Diethoxymethane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

It is a 14-day preliminary toxicity study.

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 March 2018 - 18 April 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study generally followed Good Laboratory Practice principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Principles of method if other than guideline:

The study was not designed to meet any particular regulatory requirements. No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Diethoxymethane

EC Number:

207-330-6

EC Name:

Diethoxymethane

Cas Number:

462-95-3

Molecular formula:

C5H12O2

IUPAC Name:

diethoxymethane

Test material form:

liquid

Details on test material:

Identification: Diethoxymethane
Synonym: Ethylal
CAS Number: 462-95-3
Physical State/Appearance: Clear colourless liquid
Storage Conditions: Approximately 4 °C, in the dark, under nitrogen
No correction for purity was required.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 1709141400R
- Expiration date of the batch: 14 September 2019
- Purity: 99.987%
- Appearance: Colorless liquid.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Under nitrogen, refrigerated (2 to 8°C) and protected from light (highly volatile). Allowed to return to room temperature (15 to 25°C) before opening the bottle.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD) rat.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation:
Males: 70 to 77 days, Females: 84 to 91 days
- Weight at study initiation:
Males: 336 to 395 g, Females: 257 to 292 g
- Housing:
Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:
Seven days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
20-24
- Humidity (%):
40-70
- Air changes: 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: 28 March 2018 To:18 April 2018

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was mixed with a small quantity of the vehicle. Approximately 20% of the final volume of vehicle was gradually added. The formulation was quantitatively transferred and diluted to final volume and magnetically stirred in a sealed container of appropriate size ensuring there is no head space. Homogeneity and stability of the preparation were assessed in another study (KG04BG)

VEHICLE
- Concentration in vehicle:
0, 60, 120, 200 mg/mL

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once daily at approximately the same time each day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection:
The doses used in this study (0, 300, 600 and 1000 mg/kg/day) were selected in conjunction with the Sponsor.
The oral LD50 in rats appears to be 3536 mg/kg bw.
A previous acute oral toxicity study conducted on the test material found that a single dose to rats of 5000 mg/kg caused mortality within 24 hours administration (4 of 5 animals). Signs of toxicity included ataxia, weakness (slight to severe) and prostration. Ataxia, weakness (slight to severe) was observed in the other animals administered with test article at a dose of 1250 or 2500 mg/kg, but there were no deaths at these levels.
Based on these results, to aid in determining dose levels for the subsequent OECD 421 study, dose levels of 1000 mg/kg/day (limit dose) 600 and 300 mg/kg/day were studied to define a high dose to induce toxicity but not death or of a severity to compromise interpretation of the results and a low dose, free of adverse effects with repeated administration according to the study design. The intermediate dose was approximately the geometric mean between the high and low dose levels.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded three days before treatment commenced, on the day that treatment commenced (Day 1), on Day 4, 8 and 11 of the study and before necropsy on Day 15.

Sacrifice and pathology:

All 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.
The retained tissues were checked before disposal of the carcass.

The organs weighed and tissue samples fixed are detailed as follows:
Abnormalities: Organs weighed and samples fixed.
Kidneys: Bilateral organs weighed individually and samples fixed.
Liver: Organs weighed and samples fixed.
Spleen: Organs weighed and samples fixed.

The list of tissues preserved was intended to satisfy any possible future requirement for further examination.

Statistics:

No statistical analysis of the data was performed on this study.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Table 1

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Table 2

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Table 3

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

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The analysis of absolute organ weights for animals killed after 14 days of treatment indicated, when compared to the Controls, an increase in the weight of the liver in males and females treated with 600 mg/kg/day of Ethylal, and an increase of kidney weight in males treated with 600 or 1000 mg/kg/day of Ethylal, but no dose response was apparent.
Table 4

Gross pathological findings:

no effects observed

Description (incidence and severity):

Table 5

Any other information on results incl. tables

 

	Control	Ethylal
Dose group	1	2	3	4
Dose (mg/kg/day)	0	300	600	1000

Table 1: Signs associated with dosing - group distribution of observations

				Number of animals affected
Category	Observation	Day	Group/Sex: Initial no:	1M 3	2M 3	3M 3	4M 3	1F 3	2F 3	3F 3	4F 3
Abnormal gait	Unsteady	1 2		0 0	0 0	0 0	3 1	0 0	0 0	0 0	2 0
Behavior	Decreased activity	1 2		0 0	0 0	1 0	3 1	0 0	0 0	1 0	3 0
Coat	Piloerection	2		0	0	0	1	0	0	0	0
Eyelids	Partially closed, Bilateral	1		0	0	0	0	0	0	1	0

 

Table 2: Body weight - group mean values (g)

Group /sex		Day P4	Day 1	4	8	11	Change 1-15
1M	Mean	350	367	380	382	398	49
	SD	25.3	26.8	25.4	41.8	33.5	9.3
	N	3	3	3	3	3	3
2M	Mean	343	361	374	386	395	54
	SD	8.3	3.4	9.2	2.5	6.6	2.6
	N	3	3	3	3	3	3
3M	Mean	343	361	376	392	406	61
	SD	11.6	13.1	14.8	14.2	19.0	10.9
	N	3	3	3	3	3	3
4M	Mean	350	367	375	392	400	49
	SD	21.8	24.4	19.1	22.4	25.9	12.7
	N	3	3	3	3	3	3

