Ethanol, 2,2'-oxybis-, 1,1'-diformate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on June 25, 2018.

Deviations:

yes

Remarks:

findings on sexual organs gross pathology and histopathology, oestrous cycle and spermatogenesys assessment were also recorded

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-oxybis-ethanol diformate

EC Number:

601-722-4

Cas Number:

120570-77-6

Molecular formula:

C6H10O5

IUPAC Name:

2,2'-oxybis-ethanol diformate

Details on test material:

- Name of test material (as cited in study report): 2,2-Oxybis-ethanol diformate (Diethylene Glycol Diformate)
- Analytical purity: no data

For OECD 471, OECD 473, OECD 490, OECD 414 and OECD 408
Name of test material (as cited in the tstudy report): Diethylene glycol diformate
CAS No. 120570-77-6
Chemical Name (IUPAC) : 2,2’-oxybis-ethanol diformate:
Physical Appearance : Clear pale-yellow liquid, free from suspended matter
Purity (as per CoA) : 96.1%
Batch No. : 26088
Manufactured Date : 07 July 2019
Retest Date : 06 July 2021
Recommended Storage Condition: Ambient(+18 to +36ºC)
pH: 2.8 at 30% water solution
density: 1.19 at 20°C

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Source :
Hylasco Biotechnology Pvt. Ltd.
Plot 4B, AKP, Turkapally Village,
Shameerpet Mandal, RR Dist,
Telangana 500078

Sex:

male/female

Details on test animals or test system and environmental conditions:

No. of groups: 6

No. of rats/group:
• Main groups: 10 males + 10 females
• Recovery groups: 5 males + 5 females
Total = 100 (50 males + 50 females)

Age at the start of treatment: 7 weeks

Body weight range at the start of treatment:
Males : 245.55 to 299.18 g
Females : 185.09 to 223.57 g

Acclimatization :
After clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before start of the treatment. During acclimatization period, rats were observed once daily for any abnormalities. Only nulliparous and nonpregnant females were used in the study.

Husbandry Conditions:
Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 19 to 24°C and relative humidity was between 56 to 68 %. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12-15 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings

Housing:
Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. Polycarbonate rat huts were provided to the animals as
environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area (SC-22).

Bedding
Steam sterilized corn cob was used as bedding and changed along with the cage atleast twice a week. Contaminant analysis report for bedding material (corn cob) is presented

Diet
Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum to the rats.

Water
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes. The feed and water provided to the rats was tested for contaminants. Analysis and contaminant analysis reports of feed, water and bedding material were included in the report as Annexures 2 to 4. Based on the latest analytical certificate/s available, there were no known contaminants in the food, water and bedding that are expected to interfere with the results of this study.

Grouping
Animals were randomly distributed to different groups by body weight stratification method using ProvantisTM software (Version 10.1.0.1, Instem LSS, Staffordshire ST15OSD, United Kingdom). Rats with extreme body weights were discarded. Grouping was done one day prior to start of treatment during acclimatization.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

- Concentration in vehicle: 0, 10, 30 and 100 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg
- Purity: see details in the test item box

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration and stability of the test item in the vehicle were established at 0.1 and 150 mg/mL concentrations under Eurofins Advinus Study No. G18368.
Based on the results, the test item was stable in the vehicle up to 5 hours when stored at room temperature

Dose Formulation Analysis of the Test Item
The results indicated the percent agreement of the analyzed concentrations were in the range, 85% to 115% of the theoretical concentrations and the overall %
RSD from six replicates at each dose level was less than 10.0% of theoretical concentrations

Duration of treatment / exposure:

90 days

Frequency of treatment:

The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period
of 90 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once orally for 90 consecutive days.
The vehicle or the dose formulations were not administered to recovery groups for 28 days following the 90-day treatment period.
The dose formulation and the vehicle were administered at an equivolume of 10 mL/kg/day. The dose volume was calculated for individual animals on the first
day of the treatment period and was adjusted according to the most recent body weights recorded during the treatment period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

No. of rats/group:
• Main groups: 10 males + 10 females
• Recovery groups: 5 males + 5 females
Total = 100 (50 males + 50 females)

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels of 100 (G2), 300 (G3) and 1000 (G4/G4R) mg/kg/day were selected for this study based on the available literature in consultation with the Sponsor
In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and vehicle control recovery groups were handled in a manner similar to the treatment groups except for test item administration.

