Dipropoxymethane

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 mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

OECD 490 for Ethylal and OECD 476 for Butylal

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The hypothesis for the relevant (eco)toxicological endpoints (see section 1.3) is that toxicity of the substances are proportional to the chain length. Hence, the toxic potential of Propylal is anticipated to be in between the toxic potential of Ethylal and Butylal.

For the short-term toxicity to fish endpoint, experimental study is available for Ethylal, but not Butylal, for which an ECOSAR prediction is available. Based on this information, ECOSAR is deemed more appropriate for this endpoint than the read-across approach. More detailed justification for this endpoint is provided in the following sections.

The selected approach corresponds to ECHA’s Read-Across Assessment Framework (RAAF) scenario #4 (RAAF, 2017): Catalogue approach, read-across hypothesis based on different compounds which have the same type of effect(s). There are differences in strength of the effect(s) and they may form a regular pattern. The prediction is based on the regular pattern within the category, when the members are ordered by a variable related to the structural differences in the category, or on a worst-case approach.

In accordance with the RAAF, a conclusion on the adequacy and scientific robustness of the information will be provided in the conclusion for each endpoint, using the assessment options (AOs) provided in the RAAF.

ECHA has identified assessment elements (AEs) for using the catalogue approach which are mentioned below.

• AE C.1 - Common - Substance characterisation
• AE C.2 – Common - Structural similarity and category hypothesis
• AE C.3 – Common - Link of structural similarities and structural differences with the proposed regular pattern
• AE C.4 – Common - Consistency of effects in the data matrix
• AE C.5 – Common - Reliability and adequacy of the source study(ies)
• AE 4.1 - Scenario-specific - Compounds the test organism is exposed to
• AE 4.2 - Scenario-specific - Common underlying mechanism, qualitative aspects
• AE 4.3 - Scenario-specific - Common underlying mechanism, quantitative aspects
• AE 4.4 - Scenario-specific - Exposure to other compounds than to those linked to the prediction
• AE 4.5 - Scenario-specific - Occurrence of other effects than covered by the hypothesis and justification
• AE C.6 – Common - Bias that influences the prediction


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For assessment of (eco)toxicological endpoints of Propane,1,1'-[methylenebis(oxy)]bis- (Propylal, CAS# 505-84-0, EC# 208-021-9, target substance), read-across is performed to close structural analogues:
- 1,1'-[methylenebis(oxy)]diethane (Ethylal, CAS# 462-95-3, EC# 207-330-6, source substance 1) and;
- 1,1'-[Methylenebis(oxy)]dibutane (Butylal, CAS# 2568-90-3, EC# 219-909-0, source substance 2).
The target and source substances are all mono-constituent substances which belong to the family of acetals. Acetals belong to a specific chemical family, resumed in the generally term solvent, distinct from ethers. Acetals are molecules with two single-bonded oxygen atoms attached to the same carbon atom. Acetals are formally derived from an aldehyde or ketones. Formation of an acetal occurs when the hydroxyl group of a hemiacetal becomes protonated and is lost as water. The carbocation ion that is produced is then rapidly attacked by a molecule of alcohol. Loss of the proton from the attached alcohol gives the acetal. It can be noticed that, all the chemicals under investigation are characterised by the sole acetal functional group, thus they exhibit a very close structural similarity.
The structural differences between the substances are limited to the number of carbon atoms in the chain, and the category members can be ordered from C5 to C9 chain alkane (Table 1): Ethylal (C5), Propylal (C7) and Butylal (C9). There are no differences in functional groups.

Substances:
Source 1: 1,1'-[methylenebis(oxy)]diethane (Ethylal) (CAS: 462-95-3)
Target: Propane,1,1'-[methylenebis(oxy)]bis- (Propylal) (CAS: 505-84-0)
Source 2: 1,1'-[Methylenebis(oxy)]dibutane (Butylal) (CAS: 2568-90-3)

Purity/Impurities:
The typical concentration of Ethylal, Propylal, and Butylal are 99.96, 99.85, and 99.75%, respectively. No impurities of the three substances have been considered relevant for hazard identification because of the very low concentrations.


