ethyl 5,5-diphenyl-2-isoxazoline-3-carboxylate

Endpoint summary

Description of key information

Acute oral, OECD 401, rat (male): LD50 = 1611 mg/kg bw

Acute inhalation, OECD 403, rat, nose only, dust (limit test): LC50 >5.040 mg/L (5040 mg/m³)

Acute dermal, OECD 402, rat (limit test): LD50 >2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Feb - 05 May 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

Adopted in 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-1 (Acute Oral Toxicity)

Version / remarks:

Adopted in 1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

Version / remarks:

Adopted in 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Agricultural chemicals, Laws and Regulations Japan, MAFF (p. 19 - 20)

Version / remarks:

Adopted in 1985

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

WISKf(SPF71)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding colony, Hoechst AG, Kastengrund, Germany
- Age at study initiation: males: approx. 6 weeks, females: approx. 7 weeks
- Body weight at study initiation: males: 183 g (mean), 169 - 199 g (range); females: 173 g (mean), 162 - 180 g (range)
- Fasting period before study: 16 h before treatment to 3 - 4 h after treatment
- Housing: Makrolon cages (Type 4) on soft wood granulate in groups of 5 animals
- Diet: Altromin 1324 rat diet (Altromin GmbH, Lage/Lippe, Germany), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: at least 1 day

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes: fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 6.25, 12.00, 20.00, 25.00, and 50.00% (w/v)
- Amount of vehicle (if gavage): 10 mL/kg bw

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION: The test substance was suspended in the above stated concentrations in sesame oil with mortar and pestle and distributed homogeneously by means of a magnetic stirrer.

Doses:

625, 1250, 2000, 2500, and 5000 mg/kg bw

No. of animals per sex per dose:

5 males (625 mg/kg bw dose group)
5 males and 5 females (1250 - 5000 mg/kg bw dose groups)

Control animals:

other: not required

Details on study design:

- Duration of observation period following administration: 15 days (16 days for 1250 mg/kg bw dose group for technical reasons)
- Frequency of observations and weighing: Animals were observed for mortality/moribundity and clinical signs 10, 30, 60 min and 2, 4, and 6 h after dosing, twice daily from Day 2 to 6 and daily thereafter until the end of the observation period. Individual body weights were determined weekly.
- Necropsy of survivors performed: yes

Statistics:

The LD50, the 95% limits of confidence and the equation of the probit lines were established on the basis of the mortality rates by probit analysis. The LD50 values were calculated for males and females separately. To get one probit line, a goodness-of- fit-test (Pearson) was performed with a 5% level of significance. Then the limits of confidence were calculated according to the method of FIELLER.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

1 740 mg/kg bw

Based on:

test mat.

95% CL:

>= 1 251 - <= 2 245

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

1 611 mg/kg bw

Based on:

test mat.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

1 904 mg/kg bw

Based on:

test mat.

Mortality:

625 mg/kg bw: 0/5 males died
1250 mg/kg bw: 3/5 males (48 - 72 h post-dose) and 1/5 females (6 days post-dose) died
2000 mg/kg bw: 2/5 males (48 h post-dose) and 2/5 females (48 h and 13 days post-dose) died
2500 mg/kg bw: 4/5 males (48 - 72 h post-dose) and 4/5 females (48 - 96 h post-dose) died
5000 mg/kg bw: 5/5 males (48 h post-dose) and 5/5 females (48 - 96 h post-dose) died

Clinical signs:

other: Clinical signs of intoxication did not significantly differ in type, incidence, and severity between male and female animals. Incidence and severity of clinical signs were dose-dependently increased with ascending dose. They began to emerge 1 h after admi

Gross pathology:

625 mg/kg bw:
Necropsy revealed no test substance-related findings.

1250 mg/kg bw:
- animals found dead: general autolysis, small intestine full of a reddish-black mass, positive in the faecal occult blood test
- animals sacrificed at termination: Necropsy revealed no test substance-related findings.

2000 mg/kg bw:
- animals found dead: general autolysis, liver with light discolouration, small intestine discoloured by test substance and full of test substance
- animals sacrificed at termination: Necropsy revealed no test substance-related findings.

2500 mg/kg bw:
- animals found dead: general autolysis, liver with light discolouration and lobular demarcation, lungs discoloured red, small intestine full of test substance, and reddish black mass
- animals sacrificed at termination: Necropsy revealed no test substance-related findings.

5000 mg/kg bw:
- animals found dead: general autolysis, liver with light discolouration and lobular demarcation, lungs discoloured orange, stomach full of test substance, small intestine full of test substance, and reddish black mass

Table 1. Table for acute oral toxicity.

