Dipropoxymethane

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

From 20 September 2018 to 25 June 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 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by 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: males: 69 to 76 days old; females: 83 to 89 days old
- Weight at study initiation: (P) Males: 283-373 g; Females: 251-304 g
- Fasting period before study: no
- Housing: Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter
- Diet: ad libitum SDS VRF1 Certified pelleted diet
- Water: ad libitum Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: Males: six days prior to the commencement of treatment. Females: 20 days prior to the commencement of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 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 light : 12 hours dark
IN-LIFE DATES: From: 10 October 2018 To: 22 December 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Starting with the lowest concentration, the required amount of test item was weighed. Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous.

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5ml/kg

Details on mating procedure:

- M/F ratio per cage: 1:1 from within the same treatment groups
- Length of cohabitation: Up to two weeks
- Proof of pregnancy: Ejected copulation plugs in cage tray and sperm in the vaginal smear. Day 0 of gestation: When positive evidence of mating was detected
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first and last weeks of treatment were analyzed for achieved concentration of the test item.
The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 μg/mL to 100 μg/mL.
For the First and Last Week of dosing, all groups were sampled (4 × 1 mL, accurately weighed) from the middle of the formulation by Pharmacy personnel.
Two samples from each group were analyzed in accordance with the analytical procedure. The remaining samples were retained for contingency. Samples were disposed of once results were obtained.
Procedural recoveries were prepared as a quality control measure and were not used to correct for the formulation samples.
The mean concentrations of Butylal in test formulations analyzed during the study and the deviation of the mean result from the nominal value are detailed in Table 1.
The mean concentrations were within +10/-15% of the nominal concentration, confirming accurate formulation. The percentage difference from mean values remained within 2%, confirming precise analysis.
Procedural recovery values were within the validated range confirming the continued accuracy of the analytical procedure.

Duration of treatment / exposure:

Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Males: Two weeks pre-pairing up to necropsy after minimum of four weeks
Females: Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation

Frequency of treatment:

Once daily at approximately the same time each day

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels of 0, 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on findings from a preliminary 4-week repeat dose toxicity study in the Sprague-Dawley rat.
In the 4-week preliminary study dose levels of 500, 750, and 1000 mg/kg/day had no effect on general condition, body weight, food consumption or macropathology. Effects were limited to high liver weights for males that received 1000 mg/kg/day and for females at all dose levels.
It was therefore considered that a high dose of 1000 mg/kg/day would be tolerated in this OECD 421 screening study. The low and intermediate doses were 100 and 300 mg/kg/day, providing approximate 3-fold dose increments and allowing investigation of any dose-related response.
- Rationale for animal assignment: On arrival and non-selective allocation to cages

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded at the following times in relation to dose administration: Pre-dose observation, One to two hours after completion of dosing, As late as possible in the working day. A detailed physical examination was performed on each animal to monitor general health according to the following schedule: F0 males weekly; F0 females: Week 1 and 2 - weekly, Gestation phase - Days 0, 7, 14 and 20, Lactation phase - Days 1, 7 and 13

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of animals was recorded as follows
F0 males: Weekly during acclimatization (not reported), Before dosing on the day that treatment commenced (Day 1) and weekly thereafter; On the day of necropsy.
F0 females: Weekly during acclimatization (not reported), Before dosing on the day that treatment commenced (Day 1) and weekly before pairing, Days 0, 7, 14 and 20 after mating, Day 1, 7 and 13 of lactation, On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Weekly before pairing.
For females after mating food consumption was recorded as follows:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
- For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
- After pairing until mating.
- For four days before scheduled termination (nominally Days 10 to 13 of lactation).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, thyroid hormone analysis. Particular attention was paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
yes
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content

Postmortem examinations (parental animals):

SACRIFICE
Sequence: To allow satisfactory inter-group comparison

GROSS NECROPSY
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected for thyroid hormone analysis: Scheduled kill - Day 13 of age
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
F1 offspring on Day 4 of age: Externally normal offspring were discarded without examination. Externally abnormal offspring were subject to an external macroscopic examination and retained pending possible future examination.
F1 offspring on Day 13 of age: All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities were retained in appropriate fixative. Thyroid glands were preserved from two offspring per litter, one male and one female in each litter, where possible.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including estrous cycles before treatment, pre coital interval, mating performance, gestation length and index and stage of estrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
A parametric analysis was performed if Bartlett's test for variance homogeneity (Bartlett 1937) was not significant at the 1% level. A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations.

