Bis(2-ethoxyethyl) ether

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 9, 2021 to May 9, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

OECD TG compliant GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

Remarks:

There were no deviations from the OECD TG. However, between Day 60-63, slightly higher volume of doses were administered. The deviations were minor. No effect on study interpretation.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethoxyethyl) ether

EC Number:

203-963-7

EC Name:

Bis(2-ethoxyethyl) ether

Cas Number:

112-36-7

Molecular formula:

C8 H18 O3

IUPAC Name:

1-ethoxy-2-(2-ethoxyethoxy)ethane

Test material form:

liquid

Remarks:

Colorless transparent

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sponsor
- lot/batch number of test material: 1B25AB
- Purity: 99.9%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Cool and dry (+2 to +8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: dose formulations at the dose of 5 mg/mL and 100 mg/mL are stable for 6 hours and 48 hours at room temperature (reference: Study No.: BIO-ANM 1807)


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the recommended species by regulatory agencies for conducting toxicity assessment studies among rodents.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals (Bioneeds)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Housing: Two animals of same sex were housed in a standard polysulphonate cage (size: L 43 x B 28 x H 21 cms) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.

- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: The contaminant analysis test report for food, water and bedding material nearest to the experiment period are included in the report.

ENVIRONMENTAL CONDITIONS
- Temperature: 19.6°C to 22.9°C
- Humidity: 47% to 65%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered through oral (gavage) route using stainless steel cannula as it is the probable route in human.

Vehicle:

water

Remarks:

Distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed in a beaker and small quantity of vehicle was added and mixed with a glass rod. Thereafter the entire quantity of the formulation was transferred into measuring cylinder. A small quantity of vehicle was added to rinse the beaker and this was transferred into the measuring cylinder. Finally, the volume was made up to required quantity with vehicle to get desired concentration of 10, 30 and 100 mg/mL of test item for low, mid and high dose groups respectively.


VEHICLE
- Concentration in vehicle: 0, 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): 7-23, JA072
- Purity: not relevant

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis for concentration verification was done by Analytical Chemistry Department of Bioneeds India Private Limited as per validated analytical methods detailed in the Study No. BIO-ANM 1807.

Formulation analysis for concentration verification was performed for all the dose formulations on day 1 (or prior to first dosing) and during month 2 and month 3 of the treatment. The prepared formulations were sampled in duplicate sets from each dose level of prepared formulations including vehicle control.. Exact volumes of test item formulation samples were included in study report.

The collected samples were transferred to Analytical Chemistry Department of Bioneeds India Private Limited for dose formulation analysis at established stability conditions. One set of aliquot of each formulation was analysed.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once/day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Formulations are considered acceptable, mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is ≤10.0%.

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Formulations are considered acceptable, mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is ≤10.0%.

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Formulations are considered acceptable, mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is ≤10.0%.

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Formulations are considered acceptable, mean results are within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) is ≤10.0%.

No. of animals per sex per dose:

10 (Recovery groups (5/sex additional))

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A dose range finding study (study no. BIO-CTX 401) was conducted with the dose levels of 100, 300 and 1000 mg/kg/day. The results of the study indicated that there were no adverse changes in any of the parameters up to the dose levels of 1000 mg/kg/day. Hence, in the current study, same dose levels of 100, 300 and 1000 mg/kg/day were selected as low (G2), mid (G3) and high (G4/G4R) doses.

- Fasting period before blood sampling for clinical biochemistry:
10-12 hours before blood collection


- Rationale for selecting satellite groups: recovery groups


- Post-exposure recovery period in satellite groups: 28 days post exposure


- Dose range finding studies:
A dose range finding study (study no. BIO-CTX 401) was conducted with the dose levels of 100, 300 and 1000 mg/kg/day. The results of the study indicated that there were no adverse changes in any of the parameters up to the dose levels of 1000 mg/kg/day.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the treatment and at weekly intervals thereafter during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 before test item administration and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during Week 13 for vehicle control and high dose main group animals and during Week 17 for recovery group animals. No treatment related ocular changes were observed in high dose group animals. Hence, the examination was not extended to lower dose group animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 91 and 119 for main and recovery groups, respectively.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- Parameters checked: See attachments

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: same as hematology
- Animals fasted: same as hematolog
- Parameters checked: See attachments

