Ethyl 4-methyl-2-oxocyclohexanecarboxylate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 May 2017 - 14 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

September 2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

May 2008

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Appendix to Director General Notification, No. 12-Nousan-8147. Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan (JMAFF)

Version / remarks:

November 2000, including the most recent revisions

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 4-methyl-2-oxocyclohexanecarboxylate

EC Number:

236-910-1

EC Name:

Ethyl 4-methyl-2-oxocyclohexanecarboxylate

Cas Number:

13537-82-1

Molecular formula:

C10H16O3

IUPAC Name:

ethyl 4-methyl-2-oxocyclohexane-1-carboxylate

Test material form:

liquid

Details on test material:

Physical appearance: Clear colourless liquid
Storage conditions: In refrigerator (2-8°C) in clear glass bottle

Specific details on test material used for the study:

Stability at higher temperatures: Yes, maximum temperature: 50°C, maximum duration: 1440 minutes
Vapour pressure: 1.18 Pa
pH (1% in water, indicative range): 5.11 – 5.25
Specific gravity / density: 1.04 g/mL

Test animals

Species:

rat

Strain:

other: Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9-11 weeks old
- Weight at study initiation: males: 251-300 g; females: 175-221 g
- Fasting period before study: not specified
- Housing: group housed (max 5 animals of the same sex and same exposure group together) in polycarbonate cages containing sterilized sawdust as bedding material
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap-water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24 (actual: 20-21)
- Humidity (%): 40-70 (actual: 50-71)
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 May 2017 To: 14 September 2017

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 1 - < 4 µm

Geometric standard deviation (GSD):

> 1.5 - < 3

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chamber based on the directed flow nose only inhalation chamber (Am. Ind. Hyg Assoc. J. 44(12): 923-928, 1983)
- Exposure chamber volume: not indicated
- Method of holding animals in test chamber: polycarbonate restraining tubes
- Source and rate of air: at least 1 L/min (theoretical air flow in each animal port); mean total air flow: 22 L/min (first test group) and 28 L/min (second test group)
- System of generating aerosols: nebulization of the test item with pressurized air: the test item was transferred to a Collison nebulizer by means of a rotating pump (type VL500 digit, VERDER Lab Tec GmbH & Co. KG, Haan, Germany) in order to maintain a constant level of freshly available test item. The primary aerosol was subsequently diluted with pressurized air before it entered the exposure chamber
- Method of particle size determination: samples were drawn with a flow of 2 L/min. from the test atmosphere through a tube mounted in one of the free animal ports of the exposure chamber. The samples were collected with an 8 stage Marple personal cascade impactor containing fiber glass filters (TE-290-GF. Tisch Environmental, Cleves, Ohio, USA) and a fiber glass back-up filter (SEC- 290-F1, Westech, Upper Stondon, Bedfordshire, England). Amounts of test item collected were measured gravimetrically.
- Treatment of exhaust air: filtered and released to the exhaust of the fume hood
- Temperature and humidity in air chamber: 20.8-21.6 °C and 5-10%

TEST ATMOSPHEREn (see table 1 and 2 for MMAD and GSD)
- Brief description of analytical method used: Samples were drawn from the test atmosphere through a tube mounted in one of the free animal ports of the exposure chamber. Samples were drawn through a glass fiber filter. Sample volumes were measured by means of a dry gas meter. The collected amount of test item in the air sample was measured gravimetrically.
- Samples taken from breathing zone: yes, a total of 15 and 18 representative samples were taken for determination of the actual concentration during the first and second treatment group, respectively.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: trial generation results showed that the test item was not volatile and the test atmosphere consisted mainly of aerosol with a negligibly small (if any) vapor part. Trial generation results showed that it was not possible to generate a suitable test atmosphere sufficiently stable at the highest target concentration. Instead, the generation was performed at the technically maximum attainable concentration.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

5 mg/L for both test groups

No. of animals per sex per dose:

3 animals were initially exposed, 2 animals were additionally exposed

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
During exposure: mortality, behavioral signs of distress and effects on respiration at least three times a day
After exposure: mortality: twice daily; clinical signs: at least two times on the day of exposure and once daily thereafter
Body weights (individually): on day 1, 2, 4, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: descriptions of all internal macroscopic abnormalities were recorded.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality occurred.

Clinical signs:

other: During exposure, slow breathing was seen. After exposure, lethargy, hunched posture, labored respiration, chromodacryorrhea (nose) and ptosis were seen for the animals on days 1 and/or 2.

Body weight:

Overall body weight gain in males and females was within the range expected for rats of this strain and age used in this type of study, except for one female which showed body weight loss up to Day 8. This female regained weight during the second week.

