Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and 4-(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

data is from experimental report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Principles of method if other than guideline:

Acute dermal toxicity study of the given test chemical was performed in rats.

GLP compliance:

yes

Test type:

other: not specified

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(3E)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)but-3-en-2-one; (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one

EC Number:

907-706-6

Molecular formula:

C13H20O

IUPAC Name:

(3E)-4-(2,6,6-trimethylcyclohex-1-en-1-yl)but-3-en-2-one; (3E)-4-(2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-one

Details on test material:

SOURCE OF TEST MATERIAL
- Identity: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one 100%
- EC no: 907-706-6
- Lot/batch No.of test material: 9000533813
- Expiration date of the lot/batch: April 14, 2004
- Purity: 85.1% (sum of 2 isomers)
- Colour: colourless to pale yellow

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:On the day of the experiment, the test item was dissolved in ethanol (purity > 99 %,MERCK, D-64293 Darmstadt).

Specific details on test material used for the study:

- Name of test material: Reaction mass of 4-(2,6,6-trimethylcyclohex-2-ene-1-yl)-but-3-ene-2-one and -(2,6,6-trimethylcyclohex-1-ene-1-yl)-but-3-ene-2-one
- Molecular formula: C13H20O
- Molecular weight: 192.3 g/mole
- Smiles : C1([C@@H](C(=CCC1)C)\C=C\C(C)=O)(C)C
- Inchl: 1S/C13H20O/c1-10-6-5-9-13(3,4)12(10)8-7-11(2)14/h6-8,12H,5,9H2,1-4H3/b8-7+
- Substance type: Organic
- Physical state: Liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house animals, bred at Animal House, sa-FORD.CPCSEA Registration No. 1256/bc/09/CPCSEA.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Weight at study initiation: Male:Minimum: 229 g and Maximum: 262 g
(Prior to Treatment) Female:Minimum: 203 g and Maximum: 241 g
- Fasting period before study: The food was withheld prior to dosing and 3-4 hours post dosing but drinking water was provided ad libitum.
- Housing: The animals were housed individually in polycarbonate cages.All cages were provided with corn cobs (Sparconn Life Sciences,Bangalore) SPAR – 24/ 2013.
- Diet (e.g. ad libitum): All animals were provided conventional laboratory rodent diet (Nutrivet Life Sciences, Pune) ad libitum. Batch No.: 400012.
- Water (e.g. ad libitum): Aqua guard filtered tap water was provided ad libitum via drinking bottles
- Acclimation period:All animals were acclimatized to the test conditions for 5 days prior to administration of the test item.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature was maintained at 21.10 to 24.40 degree centigrade.
- Humidity (%): Room humidity was maintained at 36.60% to 57.30%.
- Air changes (per hr): More than 12 changes per hour
- Photoperiod (hrs dark / hrs light): An artificial light and dark cycle of 12 hours each was provided to the room.

Administration / exposure

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: over clipped dorsal area of rat skin. Approx. 10% of body surface area of rat
- % coverage: porous gauze dressing
- Type of wrap if used: The test site was further covered in a suitable manner to retain the gauze dressing

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, At the end of the exposure period, residual test item was removed by using distilled water.
- Time after start of exposure:24hr

TEST MATERIAL
- Concentration (if solution): 2000mg/kgbw
- Constant volume or concentration used: yes

Duration of exposure:

24hr

Doses:

2000mg/kg bw

No. of animals per sex per dose:

Total : 10
male : 5
female: 5

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:daily
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs:After test item administration, individual animals were frequently observed at 1, 2, 3 and 4 hours post dosing on day 0 (day of dosing). Subsequently, all animals were observed once a day during the 14 day observation period.
Local Signs/Skin Reactions: All animals were observed once daily during days 1-14 (in common with clinical signs).
body weight:All rats were weighed on days 0 (prior to dosing), 7 and 14.
Mortality:Animals were observed twice daily for any mortality during the experimental period
Pathology:At the end of 14 day observation period, all the surviving rats were euthanised by overdose of CO2 and subjected to gross pathology examination, for external and internal observations.

Statistics:

No statistical analysis was performed since the study was terminated with limit test

Results and discussion

Preliminary study:

not specified

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed

Mortality:

No mortality was observed at limit dose of 2000 mg/kg body weight of test item during the 14 day observation period

Clinical signs:

other: No systemic or local signs of toxicity were observed at limit dose of 2000 mg/kg body weight of test item during the experimental period

Gross pathology:

The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality

Other findings:

not specified

Any other information on results incl. tables

Individual Animal Body Weight (g) andBody Weight Changes(%)

 

Dose:2000 mg/ kg bodyweight                                                                             

 Density:0.9407

Animal No.	Sex	Dose (ml) Applied*	Body Weight (gram)			Body Weight Change (%)
			Day 0	Day 7	Day 14	Day 0-7	Day 0-14
1	Male	0.49	229	247	256	7.86	11.79
2		0.50	235	256	278	8.94	18.30
3		0.49	230	245	272	6.52	18.26
4		0.53	247	262	296	6.07	19.84
5		0.56	262	289	310	10.31	18.32
6	Female	0.51	241	240	258	-0.41	7.05
7		0.43	203	205	211	0.99	3.94
8		0.43	203	206	213	1.48	4.93
9		0.50	236	241	245	2.12	3.81
10		0.48	226	231	232	2.21	2.65

Key:* = Based on density of test item and day 0 body weight taken prior to dose application.