3. ANALOGUE APPROACH JUSTIFICATION
In overall conclusion, according to Regulation No 1907/2006 read-across between substances can be performed if the physicochemical, ecotoxicological and toxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity. The source and target substances have very similar structures and physicochemical properties.
The target and source substances are all mono-constituent substances which belong to the family of acetals which are molecules with two single-bonded oxygen atoms attached to the same carbon atom. The structural differences between the substances are limited to the number of carbon atoms in the chain, Ethylal having the shortest chain length, followed by Propylal and then Butylal. There is no difference in functional groups. The substances are very pure with typical concentrations of 99.85, 99.96, and 99.75, for Ethylal, Propylal and Butylal, respectively. There are no impurities at relevant concentrations.
No relevant toxicokinetic studies were available for the target or source substances. Therefore, the physicochemical properties of the substances were assessed to conclude on the toxicokinetic behaviour. The molecular weight of the Ethylal, Propylal and Butylal are similar with values of 104.148, 132.2 and 160.25 g/mol, respectively, which is favourable for absorption since the molecular weights are below 500 g/mol. In addition, all substances are liquid and water soluble and are therefore more readily taken up than dry particulates. Water solubility increases with smaller nonpolar chain lengths: 70 g/L (at 18 °C) for Ethylal, 3.65 g/L (at 20 °C) for Propylal and 0.225 g/L (at 20 °C) for Butylal. Consequently, the Log Pow values increase with longer nonpolar chain lengths: 0.84 for Ethylal, 2.04 at 21 °C for Propylal and 2.77 at 20 °C for Butylal. The vapour pressure values increase with smaller chain lengths: 17000 Pa (at 20 °C) for Ethylal, 2000 Pa (at 20 °C) for Propylal and 111 Pa (at 25 °C) for Butylal. Based on these values, substances are anticipated to behave more dynamically with smaller chain lengths. However, absorption via the dermal and inhalation route is limited with smaller chain lengths, because they may be too hydrophilic to cross the biological membranes. Based on the available toxicity data, the toxicological profiles are similar. Available acute oral toxicity data for Ethylal, Propylal and Butylal, show that the test substances trigger clinical signs of toxicity, thus, the bioavailability of the test substance via the oral route is supported. The bioavailability of Ethylal and Butylal by the inhalation route is also supported by the findings of clinical signs of toxicity in acute inhalation toxicity studies. For Ethylal, lethargy, gait disturbance, narcosis and death were recorded in rats treated at high vapour doses of the test substance (20000 ppm). For Butylal, irritation of the mucous membrane, intense respiration, high stepping almost staggering gait, and tremor of the whole body were observed at a vapour dose of 11.24 mg/L air. Both source substances, Ethylal and Butylal, were found to be slightly irritating to the skin but were not classified. Propylal and Ethylal were found to be slightly irritating to the eyes but were also not classified. All substances were not sensitising to the skin. In the reproductive and developmental toxicity studies for Ethylal and Butylal, no toxic effects were related to the treatment for parental animals and for the embryo-foetal development. The NOAEL for the offspring development was found for Ethylal at 1000 mg/kg bw/day and for Butylal at 300 mg/kg bw/day. For Butylal, toxic effects were limited to offspring derived from the group that received 1000 mg/kg bw/day which had low absolute weight and body weight gain from Day 1 of age with a percentage difference between control and the highest administered dose for male animals 20% and for female animals 16%. However, due to the absence of any effect on offspring survival, general condition or thyroid hormones, this effect on offspring body weight is considered to not warrant the classification of the test item as a reproductive toxicant.
Based on the QSAR data, all of the substances had no structural alerts and were classified as Low (Class I) in the toxic hazard classification by Cramer.
Considering ecotoxicity, the increasing toxic effect with increasing length of the chain (i.e. the toxicity level is Ethylal< Propylal < Butylal) is found for all three trophic levels (fish, invertebrates and algae) in experimental studies and ECOSAR predicted results. Therefore, the effect value from the short-term toxicity to fish study on Ethylal cannot be read-acrossed to Propylal. No experimental study is available on Butylal, which is expected to be significantly more toxic than Propylal. Based on this information, ECOSAR is deemed more appropriate for this endpoint than the read-across approach. Therefore, the 96-h LC50 value of Propylal to fish is assessed to be 175 mg/L according to ECOSAR (v2.0). The QMRF and QPRF are attached to this endpoint of the dossier.
In conclusion, the read-across hypothesis is supported by comparable structural characteristics with a trend in chain length and similar toxicological behaviour of the substances in the group. Adequate, reliable and available scientific information indicates that the substances have similar toxicological profiles and that data for the source substances are reliable to predict the toxicity of the Propylal for which the experimental data is lacking. Therefore, information from the following endpoints assessed for Ethylal and Butylal can be used as read-across source substances, for Propylal with a high level of confidence (AO 5 in accordance with the ECHA RAAF document (2017)):
Mammalian toxicology:
- Acute inhalation study: LC50 > 5.62 mg/L (5620 mg/m³) (Butylal, BASF Test, 4-hour vapour)
- Repeated dose toxicity study – oral: NOAEL = 1000 mg/kg bw/day (Butylal, OECD TG 408)
- Repeated dose toxicity study – inhalation: NOAEL = 3860 ppm (Ethylal, OECD TG 413, 6-hour vapour)
- In vitro micronucleus assay: Negative (Ethylal, OECD TG 487)- In vitro gene mutation in mammalian cells: Negative (Butylal, OECD TG 479)
- Screening for reproductive and developmental toxicity: NOAEL = 1000 mg/kg bw/day (parental) and 300 mg/kg bw/day (offspring) (Butylal, OECD TG 421)
Based on this information, it is concluded that Propylal does not have to be classified for acute inhalation toxicity, it has a low toxic potential following oral and inhalation repeated dose exposure, it is not reprotoxic, and furthermore, it is not genotoxic.


