Ethylene di(acetate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 May – 25 Sep 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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 21 Sep 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Government of India, Department of Science and Technology, New Delhi, India

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene di(acetate)

EC Number:

203-881-1

EC Name:

Ethylene di(acetate)

Cas Number:

111-55-7

Molecular formula:

C6H10O4

IUPAC Name:

2-(acetyloxy)ethyl acetate

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: + 15 to 25 °C
- Solubility and stability of the test substance in the vehicle were confirmed by analytical methods

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanTac: WH

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Pregnapur, Gajwel Mandal, Medak District, Telangana, India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 83.55 - 131.46 g (males) and 82.11 - 109.69 g (females)
- Housing: Two rats of the same sex per cage in sterilised standard polysulfone cages (size: 425 x 266 x 185 mm) with stainless steel top grill having facilities for pelleted food and drinking water. The last animal in each recovery group and sex was housed individually. Polycarbonate rat huts were included as environmental enrichment. The bedding consisted of steam sterilized corn cob.
- Diet: Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet Pellet (Envigo, Madison, Wisconsin) ad libitum
- Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier (Eureka Forbes Ltd., Mumbai, India), ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: The quality of food and water provided to the animals as well as the absence of contaminants in bedding confirmed by analytical methods.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 56 - 68
- Air changes (per hr): 12.5 - 13.3
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 25 May 2018 To 25 Sep 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared freshly the day of dosing, except for Day 2, when the formulations prepared on Day 1 were administered. Required amounts of the test item were weighed in a beaker for each dose level separately. A small volume of vehicle (corn oil) was added and stirred until a uniform suspension was prepared. The final volume was made up to the mark using vehicle to get the required final concentration.

- VEHICLE: corn oil
- Justification for use and choice of vehicle: Based on a solubility test, the test item forms uniform suspensions in corn oil. Hence, corn oil was used as vehicle for dose formulation preparations.
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicates on Day 1, during month 2 (Day 51) and month 3 (Day 85) of the treatment period. For each set, duplicate samples were drawn from top, middle and bottom layers from each preparation and in case of control, duplicate samples from middle layer were drawn. The test item in the dose formulation was determined using Gas Chromatograph with Flame Ionisation Detection (FID) detector. The test item was used as the analytical standard. The results were considered acceptable, if the overall mean result of all layers was within ± 15.0% of the theoretical concentration and the relative standard deviation was equal or less than 10.0%. The results of dose formulations were within the acceptable limits for all dose groups.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 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.:

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

No. of animals per sex per dose:

10 (main treatment group)
5 (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were based on the results of a preliminary range-finding study, in which animals were orally exposed to 50, 200, 500 and 1000 mg/kg bw/day for 14 days. There were no adverse effects observed in any dose group. Therefore 100, 300 and 1000 mg/kg bw/day were selected as the dose levels for the main study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations included the ease of removal from the 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/ranks.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to test item administration on Day 1 and at weekly intervals thereafter (± 1 day) during treatment period.
- Detailed clinical examination included: observation for changes in skin, fur, eyes, mucous membranes, occurrence of secretions, excretions and autonomic reactivity (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).

BODY WEIGHT: Yes
- Time schedule for examinations: prior to test item administration on Day 1 and weekly thereafter (± 2 days) for all groups during treatment and recovery period. Fasting body weight was recorded prior to necropsy of the respective groups.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start of treatment and at the end of the treatment period. Before examination, mydriasis was induced using a 1% solution of Tropicamide.
- Dose groups that were examined: all animals of the main treatment group and of the recovery group

HAEMATOLOGY AND COAGULATION: Yes
- Time schedule for collection of blood: Blood was collected by retro-orbital sinus puncture on Day 91 from the main treatment group rats and on Day 119 from the recovery group rats. For coagulation analysis an additional set of plasma samples was generated.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in Table No. 1 were examined. Heamatology parameters were analysed using Advia 2120i Heamatology system (Siemens Healthcare Diagnostics Inc., NY, USA). Coagulation parameters were analysed using a STart max coagulation analyser (Diagnostica stago, Asnieres, France)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected by retro-orbital sinus puncture on Day 91 from the main treatment group rats and on Day 119 from the recovery group rats. Plasma was separated after centrifugation of whole blood samples at 4 °C.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters listed in Table No. 2 were analysed using the Dimension RxL MaX Clinical Chemistry System (Dade Behring Inc Newark, USA).

