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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 MARCH 2018 to 15 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

219-909-0

EC Name:

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

Cas Number:

2568-90-3

Molecular formula:

C9H20O2

IUPAC Name:

1-(butoxymethoxy)butane

Test material form:

liquid

Details on test material:

SOURCE OF TEST MATERIAL
- Batch No.of test material:
1802161800R
- Expiration date of the batch:
16 February 2020
- Purity: 99.9752 %
- Appearance: Clear Liquid.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At ambient temperature (15 to 25°C), in the dark.

Test animals

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

Administration / exposure

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 (Table 3). 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

5 ml/kg

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

5 ml/kg - 20 mg/ml

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

5 ml/kg - 60 mg/ml

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

5 ml/kg - 200 mg/ml

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.

Examinations

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
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 3 were examined.

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 (see table 4)

HISTOPATHOLOGY: Yes (see table 4)

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.

Results and discussion

Results of examinations

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).

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.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Local NOAEL

2

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

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

It is concluded that the oral administration of Butylal to Sprague-Dawley rats for 13 weeks at doses of 100, 300 or 1000 mg/kg/day was well-tolerated in-life, but at 1000 mg/kg/day caused local irritation to the gastric epithelium resulting in minimal hyperplasia of the nonglandular epithelium. The local effects of ulceration in the stomach were adaptive but adverse; however the systemic effects seen were considered not adverse. Therefore, the systemic no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg/day and the local no-observed-adverse-effect-level (NOAEL) was considered to be 300 mg/kg/day.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of Butylal, an industrial chemical, when administered orally, by gavage to Crl:CD(SD) rats for 13 weeks.

Three groups, each comprising 10 male and 10 female Sprague-Dawley (CD) rats, received Butylal at doses of 100, 300 or 1000 mg/kg/day. 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, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

Results

The appearance and behavior 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/day exhibited a significantly higher body weight gain in the first 6 weeks on 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/day.

There were no treatment related signs during ophthalmic examination.

At the hematological 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 was significantly higher than control in males at 300 or 1000 mg/kg/day and in females at 1000 mg/kg/day; cholesterol concentration was significantly higher than control in animals of both sexes at 300 or 1000 mg/kg/day; and, triglyceride concentration was significantly higher than control in males at 300 or 1000 mg/kg/day and in females at 1000 mg/kg/day. Other statistically significant changes in blood chemistry were considered unrelated to treatment because they did not show a dose-response relationship or were observed in only one sex.

Examination of the composition of the urine in Week 13 indicated an increase in urinary protein in males receiving 1000 mg/kg/day.

After 13 weeks of treatment, adjusted liver weights were slightly increased in both sexes given 1000 mg/kg/day. In males at 1000 mg/kg/day, adjusted heart weights were significantly reduced; and, in females at 1000 mg/kg/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 two males treated at 1000 mg/kg/day. At the histopathologic examination, these same two 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/day in both sexes.

Centrilobular hypertrophy (minimal) of the liver was observed in females at 300 and 1000 mg/kg/day; similar changes were not reported in males. Thyroid follicular cell hypertrophy was increased in males at 300 and in both sexes at 1000 mg/kg/day. In males, all animals at 1000 mg/kg/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/day, but not seen in females.

Conclusion

It is concluded that the oral administration of Butylal to Sprague-Dawley rats for 13 weeks at doses of 100, 300 or 1000 mg/kg/day was well-tolerated in-life, but at 1000 mg/kg/day caused local irritation to the gastric epithelium resulting in minimal hyperplasia of the nonglandular
epithelium. The local effects of ulceration in the stomach were adaptive but adverse; however the systemic effects seen were considered not adverse. Therefore, the systemic no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg/day and the local no-observed-adverse-effect-level (NOAEL) was considered to be 300 mg/kg/day.