4. DATA MATRIX
See Attached justification

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Substance tested: Ethylal

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

Substance tested Butylal

GLP compliance:

yes (incl. QA statement)

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

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.

Key result

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:

Microsomal Enzyme Fraction

Lot Number 30.06.17 was used in this study, and was pre-prepared in house (outside the confines of the study) following standard procedures.

The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6 P (Glucose-6-Phosphate) (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.





Test Item Preparation

Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to dilutions being prepared. The test item had a molecular weight of 160.26 therefore, the maximum recommended dose level was 1603 μg/mL, and no correction for the purity (99.91%) of the test item was applied. However, due to formulation issues at a higher concentration, the maximum achievable dose level was 801.5 μg/mL

There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated (Scott et al., 1991). The pH and osmolality readings are in the following table:



Concentration

µg/mL

 

0

 

3.13

 

6.26

 

12.52

 

25.5

 

50.09

 

100.19

 

200.38

 

400.75

 

801.5

pH

7.42

7.40

7.39

7.39

7.37

7.39

7.39

7.39

7.39

7.39

Osmolality mOsm

436

-

443

-

452

-

445

437

432

428



- = not determined

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP.





Calculations

The cloning efficiency (CE), % control, mutant plate counts, mutant frequency/10^6 (MF10^-6) and mutant frequency/10^6 survival rate (MFSV) were calculated using the following formulae:

CE% = (meanCE counts/200)x100 %

Control = (CE% of Dose IDx/CE% of Dose ID0)x100

MF 10^-6 for each dose = Total mutant plate counts/2

MFS 10 ^-6 for each dose = (MF 10-6/Day 7 CE%)x100

Where:

Concentration ID0 = Vehicle control values

Concentration IDx = Concentration values

Small errors may occur when calculating mean cell concentrations and volumes for diluting; and in the calculation of means for cloning efficiency and mutant frequency; if these errors are ≤5% they are regarded to be within reasonable experimental error and considered not to affect the integrity of the study.



Assay Acceptance Criteria

The following criteria will be used to determine a valid assay:

i) The average absolute cloning efficiency of the Day 7 negative controls should exceed 50%. All assays below 50% cloning efficiency will be unacceptable.

ii) The background (spontaneous) mutant frequency of the vehicle controls is generally within the historical range. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays.

iii) The concurrent positive controls should induce responses that are comparable with those generated in the historical positive control range and produce a statistically significant increase compared with the concurrent negative control.

iv) The criteria for selection of the maximum concentration have been met. Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended concentration (10 mM, 2 mg/mL or 2 μL/mL whichever is lower, or 5 mg/mL for a UVCB*), or include the lowest precipitating concentration. Where a test item is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic concentration should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic. Treatments that reduce relative clonal survival to less than ten percent will not be scored for mutant frequency in the assay.

v) Adequate numbers of cells and concentrations are analyzable. Mutant frequencies are normally derived from sets of ten dishes/flasks for the mutant colony count and three dishes for cloning colony counts. To allow for contamination losses it is acceptable to score a minimum of eight mutant selection dishes and two cloning efficiency flasks.

vi) Five concentrations of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analyzed duplicate concentrations is considered necessary in order to accept a single assay for evaluation of the test item.

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 absence of mutagenic effects in bacterial cells has been established for all substances. In addition, no differences in the genotoxicity profilers of the OECD Toolbox were observed. Based on the structural similarity, and no differences in functional groups, or formation of genotoxic metabolites, read-across for genotoxicity studies is applied with a high level of confidence (AO 5 in accordance with the ECHA RAAF document (2017)).
The genotoxic potential of Propylal for in vitro gene mutation in mammalian cells is supplemented with the available OECD TG 490 and OECD TG 476 studies for Ethylal and Butylal, respectively. Based on data of the source substances, sufficient information is available to conclude that the target substance does not have any genotoxic potential.

