Dipropoxymethane

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Remarks:

read-across with Butylal

Adequacy of study:

key study

Study period:

From 15 MARCH 2018 to 15 November 2021

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 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD) rat

Details on species / strain selection:

The rat was chosen as the test species because it is accepted regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 41 to 47 days
- Weight at study initiation: Males: 192 to 250g; Females: 149 to 198g
- Fasting period before study: No
- Housing: Four or three of the same sex per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:12 days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY:
Food: Teklad 2014C Diet; Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Water: Potable water from the public supply via polycarbonate bottles with sipper tubes; Certificates of analysis are routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15 March 2018 To: 15 to 16 October 2018

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen to simulate the conditions of human exposure.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test material was weighed out into a suitable container. Approximately 50% of the final volume of vehicle was added to the test material and mixed with a magnetic stirrer until homogenous. The solution was then made up to the required volume with vehicle. The formulation was transferred to a final container and mixed with a magnetic stirrer until homogenous.
A series of solutions at the required concentrations were prepared by dilution of individual weighings of the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A representative sample of test formulation (1 mL, accurately weighed) was dissolved with the aid of swirling in a suitable volume of acetone. The extract was diluted using acetone, where necessary, to provide a solution containing Butylal at an expected concentration within the range 20 μg/mL to 80 μg/mL.
An accurate volume of internal standard solution (100 μL) was added to each test sample solution (1000 μL) and mixed by vortex to provide a final concentration of ca. 50 μg/mL of tetradecane.
The concentration of Butylal in the final solution was quantified by GC using flame ionisation detection (FID) as detailed in the chromatographic section. The analytical procedure was successfully validated for Butylal in corn oil formulations with respect to the specificity of chromatographic analysis, limits of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day and refrigeration for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 5% of the initial time zero value and the coefficient of variation was less than 3%.
Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method. Results were not corrected for recoveries.
The mean concentrations were within 5% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 4%, confirming precise analysis. The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure. Results were not corrected for recoveries.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days each week

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

5 ml/kg - 200 mg/ml

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

5 ml/kg - 60 mg/ml

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

5 ml/kg - 20 mg/ml

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

5 ml/kg

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 100, 300 and 1000 mg/kg/day) were selected in conjunction with the Sponsor. Doses were selected following the completion of the preliminary toxicity study, conducted in CD rats, in which three groups of five males and females were treated at 500, 750 or 1000 mg/kg/day. Treatment was well tolerated with no clear treatment related in-life findings or death at any dose. Post-life investigations revealed higher liver weights in males (at 750 mg/kg/day or above) and females (at all treated levels); however, there were no changes at macroscopic examination considered to be related to treatment. It was therefore considered that a high dose of 1000 mg/kg/day would be tolerated in this 13-week toxicity study. The low and intermediate doses were 100 and 300 mg/kg/day.

- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: yes
- Dose range finding studies: yes, conducted in CD rats, in which three groups of five males and females were treated at 500, 750 or 1000 mg/kg/day

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of animal ill-health amongst the occupants.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

FOOD CONSUMPTION EXAMINATION: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION EXAMINATION: Yes
During Weeks 1 to 12 of treatment, water consumption was recorded by weight (over a 3-day period on each occasion) for each cage of animals, using water bottles fitted with sipper tubes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretreatment and on Week 12
- Dose groups that were examined: All animals for pretreatment and dose groups 0 and 1000 mg/kg/day on Week 12

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: All animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Assessments were performed (before dosing) during Week 12 of treatment.
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity, grip strength and motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analyzed at each timepoint separately:
Grip strength and motor activity
Body weight, using gains over appropriate study periods
Hematology
Blood chemistry
Urinalysis
Organ weights, absolute or adjusted for terminal body weight

The following sequence of statistical tests was used for grip strength, motor activity, body weight, organ weight and clinical pathology data.

A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. The F1 approximate test was applied.