Examinations

Observations and examinations performed and frequency:

Clinical Signs and Mortality
Each rat was observed twice daily for morbidity and mortality except during holidays wherein the observation was done once daily as there were no clinical signs observed. Routine observations for checking general clinical signs were performed once a day during treatment (post-dose) and recovery period.

Detailed Clinical Examination
Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (± 1 days) during treatment and recovery periods.
During detailed clinical examination, all rats were observed for changes in skin,fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards).
On the days of detailed clinical examination, observation for general clinical signs (first post-dose) was not performed except on Day 1.

Ophthalmological Examination
Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups and at the end of recovery period for recovery groups. Before examination, mydriasis was induced using a 1 % solution of Tropicamide.

Functional Observation Battery Tests (FOB)
The following neurological examination was performed during the 13th week (Day 89) of treatment period for main groups and towards the end of recovery period (Day 116) for recovery groups.

FOB 1. Home Cage Observations
Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.

FOB 2. Observations during Removal of Animal from Home Cage and Handling
The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that
can best be performed when the rat is being held. Each rat was observed for the following examinations:
- ease of removal from home cage
- handling reactivity
- palpebral closure
- eye examination
- piloerection
- lacrimation
- salivation
- skin/fur examination
- perineum wetness
- respiration
- muscle tone and
- extensor thrust response
The observations were recorded using scores.

FOB 3. Open Field Observation
Rat was placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group. During this observation period, rat was evaluated as it moves about freely/unperturbed and the following observations were made and
observations were recorded using score/ranks:
- gait
- posture
- tremors
- mobility score
- arousal level
- clonic or tonic movements
- stereotypic behaviour
- bizarre behaviour
- urination
- defecation
- rearing
- abnormal vocalizations

FOB 4. Functional Tests
Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
Motor Activity
The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, ambulatory counts were monitored. The Opto-Varimex 4 motor activity measurement system provided the data at 1 minute interval and the data was analyzed in blocks of 10 minutes interval and the same was reported.

Sensory Reactivity Measurements
After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
- approach response
- touch response
- click response
- tail-pinch response
- pupil response
- aerial righting reflex

Landing Hindlimbs Footsplay
The landing hind limbs foot splay was performed by dropping the rat onto a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat was gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values is presented in the report along with the individual footsplay values.

Grip Performance
Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Averages of three trials for both forelimb and hindlimbs are calculated and
presented in the report along with the individual grip strength values.

FOB 5. Physiological Observations
Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured.

Body Weight
Individual body weights (g) were recorded prior to test item administration on Day 1 and weekly thereafter (± 1 day) for all groups of rats during treatment and recovery period except for week 13 wherein the animals were weighed on day 5 of that week (Day 90). Fasting body weight was recorded prior to sacrifice for all animals.

Food Consumption
The food consumption was measured at weekly intervals during treatment and recovery period except on week 13, wherein the food consumption was measured on Day 5 of that week. The cage wise average food consumption (g/rat/day) was calculated and presented in the report.

Oestrous Cycle Evaluation
Vaginal smear was examined in female rats and the stage of estrous cycle was recorded daily for two weeks prior to necropsy for main and recovery groups to evaluate the estrous cycle length and normality. The time interval (in days) from the diestrus of an estrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.

