1,1'-[methylenebis(oxy)]dibutane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to column 1 of REACH Annex VII and VIII, the following in vitro tests are required and have been performed :

8.4.1 - an Ames test (OECD 471) with butylal is available with a negative result.

8.4.2 - a Chromosomal aberrationa test (OECD 473) with butylal is available with a negative result.

8.4.3 - a HPRT test (OECD 476) with butylal is available with a negative result.

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

Study period:

From 1998-02-06 to 1998-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP lab following OECD guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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):

None

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

None

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

None

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (see Point 1 under "Any other information on materials and methods incl. table")

Test concentrations with justification for top dose:

The test substance was dissolved in the vehicle at a concentration
. of 50 mg/ml for the preliminary toxicity test and the first mutagenicity experiment,
. of 50, 10 and 5 mg/ml for the second mutagenicity experiment.
The preparations were made immediately before use.

Vehicle / solvent:

The vehicle was dimethylsulfoxide (DMSO), batch Nos. K22294850 549 and I743850 731 (Merck Clévenot, 77500 Chelles, France).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Six known mutagens, dissolved in dimethylsulfoxide (except for mitomycin C which is dissolved in distilled water), were used to check the sensitivity of the test system. Please refer to Table 1 of "Any other information on materials and methods incl. tabl

Details on test system and experimental conditions:

Treatment
The experiments were performed according to:
. direct plate incorporation method (preliminary toxicity test, both experiments without S9 mix, first experiment with S9 mix): test substance solution (0.05 to 0.1 ml), S9 mix (0.5 ml) when required and bacterial suspension (0.1 ml) were mixed with 2 ml of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
. preincubation method (second experiment with S9 mix): test substance solution (0.05 to
0.1 ml), S9 mix (0.5 ml) and bacterial suspension (0.1 ml) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.

After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Artek counter, model 880, O.S.I., 75015 Paris, France).

Preliminary toxicity test
To assess the toxicity of the test substance to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and without S9 mix.
The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Mutagenicity experiments
In two independent experiments, five dose-levels of BUTYLAL (three plates/dose-level) were tested on each strain, with or without S9 mix.

In each experiment, the following controls were included using triplicate plates:
. vehicle controls: each bacterial tester strain treated with the vehicle,
. positive controls: each bacterial tester strain treated with appropriate reference mutagens.

The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

Evaluation criteria:

Treatment of results
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test substance/mutants obtained in the presence of the vehicle), are presented in a table.

Acceptance criteria
This study would be considered valid since the following criteria are fully met:
.the number of revertants in the vehicle controls is consistent with our historical data
.the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.

Evaluation criteria
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

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:

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

Remarks:

determined in the preliminary test

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

determined in the preliminary test

Vehicle controls validity:

valid

Untreated 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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

determined in the preliminary test

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
The test substance was freely soluble in the vehicle (DMSO) at 50 mg/ml.
Consequently, with a maximum dose volume of 100 µl/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.
Without S9 mix, slight to marked toxicity was observed in the TA 98 strain at dose-levels ≥ 500 µg/plate. In the TA 100 strain, moderate to total toxicity was induced at the same dose-levels. At dose-levels ≥ 100 µg/plate, slight to total an slight to moderate toxicity were noted in the TA 102 and WP2 uvrA strains, respectively.
With S9 mix, slight toxicity was observed in the TA 98 and WP2 uvrA strains, at 5000 µg/plate and at dose-levels ≥ 2500 µg/plate, respectively.
Moderate to total toxicity was induced in the TA 100 strain at dose-levels ≥ 1000 µg/plate and in the TA 102 strain at dose-levels  500 µg/plate.

MUTAGENICITY EXPERIMENTS
The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test substance was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.

Experiments without S9 mix:
The selected treatment levels were as follows:
• 62.5, 125, 250, 500 and 1000 µg/plate, for the TA 1535 strain in both experiments and for the TA 1537 strain in the second experiment,
• 31.25, 62.5, 125, 250 and 500 µg/plate, for the 5 remaining strains in the first experiment and for the 4 remaining strains in the second experiment.

