2-{4-[2-(4,4-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)propan-2-yl]phenyl}ethyl 4-methylbenzenesulfonate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 28, 2002 - June 11, 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

no

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-{4-[2-(4,4-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)propan-2-yl]phenyl}ethyl 4-methylbenzene-1-sulfonate

EC Number:

606-467-2

Cas Number:

202189-76-2

Molecular formula:

C23H29NO4S

IUPAC Name:

2-{4-[2-(4,4-dimethyl-4,5-dihydro-1,3-oxazol-2-yl)propan-2-yl]phenyl}ethyl 4-methylbenzene-1-sulfonate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: sponsor
- Batch No.of test material: F-24 Reprec.
- Purity test date: May 22, 2002
- Purity: 96.8%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature, protected from moisture and in the absence of light.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 3-4 weeks old
- Weight at study initiation: 72.7 - 77.4 g (males); 72.6 - 79.9 g (females)
- Housing: The animals were house in Makrolon type III cages (930 cm2 surface) with a stainless steel mesh lid and a poplar sawdust bedding, at the rate of 3 animals per cage. The sawdust and the tray were changed three times a week. The mesh lid and the feeding bottle were changed once a week
- Diet: ad linitum, foodstuff UAR-R04-C (Panlab, S.L., Barcelona. Batch 10517. Expiration date: May 17, 2003) on a controlled maintenance diet.
- Water: ad libitum, tap-water from public distribution system (Consorcio de Aguas Bilbao Bizkaia) ad libitum. During the study, the physical and chemical properties of the water were analyzed twice, obtaining satisfactory results.
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22ºC (+/- 3ºC)
- Humidity: 30-70%
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.
- Microbiological controls on the air and the surface of the room have been carried out resulting in 54 UFC/m3 in air, 0 UFC/plate on the wall and 7 UFC/plate in soil.

IN-LIFE DATES: From: May 28, 2002 To: June 11, 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Xanthan gum suspended 25% (w/v) in tap water

Remarks:

Xhantan gum supplied by the sponsor's Raw Materials Warehouse

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle: 8.84 g
- Lot/batch no.: L0992

MAXIMUM DOSE VOLUME APPLIED: 1 mL/100g bw

DOSAGE PREPARATION: 8.84 g of vehicle were progressively added to 2.066 g of the test item. Test item was suspended by magnetic stirring in the vehicle. Gross particles were grounded using a metal spatula to improve test item dispersion.

Doses:

2000mg/kg bw

No. of animals per sex per dose:

3

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: At 1h, 2h, 3h, 5h, then, observations were done at least once a day for 14 days. Animals were weighed on days 1, 2, 3, 7, 9, 11 and 14.
- Necropsy of survivors performed: yes. On D14 after the administration, each animal was sacrificed and the necropsy was performed. It was examined the organs and tissues of the cranial cavity, thoracic and abdominal cavity, taking special care on each organ after extraction.
- Other examinations performed:
Mortality: animals were observed daily in order to register mortality
Clinical signs: In order to register the beginning and evolution of the possible toxic effects of the test item, animals were observed during the following 5 hours after administration, (0h, 1h,2h, 3h, 5h), then, observations were done at least once a day for 14 days. Parameters observed on animals inside the cage: physical activity, posture while walking, posture at rest and sense of fear. Parameters observed on each animal outside the cage: nervousness/agitation, fear, tremors; hair, eyes, nostrils and stools appearance; posture while walking and posture at rest.
Body weight: Body weight was assed in day 1,2,3,7,9, 11 and 14. The mean body weight has been calculated based on the individual body weights recorded in each weighing.
Body temperature: the body temperature was assessed with a temperature probe that was introduced approximately 2-3 cm into the rectum of every animal in a total of a 6 times during the test.
Locomotor activity: The locomotor activity has been assessed based on the number of frames traversed by each animal for 2 minutes. The test was performed in a total of 9 times during the test.
Hematology: At the end of the test, before necropsy, blood samples were obtained from the jugular veins of all surviving animals, after anesthesia by inhalation of ethyl ether. The fasting period before blood drawn was of 19-20 hours (animals were not deprived of drinking water). The parameters examined were red blood cells count (RBCs), white blood cells count (WBCs), hemoglobine (HB), hematocrit (HCT), mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT).
Food intake: observations were done on day 2, 3, 6, 11 and 14.