Reproductive indices:

Estrous Cycle
The incidence and percentage females showing the following classifications of estrous cycles before treatment commenced and during treatment are presented:
Regular: All observed cycles of 4 or 5 days (divided into cycles of 4, 4 and 5 and 5 days)
Irregular: At least one cycle of 2, 3 or 6 to 10 days
Acyclic: At least 10 days without estrus

Vaginal smearing prior to termination is presented in terms of numbers of females that showed estrus during this period and the cycle stage at termination.
Pre-Coital Interval
Individual intervals were tabulated for females only, for the time elapsing between initial pairing and mating. Percentage of females with pre-coital intervals calculated for durations of 1-4, 5-8, 9-12 and 13-14 days of pairing.
Mating Performance and Fertility
Individual data were tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number of animals mating x 100) / Animals paired

Conception rate (%) = (Number of animals achieving pregnancy x 100) / Animals mated
Fertility index (%) = (Number of animals achieving pregnancy x 100) /Animals paired

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = (Number of live litters born x 100) / Number pregnant

Offspring viability indices:

The following were calculated for each litter:
Post-implantation survival index (%) = (Total number of offspring born x 100) / Total number of uterine implantation sites

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering x 100) / Total number of offspring born

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) x 100) / Number live offspring on Day 1 after littering

Lactation index (%) = (Number of live offspring on Day 13 after littering x 100) / Number of live offspring on Day 4 (after blood sampling)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs at routine physical examination that could be related to treatment.
Signs associated with dose administration were limited to a low incidence of increased salivation with associated chin rubbing for animals receiving 1000 mg/kg/day; these signs are often seen in association with dosing by oral gavage and are considered to relate to the palatability of the formulations rather than a direct effect of treatment with the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain for males during treatment and for females before pairing was unaffected by treatment.
During early gestation (GD0-7) females receiving 1000 mg/kg/day showed significantly low weight gain when compared with Controls (p<0.05); however subsequent weight gain and the overall weight gain during gestation was essentially similar to Controls.
During lactation the absolute body weights for females receiving 1000 mg/kg/day on LD1 and LD7 were slightly but significantly low when compared with Controls (p<0.05). Body weight gain (LD7-13 and LD1-13) for females at 1000 mg/kg/day was high when compared with Controls although the difference did not attain statistical significance.
Body weight gain at 100 and 300 mg/kg/day was unaffected by treatment during both gestation and lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for the two-week period before pairing was unaffected by treatment.
During GD14-19 food consumption for females receiving 1000 mg/kg/day was low when compared with Controls with the difference attaining statistical significance (p<0.01).
During lactation food consumption for females receiving 1000 mg/kg/day was slightly low at approximately 94% of Controls; however, the differences for each recording period did not attain statistical significance.
Food consumption at 100 and 300 mg/kg/day was unaffected by treatment during both gestation and lactation.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Individual serum TSH concentrations were found to be variable.
Both male and female mean serum TSH concentrations were slightly low in the groups that received Butylal via oral gavage when compared with the Control Group. However, these differences did not attain statistical significance.
Thyroxine levels in females that received 1000 mg/kg/day and in F1 offspring on Day 13 of age at 300 or 1000 mg/kg/day were slightly low when compared with Controls; this was not evident in F0 males or F1 female offspring on Day 4 of age. The data was highly variable and the differences from Control were slight, therefore there was no conclusive evidence that could attribute this to treatment with Butylal.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The microscopic examination performed after at least 4 weeks of treatment revealed no test item related lesions in the tissues examined (testes, epididymides and ovaries).
Incidental Findings:
All histological changes were considered to be unrelated to treatment.
One Group 3 female had a mammary adenocarcinoma which is an incidental finding and not unusual in female rats of this age.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles during treatment, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by parental treatment. At termination all females were confirmed to be in diestrus.

Reproductive performance:

no effects observed

Description (incidence and severity):

Estrous cycles during treatment, pre-coital interval, mating performance, fertility, gestation length and gestation index were unaffected by parental treatment. At termination all females were confirmed to be in diestrus.