A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no clear effect of treatment on body weight or overall body weight gain in males or females treated with Butylal.
When compared to Control, body weights in females receiving 1000 mg/kg/day were significantly increased in the first half of the treatment period, which peaked in Week 6 (difference of 30 g); however, there was no dose-response and the difference resolved by Week 13. No treatment related changes were noted in males

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Overall food intake of females at 1000 mg/kg/day was slightly increased compared to Control
(1.1X of Control). The difference in food intake was small but more pronounced in the first
half of the treatment period (Weeks 1 to 6).

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

When compared to Controls, females treated at 1000 mg/kg/day showed consistently higher water consumption (1.4X).
There was no effect of treatment on water consumption in males and females treated at 100 or 300 mg/kg/day or in males at 1000 mg/kg/day.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

The hematological analysis performed in Week 13 of treatment did not reveal any differences of toxicological significance for animals treated with Butylal compared to Controls.
All differences from Control were minor, lacked dose response, were confined to one sex and were therefore attributed to normal biological variation. Differences included the following. A statistically significant decrease of prothrombin time in male receiving 1000 mg/kg/day and activated partial thromboplastin time; but the majority of values were within the concurrent control range, had no dose response to treatment, and no significant decreases seen in treated females. The red cell distribution width was slightly, but significantly, reduced in males receiving 1000 mg/kg/day, but there was no dose response and a similar pattern was not seen in females. Low hematocrit values were observed in treated males, but the difference compared to Controls was minimal and there was no dose response.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of blood plasma at Week 13 revealed significant increases in alanine amino-transferase in males at 300 (1.3X control) or 1000 mg/kg/day (1.4X control) and in females at 1000 mg/kg/day (1.1X control); no corresponding increase was seen in females at 300 mg/kg/day (due to an atypical value of one Control female (1F 120), the individual values indicate an increase in this group). Significantly increased cholesterol concentrations were observed in both males and females receiving 300 or 1000 mg/kg/day (1.4X or 1.6X for males and 1.3X or 1.6X for females, respectively). Increased triglyceride concentrations were reported for treated males (between 1.2 to 2.2X Control) and females (between 1.2 to 1.4X Control), however there was no dose relationship in the females. Also, an increase in potassium for males receiving 1000 mg/kg/day (1.1X Control) was seen, although no corresponding increase was seen in females.
All other inter-group differences from Control were minor, influenced by a single value, lacked dose response and/or confined to one sex and were therefore attributed to normal biological variation. Such differences included, but were not limited to, a slight decrease in aspartate transferase seen in females, (again, largely a result of an atypical value in a control female; animal 1F 120). A slight statistically significant increase in calcium concentration was observed in treated males that showed no relationship to dose; and although an increase was seen in females receiving 1000 mg/kg/day, the difference did not attain statistical significance. Increases in total protein concentrations in males receiving 1000 mg/kg/day were small and the majority of values were within the range of concurrent control values.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Examination of the composition of the urine in Week 13 indicated increased output of total protein in males receiving 1000 mg/kg/day (2.3X Control).
All other statistically significant differences from Control were minor, lacked dose-relationship, were confined to one sex or individual animal values were within the Control range and were therefore attributed to normal biological variation.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of organ weights after 13 weeks of treatment revealed slightly increased adjusted liver weights in both sexes treated at 1000 mg/kg/day (1.2X or 1.3X Control, respectively). Low adjusted thymus weights (1.2X Control) and significantly increased absolute/adjusted adrenal weights of females at 1000 mg/kg/day (0.81X and 1.3X Control).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed after 13 weeks of treatment revealed the following changes in the stomach: Depressions were seen in two males given 1000 mg/kg/day. The incidence and distribution of all other findings were considered unrelated to treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with Butylal were seen in the kidneys, liver, thyroids, adrenals and stomach.
Kidneys: Increased incidences and severity levels, compared to control animals, of accumulation of hyaline droplets, tubular basophilia and granular casts were seen in males given 1000 mg/kg/day. These findings were not observed in females.
Liver: Minimal centrilobular hypertrophy was seen in females given 300 or 1000 mg/kg/day; such findings were not reported in males.
Thyroids: Increased incidences and severity levels, compared to control animals, of follicular cell hypertrophy were seen in males given 300 mg/kg/day and in both sexes given 1000 mg/kg/day. The low incidences of this finding at all other dose levels were considered to be spontaneous, with no relationship to treatment.
Adrenals: Increased incidences and severity levels, compared to control animals, of cortical (zona fasciculata) vacuolation was seen in males given 1000 mg/kg/day. The low incidences of this finding in males at all other dose levels were considered to be spontaneous, with no relationship to treatment. This finding was not observed in females.
Stomach: Increased incidences and severity levels, compared to control animals, of hyperplasia of the nonglandular region were seen in animals given 1000 mg/kg/day. The low incidences of this finding at all other dose levels were considered to be spontaneous, with no relationship to treatment. Ulceration of the nonglandular region was also seen in two males given 1000 mg/kg/day. The two males were the same animals observed to have macroscopic depressions in the same region of the stomach.
All other histological changes were considered unrelated to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Remarks on result:

