dec-9-en-2-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 April to 17 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD TG 407 without any deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ICH Guidelines, in accordance with the mutually agreed protocol (No. P/8550/SOR-28/08).

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed 15 March 2007.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

dec-9-en-2-one

EC Number:

685-618-4

Cas Number:

35194-30-0

Molecular formula:

C10 H18 O

IUPAC Name:

dec-9-en-2-one

Test material form:

liquid

Details on test material:

- Physical state: colorless liquid
- Storage condition of test material: Room temperature, darkness.

Specific details on test material used for the study:

- Characteristics: Pale yellow liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The albino rat is one of the recommended rodent species for oral studies as per OECD guidelines. The SD strain is used because of its availability and of the existing historical data base for comaparative evaluation.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: INTOX PVT. LTD.
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 6 - 8 weeks old
- Weight at study initiation: 140 - 166 g (males) and 130 - 159 g (females)
- Fasting period before study: no data
- Housing: groups of five animals of similar sex in polypropylene cages with stainless steel grill tops, facilities for food and water bottle, and bedding of steam sterilized clean paddy husk. Animals were housed in room number AR-10.
- Diet (e.g. ad libitum): Nutrilab brand extruded pelleted rodent feed manufactured by M/s Tetragon Chemie Pvt. Ltd., Bangalore, ad libitum.
- Water (e.g. ad libitum): potable water passed through' Aquaguard"water filter, ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15 times/hour
- Photoperiod (hrs dark / hrs light): 12 h dark / 12 h light

IN-LIFE DATES: From: 05 May to 17 June, 2008.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing formulations of test article were prepared shortly before dosing on each day. The test substance was diluted with corn oil. Formulations for the doses of 250, 500 and 1000 m/Kg bw/day were prepared at the concentration of 50 mg/mL, 100 mg/mL and 200 mg/mLof the test substance to allow a constant dosage volume of 5 mL/Kg bw.
VEHICLE
- Justification for use and choice of vehicle : had been prooved as appropriate in previous studies (7 days repeated dose toxicity study).
- Amount of vehicle: dose volume = 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test formulations were subjected for verification of concentration twice during the study period. The concentration of test article measured in the formulations prepared for dosing on day 5 and day 21 of study, was close to intended dose and was within acceptable limits.
As the test article was freely miscible in the vehicle i.e. corn oil, homogeneity of test article in dosing formulations was not required.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 6/High dose Recovery

Doses / concentrations 2

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control group 1 (5 mL/kg corn oil)

Doses / concentrations 3

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control group 2/Recovery (5 mL/kg corn oil)

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Group 3/Low dose

Doses / concentrations 5

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Group 4/Mild dose

Doses / concentrations 6

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 5/High dose

No. of animals per sex per dose:

5 animals/sex/dose for the following groups : 0, 250, 500 and 1000 mg/kg body weight/day
Additional satellite group of 5 animals/sex/dose for the following groups: 0 and 1000 mg/kg body weight/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on results of 7 day dose range finding study conducted separately.
- Rationale for animal assignment: random
- Post-exposure recovery period in satellite groups: 14 days after the end of administration.

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of administration and once every week during the administration period and recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded before initiation of treatment (day 0), weekly during treatment, recovery period and before necropsy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: was recorded weekly during treatment and recovery period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of treatment (day 29/30) and at the end of recovery period (day 43).
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals: all animals
- Parameters checked in Table 7.5.1/2 were examined.

CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood: at termination of treatment (day 29/30) and at the end of recovery period (day 43).
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals: all animals.
- Parameters checked in Table 7.5.1/2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at termination of the treatment period (day 27/28) and at the end of reversal period (day 42).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: yes
- Parameters checked in Table 7.5.1/2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the fourth week of treatment (day 23/24)
- Dose groups that were examined: 0, 250, 500 and 1000 mg/kg bw/day
- Battery of functions tested: grip strength / locomotor activity and Sensory function test.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Blood samples were collected from animals on the next day of the end of treatment period and on the next day of the recovery period, after which the animals were euthanized by exsanguination, and macroscopically observed for the body surface, mucous membrane of the opening, and visceral organs.

Organ weight:
Weight of the following organs were weighed (absolute weight): kidneys, liver, adrenals, testes, epididymides/uterus, thymus, spleen, brain, heart and lung.
Values of these organ weights as percent of body weight, on the day of necropsy, were also estimated (relative organ weights).