Sacrifice and pathology:

Clinical Pathology Investigations

Blood Collection
At the end of the treatment period (Day 90) and at the end of recovery period (Day 118), all rats were fasted overnight (water allowed) and approximately 4.0
mL of blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture

Haematology
The following haematological parameters were determined using Advia 2120i Hematology system (Siemens Healthcare Diagnostics Inc., NY, USA).
No. Parameter Abbreviations Units2
1 Red Blood Corpuscles RBC 1012/L
2 Haemoglobin HGB g/L
3 Haematocrit HCT L/L
4 Mean Corpuscular Volume MCV fL
5 Mean Corpuscular Haemoglobin MCH pg
6 Mean Corpuscular Haemoglobin Concentration MCHC g/L
7 Reticulocytes count Retic 1012/L
8 White Blood Corpuscles WBC 109/L
9 Differential leukocyte count1 DLC 109/L
10 Platelets PLT 109/L
11 Red Blood Cell Morphology
a. Red Cell Distribution Width
b. Haemoglobin Distribution Width
c. Hyperchromic cells
d. Hypochromic cells
e. Macrocytes
f. Microcytes
g. RBC Fragments
h. RBC Ghosts

1: Differential Leukocyte parameters and their respective abbreviations: Neutrophils (Neut),
Lymphocytes (Lymp), Monocytes (Mono), Eosinophils (Eosi) and Basophils (Baso).
2: Abbreviations: L: Litre, g: Gram, pg: Picogram, fL:Femtolitre.

Additionally, blood smears were prepared from the hematology (K2EDTA tube) sample and stained with Giemsa stain (solution). In the absence of any treatment
related effects, the blood smears were not subjected for microscopic evaluation. All the blood smear slides were discarded at the time of final report preparation.

Coagulation
Blood samples collected for coagulation analysis were centrifuged at 2500 times gravity (xg), at 15°C for 10 minutes for separation of plasma and analysed for
the following parameters in plasma sample using STart Max coagulation analyzer (Diagnostica stago, 92600 Asnieres, France):
Sl. No. Parameter Abbreviations Units
1 Prothrombin Time PT Sec
2 Activated Partial Thromboplastin Time APTT Sec

Clinical Chemistry
Plasma was separated after centrifugation of the whole blood samples at 5000 rpm, 4°C for 5 minutes and analysed using Dimension RxL MaX Clinical
Chemistry System (Dade Behring Inc. Newark, DE 19714, USA) for the following parameters:
Sl. No. Parameter Abbreviations Units
1 Alanine Aminotransferase ALT U/L
2 Alkaline Phosphatase Alp U/L
3 Albumin ALB g/L
4 Albumin/Globulin ratio (calculated value) A/G -
5 Aspartate Aminotransferase AST U/L
6 Blood Urea Nitrogen BUN mmol/L
7 Chloride Cl mEq/L
8 Creatinine Creat μmol/L
9 Calcium Ca mmol/L
10 Glucose Glu mmol/L
11 Gamma Glutamyl Transpeptidase GGT U/L
12 Globulin (calculated value) GLOB g/L
13 HDL Cholesterol HDL.Chol mmol/L
14 Inorganic Phosphorous Pi mmol/L
15 LDL Cholesterol2 LDL.Chol mmol/L
16 Potassium K mEq/L
17 Sodium Na mEq/L
18 Total Cholesterol T. Chol mmol/L
19 Total Plasma Protein T. Pro g/L
20 Triglycerides Trig mmol/L
21 Total Bilirubin T. Bil μmol/L
22 Direct Bilirubin1 D.Bil μmol/L
23 Indirect Bilirubin (calculated)1 Ind.Bil μmol/L
1: Note: Direct and indirect bilirubin were not determined as total bilirubin values were <10 μmol/L
2: LDL Cholesterol was calculated using total Cholesterol, Triglyceride and HDL cholesterol values.

Hormone Analysis
At the end of the treatment and recovery periods, blood was collected from all rats along with blood collection for clinical pathology investigation for the
determination of total T3, T4 and TSH hormones.
Blood samples were collected in plain labeled tubes and kept on bench top for 20 minutes before centrifugation. Serum was separated by centrifuging the
whole blood samples at 5000 rpm for 5 minutes at 4° C. The serum samples were placed in labeled plastic tubes and stored at ~70 °C until they were
analyzed. The left over samples from hormone analysis were discarded after data review.

Thyroid Profile Hormones:
The following thyroid hormones were estimated by Enzyme Linked Immuno Sorbent Assay (ELISA) using BIO-RAD microplate washer PW 40 and BIORAD
model 680 readers (Bio-Rad Laboratories India Pvt. Ltd., Gurugram, Haryana, India).