Individual Animal Clinical Signs and Symptoms

 

Dose:2000 mg/kg body weight

Animal No.	Sex	Hour(s) - Day 0				Day
		1	2	3	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
1	Male	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
2		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
3		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
4		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
5		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
6	Female	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
7		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
8		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
9		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
10		1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1

Key: 1 = Normal

Individual Animal Mortality Record

 

Dose:2000 mg/kg body weight

Animal No.	Sex	Days of Observation (0 to 14)
		Morning Observations	Evening Observations
1	Male	No mortality and morbidity	No mortality and morbidity
2		No mortality and morbidity	No mortality and morbidity
3		No mortality and morbidity	No mortality and morbidity
4		No mortality and morbidity	No mortality and morbidity
5		No mortality and morbidity	No mortality and morbidity
6	Female	No mortality and morbidity	No mortality and morbidity
7		No mortality and morbidity	No mortality and morbidity
8		No mortality and morbidity	No mortality and morbidity
9		No mortality and morbidity	No mortality and morbidity
10		No mortality and morbidity	No mortality and morbidity

Summaryof Animal Body Weight (g) and Body Weight Changes (%)

 

Dose:2000 mg/kg body weight

Sex		Body Weight (gram)			Body Weight Changes (%)
		Day 0	Day 7	Day 14	Day 0-7	Day 0-14
Male	Mean	231.20	242.20	257.10	7.94	17.30
	SD	18.12	25.01	33.03	1.74	3.15
	n	10	10	10	5	5
Female	Mean	221.80	224.60	231.80	1.28	4.48
	SD	17.99	17.87	20.29	1.07	1.65
	n	5	5	5	5	5

Keys:SD= Standard deviation, n = Number of animals

Gross Necropsy Observation

 

 Dose:2000 mg/kg body weight                                               

Mode of Death:Terminal Sacrifice

Animal No.	Sex	Gross Observation
		External	Internal
1	Male	No abnormalities detected	No abnormalities detected
2		No abnormalities detected	No abnormalities detected
3		No abnormalities detected	No abnormalities detected
4		No abnormalities detected	No abnormalities detected
5		No abnormalities detected	No abnormalities detected
6	Female	No abnormalities detected	No abnormalities detected
7		No abnormalities detected	No abnormalities detected
8		No abnormalities detected	No abnormalities detected
9		No abnormalities detected	No abnormalities detected
10		No abnormalities detected	No abnormalities detected

Applicant's summary and conclusion

Interpretation of results:

other: Not classified

Conclusions:

The LD50 value was considered to be >2000 mg/kg body weight, when female wistar rats were treated with the given test chemical by dermal application.

Executive summary:

Acute dermal toxicity study of the given test chemical was performed as per OECD No.402 in Wistar Rats. Five male and five female healthy young adult rats were randomly selected. Rats free from injury and irritation of skin were selected for the study. Twenty four hours prior to dermal application of test item, approximately 10% of body surface area of each rat was clipped. A limit dose of 2000 mg/ kg body weight of test item was applied by single dermal application and observed for 14 days after treatment. On test day 0, as such amount of test item, calculated based on density (0.9407) and body weight was applied directly on the intact skin of clipped area of rats; the surgical gauze patch was put on to the intact skin of clipped area. This gauze patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with non-irritating adhesive tape. After the 24-hour application period, the dressings were removed and the skin was gently wiped with distilled water. The skin reactions were assessed. The animals were observed daily for mortality and clinical signs, during the acclimatization period. All animals were observed for clinical signs at approximately 1, 2, 3 and 4 hours after treatment on day 0 and once daily during test days 1‑14. Mortality was recorded after application on test day 0 and twice daily during days 1-14 (at least once on the day of sacrifice). Local signs / Skin reactions were observed daily from test days 1-14 (in common with clinical signs). Body weights were re­corded on day 0 (prior to application) and on day 7 and 14. All animals were necropsied and examined macroscopically. No mortality was observed in any animal till the end of the experimental period. No clinical signs and any skin reaction were observed throughout the experimental period in all treated animals. The male and female animals were observed with body weight gain compared to day 0 throughout the experiment. The external and internal gross pathological observation of all terminally sacrificed animals did not show any pathological abnormality. Hence the LD50 value was considered to be >2000 mg/kg body weight, when female wistar rats were treated with the given test chemical by dermal application.