HISTOPATHOLOGY: Yes. Tissues that were subjected to microscopic examination in this study are: Brain-cerebrum, cerebellum, midbrain, spinal cord, eye, thyroid, parathyroid, spleen, thymus, adrenals, pancreas, trachea, lung, heart, aorta, oesophagus, stomach, duodenum, jejunum, terminal ileum, colon, rectum, liver, kidneys, urinary bladder, prostate, seminal vesicle, epididymides, testes, ovaries, uterus, skin, sciatic nerve, bone marrow, mammary gland (females) mesenteric lymph node and skeletal muscle. These tissues were embedded in paraffin wax, sectioned at five micrometers and stained with haematoxylin and eosin.
Histopathological examination was conducted of all animals from the control and the high dosage level groups sacrificed at termination. As no treatment related changes were observed in high dose group animals, histopathological examination was not conducted on animals from lower dose groups and recovery groups.

Statistics:

The body weight, haematology , clinical chemistry and organ weight data of different groups was compared by Bartlett's test for homogeneity. The data with homogeneous intra-group variances was subjected to one-way analysis of variance (ANOVA-Snedecor and Cochran, 1980). When 'F' value was significant, Dunnett's pair wise comparison (Scheffe, 1953) of means of treated groups with control mean was doen individually.
All analysis and comparisons were evaluated at 5% (P<0.05).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test article-related changes in hematology.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test article-related changes in clinical biochemistry.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes were observed in the test items in any treatment group throughout the administration period and the recovery period.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Relative weights of liver were significantly higher in group 5 males as compared to control animals. The changes observed were not considered to be the treatement related due to lack of dose related effect and no corresponding histological changes were observed in these organs.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY:

There was no incidence of any treatment related mortality amongst the rats treated with test article at 250, 500 and 1000 mg/kg bw/day. No mortality was observed in vehicle control group animals. All treated animals survived throughout the treatment period of 28 days and also during recovery period.
In animals treated at and up to the dose of 1000 mg/kg bw/day, the daily general clinical examinations and weekly detailed clinical examinations did not reveal any treatment related incidence of clincial abnormalities.

BODY WEIGHT AND WEIGHT GAIN:

Body weight gain by male and female rats treated at 250, 500 and 1000 mg/kg bw/day was found to be comparable to that by the control rats throughout the treatment period.
Also during recovery period, the weigyt gain by rats from high dose group was found to be comparable to that by the control group rats.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):

The values of average daily food consumption by male and female rats treated with test article and up to 1000 mg/kg bw/day, remained comparable to those of the control group rats.
The average daily food consumption per rat per day, computed over the period of 4 weeks, by male rats receiving test article at 250, 500 and 1000 mg/kg bw/day was 99%, 98% and 96% respectively of that by control rats. Similarly, the average daily food consumption by female receiving the test article at 250, 500 and 1000 mg/kg bw/day was 104%, 104% and 102% respectively of that by control rats. After cessation of treatment the values of food intake during the recovery period were found to be comparable among the vehicle control and the high dose groups.

HAEMATOLOGY:

At the end of treatment period and also at the end of recovery period, the group mean values of haematological parameters such as haemoglobin, haematocrit, total and differentila leucocyte counts, total RBC count, RBC indices, platelet count and reticulocyte count of male and female rats, treated with test article at and up to the level of 1000 mg/kg bw/day, were found to be comparable with those of the control animals.
Although a statistically significant change seen in few parameters, but it was not considered to be the treatment related change due to lack of dose related effect. With the exception of few individual values, all mean and individual values obtained were within the range of historical control values. Few individual deviations were noted both in treatment and control groups and hence none were considered of toxicological significance.

CLINICAL CHEMISTRY:

The test article, up to the dose level of 1000 mg/kg bw/day, did not induce any changes in the plasma levels of total protein, albumin, globulin, alanine, aminotransferase, asparate aminotransferase, alkaline phosphatase, glucoe, urea nitrogen, creatine, cholesterol, triglycerides, total bilirubin, sodium, potassium, calcium and phosphorous in male and female rats, at termination of the treatment and at the end of recovery period.
Although a statistically significant change seen in few parameters, but it was not considered to be the treatment related change due to lack of dose related effect. It was noted that, with the exception of few individual values, all mean and individual values obtained were within the range of historical control values. Few individual were noted both in treatment and control groups and hence none were considered of toxicological significance.

URINALYSIS:

The data on urinalysis evaluated at termination of treatment and also at the end of recovery period did not indicate any abnormalities due to treatment with the test article. The data in treated animals and control animals was found to be comparable.

NEUROBEHAVIOUR:

The neurological examinations (functional observations) conducted in the fourth week of the study did not reveal any remarkable and treatment related incidence of cilinical abnormalities. Also no findings, indicative of a neurotoxic potential of the test article, were encountered during these examinations.

