Ethyl formate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from publication

Data source

Reference

Reference Type:

publication

Title:

A 90-Day Inhalation Toxicity Study of Ethyl Formate in Rats

Author:

Mi Ju Lee and Hyeon-Yeong Kim

Year:

2017

Bibliographic source:

Toxicol. Res. Vol. 33, No. 4, pp. 333-342 (2017),

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Principles of method if other than guideline:

Repeated dose inhalation toxicity study of Ethyl Formate in Rats

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl formate

EC Number:

203-721-0

EC Name:

Ethyl formate

Cas Number:

109-94-4

Molecular formula:

C3H6O2

IUPAC Name:

ethyl formate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Ethyl formate
- Molecular formula (if other than submission substance): C3H6O2
- Molecular weight (if other than submission substance): 74.0785 g/mol
- Substance type: Organic
- SMILES:CCOC=O
- Physical state: liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Japan SLC., Inc. (Hamamatsu, Japan) via Joongang Experimental Animal Co., Ltd. (Seoul, Korea)
- Age at study initiation: 7 weeks
- Weight at study initiation:
- Fasting period before study:
- Housing: Animals were housed individually in wire-bottomed stainless-steel mesh cages placed in exposure
Chambers.
- Diet (e.g. ad libitum): Commercial
rodent chow (PicoLab Rodent Diet 5053, LabDiet, USA) ad libitum, ad libitum
- Water (e.g. ad libitum): sterilized tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 12-15 air changes/hr
- Photoperiod (hrs dark / hrs light): artificial lighting from 08:00 to 20:00

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body
exposure chambers (WITC-00-M, HCT Co., Korea)
- Method of holding animals in test chamber: Whole-body
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols: LVg-04-A
- Temperature, humidity, pressure in air chamber:23 °C
- Air flow rate:
- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: The ethyl formate levels were detected using gas chromatography (TRACE1310,
Thermo Scientific, China) with the following apparatus: detector, flame ionization detector; column, TraceGold TG- 5MS 5% diphenyl-95% dimethyl polysiloxane capillary column (30 m length, 0.25 mm i.d., 0.25 mm film thickness).
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: fresh air
- Composition of vehicle:
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle: 0, 66, 330, and 1,320 ppm
- Lot/batch no. of vehicle (if required):
- Purity of vehicle:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

using gas chromatography

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hr/day, 5 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L air (analytical)

Doses / concentrations 2

Dose / conc.:

0.2 mg/L air (analytical)

Doses / concentrations 3

Dose / conc.:

1 mg/L air (analytical)

Doses / concentrations 4

Dose / conc.:

4 mg/L air (analytical)

No. of animals per sex per dose:

Total: 80
0 mg/L : 10 male, 10 female
0.2 mg/L : 10 male, 10 female
1 mg/L : 10 male, 10 female
4 mg/L : 10 male, 10 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Concentrations of 0, 0.2, 1 and 4 mg/L was selected based on a prior study and the OEL.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before, during, and after the exposure period
- Cage side observations checked in table [No.?] were included.: Mortality were observed.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before, during, and after the exposure period

BODY WEIGHT: Yes
- Time schedule for examinations: All the rats were weighed individually on the first exposure day and once a week after that.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: On week 12.
- Dose groups that were examined: All doses were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On day 90 of treatment.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight
- How many animals: All 80 animals were examined.
- Parameters checked in table [No.?] were examined.: Prothrombin time (PT)
and activated partial thromboplastin time (APTT), total red blood cell (RBC) count, hemoglobin (HGB) concentration, hematocrit (HCT), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), platelet (PLT) count, total white blood cell (WBC) count, differential WBC count (neutrophil, lymphocyte, monocyte, eosinophil, and basophil), reticulocyte (Reti) count, PT, and APTT were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day 90 of treatment.
- Animals fasted: Yes, overnight
- How many animals: All 80 animals were examined.
- Parameters checked in table [No.?] were examined.: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen
(BUN), creatinine (Crea), total bilirubin (T-Bili), total protein (TP), albumin (Alb), A/G ratio, total cholesterol (TChol), triglyceride (TG), glucose (Glu), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphorus (P), and sodium (Na) were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: on week 13
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.: pH, protein, glucose, ketone body, bilirubin, occult blood, leukocyte, nitrite, urobilinogen, and specific gravity were examined.

NEUROBEHAVIOURAL EXAMINATION: No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:
Organ weight:
Absolute and relative adrenals, brain, heart, kidneys, liver, spleen, testes, thymus, epididymides, lung, thyroids, ovaries, and uterus weight were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
External body surfaces and all orifices were examined.