other: Systemic NOAEL

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Local NOAEL

Critical effects observed:

no

Conclusions:

The oral and inhalation repeated dose toxicity data for Ethylal and Butylal can be used as source data for Propylal. The absorption via the oral route of Propylal, Ethylal and Butylal are considered to be similar based on the similarities in purity and physicochemical properties. Additionally, the substances share a similar mode of action based on signs of systemic toxicity seen in the acute toxicity studies. In the 28-day repeated dose toxicity studies for Ethylal and Butylal, the toxic effects were related to histopathology and blood chemistry, and water consumption and organ weights, respectively. The NOAELs derived in the 28-day toxicity studies for Ethylal and Butylal were 100 and 1000 mg/kg bw/day, respectively. For Butylal, a 13-week study also concluded the NOAEL to be 1000 mg/kg bw/day. For inhalation toxicity, toxic effects were seen at doses of 750 ppm Ethylal but such effects were of very low severity and frequency. For Ethylal, toxic effects were observed at the highest dose of 4000 ppm following 13-week aerosol exposure in rats, thus, the NOAEL was estimated to be 3860 ppm. The data obtained from the repeated dose studies can be used as source information for Propylal. The read-across is applied with a high level of confidence (AO 5 in accordance with the ECHA RAAF document (2017)).

Executive summary:

For Ethylal, there are two repeated oral dose toxicity studies available. In a 14-day preliminary toxicity study, the systemic toxic potential of orally dosed Ethylal in Sprague Dawley rats was assessed to select suitable doses for a subsequent reproduction/developmental toxicity screening test (OECD TG 421) (Kl2) (see Section 5.4.1). This study was not conducted according to a particular OECD TG and did not have GLP compliance. Three rats per sex per dose received the test substance at doses of 300, 600 or 1000 mg/kg bw/day. A vehicle (corn oil) control group was included. During the study, clinical signs, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken. All animals survived to the scheduled sacrifice. Post dose signs of decreased activity and unsteady gait were seen on day 1 of treatment with 1000 mg/kg bw/day, and in one male on day 2 at this dose level. Slightly higher kidney and liver organ weights in animals treated at 600 mg/kg bw/day were observed. In one male at 1000 mg/kg bw/day slightly higher kidney weight was reported. Based on the results of this study, treatment with the test substance was well tolerated at oral doses up to 1000 mg/kg bw/day for 14 days. Therefore, it was concluded that 1000 mg/kg bw/day would be a suitable high dose level for the subsequent study.

 

In the second repeated oral dose toxicity study (Donnelly, 1996, Kl2), groups of male and female rats were exposed to Ethylal by gavage at concentrations of 0, 100, 300 or 1000 mg/kg bw/day for 28 days. No guideline was followed. The NOAEL for females was estimated to be 100 mg/kg bw/day and could not be established for males. In the 1000 mg/kg bw/day group, lesions in the kidney tubules were observed which were characterised by degeneration (moderate severity) and necrosis of the tubular epithelium of the proximal tubules. Mean creatinine levels for this group were also significantly higher than for the control group, but blood urea nitrogen levels were unaffected. Tubular degeneration of minimal severity was observed in the 100 and 300 mg/kg bw/day groups but without changes in serum chemistry parameters. The incidence of tubular degeneration in the female 100 mg/kg bw/day group was comparable to the incidence in the control group.