ORGAN WEIGHTS:

The values of absolute and relative weights of kidneys, liver, adrenals, testes, epididymides, uterus, thymus, spleen, brain, heart and lungs of male/female rats treated with the test article and up to 1000 mg/kg bw/day were found to be comparable to those of the control group rats at termination of the treatment and at the end of recovery period.
Statistically significant changes were observed in a couple of organs. Absolute weights of liver and spleen in recovery group females were lower as compared to control groups. Relative weights of liver were significantly higher in group 5 males as compared to control animals. The changes obserevd were not consdered to be the treatment related due to lack of dose realted effect and no corresponding histological cahnges were observed in these organs.

GROSS PATHOLOGY:

The test article, at and up to the dose level of 1000 mg/kg bw/day, did not induce any treatment related gross pathological alterations in any of the tissues of treated rats, as evident at the detailed necropsy examination carried out at terlination of the study and also at the end of recovery period.

HISTOPATHOLOGY:

Histopathological examinations of the protocol listed tissues of males and females rats from the control group and those treated with the test article at the dose level of 1000 mg/kg bw/day did not reveal any significant and treatment histopathological alterations.
Few incidental and non-specific lesions obserevd were chronic inflammatory foci in liver, lymphocyte aggregation, foam cells and haemorrhages in lungs, submucosal lymphocytic infiltration in stomach, colon rectum and trachea, glandular cyst in stomach and dilatation of uterus. All the microscopic changes noticed in this study appeared to be incidental as their frequencies were similar to control group animals. Some of the changes were again considered as incidental as their frequency is very low and not dose dependent.

Effect levels

Key result

Dose descriptor:

NOEL

Remarks:

Systemic effects

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No effects were observed up to 1000 mg/kg bw/day

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 7.5.1/3: Results of analysis of test article concentration in formulation

Study Day	Concentration of the test substance in Formulation for Target Concentration
	Dose (mg/kg bw)	Target concentration of active ingredient (mg/mL)	Result active ingredient (mg/mL)
5	250	50	52
	500	100	102
	1000	200	199
21	250	50	46
	500	100	99
	1000	200	203

Applicant's summary and conclusion

Conclusions:

Under the test condtions, the no-observed-effect-level (NOEL) of the test substance in male and female rats, following oral administration for 28 days was found to be more than 1000 mg/kg bw/day. No effects were observed up to 1000 mg/kg bw/day. The test substance is not classified for damage to organs through prolonged oral dose repeated exposure according to the criteria of the Annex I of the Regulation (EC) No 1272/2008 (CLP) and the GHS.

Executive summary:

In a repeated dose toxicity study performed according to the OECD test guideline No. 407 and in compliance with GLP, the test substance was administered daily by oral gavage to Sprague Dawley rats of both sexes at dose levels of 250, 500 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, corn oil at 5 ml/kg, only. The groups comprised 5 animals per sex which were sacrificed after the end of the treatment period. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. The rats were examined daily for signs of toxicity, morbidity and mortality. They were subjected to detailed clinical examination before initiation of the study and weekly thereafter during the treatment period, reversal period, and at termination. In the fourth week of treatment, they were additionally examined for assessment of sensory reactivity, assessment of grip strength and motor activity. Body weight and food consumption were recorded weekly. Laboratory investigations were performed on blood and urine at termination of the study. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

 There was no incidence of treatment related mortality in rats treated with test article at 250, 500 or 1000 mg/kg bw/day. The test article did not induce any treatment related clinical abnormalities in rats treated up to the dose of 1000 mg/kg bw/day. No mortality or abnormal clinical signs were observed in animals treated with vehicle control article.

The neurological examination did not rveal any neurotoxic potential of the test article up to its highest level of exposure.

Test article did not have any adverse effect on the body weight gain and average daily food consumption by the male and female rats treated up to the dose level of 1000 mg/kg bw/day.

No effect on the hematological and the clinical chemistry parameters of male and female rats treated at and up to the dose level of 1000 mg/kg bw/day.

The test article, at and up to 1000 mg/kg bw/day, did not affect the qualitative and microscopic urinalysis parameters of the treated rats.

No significant alterations in the absolute and relative organ weights. No treatment related gross and microscopic pathological alterations in the tissues of male nad female rats treated up to the level of 1000 mg/kg bw/day.

Based on the results of this study, the no-observed-effect-level (NOEL) of the test substance in SD rats, following oral administration for 28 days was found to be more than 1000 mg/kg bw/day. 

Under the test conditions, the test substance is not classified according to the criteria of the Regulation (EC) No.1272/2008 (CLP) and to the GHS.

This study is acceptable and satisfies the requirement for repeated dose toxicity endpoint.