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:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs that were considered to relate to treatment

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of implantations for females receiving 1000 mg/kg/day was slightly but significantly high when compared with Controls; subsequent litter size was slightly high but these differences did not attain statistical significance. This difference was not considered to be of any toxicological significance.
Litter size at 100 or 300 mg/kg/day and offspring survival at all dose levels was unaffected by parental treatment.
The mean percentage of male offspring in the treated groups was slightly but significantly low when compared with Controls on Day 1 and 4 of age (the values on Day 4 and 13 after females are selected to provide blood samples of T4 analysis are not considered relevant); however this is considered to be unrelated to treatment as the Control mean was atypically high.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day the absolute mean body weight for offspring on Day 1 of age (p<0.01) and subsequent mean weight gain up to termination on Day 13 of age was low when compared with Controls (80% of Controls for male offspring, p<0.05, and 84% of Controls for female offspring); the mean values at 1000 mg/kg/day exceeded the historical control range.
Offspring body weight at 100 or 300 mg/kg/day was unaffected by parental treatment.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Ano-genital distance for both male and female offspring was unaffected by parental treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were apparent for male offspring on Day 13 of age

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring that either died prematurely or at scheduled termination did not reveal any findings that could be related to parental treatment

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The oral reproductive and developmental 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 screening for reproductive and developmental toxicity studies for Ethylal and Butylal, no parental toxicity was observed. The NOAEL derived for both Ethylal and Butylal the NOAEL was found to be 1000 mg/kg bw/day for the parental development. The NOAEL for the offspring development was found for Ethylal at 1000 mg/kg bw/day and for Butlal at 300 mg/kg/day. For Butylal, toxic effects were limited to offspring derived from the group that received 1000 mg/kg/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%. This effect on offspring body weight was in the absence of any effect on offspring survival, general condition or thyroid hormones and within the context of this screening study is considered to not warrant the classification of the test item as a reproductive toxicant. In addition, an OECD TG 414 study with Ethylal did not indicate adverse effects on development, a NOAEL of 1000 mg/kg bw/day was derived for the embryo-fetal development. The data obtained from the screening for reproductive and developmental toxicity 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 reproductive/developmental toxicity studies available. The first study was conducted according to OECD TG 414 following GLP (Low, 2020, Kl1). In this study, the maternal and the embryo-foetal NOAEL is estimated to be to be 1000 mg/kg bw/day. Four groups of 20 females received Ethylal at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, corn oil at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and foetal  examination. Clinical observations, body weight and food consumption were recorded. Adult females were examined; macroscopic and microscopic pathology investigation were undertaken on Day 20 after mating and the gravid uterus weight and thyroid weight were recorded. Anogenital distance were measured for all foetuses and were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. Following initial treatment (gestation Day 6), signs associated with dosing Ethylal at 1000 mg/kg/day included unsteady gait in 11 animals, decreased activity in 5 animals, flattened posture, or gait, in 4 animals and partially closed eye lids in 3 animals. At 1000 mg/kg/day, chin rubbing was seen towards the end of treatment and at 330 mg/kg/day, or above, salivation was seen throughout the treatment period. There was no clear difference in the overall (gestation day 6 to 20) body weight change for females treated with Ethylal at any dose level. Although body weight loss/stasis was seen in females receiving 330 or 1000 mg/kg/day during gestation Day 6 to 7,there was no effect of treatment on gravid uterine weight. Food consumption of females treated at 1000 mg/kg/day was statistically significantly low prior to the start of treatment and remained low throughout treatment, although statistical significance was not attained on the last recording occasion. There was no effect of treatment on the macroscopic appearance of organs in the adults or the external appearance of foetuses at necropsy. The microscopic examination of the thyroid and parathyroid revealed no test item-related lesions. There were no clear effects of treatment on early or late resorptions, numbers of live young, sex ratio or pre/post implantation losses. There were no effects of treatment on placental, litter or foetal weights. There was no effect of treatment on ano-genital distances. There was a slight dose related decrease in levels of T3 at 330 or 1000 mg/kg/day, a slight increase in T4 levels at 330 or 1000 mg/kg/day and a slight increase in TSH levels at 1000 mg/kg/day. The incidence of major and minor abnormalities and skeletal variants in foetuses show no relationship to treatment.