Executive summary:

For Ethylal, a recent in vitro mammalian cell gene mutation test using the TK gene is available which has been performed according to OECD TG 490 and GLP (Brown,   2018, Kl1). The concentrations of Ethylal applied were 32.56, 65.13, 130.25, 260.5, 521, 1042 μg/mL and three exposure groups were used including a 4-hour exposure with and without metabolic activation (S9-mix) and a 24-hour exposure without metabolic activation. Under the conditions of this study, the test substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor of 126 x 10^-6 in either of the three exposure groups, consequently it is considered to be non-mutagenic in this assay.

 

For Butylal, a hypoxanthine-guanine phosphoribosyl transferase (HPRT) test according to OECD TG 476 following GLP is available (Brown, 2018, Kl1). In the main test Chinese hamster (V79) cells were treated with the test substance at eight concentrations, in duplicate, together with vehicle and positive controls in both the absence and presence of metabolic activation (S9-mix). The concentrations of Butylal applied were 0, 6.25, 12.5, 25, 50, 100, 200 μg/mL for the 4-hour exposure without metabolic activation, and 0, 3.13, 6.25, 12.5, 25, 50, 100, 200, 400 μg/mL for the 4-hour exposure with metabolic activation (2 %). 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. Therefore, Butylal was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Experimental start date 09 March 2018 Experimental completion date 29 March 2018

Reliability:

1 (reliable without restriction)

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The hypothesis for the relevant (eco)toxicological endpoints (see section 1.3) is that toxicity of the substances are proportional to the chain length. Hence, the toxic potential of Propylal is anticipated to be in between the toxic potential of Ethylal and Butylal.

For the short-term toxicity to fish endpoint, experimental study is available for Ethylal, but not Butylal, for which an ECOSAR prediction is available. Based on this information, ECOSAR is deemed more appropriate for this endpoint than the read-across approach. More detailed justification for this endpoint is provided in the following sections.

The selected approach corresponds to ECHA’s Read-Across Assessment Framework (RAAF) scenario #4 (RAAF, 2017): Catalogue approach, read-across hypothesis based on different compounds which have the same type of effect(s). There are differences in strength of the effect(s) and they may form a regular pattern. The prediction is based on the regular pattern within the category, when the members are ordered by a variable related to the structural differences in the category, or on a worst-case approach.

In accordance with the RAAF, a conclusion on the adequacy and scientific robustness of the information will be provided in the conclusion for each endpoint, using the assessment options (AOs) provided in the RAAF.

ECHA has identified assessment elements (AEs) for using the catalogue approach which are mentioned below.

• AE C.1 - Common - Substance characterisation
• AE C.2 – Common - Structural similarity and category hypothesis
• AE C.3 – Common - Link of structural similarities and structural differences with the proposed regular pattern
• AE C.4 – Common - Consistency of effects in the data matrix
• AE C.5 – Common - Reliability and adequacy of the source study(ies)
• AE 4.1 - Scenario-specific - Compounds the test organism is exposed to
• AE 4.2 - Scenario-specific - Common underlying mechanism, qualitative aspects
• AE 4.3 - Scenario-specific - Common underlying mechanism, quantitative aspects
• AE 4.4 - Scenario-specific - Exposure to other compounds than to those linked to the prediction
• AE 4.5 - Scenario-specific - Occurrence of other effects than covered by the hypothesis and justification
• AE C.6 – Common - Bias that influences the prediction


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
For assessment of (eco)toxicological endpoints of Propane,1,1'-[methylenebis(oxy)]bis- (Propylal, CAS# 505-84-0, EC# 208-021-9, target substance), read-across is performed to close structural analogues:
- 1,1'-[methylenebis(oxy)]diethane (Ethylal, CAS# 462-95-3, EC# 207-330-6, source substance 1) and;
- 1,1'-[Methylenebis(oxy)]dibutane (Butylal, CAS# 2568-90-3, EC# 219-909-0, source substance 2).
The target and source substances are all mono-constituent substances which belong to the family of acetals. Acetals belong to a specific chemical family, resumed in the generally term solvent, distinct from ethers. Acetals are molecules with two single-bonded oxygen atoms attached to the same carbon atom. Acetals are formally derived from an aldehyde or ketones. Formation of an acetal occurs when the hydroxyl group of a hemiacetal becomes protonated and is lost as water. The carbocation ion that is produced is then rapidly attacked by a molecule of alcohol. Loss of the proton from the attached alcohol gives the acetal. It can be noticed that, all the chemicals under investigation are characterised by the sole acetal functional group, thus they exhibit a very close structural similarity.
The structural differences between the substances are limited to the number of carbon atoms in the chain, and the category members can be ordered from C5 to C9 chain alkane (Table 1): Ethylal (C5), Propylal (C7) and Butylal (C9). There are no differences in functional groups.