Dose [mg/kg bw]	Toxicological results*	Duration of clinical signs	Time of death	Mortality (%)
Males
625	0/5/5	1 h – Day 2	---	0
1250	3/5/5	2 h – Day 3	Day 2 + Day 3	60
2000	2/5/5	2 h – Day 2	Day 2	40
2500	4/5/5	2 h – Day 5	Day 2 + Day 3	80
5000	5/5/5	1 h - death	Day 2	100
Females
650	0/0/0	---	---	---
1250	1/4/5	4 h - Day 6	Day 6	20
2000	2/5/5	2 h - Day 14	Day 2 + Day 13	40
2500	4/5/5	2 h - Day 4	Day 2 + Day 4	80
5000	5/5/5	2 h - death	Day 2 - Day 4	100
LD50 = 1740 mg/kg bw

* number of dead animals/number of animals with clinical signs/number of animals used  

Interpretation of results:

other: Acute Oral Cat. 4 (H302) according to Regulation (EC) No 1272/2008

Conclusions:

In the present acute oral toxicity study in rats a LD50 value of 1740 mg/kg bw for both sexes was determined.
The LD50 value was 1611 mg/kg bw for males and 1904 mg/kg bw for females, respectively.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

1 611 mg/kg bw

Quality of whole database:

The study was conducted according to the appropriate test guideline and in compliance with GLP (RL1).

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Sep - 07 Oct 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

Current version adopted in 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

Guideline in place during study conduct: adopted in 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-3 (Acute inhalation toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (MAFF), Testing Guidelines for Toxicology Studies, 59 NohSan No. 4200

Version / remarks:

Adopted in 1985

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom

Test type:

traditional method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ltd., Margate, UK
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: males: 272 – 317 g, females: 215 – 232 g
- Fasting period before study: no
- Housing: 5 animals of the same sex per cage in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK), except during exposure period
- Diet: Rat and Mouse Expanded Diet No. 1 (Special Diets Services Ltd., Witham, UK), ad libitum (except during exposure period)
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

1.7 µm

Geometric standard deviation (GSD):

0.55

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: cylindrical exposure chamber
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber 'O' ring. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air (airflow): oil free compressor, 20 L/min, providing 40 air changes per hour
- Method of conditioning air: Compressed air was supplied by means of an oil free compressor and was passed through a water trap and respiratory quality filters before it was introduced to the dust feed.
- System of generating particulates/aerosols: A dust atmosphere was produced from the test substance using a 'Wright's Dust Feed' mechanism located at the top of the exposure chamber and driven by a variable speed motor. The dust feed was connected to a metered compressed air supply.
- Method of particle size determination: The particle size of the generated atmosphere of the test substance inside the exposure chamber was determined three times during the exposure period using a cascade impactor. This device consisted of six impactor stages with stainless steel collection substrates (10, 6, 3.5, 1.6, 0.9 and 0.5 µm cut-off points) and a back-up glass fibre filter housed in an aluminium sampler. The sampler was temporarily sealed in a sampling port in the animals' breathing zone. Exposure chamber air was drawn through the cascade impactor using a vacuum pump for a suitable time period. The collection substrates and back-up filter were weighed before and after sampling and the weight of test substance, collected at each stage, calculated by difference.
- Treatment of exhaust air: The extract from the exposure chamber passed through a 'scrubber' trap and was connected with a high efficiency filter to a metered exhaust system.
- Temperature and humidity in air chamber: 20 - 21 °C, 41 - 57%. The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Kane-May Ltd., Welwyn Garden City, UK) located in a vacant port in the animals' breathing zone of the chamber and recorded every 30 min throughout the 4 h exposure period.

TEST ATMOSPHERE
- Brief description of analytical method and equipment used: The chamber atmosphere was sampled once after chamber equilibration and in 15-min intervals thereafter till the end of the exposure. The method used employed glass fibre filters (Gelman type A/E 25 mm) placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals' breathing zone. Exposure chamber air was drawn through the filter at a measured rate using a vacuum pump for a suitable time period. Each filter was weighed before and after sampling in order to calculate the weight of collected test substance. The difference in the two weights divided by the volume of atmosphere sampled was used for real-time monitoring of chamber concentration. At 30-min intervals a filter was placed in a pre-labelled glass container, extracted with acetonitrile, and submitted for chemical analysis by HPLC (column: Prodigy ODS (250 x 4.6 mm id), mobile phase: acetonitrile: 0.1% orthophosphoric acid:propan-2-ol (52.5:45:2.5 v/v), flow rate: 1.5 mL/min, UV detector wavelength: 230 nm, injection volume: 10 µL, retention time: ~ 15 min)
- Samples taken from breathing zone: yes
- Time needed for equilibrium of exposure concentration before animal exposure : theoretical chamber equilibration time (T99): 7 min