PLASMA/SERUM HORMONES/LIPIDS: Yes (T4, T3, TSH)
- Time of blood sample collection: at termination
- Animals fasted: Not specific

URINALYSIS: Yes
- Time schedule for collection of urine: Day 91 and 119 (main and recovery, respectively)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided)
- Parameters checked: see attachment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 13 for vehicle and high dose main group; Week 17 for recovery group.
- Dose groups that were examined: See above
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

The data was subjected to statistical analysis. The computer printout of the data (in the form of appendix) was verified with the original raw data. After verification, the data was subjected to statistical analysis using SPSS software version 27. Body weight, percent change in body weight (with respect to Day 1 body weight), feed consumption, organ weights and ratios, hematological and clinical chemistry estimations and urine analysis parameters (pH, specific gravity and urobilinogen), FOB parameters were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment and recovery groups comparing with the respective vehicle control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). Statistically significant changes obtained from the aforementioned tests was designated by the superscripts throughout the report as stated below:
*: Statistically significant (P<0.05)

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):

statistically significant lower percent change in body weight with respect to day 1 was noted during days 1-8 (G4RF). The noted variation is considered incidental in the absence of change in body weights and no concomitant variation in feed consumption was noted.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

statistically (p<0.05) significant higher feed consumption during week 4 to week 6 in G2 females was noted. The noted variation is considered incidental as the noted changes were inconsistent and there were no concomitant variations in body weights.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

In main group males, increase in RBC (Erythrocyte count) in G2 (+6.4%), percentage eosinophils in G3: (+90.2%) and decrease in MCHC (Mean corpuscular hemoglobin concentration) in G4: (-3.9%), decrease in absolute neutrophils in
G4: (-51.6%) and decrease in APTT (Activated Partial Thromboplastin Time) in
G2: (-20.5%); G3: (-23.0%); G4: (-27.7%); In females decrease in percent reticulocyte count in G4: (-29.3%), absolute reticulocyte count in G4: (-29.6%) and increase in percentage monocytes in G2: (+54.9%); G4: (+58.0%) and increase in APTT (Activated Partial Thromboplastin Time) in G2: (+19.8%) were noted.
Decrease in APTT in males is toxicologically not significant. All the other noted variations are considered incidental as they lack dose dependency and/or the significance were minimal in nature and random in occurance.
In recovery males, decrease in absolute neutrophils in G4R: (-29.3%) was noted and in recovery females, increase in percentage basophils in G4R: (+46.2%) and decrease in MCV (Mean corpuscular volume) in G4R: (-7.9%) and decrease in MCH (Mean corpuscular haemoglobin) in G4R:(-8.1%) was noted. All the noted variations in recovery groups are also considered incidental as similar incidences were not noted at the end of treatment period and differ between sexes. Further, the variations noted could be due to random biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In main group males, increase in calcium G4: (+5.1%), albumin G4: (+6.6%), albumin globulin ratio G3: (+11.3%), chloride G3: (+1.6%); G4: (+1.7%) and potassium G4: (+7.4%); in females increase in total protein G2: (+5.7%); G3: (+6.9%), albumin G3: (+6.8%), globulin G2:(+9.2%), urea in G3: (+19.8%), LDL (Low Density Lipoprotein) in G4: (+34.3%) and BUN (Blood Urea Nitrogen) in G3: (+19.8%) and decrease in creatinine in G4: (-8.2%) and calcium in G4: (-5.3%) was noted. All changes are considered incidental because of their minimal nature and also few varaitions lack dose dependency and some were not associated with any microscopic changes in associated organs.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

in main group males statistically significant increase in specific gravity in (G4) and decrease in pH in G4 was noted which is considered incidental in the absence of any microscopic changes in kidneys. In females decrease in pH in (G3), increase in specific gravity in (G3), was noted which is considered incidental as it lacks dose dependency.
In recovery males, statistically significant increase in specific gravity in (G4R), was noted which is considered incidental because of its isolated occurrence and no such variation was noted in other sex.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