Substances:
Source 1: 1,1'-[methylenebis(oxy)]diethane (Ethylal) (CAS: 462-95-3)
Target: Propane,1,1'-[methylenebis(oxy)]bis- (Propylal) (CAS: 505-84-0)
Source 2: 1,1'-[Methylenebis(oxy)]dibutane (Butylal) (CAS: 2568-90-3)

Purity/Impurities:
The typical concentration of Ethylal, Propylal, and Butylal are 99.96, 99.85, and 99.75%, respectively. No impurities of the three substances have been considered relevant for hazard identification because of the very low concentrations.


3. ANALOGUE APPROACH JUSTIFICATION
In overall conclusion, according to Regulation No 1907/2006 read-across between substances can be performed if the physicochemical, ecotoxicological and toxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity. The source and target substances have very similar structures and physicochemical properties.
The target and source substances are all mono-constituent substances which belong to the family of acetals which are molecules with two single-bonded oxygen atoms attached to the same carbon atom. The structural differences between the substances are limited to the number of carbon atoms in the chain, Ethylal having the shortest chain length, followed by Propylal and then Butylal. There is no difference in functional groups. The substances are very pure with typical concentrations of 99.85, 99.96, and 99.75, for Ethylal, Propylal and Butylal, respectively. There are no impurities at relevant concentrations.
No relevant toxicokinetic studies were available for the target or source substances. Therefore, the physicochemical properties of the substances were assessed to conclude on the toxicokinetic behaviour. The molecular weight of the Ethylal, Propylal and Butylal are similar with values of 104.148, 132.2 and 160.25 g/mol, respectively, which is favourable for absorption since the molecular weights are below 500 g/mol. In addition, all substances are liquid and water soluble and are therefore more readily taken up than dry particulates. Water solubility increases with smaller nonpolar chain lengths: 70 g/L (at 18 °C) for Ethylal, 3.65 g/L (at 20 °C) for Propylal and 0.225 g/L (at 20 °C) for Butylal. Consequently, the Log Pow values increase with longer nonpolar chain lengths: 0.84 for Ethylal, 2.04 at 21 °C for Propylal and 2.77 at 20 °C for Butylal. The vapour pressure values increase with smaller chain lengths: 17000 Pa (at 20 °C) for Ethylal, 2000 Pa (at 20 °C) for Propylal and 111 Pa (at 25 °C) for Butylal. Based on these values, substances are anticipated to behave more dynamically with smaller chain lengths. However, absorption via the dermal and inhalation route is limited with smaller chain lengths, because they may be too hydrophilic to cross the biological membranes. Based on the available toxicity data, the toxicological profiles are similar. Available acute oral toxicity data for Ethylal, Propylal and Butylal, show that the test substances trigger clinical signs of toxicity, thus, the bioavailability of the test substance via the oral route is supported. The bioavailability of Ethylal and Butylal by the inhalation route is also supported by the findings of clinical signs of toxicity in acute inhalation toxicity studies. For Ethylal, lethargy, gait disturbance, narcosis and death were recorded in rats treated at high vapour doses of the test substance (20000 ppm). For Butylal, irritation of the mucous membrane, intense respiration, high stepping almost staggering gait, and tremor of the whole body were observed at a vapour dose of 11.24 mg/L air. Both source substances, Ethylal and Butylal, were found to be slightly irritating to the skin but were not classified. Propylal and Ethylal were found to be slightly irritating to the eyes but were also not classified. All substances were not sensitising to the skin. In the reproductive and developmental toxicity studies for Ethylal and Butylal, no toxic effects were related to the treatment for parental animals and for the embryo-foetal development. The NOAEL for the offspring development was found for Ethylal at 1000 mg/kg bw/day and for Butylal at 300 mg/kg bw/day. For Butylal, toxic effects were limited to offspring derived from the group that received 1000 mg/kg bw/day which had low absolute weight and body weight gain from Day 1 of age with a percentage difference between control and the highest administered dose for male animals 20% and for female animals 16%. However, due to the absence of any effect on offspring survival, general condition or thyroid hormones, this effect on offspring body weight is considered to not warrant the classification of the test item as a reproductive toxicant.
Based on the QSAR data, all of the substances had no structural alerts and were classified as Low (Class I) in the toxic hazard classification by Cramer.
Considering ecotoxicity, the increasing toxic effect with increasing length of the chain (i.e. the toxicity level is Ethylal< Propylal < Butylal) is found for all three trophic levels (fish, invertebrates and algae) in experimental studies and ECOSAR predicted results. Therefore, the effect value from the short-term toxicity to fish study on Ethylal cannot be read-acrossed to Propylal. No experimental study is available on Butylal, which is expected to be significantly more toxic than Propylal. Based on this information, ECOSAR is deemed more appropriate for this endpoint than the read-across approach. Therefore, the 96-h LC50 value of Propylal to fish is assessed to be 175 mg/L according to ECOSAR (v2.0). The QMRF and QPRF are attached to this endpoint of the dossier.
In conclusion, the read-across hypothesis is supported by comparable structural characteristics with a trend in chain length and similar toxicological behaviour of the substances in the group. Adequate, reliable and available scientific information indicates that the substances have similar toxicological profiles and that data for the source substances are reliable to predict the toxicity of the Propylal for which the experimental data is lacking. Therefore, information from the following endpoints assessed for Ethylal and Butylal can be used as read-across source substances, for Propylal with a high level of confidence (AO 5 in accordance with the ECHA RAAF document (2017)):
Mammalian toxicology:
- Acute inhalation study: LC50 > 5.62 mg/L (5620 mg/m³) (Butylal, BASF Test, 4-hour vapour)
- Repeated dose toxicity study – oral: NOAEL = 1000 mg/kg bw/day (Butylal, OECD TG 408)
- Repeated dose toxicity study – inhalation: NOAEL = 3860 ppm (Ethylal, OECD TG 413, 6-hour vapour)
- In vitro micronucleus assay: Negative (Ethylal, OECD TG 487)- In vitro gene mutation in mammalian cells: Negative (Butylal, OECD TG 479)
- Screening for reproductive and developmental toxicity: NOAEL = 1000 mg/kg bw/day (parental) and 300 mg/kg bw/day (offspring) (Butylal, OECD TG 421)
Based on this information, it is concluded that Propylal does not have to be classified for acute inhalation toxicity, it has a low toxic potential following oral and inhalation repeated dose exposure, it is not reprotoxic, and furthermore, it is not genotoxic.