In the TA 1535 strain, slight to moderate toxicity was observed at dose-levels ≥ 500 µg/plate in the first experiment. In the second experiment with this strain as well as with the TA 1537 strain, only slight toxicity was noted at 1000 µg/plate. Slight toxicity was noted at 500 µg/plate in the TA 98 strain (first experiment) and in the TA 102 strain (second experiment). In the first experiment with the TA 102 strain, moderate toxicity was observed at dose-levels ≥ 250 µg/plate.
In both the TA 100 and WP2 uvrA strains, slight toxicity was induced at dose-levels ≥ 250 µg/plate, in the first experiment only.
No increase in the number of revertants, in comparison with the vehicle control values, was observed in all tester strains in both experiments.

Experiments with S9 mix:
The selected treatment levels were as follows:
• 31.25, 62.5, 125, 250 and 500 µg/plate, for the TA 102 strain in the first experiment as well as for the TA 1535, TA 1537 and TA 98 strains in the second experiment,
• 62.5, 125, 250, 500 and 1000 µg/plate, for the TA 100 strain in both experiments, for the TA 1535 strain in the first experiment and for the TA 102 strain in the second experiment,
• 312.5, 625, 1250, 2500 and 5000 µg/plate, for the TA 1537, TA 98 strains in the first experiment and for the WP2 uvrA strain in both experiments.

Slight toxicity was observed in the TA 1535 and TA 100 strains at 1000 µg/plate in the first experiment. In the second experiment (preincubation method), slight toxicity was induced in the TA 1535 strain at 500 µg/plate and moderate to total toxicity was noted in the TA 100 strain at dose-levels  500 µg/plate.
Slight to marked toxicity was observed in both TA 1537 and TA 98 strains at dose-levels ≥ 1250 µg/plate in the first experiment. With the preincubation method, slight toxicity was noted at 500 µg/plate in these two tester strains.
In the TA 102 strain, slight toxicity was observed at 1000 µg/plate (second experiment).
Slight to moderate toxicity was induced in the WP2 uvrA strain in the first experiment at dose-levels ≥ 1250 µg/plate. In the second experiment with this strain, slight to marked toxicity was observed at dose-levels ≥ 625 µg/plate.

The test substance did not induce any significant increase in the number of revertants, in both experiments, in any of the six strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions, the test substance BUTYLAL does not show mutagenic activity in the bacterial reverse mutation test on Salmonella typhimurium and Escherichia coli.

Executive summary:

The objective of this study was to evaluate the potential of the test substance BUTYLAL to induce reverse mutation inSalmonella typhimuriumandEscherichia coli.

 

 

Methods 

A preliminary toxicity test was performed to define the dose-levels of BUTYLAL to be used for the mutagenicity study. The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Both experiments were performed according to the direct plate incorporation method except the second with S9 mix, which was performed according to the preincubation method (60 minutes,).

 

Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and
TA 102 and one strain ofEscherichia coli: WP2 uvrA were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test substance BUTYLAL was dissolved in dimethylsulfoxide (DMSO).

 

The dose-levels of the positive controls were as follows:

 

without S9 mix:

. 1 µg/plate of sodium azide (NaN₃): TA 1535 and TA 100 strains,

. 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

. 0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

. 0.5 µg/plate of mitomycin C (MMC): TA 102 strain,

. 2 µg/plate of N-ethyl-N-nitro-nitrosoguanidine (ENNG):WP2 uvrA strain.

 

with S9 mix:

. 2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

. 10 µg/plate of 2-Anthramine (2AM): TA 102 andEscherichia coliWP2 uvrA strains.

 

 

Results 

The test substance was freely soluble in the vehicle at 50 mg/ml.

Consequently, with a maximum dose volume of 100 µl/plate, the dose-levels for the preliminary toxicity test were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Since the test substance was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.

 

Experiments without S9 mix:

The selected treatment levels were as follows:

·      62.5, 125, 250, 500 and 1000 µg/plate, for the TA 1535 strain in both experiments and for the TA 1537 strain in the second experiment,

·      31.25, 62.5, 125, 250 and 500 µg/plate, for the 5 remaining strains in the first experiment and for the 4 remaining strains in the second experiment.

 

Slight to moderate toxicity was observed in all tester strains, depending on the dose-levels.

 

No increase in the number of revertants, in comparison with the vehicle control values, was observed in all tester strains in both experiments.