TEST ATMOSPHERE
- Particle size distribution: 91.8% < 4 µm

Analytical verification of test atmosphere concentrations:

yes

Remarks:

HPLC

Duration of exposure:

4 h

Concentrations:

5040 mg/m³, 5.04 mg/L

No. of animals per sex per dose:

5

Control animals:

other: not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 h after termination of exposure, and subsequently once daily till study termination. Body weights were recorded prior to treatment and weekly thereafter.
- Necropsy of survivors performed: yes
- Examinations performed: clinical signs, body weight, detailed macroscopic examination of the respiratory tract

Statistics:

Means and standard deviations were calculated.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.04 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 040 mg/m³ air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: After exposure, all animals showed wet fur, hunched posture, and piloerection.

Remarks:

For details see "Other findings".

Body weight:

No effect on body weight was noted.

Gross pathology:

No abnormalities were detected at necropsy, with the exception of one male which showed dark foci on the lungs. This isolated finding was not considered to be related to treatment with the test substance.

Other findings:

- Clinical observations: During exposure, wet fur was commonly observed and in the females signs of decreased respiratory rate and an isolated incident of laboured respiration were noted. After exposure, all animals showed wet fur, hunched posture and piloerection. There were incidents of increased (2/5 males, 1/5 females) or reduced (1/5 males) respiratory rate, ptosis (2/5 males, 3/5 females) and red/brown staining around the eyes and/or snout (1/5 males, 1/5 females). 1 h post exposure, signs of wet fur had diminished. 1 day post exposure 9/10 animals showed no abnormalities while one female continued to show hunched posture. All animals had recovered on Day 2. No further abnormalities were observed.

Table 1. Table for acute inhalation toxicity.

 

Target concentration [mg/L air]	Toxicological results*	Duration of clinical signs	Time of death	Mortality (%)
Males
5.04	0/5/5	Day 0-1	---	0
Females
5.04	0/5/5	Day 0-2	---	0
LC50 > 5.04 mg/L air

                                                                                           

* first number = number of dead animals                                   

 second number = number of animals with clinical signs           

 third number = number of animals used                                 

 

Interpretation of results:

other: CLP: not classified

Conclusions:

Based on the results of the present study, no classification for acute inhalation toxicity according to Regulation (EC) 1272/2008 is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The study was conducted according to the appropriate test guideline and in compliance with GLP (RL1).

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Feb - 09 Mar 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

Current version adopted in 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

Guideline in place during study conduct: adopted in 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-2 (Acute Dermal Toxicity)

Version / remarks:

Adopted in 1988

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Agricultural chemicals, Laws and Regulations Japan, MAFF (p. 20 - 21)

Version / remarks:

1985

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

Hoe: WISKf(SPF71)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding colony, Hoechst AG, Kastengrund, Germany
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: males: approx. 8 weeks, females: approx. 11 weeks
- Body weight at study initiation: males: 225 g (mean), 220 - 230 g (range); females: 227 g (mean), 222 - 229 g (range)
- Housing: single housed in Makrolon cages (Type 3) on soft wood granulate
- Diet: Altromin 1324 rat diet (Altromin GmbH, Lage/Lippe, Germany), ad libitum
- Water: tap water in drinking water quality, ad libitum
- Acclimation period: at least 1 day
- Microbiological status : SPF bred animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes: fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 Feb 1994 To: 09 Mar 1994

Type of coverage:

occlusive

Vehicle:

other: sesame oil

Details on dermal exposure:

TEST SITE
- Area of exposure: 30 cm² shaved skin of the dorsal area of the trunk
- Type of wrap if used: The treated skin area was covered with a porous gauze and an aluminium foil (6 x 8 cm), which was held in place with an elastic plaster bandage fixed around the animal's body in order to avoid evaporation of the test substance and ingestion of the test substance by the animals.

REMOVAL OF TEST SUBSTANCE
- Washing: residual test substance was removed with warm water
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount applied: 0.5 g
- Constant volume or concentration used: yes, limit test
- For solids, paste formed: yes, 0.5 g test substance were moistened with 0.35 mL sesame oil

VEHICLE
- Amount applied: 0.35 mL

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations and weighing: animals were observed 15, 30, 60 min and 2, 4, and 6 h after dosing, twice daily from Day 2 to 6 and daily thereafter until the end of the observation period; individual body weights were determined weekly
- Necropsy of survivors performed: yes
- Examinations performed: clinical signs, body weight, assessment of macroscopic changes at necropsy

Statistics:

Mean body weights and standard deviations were calculated.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Mortality:

No mortality occurred during the study period.