Results and discussion

Preliminary study:

A preliminary study was performed by administration of a single dose of 1000 mg/kg in mouse. No mortality occurred during the study. Therefore, the dose determined for the main study was 2000 mg/kg.

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred during the study.

Clinical signs:

other: No clinical signs observed during the study, with the exception of tha animal AH3 that presented, after 2 hours of the administration, a remarkable nervousness/agitation and alteration of the body posture (dorsal bending and pelvic elevation) and that was

Gross pathology:

No macroscopic lesions were found that could be attributed to a toxic effect of the test item.

Other findings:

Feed intake: In both males and females, there was a slight decrease in feed intake during the 24 hours after administration, about 10%, which in females recovered after 48 hours and in males the recovery occurred progressively until day 5 of the study.

Hematology: A slight increase in the number of platelets (10-12%) was observed in both males and females

Any other information on results incl. tables

Body temperature (ºC) – Males

	Time after administration
Animal	5-6 hours	24 hours	48 hours	Day 7	Day 9	Day 14
TM1	35.9	37.2	36.5	37.8	37.3	37.5
TM2	36.4	37.0	36.9	37.7	38.0	37.7
TM3	36.0	37.3	36.6	37.7	38.1	38.4
Group T: Mean temperature	36.1	37.2	36.7	37.7	37.8	37.9
AM1	35.8	37.4	37.1	37.8	37.6	37.9
AM2	36.1	37.3	37.0	37.1	37.7	38.1
AM3	35.5	37.0	36.9	37.1	37.7	38.1
Group A: Mean temperature	35.8	37.2	37.0	37.3	37.7	38.0
Difference A - T (%)	- 0.8	0	+ 0.8	- 1.1	- 0.3	+ 0.3

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

 

Body temperature (ºC) – Females

	Time after administration
Animal	5-6 hours	24 hours	48 hours	Day 7	Day 9	Day 14
TH1	36.7	37.1	37.7	37.3	38.1	38.2
TH2	37.1	37.4	38.1	38.2	38.1	38.7
TH3	36.7	37.1	38.0	37.7	37.7	38.3
Group T: Mean temperature	36.8	37.2	37.9	37.7	38.0	38.4
AH1	36.5	37.7	37.6	37.8	37.7	38.7
AH2	36.2	37.5	38.1	37.5	37.8	38.5
AH3	36.7	37.4	38.2	37.6	38.3	38.4
Group A: Mean temperature	36.5	37.5	38.0	37.6	37.9	38.5
Difference A - T (%)	- 0.8	+ 0.8	+ 0.3	- 0.3	- 0.3	+ 0.3

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

 

Locomotor activity (No. of frames traversed in 2 minutes) – Males

	Time after administration
Animal	5-6 hours	24 hours	30 hours	48 hours	3 days	7 days	9 days	11 days	14 days
TM1	2	6	17	5	12	22	3	7	2
TM2	3	5	26	6	16	3	14	5	9
TM3	3	18	2	12	41	39	52	41	25
Group T: Mean activity	2.7	9.7	15.0	7.7	23.0	21.3	23.0	17.7	12.0
AM1	3	28	20	29	41	45	26	25	35
AM2	11	28	26	13	19	16	22	18	19
AM3	2	9	8	4	22	7	44	39	39
Group A: Mean activity	5.3	21.7	18.0	15.3	27.3	22.7	30.7	27.3	31.0

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

 

 

Locomotor activity (No. of frames traversed in 2 minutes) – Females

	Time after administration
Animal	5-6 hours	24 hours	30 hours	48 hours	3 days	7 days	9 days	11 days	14 days
TH1	4	3	16	3	6	17	30	55	66
TH2	14	13	2	17	19	32	33	26	32
TH3	1	5	41	31	21	37	13	41	43
Group T: Mean activity	6.3	7.0	19.7	17.0	15.3	28.7	25.3	40.7	47.0
AH1	3	10	17	1	36	44	30	50	64
AH2	2	14	23	3	2	28	6	23	54
AH3	3	28	37	17	42	46	52	67	57
Group A: Mean activity	2.7	17.3	25.7	7.0	26.7	39.3	29.3	46.7	58.3