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment period (Day 91) and at the end of the recovery period (Day 119).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table No. 3 were examined. In addition, urine was also subjected to microscopic examination for sediments such as crystals, epithelial cells and casts.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 13th week of treatment period (Day 86 and 87) for main groups and towards the end of week 17 (Day 114) of recovery period for recovery groups.
- Dose groups that were examined: all animals of the main treatment group and of the recovery group.
- Battery of functions tested: sensory activity (approach response, touch response, click response, tail-pinch response, pupil response, aerial righting reflex) / neuromuscular observations (landing hind limbs footsplay, grip strength) / motor activity
- Open field observations included: gait, posture, tremors, mobility score, arousal level, clonic or tonic movements, stereotypic behavior, urination, defecation, rearing and abnormal vocalizations

IMMUNOLOGY: No

OTHER:
BODY TEMPERATURE:
Body temperature (rectal temperature) of each animal was measured at the end of each functional test

Sacrifice and pathology:

SACRIFICE: Terminally sacrificed adult animals were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia.
- Animals of the treatment group were sacrificed at the end of treatment period on Day 91.
- Animals of the recovery group were sacrificed at the end of the recovery period on Day 119.

GROSS NECROPSY: Yes
Gross necropsy consisted of examination of external surfaces of the body, all orifices, cranial, thoracic and abdominal cavities and their contents. The tissues and organs listed in Table 4 were macroscopically analysed from all animals.


HISTOPATHOLOGY / ORGAN WEIGHTS: Yes
Histopathological examination was carried out on all preserved organs and tissues of the vehicle control group and the high dose group (1000 mg/kg bw/day) rats and on all gross lesions. Kidneys and adrenals were examined in the lower dose groups (100 and 300 mg/kg bw/day) and in male recovery animals.

Statistics:

ProvantisTM was used to capture data for the parameters body weight, organ weight, hematology, coagulation, clinical chemistry and terminal fasting body weight. The data was analysed using ProvantisTM built-in statistical tests, as well as the data on body weight gain, food consumption and organ weight ratios. The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. The following tests were applied:
- Shapiro-Wilk test to test all quantitative variables like neurological observations (neuromuscular observation/ body temperature/ body weights) for normality of variances within the group
- Levene’s test to test all quantitative variables like neurological observations (neuromuscular observation/ body temperature/ body weights) for homogeneity of variances within the group
- one-factor ANOVA modeling by treatment groups (non-optimal, non normal or heteroschedastic data was transformed before performing ANOVA)
- Dunnett’s test for comparison of means between treatment groups and control group
- F-test for statistical significance
The data pertaining to males and females were evaluated separately. All analyses and comparisons were evaluated at the p < 0.05.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: significantly higher body weights in males during days 71-77
300 mg/kg bw/day: significantly lower body weight gain during days 22-29 in females; significantly higher body weight gain during days 29-36 in females
1000 mg/kg bw/day: significantly higher body weight gain during days 1-8, absolute weight gain during days 1-90; significantly lower absolute weight gain during days 90-97 in the recovery group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Sporadic incidences of significantly lower and higher food consumption were observed for the following dose groups:
100 mg/kg bw/day: higher food consumption on treatment days 43-50 and 71-77 in female rats
300 mg/kg bw/day: higher food consumption in male rats during treatment days 15-50 and 71-70
1000 mg/kg bw/day: lower food consumption in male animals during days 77-85 (in both, the treatment and the recovery group); higher food consumption in female rats during treatment days 29-36, 43-77 and 85-90
As the mean body weights were not altered by the treatment, the significant differences were not considered toxicologically relevant.

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):

All adverse effects of statistical significance observed in haematology parameters were considered incidental in nature, as the changes were of minimal magnitude and/or not dose related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: increased Bood Urea Nitrogen in male rats at the end of treatment (43%) and till the end of recovery period (39%).

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: increased kidney weight in male rats

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All single incidences of randomly distributed gross findings in different groups were considered incidental/spontaneous and not related to the test item. The solidary incidence of kidney enlargement was found to be neoplasm. The occurence of nephroblastoma has been occasionally reported in young rats.

Neuropathological findings:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: tubular damage of kidneys in male rats, adverse

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no treatment related clinical signs and mortalities observed at any of the doses tested in both sexes.

BODY WEIGHT AND WEIGHT GAIN
The mean body weights were not significantly different from the vehicle control group at all the tested doses in both sexes during the treatment and recovery period. Significantly higher body weight gain was observed during days 71 - 77 in 100 mg/ kg bwt/day dose group males. In females, significantly higher body weight gain during days 29 - 36 and lower body weight gain during days 22 - 29 was observed at 300 mg/kg bw/day. Significantly higher body weight gain during days 1 - 8, absolute weight gain during days 1-90 at 1000 mg/kg bw/day and significantly lower absolute weight gain during days 90 - 97 in the 1000 mg/kg bw/day recovery group was observed. The above significant differences were considered toxicologically not relevant as the mean body weights were comparable to vehicle control.