4. DATA MATRIX
See Attached justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: Ethylal
Chemical name: Diethoxymethane
CAS Number: 462-95-3
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 °C in the dark under nitrogen
Intended use/Application: Not supplied
No correction for purity was made.

Species / strain / cell type:

primary culture, other: whole blood

Details on mammalian cell type (if applicable):

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

The details of the donors used are:
Preliminary Toxicity Test: male, aged 29 years
Main Experiment: female, aged 25 years

Cell Culture
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% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

standard metabolizing system (S9)

Test concentrations with justification for top dose:

Preliminary Toxicity Test
The dose range of test item used was 0, 4.07, 8.14, 16.28, 32.56, 65.13, 130.25, 260.5, 521 and 1042 μg/mL.

Main Experiment
The dose range of test item used for all three exposures was 0, 65.13, 130.25, 260.5, 521, 781.5, 1042 μg/mL.

Vehicle / solvent:

The test item was miscible in aqueous media (MEM) at 10.42 mg/mL by mixing on a vortex for approximately 10 seconds in a solubility check performed in-house. The highest concentration of the test item tested in this study was 10.42 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium

True negative controls:

no

Positive controls:

yes

Remarks:

5 μg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium

True negative controls:

no

Positive controls:

yes

Remarks:

0.075 μg/mL for 24-hour continuous exposure

Positive control substance:

other: Demecolcine

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 μg/mL for 4-hour exposure

Positive control substance:

mitomycin C

Remarks:

Absence of S9-mix

Details on test system and experimental conditions:

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 0, 4.07, 8.14, 16.28, 32.56, 65.13, 130.25, 260.5, 521 and 1042 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used for all three exposures was 0, 65.13, 130.25, 260.5, 521, 781.5, 1042 μg/mL.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
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 labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.

Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.

When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear 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 judgement and/or further investigations.
Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Key result

Species / strain:

primary culture, other: whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test (Table 1)
The dose range for the Preliminary Toxicity Test was 4.07 to 1042 μg/mL. The molecular weight of the test item was given as 104.15, therefore, the maximum dose level was 1042 μg/mL, which was calculated to be equivalent to the 10mM concentration (the maximum recommended dose level).
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level tested in any of the exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to the maximum concentration in all three exposure groups. The test item induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for the Main Experiment was, therefore, based on the maximum recommended dose level (1042 μg/mL) for all three exposure group.