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

 

Individual weight (g) – Males

	Day of the study
Animal	1	2	3	7	9	11	14
TM1	74.5	85.2	90.9	120.1	134.4	147.1	170.8
TM2	77.4	91.2	98.7	133.0	154.3	174.0	200.6
TM3	76.7	87.5	97.7	129.1	144.4	158.1	181.0
Group T: Mean weight	76.20	87.97	95.77	127.40	144.37	159.73	184.13
AM1	72.7	83.3	89.7	123.2	144.6	163.7	191.0
AM2	76.4	91.7	96.8	129.4	144.1	166.2	192.8
AM3	74.6	85.1	93.7	120.1	134.9	149.5	165.6
Group A: Mean weight	74.57	86.70	93.40	124.23	141.20	159.80	183.13

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

 

Individual weight (g) – Females

	Day of the study
Animal	1	2	3	7	9	11	14
TH1	75.6	87.6	94.2	119.8	127.1	136.0	146.8
TH2	72.6	85.4	91.6	116.7	126.9	135.4	151.0
TH3	73.7	83.8	89.1	113.0	123.8	131.5	144.3
Group T: Mean weight	73.97	85.60	91.63	116.50	125.93	134.30	147.37
AH1	79.9	87.3	92.3	120.1	134.2	141.1	159.9
AH2	74.2	85.7	92.8	119.3	129.0	140.8	149.9
AH3	75.0	82.7	89.0	114.4	121.6	129.0	141.7
Group A: Mean weight	76.37	85.23	91.37	116.93	128.27	136.97	150.50

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

 

  

Evolution (Statistical analysis) – Males

	Day of the study
	1		2		3		7		9		11		14
	T	A	T	A	T	A	T	A	T	A	T	A	T	A
Mean weight (g)	76.20	74.57	87.97	86.70	95.77	93.40	127.40	124.23	144.37	141.20	159.73	159.80	184.13	183.13
Difference with respect to T	-	- 2.14	-	- 1.44	-	- 2.47	-	- 2.49	-	- 2.20	-	+ 0.04	-	- 0.54
DE	1.51	1.85	3.03	4.42	4.24	3.56	6.62	4.74	9.95	5.46	13.52	9.01	15.15	15.21
N	3	3	3	3	3	3	3	3	3	3	3	3	3	3
CV (%)	2	2	3	5	4	4	5	4	7	4	8	6	8	8
P^(1)value with respect to group T	-	0.4	-	0.7	-	1.0	-	1.0	-	1.0	-	1.0	-	1.0

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

(1): Probability by comparing group T and A by the 2-tailed Mann-Whitney U test, with a confidence interval of 95%

 

 

Evolution (Statistical analysis) – Females

	Day of the study
	1		2		3		7		9		11		14
	T	A	T	A	T	A	T	A	T	A	T	A	T	A
Mean weight (g)	73.97	76.37	85.60	85.23	91.63	91.37	116.50	116.93	125.93	128.27	134.30	136.97	147.37	150.50
Difference with respect to T	-	+ 3.14	-	- 0.43	-	- 0.28	-	+ 0.38	-	+ 1.82	-	+ 1.95	-	+ 2.08
DE	1.52	3.09	1.91	2.34	2.55	2.06	3.40	4.81	1.85	6.33	2.44	6.90	3.39	9.11
N	3	3	3	3	3	3	3	3	3	3	3	3	3	3
CV (%)	2	4	2	3	3	2	3	4	1	5	2	5	2	6
P^(1)value with respect to group T	-	0.4	-	1.0	-	1.0	-	1.0	-	0.7	-	0.7	-	1.0

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

(1): Probability by comparing group T and A by the 2-tailed Mann-Whitney U test, with a confidence interval of 95%