 

For Butylal, there are two repeated oral dose toxicity studies available. The 28-day study was conducted following GLP however, no particular OECD TG was followed (Low, 2018, Kl2). In this study, the NOAEL is estimated to be 1000 mg/kg bw/day for male and female rats. Groups of male and female Sprague-Dawley (CD) rats were exposed to Butylal by gavage at concentrations of 500, 750 or 1000 mg/kg bw/day for 4 weeks. Similarly constituted control group received the vehicle, corn oil, at the same volume dose as treated groups. During the study, clinical condition, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken. No statistical analysis was performed on this study. The clinical condition of the animals was unaffected by treatment, there were no toxicologically significant signs observed following dose administration and there were no premature deaths. There was no effect of treatment on body weight or food consumption. On two or three occasions in week 4 of treatment, water consumption was apparently greater (by visual assessment) in cages with animals treated at 750 or 1000 mg/kg bw/day, respectively. The analysis of organ weights revealed high adjusted liver weights in males given 750 or 1000 mg/kg bw/day and females given 500, 750 or 1000 mg/kg bw/day. There were no macroscopic findings considered to be related to treatment. The increased liver weights may reflect a general adaptive response to a xenobiotic. It was concluded that oral administration of Butylal to Spargue-Dawley rats for 4 weeks was well-tolerated and did not cause any adverse response. The high dose level of 1000 mg/kg/day was therefore considered suitable for administration in the subsequent 13-week toxicity study.

 

The 13-week study was conducted according to OECD TG 408 following GLP (Low, 2021, Kl1). In this study, the systemic NOAEL was calculated to be 1000 mg/kg bw/day and the local NOAEL was calculated to be 300 mg/kg bw for male and female rats. Groups of 10 male and female rats were exposed to Butylal by oral gavage at concentrations of 100, 300 or 1000 mg/kg bw/day for 13 weeks. A similarly constituted control group received the vehicle, corn oil, at the same volume dose as treated groups. During the study, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption, ophthalmic examination, haematology, blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken. The appearance and behaviour of the animals, sensory reactivity, grip strength and motor activity were unaffected by treatment and there were no unscheduled deaths. Body weight was not clearly affected by treatment. Although females at 1000 mg/kg bw/day exhibited a significantly higher body weight gain in the first 6 weeks of the study, this was without a dose response, was not significantly different after 13 weeks of treatment and was not seen in males. Water consumption and food intake were slightly high for females treated at 1000 mg/kg bw/day. There were no treatment related signs during ophthalmic examination. At the haematological examination performed in week 13, there were no toxicologically significant effects of treatment. At the biochemical examination of the blood plasma, alanine amino-transferase activity and triglyceride concentration were significantly higher than control in males at 300 or 1000 mg/kg bw/day and in females at 1000 mg/kg bw/day; and cholesterol concentration was significantly higher than control in animals of both sexes at 300 or 1000 mg/kg bw/day. Examination of the composition of the urine in week 13 indicated an increase in urinary protein in males receiving 1000 mg/kg bw/day. Adjusted liver weights were slightly increased in both sexes given 1000 mg/kg bw/day. In males at 1000 mg/kg bw/day, adjusted heart weights were significantly reduced; and, in females at 1000 mg/kg bw/day, adjusted thymus weights were significantly lower whilst adjusted adrenal weights were significantly increased. Macroscopic examination performed after 13 weeks revealed depressions in the stomach of 2 males treated at 1000 mg/kg bw/day. At the histopathologic examination, these same 2 males are the ones that exhibited ulceration of the non-glandular region of the stomach. Additional histopathological findings included treatment-related epithelial hyperplasia of the non-glandular stomach at 1000 mg/kg bw/day in both sexes. Centrilobular hypertrophy (minimal) of the liver was observed in females at 300 and 1000 mg/kg bw/day; but not males. Thyroid follicular cell hypertrophy was increased in males at 300 mg/kg bw/day and in both sexes at 1000 mg/kg bw/day. All males at 1000 mg/kg bw/day showed hyaline droplet accumulation in the kidneys; additional findings (tubular basophilia and granular casts) were also observed in some of the males at this dose; no treatment-related histopathological findings of the kidneys were seen in females. Adrenal cortical vacuolation was increased in males at 1000 mg/kg bw/day, but not in females. In conclusion, oral administration of Butylal to Sprague-Dawley rats for 13 weeks at doses of 1000 mg/kg bw/day caused local irritation to the gastric epithelium resulting in minimal hyperplasia of the non-glandular epithelium. The local effects of ulceration in the stomach were adaptive but adverse; however the systemic effects seen were considered not adverse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