 

Experiments with S9 mix:

The selected treatment levels were as follows:

·      31.25, 62.5, 125, 250 and 500 µg/plate, for the TA 102 strain in the first experiment as well as for the TA 1535, TA 1537 and TA 98 strains in the second experiment,

·      62.5, 125, 250, 500 and 1000 µg/plate, for the TA 100 strain in both experiments, for the TA 1535 strain in the first experiment and for the TA 102 strain in the second experiment,

·      312.5, 625, 1250, 2500 and 5000 µg/plate, for the TA 1537, TA 98 strains in the first experiment and for the WP2 uvrA strain in both experiments.

 

Slight to total toxicity was observed in all tester strains, depending on the dose-levels and the experimental conditions.

The test substance did not induce any significant increase in the number of revertants, in both experiments, in any of the six strains.

The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

 

Conclusion

 Under the experimental conditions, the test substance BUTYLAL does not show mutagenic activity in the bacterial reverse mutation test on Salmonella typhimurium and Escherichia coli.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 09 June 2017 Experimental completion date 22 September 2017

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

Identification: Butylal
Physical State / Appearance: Clear colourless liquid
CAS Number: 2568-90-3
Storage Conditions: Approximately 4οC in the dark

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell Line
The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).
The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

Cell Culture
Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM), Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The concentrations in the main experiment were based on results from a preliminary cytotoxicity test and were as follows:

4-hour without S9
0, 6.25, 12.5, 25, 50, 100, 200, EMS 500 and 750
4-hour with S9 (2%)
0, 3.13, 6.25, 12.5, 25, 50, 100, 200, 400

Vehicle / solvent:

Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

500 and 750 μg/mL for 4-hour exposure

Positive control substance:

ethylmethanesulphonate

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

1 and 2 μg/mL for 4-hour exposure

Positive control substance:

other: Dimethyl benzanthracene

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Test Procedure
Preliminary Cytotoxicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 1 x 10^7 cells/225 cm2 flask approximately 24 hours before dosing. This was demonstrated to provide at least 20 x 10^6 available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). The dose range of test item was 3.13 to 801.5 μg/mL for both of the exposure groups.
Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of MEM with 10% FBS and incubated for 6 to 7 days at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies (CE).
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment.

Mutagenicity Test – Main Experiment
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 1 x 107 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with up to eight test item concentrations, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). The concentrations range of test item used was 6.25 to 200 μg/mL in the absence of metabolic activation and 3.13 to 400 μg/mL in the presence of metabolic activation.
At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10^6 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10^5 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.
The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical 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.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful.

Statistics:

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test
A concentration range of 3.13 to 801.5 μg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was limited by the formulation of the test item.
A precipitate of the test item was observed at 200.38, 400.75 and 801.5 μg/mL.
There was no evidence of any marked concentration related reductions in cloning efficiency in both the absence and presence of metabolic activation. During the washing phase the two highest concentrations 400.75 and 801.5 μg/mL were discarded due to excessive precipitating dose levels.
The maximum concentration selected for the main mutagenicity experiment was therefore limited by the incidence of precipitate in the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines.

Mutagenicity Test – Main Experiment
The excessive levels of precipitate observed at 400 μg/mL in the presence of metabolic activation, resulted in this concentration not being plated for cloning efficiency and mutant frequency. The concentrations of 6.25 and 12.5 μg/mL in the absence of metabolic activation were considered to be surplus to requirements, as four analysable dose levels including the lowest precipitating dose level were selected to meet the requirements of the OECD 476 guideline. In the presence of metabolic activation the concentrations of 3.13 and 6.25 μg/mL were also considered to be surplus to requirements and therefore not plated for cloning efficiency and mutant frequency. The dose level of 400 μg/mL was also considered to be surplus to requirements due to excessive precipitate and therefore not plated for cloning efficiency and mutant frequency.
A precipitate of the test item was observed at 200 μg/mL in the absence of metabolic activation and 200 and 400 μg/mL in the presence of metabolic activation.
The Day 0 and Day 7 cloning efficiencies for the exposure groups in the absence and presence of metabolic activation are presented in Tables 2 and 3. There were no marked concentration related reductions in the Day 0 cloning efficiency values the absence of metabolic activation. There were no marked concentration related reductions in the Day 0 cloning efficiency values the presence of metabolic activation. There was no evidence of any marked reductions in the Day 7 cloning efficiencies in any of the surviving concentration levels, therefore indicating that residual toxicity had not occurred.
The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.
The vehicle control values were all considered to be within an acceptable range, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

The concentrations of the controls and the test item are given in the table below:

 