 

 

Feed intake (g) – Males and Females

		Day of the study
Animal		2	3	7	11	14
MALES	Mean intake Group T	15.33	16.17	18.98	22.31	22.74
	Mean intake Group A	13.83	15.43	18.45	23.03	23.46
Difference from A to T (%)		- 9.78	- 4.54	- 2.81	+ 3.11	+ 3.03
FEMALES	Mean intake Group T	16.70	14.60	15.97	17.34	15.87
	Mean intake Group A	15.07	15.73	16.98	19.14	17.94
Difference from A to T (%)		- 9.78	+ 7.20	+ 5.99	+ 9.40	+ 11.58

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

 

 

 Hematology – Males

	Parameter
Animal	GB (10^3/ml)	GR (10^6/ml)	HGB (g/dL)	HCTO (%)	VCM (fL)	HCM (pg)	CHCM (g/dL)	PLT (10^3/ml)
TM1	7.2	6.00	11.8	37.7	62.9	19.7	31.3	1046.5
TM2	5.2	5.85	11.3	36.4	62.2	19.3	31.0	978.5
TM3	8.3	5.98	12.2	38.2	63.9	20.3	31.8	926.5
Group T: Mean value	6.9	5.94	11.7	37.4	63.0	19.8	31.4	983.8
AM1	6.9	6.11	11.9	37.3	61.1	19.4	31.8	1047
AM2	8.3	6.21	12.4	39.7	63.9	19.9	31.2	392.5
AM3	6.7	6.39	12.5	40.2	62.9	19.6	31.1	1158
Group A: Mean value	6.8	6.25	12.2	38.7	62.0	19.5	31.4	1102.5
Difference from A to T (%)	- 1.4	+ 5.0	+ 4.1	+ 3.4	- 1.6	- 1.5	0	+ 10.8

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

 

 

Hematology – Females

	Parameter
Animal	GB (10^3/ml)	GR (10^6/ml)	HGB (g/dL)	HCTO (%)	VCM (fL)	HCM (pg)	CHCM (g/dL)	PLT (10^3/ml)
TH1	6.2	6.55	12.4	38.9	59.4	19.0	31.9	987.0
TH2	5.3	6.35	12.6	39.6	62.3	19.8	31.7	1008.0
TH3	6.5	6.08	12.2	38.3	63.0	20.0	31.8	957.5
Group T: Mean value	6.0	6.32	12.4	38.9	61.5	19.6	31.8	948.2
AH1	6.4	6.71	12.8	41.1	61.3	19.1	31.2	1132.0
AH2	6.4	6.23	12.6	38.2	61.3	20.3	33.0	1110.5
AH3	4.0	6.12	12.9	40.0	65.4	21.1	32.3	1142.5
Group A: Mean value	6.4	6.47	12.7	39.6	61.3	19.7	32.1	1121.3
Difference from A to T (%)	+ 6.3	+ 2.3	+ 2.4	+ 1.8	- 0.3	+ 0.5	+ 0.9	+ 12.2

(T) = group T; dose (mg/kg): 0

(A) = group A; dose (mg/kg): 2000

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat.

Executive summary:

An acute toxic class method test on rats was performed according to OECD Guideline 423 and test method B.1. The test item was administered, as supplied, to a group of 6 male and female Wistar rats (3-4 weeks old, 3 per group) at the dose of 2000 mg/kg body weight by oral gavage. The selection of the dose was made based on the preliminary results obtained, in which the administration of a dose of 1000 mg / kg in mouse did not cause the death of any of the animals administered with the test item.

No clinical signs observed during the study, with the exception of the female AH3 that presented a remarkable nervousness/agitation and alteration of the body posture (dorsal bending and pelvic elevation) and that was maintained until the animal was euthanized. Hematological analysis revealed mild thrombocytosis in both males and females. Necropsies and macroscopic study of organs and tissues have not revealed the presence of macroscopic alterations attributable to a toxic effect of the test item. No mortality occurred during the study, therefore the LD50 of the test item is higher than 2000 mg/kg body weight by oral route in the rat.