 

The second reproductive/developmental toxicity study was conducted according to the OECD TG 421 following GLP (Whitehead, 2019, Kl1). In this study, groups of male and female rats were exposed to Ethylal by gavage at concentrations of 300, 600 or 1000 mg/kg/day. The NOAEL for Ethylal was estimated to be 1000 mg/kg bw/day in the absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose (5 mL/kg/day) as the treated groups. During the study, clinical condition, body weight, food consumption, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis. The mean concentrations of Ethylal in test formulations analysed confirmed accurate and precise formulation. Administration of Ethylal at dose levels of 300, 600 and 1000 mg/kg/day was well tolerated with no effects that could be attributed to treatment on clinical condition, oestrous cycles, organ weights, macroscopic examination or histopathology. In addition there were no effects on pre-coital interval, mating performance, fertility, gestation length, offspring survival or development. Body weight performance and food consumption was not adversely affected by treatment before pairing, during gestation and during lactation when compared with Controls. There was no test item related finding seen in this study, following the histopathological examination of testes and epididymides in males, ovaries of females or abnormalities in both sexes given Ethylal. There was no effect of treatment on litter size or the survival, growth, anogenital distance or development of the offspring. T4 levels in F0 males and Day 13 offspring were unaffected by treatment.

 

For Butylal, one reproductive/developmental oral dose toxicity study available. The study was conducted according to OECD TG 421 following GLP (Renaut, 2022, Kl1). In this study, the NOAEL is estimated to be  1000 mg/kg bw/day for parental systemic toxicity and reproductive parental toxicity. For offspring development, 1000 mg/kg bw/day was considered to be the lowest observed adverse effect level (LOAEL) with a NOAEL of 300 mg/kg bw/day. Groups of male and female Sprague-Dawley (CD) rats were exposed to Butylal by gavage at concentrations of 100, 300 or 1000 mg/kg bw/day.  Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose (5 mL/kg/day) as the treated groups. During the study, clinical condition, body weight, food consumption, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, thyroid hormone analysis, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age. Blood samples were collected from selected offspring on Day 4 and Day 13 of age for thyroid hormone analysis. Treatment of Butylal to parental animals at 100, 300 or 1000 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, thyroid hormones, oestrus cycles, mating performance, fertility, macropathology or histopathology. The clinical condition and survival of the subsequent F1 offspring were also unaffected by parental treatment. Adverse findings were limited to offspring derived from the group that received 1000 mg/kg/day which had low absolute weight and body weight gain from Day 1 of age. In terms of percentage, the difference between the body weight change from day 1 to the last day of the experiment between the control and the highest concentration was found to be for male animals 20% and for female animals 16%. This effect on offspring body weight was in the absence of any effect on offspring survival, general condition or thyroid hormones and within the context of this screening study is considered to not warrant the classification of the test item as a reproductive toxicant.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Toxicity to reproduction: other studies

Description of key information

It was observed from this exploratory investigation that embryos cultured in the presence of propylal exhibited no adverse effects on growth, development or morphology. It was concluded, therefore, that propylal showed no potential embryo toxicity or teratogenicity.

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 5 April 2016, Experimental completion date: 20 May 2016.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: INVITTOX protocol no. 123.

Deviations:

yes

Remarks:

please see any other information on methods and materials section

GLP compliance:

yes (incl. QA statement)

Type of method:

in vitro

Specific details on test material used for the study:

Test substance: Propylal
Test substance identity (including alternative names): Dipropoxymethane
CAS number: 505-84-0
Intended use: solvent
Appearance: Colourless liquid
Storage conditions: Refrigerated (2-8°C), protected from light.
Batch number: 1602181700R
Expiry date: 18 February 2017
Purity: 99.6658%

Species:

rat

Strain:

other: Crl:CD (SD)

Details on test animals or test system and environmental conditions:

Animal supply, acclimatisation and allocation
Strain/Species Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd.
Number of animals ordered 80 females.
Duration of acclimatisation At least 1week before commencement of pairing.
Age of the animals ordered Approximately 9 weeks old.

Mating
Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.

Allocation and identification
Allocation On the day of positive evidence of mating (Day 0).
Identification of animals Each animal was assigned a number and identified uniquely within the study by a tail tattoo.

Environmental control
Rodent facility Full barrier - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 19-23ºC and 40-70%, respectively.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light : 12 hours dark, with lights on at 01:00 GMT.
Window blinds remained open for the first three days allowing light ingress during the dark period. Blinds were closed prior to 13:00 GMT on 1st April 2016.
Electricity supply Public supply with automatic stand-by generators.

Animal accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatisation and gestation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Bedding (solid bottom cages) Wood based bedding which was changed at appropriate intervals each week.