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals.

Other findings:

- Actual exposure concentrations: for the initially exposed group, the time weighted mean actual concentration was 5.0 ± 0.1 mg/L. The nominal concentration was 7.2 mg/L and the generation efficiency was 70%. For the additionally exposed group, the time weighted mean actual concentration was 5.1 ± 0.1 mg/L. The nominal concentration was 6.8 mg/L and the generation efficiency was 76%.
- Particle size: the Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were determined twice during each exposure period (see table 1 and 2).

Any other information on results incl. tables

Table 1 Aerodynamic particle size distribution in the test atmosphere (first test group)

Measurement 1:

Stage	Cut point (mm)		Mass sampled (mg)	Relative mass (%)	Cumulative mass (% of total sampled)
1	21.0		0.10	1.27	98.73
2	15.0		0.00	0.00	98.73
3	10.0		0.56	7.13	91.59
4	6.0		1.37	17.45	74.14
5	3.5		2.56	32.61	41.53
6	2.0		2.41	30.70	10.83
7	0.9		0.61	7.77	3.06
8	0.5		0.22	2.80	0.25
Back up	0.25		0.02	0.25	0.00
MMAD^1(mm):		3.7
gsd^2:		1.9

Measurement 2:

Stage	Cut point (mm)		Mass sampled (mg)	Relative mass (%)	Cumulative mass (% of total sampled)
1	21.0		0.03	0.37	99.63
2	15.0		0.08	0.99	98.65
3	10.0		0.57	7.02	91.63
4	6.0		1.25	15.39	76.23
5	3.5		2.76	33.99	42.24
6	2.0		2.56	31.53	10.71
7	0.9		0.71	8.74	1.97
8	0.5		0.12	1.48	0.49
Back up	0.25		0.04	0.49	0.00
MMAD^1(mm):		3.7
gsd^2:		1.9

¹ Mass Median Aerodynamic Diameter; ² Geometric standard deviation

 

Table 2 Aerodynamic particle size distribution in the test atmosphere (second test group)

Measurement 1:

Stage	Cut point (mm)		Mass sampled (mg)	Relative mass (%)	Cumulative mass (% of total sampled)
1	21.0		0.08	0.63	99.37
2	15.0		0.04	0.31	99.06
3	10.0		0.31	2.44	96.62
4	6.0		1.63	12.81	83.81
5	3.5		4.93	38.76	45.05
6	2.0		4.26	33.49	11.56
7	0.9		1.31	10.30	1.26
8	0.5		0.16	1.26	0.00
Back up	0.25		0.00	0.00	0.00
MMAD^1(mm):		3.7
gsd^2:		1.8

Measurement 2:

Stage	Cut point (mm)		Mass sampled (mg)	Relative mass (%)	Cumulative mass (% of total sampled)
1	21.0		0.03	0.22	99.78
2	15.0		0.00	0.00	99.78
3	10.0		0.35	2.58	97.20
4	6.0		1.78	13.11	84.09
5	3.5		5.30	39.03	45.07
6	2.0		4.45	32.77	12.30
7	0.9		1.49	10.97	1.33
8	0.5		0.18	1.33	0.00
Back up	0.25		0.00	0.00	0.00
MMAD^1(mm):		3.5
gsd^2:		1.7

¹ Mass Median Aerodynamic Diameter; ² Geometric standard deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not classified according to Regulation (EC) No. 1272/2008

Conclusions:

In an acute inhalation study, performed according to OECD 403 and GLP, the LC50 of FRET 13-0545 in rats was established to exceed 5 mg/L. Based on these results, the test item is not classified according to GHS and Regulation (EC) No. 1272/2008.

Executive summary:

In an acute inhalation study, performed according to OECD 403 and GLP, FRET 13-0545 was administered as an aerosol by nose only inhalation for 4 hours to one group of three male and three female Wistar rats. This group was expanded with another two animals of each sex on a later time span to meet the guideline requirements.

For the initially exposed animals, the time-weighted mean actual concentration was 5.0 ± 0.1mg/L, and the MMAD was 3.7 μm (gsd 1.9)(twice). For the additionally exposed animals, the time-weighted mean actual concentration was 5.1 ± 0.1mg/L, the MMAD was 3.7 μm (gsd 1.8) and 3.5 μm (gsd 1.7) (measured twice).

The inhalation LC50, 4h value of FRET 13-0545 in Wistar rats was established to exceed 5 mg/L.

Based on these results FRET 13-0545 does not have to be classified and has no obligatory labelling requirement for acute inhalation toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015)

(including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).