Micronucleus Test – Main Experiment
As during the Preliminary Toxicity Test, there were binucleate cells suitable for scoring at the maximum dose level of test item, 1042 μg/mL, for all three exposure groups.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level tested in any of the exposure groups.
The CBPI data confirm the qualitative observations in that no dose-related inhibition of CBPI was observed Tables 2 to 6).
The maximum dose level selected for analysis of binucleate cells was the maximum recommended dose level (1042 μg/mL) for all three exposure groups.
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

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

 

Group	Final concentration of test item Ethylal (µg/mL)
4-hour without S9	0*,65.13, 130.25, 260.5, 521*, 781.5*, 1042*,MMC0.2*
4-hour with S9 (2%)	0*, 65.13, 130.25, 260.5, 521*, 781.5*, 1042*,CP5*
24-hour without S9	0*, 65.13, 130.25, 260.5, 521*, 781.5*, 1042*,DC0.075*

 

*   = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC  = MitomycinC

CP = Cyclophosphamide

DC            =Demecolcine

 

 

Table 1: CBPI - Preliminary Toxicity test

4-hour exposure without S9						4-hour exposure with S9						24-hour exposure without S9
Dose Level μg/mL	Nucleate Cells/500 Cells			CBPI	% Cytostasis	Dose Level μg/mL	Nucleate Cells/500 Cells			CBPI	% Cytostasis	Dose Level μg/mL	Nucleate Cells/500 Cells			CBPI	% Cytostasis
	Mono	Bi	Multi				Mono	Bi	Multi				Mono	Bi	Multi
0	248	228	24	1.55	0	0	229	244	27	1.60	0	0	83	313	104	2.04	0
4.07	-	-	-	-	-	4.07	-	-	-	-	-	4.07	-	-	-	-	-
8.14	-	-	-	-	-	8.14	-	-	-	-	-	8.14	-	-	-	-	-
16.28	-	-	-	-	-	16.28	-	-	-	-	-	16.28	-	-	-	-	-
32.56	-	-	-	-	-	32.56	-	-	-	-	-	32.56	-	-	-	-	-
65.13	-	-	-	-	-	65.13	-	-	-	-	-	65.13	-	-	-	-	-
130.25	-	-	-	-	-	130.25	-	-	-	-	-	130.25	-	-	-	-	-
260.5	244	229	27	1.57	0‡	260.5	270	210	20	1.50	17	260.5	101	306	93	1.98	6
521	296	193	11	1.43	22	521	201	282	17	1.63	0‡	521	54	354	92	2.08	0‡
1042	228	255	17	1.58	0‡	1042	259	224	17	1.52	13	1042	66	345	89	2.05	0‡

-     = Not selected for scoring

‡    = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control

 

Table 2: CBPI Data - Main Experiment - 4HOUR Exposure With and Without Metabolic Activation (S9)

4-Hour exposure without S9								4-hour exposure with S9
Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis	Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis
		M ono	Bi	Multi						M ono	Bi	Multi
0	A	125	316	59	1.87	1.89	0	0	A	227	241	32	1.61	1.65	0
	B	109	333	58	1.90				B	203	251	46	1.69
65.13	A	-	-	-	-	-	-	65.13	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
130.25	A	-	-	-	-	-	-	130.25	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
260.5	A	-	-	-	-	-	-	260.5	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
521	A	150	306	44	1.79	1.80	10	521	A	231	244	25	1.59	1.59	10
	B	150	302	48	1.80				B	233	244	23	1.58
781.5	A	125	331	44	1.84	1.84	5	781.5	A	217	253	30	1.63	1.59	9
	B	113	353	34	1.84				B	258	209	33	1.55
1042	A	144	325	31	1.77	1.76	15	1042	A	265	209	26	1.52	1.58	12
	B	172	286	42	1.74				B	218	247	35	1.63
MMC 0.2	A	268	227	5	1.47	1.48	46	CP 5	A	356	142	2	1.29	1.31	53
	B	258	238	4	1.49				B	346	146	8	1.32

MMC    = Mitomycin C

CP      = Cyclophosphamide

-        = No selected for scoring

 

 

Table 3: CBPI Data - Main Experiment - 24-Hour Exposure Without Metabolic Activation (S9)

