Registration Dossier

Administrative data

Endpoint:
short-term repeated dose 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 study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Principles of method if other than guideline:
Repeated dose dermal toxicity study was performed to determine the toxic nature of the test chemical upon repeated exposure by dermal route
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: 2, 2, 2-trichloro-1-phenylethyl acetate
- Molecular formula: C10H9Cl3O2
- Molecular weight: 267.54
- Substance type: Organic
- Physical state: Solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female

Administration / exposure

Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
DOSE FORMULATION
Dose preparation of the 2, 2, 2-trichloro-1-phenylethyl acetate was done freshly, few minutes prior to dosing. The test substance 2,2,2-Trichloro-1-phenyl ethyl acetate was mixed with distilled water to obtain paste.

ADMINISTRATION OF TEST ARTICLE
The test substance 2, 2, 2-trichloro-1-phenylethyl acetate was applied uniformly daily over an area approximately 10 per cent of the total body surface area in graduated doses (125, 500 & 1500 mg/kg b.wt) for a period of 28 days. Between applications the test substance is held in contact with the skin with a porous gauze dressing and non-irritating tape. The test site was covered in a suitable manner to retain the gauze dressing and test substance and ensure that the animals cannot ingest the test substance.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily (6 hours per day on a 7-day per week )
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 125, 500 & 1500 mg/kg b.wt
Basis:
nominal per unit area
No. of animals per sex per dose:
Total nos of animal 25 male & 25 female
Group 1 - 5 males and 5 females
Group 2 - 5 males and 5 females
Group3 - 5 males and 5 females
Group 4 - 5 males and 5 females
Group 5 - 5 males and 5 females (reversal group)
Control animals:
yes, concurrent vehicle
Details on study design:
STUDY DESIGN:
The toxicity of the test compound following dermal application was assessed. Five male & five female rats were used per step for each dose level. The rats were observed for incidence of mortality and signs of intoxication for 28 days after the administration of test article.

Group Dose (mg/kg) Treatment 28-Days
Male Female
Group-I Vehicle control 5 5
Group-II 125 mg/kg b. wt 5 5
Group-III 500 mg/kg b. wt 5 5
Group-IV 1500 mg/kg b. wt 5 5
Group-V 1500 mg/kg b. wt 5 5
Total nos of animal 25 male & 25 female
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS : yes
changes in skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern.

DETAILED CLINICAL OBSERVATIONS: Yes
- The treated animals were closely observed for clinical signs of intoxication, first 4 hours and every 1 hrs interval for 24 hrs after dosing and thereafter twice a day for 28 days. All the rats were observed at least twice daily to observe any clinical signs or behavioral changes. These observations included changes in skin and fur, in the eyes and mucous membranes, respiratory, circulatory, central and autonomic nervous systems, somatomotor activity and Behavioral changes. The clinical sign will be graded as 0 = Normal, + = Mild, ++= Moderate, +++ =High and ++++ = Severe.

DERMAL IRRITATION (if dermal study): Yes / No / No data
- Time schedule for examinations:

BODY WEIGHT: Yes
- The body weight of each rat was recorded before the start of experiment and 7th, 14th, 21st and 28th day of experiment. The mean body weights of different groups and sex were calculated from the individual weights.

ORGAN Weight: Yes
Absolute organ weight
The organ weight of each rat observed on day 28th - Adrenals, Brain, Ovaries, Heart, Testes, Spleen, Kidney, Liver and Lungs

FOOD CONSUMPTION: Yes
- The food intake of each cage was recorded on week prior to the beginning of treatment and 7th, 14th, 21st and 28th day of experiment. The mean value of food intake weekly per cage was calculated.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: All the animals kept overnight fast before blood collection to minimize variability in blood glucose level. The blood sample was collected from each rat on day 28th of treatment form orbital plexus of each animal in sterilized EDTA and heparin vial.
The blood samples were taken from each animal between 7:30 and 10:00 hrs in order to reduce biological variation caused by circadian rhythms.

- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: No data
- Parameters checked : Haemoglobin (Hb) (g/dl), Packed cell volume (PCV) (%), Total red cell count (Total RBC) (x106/cmm), Total white cell count (Total WBC) (x103/cmm), Platelet Count, Total (Platelets) (x103/cmm), Mean corpuscular volume (MCV) (fl), Mean corpuscular haemoglobin (MCH) (pg), Mean corpuscular haemoglobin concentration (MCHC) (g/dl), Clotting time measurement (seconds) was performed manually using standard techniques., Differential WBC counts: Were determined by microscopy of blood smear, stained with Wright's stain, counting 100 cells – Neutrophils (N) % Lymphocytes (L) %, Eosinophils (E) % Monocytes (M) %