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

Adequacy of study:

key study

Study period:

From 04 APRIL 2019 to 11 SEPTEMBER 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

Some observed chamber temperatures and humidity were below the recommended range (recorded values 14.5 - 16.6 °C). These deviations had no impact on study integrity.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie - 1808131400R and 1904111400R
- Expiration date of the lot/batch: 13 August 2020 and 11 April 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2 to 8°C), under nitrogen and protected from
light

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The RccHan™®:WIST strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited (UK).
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks.
- Weight at study initiation: Males: 193 to 240 g; Females: 143 to 178 g
- Housing: Five of the same sex, unless reduced by mortality or isolation
- Diet: Teklad 2014C Diet ad libitum (removed overnight before blood sampling for hematology or blood chemistry)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (except during exposure)
- Acclimation period: At least 11 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24ºC
- Humidity (%): Monitored and maintained within the range of 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours dark : 12 hours light.

IN-LIFE DATES: From: 04 APRIL 2019 To: 15 to 17 JULY 2019

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

clean air

Remarks on MMAD:

Due to the physical nature of the test material atmosphere it was not possible to capture the samples required to determine a particle size distribution

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through nose-only chamber; Aluminum alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber
- Method of holding animals in test chamber: Plastic nose-only restraint tube
- Source and rate of air: From in-house compressed air system – breathing quality
- System of generating particulates/aerosols: Glass sintered vaporizer; The test item was supplied to the generator, via a feed line, from a syringe driven at a constant rate by a syringe pump
- Temperature, humidity in air chamber: 19 – 25°C; 30 - 70%
- Air flow rate: Generator flow: 19L/minute; Extract flow: 20L/minute
- Treatment of exhaust air: Drawn by in-house vacuum system and filtered locally

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test sample (Solvent trap collected in trapping solvent): Transfer to volumetric flask using extraction solvent. Further dilute, if necessary, using extraction solvent to provide a solution containing Ethylal at a nominal concentration in the range 20 – 200 µg/mL. Inject onto the GC in duplicate.

Duration of treatment / exposure:

13 weeks 6 hours daily

Frequency of treatment:

exposure for 5 days per week

Dose / conc.:

3 860 mg/L air (analytical)

Dose / conc.:

1 520 mg/L air (analytical)

Dose / conc.:

505 mg/L air (analytical)

Dose / conc.:

0 mg/L air (analytical)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previous inhalation toxicity study, three groups of rats were exposed to ethylal for 2 weeks (6 hours/day, 5 days/week) at mean achieved aerosol concentrations of 490, 1520 and 3930 ppm respectively. A fourth group received a single 4-hour exposure at an achieved concentration of 4810 ppm.
Exposures at mean achieved concentrations of 490, 1520 or 3930 ppm resulted in clinical signs of unsteady gait/swaying, salivation and piloerection. These signs were transient after dosing and were not considered to be adverse for these groups. Pathological examination did not show any test item related changes at 490 or 1520 ppm. Minimal olfactory epithelial degeneration was evident in two males and one female exposed at 3930 ppm.
A single exposure to ethylal at an achieved concentration 4810 ppm was stopped after 4 hours, due to adverse clinical signs. Signs seen on return to home cage included: unresponsiveness to external stimuli, decreased activity, breathing abnormalities, reduced body temperature and abnormal posture. The condition of these animals did not improve by 90 minutes post exposure and they were euthanized on grounds of animal welfare. Pathological examination showed moderate olfactory epithelial degeneration in all animals.
For this study, the highest target concentration of 4000 ppm was expected to result in treatment related effects, but not cause deaths. The lower target concentrations of 500 and 1500 ppm were chosen to explore the dose relationship of any effects seen over 13 weeks.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded twice weekly one week before treatment commenced to Week 4, weekly from Weeks 5 to 13 and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the week before treatment started and for each week throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows: pretreatment for all animals and at week 13 for all animals of groups 1 and 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes
- How many animals: all animals.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at termination
- Dose groups that were examined: all groups
- Number of animals: all animals