FOOD CONSUMPTION/EFFICIENCY AND COMPOUND INTAKE
Sporadic incidences of significantly lower and higher food consumption were observed. As the mean body weights were not altered by the treatment, the significant differences were not considered toxicologically relevant.

OPHTHALMOSCOPIC EXAMINATION
There were no abnormalities in the eyes of the experimental rats detected.

HAEMATOLOGY
There were no test item-related changes observed in the haematology parameters. All adverse effects of statistical significance observed in haematology parameters were considered incidental in nature, as the changes were of minimal magnitude and/or not dose related.

CLINICAL CHEMISTRY
The test item-related increase in Blood Urea Nitrogen (BUN) was observed in 1000 mg/kg bwt/day males at the end of treatment (43%) and it persisted till the end of recovery period (39%). All other differences observed in biochemical parameters between vehicle control and treatment groups, including the changes that reached statistical significance were considered as incidental due to lack of dose relation and/or the changes were of minimal magnitude. The individual animal values were within the range of normal biological variation.

URINALYSIS
No test item-related changes were observed in any of the urine parameters analysed.

NEUROBEHAVIOUR
Open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls. There were no treatment related changes in functional performance, motor activity and sensory reactivity.

ORGAN WEIGHTS
In male animals treated at 1000 mg/kg bw/day the weight of kidneys increased in relation to treatment. The weight increase was also noted at the end of the recovery period and associated with the microscopic lesions of crytal material deposits, tubular damage and inflammatory response. The percentage intergroup differences for these changes are listed in Table 5. All other weight changes were considered incidental and not related to test item administration as the changes were not consisitent or there were no microscopic correlates at 1000 mg/kg bw/day.

GROSS PATHOLOGY
All single incidences of randomly distributed gross findings in different groups were considered incidental/spontaneous and not related to the test item. The solidary incidence of kidney enlargement was found to be neoplasm. The occurence of nephroblastoma has been occasionally reported in young rats.

HISTOPATHOLOGY
At 1000 mg/kg bw/day tubular damage of kidneys was observed in male animals. Histopathological findings included: crystalline deposits with tubular basophilia, mono nuclear cell infiltration and dilatation of tubules. All changes were observed in both kidneys. Single incidences of unilateral tubular basophilia finding were considered as incidental, not related to test item administration. The crystalline material deposits could be accumulation of the test material causing dilatation of tubules. These findings were predominately seen in the cortex although cortico medullary tubules were also seen in some animals. In addition, the peritubular fibrosis involving interstitial tissue was also noted in spite of marginal recovery in the kidney weights. Detailed information is provided in Table No. 6. All other microscopic finidings were considered incidental/spontanous changes which are common for this age group rats and were similar in control and high dose treated animals.

Effect levels

1

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

3

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effect observed

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Table 5: Percentage group differences in kidney weight changes in male animals

Sex	Males
Group	G2	G3	G4	G4R
Dose (mg/kg bwt/day)	100	300	1000	1000
No. of rats	10	10	10	5
Kidneys – absolute weights	─	─	↑26%	↑18%
– relative to body weights	─	─	↑29%	↑4%
– relative to brain weights	─	─	↑28%	↑15%

↑: Increase

─: values are not considered meaningfully different from controls.

R: Recovery group

Table 6: Histopathological findings in kidneys of male rats:

Sex	Males
Group	G1	G2	G3	G4	G1R	G4R
Dose (mg/kg bwt/day)	0	100	300	1000	0	1000
No. of rats	10	10	10	10	5	5
Kidneys	10	10	10	10	5	5
Crystalline deposits; tubular; bilateral	0	0	0	5	0	4
Minimal	─	─	─	3	─	2
Mild	─	─	─	2	─	1
Moderate	─	─	─	─	─	1
Basophilia; tubular; bilateral	0	1	0	10	1	5
Minimal	─	1	─	─	1	2
Mild	─	─	─	6	─	1
Moderate	─	─	─	4	─	2
Infiltration; mononuclear cell; bilateral	0	0	0	5	0	2
Minimal	─	─	─	4	─	1
Mild	─	─	─	1	─	1
Dilatation; tubular; bilateral	0	0	0	9	0	4
Minimal	─	─	─	3	─	1
Mild	─	─	─	6	─	2
Moderate	─	─	─	─	─	1
Fibrosis; peritubular; bilateral	0	0	0	0	0	4
Minimal	─	─	─	─	─	1
Mild	─	─	─	─	─	3