Environmental enrichment
Aspen chew block Provided to each animal throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet supply
Diet SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Route of administration:

other: Roller bottle culture technique

Vehicle:

corn oil

Details on exposure:

The roller bottle culture technique was used (New 1978). Embryos were cultured in the presence of each test substance for a period of approximately 48 hours at a temperature of 37.0 ± 1.0°C.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

48 hours

Duration of test:

48 hours

Dose / conc.:

0 other: µg/mL

Dose / conc.:

30 other: µg/mL

Dose / conc.:

100 other: µg/mL

Dose / conc.:

300 other: µg/mL

Dose / conc.:

1 000 other: µg/mL

No. of animals per sex per dose:

3 embryos per dose for preliminary study.
7 embryos per dose for main study

Control animals:

yes, concurrent vehicle

Details on study design:

A preliminary test (0, 1, 10, 100 and 1000 µg/mL) was used to establish suitable treatment levels for the Main study. Propylal and Butylal showed little or no adverse effect on embryonic growth or development at dose levels up to 1000 μg/mL

Culture medium
The culture medium consisted of equal volumes of homologous rat serum, heat inactivated at 56.0 ± 0.5°C for 40 minutes, and HEPES-buffered Eagle’s minimum essential medium.

Maternal euthanasia
Dams were anaesthetised with a mixture of isofluorane and oxygen on the afternoon of the ninth day of gestation and, after removal of the uterus, were killed by exsanguination and/or cardiac section.

Embryo preparation
Decidua were subsequently released from the uterus and the embryos dissected out. The parietal yolk sac was torn open and removed. Only overtly healthy embryos with the visceral yolk sac and ectoplacental cone intact were cultured.

Culture methods
Embryos were incubated at 37.0 ± 1.0°C in 30 mL glass bottles which were rotated continuously at 60 rev/min throughout the period of culture. Each bottle contained up to five embryos with 1 mL of culture medium per embryo and a gas phase. The gas phase was 5% O2, 5% CO2 and 90% N2 for approximately 20 hours followed by 20% O2, 5% CO2 and 75% N2. Each bottle was identified by indelible marker pen showing group number, compound name and dose level. Cultures were terminated after approximately 48 hours.
Temperature was monitored continuously but recorded daily. Since these data show that there were no significant deviations from target values they are not presented.

Dose descriptor:

NOAEL

Effect level:

> 1 000 other: μg/mL

Based on:

test mat.

Basis for effect level:

other: embryonic growth, development and morphology

Preliminary study

Embryos cultured in the presence of Propylal at doses up to 1000 μg/mL exhibited no adverse effect on embryonic growth, development or morphology. Findings at 1 μg/mL were not attributed to treatment.

Main Study

Embryos cultured in the presence ofPropylalat doses up to 1000 μg/mL exhibited no adverse effect on embryonic growth, developmentor morphology. Morphological observations were generally of a minor nature and were therefore considered incidental.

Discussion

Low incidences of minor morphological observations were recorded; these were mostly haemorrhages and common pericardial defects, which were considered unlikely to be related to treatment. 

For preliminary study cultures started on 19 April 2016, stoppers had fallen out of four bottles prior to the start of culture. The medium within these bottles appeared bright pink. Since there was insufficient time to prepare fresh medium these bottles were assigned to the lowest concentration level for material dosed on that day. Effects seen could be attributed to the change in the culture medium as they were not seen at the higher dose levels; hence for propylal, findings at 1 μg/mL were not attributed to treatment

No other effects were seen following treatment with propylal, hence this material was considered not to show developmental toxicity in vitro.

Conclusions:

It was observed from this exploratory investigation that embryos cultured in the presence of propylal exhibited no adverse effects on growth, development or morphology. It was concluded, therefore, that propylal showed no potential embryo toxicity or teratogenicity.

Executive summary:

Summary

The influence of Propylal, upon growth and development in vitro was assessed in Day 9.5 embryos from rats of the CD strain. Each test substance was added to the culture medium at concentrations of 1, 10, 100 and 1000 mg/mL in groups of 3 embryos in the preliminary study. In the main study groups of 7 embryos were administered each test substance at concentration levels of 30, 100, 300 and 1000mg/mL. The negative Control group received the vehicle corn oil at the same volume-dose. All embryos were evaluated after approximately 48 hours in culture.

Results

Propylal: No adverse effect on embryonic growth, development or morphology was observed at doses up to 1000 μg/mL.

Conclusion

It was observed from this exploratory investigation that embryos cultured in the presence of propylal exhibited no adverse effects on growth, development or morphology. It was concluded, therefore, that propylal showed no potential embryo toxicity or teratogenicity.