24-Hour exposure without S9
Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis
		Mono	Bi	Multi
0	A	82	383	65	1.97	1.96	0
	B	96	340	64	1.94
65.13	A	-	-	-	-	-	-
	B	-	-	-	-
130.25	A	-	-	-	-	-	-
	B	-	-	-	-
260.5	A	-	-	-	-	-	-
	B	-	-	-	-
521	A	91	346	63	1.94	1.96	0‡
	B	89	336	75	1.97
781.5	A	75	366	59	1.97	1.99	0‡
	B	74	345	81	2.01
1042	A	69	366	65	1.99	1.97	0‡
	B	88	346	57	1.94
DC 0.075	A	196	259	45	1.70	1.66	31
	B	237	218	45	1.62

DC  = Demecolcine

-     = Not selected for scoring

‡    = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control

 

 

Table 4: CBPI and Micronucleus Data - Main Experiment - 4-Hour Exposure Without Metabolic Activation (S9)

Exposure Time +/-S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
4Hr-S9	0	A	125	316	59	1.87	0	5	0	0	0.50	0.65
		B	109	333	58	1.90		7	1	0	0.80
	521	A	150	306	44	1.79	10	3	0	0	0.30	0.40
		B	150	302	48	1.80		5	0	0	0.50
	781.5	A	125	331	44	1.84	5	6	0	0	0.60	0.50
		B	113	353	34	1.84		4	0	0	0.40
	1042	A	144	325	31	1.77	15	4	1	0	0.50	0.45
		B	172	286	42	1.74		3	1	0	0.40
	MMC 0.2	A	268	227	5	1.47	46	62	5	0	6.70	9.7***
		B	258	238	4	1.49		116	8	3	12.70

MMC   = Mitomycin C

***    = P<0.001

 

Table 5: CBPI and Micronucleus Data - Experiment - 4-Hour Exposure With Metabolic Activation (S9)

Exposure Time +/-S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
4Hr+S9	0	A	227	241	32	1.61	0	5	0	0	0.50	0.45
		B	203	251	46	1.69		4	0	0	0.40
	521	A	231	244	25	1.59	10	1	1	0	0.20	0.25
		B	233	244	23	1.58		3	0	0	0.30
	781.5	A	217	253	30	1.63	9	5	1	1	0.70	0.55
		B	258	209	33	1.55		4	0	0	0.40
	1042	A	265	209	26	1.52	12	4	0	0	0.40	0.50
		B	218	247	35	1.63		6	0	0	0.60
	CP 5	A	356	142	2	1.29	53	35	2	0	3.70	4.15***
		B	346	146	8	1.32		44	2	0	4.60

CP   = Cyclophosphamide

*** = P<0.001

 

Table 6: CBPI and Micronucleus Data - Main Experiment - 24-Hour Exposure Without Metabolic Activation (S9)

Exposure Time +/- S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
24Hr-S9	0	A	82	383	65	1.97	0	6	0	1	0.70	0.55
		B	96	340	64	1.94		3	0	1	0.40
	521	A	91	346	63	1.94	0‡	5	0	0	0.50	0.65
		B	89	336	75	1.97		6	0	2	0.80
	781.5	A	75	366	59	1.97	0‡	7	0	0	0.70	0.90
		B	74	345	81	2.01		11	0	0	1.10
	1042	A	69	366	65	1.99	0‡	0	0	0	0.00	0.40
		B	88	346	57	1.94		2	0	6	0.80
	DC 0.075	A	196	259	45	1.70	31	40	9	5	5.40	5.75***
		B	237	218	45	1.62		36	19	6	6.10

DC   = Demecolcine

***  = P<0.001

‡    = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control

Conclusions:

The absence of mutagenic effects in bacterial cells has been established for all substances (Butylal and Ethylal). In addition, no differences in the genotoxicity profilers of the OECD Toolbox were observed. Based on the structural similarity, and no differences in functional groups, or formation of genotoxic metabolites, read-across for genotoxicity studies is applied with a high level of confidence (AO 5 in accordance with the ECHA RAAF document (2017)).

The genotoxic potential of Propylal in the in vitro micronucleus assay is supplemented with the available in vitro micronucleus assay (OECD TG 487) for Ethylal. Based on data of the source substance, sufficient information is available to conclude that the target substance does not have any genotoxic potential.

Executive summary:

For Ethylal, an in vitro micronucleus assay has been performed according to OECD TG 487 following GLP (Bowles, 2018, Kl1). Duplicate cultures of human lymphocytes, treated with the test substance, were evaluated for micronuclei in binucleate cells at the following dose levels: 65.13, 130.25, 260.5, 521, 781.5, 1042 μg/mL, together with vehicle and positive controls. Three exposure conditions were used including a 4-hour exposure in the presence and absence of metabolic activation (S9-mix) at a final concentration of 2 %, and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. Ethylal was non-toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the maximum recommended dose level. Under the conditions of this study, the test substance was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)