Clinical signs:

other: No clinical signs of toxicity were observed during the 15-day observation period in 5/5 male and 4/5 female animals. 1/5 females showed alopecia and an encrusted skin surface on its rump, which did not affect the treated skin area. These findings were con

Gross pathology:

Necropsy revealed no substance-related findings.

Other findings:

No local effects on the treated skin area were observed.

Interpretation of results:

other: CLP: not classified

Conclusions:

Based on the results of the present study, no classification for acute dermal toxicity according to Regulation (EC) 1272/2008 is warranted.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The study was conducted according to the appropriate test guideline and in compliance with GLP (RL1).

Additional information

Acute oral toxicity

One acute oral toxicity study, conducted with the registered test substance, is available. The study was performed according to OECD guideline 401 and GLP (M-133573-01-1, 1994). 45 fasted Wistar rats (5/sex/group) received the test substance by oral gavage at a single dose of 625 (males only), 1250, 2000, 2500, and 5000 mg/kg bw. The test substance was suspended in sesame oil. The mortality rates were comparable in both sexes. Lethality generally occurred between Day 2 and 6 of the study, only one female animal died on Day 13. The mortality rates were 0/5, 3/5, 2/5, 4/5, and 5/5 for males and 1/5, 2/5, 4/5, and 5/5 for the females, respectively, counted from the lowest to the highest dose group tested. Clinical signs were comparable in both sexes but they were more protracted in females and increased in incidence and severity in a dose-dependent manner. They began to emerge 1 h after administration and generally persisted up to Day 3 (males) or Day 14 (females) of the study. Nonspecific clinical signs comprised decreased spontaneous activity, drawn in flanks, stilted gait and squatting posture; these signs were accompanied by irregular respiration, increased respiration rate, coat bristling and miosis. The body weight gains were only initially lowered in three female animals at 2000 mg/kg bw but they returned to normal by the end of the study. Necropsy of the decedent animals revealed light discolouration and lobular demarcation of the liver, changes in the small intestine indicative for haemorrhages and red- or orange-discoloured lungs. The animals killed at the end of the experiment were free of macroscopically visible changes. Based on the mortality rates in this study the LD50 value was calculated by probit analysis for the male and female Wistar rat to be 1740 mg/kg bw. The LD50 value was 1611 mg/kg bw for females and 1904 mg/kg bw for males, respectively.

 

Acute inhalation toxicity

One study is available for acute inhalation toxicity. The study is GLP-conform and was performed with the registered test substance according to OECD guideline 403 (M-147615-01-1, 1998). In this study, 5 rats/sex were nose-only exposed for a period of 4 h to dust of the test substance (MMAD 1.7 µm) at a concentration of 5040 mg/m³ (limit test). No mortality occurred within the 14-day observation period. Common clinical signs noted during the study were wet fur, hunched posture, and piloerection. In addition, signs of increased or decreased respiratory rate, ptosis, red/brown staining around the eyes and snout were observed in some animals, and an isolated incident of laboured respiration was noted. Animals recovered normal one or two days post exposure. With the exception of one male, which showed dark foci on the lungs, no macroscopic abnormalities were detected at necropsy. This isolated finding was not considered to be related to treatment with the test substance. The LC50 was determined to be > 5040 mg/m³ for exposure to dust of the test substance for 4 h.

 

Acute dermal toxicity

One study addressing acute dermal toxicity of the registered test substance is available, which was conducted with the registered test substance according to OECD TG 402 and GLP (M-133138-01-1, 1994). The study was conducted as limit test with a single dose of 2000 mg/kg bw applied to the dorsal area of the skin of 5 rats/sex under occlusive conditions for 24 h. No mortality occurred in the dose group tested during the 15 days observation period. Neither treatment-related clinical signs of systemic toxicity nor treatment-related local findings were observed. Body weight gains were slightly impaired during the first week in two female animals only, which returned to normal during the second week of observation until study termination. No treatment -related changes were observed during necropsy. Based on the results of this study the LD50 value for the male and female Wistar rat is >2000 mg/kg bw.

Justification for classification or non-classification

The available data on acute oral toxicity of the test substance meet the criteria for classification as Acute Tox. Cat. 4 for the oral route (H302) according to Regulation (EC) 1272/2008.

The available data on acute dermal and acute inhalation toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.