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

All statistical analyses were carried out separately for males and females using the individual
animal as the basic experimental unit.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Unsteady gait was occasionally observed for rats receiving ethylal at all exposure levels. In addition, swaying, decreased activity, lacrimation and wet rales were occasionally observed for a proportion of rats exposed to 3860 ppm. These signs were observed on return to home cage and resolved by the end of the working day.

On Week.Days 7.1 to 7.3, clinical signs evident for rats receiving the highest exposure level of ethylal were more marked than those seen after other exposures. Signs seen after exposure, for a proportion of rats, included locomotor and responsiveness effects (decreased activity, unsteady gait, unresponsiveness, flattened posture) and breathing effects (slow and/or shallow breathing and wet rales). Animal 138 (a high exposure level female) was removed from exposure on Week.Day 7.2, after approximately 4.5 hours, due to welfare concerns regarding clinical signs. Clinical signs evident for this animal included abnormal gait (unsteady and elevated), hunched posture, shallow breathing, decreased activity and pale ears. This rat had recovered by the end of the working day.
Signs associated with the administration procedure included red staining of the head and eyes, and/or wet fur on occasion. These were seen for rats of all groups, including control, on return to the home cage and at the end of the working day. These are signs considered to be due to the method of restraint and are not test item related.
Table 9 includes individual observation following detailed weekly physical examination

Mortality:

mortality observed, treatment-related

Description (incidence):

There was one unscheduled death due to overexposure to ethylal. Animal 134, a female receiving the highest exposure level of ethylal was found dead at the end of exposure on Week.Day 7.1. Macroscopic observations included incomplete deflation of the lungs, depressions and pale areas in the glandular mucosa of the stomach (not confirmed microscopically), and gaseous distension of the stomach, duodenum and jejunum. Incomplete deflation of the lungs and gaseous distension of the GIT were considered to be post-mortem changes. Other microscopic changes consisted in unilateral pyogranulomatous inflammation within the glandular epithelium of the nose (with presence of the bacteria and Splendore-Hoeppli material) and minimal bilateral dilation if the renal pelvis.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Differences in mean bodyweight gain were small compared with intragroup variation and were inconsistent between exposure levels and/or sexes. Small weight losses in the final week, for all groups, are considered to be associated with blood sampling techniques.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Minor fluctuations in food consumption lacked dose relationship, were inconsistent between the sexes and are considered to be unrelated to treatment

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All differences from control, that attained statistical significance, were small compared with intragroup variation, lacked dose relationship and/or were inconsistent between sexes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

All differences from control, that attained statistical significance, were small compared with intragroup variation, lacked dose relationship and/or were inconsistent between sexes.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Mean liver weights (absolute and adjusted for terminal bodyweight) were higher than control for males exposed to 1520 ppm and both sexes exposed to 3860 ppm (up to 1.23X control).
Mean kidney weights (absolute and adjusted for terminal bodyweight) were higher than control for females exposed to 1520 ppm and both sexes exposed to 3860 ppm (up to 1.21X control).
Mean adrenal weights (absolute and adjusted for terminal bodyweight) were higher than control for both sexes exposed to 1520 or 3860 ppm (up to 1.26X control).
All other differences from control were small, lacked dose relationship and/or were inconsistent between sexes.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to exposure to ethylal were seen in thyroid glands and liver.
In the thyroids, minimal hypertrophy of the follicular cells was observed for one control male and 5 out of 10 males exposed to 3860 ppm.
In the liver, diffuse minimal centrilobular hepatocellular hypertrophy was recorded for four males out of 10 exposed to 3860 ppm.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on BAL parameters.
All differences from control were small, lacked dose relationship and/or were inconsistent between sexes.