No. Parameters Abbreviations Unitsc
1 Rodent Thyroid Stimulating Hormone TSH ng/mL
2 Rodent Thyroxin T4 ng/mL
3 Rodent Triiodothyronine T3 ng/mL
c: Abbreviations: ng/mL: nano grams/ per milli litre
The kits manufactured by Endocrine Technologies Inc., USA were used for the
assay.

Urinalysis
Urine was collected from all rats at the end of the treatment period for main groups and at the end of the recovery period for recovery groups in urine
collection tubes. Each rat was placed overnight in a specially fabricated cage (water allowed) and the next morning the collected urine was sent for analysis.
Urine was analyzed for:
No. Parameters
1 Specific gravity(1)
2 Nitrite(2)
3 pH(2)
4 Proteins(2)
5 Glucose(2)
6 Ketone bodies(2)
7 Urobilinogen(2)
8 Bilirubin(2)
9 Appearance (colour and clarity)(3)
10 Volume (approximate)(3)
(1): Measured using Refractometry method [Refractometer – Digital Handheld “Pocket” Urine Specific Gravity Refractometer by (PAL-10S, ATAGO Co., Ltd 32-10) Honcho, Itabashi, Tokyo, Japan].
(2): Analyzed using Multistix 10 SG strips, read with Clinitek Advantus® analyzer (Siemens Healthcare diagnostics INC., USA)
(3): Recorded manually.
Urine was also subjected to microscopic examination for sediments such as crystals, epithelial cells, erythrocytes, leukocytes and casts.

Anatomic Pathology

Necropsy
All rats from main groups at the end of the treatment period and from recovery groups at the end of recovery period were subjected to detailed necropsy (examination of external surfaces of the body, all orifices; cranial, thoracic and abdominal cavities and their contents) and findings were recorded. Terminal fasting body weights were recorded for all animals immediately prior to terminal sacrifice and used in calculation of relative organ weights. Rats sacrificed at term were fasted overnight (water allowed), euthanized with isoflurane, exsanguinated and subjected for gross examination.

Tissue Collection, Weighing and Preservation
On completion of gross pathology examination, the tissues and organs noted below were collected from all rats and weighed from all terminally sacrificed rats. The organ weight ratios (organ to body weight and organ to brain weight) as percentage of fasting body weight and brain weight were determined. The paired organs were weighed together and combined weights were presented.
The tissues were preserved in 10% Neutral Buffered Formalin (NBF) except for the testes and eyes.

Microscopic Examination
1. Aorta
2. Bone marrow smear
3. Brain including medulla/pons, cerebellum and cerebrum (weight)
4. Cecum
5. Cervix
6. Colon
7. Duodenum
8. Epididymides (weight)
9. Esophagus
10. Eyes
11. Femoral muscle (Skeletal Muscle)
12. Bone, femur/joint, femorotibial
13. Glands, adrenal (weight)
14. Glands, salivary
15. Gross lesions/masses/nodules
16. Gut associated lymphoid tissue
17. Heart (weight)
18. Ileum
19. Jejunum
20. Kidneys (weight)
21. Larynx
22. Liver (weight)
23. Lungs (with bronchi and bronchioles)
24. Lymph nodes, mandibular
25. Lymph nodes, mesenteric
26. Mammary gland
27. Nerves, sciatic
28. Nerve, optic
29. Ovaries (weght)
30. Oviducts
31. Pancreas
32. Pharynx
33. Pituitary (weight)
34. Prostate (weigth)
35. Rectum
36. Seminal vesicles and coagulating glands (weight)
37. Skin
38. Spinal cord (cervical, thoracic and lumbar)
39. Spleen (weight)
40. Sternum with marrow
41. Stomach
42. Testes (weight)
43. Thymus (weight)
44. Thyroid and Parathyroid (weight)
45. Trachea
46. Tongue
47. Urinary bladder
48. Uterus (weight)
49. Vagina

Histopathology
Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose group animals (G4), initially.
Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions were also
examined microscopically. Further, kidney was suspected of showing test itemrelated histopathological changes in high dose (G4) group (males and females)
and the same was examined in the respective lower dose groups and recovery groups.
The tissues were processed for routine paraffin embedding and 4-5 micron sections were stained with Mayer’s Haematoxylin and Eosin stain. Unused
tissues were archived.