Exposure Group	Final concentration of Butylal (µg/mL)
4-hour without S9	0*, 6.25, 12.5, 25*, 50*, 100*, 200*, EMS 500* and 750*
4-hour with S9(2%)	0*, 3.13, 6.25, 12.5*, 25*, 50*, 100*, 200*, 400, DMBA 1.0* and 2.0*

* = Concentrations plated out for cloning efficiency andmutantfrequency

EMS= Ethyl methanesulphonate

DMBA = Dimethyl benzanthracene

Conclusions:

The test item, Butylal did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

Introduction

The purpose of this study is to assess the potential mutagenicity of a test item, supplied by the Sponsor, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Methods

In the main test chinese hamster (V79) cells were treated with the test item at up to eight concentrations, in duplicate, together with vehicle (MEM culture media) and positive controls in both the absence and presence of metabolic activation.

The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate of test item in both the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines. The concentrations of test item plated for cloning efficiency and expression of mutant colonies were as follows:

Exposure Group	Final concentration of Butylal (µg/mL)
4-hour without S9	0*, 6.25, 12.5, 25*, 50*, 100*, 200*,
4-hour with S9 (2%)	0*, 3.13, 6.25, 12.5*, 25*, 50*, 100*, 200*, 400,

*            = Concentrations plated out for cloning efficiencyand mutant frequency

Results

The vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item, Butylal did not induce any toxicologically significant or concentration-related increases in mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

Conclusion

The test item, Butylal was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

 

 

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 July to 10 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by Sponsor, Batch no 1606071800R
- Expiration date of the lot/batch: 07 June 2017
- Purity test date: 07 June 2016
- Purity: 99.9425%
- Physical state/appearance:Clear colourless liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C under nitrogen, in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING :
- Prior to testing the substance was stored at approximately 4°C under nitrogen, in the dark. Prior to each experiment, the test item was accurately weighed, formulated in DMSO and appropriate serial dilutions prepared
- Final dilution of a dissolved stock liquid: The test item was insoluble in Eagle's minimal essential medium with HEPES buffer (MEM) at 16.03mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 80.2 mg/mL, after being warmed at 40°C for 10 minutes and vortexed for 10 seconds in solubility checks performed in-house. This gave a maximum achievable dose level of 802 μg/mL; which matched that achieved with acetone. As DMSO is preferred over acetone as a solvent due to the sensitivity of human lymphocytes to acetone the maximum dose level was reduced to 802 μg/mL.


FORM AS APPLIED IN THE TEST
Liquid (in solution in DMSO)

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: non-smoking human (male) volunteers
- Suitability of cells: suitable
- Normal cell cycle time (negative control): ca. 16 hours

For lymphocytes:
- Sex, age and number of blood donors: 3 male donors, ages below
Preliminary Toxicity Test: male, aged 28 years
Main Experiment 4-hour with S9 and 24 hour continuous: male, aged 25 years
Main Experiment 4-hour without S9: male, aged 21 years
- Whether whole blood or separated lymphocytes were used: whole blood cultures cells
- Whether blood from different donors were pooled or not: No
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 - prepared in-house
- method of preparation of S9 mix - prepared using standardised in-house procedures (Certificate of Efficacy presented in the report)
- concentration or volume of S9 mix and S9 in the final culture medium - 2% S9 when dosed at a 10% volume of S9-mix into culture media
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) Certificate of Efficacy presented in the report

Test concentrations with justification for top dose:

The maximum achievable solubility of Butylal in DMSO was 80.2 mg/mL, after being warmed at 40 oC for 10 minutes and vortexed for 10 seconds, which gave a maximum achievable dose level of 802 μg/mL in the culture medium. Appropriate serial dilutions were performed to give dose levels of 0, 3.13, 6.27, 12.53, 25.06, 50.13, 100.25, 200.5, 401 and 802 μg/mL for the preliminary study. The dose range of test item used for the three main studies was 25 to 300μg/mL.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: The test item was insoluble in Eagle's minimal essential medium with HEPES buffer (MEM) but was soluble in dimethyl sulphoxide (DMSO) at 80.2 mg/mL which was sufficient to give the desired maximim achievable dose. This dose could also be achieved with acetone but DMSO is preferred over acetone as a solvent due to the sensitivity of human lymphocytes to acetone.