CLINICAL CHEMISTRY: Yes
- Total Protein (g/dl) , Total Cholesterol (mg/dl), Albumin (g/dl) , Creatinine (mg/dl), Alanine aminotransferase (ALT) (IU/L) , Blood Urea Nitrogen (BUN) (mg/dl), Aspartate aminotransferase (AST) (IU/L) , Bilirubin (mg/dl), Glucose (mg/dl) , Alkaline phosphatase (ALP) (IU/L)

URINALYSIS: Yes
Urinalysis was performed on all animals (except reversal groups) in last week of the study before termination of the treatment period (week 4). Urinalysis was performed on animals of reversal groups at end of the reversal period.Urine samples were collected using a battery of specially designed stainless steel urine collection cages. Each rat was housed in this cage. Urine samples were collected over a period of 4 hours. Food and water was not offered during this period.
Qualitative tests:
Colour, Appearance, Specific gravity, pH, Protein and/or Albumin, Glucose Ketone, Volume Bilirubin Urobilinogen Occult Blood
Microscopic examination: Epithelial cells (E), Leucocytes (L), Erythrocytes (R), Casts (C), Crystals and other abnormal constituents

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER: No data
Sacrifice and pathology:
Gross pathology-
During necropsy all the organs viz; Adrenals, Brain, Heart, Kidneys, Liver, Lungs, intestine, Spleen, lymph nodes and Testes of body were examined for gross pathological changes.

Histopathology-
Samples of following organs and tissues were processed for histopathological examination Adrenals, Brain, Heart, Kidneys, Liver, Lungs, intestine, Spleen and Testes.
Other examinations:
No data
Statistics:
The data obtained from present investigation were analyzed by One Way ANOVA and Tukey HSD Test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Other effects:
not specified
Details on results:

Body weight:
The body weight of each group recorded on day 7th, 14th, 21st, 28th (post treatment) showed significant decrease in body weight at the tested dose levels 500 & 1500 mg/kg. Whereas, no significant change were recorded at the lowest dose level 125 mg/kg b.wt. Furthermore, reversal group showed significant increase in body weight during reversal period.

Food intake:
The food intake gm/cage recorded on day 7th, 14th, 21st and 28th (post treatment) did not show significant change upto the highest tested dose level 1500 mg/kg b.wt of test compound 2,2,2-Trichloro-1-phenyl ethyl acetate as compared the control group.

Clinical signs:
The test compound 2, 2, 2-Trichloro-1-phenyl ethyl acetate did not produce any clinical signs of toxicity upto the tested dose level 500 mg/kg b.wt in wistar albino rat. Whereas, reduced in cage side activity, respiratory distress and mild degree of convulsion at the tested dose level 1500 mg/kg b.wt. Furthermore, no clinical sign of toxicity was observed in vehicle control group.

Mortality:
The test compound 2,2,2-Trichloro-1-phenyl ethyl acetate did not produces any mortality upto highest tested dose level 1500 mg/kg b.wt in wistar albino rat.

Haematological study:
The haematological parameters RBC(106/µl), Hb (g/dl), PCV (%), Platelet count, MCHC (g/dl) and lymphocyte significant (p<0.05) decreased at the highest tested dose level as compared to control group whereas, WBC (103/µl), MCV (fl), MCH(Pg), neutrophils and Eosinophils showed significant (p<0.05) decrease at the highest tested dose level as compared to control group. Furthermore reversal group exhibited significant change after reversal period of observation.

Clinical Chemistry:
The blood chemistry parameters like blood glucose (mg/dl), Alanine aminotransferase (ALT IU/L), Aspartate aminotransferase (AST IU/L), total bilirubin (mg/dl), Bilirubin Direct (mg/dl), total cholesterol, Creatinine (mg/dl) Alkaline phosphatase (ALP) (IU/L), uric acid and blood urea nitrogen (BUN, mg/dl) showed significant (p>0.05) increase at the highest tested dose level 1500 mg/kg b.wt as compared to control group. Whereas, total protein (gm/dl) and albumin (gm/dl) showed significant (p<0.05) decrease at the highest tested dose level (1500 mg/b.wt) as compared to the control group.

Urine analysis:
The test compound 2,2,2-Trichloro-1-phenyl ethyl acetate showed slight physical change in colour dark yellowish, clarity decreased and pH increases at the highest tested dose level 1500 mg/kg b.wt as compared to the control group. Furthermore, no change was recorded in reversal group. (Table-7)
The test compound did not show any significant chemical change such as glucose, protein and bilirubin etc upto highest tested dose level 1500 mg/kg b.wt as compared to the control group.

Necropsy findings:
The necropsy of each wistar albino rats of all groups were conducted on day 29th congestion in lung not show any gross pathological changes upto the highest tested dose level 1500 mg/kg b.wt of test compound as compared to control group.

Absolute Organ Weight
The organ weight of each rat observed on day 29th Kidney and Lungs Adrenals, Brain, Ovaries, Heart, Testes, Spleen and Liver did not show significant reveal any significant change upto the highest tested dose level 1500 mg/kg b.wt as compared to the control group.