Statistics:

STATISTICAL ANALYSIS
All the data pertaining to terminal fasting body weights, organ weights (including derived data) and clinical pathology – Haematology, Coagulation,
and Clinical chemistry were captured and analysed using built-in statistical algorithms in the Provantis.
The statistical analysis of neurological observations (neuromuscular observation/body temperature/body weights) and thyroid hormone data (T3, T4
and TSH) was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor
ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before performing ANOVA.
Comparison of means between treatment groups and control group was done using Dunnett’s test whenthe overall treatment, ‘F’ test was found significant. In
case of recovery groups, data were analysed by Student‘t’ test. Comparison of means between treatment recovery group(s) and vehicle control recovery group was performed.
The data pertaining to males and female rats was evaluated separately.
All analyses and comparisons were evaluated at the 5% (p<0.05) level.
Statistically significant differences (p<0.05), were denoted by asterisk (*) throughout the report.
*: Significantly higher/lower than the respective control group

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs or mortalities observed during the treatment and recovery period at any of the doses tested in either sex

Mortality:

no mortality observed

Description (incidence):

There were no clinical signs or mortalities observed during the treatment and recovery period at any of the doses tested in either sex

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weights measured at the end of Functional tests were not statistically different when compared to vehicle control.
Treatment did not affect the mean body weights and net body weight gains at all the tested doses. However, incidence of significantly lower absolute weight gain during Days 1-8 in males at 300 mg/kg/day mid dose and during Days 50-57 in the high dose recovery males were observed. In females, absolute weight gains were significantly higer during Days 15-22 at low and mid dose group. These significant differences were not considered toxicologically relevant as the mean body weights and total body weight gains were not altered by the treatment. Thus, the treatment did not affect the mean body weights and body weight gains during treatment and recovery period at any of the doses tested in either sex.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption was significantly higher in males during Days 63-71 in low, mid and high doses and during Days 57-63 in low and mid dose groups. In the high dose recovery group, significantly higher food consumption was observed during Days 29-35 and 71-78 during the treatment period in males.

In females, the food consumption was significantly higher during Days 15-22 in the high dose group and during Days 22-29 and 29-35 in the mid and high dose groups. In the mid dose group, significantly higher food consumption was observed during Days 71-78 in mid dose during the treatment period.

These significant differences were not considered toxicologically relevant as the body weights were not altered by the treatment.

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examination carried out with an ophthalmoscope prior to start of treatment, at the end of the treatment and recovery period did not reveal any abnormalities in the eyes of the experimental rats.

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related variations were observed in the hematology parameters as compared to control groups.
All the statistically significant differences noted with in the treated groups relative to controls were considered incidental, as these findings were either minimal or not consistent/dose-dependent.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no changes in clinical chemistry attributed to test item administration over the course of the study.
All the statistically significant differences in biochemical parameters noted in the treated groups were considered to be incidental.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No significant differences were noted in any of the parameters in treated rats as compared to vehicle controls.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Home cage and Handling observations:
No treatment-related abnormalities were observed in any of the tested dose groups in either sex.

Open field observations:
No treatment-related abnormalities were observed in any of the doses tested in either sex.

Sensory observations:
No treatment-related abnormalities were observed in any of the groups in either sex.

Motor Activity:
The following statistically higher significant differences were observed in the motor activity in rats when compared to concurrent vehicle control groups:
Males:
• Stereotypic time during interval 2 at 100, 300 and 1000 mg/kg/day.
• Ambulatory time during intervals 1, 2 and total counts at all doses.
• Horizontal counts and total horizontal counts at all doses.
• Ambulatory counts during intervals 1 and total counts at all doses and Ambulatory counts during internal 3 at 300 mg/kg/day.
Recovery Males:
• Ambulatory time at interval 1 at high dose recovery group.
• Horizontal and ambulatory counts during interval 1 at high dose recovery group.
Females:
• Horizontal and ambulatory counts during interval 1 at all the doses 100, 300 and 1000 mg/kg/day tested.
• Horizontal, ambulatory counts during interval 3 and total horizontal and ambulatory counts at doses of 100 and 1000 mg/kg/day.
Recovery Females:
• Horizontal and ambulatory counts during interval 1 and total Horizontal and ambulatory counts at high dose recovery group.