HISTOPATHOLOGY: Yes
Organ/tissues were preserved in 10% neutral buffered formalin:
Organ examined: adrenals, aorta, bone
marrow, brain, cecum, colon, duodenum, epididymides, esophagus, eyes, femur, Harderian glands, heart, ileum, jejunum, kidneys, larynx, liver, lung, lymph nodes (hilar and mesenteric), mammary gland, nasopharyngeal tissue, nerves (optic and sciatic), pancreas, parathyroids, pituitary, prostate, rectum, salivary glands (submandibular, sublingual,
and parotid), seminal vesicles, skeletal muscle, skin, spinal cord (cervical, lumbar, and thoracic), spleen, sternum, stifle joint, stomach, testes, teeth, thymus, thyroids, trachea, urinary bladder, ovaries, and uterus. The eyes/optic nerve and testes were preserved in Davidson’s solution

Statistics:

Statistical analysis was performed for body weight, food consumption, hematology and serum biochemistry parameters, organ weights, and organ/body weight. For the control and ethyl formate-exposed groups, the homogeneity of the variance of numerical data was determined using Levene’s test. Groups with homogenous and heterogeneous data were compared using one-way analysis of variance (ANOVA) and non-parametric Kruskal- Wallis test, respectively. If statistical significance was observed (p < 0.05), Dunnett’s test (for ANOVA) or the Steel test (for Kruskal-Wallis) was used for multiple comparisons of the control group with each dose group.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

when treated with 4 mg/L, decrease in locomotor activity of the male and female rats were observed as compared to control, but recovered after the exposure ended.

Mortality:

no mortality observed

Description (incidence):

No mortality were observed during the study in treated rats as compared to control.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When treated with 4 mg/L, Body weight gain significantly decreased in male and female rats from week 1 were observed as compared to control rats. Increased body weight gain on week 4 were observed in male rat as compared to control.

When treated with 1 mg/L, increased body weight gain on week 4 in male rats were observed as compared to control.
The increased body weight of the male rats exposed to 330 ppm at week 4 was
not considered to be ethyl formate-related change because they were isolated changes.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

When treated with 4 mg/L, significantly decreased in male and female rat were observed from week 1 and week 3, respectively as compared to control.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

When treated with 4 mg/L, increased HGB and HCT level in male and decreased in Reti level n female rat were observed as compared to control.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

When treated with 4 mg/L, Ca and TG levels decreased in male and female rats respectively.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

When treated with 4 mg/L, incerase in Ketone body in male and femlae rat and Urobilinogen level in female rat were observed as compared to control.

When treated with 1 mg/L, incerase in Ketone body in femlae rat were observed as compared to control.
Reduced body weight and food consumption are occasionally accompanied by increase in urinary ketone levels, because energy metabolism under those conditions shifts from gluconeogenesis to incomplete oxidation of fatty acids.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When treated with 4 mg/L, absolute and relative adrenal weight increased in male and female rats and relative brain, kidney, and lung in male and female rat as compered to control.
Absolute or relative thymus weight or both decreased in male and female rats and absolute heart, kidney, liver, lung, and spleen weights decreased as compered to control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All the macroscopic findings of the eye, liver, and ovaries were considered to be incidental and of no toxicological significance.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

When treated with 4 mg/L, Degeneration, Squamous metaplasia and Lesions were mainly noted in the dorsal meatus and occasionally in the septum and turbinate in male and female rats as compared to control.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

Dose descriptor:

NOAEC

Effect level:

1 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Remarks on result:

other: No effect observed

Target system / organ toxicity

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEC was considered to 1 mg/L when Sprague-Dawley male and female rats were inhaled with test substance for 90 days.

Executive summary:

In a Repeated dose inhalation toxicity study, Sprague-Dawley male and female rats were inhaled with test substance in the concentration of 0, 0.2, 1 and 4 mg/L for 6 hr/day, 5 days by Whole body exposure for 90 days. Decrease in locomotor activity of the male and female rats were observed as compared to control, but recovered after the exposure ended at 4 mg/L. No mortality was observed during the study in treated rats as compared to control. Body weight gain significantly decreased in male and female rats from week 1 were observed as compared to control rats. Increased body weight gain on week 4 were observed in male rat as compared to control at 4 mg/L and increased body weight gain on week 4 in male rats were observed as compared to control at 1 mg/L. The increased body weight of the male rats exposed to 330 ppm at week 4 was not considered to be ethyl formate-related change because they were isolated changes. Significantly decreased in male and female rat were observed from week 1 and week 3, respectively as compared to control at 4 mg/L. Similarly, increased HGB and HCT level in male and decreased in Reti level n female rat were observed as compared to control and Ca and TG levels decreased in male and female rats respectively at 4 mg/L. Increase in Ketone body in male and female rat and Urobilinogen level in female rat were observed as compared to control. Increase in Ketone body in female rat were observed as compared to control at 1 mg/L. Reduced body weight and food consumption are occasionally accompanied by increase in urinary ketone levels, because energy metabolism under those conditions shifts from gluconeogenesis to incomplete oxidation of fatty acids. In addition, absolute and relative adrenal weight increased in male and female rats and relative brain, kidney, and lung in male and female rat as compared to control and Absolute or relative thymus weight or both decreased in male and female rats and absolute heart, kidney, liver, lung, and spleen weights decreased as compared to control at 4 mg/L. Degeneration, Squamous metaplasia and Lesions were mainly noted in the dorsal meatus and occasionally in the septum and turbinate in male and female rats as compared to control at 4 mg/L. Therefore, NOAEC was considered to 1 mg/L when Sprague-Dawley male and female rats were inhaled with test substance for 90 days.