- Justification for percentage of solvent in the final culture medium: There was no significant change in pH when the test item, at the concentration of 802 μg/mL ie the maximum achievable dose, was dosed into media and the osmolality did not increase by more than 50 mOsm therefore no effect on the final culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin C was used in the absence of S9 mix and Cyclophosphamide in the presence of S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments :

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time

- If cytokinesis blocked method was used for micronucleus assay: N/a

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4ºC to ensure complete fixation prior to slide preparation.
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985).
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported.

- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not performed
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
cells with 69 chromosomes or more were scored as polyploid cells
- Determination of endoreplication:
incidence of cells with endoreduplicated chromosomes assessed during the microscopic examination of slides was reported

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related

Evaluation criteria:

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline OECD TG 473.
• The required number of cells and concentrations were analyzed.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no significant change in pH when the test item was dosed into media
- Data on osmolality: osmolality did not increase by more than 50 mOsm when test item dosed into the media
- Possibility of evaporation from medium: no
- Water solubility: n/a
- Precipitation and time of the determination: In the preliminary toxicity test precipitation was observable at and above 401 μg/mL in all three exposure groups


STUDY RESULTS
- Concurrent vehicle and positive control data - Yes

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI)
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index.
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes) if seen

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Historical control data is presented in an Appendix to the report
- Negative (solvent/vehicle) historical control data: Historical control data is presented in an Appendix to the report

Remarks on result:

other: non-clastogenic to human lymphocytes in vitro

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 200 μg/mL in both of the exposure groups without metabolic activation. In the presence of metabolic activation (S9), the maximum dose level of the test item with metaphases suitable for scoring was 150 μg/mL.

No precipitate was observed at the end of exposure in blood-free cultures in all exposure groups. Haemolysis was observed at and above 50 μg/mL in all three exposure groups.

The mitotic index data for the Main Experiment confirm the qualitative observations in that modest dose-related inhibition of mitotic index was observed in all exposure groups. In the 4(20)-hour exposure group in the absence of S9, near optimum 47% mitotic inhibition was achieved at 200 μg/mL. In the presence of S9, dose-related inhibition of mitotic index was observed with near optimum 49% inhibition at 150 μg/mL. An inhibition of mitotic index of 62% was noted at 200 μg/mL in the 24-hour continuous exposure group whilst this exceeds the optimum level it was scored for aberrations because the next dose level down exhibited no toxicity.

The maximum dose levels selected for metaphase analysis were selected based on toxicity and were 200 μg/mL for both exposure groups without S9 and 150 μg/mL in the presence of S9.

The main experiment assay was considered valid as it met all of the following criteria:

The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix. Due to toxicity in the B culture of Cyclophosphamide 2 μg/mL dose level only 70 cells were scored. However this was considered acceptable as both the A and B cultures showed an identical significant dose response.

The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.

The required number of cells and concentrations were analyzed.

The test item induced a very modest statistically significant increase in the frequency of cells with aberrations in the absence metabolic activation following 24-hours exposure only. However, the response was within the current historical control range for vehicle controls and compared to a very low vehicle control value. The majority of the aberrations were break type aberrations which are often observed where excessive toxicity is recorded indicating a cytotoxic response. It was therefore considered that the response was of no biological or toxicological significance.

It was also considered that the test item had been adequately tested under the conditions used in this study.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Conclusions:

Butylal did not induce any toxicologically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

An in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells was performed with the test item Butylal in order to supplement microbial system data as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations.

During this experiment duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on toxicity. The dose levels selected for the Main Test were as follows:

Group                                          Final concentration of test item Butylal (µg/mL)

4(20)-hour without S9                    25, 50, 75, 100, 150, 200, 300

4(20)-hour with S9 (2%)                25, 50, 75, 100, 150, 200, 300

24-hour without S9                         25, 50, 75, 100, 150, 200, 300

The results of the experiment demonstrated that all vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that achieved optimum toxicity or greater.

It was concluded that the test item, Butylal was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

As stated in Annex IX of REACH Regulation (column 2, point 8.4), in absence of positive results in any of the in vitro genotoxicity studies in Annex VII or VIII and in absence of results available from an in vivo study already, in vivo somatic cell genotoxicity study are not required.

 

Three reliable in vitro genetic toxicity studies were performed on butylal and gave negative results.In vivo study is consequently not required.

Justification for classification or non-classification

Based on the negative result in the 3 in vitro assays, this substance is not classified for mutagenicity.