statistically significant increase in movement count in (G4 males) was noted. In the absence of any adverse changes in any of the other neuromuscular parameters, the noted changes are considered incidental and also there were no clinical signs observed during the experimental period that could be attributed to the variations noted.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Relative organ weight: In main group females, decrease in weight of lungs in G3 was noted.
Absolute organ weight: In recovery females, increase in absolute weight of brain in G4R was noted.
Relative organ weight: In recovery males, increase in weight of brain in G4R and increase in weight of liver in G4R was noted and in recovery females increase in weight of brain in G4R was noted.
The variations noted were not associated with any macroscopic changes and hence considered incidental.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Few microscopic findings observed in the study such as ultimobranchial cyst/s in thyroid gland, epithelial cyst/s in thymus, ectopic adrenocortical tissue in adrenal gland and all other findings were considered incidental as they occurred randomly across the groups including concurrent controls and/or were expected for laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No treatment related changes in thyroid hormones (T3, T4 and TSH) were noted. In females decrease in T3 in G4F was noted. In recovery males decrease in T3 in G4RM and in recovery females increase in TSH in G4RF were noted. These variations are considered incidental as there were no macroscopic changes in noted groups and also no microscopic changes in high dose main groups.


Vaginal smear examination revealed normal stages of estrous cyclicity both in main and recovery females.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No adverse effects observed at the highest dose tested.

Target system / organ toxicity

Key result

Critical effects observed:

no

System:

other: No adverse effects were observed in this study

Any other information on results incl. tables

See attachment titled 'Summary_Tables' for all results in a summary form.

Applicant's summary and conclusion

Conclusions:

Based on the observed results and experimental conditions used in the study, the repeated oral administration of test item, DI ETHYL DI GLYCOL (DEDG) to Sprague Dawley rats for 90 consecutive days, did not result in any changes in growth, feed consumption, ocular changes, organ weights and gross or histopathological changes. Hence, the No Observed Adverse Effect Level (NOAEL) of the test item, DI ETHYL DI GLYCOL was found to be 1000 mg/kg/day when administered for a period of 90 consecutive days repeatedly by oral (gavage) route to Sprague Dawley rats under the experimental conditions and the doses employed.

Executive summary:

The objective of this study was to assess the toxic potential of the test item, DI ETHYL DI GLYCOL (DEDG) when administered for a period of 90 consecutive days repeatedly by oral (gavage) to Sprague Dawley rats. This study provides information on major toxic effects, target organs, possibility of cumulative effects and also to assess the reversibility of effects (after 28 days recovery period) and an estimate of the No Observed Adverse Effect Level (NOAEL).

A total of 100 Sprague Dawley rats (50 males + 50 females) were distributed to 4 main and 2 recovery groups. Each main group (G1 and G4) consisted of 10 rats/sex and each recovery group (G1R and G4R) consisted of 5 rats/sex. The animals allocated to groups G2, G3, G4/G4R were administered with oral doses of 100, 300 and 1000 mg/kg body weight/day of DI ETHYL DI GLYCOL. The animals allocated to group G1/G1R received vehicle (distilled water) alone. The vehicle/test item formulations were administered by oral (gavage) route at an equivolume of 10 mL/kg body weight.

The test item formulations were stable for 6 hours and 48 hours at room temperature. Formulation analysis for concentration verification was performed for all the dose formulations prior to initiation of treatment, month 2 and month 3 of the treatment period. As per the results, the dose formulations were within the acceptable limits of ±10% to the nominal concentrations with less than 10%. All animals were observed once daily for clinical signs and twice daily for mortality; weekly for detailed clinical examination, weekly body weight and feed consumption; ophthalmoscopic examination during week 13 for control and high dose main groups (G1 and G4) and during week 17 for recovery groups (G1R and G4R). Neurological/Functional observational battery during week 13 for control and high dose main group animals (G1 and G4) and during week 17 for recovery group animals (G1R and G4R).

At the end of treatment and recovery period (day 91 for main groups and day 119 for recovery group), blood and urine samples were collected and analysed. Subsequently, the animals were sacrificed and subjected to gross pathological examination and the organs were collected, weighed and preserved. The tissues/organs in vehicle control (G1) and high dose (G4) group animals were subjected to histopathological examination.

No treatment related clinical signs of toxicity or mortality was observed in any of the doses tested. No treatment related changes in body weight, percent change in body weight with respect to day 1, feed consumption, functional/neurological examination and ophthalmoscopic examination were noted. No adverse treatment related changes were noted in haematology, clinical chemistry, coagulation and urinalysis parameters. There were no test item related significant variations noted in any of the thyroid hormones assessed (T3, T4 and TSH) when compared with vehicle control group.

There were no gross pathological changes noted in any of the rats. There were no test item related histopathological changes noted.