Key result

Dose descriptor:

NOAEC

Effect level:

3 860 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

Critical effects observed:

no

Conclusions:

The oral and inhalation repeated dose toxicity data for Ethylal and Butylal can be used as source data for Propylal. The absorption via the oral route of Propylal, Ethylal and Butylal are considered to be similar based on the similarities in purity and physicochemical properties. Additionally, the substances share a similar mode of action based on signs of systemic toxicity seen in the acute toxicity studies. In the 28-day repeated dose toxicity studies for Ethylal and Butylal, the toxic effects were related to histopathology and blood chemistry, and water consumption and organ weights, respectively. The NOAELs derived in the 28-day toxicity studies for Ethylal and Butylal were 100 and 1000 mg/kg bw/day, respectively. For Butylal, a 13-week study also concluded the NOAEL to be 1000 mg/kg bw/day. For inhalation toxicity, toxic effects were seen at doses of 750 ppm Ethylal but such effects were of very low severity and frequency. For Ethylal, toxic effects were observed at the highest dose of 4000 ppm following 13-week aerosol exposure in rats, thus, the NOAEL was estimated to be 3860 ppm. The data obtained from the repeated dose studies can be used as source information for Propylal. The read-across is applied with a high level of confidence (AO 5 in accordance with the ECHA RAAF document (2017)).

Executive summary:

For Ethylal, a 12-day exposure inhalation toxicity study performed according to an internal SOP similar to OECD Annex V B 8 (Sub-acute inhalation toxicity) following GLP is available (ECHA Registered Dossier, 2021a/ Katz, 1987). The NOAEL was estimated to be 750 ppm for 6-hour exposure for 12 days. Groups of 5 male and female rats were exposed to target vapour concentrations of 0, 750, 1500 or 3000 ppm for 6 hours per exposure, for 12 exposures. Exposure produced transient central nervous system depression in all groups during exposure which resolved shortly after exposure ceased. Gel-like casts or spontaneous seminal plug emissions, which may reflect changes in grooming habits during exposure, were observed beneath the cages in all treatment groups. No deaths occurred during the study. Exposure to Ethylal produced no toxicologically significant changes in body weight gain, haematology or clinical chemistry. Slight but statistically significant increases in liver weights in males and females and a small increase also statistically significant, in adrenal gland weights in females exposed to 3000 ppm were deemed likely compound-related. There were no compound-related gross or histopathological changes detected. Overall, the effects observed following exposure to 1500 and 3000 ppm Ethylal were all slight. The effects observed at 750 ppm (transient lethargy, casts (males), increased relative liver weight (males)) were of even lesser severity and frequency. A concentration of 750 ppm is the proposed NOAEL for toxicity.

 

For Ethylal, a 13-week inhalation study in Han Wistar rats performed according to OECD TG 413 following GLP is available (Horrell, 2021). Animals received doses of either 0, 500, 1500 or 4000 ppm test substance by the inhalation route (aerosols) for 13 weeks (6 hours daily exposure for 5 days per week). During the study, clinical condition, body weight, food consumption, ophthalmoscopy, haematology, blood chemistry, organ weight, bronchoalveolar lavage, macropathology and histopathology investigations were undertaken. One female receiving the highest exposure level was found dead at the end of exposure on Week 7. On Week 7, clinical signs evident for rats receiving the highest exposure level were more marked than those seen after other exposure levels, which included locomotor and responsiveness effects (decreased activity, unsteady gait, unresponsiveness, flattened posture) and breathing effects (slow and/or shallow breathing and wet rales). One animal (a high exposure level female) was removed from exposure on Week 7, due to welfare concerns. Clinical signs evident for this animal included abnormal gait (unsteady and elevated), hunched posture, shallow breathing, decreased activity and pale ears; but it had recovered by the end of the working day. In all exposure level groups, unsteady gait was occasionally observed. In addition, swaying, decreased activity, lacrimation and wet rales were occasionally observed for a proportion of rats exposed to 3860 ppm which resolved by the end of the working day. Pathological examination revealed follicular cell hypertrophy in the thyroid of five males exposed to 3860 ppm and hypertrophy of the centrilobular hepatocytes of the liver in four of the same males. These findings are considered rodent-specific, adaptive and non-adverse. Overall, the NOAEL was considered to be 3860 ppm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

16 665 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

42

Species:

rat