Group /sex		Day P4	Day 1	4	8	11	15	Change 1-15
1F	Mean	264	275	268	279	280	284	9
	SD	4.8	12.1	4.6	3.5	3.6	8.1	4.8
	N	3	3	3	3	3	3	3
2F	Mean	259	274	276	283	285	288	15
	SD	12.0	12.0	8.6	7.1	9.1	8.8	17.8
	N	3	3	3	3	3	3	3
3F	Mean	269	271	277	284	289	297	26
	SD	6.1	13.9	12.2	18.0	18.3	22.6	10.1
	N	3	3	3	3	3	3	3
4F	Mean	271	278	282	289	292	300	22
	SD	20.7	11.9	19.4	19.5	28.1	26.9	15.0
	N	3	3	3	3	3	3	3

 

Table 3: Food consumption - group mean values (g/animal/day)

Group /sex		Day P4-P7	Day 1-4	4-8	8-11	11-15	Mean 1-15
1M	Mean	28	23	22	22	23	23
	SD	-	-	-	-	-	-
	N	1	1	1	1	1	1
2M	Mean	28	23	23	24	23	23
	SD	-	-	-	-	-	-
	N	1	1	1	1	1	1
3M	Mean	31	26	25	26	25	26
	SD	-	-	-	-	-	-
	N	1	1	1	1	1	1
4M	Mean	30	24	24	25	24	24
	SD	-	-	-	-	-	-
	N	1	1	1	1	1	1

Group /sex		Day P4-P7	Day 1-4	4-8	8-11	11-15	Mean 1-15
1F	Mean	20	13	18	17	17	16
	SD	-	-	-	-	-	-
	N	1	1	1	1	1	1
2F	Mean	22	17	17	17	17	17
	SD	-	-	-	-	-	-
	N	1	1	1	1	1	1
3F	Mean	20	17	17	16	17	17
	SD	-	-	-	-	-	-
	N	1	1	1	1	1	1
4F	Mean	24	17	18	18	18	18
	SD	-	-	-	-	-	-
	N	1	1	1	1	1	1

 

Table 4: Organ weights - group mean absolute values (g) for animals killed after 14 days of treatment

Group /sex		Terminal Bodyweight	Kidneys	Liver	Spleen
1M	Mean	414	2.914	17.390	0.747
	SD	32	0.330	1.717	0.082
	N	3	3	3	3
2M	Mean	411	3.085	18.481	0.702
	SD	2	0.255	1.080	0.037
	N	3	3	3	3
3M	Mean	421	3.325	19.236	0.684
	SD	24	0.202	2.273	0.047
	N	3	3	3	3
4M	Mean	419	3.242	18.257	0.805
	SD	25	0.126	1.352	0.078
	N	3	3	3	3

Group /sex		Terminal Bodyweight	Kidneys	Liver	Spleen
1F	Mean	285	2.019	11.556	0.591
	SD	6	0.081	1.168	0.077
	N	3	3	3	3
2F	Mean	291	2.268	12.093	0.658
	SD	7	0.180	0.435	0.118
	N	3	3	3	3
3F	Mean	298	2.105	13.703	0.638
	SD	22	0.244	1.666	0.030
	N	3	3	3	3
4F	Mean	300	1.947	12.125	0.656
	SD	26	0.246	1.112	0.141
	N	3	3	3	3

 

Table 5: Macropathology - group distribution of findings for animals killed after 14 days of treatment

		Number of animals affected
Tissue/Organ and Findings	Group/Sex No. of animals	1M 3	2M 3	3M 3	4M 3	1F 3	2F 3	3F 3	4F 3
Number of animals within normal limits		3	3	3	3	3	3	2	3
Skin and Subcutis Hair Loss		0	0	0	0	0	0	1	0

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, treatment with the test item, Ethylal, was well tolerated at oral doses up to 1000 mg/kg/day for 14 days. Therefore, it was concluded that 1000 mg/kg/day would be a suitable high dose level for the subsequent Reproductive/Development Toxicity Screening Study in the Sprague Dawley rat by Oral Gavage Administration (OECD 421)

Executive summary:

The purpose of this study was to assess the systemic toxic potential of Ethylal in a 14-day oral gavage study in Sprague Dawley rats, to select suitable doses for a subsequent reproduction/developmental toxicity screening test (OECD TG 421).

Three groups, each comprising three male and three female Sprague Dawley (Crl:CD(SD)) rats, received Ethylal at doses of 300, 600 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, clinical condition, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken.

 

Results

All animals survived to the scheduled sacrifice. Post dose signs of decreased activity and unsteady gait were seen on Day 1 of treatment at 1000 mg/kg/day, and in one male on Day 2 at this dose level.

There were no treatment related clinical observations, no adverse effects on bodyweight gain or food intake, and no changes in water consumption. The organ weights of the liver and kidney were slightly high in animals treated at 600 mg/kg/day and of the kidney of males at 1000 mg/kg/day. The spleen weight was unaffected by treatment.

There were no treatment related findings seen at macroscopic examination.

 

Conclusion

Based on the results of this study, treatment with the test item, Ethylal, was well tolerated at oral doses up to 1000 mg/kg/day for 14 days. Therefore, it was concluded that 1000 mg/kg/day would be a suitable high dose level for the subsequent Reproductive/Development Toxicity Screening Study in the Sprague Dawley rat by Oral Gavage Administration (OECD 421)