimental conditions.

Executive summary:

It is concluded that 1,4-Butenediol does not have potential to induce gene mutations at the hprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation (2% v/v S9 mix) under the given experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH WIGA , Sulzfeld
- Weight at study initiation: 26 g (mean)
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): Kliba Haltungsdiaet; Klingenmuehle AG
- Water (e.g. ad libitum): tap water

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: water

Duration of treatment / exposure:

single oral administration

Frequency of treatment:

once

Remarks:

Doses / Concentrations:
100, 200, 400 mg/kg in 10 ml/kg distilled water
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

- Doses / concentrations: 20 mg/kg bw cyclophosphamide
- Doses / concentrations: 0.15 mg/Kg bw vincristine

Tissues and cell types examined:

Bone marrow

Statistics:

Statistical evaluation was performed using the program system MUKERN (BASF AG).

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

According to the results of the present study, the single oral administration of 2-Butene-1,4-diol did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

 

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

 

Under the experimental conditions chosen here, the test substance does not have any chromosome-damaging (clasto- genic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.

Conclusions:

Interpretation of results (migrated information): negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

1,4 -Butenediol (B2D) should not be classified as a mutagen under the EU CLP classification criteria (Regulation (EC) 1272/2008) on the basis that B2D was not genotoxic in the Ames test (BASF, 1989), in vitro cell gene mutation assay (JRF, 2019) and did not induce chromosomal aberrations in a chromosome aberration assay (BASF, 1994).