Histopathological finding:
The test compound elicit microscopical changed at the tested dose levels 500 and 1500 mg/kg b.wt in wistar albino rats as Liver showed- Vascular congestion, infiltration of Neutrophils and necrotic changes in hepatocytes, Kidney- Moderate to severe congestion glomerulus, vascular hemorrhage and necroticchanges in tubular epithelium, Adrenal- Spread of lymphoid tissue in red pulp, Spleen-Infiltration of Neutrophils, increase white pulp and Lungs- Mild congestion and infiltration of Neutrophils in alveolar wall

Effect levels

Dose descriptor:
NOAEL
Effect level:
500 other: mg/Kg bw
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

HISTOPATHOLOGICAL STUDIES:

The test compound did not elicit any microscopical changes at the different tested dose level 125, 500 & 1500 mg/kg b.wt (Table-11).

Organs

Histopathological finding

Group-I Vehicle Control

Group-II 125 mg/kg b.wt

Group-III 500 mg/kg b.wt

Group-IV 1500 mg/kg b.wt

Group-V Highest dose reversal group

Liver

NAD

NAD

Mild vascular congestion

Vascular congestion, infiltration of Neutrophils and necrotic changes in hepatocytes

Mild vascular congestion

Kidney

NAD

NAD

Mild congestion and necrotic changes in tubular epithelium

Moderate to severe congestion glomerulus, vascular hemorrhage and necrotic changes in tubular epithelium

Mild congestion and necrotic changes in tubular epithelium

Testes

NAD

NAD

NAD

NAD

NAD

Ovary

NAD

NAD

NAD

NAD

NAD

Adrenal

NAD

NAD

NAD

Spread of lymphoid tissue in red pulp

NAD

Spleen

NAD

NAD

NAD

Infiltration of Neutrophils, increase white pulp

Infiltration of Neutrophils

Lungs

NAD

NAD

NAD

Mild congestion and infiltration of Neutrophils in alveolar wall

Mild congestion in lung

Intestine

NAD

NAD

NAD

NAD

NAD

Brain

NAD

NAD

NAD

NAD

NAD

TABLE- BODY WEIGHT OF RAT (gm)

 

Week

Groups

Group-I

Group-II

Group-III

Group-IV

Group-V

Day 0

206

202.9

212.3

201.9

198.4

1stWeek

220.8

216.2

216.7

202.8

200.3

Average Gain/Loss

7.18

6.55

2.07

0.45

0.96

2ndWeek

236.5

227.1

225.3

206.2

203.6

Average Gain/Loss

14.8

11.9

6.12

2.13

2.62

3rdWeek

248.7

238.6

233.2

210.5

208.0

Average Gain/Loss

20.7

17.6

9.84

4.26

4.84

4thWeek

258.4

251.7

241.2

212.5

211.6

Average Gain/Loss

25.4

24.1

13.6

5.25

6.65

5thWeek

-

-

-

-

216.4

Average Gain/Loss

-

-

-

-

9.07

6thweek

-

-

-

-

224.2

Average Gain/Loss

-

-

-

-

13

TABLE: SUMMARY OF CLINICAL SIGNS

Parameters

Groups

Group- I Vehicle control

Group- II 125 mg/kg b.wt

Days

Days

Day 0

 

1

2

3

4

5

6

7

1

2

3

4

5

6

7

Mortality

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Clinical signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1stweek

Mortality

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Clinical signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2ndweek

Mortality

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Clinical signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3rdweek

Mortality

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Clinical signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

4thweek

Mortality

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Clinical signs

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Applicant's summary and conclusion

Conclusions:
The No observed adverse effect level (NOAEL) of the test chemical in rats, following dermal application for 28 days was found to be more than 500 mg/kg body weight.
Executive summary:

The Sub-acute Dermal toxicity study of the test chemical was conducted in wistar albino rats. Fifty healthy wistar albino rats were acclimatized for standard laboratory condition for period of one week. After acclimatization animal were divided into five groups (one control and four treated) each having five male and five female. All the animals were prepared 24 hrs prior to application of test compound. The test substance applied uniformly 125, 500 and 1500 mg/kg b.wt for at least 6 hours per day on a 7-day per week basis. A reversal group was also maintained in same manner at the highest test dose level 1500 mg/kg b.wt for period of 42 days.All the parameters were examined. No adverse effect on general health, growth, behavioural, neurological, haematological, clinical chemistry and urinalysis parameters, organ weights and gross and microscopic changes of the tissues/organs of the rats treated up to the dose level of 500 mg/kg body weight. Based on the findings of this study, the no observed adverse effect level (NOAEL) of the test chemical in rats was considered to be 500 mg/kg body weight and the No Observed Effect Level (NOEL) was considered to be 125 mg/kg b.wt.