The above observed statistical variations in the motor activity measurements are considered to be incidental as there were no changes observed in the home cage
or open field observations. Further there were no clinical signs observed during daily clinical observation.

Neuromuscular observation:
Landing hind limb footsplay:
No treatment-related changes were observed at all the tested doses in both sexes.

Grip strength:
Significantly lower hindlimb grip strength was observed in both the sexes in the low dose group. This significant difference was not consideredtoxicologically relevant, as there were no changes observed in the home cage or open field observations or motor activity. Further, there were no clinical signs observed during daily clinical observation.

Physiological observation:
Body temperature: Significantly lower body temperature was observed in both sexes at 1000 mg/kg/day high dose group and 100 mg/kg/day low dose females.
This change was considered to be toxicologically not significant as there was no dose dependency.

Body weight: The body weights measured at the end of Functional tests were not statistically different when compared to vehicle control.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no changes in terminal fasting body weights, absolute organ weights or organ to body/brain weight ratios which were related to treatment.
Statistically significant differences in weight noted in some of the organs in the treatment groups as compared to control rats were considered incidental in the absence of any significant gross/histologic findings.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Oral administration of the test item did not induce gross lesions at any of the dose levels in either sex.
In treated rats, gross lesions involving uterus (dilatation) and testes (enlarged/small and flaccid) were associated with incidental microscopic findings.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic evaluation of the protocol tissues revealed test item-related tubular degeneration in kidneys at 1000 mg/kg/day in either sex.
Tubular degeneration showed focal to multifocal distribution involving groups of tubules in the renal parenchyma. This was not accompanied by changes in renal markers (BUN/creatinine) and findings such as inflammatory reaction, tubular casts, and tubular dilatation with no evidence of tubular basophilia/regeneration. In addition, the glomeruli were also spared. The extent of tubular degeneration was higher in males as compared to females. Tubular degeneration was not observed in the recovery groups (males and females) indicating complete reversal of the effect after 28 treatment free period.
All other microscopic lesions were considered background changes, and therefore, not regarded biologically relevant or treatment related.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

See above (non-neoplastic)

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone profile (TSH, T4 and T3 was not affected in both sexes across the treated groups when compared to the concurrent vehicle control group. Evaluation of serum Thyroid Stimulating Hormone (TSH), Thyroxine (T4) and
Triiodothyronine (T3) levels did not show any test item-related effects in either sex. Higher T4 level noted in 1000 mg/kg/day females in the absence of auxiliary changes in TSH level and/or thyroid histology was considered incidental.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

The OECD 408 was performed following a deviation including detailed monitoring of parameters relevant for reproduction, in particular:

-weight of male and femal gonads and accessory sex organs

-stage of oestrous cycle and cycle duration for females

-histopathological examination of uterus and cervix

-histopathological examination, with special emphasis to stages of spermatogenesis, in males

-histopathology of interstitial testicular structure

Results:

Weight

There were no changes in terminal fasting body weights, absolute organ weights or organ to body/brain weight ratios which were related to treatment.

Gross Pathology

In treated rats, gross lesions involving uterus (dilatation) and testes (enlarged/small and flaccid) were associated with incidental microscopic findings

Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks prior to necropsy for main and recovery groups.

The calculated mean oestrous cycle length was 4.1, 4.3, 4.2 and 4.1 days in vehicle control, low, mid and high dose groups, respectively during treatment period.

During recovery period the mean oestrous cycle length was 4.3 and 4.4 days in vehicle control recovery and high dose recovery groups.

The mean oestrous cycle length for main and recovery groups was not significantly different from respective vehicle control groups.

The time interval (in days) from the diestrus of an estrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.

