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Genetic toxicity in vitro

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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:
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
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:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
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 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)

Additional information

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).