Histopathological examination

No effects on uterus, cervix, testes at micro level

Spermatogenesys (qualitative) :

no treatment related effects

refer to the attached report for details and tables

Applicant's summary and conclusion

Conclusions:

The substance was tested for long-term toxicity by following OECD408, by oral exposure with examination of some reproductive parameters. Daily oral (gavage) administration of Diethylene Glycol Diformate to Wistar rats at dose levels of 100, 300 and 1000 mg/kg bwt/day for 90 consecutive days showed evidence of kidney toxicity (tubular degeneration) in either genders at 1000 mg/kg/day dose. Considering the microscopic changes in kidneys at 1000 mg/kg/day, No Observed Adverse Effect Level (NOAEL) is considered to be 300 mg/kg Bwt/day under the test conditions and doses employed.

Executive summary:

The substance was tested for long-term toxicity by following OECD408, by oral exposure. The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test item, Diethylene Glycol Diformate in Wistar rats when administered orally by gavage for a period of 90 consecutive days and to assess the reversibility of any effects during a subsequent 28 days recovery period.

The test item was weighed and suspended in vehicle [Milli-Q-water] and administered to rats at the graduated dose levels of 100, 300 and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4 ) / high dose recovery (G4R) group rats, respectively.

The rats in the vehicle control (G1)/ vehicle control recovery (G1R) groups received vehicle [Milli-Q-water] alone. The dose volume administered was 10 mL/kg body weight. Each main groups in the experiment was comprised of 10 males and 10 female rats and recovery groups comprised of 5 males and 5 female rats.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not done at the test facility. The stability of test item in the vehicle (Milli-Q-Water) was carried out under Eurofins Advinus Study No.G18368 at 0.1 and 150 mg/mL concentrations. Based on the results, the test item was found to be stable for 5 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analyzed for active ingredient (a.i.) concentration on test Day 1, during 2nd (Day 34) and 3rd (Day 63) month of the treatment. The results indicated that the analyzed concentrations were within ± 15% of variations from the theoretical concentrations and % RSD from six replicates at each dose level was <10.0%.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured weekly during in-life phase of the experiment.

Neurological examinations were conducted towards the end of treatment for main groups (Day 89) and recovery period for recovery groups (Day 116). The clinical laboratory

investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination.

All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratios were derived as percent fasting body weights and brain weight. Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose group animals (G4), initially.

Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions were also examined microscopically. Further, kidney was suspected of showing test itemrelated histopathological changes in high dose (G4) group (males and females) and the same was examined in the respective lower dose groups and recovery groups.

Under the experimental conditions employed, the following results were obtained:

• Clinical Signs and Mortality: No Clinical signs or mortalities occurred at any of the doses tested.

• Ophthalmological Examination: Ophthalmological examination did not reveal any ocular abnormalities.

• Neurological Findings: No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.

• Body Weights and Food Consumption: Treatment did not affect the mean body weights, body weight gain and food consumption at all the tested doses in either sex.

• Haematology, Coagulation, Clinical Chemistry and urine parameters: There were no test item related alterations observed at any of the tested dose levels in either sex.

• Thyroid Hormone Profile: Thyroid hormone profile (TSH, T4 and T3) was not affected in both sexes across the treated groups when compared to the concurrent vehicle control group.

• Terminal Fasting Body Weights and Organ Weights: No significant changes observed in terminal fasting body weights and organ weights attributed to test item at any of the tested dose levels in either sex.

• Gross pathology: There were no test item-related gross pathological changes observed in both sexes.

• Histopathology: Microscopically, tubular degeneration showed focal to multifocal distribution involving groups of tubules in the renal parenchyma at 1000 mg/kg/day. This was not accompanied by changes in renal markers (BUN/creatinine) and findings such as inflammatory reaction, tubular casts, and tubular dilatation with no evidence of tubular basophilia/regeneration.

In addition, the glomeruli were also spared. The extent of tubular degeneration was higher in males as compared to females. Tubular degeneration was not observed in the recovery groups (males and females) indicating complete reversal of the effect after 28 treatment free period.

Lower dose groups remained unaffected by administration of the test item.